key: cord-0046168-k0uby99n
authors: Nabel, Gary J.
title: The development of gene-based vectors for immunization
date: 2020-06-22
journal: Vaccines
DOI: 10.1016/b978-1-4160-3611-1.50066-0
sha: 7bd6e3b10fab47eb6060edd3cba1befda32e5624
doc_id: 46168
cord_uid: k0uby99n

nan

The development of DNA vaccines has evolved since the initial description of the ability of naked DNA to support gene expression after intramuscular injection. 3, 4 The concept behind these vaccines is that expression of specifi c viral genes under the control of eukaryotic enhancer-promoters and polyadenylation signals allows appropriate expression of specifi c viral gene products which can be processed and presented as foreign antigens. The genes encoded by DNA vaccines can be readily modifi ed and regulatory sequences can be adjusted to optimize level, duration and potency of the immunogen. 5 When injected into muscle, DNA is taken up by cells surrounding the injection site and internalized. After uptake and transport to the nucleus, transcription, translation, and post-translational modifi cation allow for the synthesis of a specifi ed gene product. In contrast to inactivated virus particles or recombinant protein vaccines produced in bacteria, yeast or mammalian cells, proteins expressed from gene-based DNA vaccines are more likely to assume a native conformation, and their expression within cells allows for more native processing and presentation of antigens that can stimulate CD4 and CD8 responses in vivo. Because they are in native form, the antibodies generated against these immunogens are theoretically more likely to be cross-reactive with native viral gene products from the pathogen. In addition, because DNA is rapidly degraded in the body, the plasmid DNA vaccines can provide an advantage in terms of safety, in contrast to live-attenuated viruses, with the possibility of chronic infection and immune stimulation.

The application of DNA-based genetic immunization has now been demonstrated in a variety of animal models. [6] [7] [8] In addition, in animals it has been shown to be effective in inducing protective immunity against infl uenza virus, 4 malaria, [9] [10] [11] [12] tuberculosis, 13 Ebola virus, 14 rabies, 15 lymphocytic choriomeningitis virus, 16,17 herpes simplex virus 18 and lentiviruses 19 in addition to other pathogens. Studies in nonhuman primates and humans have indicated that the approach is effective in inducing CTL responses ( 20 and Graham et al, unpublished data) .

DNA vaccines have also been used successfully alone or in combination with other gene-based approaches to develop protective immunity against pathogenic SHIV and SIV challenge. 19,21-27 Various prime-boost strategies have utilized Gary J. Nabel Vaccines can confer immune protection against infectious agents through divergent arms of the adaptive immune response. The elaboration of antibodies through the humoral immune system has been highly effective in the neutralization of many bacteria, viruses, fungi and parasites. The cell-mediated immune response also plays a major role in containment of infectious agents. T lymphocytes comprise a diverse set of cells, and their functional activity is dependent upon helper T cells, which elaborate a variety of cytokines and stimulate B cells to produce antibodies and induce the formation of cytolytic T lymphocytes (CTL). CTLs recognize processed antigen on major histocompatibility complex (MHC) molecules and lyse infected cells.

Both humoral and cellular immunity are the targets of vaccine-induced immunological responses, each with its own effector functions that can inactivate pathogens in different ways (Table 62-1) . While the humoral immune response is wellknown to confer protection, the role of CTL in protective immunity against viral infections has been recognized more recently. The function and specifi city of these cells has provided the foundation for understanding MHC restriction and its importance in protection against viral infection. 1,2 Such cellular immune responses help control infectious diseases, particularly when it is diffi cult to generate neutralizing antibodies, as in HIV/AIDS, malaria and tuberculosis. Humoral immunity is more readily induced with purifi ed proteins or inactivated viruses together with appropriate adjuvants; gene-based vaccines appear to be particularly effective at inducing T cell responses, both CD4 and CD8. At the same time, some genebased vaccines can induce humoral immune responses when used with specifi c vectors or in specifi c prime-boost combinations.

The majority of adjuvants that have been utilized in vaccine development have affected humoral immunity and appear to enhance antibody responses without inducing cellular immunity. In contrast, the gene-based delivery of vaccine vectors can stimulate both humoral and cellular immunity, thus providing greater selective pressure on infectious agents in vaccines. In this chapter, the major genebased vaccines progressing into clinical trials are summarized, together with the advantages and disadvantages of each individual vector and their infl uence on different effector arms of the immune system. While there is considerable experience with inactivated viruses and protein-based vaccines, the development of gene-based vaccine vectors is only beginning. The advantages of their ability to induce cellular immunity, immunogenicity, safety, mode of antigen presentation, and other attractive features are countered by limitations in knowledge about clinical effi cacy, production methodologies, DNA vaccination as the initial vaccine constituent and replication-defective viral vectors, including modifi ed vaccinia Ankara virus (MVA), 21,28 rAd 22,23,27,29 or proteins to boost the initial response. This approach avoids repeat exposure to the same viral vector and takes advantage of the ability of DNA vaccines to evade anti-vector immunity and to induce immune responses to subdominant T cell epitopes that might otherwise not be stimulated. In the case of DNA/rAd prime-boost vaccination, this vaccination approach induces greater breadth of the CD4 response which in turn supports a greater magnitude CD8 response that does not change in specifi city. 30 There is one Phase II study with a DNA prime-rAd boost vaccine for HIV infection that has been conducted internationally.

A potential limitation of DNA vaccine technology is its low immunogenicity in humans. Though immune responses can be induced in primates, their potency appears reduced relative to rodent species. In part, this may be due to the relatively lower dose of DNA in mass per body weight or surface area; however, improvements in expression vector technology and in the development of DNA adjuvants offer the potential for improvements in this area ( Fig. 62-1 ). One successful approach has involved improvement of transcriptional and translational effi cacy using modifi ed codons preferred in the host species. 31, 32 In addition, the development of improved enhancer/ promoter regions can allow for even higher expression 5 and these vaccines have advanced into multiple human Phase I studies, alone or in combination with other gene-based vectors. Advancements of this approach for human use will require further improvements, both in delivery technology and DNA adjuvants, of which some representative approaches are described ( Fig. 62-1 ).

Advances in molecular virology have facilitated an understanding of the regulation of viral replication, gene expression, and molecular pathogenesis. At the same time, this understanding has enabled the development of novel viral vectors useful for vaccination. A variety of such vectors have now been advanced in preclinical and clinical studies ( Fig. 62-1 ). Depending on their ability to target antigen presenting cells, ability to develop packaging lines, inherent immunogenicity of both the vector and insert, and other factors (Table 62 -2), these viral vectors are helping to improve vaccine effi cacy in a variety of infectious disease models. The properties of the more promising vectors and current progress in their development are summarized in the following sections.

Among the viral vectors that have shown promise for their ability to elicit protective immunity, recombinant adenoviral vectors (rAd) have now demonstrated immunogenicity and protective immunity in a variety of animal models. Similar to DNA vaccines, these vectors transduce cells which can synthesize native gene products and appear to be quite potent in their ability to induce not only helper but specifi cally cytolytic T cell immunity; from 45-90% in various human studies. The majority of clinical vectors have been derived from adenovirus serotype 5 (Ad5), although there are more than 51 known human serotypes in six subfamilies (A-F). Ad5 is derived from the C subfamily and is the most common and best-studied serotype; however, the relatively high prevalence of immunity to Ad5 in human populations may pose limitations to the use of these vectors. Pre-existing anti-Ad5 immunity may inhibit the response to rAd5 vaccine immunization. For this reason, alternative serotypes and chimeric vectors have been developed to circumvent this potential limitation. The attraction to rAd5 for immunization has followed from its success with a variety of preclinical animal models and Phase I/II human trials. With respect to animal models, the replication-defective adenovirus has been shown to elicit potent immune responses and protection against Ebola virus, either administered alone as a single injection or in prime-boost combinations. 29,33 It is interesting to note that the prime-boost approach induces more potent and durable immunity suitable for a preventive vaccine, while a single rAd vaccination induces a more rapid response that is suffi cient for protection ( Fig. 62-2 ). This latter approach may be useful in containing acute outbreaks of Ebola infection and could be applicable to other pathogens. 33 In addition, both recombinant Ad5 vaccines, as well as DNA prime/recombinant Ad5 boost combinations, have been shown to confer partial protection in rhesus macaques against multiple HIV isolates, including SHIV-89.6P, 22,23 SIVmac239 25 and SIVmac251. 24,26,27 Replication-defective adenovirus has also been used in a variety of additional animal models of infectious disease, including plague, anthrax, infl uenza and malaria. 34 Phase I and II clinical studies with replication-defective adenoviral vectors for HIV-1 have undergone analysis independently by the Merck research laboratories and the NIH Vaccine Research Center in NIAID. The clinical utility of these vaccines has yet to be defi ned; however, the preliminary data suggest that rAd5 vaccines elicit potent cellular immune responses in humans. 25 In addition, the DNA prime-rAd5 boost combinations appear to promote even further stimulation, which has proven more effi cacious in animal models of SIV challenge. A more comprehensive Phase IIB clinical trial has begun and should provide information regarding potential effi cacy of the Merck vaccine. In addition, the VRC DNA-rAd5 vaccine has completed Phase II testing and may undergo effi cacy testing in the near future.

The effect of pre-existing antivector immunity and alternative adenovirus serotypes Despite the ability of rAd5 to induce potent and sustained immune responses against a variety of infectious pathogens, concerns remain that preexisting immunity against rAd5 may compromise its effi cacy. This immunity has been found in particular in certain regions of Africa, where Ad5 seroprevalence is greater than 90% with a high degree of neutralizing antibody. While both cellular and humoral immune responses contribute to anti-Ad5 immunity, it is likely that the Ad5 neutralizing antibodies play the major role in suppressing rAd5-induced immunogenicity, and such immune responses have been observed in humans. This pre-existing immunity can reduce the immunogenicity of Ad vaccines in mice, 35, 36 rhesus monkeys 37 and potentially in humans. 38, 39 But it is not clear that pre-existing immunity in humans will block vaccine immunogenicity. The reduction in the Gag-specifi c response induced by rAd5 in Ad5 seropositive recipients seen in the initial Merck rAd5 HIV vaccine trial was less striking when the expression and immunogenicity of the vector were improved. Similarly, in VRC trials of DNA priming followed by rAd5 boosting, signifi cant immune responses are observed in rAd5 seropositive individuals.

Several strategies have been developed to overcome the potential problem of rAd immunity. Novel methods to deliver existing recombinant Ad vectors are being explored. For example, it is possible that the administration of higher doses of recombinant Ad5 vectors may overcome anti-Ad5 immunity, although this strategy may be limited by increased toxicity with dose escalation. Ad boosting after DNA priming may potentially overcome its immunosuppression, too. 35, 36 The effi cacy of this approach in humans remains to be determined. Finally, the administration of Ad5 vectors through mucosal routes may help to circumvent this problem. 40 However, the safety of this approach, particularly for intranasal delivery, has yet to be determined. 41 In addition, several investigators have explored the possibility of coating rAd5 particles with chemicals such as polyethylene glycol that may block access of antibodies to the viral surface. 41a Alternative approaches to evasion of Ad5 immunity include engineering of the vectors to evade dominant Ad5 immune responses. A variety of chimeric fi ber or hexon proteins have been described that maintain immunogenicity and can evade neutralizing antibodies, both against the fi ber [42] [43] [44] [45] or through the use of hexon chimeras which appear to be the targets of the major neutralizing antibody response. 46, 47 Another approach to antivector immunity involves the development of novel vectors from alternative serotypes. To develop such vectors, investigators have evaluated rAd vectors from low seroprevalence human adenoviruses, as well as from nonhuman primates. Recombinant Ad vectors from human serotypes have been well described. [48] [49] [50] Seroprevalence of the 51 Ad serotypes suggests that the Ad11 and Ad35 subfamilies as well as adenoviruses from subfamily D, including Ad26, are uncommon in humans 51 and may therefore offer advantages over Ad5 as vectors. Novel vectors based on rAd35 and rAd11 have been developed, and preclinical studies suggest that they are resistant to anti-Ad5 immunity in mice. 52, 53 The utility of these vectors has been compared to rAd5. While some of the alternative vectors show less seropositivity, they are often also less immunogenic in preclinical animal studies; how they are able to perform in human studies compared to Ad5 vectors in the presence of rAd5 immunity has yet to be determined. In addition to the replication-incompetent Ad vectors, replicationcompetent vectors from Ad4 and Ad7 have been used as vaccine vectors, either for immunization against adenovirus infection or as recombinant vectors, for example, against HIV. 54,55 These vaccines offer not only alternative serotypes but also deliver the immune stimulus to the gut mucosa, which may have potentially desirable effects in protection against some diseases. Finally, recombinant Ad vectors have been developed from alternative species, including sheep, pigs, cows and chimpanzees. [56] [57] [58] [59] [60] [61] In conclusion, the immunogenicity of rAd vectors has prompted their development as candidate vaccines for a variety of infectious diseases. These vectors are well tolerated and highly immunogenic at moderate doses. Whether the frequency of preexisting Ad5 immunity may compromise their utility in humans remains to be determined; however, a variety of strategies are under development to overcome this effect should it be found. Novel delivery vectors, as well as molecularly engineered rAd5 with development of alternative Ad serotypes from humans or other species should provide a number of options to expand their use in the future.

The effi cacy of vaccinia virus as a vaccine vector represents one of the most well-documented examples of a vaccination against infectious diseases. Based on safety issues observed in the use of vaccinia strains against smallpox, 62-65 a number of alternative vaccinia virus strains have been developed as immunization vehicles. To avoid these complications, several highly attenuated virus vaccine vectors have been described, as well as avipox and fowlpox vectors. These strains are listed in Table  62 -3. The development of such attenuated vaccinia viruses also promoted their use as delivery vectors for gene products against specifi c pathogens other than smallpox and the use of these vectors has now been explored extensively in a variety of infectious disease models.

One of the two major attenuated strains of poxvirus is modifi ed vaccinia Ankara (MVA), developed by repeated passaging of the Ankara strain on primary chicken embryo fi broblasts. This resulted in the ability of the virus to replicate effi ciently on a variety of non-avian cell types because of multiple genetic changes, which facilitates its propagation and use as a vector. An alternative attenuated strain, the New York vaccinia strain, NYVAC, was developed by genetic modifi cation of the viral genome, including the deletion of 18 open reading frames associated with virulence and host range in the Copenhagen strain. 66-69 NYVAC, like MVA, is attenuated in animal models and shows favorable safety and immunogenicity in animals and humans. 67, 70, 71 This virus also shows block at an early stage of replication, though it is able to replicate productively in African green monkey kidney cells and primary chicken embryo fi broblasts (CEF).

The avipox vectors include fowlpox and canarypox as well as ALVAC. ALVAC is derived from a plaque-purifi ed virus isolated from an existing canarypox strain, canapox. 72 ALVAC is able to express inserted transgenes and has been shown to be immunogenic in both animal and early clinical trials. 70, 71, [73] [74] [75] [76] These vectors have been evaluated both alone and in primeboost combinations in a variety of infectious disease and cancer models (reviewed in ref. 70 ). Poxviruses are notable for their large genome size and their ability to express recombinant genes without an effect on their replication capacity. Polyvalent recombinants have been used to immunize experimental animals and have proven useful in a variety of infectious disease models, including rabies, measles, SIV, canine distemper, RSV, 77 In addition, these vectors have been studied in a variety of HIV challenge models, both in preclinical studies and in humans [78] [79] [80] [81] [82] [83] and human studies have been undertaken with vaccinia, 84-92 NYVAC 93-96 and ALVAC. 93, 94, [96] [97] [98] [99] [100] [101] To date, these vectors have shown marginal effi cacy that has limited their ability to be tested for effi cacy in human studies, with CTL response rates generally <35%, although ALVAC-Env(clade E)Gag/Pol(clade B) is currently under evaluation in a Phase III study in Thailand. Such poxvirus vectors have also been evaluated in cancer immunotherapy protocols. While attenuated poxvectors have been evaluated in a variety of human studies, it is clear that it has been more challenging to develop these vaccines for human studies. In part this may be due to the fact that recombinant transgenes represent a small minority of gene products expressed in this otherwise large vector. Thus, there is no certainty that the immune response will be focused to the foreign transgene rather than to gene products synthesized endogenously by the poxvirus. In addition, similar to rAd, the concern of antivector immunity remains for this virus as well, though it may be a lesser concern for canarypox vectors.

Although poxvirus vectors show thermostability, ability to incorporate a large foreign transgene, a lack of persistence or genomic integration, and success in smallpox eradication, the diffi culties in manufacturing virus in high yields from primary chicken embryo fi broblast cells, as well as their antigenic complexity, reactogenicity and poor immunogenicity has limited their utility in human trials. Whether additional modifi cations of these vectors can be made to facilitate human trials remains unknown. If such modifi cations of the vector platform can be achieved, this vector may have an opportunity to contribute to the development of a variety of successful vaccines.

The adeno-associated viruses were defi ned initially by their presence as 'helper' viruses that facilitated the propagation of wild-type adenovirus in cell culture. In contrast to the large genome sizes of rAd and vaccinia vectors, this virus is much more limited in size, with insert size of approximately 5 kb. Similar to other replication-defective viruses, these particles can be produced in packaging lines that provide complementary structural proteins made constitutively by the cell rather than the virus. A variety of serotypes have been defi ned, 102 and an HIV vaccine expressed in AAV2 has been analyzed in Phase I human studies, without evidence of strong immunogenicity. Alternative serotypes, including AAV1, are currently under development and may be assessed both alone and in primeboost combinations for effi cacy in humans.

The alphaviruses represent negative-stranded RNA viruses that can be modifi ed to express foreign recombinant genes rather than produce pathogenic infections often seen with prototypes such as Venezuelan equine encephalitis virus (VEE), 103, 104 Sindbis virus 105, 106 and Semliki Forest virus (SFV). Replication-defective HSV can be produced using packaging cell lines similar to those described for replication-defective rAd5, AAV, or alphavirus vectors. These vaccines have been developed not only to deliver foreign genes as potential immunogens but also as vectors against HSV itself, including both HSV1 and HSV2. 107 More recently, vesicular stomatitis virus, dengue virus type 4, and yellow fever virus have been modifi ed to express heterologous viral genes for vaccines for infectious disease targets including HIV, West Nile virus, fi loviruses and other pathogens. [108] [109] [110] [111] [112] [113] [114] Cell substrates

The progress of more recent viral vectors has been dependent upon the development of appropriate packaging cell lines and cell substrates for viral production. Changes in regulatory requirements that allowed the advancement of transformed cell lines for virus production have proven invaluable in facilitating this effort. For recombinant adenoviral production, the PERC6 and GV11 cell lines have supported production of clinical-grade adenovirus type 5 that have progressed into trials for HIV, Ebola virus and malaria, and are under study for other infectious agents, such as Marburg virus and tuberculosis. Once approved, these cell lines can be used for diverse vectors, and the PERC6 cell line has now been used to develop a number of vaccines, including those for West Nile and infl uenza viruses. In these latter cases, the propagated virus is subsequently inactivated before administration to humans.

For the generation of replication-defective viral vectors, these cell lines allow the production of vectors that can be used in human vaccine studies. Of the viruses developed for such vaccines, representative members, summarized in Figure 62 -1B, include recombinant Ad, poxviruses, measles, Venezualan equine encephalitis (VEE) virus and AAV, all of which have progressed into human trials. The development of transformed and continuously propagatable cell lines, in contrast to the previous standard, avian leucosis free primary chick embryo fi broblasts, represents a major advance in vaccine production technology, largely because such cell lines facilitate the production of replication-defective viral vectors in stably transfected cell lines. Such lines also offer potentially improved yields and stable production capacity. The development of such lines has taken years to implement because of regulatory concerns regarding adventitious agents, tumorigenicity and other safety/consistency considerations. Such oversight and evaluation of the strengths and limitations of these cell substrates continues, 115 based on guidelines several years ago, 116, 117 with an increasing number of such lines becoming better characterized and available.

Because many infectious agents replicate at mucosal membranes and transit through the gastrointestinal tract for primary infection, the ability to elicit effective immune responses at these sites is desirable. A variety of bacteria are able to replicate at mucosal sites of natural infection, and it has been proposed that attenuation of these microorganisms and modifi cation to facilitate the delivery of antigen might allow the development of improved vaccines to protect against pathogens that enter through the mucosa. Development of live bacterial vectors has therefore focused on both their ability to induce mucosal IgA responses as well as cytolytic T cell responses at mucosal sites. The delivery of antigens into mammalian cells to stimulate antibody responses does not require the types of novel gene-based vaccines summarized in this chapter. On the other hand, the synthesis of proteins within mammalian cells delivered by bacterial vectors has the potential to induce the cellular immunity that is the goal of many gene-based viral and nonviral vaccines. These approaches have been reviewed in detail elsewhere [118] [119] [120] and are summarized briefl y here.

Among the live bacterial vectors used for antigen delivery, there are attenuated mucosal pathogens, such as Listeria monocytogenes, Salmonella, Vibrio cholera, Shigella, Mycobacteria bovis, Yersinia enterocolitica and Bacillus anthracis. In addition, there are commensal strains such as S. gordonii, lactobacilli and staphylococci that have been used for the induction of humoral 62 Chapter 

and cellular responses. For gene-based vaccination, Listeria monocytogenes has been a particular focus of research. This gram-positive intracellular pathogen has been studied as a model to understand class I MHC-restricted immune responses. These responses are normally seen against the bacterial proteins or co-expressed antigens. This microorganism utilizes a specialized system to introduce proteins into cells and facilitate processing and presentation through MHC Class I, and different mutations have been used to develop attenuated strains that retain the ability to deliver antigens. Similarly, salmonella bacterial strains are intracellular pathogens that become restricted to the endosomal compartment of eukaryotic cells where they are resistant to lysis. 121 A variety of mutations have been introduced into salmonella to generate several different live vaccine carriers, and these vaccine prototypes have undergone further development for vaccine delivery. Among the other bacterial carriers, Mycobacteria bovis Calmette-Guerin (BCG) has been a widely used bacterial vaccine; for example, recently this organism has been used to express HIV antigens. 122, 123 In some instances, expression of mammalian genes has required modifi cation of codons more consistent with the host cell type, which has improved immunogenicity. At the present time, however, the ability of such microorganisms to induce cellular immunity has been limited.

An area of intense interest has been the use of live bacterial vectors for the delivery of DNA vaccines. In this instance, the aim is for the bacteria to deliver plasmid DNA into the cytoplasm of infected cells; such organisms as Shigella and Listeria have been used for this purpose. 124, 125 In addition, attenuated Salmonella has been evaluated and has shown some promise in both infectious disease and tumor models in experimental animals. [126] [127] [128] While the use of such bacterial vectors has been attractive in theory, it has been more diffi cult to reduce this method to practice. Among the concerns is the possibility of reversion or reactogenicity of these potentially pathogenic bacteria to wild type forms, the stability of the recombinant bacteria, as well as the possibility that pre-existing immunity from exposure to natural pathogens may limit their infectivity. A variety of host genetic factors can modulate the immune response induced by the bacterial carrier, and variability in the innate immune responses to such pathogens may limit their consistency in vivo. Finally, perhaps the most challenging problem has been the ability to effect a gene transfer from bacteria into mammalian cells. It is likely that very specialized transport pathways are required for the successful implementation of this technology, and additional improvements in the future will be necessary to improve the effi cacy of this approach, which remains limited in its present form.

While substantial work has progressed in animal models of vaccine effi cacy, the ultimate value of gene-based vaccination has yet to be shown in human studies. Several trials using the poxvirus technology have advanced into clinical evaluation. These include canarypox, which has progressed through Phase II studies in the United States for HIV vaccine evaluation, and has advanced into a proof-of-concept effi cacy trial currently in progress in Thailand. In addition, both modifi ed vaccinia Ankara and NYVAC have been evaluated in phase I human studies. Because the production technology for poxviruses is well known, and GMP procedures for amplifi cation of these viruses followed protocols similar to those developed for vaccinia virus, the path into clinical studies has been relatively straightforward, as have the several trials of modifi ed vaccinia Ankara, which has been evaluated both as a vaccine for HIV, alone or in prime-boost combinations, and as a potentially safer next-generation vaccine for smallpox.

Other 129 and a DNA vaccine for infectious hematopoietic necrosis virus, developed by Merial for use in farm-raised fi sh. An additional vaccine is being developed against viral hemorrhagic septicemia virus in farmed salmon. In these studies, a single injection of microgram amounts of DNA induces rapid and long-lasting immune protection. 130 A recombinant yellow fever vaccine has advanced into effi cacy studies as well. 131 The precedent set by these studies provides hope that additional gene-based vaccines will become available for human use and may contribute to the development of protective immunity for a variety of challenging infectious diseases that have thus far eluded the grasp of vaccine-induced immunity. 

Major transplantation antigens, viruses, and specifi city of surveillance T cells

MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specifi city, function, and responsiveness

Direct gene transfer into mouse muscle in vivo

Heterologous protection against infl uenza by injection of DNA encoding a viral protein

A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodefi ciency virus type 1 DNA vaccines in mice and nonhuman primates

Genetic immunization is a simple method for eliciting an immune response

DNA inoculation induces protective in vivo immune responses against cellular challenge with HIV-1 antigen-expressing cells

Vaccination of mice and nonhuman primates using HIV-gene-containing DNA

Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8 + cell-, interferon gamma-, and nitric oxide-dependent immunity

DNA vaccines against malaria: immunogenicity and protection in a rodent model

Protection of mice against Plasmodium yoelii sporozoite challenge with P. yoelii merozoite surface protein 1 DNA vaccines

Vaccination against tuberculosis by injection

Immunization for Ebola virus infection

Overcoming immunity to a viral vaccine by DNA priming before vector boosting

Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodefi ciency virus type 1 vaccines

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodefi ciency virus type 1 gag gene

Development of an HIV-1 vaccine based on replication-defective adenovirus. Presented at the Keystone Symposium on HIV Vaccine Development

Impact of pre-existing immunity on the immunogenicity of Ad5-based vaccines. Presented at AIDS Vaccines

Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier

Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodefi ciency virus vaccine and localization in the central nervous system

PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo

Adenovirus type 5 and 7 capsid chimera: fi ber replacement alters receptor tropism without affecting primary immune neutralization epitopes

Improved adenovirus vectors for infection of cardiovascular tissues

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease

An adenoviral type 5 vector carrying a type 35 fi ber as a vaccine vehicle: DC targeting, cross neutralization, and immunogenicity

Circumvention of immunity to the adenovirus major coat protein hexon

Construction and characterization of hexonchimeric adenoviruses: specifi cation of adenovirus serotype

Circumventing the immune response to adenovirus-mediated gene therapy

Seroswitch' adenovirus-mediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype

Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype

Replication-defi cient human adenovirus type 35 vectors for gene transfer and vaccination: effi cient human cell infection and bypass of preexisting adenovirus immunity

Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of preexisting anti-Ad5 immunity

Immunogenicity of heterologous adenovirus prime-boost regimens involving Ad11 and Ad35. Presented at AIDS Vaccines

Protection against mucosal simian immunodefi ciency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting

Live recombinant vectors for AIDS vaccine development

Ovine adenovirus vectors overcome preexisting humoral immunity against human adenoviruses in vivo

Replication-defective bovine adenovirus type 3 as an expression vector

Porcine adenovirus-3 as a helper-dependent expression vector

Replicationdefective vector based on a chimpanzee adenovirus

Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene

Novel, chimpanzee serotype 68-based adenoviral vaccine carrier for induction of antibodies to a transgene product

Complications of smallpox vaccination

Disseminated vaccinia in a military recruit with human immunodefi ciency virus (HIV) disease

Smallpox and Its Eradication. World Health Organization

Live attenuated vaccinia and other poxviruses as delivery systems: public health issues

NYVAC: a highly attenuated strain of vaccinia virus

Safety and immunogenicity of recombinants based on the genetically-engineered vaccinia strain

Poxvirus-based vectors as vaccine candidates

p53 as a target for cancer vaccines: recombinant canarypox virus vectors expressing p53 protect mice against lethal tumor cell challenge

Poxvirus-based vaccine candidates for cancer, AIDS, and other infectious diseases

Applications of pox virus vectors to vaccination: an update

Highly attenuated poxvirus vectors

Immunisation with canarypox virus expressing rabies glycoprotein

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety

Vaccine therapy in early HIV-1 infection using a recombinant canarypox virus expressing gp160MN (ALVAC-HIV): a double-blind controlled randomized study of safety and immunogenicity

Canarypox virus-based vaccines: prime-boost strategies to induce cell-mediated and humoral immunity against HIV

Gene gun intradermal DNA immunization followed by boosting with modifi ed vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective effi cacy in the infl uenza and malaria models

A group specifi c anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS

Poxvirus-based vectors as vaccine candidates

Vaccination of vaccinia-naive adults with human immunodefi ciency virus type 1 gp160 recombinant vaccinia virus in a blinded, controlled, randomized clinical trial. The AIDS Vaccine Clinical Trials Network

Potential improvement for poxvirus-based immunizations vehicles

Multienvelope HIV vaccine safety and immunogenicity in small animals and chimpanzees

Containment of simian immunodefi ciency virus infection in vaccinated macaques: correlation with the magnitude of virus-specifi c pre-and postchallenge CD4+ and CD8+ T cell responses

Largescale production and purifi cation of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity

Removal of cryptic poxvirus transcription termination signals from the human immunodefi ciency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus

Recombinant virus vaccine-induced SIV-specifi c CD8+ cytotoxic T lymphocytes

Immunization with a modifi ed vaccinia virus expressing simian immunodefi ciency virus (SIV) Gag-Pol primes for an anamnestic Gag-specifi c cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge

Reduction of simian-human immunodefi ciency virus 89.6P viremia in rhesus monkeys by recombinant modifi ed vaccinia virus Ankara vaccination

Enhanced simian immunodefi ciency virusspecifi c immune responses in macaques induced by priming with recombinant Semliki Forest virus and boosting with modifi ed vaccinia virus Ankara

Effect of vaccination with recombinant modifi ed vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys

Induction of simian immunodefi ciency virus (SIV)-specifi c CTL in rhesus macaques by vaccination with modifi ed vaccinia virus Ankara expressing SIV transgenes: infl uence of preexisting anti-vector immunity

Comparison of vaccine strategies using recombinant env-gag-pol MVA with or without an oligomeric Env protein boost in the SHIV rhesus macaque model

Immunogenicity and protective effi cacy of a human immunodefi ciency virus type 2 recombinant canarypox (ALVAC) vaccine candidate in cynomolgus monkeys

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodefi ciency virus CD8+ and CD4+ T-cell responses from chronically infected individuals

Potentiation of simian immunodefi ciency virus (SIV)-specifi c CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen

Cross-protection against mucosal simian immunodefi ciency virus (SIVsm) challenge in human immunodefi ciency virus type 2-vaccinated cynomolgus monkeys

Induction of cytotoxic T lymphocytes by recombinant canarypox (ALVAC) and attenuated vaccinia (NYVAC) viruses expressing the HIV-1 envelope glycoprotein

Memory cytotoxic T lymphocyte responses in human immunodefi ciency virus type 1 (HIV-1)-negative volunteers immunized with a recombinant canarypox expressing gp 160 of HIV-1 and boosted with a recombinant gp160

Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers

Induction of neutralizing antibodies and gag-specifi c cellular immune responses to an R5 primary isolate of human immunodefi ciency virus type 1 in rhesus macaques

ALVAC-SIVgag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodefi ciency virus SIVmacinduced immunodefi ciency

AAV vectors: is clinical success on the horizon?

Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine effi cacy

Vaccination of macaques against pathogenic simian immunodefi ciency virus with Venezuelan equine encephalitis virus replicon particles

Sindbis virus vectors for expression in animal cells

Evaluation of recombinant alphaviruses as vectors in gene therapy

Replication-defective viruses as vaccines and vaccine vectors

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants

West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective effi cacy

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses

Priming with plasmid DNAs expressing interleukin-12 and simian immunodefi ciency virus gag enhances the immunogenicity and effi cacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus

A single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector

A live, attenuated recombinant West Nile virus vaccine

A single intranasal inoculation with a paramyxovirusvectored vaccine protects guinea pigs against a lethal-dose Ebola virus challenge

Vaccine cell substrates

Cell line issues: historical and future perspectives

Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals. United States Food and Drug Administration, Department of Health and Human Services

Use of live bacterial vaccine vectors for antigen delivery: potential and limitations

Progress towards the use of Listeria monocytogenes as a live bacterial vaccine vector for the delivery of HIV antigens

Novel bacterial systems for the delivery of recombinant protein or DNA

Phagolysosome formation, cyclic adenosine 3′:5′-monophosphate and the fate of Salmonella typhimurium within mouse peritoneal macrophages

Bacterial vaccine vectors and bacillus Calmette-Guerin

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guerin vector candidate vaccine for human immunodefi ciency virus type 1 in small animals

Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization

Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes

Oral somatic transgene vaccination using attenuated S. typhimurium

Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fi brosarcoma

Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis

A multiclade HIV-1 DNA vaccine elicits humoral and sustained cellular immune responses in humans in a randomized Phase I clinical trial

A DNA vaccine for Ebola virus is safe and immunogenic in a phase I clinical trial

A West Nile Virus vaccine induces neutralieing antibody in healthy adults in a phase I clinical trial

West Nile Virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays

DNA vaccines for aquacultured fi sh

Prospects for development of a vaccine against the West Nile virus

Poxviruses as immunization vehicles