key: cord-0914519-28h18zrg
authors: Sánchez-Sampedro, Lucas; Perdiguero, Beatriz; Mejías-Pérez, Ernesto; García-Arriaza, Juan; Di Pilato, Mauro; Esteban, Mariano
title: The Evolution of Poxvirus Vaccines
date: 2015-04-07
journal: Viruses
DOI: 10.3390/v7041726
sha: 902ec7158906ac390bdc04cd55350a12c8a39281
doc_id: 914519
cord_uid: 28h18zrg

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.

The most deadly poxvirus, VARV, was the agent that caused smallpox, a fatal disease for which records go back more than 3500 years [1] and which is suspected to have emerged in 10,000 BCE [2] . Since then, the virus has spread from person to person and from country to country, causing the most severe epidemics in human history, with a death rate of about 30% of those infected. More than 30 years have elapsed since WHO declared smallpox eradicated, but this achievement would have been impossible without the discovery of vaccination and the evolution of poxviruses as vaccines.

The first strategy aiming to fight the smallpox disease was the use of VARV itself as the immunization agent. Variolation, an oriental practice that consisted of inoculating small amounts of material from an infected person to a healthy individual to prevent a natural infection, was used for centuries in the Orient and introduced in Europe by the physician Emmanuel Timoni [3] , who described the technique in 1714; the practice was later introduced by Lady Mary Wortley Montagu in the United Kingdom in 1721 [4] .

In 1798 the English physician Edward Jenner established a much safer practice, demonstrating that another poxvirus, CPXV-which infected cattle-could be used to prevent smallpox infections in humans. This procedure became known as vaccination, derived from "vacca," the Latin word for cow. In 1881 Louis Pasteur proposed that vaccination should be the generic term used for preventive inoculations against any animal or human diseases [5] .

From 1803 to 1806 the Jennerian vaccination practices and viral strains were expanded to the New World in the Royal Philanthropic Expedition of the Vaccine carried out by the Spanish surgeon Francisco Xavier Balmis, in a programmed expedition under the auspices of King Carlos IV of Spain. In this first expedition in 1803 in the ship Marí a Pita, the vaccine was transported to America through arm-to-arm propagation in 22 orphan children. At La Guayra (now Venezuela) the expedition was divided into two groups, one under Salvany, who extended vaccination to South America, and the other led by Balmis, who continued to Cuba and Mexico. From Acapulco the vaccine traveled to Manila (in the Philippines) and then from Macau into China [6, 7] . The Spanish expedition was the first-ever mass vaccination campaign, 150 years before the WHO was established. The expedition was successful, and Jenner wrote: "I don't imagine the annals of history furnish an example of philanthropy so noble, so extensive as this."

Over time, CPXV and also HSPV were passed through cattle, rabbits, horses and humans and used for vaccinating against smallpox all over the globe. However, at a certain evolutionary point, those viruses were superseded by VACV, another poxvirus whose origin remains unknown, but which eventually became the most studied poxvirus and has been used extensively as a research tool.

The latter half of the 19th century saw the emergence of microbiology and immunology as scientific disciplines. Many of the pioneers in these new sciences used VACV for their studies and vaccine production was introduced into laboratories and taken over by scientists rather than local physicians. This entailed an improvement in the quality of the vaccines, the methods for the distribution and the public health infrastructure, which led to the elimination of endemic smallpox from the industrialized countries of Europe and North America by the early 1950s [8] . Modifications to traditional production and international quality control of vaccines were introduced shortly after the Intensified Smallpox Eradication Program in 1967. Thanks to this program, the last natural case of smallpox occurred in Somalia in 1977, and in 1980 the WHO declared the disease eradicated [9] . To date, smallpox is the only human infectious disease that has been successfully eradicated.

In the early 1980s, recombinant DNA technology revolutionized molecular biology, allowing the insertion of foreign DNA into poxvirus genomes. Early studies by Woodroffe and Fenner indicated in 1960 that homologous recombination could occur between the genomes of two replicating poxviruses [10] . Twenty-two years later, marker rescue studies demonstrated that fragments of genomic [11] and cloned [12] DNA could recombine with the genome of VACV in infected cells. Furthermore, poxvirus expression vectors were described simultaneously in 1982 by the laboratories of Enzo Paoletti [13] and Bernard Moss [14] , and recombinant DNA technology quickly became widely used for vaccine development as well as for research in numerous other fields. Thus, the ability to insert heterologous genes into poxvirus genomes deeply improved their vaccination capabilities. Poxviruses were no longer used only as successful smallpox vaccines, but also as vaccines against a wide range of heterologous diseases, namely the hepatitis B surface antigen [15] , the hemagglutinin of the influenza virus [16, 17] , the glycoprotein D of herpes virus [18] and the rabies virus glycoprotein [19] , the first foreign antigens and heterologous diseases explored. It is of importance that, as all chordopoxviruses have a similar arrangement of genes, interchangeable promoters and conserved RNA polymerase and transcription factors, the principles developed for VACV expression vectors could be applicable to other poxviruses [20] .

In 1990, the genome sequencing of Copenhagen, one of the most studied strains of vaccinia, was published by Paoletti and co-workers [21] . With this knowledge and the ability to insert and delete selective genes, poxviruses have been modified à la carte in order to improve their safety and immunogenicity or even their ability to selectively kill tumor cells.

In this article, we review how different poxviruses have evolved in nature and in controlled laboratory environments to generate a wide variety of strains that are being used as vaccine candidates against homologous diseases such as smallpox, heterologous diseases such as rabies, HIV/AIDS, hepatitis C, tuberculosis, malaria and leishmaniasis, among others, or against other complex diseases like cancer. We describe the sequences by which different poxvirus-based vaccines evolved with time, and how genetic manipulation of the poxvirus genome led to the development of vaccine candidates with wide application against human and animal diseases.

In order to trace, step-by-step, the evolution of poxviruses as vaccine vectors, one of the starting points should be the identification of the original virus used by Jenner and colleagues at early stages of vaccination. In 1796 Jenner vaccinated an eight-year-old boy, James Phipps, with a cowpox lesion from the milkmaid Sarah Nelmes and proved it was effective after challenge against smallpox [22] . From that experiment, the practice of arm-to-arm vaccinations in humans expanded around the world, using cattle to amplify the viral stocks. However, vaccination with the feasible original CPXV was displaced, eventually, with VACV, whose natural host and origin has not been identified yet.

Several hypotheses arose in the past about the origin of VACV and its derivation from the original "variolae vaccinae." It had been proposed that VACV could have derived from VARV, from CPXV, or could be a hybrid of VARV and CPXV viruses that had been genetically selected after the use of contaminated vaccine [23] .

VARV could have been altered and transformed into VACV after several years of vaccination from human to human or during passages in animals, but VARV host range genes and studies in animals indicate that this hypothesis should be dismissed [24, 25] . VARV has a restricted human replication phenotype that contrasts with the wide host range of VACV [26] . On the other hand, CPXV probably exhibits the broadest host range and the greatest genetic diversity among these poxviruses. Nonetheless, originally the differences between CPXV and VACV were considered too great to make that origin probable [27] , and more recent DNA sequencing data follow the same thread of thought [28] .

Several factors exacerbated the difficulty of identifying the origin of VACV. The old smallpox vaccines were rarely subjected to clonal purification, and those methods of propagation produced mixtures of viruses called quasispecies [29] . In fact, deep genome sequencing has suggested that modern vaccines are comprised of a complex mixture of different vaccinia viruses [30, 31] . Furthermore, the practice of co-cultivating smallpox vaccines with other viruses, including VARV, could have produced recombinant viruses and obscured the origin of VACV strains [8] . In this context, there are few evidences of horizontal gene transfer between orthopoxviruses. One example is the presence of two CPXV-like genes in the Lister VACV strain [32] , and another one is represented by a small region of sequenceencoding HSPV-like single nucleotide polymorphisms (SNPs) in the DPP17 Dryvax subclone [29] .

Among orthopoxviruses, CPXV isolates-which can be split into five different monophyletic lineages-have the largest genomes, averaging 220 kbp, around 30 kbp larger than VACV; only the genome of HSPV is larger than 200 kbp [33] . These five subtypes of CPXV encode all of the genes present in all other orthopoxviruses, leading to the suggestion that the modern orthopoxviruses have evolved from CPXV through reductive evolution [34] .

Historic literature states that the original vaccine strains were derived from CPXV samples; however, Edward Jenner also believed that his vaccine was originally the agent that caused an infection in the heels of horses that he called "grease" and that was suitable for human use after passaging through cows [22, 23] . It has also been argued that it could be possible that Jenner confused "grease" (dermatitis verrucosa) with horsepox (variola equina). In fact, equination was used side by side with vaccination at the early stages of smallpox epidemics. There are several examples of early vaccination with HSPV, for example by Dr. De Carro in Vienna or Dr. Sacco in Milan, who communicated the practice to Jenner [35, 36] , or by Dr. Loy, who also showed that horses were susceptible to a poxvirus that conferred immunity. Both CPXV and HSPV caused the human immune system to react as if it has been exposed to the smallpox virus, creating long-term immunity because all orthopoxviruses are antigenically related and immunization with nearly any orthopoxvirus can protect against challenge with another orthopoxvirus [37] .

Phylogenetic analysis of the conserved regions of poxvirus genomes indicated that HSPV is closely related to sequenced isolates of VACV and rabbitpox virus [38] . Furthermore, it is of interest that experimental infection of horses with VACV produces clinical signs of HSPV [39] .

In the most recent article studying poxvirus evolution, the authors claim that the most probable route by which VACV strains might have evolved is from a stock of virus containing an ancestral HSPV-like virus. This conclusion arose after identifying a gene, DVX_213, which seems to have been subject to widespread negative selection in VACV strains [28] . Nonetheless, in order to better understand the relationship between HSPV and VACV it is important to obtain more genomic sequencesofHSPV, an endeavor that is extremely challenging because HSPV seems to be extinct [5] .

Vaccination is a procedure that has been performed for over 200 years in different countries around the world and without international standardization. In fact, reliable assay methods for quality control were not developed until the 1950s and not implemented until the 1960s. As a consequence, the VACV strains used in different countries differed in their biological properties [8] . These first VACV strains received their names regarding the health agency or the country or region of origin and the most widelyused are summarized in Table 1 . In addition, a large number of manufacturers-71 distributed around the world-were involved in the global effort to eradicate smallpox. In the United States all the vaccines against smallpox were derived from a stock supplied to the New York City Health Department in 1856, NYCBH. Using this strain, Dr. Rivers developed two attenuated strains of the virus, CVI-78 and CVII, by passing NYCBH through rabbit testes and chick embryos [40] . These caused less reactogenicity in comparison with the parental strain, but their ability to mediate protection against smallpox was questioned [41, 42] . This strain was also distributed to other laboratories, where it received different names, such as IHD, LED-0, Noguchi or WR. It was passaged in different organs such as the brain or testes of rabbits or mice, thus modifying its biological properties. Several studies showed that strains such as WR or IHD presented increased levels of pathogenicity [43] and adverse effects in humans, limiting their use as vaccines of choice in the early steps of the fight against smallpox [8] .

Another vaccine was derived from NYCBH after 22 to 28 passages in cows in Wyeth Laboratories (Marietta, PA, USA). This vaccine was called Dryvax ® and is a non-clonal vaccine that was prepared in calf lymph and distributed as a freeze-dried vaccine. The nature of non-clonal origin of Dryvax ® has been recently characterized. Genetic analysis shows that Dryvax ® is a complex of different VACV sub-strains that has been classified in four subgroups according to genome structure analysis [29] . This vaccine is still one of the current USA-licensed smallpox vaccines.

A second vaccine derived from NYCBH and licensed in the USA is Aventis Pasteur Smallpox Vaccine (APSV). APSV was manufactured from 1956 to 1957 and was maintained as a frozen preparation, but clinical studies with this vaccine were stopped when myocarditis cases arose in the vaccination trials [44] .

NYCBH was also believed to be the parental strain of EM-63 vaccine, a strain derived from Ecuador that was used in the former USSR and was also widely used in the eradication of smallpox in India [45, 46] .

The Lister strain, prepared on the skin of sheep, was widely used for vaccination against smallpox from 1892 because it produced pocks on the chorioallantoic membrane that were easier to count in comparison with the other VACV strains, and because the WHO International Reference Center later produced seed lots of this strain for distribution to vaccine producers in developing countries [8] . The commercial Lancy-Vaxina (Berna Biotech) is derived from this strain, and the formulation of the vaccine is a lyophilized product prepared from calf lymph [47] .

Tian Tan virus was the most extensively VACV strain used to vaccinate against smallpox in China since 1926. The name of "Tian Tan" was acquired because the virus was isolated in Beijing's Temple of Heaven, where the Central National Epidemic Bureau was initially housed. The virus was used in China from 1926 to 1954 and from 1960 to 1980, being the Russian strain of vaccinia used between 1955 and 1960 [48] . The legend tells that the vaccine was isolated by Mr. Qi Changqing from a patient with smallpox and then passaged in the skin of monkeys, rabbits and cows. However, this story lacks scientific credibility because VARV infection is restricted to humans and does not contain the host range genes needed to infect monkeys, rabbits or cows. Moreover, recent genomic sequencing demonstrates that Tian Tan is clearly a VACV that shares a common origin with Copenhagen strain, and it is different from monkeypox (MPXV), VARV or HSPV [48] .

The intensified Eradication Campaign against smallpox started in 1967 and no particular strain was officially recommended but, in response to inquiries, the Smallpox Eradication Unit advised that either the Lister or the NYCBH strains should be used. Additionally, the potency and safety of the different vaccines were standardized; lyophilization was recommended and vaccine batches might contain at least 1 × 10 8 pock-forming units per mL [8] . Other VACV strains used in early vaccination campaigns against smallpox were Ankara (used in Turkey), Aosta (Italy), Bohemia (Czechoslovakia), Bordeaux (Africa and Portugal), Massachusetts 999 (Argentina), Gam (USSR), MRIVP (USSR), Per (USSR), Williamsport (USA), LMC (UK), Hamburg (Germany), Sweden (Sweden), Finland (Finland), Patwadanger (India), Vienna (Bulgaria), Spain (Spain), Tom (USSR) and Chambon (France and Africa) [8, 49] .

The last natural infection of smallpox occurred in Somalia in 1977. Eradication was possible due to vaccination, but also because to date no animal reservoir for smallpox other than humans exists [50] . Since the eradication, VARV is officially retained at two WHO collaborative centers: the Centers for Disease Control and Prevention (CDC), in Atlanta (Georgia, USA), and the State Research Center of Virology and Biotechnology (VECTOR), in Novosibirsk (Russian Federation).

Although smallpox has been eradicated as a public health threat it could potentially reemerge as a bioterrorist threat. The risk scenario includes other animal poxviruses and genetically engineered manipulations of poxviruses. Thus, for preventing this potential risk and due to the side effects of the first-generation VACV vaccines, safer VACV strains had to be improved and developed for the post-eradication era.

In order to standardize procedures, control possible microbial contamination and avoid sensibilization to the allergenic animal proteins that accompanied the vaccine, the use of live animals for the growth of the different vaccines was substituted by tissue culture systems or embryonated chicken eggs. These second-generation vaccines are listed in Table 2 .

Lister was the first VACV strain used for the production of cell-cultured derived smallpox vaccines, being passaged in rabbit kidney cells, in the chorioallantoic membrane of chicken embryos (CE) or in primary cells derived from chicken embryos.

Thus, the first second-generation Lister-based vaccine, RIVM, was produced in 1960 using rabbit kidney cells [51] . The virus was passaged directly from calf lymph vaccine to cells, and no further passages were performed for the generation of this vaccine. Freeze-dried vaccine demonstrated similar take rates and neutralizing antibodies to the calf lymph-derived vaccine [8] . This vaccine has been used in clinical trials in Netherlands and Indonesia without producing severe complications [52]. . This vaccine was also prepared on chicken embryo fibroblast (CEF) cells in Japan before smallpox eradication and showed an adequate safety profile, but the effectiveness was not well documented [45] .

Several second-generation VACV vaccines were also prepared using NYCBH as the seed strain. The first candidate was grown in cell cultures in 1968 and was used in clinical trials in the U.S. Army that had to be stopped due to the absence of adequate "take" rates observed [2] . Nonetheless, from that stock, another cell-cultured stock was developed in MRC-5 cells and received the name of Cell-Cultured Another vaccine candidate derived from NYCBH is ACAM2000 (from Acambis), a vaccine derived from a clone isolated from Dryvax ® . Originally six clones were isolated and their safety was evaluated in suckling mice and in rabbits. Significant differences in neurovirulence were observed among the different clones; CL2 is a clone with reduced neurovirulence that still maintains the same lesion size when compared with the Dryvax ® vaccine [59]. This clone was selected and grown first in MRC-5 cells (ACAM1000) and, later on, in VERO cells generating the ACAM2000 vaccine [60] . Preclinical studies demonstrated that this strain was less neurovirulent in comparison with Dryvax ® , but demonstrated similar immunogenicity in phase I clinical trials. Nonetheless, in phase II and III clinical trials, Dryvax ® and ACAM2000 caused myocarditis associated with the vaccination [59,60]. The Food and Drug Administration (FDA) approved ACAM2000 in 2007 as a vaccine against smallpox for human use and Sanofi Pasteur manufactured the vaccine.

NYCBH has also been used as a parental seed strain for the development of another second-generation VACV vaccine termed CJ-50300, which was obtained after passages in MRC-5 cells in South Korea. Compared to the first-generation Lancy-Vaxina vaccine, it showed similar reactogenicity, immunogenicity and neurovirulence in preclinical trials [47] . Moreover, a phase I clinical trial showed overall rates of 100% in cutaneous "take" reaction and humoral and cellular immunogenicity in CJ-50300 vaccinees, with no serious adverse reactions being observed. However, one case of possible generalized vaccinia infection occurred in one of the studied groups [61].

Other strains have been also derived from NYCBH such as Western Reserve (WR), a neurovirulent strain that has a wide history of passages; first in rabbits, followed in mice and in cell cultures [43] , and Duke (isolated from a vaccinated patient that received Dryvax ® vaccine [63]). As new research proves, VACV IHD-J, "International Health Department," also shares a common ancestor with Dryvax ® , i.e., NYCBH [28] .

All these studies with second-generation vaccines demonstrate that although cell-cultured vaccines improved the control and the standardization that were lacking in previous vaccines, the use of replication-competent strains of VACV represents associated risks and serious adverse events that still have to be controlled.

Several first-and second-generation poxvirus strains expressing different heterologous antigens have been used as vaccine candidates against a wide range of diseases. Table 3 summarizes the most relevant recombinant poxviruses used for these purposes.

Other members of the poxvirus family have also been extensively used as vaccine vectors against homologous and heterologous diseases (see Tables 4 and 5 ). There are several examples of vaccines based on avipoxvirus, suipoxvirus, capripoxvirus, leporipoxvirus and parapoxvirus, which belong to the Chordopoxvirinae subfamily. Avipoxviruses (APVs) belong to the Chordopoxvirinae subfamily of the Poxviridae family. They infect and cause diseases in poultry, pets and wild birds, are transmitted via biting insects and aerosols and are usually named on the basis of the bird species from which the virus was first isolated and characterized [98] . APV infections have been reported to affect over 232 species in 23 orders of birds [99] . However, the knowledge of the molecular and biological properties of APVs is largely restricted to canarypox virus (CNPV) and fowlpox virus (FWPV), for which full genome sequences are available [100, 101] . Despite the shorter FWPV genome, molecular comparisons show that CNPV and FWPV share 55-71% amino acid identity, significant gene-sequence rearrangements, deletions and insertions [101] . CNPV exhibits a broader tissue tropism in the permissive avian hosts than FWPV, generally associated with higher mortality rates [102] . Both viruses have been described as unable to replicate and disseminate infection in non-human primates and humans [103] , but some studies have shown replication of FWPV in non-permissive mammalian cell cultures by the presence of infectious viral particles [104] or the occasionalpresence of immature forms and mature intracellular virus in infected cells [105] . However, a recent study has demonstrated that despite the detection of mature virions in FWPV-infected VERO cells, the new progeny was not infectious [106] . Due to their natural host-range restriction to avian species [103, 105, 107] , their efficient expression of heterologous genes also in human cells [108] , and their ability to induce antigen-specific humoral and cellular immune responses [109, 110] , CNPV and FWPV represent alternative and safer vectors. In this context, several recombinant APVs have been evaluated as vaccine candidates against a wide range of infectious diseases and other APV-based vaccines have been licensed for commercial veterinary use against some animal infections; it is likely that such vaccines will also be used against human diseases in the future [111] . Table 4 summarizes the most relevant recombinant avipoxviruses used as vaccine vectors against different diseases. Furthermore, other poxvirus vectors of the Chordopoxvirinae subfamily, such as the orthopoxvirus raccoon poxvirus, parapoxvirus, capripoxvirus, suipoxvirus and myxomavirus have been widely used as vaccine candidates against several animal and human diseases (see Table 5 ), showing good levels of safety and immunogenicity. 

Given the unsatisfactory safety profile of VACV second-generation vaccines, attention has shifted to third-generation vaccines, obtained after serial passages in cell culture [37] . Thus, multiple extensive passages of a parental vaccine strain in cultured cells is a useful strategy for attenuating VACV through the generation of random mutations and deletions. Examples of this strategy are different VACV strains that are used as vaccine candidates, such as Lister clone 16m8 (LC16m8), Dairen I strain (DIs), M65 and M101, Modified Vaccinia Virus Ankara (MVA) and several attenuated avipoxviruses.

LC16m8 was obtained in the late 1970s in Japan by passaging the parental Lister strain 36 times in primary rabbit kidney (PRK) epithelial cells at low temperature (30 °C), followed by isolation of one clone (LC16) that grows to the lowest titer in monkey kidney VERO cells; this was passaged six additional times in PRK cells to obtain the clone LC16m0 from the latter stock. Then, this clone was passaged three more times in PRK cells to generate the clone LC16m8 from the final stock [246, 247] . LC16m8 replicates poorly in VERO cells, and formed small plaques in chick chorioallantoic membranes (CAM), PRK and RK13 cells. Thus, while LC16m8 can grow and produce infectious particles, it spreads poorly in cell culture. Compared to the original Lister strain, LC16m8 is temperature-restricted and displays limited host range, lower pathogenicity and adverse effects in animal models [247, 248] .

LC16m8 contains a frame-shifting single nucleotide deletion in the B5R gene [30, 249] , which encodes an extracellular enveloped virus (EEV) protein (B5) essential for EEV formation. Analysis of the LC16m8 full-genome sequence showed that there are no large deletions compared to the parental Lister strain [30] .

LC16m8 has been shown to induce protective immunity against orthopoxvirus challenge in mice [30, 250, 251] , rabbits [250] and non-human primates [252, 253] . Moreover, LC16m8 is a safe and immunogenic attenuated smallpox vaccine in immunodeficient mice [254] and vaccinia-naive humans [255, 256] . However, there are two important main concerns about this vaccine. First, since the key attenuating mutation in B5R is a one base deletion that results in a frame-shift and early truncation of the B5 protein, the virus can revert back to wild type during growth [257] -although, to avoid this phenomenon, a new version of LC16m8, with a complete deletion in the B5R gene, has been generated [257] . The second concern is related to the fact that VACV B5 protein is the primary target antigen for generating neutralizing antibodies against EEV [258] . Thus, due to the presence of a mutation in B5R, LC16m8 failed to induce either EEV-neutralizing antibodies or antibodies to B5 in humans [259] , a feature that may make LC16m8 a less efficient vaccine for protection against poxviruses. It remains to be seen whether this strain induces similar levels of neutralizing antibodies against VARV than other vaccine strains such as Dryvax ® .

Nevertheless, the combination of the deletion in the B5R gene (which causes the lack of anti-VACV vector immunity) with the insertion of heterologous antigens in the VACV TK or HA loci is a good strategy for using LC16m8 as a vaccine vector against infectious diseases. Thus, it has been reported that an attenuated recombinant LC16m8 expressing clade B HIV-1 Env [260] or SARS-CoV spike protein [261] was able to induce robust HIV-1-specific humoral and T cell immune responses or SARS-specific neutralizing antibodies in vaccinated mice and rabbits, respectively.

Thus, LC16m8 is one of the safest live, attenuated, replication-competent vaccines; it is the sole smallpox vaccine licensed in Japan and was recently recommended by the WHO as one of the preferred WHO smallpox vaccines to stockpile. Furthermore, it is a promising vaccine vector against infectious diseases.

VACV DI strain was generated after 13 successive passages of parental Dairen strain in one-day-old eggs [262] . DIs forms small plaques in CAM, growing well only in chick embryo fibroblast (CEF) cells, but is unable to grow in most mammalian cells. DIs is a highly restrictive host range mutant that contains a great deletion of 15.4 Kb in the left terminal region of the VACV genome, which results in the loss of 19 putative ORFs from genes C9L to K5L, including host-range genes K1L and C7L [263] .

Insertion of HIV-1 Gag gene in the deleted region of DIs induced high levels of cytotoxic T lymphocytes in immunized mice [263] . Furthermore, a recombinant VACV DIs expressing simian immunodeficiency virus (SIV) Gag and Pol antigens induces SIV-specific cellular and humoral immuneresponsesin mice [264, 265] or immunized non-human primates [266, 267] . These results suggest that recombinant VACV DIs is a safe, efficient, transient replication-deficient viral vector, which can be used in a vaccine regimen for HIV-1 vaccine development. . Because of the inability to replicate in human cells, MVA would likely be safe to administer to people who have conditions that would not allow routine smallpox vaccination. In fact, MVA was used as a safe highly attenuated smallpox vaccine in the last decades of the smallpox eradication campaign (1968) (1969) (1970) (1971) (1972) (1973) (1974) (1975) (1976) (1977) (1978) (1979) (1980) , being inoculated into more than 120,000 people in Germany with no adverse side effects [274,280], although its efficacy against smallpox remains untested.

Since then, MVA has been widely studied as a third-generation smallpox vaccine [281], able to induce antibody responses similar to Dryvax ® [282] as well as protection in mouse [283] and non-human primate challenge models [56, [284] [285] [286] . However, high doses or multiple doses of MVA have to be administrated to elicit immune protection, compared with other smallpox vaccines such as Dryvax ® [283, 287, 288] . Nevertheless, this protection elicited by MVA is more rapid than the one induced by the fully replication-competent vaccine Dryvax ® [286] , mainly due to the induction of a more rapid immunity and an activation of the innate immune responses. Furthermore, MVA lacks several VACV immunomodulatory genes involved in evasion of the host immune responses, such as soluble receptors for type I and II IFNs, cytokines and chemokines [277, 289] , whose absence allows an enhanced antigen presentation and immunogenicity. In fact, deletion of innate immune evasion genes leads to an increase in proinflammatory cytokines and migration of immune cells [290] [291] [292] , which have a great influence on their ability to elicit adaptive immunity.

Thus, MVA has been evaluated as a smallpox vaccine in different animal models and several human clinical trials and was found to be safe and immunogenic without developing clinical disease [2, 283, 285, 287, [293] [294] [295] [296] [297] [298] [299] [300] . Although the MVA vaccine has not been tested directly in humans for efficacy against VARV, it has being tested against monkeypox triggering protection. In terms of MVA's registration as a smallpox vaccine, the European Medicines Agency registered the vaccine as Imvanex and Health Canada also registered the vaccine for persons 18 years and older, while in the USA it is under evaluation by the FDA.

Among poxviruses, MVA is the tip of the iceberg, being one of the most promising poxvirus vectors (reviews in [37, [301] [302] [303] [304] [305] [306] [307] [308] [309] [310] ). Enormous effort has been put into the use of MVA as a vaccine vector, with several preclinical and human clinical trials developed using MVA as a vaccine candidate against an extensive number of infectious diseases, such as HIV/AIDS, malaria, tuberculosis, hepatitis C and cancer, among many others. Table 6 summarizes the use of MVA in preclinical and human clinical trials as a vaccine candidate against different viral, bacterial and parasitic infectious diseases. 

HIV-1 Env, Gag, Tat-Rev and Nef-RT (clade C) clinical [314] HIV-1 Env, Gag-Pol, Nef-Tat (clades B/C) clinical [315] HIV-1 Gag, PR, RT, Env (clade B) clinical [316] HIV-1 Env/Gag, Tat/Rev/Nef-RT (clade B) clinical [127]

HIV-1 Env, Gag-Pol-Nef (clade B) clinical [305] 21 CTL and 18 HTL epitopes from HIV-1 Gag, Pol, Vpr, Nef, Rev and Env clinical [317] HIV-1 Nef clinical [318] Influenza A NP+M1 clinical [319] HA from H5N1 clinical [320] Hepatitis B

HBs clinical [321] 30 CTL and 16 HTL epitopes preclinical [322] Hepatitis C NS3, NS4 and NS5B (genotype 1b) preclinical and clinical [323, 324] E1 and E2 (genotype 1b) preclinical [325] C, E1 and E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (genotype 1a) preclinical [326] Chikungunya C, E3, E2, 6K and E1 preclinical [327] E3 and E2 preclinical [328] E3-E2, 6K-E1 or E3-E2-6K-E1 preclinical [ Measles HA preclinical [352] F and HA preclinical [94, 353] CMV Soluble GP B (gB) preclinical [354] UL55 (surface glycoprotein), UL83 (tegument protein) and UL123/e4 (nuclear protein) preclinical [355] pp65 (tegument protein) and CMV immediate early gene IE1 preclinical [356] pp65-2, gB and IE1 (Rhesus CMV) preclinical [357, 358] pp65, IE1, IE2 preclinical [359] pp65 preclinical [360] glycoproteins gH/gL, UL128, UL130 and UL131A (UL128C) preclinical [361] gH, gL, UL128, UL130 and UL131A preclinical [362] BoHV-1 Secreted GP D preclinical [363] EHV-1 Complement-receptor GP C preclinical [364] HSV GP D (gD) (HSV-2) preclinical [365] Parainfluenza virus F and/or HN glycoproteins (parainfluenza virus 3) preclinical [366] [367] [368] Bacterial diseases

Mycobacterial mycolyl-transferase antigen 85A clinical [369] [370] [371] Babesia bovis MSA-2c, RAP-1 and HSP20 proteins preclinical [372] Bubonic plague Yersinia pestis low-calcium response V antigen preclinical [373] Parasitic diseases 

Similar to VACV-derived strains, when considering the development of an APV-derived vector for production of a vaccine for birds, the use of an attenuated strain is recommended to reduce the risk and consequences of environmental spread to other avian species. Attenuated derivatives of FWPV (such as TROVAC or FP9) and CNPV (such as ALVAC) have been extensively tested, demonstrating their safety in a variety of species, including immunocompromised animals and human volunteers. Despite the fact that their multiplication is restricted to avian species, attenuated strains of APVs have been demonstrated to be efficacious and extremely safe vectors for mammals. Indeed, it was discovered that inoculation of APV-based recombinants into mammalian cells resulted in expression of the foreign gene and that inoculation into mammals resulted in the induction of protective immunity [103, 172] (see Table 7 ). 

For the generation of TROVAC, the attenuated fowlpox vaccine strain FP-1 [103] , derived from the Duvette strain, was subjected to four successive plaque purifications. Then, one plaque isolate was further amplified in primary CEF cells and a viral stock, designated as TROVAC, was generated and deposited with the American Type Culture Collection (ATCC; Accession number: VR-2553) [392] .

FP9 is a highly attenuated form of FWPV derived from the wild-type fowlpox virus HP-1 by 438 serial passages in CEF cells [393] . The FP9 genome has been fully sequenced and found to harbor several deletions/insertions and gene modifications when compared with the sequence of wild-type FWPV strains [394] .

ALVAC is a plaque-purified clone derived from an attenuated CNPV obtained from the wild-type strain after 200 serial passages in CEF cells [395] . It has been extensively evaluated in preclinical studies with non-human primates [396] [397] [398] [399] [400] , widely used in human clinical trials as an HIV/AIDS vaccine candidate [109, 401] , and licensed for veterinary use [111] . ALVAC-based vectors have been reported to be well tolerated and safe for humans [402, 403] and the first sign of efficacy of an HIV/AIDS vaccine candidate, although modest (31.2%), was obtained in a phase III clinical trial using an ALVAC-based vector [404] .

The innovative biotechnology techniques of genetic engineering that have been developed in the 1980s and 90s allow the generation of novel poxvirus vaccines through the insertion or deletion of specific genes in the poxvirus genome. In 1982, two independent groups showed for the first time that VACV can be modified to be used as an expression vector system, where foreign DNA can be inserted into non-essential regions of the VACV genome [13, 14] . Since then several recombinant poxviruses have been generated and used as effective vector systems for vaccination-expressing heterologous antigens that were able to induce strong antigen-specific cellular and humoral responses, reinforcing the use of recombinant poxviruses as vaccine candidates against a broad range of infectious diseases. Numerous replication-deficient and competent poxvirus-based vectors have been widely and successfully used as vaccine candidates in preclinical and clinical trials in the prevention and treatment of different animal and human diseases (reviews in [37, [301] [302] [303] [304] [305] [306] [307] [308] [309] [310] [477] [478] [479] [480] ). The genetic modifications normally attenuated the virus and led to an increase in immunogenicity against the VACV antigens or against the heterologous antigens expressed from the poxvirus vector.

Poxviruses encode for many proteins involved in the host innate immune evasion, with secreted proteins that bind and neutralize IFNs, cytokines and chemokines, or intracellular proteins that inhibit apoptosis or signaling pathways that lead to the production of IFNs or proinflammatory cytokines and chemokines [289] . Thus, deletion of these VACV genes involved in immune-modulation, host-range and accessory nucleotide metabolism genes is one of the techniques that has been widely used to generate novel poxvirus vectors with a more attenuated profile or novel vaccine candidates with optimized immunogenicity [481] .

One of the best examples of an attenuated VACV vector generated by the deletion of viral genes is NYVAC, a VACV strain derived from a plaque-cloned isolate (VC-2) of the Copenhagen strain (VACV-COP) by the precise deletion of 18 Open Reading Frames (ORFs). These deleted genes include 12 ORFs from C7L to K1L genes, J2R (TK), B13R, B14R, A26L, A56R (HA) and I4L and are involved in pathogenicity, virulence and host-range functions [482] . The resultant vector exhibits a dramatically reduced ability to replicate on a variety of human and mammalian cell types, is highly attenuated since it fails to disseminate in immunodeficient mice, and is unable to produce infectious virus in humans [482] [483] [484] . Despite its limited replication in human and most mammalian cells, NYVAC provides a high level of gene expression and triggers antigen-specific immune responses when delivering foreign proteins in animals and humans [485] [486] [487] [488] . For this reason, NYVAC-based recombinants are underintensepreclinical and clinical investigation as recombinant vaccines against multiple infectious diseases [305, 306, 483] (see Table 8 ). 

SIVmac239 Gag-Pol-Nef preclinical [487, [495] [496] [497] Env (clade B) preclinical [411] clade C trimeric soluble gp140(ZM96), clade C Gag(ZM96)-Pol-Nef(CN54) as VLPs preclinical [498] Env, Gag-Pol-Nef (clade C) clinical [499] [500] [501] [502] [503] Env, Gag-Pol-Nef (clade B) clinical [504, 505] AIV HA preclinical [408] HTLV-1 Env, Env + Gag preclinical [458, 506, 507] Parasitic diseases

CS, SSP2, LSA1, MSP1, SERA, AMA1, Pfs25 clinical [509] Abbreviations Most of the studies involved in the deletion of immune-modulating VACV genes have been performed in the VACV WR strain and the general results showed that deletion of many VACV genes attenuated the virus [289] , but the impact on immunogenicity was variable. Thus, deletion of some immunomodulatory VACV genes from different strains (mainly WR and MVA) increased the immunogenicity against VACV antigens, as it is described for VACV genes E3L [510] , B15R/B16R [511] [512] [513] , A41L [514] , B13R and B22R [515] , C12L [516] , A35R [517] or C6L [518] . However, deletion of other immunomodulatory genes has no effect on the virulence or pathogenicity, as in B8R [519, 520] , or does not enhance the immunogenicity against VACV, as in N2L [521] or C16L [522] . Moreover, deletions of C12L, A44L, A46R or B7R in MVA did not significantly affect VACV-specific CD8 T cell immunogenicity in BALB/c mice [511] . Furthermore, the Wyeth strain with deletions of coding regions for the B5R, B8R, B12R, B13R, B14R, B16R, B18R and B19R immunomodulatory gene products did not increase the immunogenicity of these vectors compared with the parental VACV [523] .

Recently, several deletions in the Tian Tan strain have been performed and analyzed. For example, a recombinant Tian Tan VACV expressing HIV-1 Gag, Pol and Env genes and with deletions in the C12L and A53R genes is highly attenuated and retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV-1 target genes [524] .

In addition, it has been reported that deletion in the Lister strain of the five major nonessential regions that are deleted in MVA enhances the attenuation, although the VACV-specific immune responses were similar to those of the traditional smallpox vaccine [525] . Nonetheless, introduction of the six major genomic deletions of MVA into the parental VACV CVA is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice [526] .

Combination of the insertion of a heterologous antigen in a poxvirus vector with the deletion of an immunomodulatory VACV gene is a promising novel approach to optimize the poxvirus vaccine vector by enhancing immunogenicity against the foreign antigen [481] . This strategy has been widely used for the generation of optimized recombinant MVA and NYVAC vectors expressing HIV-1 antigens (which are used as HIV/AIDS vaccine candidates) and containing single or multiple deletions in immunomodulatory VACV genes that antagonize host-specific immune responses. These new optimized recombinant MVA and NYVAC vaccine vectors lacking VACV immunomodulatory genes have been tested in mice [327, 516, [527] [528] [529] [530] [531] [532] and non-human primates [533, 534] , and the overall results showed that they induced an enhancement in the HIV-1-specific cellular and humoral immune responses.

Thus, the removal of VACV immunomodulatory genes that block the host immune responses to the infection is a useful method to enhance the antigen-specific immune responses induced by different poxvirus-based vaccine candidates. Dissection of the immune profile induced by these novel poxvirus vectors with deletions in single genes or in gene families is necessary to find an optimized poxvirus vector that could enter into future human clinical trials to test whether it can provide protection against infection. In fact, system biology profiles of NYVAC vectors expressing HIV-1 antigens and lacking the IFN inhibitors B8 and B19 revealed in human macrophages distinct gene signatures that can be correlated with immune parameters relevant in protection [535] . Gene signatures have also been defined for the HIV vaccine candidates MVA-B [291] and MVA-C [536] . A summary of poxvirus genes deleted in the context of poxvirus-based vaccine vectors against different infectious diseases is shown in Table 9 . 

One of the advantages of replication-deficient viruses is their safety profile. However, it has been postulated that the efficacy of these viruses is restricted due to the failure to replicate and the limitation in antigen accumulation during virus infection. For this reason, the restoration of replication competence in human cells, together or not with the deletion of specific immunomodulatory VACV genes, can be a strategy to improve the efficacy of poxvirus-based vectors.

In the case of the NYVAC vector, the restoration of replication capacity is obtained by the reinsertion of K1L and/or C7L host range genes in the viral genome. It has been reported that these new constructs are still attenuated but acquire new biological properties distinct from the parental NYVAC that make them potentially improved vaccine vector candidates for human applications [542, 543] (see Table 10 ). Furthermore, the gene signatures of a replication-competent NYVAC vector expressing HIV-1 genes (termed NYVAC-C-KC) in human dendritic cells have been described [542] .

Another category of genes that has been used to improve poxviruses as vaccine vectors is those that encode co-stimulatory molecules such as IL-1α, IL-2, IFN-γ, IL-12, IL-15, OX40/OX40L, B7-1, ICAM-1, LFA-3, CD80, CD86, CD83, CD40L or GM-CSF [481] . This strategy significantly enhanced the immunogenicity and efficacy of the poxvirus vector as a vaccine against different infectious diseases and has been extensively used against cancer (see Section 7). However, there is a limitation on the insertion of this immunomodulators. Ramshaw and colleagues discovered in 2001 that the insertion of mouse interleukin-4 by a recombinant ectromelia virus suppressed antiviral cell-mediated immune responses [544] . This has been further explored and the insertion of this Th2 cytokine into several poxviruses significantly increased the efficiency of the recombinant virus as a pathogen by directly inhibiting the development of Th1 immunity, which is crucial for viral clearance [545, 546] . 

The optimization of gene expression of poxvirus-based vaccines is addressed to improve the generation of immune responses to the heterologous antigen. Thus, the regulation of the antigen expression level is an alternative vaccine-design strategy adopted to induce antigen-specific immune responses [558] .

In this regard, the late-early VACV p7.5 promoter [559] was the first strategy used to induce heterologous antigen expression. The removal of poxvirus transcription termination signals from inserted genes [560] and the regulation of gene expression under the bacteriophage T7 promoter [561] , the vaccinia modified H5 (mH5) [366] and the vaccinia short synthetic early-late pS [562] promoters have been used as alternatives to p7.5 to increase the quantity of heterologous antigen expressed during infection.

Recently, it has been demonstrated that the efficiency with which an antigen is processed and presented on the surface of infected cells influences its recognition [563] . In fact, in VACV, 90% of the antigens most recognized by CD8 T cells were ranked among the top 50% in terms of mRNA expression [564] , and there is a correlation between the timing of viral antigen expression and the generation of antigen-specific immune responses [565] . For this reason, efforts towards developing new poxvirus vaccines candidates are focused on using promoters to improve the timing rather than the quantity of antigen expression.

After a deep analysis of the VACV transcriptome, two groups have defined two categories of early genes based on their temporal expression [566, 567] . Based on these studies, endogenous poxviral early promoters have been compared with the p7.5 and pS promoters. The pC11R and pF11L promoters induced high levels of early antigen expression and cellular immunogenicity similar to those of p7.5 and pS promoters [568] .

More recent studies demonstrate that it is possible to design poxvirus promoters that improve early antigen expression and antigen-specific T cell responses. In this regard, synthetic early promoters such as psFJ1-10 [569, 570] or pHyb [571] and native early promoters like PrMVA13.5-long [572] present repeated motifs, each containing an early promoter element. An alternative strategy for poxvirus promoter design is the optimization of the early promoter element after bioinformatic analysis, as indicated by the Late-Early Optimized (LEO) promoter [573] . These new promoters are able to increase the expression of heterologous antigens and their specific immune response compared to the p7.5 and pS. They represent excellent prototypes for the generation of safe poxvirus recombinant-based vaccines to potentiate the antigen expression and immune response.

Poxviruses represent strong contenders for cancer vaccine development given their ability to express large foreign genes, capacity to induce a strong cytotoxic T lymphocyte (CTL) responses, broad tissue tropism, fast replication and lysis of infected cells, potential to take advantage of the tumor microenvironment (deregulation of cell cycle control, partially blocked IFN response and apoptosis or immune evasion), and the absence of DNA integration into the host genome for safety [574] .

All these features could be a potential solution to a range of issues that characterize cancer: low immune response generated by tumor-associated antigens (TAA), strong immune-suppressor tumor environment, antiviral immune response elicited by the vector and concerns regarding the safety of the vaccine used (sites of virus infection and/or replication, toxicity of the transgene expressed, or other vaccine-associated side effects) [575, 576] .

In 1963, for the first time, a poxvirus was assayed as a potential vector to treat tumors; in this case, Purified Vaccine Lymph was used to treat various skin cancers by local injection [577] . Within the several poxvirus-based strategies deployed and analyzed at preclinical stages for cancer vaccine development, VACV, FWPV, CNPV and their combinations as vectors represent the majority seen on Table 11 .

As we have described for infectious diseases, in the development of different vaccines against cancer, various approaches involve the insertion of heterologous genes into common poxvirus strains, such as immunotherapy by the expression of TAA (e.g., MUC1 [578] , oncofetal antigen 5T4 [579] , PSA [580] , CEA [581] ), the expression of immunomodulatory genes (e.g., costimulatory molecules-B7.1, B7.2 [582] , CD80 [583] or cytokines-IL-2 [584] , IFN-β [585] , GM-CSF [586] ), the expression of suicide genes (e.g., cytosine deaminase [587] , purine nucleoside phosphorylase [588] ), and the expression of genes used for the imaging as a support for combination therapies (thyroidal sodium iodide symporter NIS [589] ).

On the other hand, a different artificial evolution of poxviruses has been performed to direct the natural oncolytic capacity of this family, generating tumor-tailored viruses that grow to a higher extent in tumor cells and microenvironment. In this way, poxviruses have been engineered by the deletion of specific genes involved in nucleotide metabolism, interferon response, the cell cycle and other cell functions abnormally regulated in tumor cells (e.g., J2R, C11R, B18R [574] ).

Likewise, combinations of all the different strategies mentioned above have been evaluated altogether in different immunization protocols. Some of these strategies are especially promising, which is reflected in the significant number of human clinical trials in phase I, II or III that have been or are being carried out targeting different types of cancers such as melanoma, breast, prostate or liver cancer [479] . 

Since Jenner first described in 1798 the application of a virus isolated from a cow to demonstrate the efficacy of vaccination against smallpox, the poxvirus family has been in constant change and under human-made adaptation. It was soon realized that this family of viruses was quite large and infected a wide range of animal species. Only the orthopoxvirus genus replicates productively in humans, with the variola strain being the cause of smallpox. Due to the health problems inherent in smallpox, one of the most dreadful diseases of human mankind with a death rate of about 30%, a major effort was dedicated to eradicate this scourge. It was not until 1980 that the WHO declared that the world was free of smallpox. Along the way, many studies aimed to understand the biology of this group of animal viruses, and major scientific discoveries emerged that had a profound effect on biology as a whole. In fact, scientific concepts as relevant as the basis of immunology and antibody responses to virus infection, the components of a virus particle (DNA, protein, lipids), the presence of a DNA-dependent RNA polymerase, the virion machinery for mRNA synthesis and modifications at the 3'-end and 5'-end, the formation of two forms of infectious virus particles, the existence of multiple viral genes with the capacity to counteract host immune responses, and the ability of the virus to accept the insertion of heterologous foreign genes in the viral genome or the removal of multiple viral genes, are among features that define the plasticity of this family of viruses, which in turn increased our knowledge of living viruses and cells.

The urgency to develop attenuated vaccines promoted the use of different animal models and cell culture systems for virus isolation. As a result a number of vaccine strains emerged in different countries. It was not until whole genome sequencing was developed that we realized the occurrence of different genetic changes within the virus genome. Being a large DNA with a high fidelity polymerase, it was not surprising that, in order to observe genetic alterations such as deletions and point mutations, multiple passages of the virus in cell cultures were needed for attenuation. This effort resulted in the isolation and identification of the now most widely used poxvirus vaccine strain candidates, mainly those derived from NYCBH (Dryvax and ACAM2000), Lister, LC16m8, Tian Tan, MVA, NYVAC and ALVAC.

After the first entire DNA sequence of the Copenhagen strain of VACV was described in 1990, the number of poxvirus whole genome sequences has increased considerably. The information provided facilitated the identification of mutations that correlated with an attenuated phenotype. Indeed, it was found that in the case of the LC16m8 strain derived from Lister, the cause of attenuation is a frame-shifting single nucleotide deletion in the B5R gene. Thus, the old approach of allowing the appearance of spontaneous mutations in the virus genome during cell passages is no longer the method of choice. In fact, as soon as knowledge developed on the biological role of viral genes, newly designed vectors were developed, like NYVAC, a vector derived from the Copenhagen strain by selective deletion of 18 open reading frames (ORFs). The DNA sequencing methods together with the easy method for removing or incorporating selected genes in the viral genome have considerably expanded our understanding of the role of viral-encoded immune modulators and the use of poxvirus vectors as vaccine candidates.

Multiple vaccine candidates have been developed based on members of the poxvirus family with the ability to express the genes of interest in hosts of different origins. The most widely used poxvirus vectors are derived from MVA, NYVAC and ALVAC, and while none of these vectors has been approved for human usage as a virus recombinant vaccine, the promising results obtained in a large number of preclinical and clinical trials presage a not-too-distant application of these recombinant viral vectors as vaccines in humans against multiple diseases. This is exemplified in the partial efficacy of ALVAC against HIV in a phase III clinical trial in Thailand, and in the recent outbreak of Ebola after efficacy results were observed in non-human primates with the prime/boost combination of adenovirus and MVA vectors expressing Ebola GP protein, a protocol that might be implemented as part of the phase I/II clinical trials that have been initiated at various sites in Africa. There is also abundant preclinical information on the proven efficacy of these vectors in other model diseases.

What can we expect the next steps in poxvirus vaccine evolution to be? The fact that the whole virus genome sequence can be reconstituted through synthesis of nucleotides, that the virus genome can be easily manipulated genetically, and that new information on the role of viral genes and interactions with the host cell are known, indicate that for vaccine purposes novel vectors with high specificity to trigger B and T cell immune responses and with high protective capacity will be developed. Indeed, novel vectors triggering high immune responses against the foreign expressed antigens have been generated, either by selective deletion of viral immune modulators, incorporation of host range genes, incorporation of cytokines/chemokines genes or of other inducers/activators of immune responses. Still it is unclear if we just need to develop vectors that trigger very potent immune responses, i.e., high ELISPOT numbers, or just to select those that trigger long-term memory B and T cell responses as an index of potency. In all cases, efficacy will be needed in model systems, as well as definition of immune correlates of protection. The implementation of system biology approaches in preclinical and clinical trials, from non-human primates to vaccinated individuals, will identify gene signatures relevant in protection and could aid in the selection of optimal immunogens. Still, much remains to be learned about the biological role played by many of the virus-encoded immune modulators. For vaccine purposes, it will be important to advance with vectors that are well characterized in terms of pathogenicity, immunogenicity and molecular signatures. Best-in-class candidates can be defined by direct head-to-head comparison on immune characteristics among vectors. Since the final aim is to develop a vaccine that fully protects against a disease, this will only be known with the progressive advance of generating optimized poxvirus vectors and studying their behavior in animal models and in clinical trials. Nonetheless, it is predicted that while in some cases a single poxvirus recombinant vector might be sufficient to fight a disease, it is likely that in most cases heterologous vector combinations, like DNA, RNA, protein or other attenuated viral vectors, will be used together in prime/boost protocols to fight more complex diseases. Overall, we have seen remarkable changes in poxvirus evolution with time, from virus isolation in animals and cell cultures to selectively targeting viral genes. As more scientific information is gained on vector biology and preclinical and clinical trials further advance, showing health benefits on vectors' behavior, we anticipate a bright future for the poxvirus-based vaccine field. 

The Greatest Killer: Smallpox in History, with a New Introduction

Smallpox vaccines: Past, present, and future

The links between the medical school of Padua and the Hellenic medical world

Jenner and the history of smallpox and vaccination

Has horsepox become extinct?

On the vaccination before and after Jenner

The royal philanthropic expedition of the vaccine

Smallpox and Its Erradication; World Health Organization

The Global Eradication of Smallpox. Final Report of the Global Commision for the Certification of Smallpox Eradication; World Health Organization

Genetic studies with mammalian poxviruses. IV. Hybridization between several different poxviruses

Molecular genetics of vaccinia virus: Demonstration of marker rescue

Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA

Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus

Vaccinia virus: A selectable eukaryotic cloning and expression vector

Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

Immunogenicity of recombinant vaccinia viruses expressing hepatitis B virus surface antigen in mice

Protection against anthrax with recombinant virus-expressed protective antigen in experimental animals

Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus

Immunization with a vaccinia recombinant expressing the F protein protects rabbits from challenge with a lethal dose of rinderpest virus

Immunological and virological characterization of improved construction of recombinant vaccinia virus expressing rinderpest virus hemagglutinin

Evaluation of recombinant vaccinia virus-Measles vaccines in infant rhesus macaques with preexisting measles antibody

Characterization of specific immune responses of mice inoculated with recombinant vaccinia virus expressing an 18-kilodalton outer membrane protein of Brucella abortus

Evaluation in non-human primates of the safety, immunogenicity and efficacy of recombinant vaccinia viruses expressing the F or G glycoprotein of respiratory syncytial virus

Immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens

Characterization of poxviruses from forest birds in Hawaii

Avian host spectrum of avipoxviruses

The genome of fowlpox virus

The genome of canarypox virus

Morphogenesis of canary poxvirus and its entrance into inclusion bodies

Fowlpox virus as a vector in non-avian species

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease

First field trial of a transmissible recombinant vaccine against myxomatosis and rabbit hemorrhagic disease

Special edition future of vaccination: Everything about attenuated vaccines. Basics of new attenuated vaccine strain LC16m8

LC16m8: An attenuated smallpox vaccine

Comparative studies of several vaccinia virus strains by intrathalamic inoculation into cynomolgus monkeys

Regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins

Highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge

Comparative evaluation of the immune responses and protection engendered by LC16m8 and Dryvax smallpox vaccines in a mouse model

Smallpox vaccine safety is dependent on T cells and not B cells

LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox

Safety of attenuated smallpox vaccine LC16m8 in immunodeficient mice

Safety and immunogenicity of LC16m8, an attenuated smallpox vaccine in vaccinia-naive adults

Clinical and immunological response to attenuated tissue-cultured smallpox vaccine LC16m8

Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8

Antibodies against the extracellular enveloped virus B5R protein are mainly responsible for the EEV neutralizing capacity of vaccinia immune globulin

Serological responses in humans to the smallpox vaccine LC16m8

Robust HIV-specific immune responses were induced by DNA vaccine prime followed by attenuated recombinant vaccinia virus (LC16m8 strain) boost

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus

A new mutant of dermovaccinia virus

Structural analysis of vaccinia virus DIs strain: Application as a new replication-deficient viral vector

A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity

Mucosal administration of completely non-replicative vaccinia virus recombinant Dairen I strain elicits effective mucosal and systemic immunity

Recombinant vaccinia DIs expressing simian immunodeficiency virus gag and pol in mammalian cells induces efficient cellular immunity as a safe immunodeficiency virus vaccine candidate

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol

Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox

Rapid protection in a monkeypox model by a single injection of a replication-deficient vaccinia virus

Clinical and immunologic responses to multiple doses of IMVAMUNE (Modified Vaccinia Ankara) followed by Dryvax challenge

Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model

Vaccinia virus immune evasion: Mechanisms, virulence and immunogenicity

Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome

Selective induction of host genes by MVA-B, a candidate vaccine against HIV/AIDS

Modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of CCL2 expression

Evaluation of modified vaccinia virus Ankara as an alternative vaccine against smallpox in chronically HIV type 1-infected individuals undergoing HAART

Prospective surveillance for cardiac adverse events in healthy adults receiving modified vaccinia Ankara vaccines: A systematic review

Safety, immunogenicity and efficacy of modified vaccinia Ankara (MVA) against Dryvax challenge in vaccinia-naive and vaccinia-immune individuals

Effect of vaccination with modified vaccinia Ankara (ACAM3000) on subsequent challenge with Dryvax

The Future of Smallpox Vaccination: Is MVA the key?

Safety and immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine

A randomized, double-blind, dose-finding Phase II study to evaluate immunogenicity and safety of the third generation smallpox vaccine candidate IMVAMUNE

Safety and immunogenicity of modified vaccinia Ankara as a smallpox vaccine in people with atopic dermatitis

Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases

Therapeutic vaccination to treat chronic infectious diseases: Current clinical developments using MVA-based vaccines

Recombinant MVA vaccines: Dispelling the myths

Clinical development of Modified Vaccinia virus Ankara vaccines

MVA and NYVAC as vaccines against emergent infectious diseases and cancer

Poxvirus vectors as HIV/AIDS vaccines in humans

Clinical applications of attenuated MVA poxvirus strain

Poxvirus vectors. Vaccine

Protective efficacy of Modified Vaccinia virus Ankara in preclinical studies

Vaccinia viruses: Vaccines against smallpox and vectors against infectious diseases and tumors

Safety and immunogenicity of modified vaccinia Ankara (ACAM3000): Effect of dose and route of administration

A phase I randomized clinical trial of candidate human immunodeficiency virus type 1 vaccine MVA.HIVA administered to Gambian infants

Broad and potent cellular and humoral immune responses after a second late HIV-modified vaccinia virus ankara vaccination in HIV-DNA-primed and HIV-modified vaccinia virus Ankara-boosted Swedish vaccinees

Safety and immunogenicity of DNA and MVA HIV-1 subtype C vaccine prime-boost regimens: A phase I randomised Trial in HIV-uninfected Indian volunteers

Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine

Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles

DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults

MVA-nef induces HIV-1-specific polyfunctional and proliferative T-cell responses revealed by the combination of short-and long-term immune assays

Coadministration of seasonal influenza vaccine and MVA-NP+M1 simultaneously achieves potent humoral and cell-mediated responses

Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine: A randomised, double-blind phase 1/2a clinical trial

Partially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection

Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections

Efficacy of immunotherapy with TG4040, peg-interferon, and ribavirin in a Phase 2 study of patients with chronic HCV infection

A heterologous prime/boost vaccination strategy enhances the immunogenicity of therapeutic vaccines for hepatitis C virus

Comparative immunogenicity analysis of modified vaccinia Ankara vectors expressing native or modified forms of hepatitis C virus E1 and E2 glycoproteins

High, broad, polyfunctional, and durable T cell immune responses induced in mice by a novel hepatitis C virus (HCV) vaccine candidate (MVA-HCV) based on modified vaccinia virus Ankara expressing the nearly full-length HCV genome

A novel poxvirus-based vaccine, MVA-CHIKV, is highly immunogenic and protects mice against chikungunya infection

A novel MVA vectored Chikungunya virus vaccine elicits protective immunity in mice

Recombinant modified vaccinia virus Ankara expressing glycoprotein E2 of Chikungunya virus protects AG129 mice against lethal challenge

Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge

An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara

Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge

A novel vaccine against Crimean-Congo Haemorrhagic Fever protects 100% of animals against lethal challenge in a mouse model

Heterologous MVA-S prime Ad5-S boost regimen induces high and persistent levels of neutralizing antibody response against SARS coronavirus

Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region

Protection against lethal Japanese encephalitis virus infection of mice by immunization with the highly attenuated MVA strain of vaccinia virus expressing JEV prM and E genes

Immunogenicity of a recombinant MVA and a DNA vaccine for Japanese encephalitis virus in swine

Multiple linear epitopes (B-cell, CTL and Th) of JEV expressed in recombinant MVA as multiple epitope vaccine induces a protective immune response

Vaccination with recombinant modified vaccinia virus Ankara protects against measles virus infection in the mouse and cotton rat model

Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques

Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus

Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection

Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques

Evaluation of recombinant modified vaccinia Ankara virus-based rhesus cytomegalovirus vaccines in rhesus macaques

A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector

Intergenic region 3 of modified vaccinia ankara is a functional site for insert gene expression and allows for potent antigen-specific immune responses

A vaccine based on the rhesus cytomegalovirus UL128 complex induces broadly neutralizing antibodies in rhesus macaques

Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

Recombinant MVA expressing secreted glycoprotein D of BoHV-1 induces systemic and mucosal immunity in animal models

Use of apathogenic vaccinia virus MVA expressing EHV-1 gC as basis of a combined recombinant MVA/DNA vaccination scheme

Evaluation of a needle-free delivery platform for prime-boost immunization with DNA and modified vaccinia virus ankara vectors expressing herpes simplex virus 2 glycoprotein D

Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model

Comparison of the immunogenicity and efficacy of a replication-defective vaccinia virus expressing antigens of human parainfluenza virus type 3 (HPIV3) with those of a live attenuated HPIV3 vaccine candidate in rhesus monkeys passively immunized with PIV3 antibodies

The immunogenicity and efficacy of intranasally or parenterally administered replication-deficient vaccinia-parainfluenza virus type 3 recombinants in rhesus monkeys

Comparing the safety and immunogenicity of a candidate TB vaccine MVA85A administered by intramuscular and intradermal delivery

The candidate TB vaccine, MVA85A, induces highly durable Th1 responses

Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: A randomised, placebo-controlled phase 2b trial

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins

Efficacy and safety of a modified vaccinia Ankara (MVA) vectored plague vaccine in mice

Safety and immunogenicity of heterologous prime-boost immunisation with Plasmodium falciparum malaria candidate vaccines, ChAd63 ME-TRAP and MVA ME-TRAP, in healthy Gambian and Kenyan adults

Evaluation of the Efficacy of ChAd63-MVA Vectored Vaccines Expressing CS & ME-TRAP Against Controlled Human Malaria Infection in Malaria Naive Individuals

Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors

ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: Assessment of efficacy against mosquito bite challenge in humans

Assessment of humoral immune responses to blood-stage malaria antigens following ChAd63-MVA immunization, controlled human malaria infection and natural exposure

Analysis of human B-cell responses following ChAd63-MVA MSP1 and AMA1 immunization and controlled malaria infection

Combining Viral Vectored and Protein-in-adjuvant Vaccines Against the Blood-stage Malaria Antigen AMA1: Report on a Phase 1a Clinical Trial

Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors

MIG and the regulatory cytokines IL-10 and TGF-beta1 correlate with malaria vaccine immunogenicity and efficacy

A human Phase I/IIa malaria challenge trial of a polyprotein malaria vaccine

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection

Heterologous priming-boosting with DNA and modified vaccinia virus Ankara expressing tryparedoxin peroxidase promotes long-term memory against Leishmania major in susceptible BALB/c Mice

A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia)

MVA ROP2 vaccinia virus recombinant as a vaccine candidate for toxoplasmosis

TcVac3 induced control of Trypanosoma cruzi infection and chronic myocarditis in mice

Marek's disease virus recombinant poxvirus vaccine

Attenuation of virulent fowl pox virus in tissue culture and characteristics of the attenuated virus

Comparison of the genome sequence of FP9, an attenuated, tissue culture-adapted European strain of Fowlpox virus, with those of virulent American and European viruses

Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A * 01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency

Systemic immunization with an ALVAC-HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian-human immunodeficiency virus SHIVKU2 RNA levels

Immunogenicity and protective efficacy of a human immunodeficiency virus type 2 recombinant canarypox (ALVAC) vaccine candidate in cynomolgus monkeys

Challenge of chimpanzees immunized with a recombinant canarypox-HIV-1 virus

ALVAC-HIV vaccines: Clinical trial experience focusing on progress in vaccine development

Safety profile of recombinant canarypox HIV vaccines

The safety and use of canarypox vectored vaccines

Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand

Efficacy of an inactivated and a fowlpox-vectored vaccine in Muscovy ducks against an Asian H5N1 highly pathogenic avian influenza viral challenge

Immunogenicity of fowlpox virus expressing the avian influenza virus H5 gene (TROVAC AIV-H5) in cats

Characterization of host responses against a recombinant fowlpox virus-vectored vaccine expressing the hemagglutinin antigen of an avian influenza virus

Safety, immunogenicity and efficacy of poxvirus-based vector vaccines expressing the haemagglutinin gene of a highly pathogenic H5N1 avian influenza virus in pigs

Efficacy of a recombinant fowl pox-based Newcastle disease virus vaccine candidate against velogenic and respiratory challenge

Containment of simian immunodeficiency virus infection in vaccinated macaques: Correlation with the magnitude of virus-specific pre-and postchallenge CD4+ and CD8+ T cell responses

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

A prime-boost approach to HIV preventive vaccine using a recombinant canarypox virus expressing glycoprotein 160 (MN) followed by a recombinant glycoprotein 160 (MN/LAI). The AGIS Group, and l'Agence Nationale de Recherche sur le SIDA

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

Induction of human immunodeficiency virus type 1 (HIV-1)-specific cytolytic T lymphocyte responses in seronegative adults by a nonreplicating, host-range-restricted canarypox vector (ALVAC) carrying the HIV-1MN env gene

Immune responses to human immunodeficiency virus (HIV) type 1 induced by canarypox expressing HIV-1MN gp120, HIV-1SF2 recombinant gp120, or both vaccines in seronegative adults. NIAID AIDS Vaccine Evaluation Group

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

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group

Safety and immunogenicity of a live recombinant canarypox virus expressing HIV type 1 gp120 MN MN tm/gag/protease LAI (ALVAC-HIV, vCP205) followed by a p24E-V3 MN synthetic peptide (CLTB-36) administered in healthy volunteers at low risk for HIV infection

AIDS Vaccine Evaluation Group 022 Protocol Team. Cellular and humoral immune responses to a canarypox vaccine containing human immunodeficiency virus type 1 Env, Gag, and Pro in combination with rgp120

Safety and immunogenicity of a canarypox-vectored human immunodeficiency virus Type 1 vaccine with or without gp120: A phase 2 study in higher-and lower-risk volunteers

Safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp120 in a diverse population of HIV-1-uninfected adults: AIDS Vaccine Evaluation Group Protocol 022A

Immunogenicity of a recombinant human immunodeficiency virus (HIV)-canarypox vaccine in HIV-seronegative Ugandan volunteers: Results of the HIV Network for Prevention Trials 007 Vaccine Study

A double-blind randomized phase I clinical trial targeting ALVAC-HIV vaccine to human dendritic cells

Safety and immunogenicity of an HIV-1 recombinant canarypox vaccine in newborns and infants of HIV-1-infected women

A canarypox vaccine expressing multiple human immunodeficiency virus type 1 genes given alone or with rgp120 elicits broad and durable CD8+ cytotoxic T lymphocyte responses in seronegative volunteers

Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults

Antibody-dependent cell-mediated cytotoxic responses in participants enrolled in a phase I/II ALVAC-HIV/AIDSVAX B/E prime-boost HIV-1 vaccine trial in Thailand

A phase 1/2 comparative vaccine trial of the safety and immunogenicity of a CRF01_AE (subtype E) candidate vaccine: ALVAC-HIV (vCP1521) prime with oligomeric gp160 (92TH023/LAI-DID) or bivalent gp120 (CM235/SF2) boost

Safety and reactogenicity of canarypox ALVAC-HIV (vCP1521) and HIV-1 gp120 AIDSVAX B/E vaccination in an efficacy trial in Thailand

Feasibility and safety of ALVAC-HIV vCP1521 vaccine in HIV-exposed infants in Uganda: Results from the first HIV vaccine trial in infants in Africa

Immunogenicity of ALVAC-HIV vCP1521 in infants of HIV-1-infected women in Uganda (HPTN 027): The first pediatric HIV vaccine trial in Africa

Lessons from the RV144 Thai Phase III HIV-1 Vaccine Trial and the Search for Correlates of Protection

Effect of previous or simultaneous immunization with canarypox expressing cytomegalovirus (CMV) glycoprotein B (gB) on response to subunit gB vaccine plus MF59 in healthy CMV-seronegative adults

A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne)

A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects

Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species

Human safety and immunogenicity of a canarypox-rabies glycoprotein recombinant vaccine: An alternative poxvirus vector system

Immunisation with canarypox virus expressing rabies glycoprotein

Protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins

Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVACand ALVAC-based CDV recombinants protect against symptomatic infection

Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines

Mucosal vaccination with recombinant poxvirus vaccines protects ferrets against symptomatic CDV infection

Assessment of the efficacy of a single dose of a recombinant vaccine against West Nile virus in response to natural challenge with West Nile virus-infected mosquitoes in horses

Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse

Protection provided by a recombinant ALVAC((R))-WNV vaccine expressing the prM/E genes of a lineage 1 strain of WNV against a virulent challenge with a lineage 2 strain

Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge

Recombinant canarypox vectored West Nile virus (WNV) vaccine protects dogs and cats against a mosquito WNV challenge

Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL

Efficacy of a canarypox virus-vectored vaccine against feline leukaemia

Reassessment of feline leukaemia virus (FeLV) vaccines with novel sensitive molecular assays

Efficacy evaluation of prime-boost protocol: Canarypoxvirus-based feline immunodeficiency virus (FIV) vaccine and inactivated FIV-infected cell vaccine against heterologous FIV challenge in cats

Equine viral vaccines: The past, present and future

Onset and duration of immunity to equine influenza virus resulting from canarypox-vectored (ALVAC) vaccination

Antibody and IFN-gamma responses induced by a recombinant canarypox vaccine and challenge infection with equine influenza virus

Characterisation of CTL and IFN-gamma synthesis in ponies following vaccination with a NYVAC-based construct coding for EHV-1 immediate early gene, followed by challenge infection

Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus-Poxvirus vaccines in vaccinia-nonimmune and vaccinia-immune humans

Induction of Japanese encephalitis virus-specific cytotoxic T lymphocytes in humans by poxvirus-based JE vaccine candidates

Highly attenuated HTLV type Ienv poxvirus vaccines induce protection against a cell-associated HTLV type I challenge in rabbits

Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus

An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses

A recombinant canarypox virus protects rabbits against a lethal rabbit hemorrhagic disease virus (RHDV) challenge

Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: Determination of CTL and CTL precursor frequencies using a recombinant canarypox virus (ALVAC)

Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep

Cross-clade immunity in cats vaccinated with a canarypox-vectored avian influenza vaccine

Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guerin

Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus

A Plasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses

A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya

Immunogenicity of the candidate malaria vaccines FP9 and modified vaccinia virus Ankara encoding the pre-erythrocytic antigen ME-TRAP in 1-6 year old children in a malaria endemic area

Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein

Safety profile of the viral vectors of attenuated fowlpox strain FP9 and modified vaccinia virus Ankara recombinant for either of 2 preerythrocytic malaria antigens, ME-TRAP or the circumsporozoite protein, in children and adults in Kenya

Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara

Safety of recombinant fowlpox strain FP9 and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers

Safety and immunogenicity of the malaria candidate vaccines FP9 CS and MVA CS in adult Gambian men

Induction of multi-antigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys, in the absence of antigen interference, with heterologous DNA prime/poxvirus boost immunization

Multistage multiantigen heterologous prime boost vaccine for Plasmodium knowlesi malaria provides partial protection in rhesus macaques

Utilizing poxviral vectored vaccines for antibody induction-progress and prospects

Prime-boost vectored malaria vaccines: Progress and prospects

Poxvirus-based vaccines for cancer immunotherapy: New insights from combined cytokines/co-stimulatory molecules delivery and "uncommon

Poxvirus vector-based HIV vaccines

Enhancing poxvirus vectors vaccine immunogenicity

Highly attenuated poxvirus vectors

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

NYVAC: A highly attenuated strain of vaccinia virus

Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of clade B

Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens

Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates

Immunization with HIV Gag targeted to dendritic cells followed by recombinant New York vaccinia virus induces robust T-cell immunity in nonhuman primates

Successful pseudorabies vaccination in maternally immune piglets using recombinant vaccinia virus vaccines

Vaccination with recombinant vaccinia virus vaccines expressing glycoprotein genes of pseudorabies virus in the presence of maternal immunity

Comparison of the efficacy of replication-defective adenovirus and Nyvac poxvirus as vaccine vectors in mice

Vaccination of pigs against pseudorabies with highly attenuated vaccinia (NYVAC) recombinant viruses

Evaluation of a recombinant vaccinia virus containing pseudorabies (PR) virus glycoprotein genes gp50, gII, and gIII as a PR vaccine for pigs

Comparison of the protective response induced by NYVAC vaccinia recombinants expressing either gp50 or gII and gp50 of pseudorabies virus

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen

Recombinant vaccine-induced protection against the highly pathogenic simian immunodeficiency virus SIV(mac251): Dependence on route of challenge exposure

Both mucosal and systemic routes of immunization with the live, attenuated NYVAC/simian immunodeficiency virus SIV(gpe) recombinant vaccine result in gag-specific CD8(+) T-cell responses in mucosal tissues of macaques

Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses

EV01: A phase I trial in healthy HIV negative volunteers to evaluate a clade C HIV vaccine, NYVAC-C undertaken by the EuroVacc Consortium

EV02: A Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone

Optimal priming of poxvirus vector (NYVAC)-based HIV vaccine regimens requires 3 DNA injections. Results of the randomized multicentre EV03/ANRS Vac20 Phase I/II Trial

DNA/NYVAC vaccine regimen induces HIV-specific CD4 and CD8 T-cell responses in intestinal mucosa

NYVAC immunization induces polyfunctional HIV-specific T-cell responses in chronically-infected, ART-treated HIV patients

HIV-specific humoral responses benefit from stronger prime in phase Ib clinical trial

Immunogenicity and protective efficacy of recombinant human T-cell leukemia/lymphoma virus type 1 NYVAC and naked DNA vaccine candidates in squirrel monkeys

Attenuated poxvirus vectors as carriers in vaccines against human T cell leukemia-lymphoma virus type I

Recombinant vaccinia viruses for the characterization of Plasmodium falciparum-specific cytotoxic T lymphocytes: Recognition of processed antigen despite limited re-stimulation efficacy

Phase I/IIa safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria

Vaccinia viruses with mutations in the E3L gene as potential replication-competent, attenuated vaccines: Scarification vaccination

Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

Inactivation of the viral interleukin 1beta receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara

Interleukin-1beta receptor expressed by modified vaccinia virus Ankara interferes with interleukin-1beta activity produced in various virus-infected antigen-presenting cells

Deletion of gene A41L enhances vaccinia virus immunogenicity and vaccine efficacy

Induction of potent humoral and cell-mediated immune responses by attenuated vaccinia virus vectors with deleted serpin genes

Improving the MVA vaccine potential by deleting the viral gene coding for the IL-18 binding protein

Deletion of the A35 gene from Modified Vaccinia Virus Ankara increases immunogenicity and isotype switching

Deletion of immunomodulator C6 from vaccinia virus strain Western Reserve enhances virus immunogenicity and vaccine efficacy

Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity

Cytokine receptors encoded by poxviruses: A lesson in cytokine biology

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence

Vaccinia virus protein C16 acts intracellularly to modulate the host response and promote virulence

Role of genes that modulate host immune responses in the immunogenicity and pathogenicity of vaccinia virus

Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes

Deletion of major nonessential genomic regions in the vaccinia virus Lister strain enhances attenuation without altering vaccine efficacy in mice

Introduction of the six major genomic deletions of modified vaccinia virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice

Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways

Immunogenic profiling in mice of a HIV/AIDS vaccine candidate (MVA-B) expressing four HIV-1 antigens and potentiation by specific gene deletions

A candidate HIV/AIDS vaccine (MVA-B) lacking vaccinia virus gene C6L enhances memory HIV-1-specific T-cell responses

Removal of vaccinia virus genes that block interferon type I and II pathways improves adaptive and memory responses of the HIV/AIDS vaccine candidate NYVAC-C in mice

Deletion of the vaccinia virus gene A46R, encoding for an inhibitor of TLR signalling, is an effective approach to enhance the immunogenicity in mice of the HIV/AIDS vaccine candidate NYVAC-C

Deletion of the viral anti-apoptotic gene F1L in the HIV/AIDS vaccine candidate MVA-C enhances immune responses against HIV-1 antigens

Deletion of specific immune-modulatory genes from modified vaccinia virus Ankara-based HIV vaccines engenders improved immunogenicity in rhesus macaques

Expanding the repertoire of Modified Vaccinia Ankara-based vaccine vectors via genetic complementation strategies

Interleukin 1-and type I interferon-dependent enhancement of the innate immune profile of a NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletion of type I and type II interferon-binding proteins

Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C

Deletion of the vaccinia virus N2L gene encoding an inhibitor of IRF3 improves the immunogenicity of modified vaccinia virus Ankara expressing HIV-1 antigens

Attenuated NYCBH vaccinia virus deleted for the E3L gene confers partial protection against lethal monkeypox virus disease in cynomolgus macaques

The NYCBH vaccinia virus deleted for the innate immune evasion gene, E3L, protects rabbits against lethal challenge by rabbitpox virus

The attenuated NYCBH vaccinia virus deleted for the immune evasion gene, E3L, completely protects mice against heterologous challenge with ectromelia virus

Induction of potent immune responses by recombinant fowlpox virus with deleted ORF73 or ORF214

Improved NYVAC-based vaccine vectors

Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors

Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis

The "supervirus"? Lessons from IL-4-expressing poxviruses

Insertion of vaccinia virus C7L host range gene into NYVAC-B genome potentiates immune responses against HIV-1 antigens

Induction of adult-like antibody, Th1, and CTL responses to measles hemagglutinin by early life murine immunization with an attenuated vaccinia-derived NYVAC(K1L) viral vector

High-dose recombinant Canarypox vaccine expressing HIV-1 protein, in seronegative human subjects

Lessons from a multisite international trial in the Caribbean and South America of an HIV-1 Canarypox vaccine (ALVAC-HIV vCP1452) with or without boosting with MN rgp120

Phase 2 study of an HIV-1 canarypox vaccine (vCP1452) alone and in combination with rgp120: Negative results fail to trigger a phase 3 correlates trial

CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

A randomized therapeutic vaccine trial of canarypox-HIV-pulsed dendritic cells vs. canarypox-HIV alone in HIV-1-infected patients on antiretroviral therapy

A randomized controlled trial of HIV therapeutic vaccination using ALVAC with or without Remune

Phase I/II randomized trial of safety and immunogenicity of LIPO-5 alone, ALVAC-HIV (vCP1452) alone, and ALVAC-HIV (vCP1452) prime/LIPO-5 boost in healthy, HIV-1-uninfected adult participants

Safety and immunogenicity of ALVAC vCP1452 and recombinant gp160 in newly human immunodeficiency virus type 1-infected patients treated with prolonged highly active antiretroviral therapy

Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria

Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines

In vitro mutagenesis of the promoter region for a vaccinia virus gene: Evidence for tandem early and late regulatory signals

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

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes

Compact, synthetic, vaccinia virus early/late promoter for protein expression

Correlates of protection efficacy induced by vaccinia virus-specific CD8+ T-cell epitopes in the murine intranasal challenge model

Definition of epitopes and antigens recognized by vaccinia specific immune responses: Their conservation in variola virus sequences, and use as a model system to study complex pathogens

Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination

Deficiency of two red-cell flavin enzymes in a population in Sardinia: Was glutathione reductase deficiency specifically selected for by malaria?

Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing

Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA

Immunogenicity and safety of the vaccinia virus LC16m8Delta vector expressing SIV Gag under a strong or moderate promoter in a recombinant BCG prime-recombinant vaccinia virus boost protocol

Effects of different promoters on the virulence and immunogenicity of a HIV-1 Env-expressing recombinant vaccinia vaccine

Immediate-early expression of a recombinant antigen by modified vaccinia virus ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses

A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses

New vaccinia virus promoter as a potential candidate for future vaccines

Targeted and armed oncolytic poxviruses: A novel multi-mechanistic therapeutic class for cancer

The role of poxviruses in tumor immunotherapy

Therapeutic cancer vaccines: Current status and moving forward

Viral Treatment of Skin Cancers

Vaccinia virus MUC1 immunization of mice: Immune response and protection against the growth of murine tumors bearing the MUC1 antigen

Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors

A recombinant vaccinia virus expressing human prostate-specific antigen (PSA): Safety and immunogenicity in a non-human primate

Viral vectors for vaccine applications

Enhanced generation of cytotoxic T lymphocytes using recombinant vaccinia virus expressing human tumor-associated antigens and B7 costimulatory molecules

Intranodal immunization with a vaccinia virus encoding multiple antigenic epitopes and costimulatory molecules in metastatic melanoma

A phase II study of Tg4010 (Mva-Muc1-Il2) in association with chemotherapy in patients with stage III/IV Non-small cell lung cancer

Treating tumors with a vaccinia virus expressing IFNbeta illustrates the complex relationships between oncolytic ability and immunogenicity

Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma

Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene

Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy

A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter

Local delivery of recombinant vaccinia virus encoding for neu counteracts growth of mammary tumors more efficiently than systemic delivery in neu transgenic mice

Intratumoral delivery of recombinant vaccinia virus encoding for ErbB2/Neu inhibits the growth of salivary gland carcinoma cells

Vaccination of prostate cancer patients with modified vaccinia ankara delivering the tumor antigen 5T4 (TroVax): A phase 2 trial

Vaccination of metastatic renal cancer patients with MVA-5T4: A randomized, double-blind, placebo-controlled phase III study

Evaluation of MVA-5T4 as a novel immunotherapeutic vaccine in colorectal, renal and prostate cancer

Heterologous prime/boost immunization with p53-based vaccines combined with toll-like receptor stimulation enhances tumor regression

Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression

The immunogenicity of the tumor-associated antigen alpha-fetoprotein is enhanced by a fusion with a transmembrane domain

Modified vaccinia Ankara expressing survivin combined with gemcitabine generates specific antitumor effects in a murine pancreatic carcinoma model

Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein

Dual stimulation of Epstein-Barr Virus (EBV)-specific CD4+-and CD8+-T-cell responses by a chimeric antigen construct: Potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma

Cancer gene therapy using tumor cells infected with recombinant vaccinia virus expressing GM-CSF

Recombinant vaccinia virus expressing interleukin-2 invokes anti-tumor cellular immunity in an orthotopic murine model of head and neck squamous cell carcinoma

Combination of intratumoral injections of vaccinia virus MVA expressing GM-CSF and immunization with DNA vaccine prolongs the survival of mice bearing HPV16 induced tumors with downregulated expression of MHC class I molecules

Immunization with a syngeneic tumor infected with recombinant vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) induces tumor regression and long-lasting systemic immunity

Construction of recombinant vaccinia virus expressing GM-CSF and its use as tumor vaccine

MVA-MUC1-IL2 vaccine immunotherapy (TG4010) improves PSA doubling time in patients with prostate cancer with biochemical failure

Immunotherapy with MVA-BN(R)-HER2 induces HER-2-specific Th1 immunity and alters the intratumoral balance of effector and regulatory T cells

Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes

Oncolytic vaccinia virotherapy for endometrial cancer

Regional versus systemic delivery of recombinant vaccinia virus as suicide gene therapy for murine liver metastases

Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus

Modified vaccinia virus Ankara as a vector for suicide gene therapy

The immunologic aspects of poxvirus oncolytic therapy

Characterization and evaluation of a new oncolytic vaccinia virus strain LIVP6.1.1 for canine cancer therapy

Vaccinia virus GLV-1h153 in combination with 131I shows increased efficiency in treating triple-negative breast cancer

Oncolytic vaccinia virus demonstrates antiangiogenic effects mediated by targeting of VEGF

T-cell engager-armed oncolytic vaccinia virus significantly enhances antitumor therapy

Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF

A mechanistic proof-of-concept clinical trial with JX-594, a targeted multi-mechanistic oncolytic poxvirus, in patients with metastatic melanoma

Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer

Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus

Chemokine expression from oncolytic vaccinia virus enhances vaccine therapies of cancer

Insertion of interleukin-2 (IL-2) and interleukin-12 (IL-12) genes into vaccinia virus results in effective anti-tumor responses without toxicity

Oncolytic poxvirus armed with Fas ligand leads to induction of cellular Fas receptor and selective viral replication in FasR-negative cancer

Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases

CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging

Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits

Making an avipoxvirus encoding a tumor-associated antigen and a costimulatory molecule

Induction of p53-specific immune responses in colorectal cancer patients receiving a recombinant ALVAC-p53 candidate vaccine

Preclinical qualification of a new multi-antigen candidate vaccine for metastatic melanoma

Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas

Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule

The influence of granulocyte macrophage colony-stimulating factor and prior chemotherapy on the immunological response to a vaccine (ALVAC-CEA B7.1) in patients with metastatic carcinoma

Adoptive T cell immunotherapy of human uveal melanoma targeting gp100

Enhanced viral and tumor immunity with intranodal injection of canary pox viruses expressing the melanoma antigen, gp100. Cancer

Myxoma Virus Expressing a Fusion Protein of Interleukin-15 (IL15) and IL15 Receptor Alpha Has Enhanced Antitumor Activity

Myxoma virus oncolytic efficiency can be enhanced through chemical or genetic disruption of the actin cytoskeleton

Oncolytic myxoma virus: The path to clinic

Recombinant vaccinia/fowlpox NY-ESO-1 vaccines induce both humoral and cellular NY-ESO-1-specific immune responses in cancer patients

Evaluation of prime/boost regimens using recombinant poxvirus/tyrosinase vaccines for the treatment of patients with metastatic melanoma

Local immunotherapy of spontaneous feline fibrosarcomas using recombinant poxviruses expressing interleukin 2 (IL2)

Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant

PROSTVAC(R) targeted immunotherapy candidate for prostate cancer

Immune impact induced by PROSTVAC (PSA-TRICOM), a therapeutic vaccine for prostate cancer

Poxviral-based vaccine elicits immunologic responses in prostate cancer patients

Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses

A triad of costimulatory molecules synergize to amplify T-cell activation

Vector-based vaccine/cytokine combination therapy to enhance induction of immune responses to a self-antigen and antitumor activity

Poxviral-based vaccine therapy targeting CEA and MUC1 in carcinoma

ORFV: A novel oncolytic and immune stimulating parapoxvirus therapeutic

Potent oncolytic activity of raccoonpox virus in the absence of natural pathogenicity

Yaba-like disease virus: An alternative replicating poxvirus vector for cancer gene therapy

The experiment performed in the laboratory of M. Esteban was funded by research grants from Spain, EU 7th Framework Program and by the Bill and Melinda Gates Foundation. We thank all members of the lab for their continuous support.

The authors declare no conflict of interest.