Search

Summary The advanced-phase HIV prevention vaccine trials done in South Africa (HVTN 702) and in Thailand (RV144), which both investigated canarypox vectors and adjuvanted gp120 proteins, gave rise to different results. The South African trial did not find vaccine efficacy, whereas the Thai trial had modest, but statistically significant, success with the modified intention-to-treat analysis prespecified in the protocols of both studies. An understanding of the differences between the studies is required to avoid the possible, but erroneous, conclusion that the results from the South African trial negatively affect the results of the Thai trial.

Efforts to breed Attwater's prairie chickens (APC; Tympanuchus cupido attwateri) in captivity to supplement wild populations of this endangered bird have been negatively affected by infections with Avipoxvirus and reticuloendotheliosis virus (REV). Because REV can be integrated into the genome of fowlpox virus (FPV) and may be transmitted in that manner, identifying the source of avipox disease in APC is important to mitigate the impact of this virus. Tissue samples from APC were collected from breeding programs in Texas from 2016 to 2020. These samples consisted of 11 skin lesions and three internal organs from a total of 14 different birds that died of unknown causes or were euthanized. Avipoxvirus was detected by PCR and isolation in embryonating chicken eggs in all skin lesion samples but was not detected in internal organs. Using sequence analysis of FPV polymerase and 4b genes, we determined that 10 out of 11 Avipoxvirus detections resided within the fowlpox clade and a single sample resided within the canarypox clade. REV sequences were detected in all FPV positive samples and in all internal organ tissues but were not detected in the sample matching the canarypox clade. Analysis of REV sequences and PCR detection showed the REV infecting APC was consistent with REV-A and had little variability on analysis of the U3 region of the long terminal repeat. The results of this study indicate control of REV in APC breeding colonies may benefit by a vaccination program targeting FPV and REV. However, a commercially available vaccine for REV is not available at this time.

Protective protein antigens from pathogenic bacteria or viruses can be cloned into other nonpathogenic organisms called vectors. If the vector cannot cause disease, then these recombinants can be used as vaccines. The most widely employed vectors used in animal vaccines are poxviruses such as vaccinia or canarypox. However, other vectors include adenoviruses, herpesviruses, and the insect viruses called baculoviruses. Vectors may also include some bacteria, yeasts, and even plants. These vectored vaccines are highly immunogenic and very safe. They have been widely and successfully used in many animal species. Vaccinia vectored vaccines have been used extensively to control rabies in wildlife.

Canine distemper virus (CDV) is one of the most significantinfectious disease threats to the health and conservation of free-ranging and captive wild carnivores. CDV vaccination using recombinant canarypox-based vaccines has been recommended for maned wolf (Chrysocyon brachyurus) after the failure of modified live vaccines that induced disease in vaccinated animals. Here, we report a CDV outbreak in a captive population of maned wolves that were previously vaccinated. Five juveniles and one adult from a group of seven maned wolves housed in an outdoor exhibit died in April&ndash, May 2013 in a zoo in the Metropolitan Region, Chile. Clinical signs ranged from lethargy to digestive and respiratory signs. Diagnosis of CDV was confirmed by histopathology, antibody assays, and viral molecular detection and characterization. The phylogenetic analyses of the nucleotide sequence of the H gene of the CDV genome identified in the two positive samples suggest a close relation with the lineage Europe 1, commonly found in South America and Chile. CDV infections in maned wolves have not been previously characterized. To the authors&rsquo, best knowledge, this is the first report of the clinical presentation of CDV in a canine species previously immunized with a recombinant vaccine.

Nipah virus (NiV) is an emergent pathogen capable of causing acute respiratory illness and fatal encephalitis in pigs and humans. A high fatality rate and broad host tropism makes NiV a serious public and animal health concern. There is therefore an urgent need for a NiV vaccines to protect animals and humans. In this study we investigated the immunogenicity of bovine herpesvirus (BoHV-4) vectors expressing either NiV attachment (G) or fusion (F) glycoproteins, BoHV-4-A-CMV-NiV-G&Delta, TK or BoHV-4-A-CMV-NiV-F&Delta, TK, respectively in pigs. The vaccines were benchmarked against a canarypox (ALVAC) vector expressing NiV G, previously demonstrated to induce protective immunity in pigs. Both BoHV-4 vectors induced robust antigen-specific antibody responses. BoHV-4-A-CMV-NiV-G&Delta, TK stimulated NiV-neutralizing antibody titers comparable to ALVAC NiV G and greater than those induced by BoHV-4-A-CMV-NiV-F&Delta, TK. In contrast, only BoHV-4-A-CMV-NiV-F&Delta, TK immunized pigs had antibodies capable of significantly neutralizing NiV G and F-mediated cell fusion. All three vectored vaccines evoked antigen-specific CD4 and CD8 T cell responses, which were particularly strong in BoHV-4-A-CMV-NiV-G&Delta, TK immunized pigs and to a lesser extent BoHV-4-A-CMV-NiV-F&Delta, TK. These findings emphasize the potential of BoHV-4 vectors for inducing antibody and cell-mediated immunity in pigs and provide a solid basis for the further evaluation of these vectored NiV vaccine candidates.

In the RV144 trial, vaccine-induced V1V2 IgG correlated with decreased HIV-1 risk. We investigated circulating antibody specificities in two phase 1 poxvirus prime-protein boost clinical trials conducted in South Africa: HVTN 097 (subtype B/E) and HVTN 100 (subtype C). With cross-subtype peptide microarrays and multiplex binding assays, we probed the magnitude and breadth of circulating antibody responses to linear variable loop 2 (V2) and conformational V1V2 specificities. Antibodies targeting the linear V2 epitope, a correlate of decreased HIV-1 risk in RV144, were elicited up to 100% and 61% in HVTN 097 and HVTN 100, respectively. Despite higher magnitude of envelope-specific responses in HVTN 100 compared to HVTN 097 (p’s

Antibodies are the primary correlate of protection for most licensed vaccines; however, their mechanisms of protection may vary, ranging from physical blockade to clearance via the recruitment of innate immunity. Here, we uncover striking functional diversity in vaccine-induced antibodies that is driven by immunization site and is associated with reduced risk of SIV infection in nonhuman primates. While equivalent levels of protection were observed following intramuscular (IM) and aerosol (AE) immunization with an otherwise identical DNA prime-Ad5 boost regimen, reduced risk of infection was associated with IgG-driven antibody-dependent monocyte-mediated phagocytosis in the IM vaccinees, but with vaccine-elicited IgA-driven neutrophil-mediated phagocytosis in AE-immunized animals. Thus, although route-independent correlates indicate a critical role for phagocytic Fc-effector activity in protection from SIV, the site of immunization may drive this Fc activity via distinct innate effector cells and antibody isotypes. Moreover, the same correlates predicted protection from SHIV infection in a second nonhuman primate vaccine trial using a disparate IM canarypox prime-protein boost strategy, analogous to that used in the first moderately protective human HIV vaccine trial. These data identify orthogonal functional humoral mechanisms, initiated by distinct vaccination routes and immunization strategies, pointing to multiple, potentially complementary correlates of immunity that may support the rational design of a protective vaccine against HIV.

A canarypox outbreak resulted in the death of about 50% of canaries (Serinus canaria domesticus) in several breeding farms in Lebanon. Infected birds showed thickened eye lid and skin scab-like lesions at the beak, foot and caudal regions and died 5–6 days post disease symptoms onset. Out of seven sick birds that were autopsied for gross pathology evaluation, one bird demonstrated turbid airsacs with absence of any lesions in the trachea, oesophagus, liver and lung. Histopathological examination showed hyperplasia, heterophils infiltration and Bollinger body formation in the skin of five out of the seven autopsied canaries. Hyperplasia and infiltration of heterophils were also observed in the airsacs of one bird. PCR analysis of specimens taken from the skin and feet, targeting the fpv167 gene of the canarypox virus, was positive for all of the analyzed canaries. PCR analysis also revealed that two birds had concurrent infection with Mycoplasma gallisepticum. Sequencing and alignment of the amplified fpv167 gene of the canarypox showed 100% similarity with the Iranian canary pox isolate IR/H913/14. Smuggling of pet birds through the borders should be strictly controlled and biosecurity measures must be adequately applied to control the circulation of the two identified pathogens.

The RV144 HIV-1 vaccine trial results showed moderate reduction in viral infections among vaccinees as well as induction of antibody-dependent cellular cytotoxicity and vaccine-specific IgG and IgG3 responses directed at variable loop regions 1 and 2 of the HIV envelope protein. However, with the recent failure of the HVTN 702 clinical trial, comprehensive profiling of humoral immune responses may provide insight for these disappointing results. One of the changes included in the HVTN 702 study was the addition of a late boost, aimed at augmenting peak immunity and durability. The companion vaccine trial RV305 was designed to permit the evaluation of the immunologic impact of late boosting with either the boosting protein antigen alone, the canarypox viral vector ALVAC alone, or a combination of both. Although previous data showed elevated levels of IgG antibodies in both boosting arms, regardless of ALVAC-HIV vector incorporation, the effect on shaping antibody effector function remains unclear. Thus, here we analyzed the antibody and functional profile induced by RV305 boosting regimens and found that although IgG1 levels increased in both arms that included protein boosting, IgG3 levels were reduced compared with the original RV144 vaccine strategy. Most functional responses increased upon protein boosting, regardless of the viral vector-priming agent incorporation. These data suggest that the addition of a late protein boost alone is sufficient to increase functionally potent vaccine-specific antibodies previously associated with reduced risk of infection with HIV. CA extern

Current HIV vaccines are only partially efficacious, presenting an opportunity to identify correlates of protection and, thereby, potential insight into mechanisms that prevent HIV acquisition. Two independent preclinical challenge studies in nonhuman primates (NHPs) previously showed partial efficacy of a mosaic adenovirus 26 (Ad26)-based HIV-1 vaccine candidate. To investigate the basis of this protection, we performed whole transcriptomics profiling by RNA sequencing (RNA-seq) in sorted lymphocytes from peripheral blood samples taken during these studies at different time points after vaccination but before challenge. We observed a transcriptional signature in B cells that associated with protection from acquisition of simian immunodeficiency virus (SIV) or the simian-human immunodeficiency virus (SHIV) in both studies. Strong antibody responses were elicited, and genes from the signature for which expression was enriched specifically associated with higher magnitude of functional antibody responses. The same gene expression signature also associated with protection in RV144 in the only human HIV vaccine trial to date that has shown efficacy and in two additional NHP studies that evaluated similar canarypox-based vaccine regimens. A composite gene expression score derived from the gene signature was one of the top-ranked correlates of protection in the NHP vaccine studies. This study aims to bridge preclinical and clinical data with the identification of a gene signature in B cells that is associated with protection from SIV and HIV infection by providing a new approach for evaluating future vaccine candidates.

The recombinant Canarypox ALVAC-HIV/gp120/alum vaccine regimen was the first to significantly decrease the risk of HIV acquisition in humans, with equal effectiveness in both males and females. Similarly, an equivalent SIV-based ALVAC vaccine regimen decreased the risk of virus acquisition in Indian rhesus macaques of both sexes following intrarectal exposure to low doses of SIVmac251. Here, we demonstrate that the ALVAC-SIV/gp120/alum vaccine is also efficacious in female Chinese rhesus macaques following intravaginal exposure to low doses of SIVmac251 and we confirm that CD14+ classical monocytes are a strong correlate of decreased risk of virus acquisition. Furthermore, we demonstrate that the frequency of CD14+ cells and/or their gene expression correlates with blood Type 1 CD4+ T helper cells, α4β7+ plasmablasts, and vaginal cytocidal NKG2A+ cells. To better understand the correlate of protection, we contrasted the ALVAC-SIV vaccine with a NYVAC-based SIV/gp120 regimen that used the identical immunogen. We found that NYVAC-SIV induced higher immune activation via CD4+Ki67+CD38+ and CD4+Ki67+α4β7+ T cells, higher SIV envelope-specific IFN-γ producing cells, equivalent ADCC, and did not decrease the risk of SIVmac251 acquisition. Using the systems biology approach, we demonstrate that specific expression profiles of plasmablasts, NKG2A+ cells, and monocytes elicited by the ALVAC-based regimen correlated with decreased risk of virus acquisition., Author summary The ALVAC-HIV/gp120/alum regimen tested in 8,197 human volunteers (61.4% males, 38.6% females) in the RV144 trial decreased the risk of HIV infection similarly in both sexes. The ALVAC-SIV/gp120/alum vaccine also reduced the risk of intrarectal SIVmac251 acquisition in both female and male vaccinated macaques at an average of 44% per exposure. In the current work, we tested whether this vaccine modality could also reduce the risk of intravaginal SIVmac251 exposure. In order to detect correlates of risk, we administered the virus by the intravaginal route and tested another vaccine regimen based on the vaccinia derivative poxvirus NYVAC in parallel. We demonstrate here that the ALVAC-SIV/gp120/alum regimen decreases the risk of vaginal SIVmac251 acquisition (50% vaccine efficacy) and, importantly, we confirmed that subsets of monocytes and CD4+ T cells are correlates of risk of acquisition. In addition, we uncovered cytotoxic vaginal NKG2A+ cells and gut-homing α4β7 positive plasmablasts as novel correlates of risk of intravaginal virus acquisition. In contrast, NYVAC-SIV vaccination induced high levels of activated T cells and did not protect against SIVmac251 acquisition. We examined the contrasting immune responses to better understand the correlate of protection and found that the unique ability of ALVAC-SIV to activate early interferon responses and the inflammasome during priming differentiates the two poxvirus vectors. This work demonstrates the reproducibility of the efficacy observed in the ALVAC-based regimen and defines novel correlates of risk in the rigorous SIVmac251 macaque model, establishing a benchmark for future improvement of this vaccine approach.

Avipoxviruses (APVs) are large DNA viruses that are detected widely in many species of birds. Little information is available regarding genetic variations in these host-specific viruses. In the present study, nine canarypox virus and five pigeonpox virus isolates were collected from northeastern Iran and isolated via the chorioallantoic membrane of chicken embryos. Further investigations were conducted using analysis of virus growth in chicken embryo fibroblasts, histopathology, electron microscopy, and molecular techniques such as polymerase chain reaction (PCR) combined with sequencing and phylogenetic analysis to investigate variations in the highly conserved P4b gene of poxviruses. Virus replication and pock lesions were evident, and microscopic examination revealed eosinophilic intracytoplasmic inclusion bodies and biconcave enveloped virus particles with randomly arranged surface filaments, which are characteristic features of poxviruses. PCR results confirmed the presence of an APV-specific 578-bp fragment in all of the samples. Sequence analysis and phylogenetic analysis of 578-bp P4b fragments of eight isolates confirmed that our canary and pigeon isolates clustered with previously reported isolates. The similarity between the nucleotide sequences of most of our isolates and those isolated previously in other countries could be due to the high degree of conservation of these fragments. However, the FZRC6V isolate from a canary in this study did not have a canarypox virus origin according to the sequence analysis, and might have originated from cross-infection with different strains of avipoxviruses.

Among all the emerging and re-emerging animal diseases, influenza group is the prototype member associated with severe respiratory infections in wide host species. Wherein, Equine influenza (EI) is the main cause of respiratory illness in equines across globe and is caused by equine influenza A virus (EIV-A) which has impacted the equine industry internationally due to high morbidity and marginal morality. The virus transmits easily by direct contact and inhalation making its spread global and leaving only limited areas untouched. Hitherto reports confirm that this virus crosses the species barriers and found to affect canines and few other animal species (cat and camel). EIV is continuously evolving with changes at the amino acid level wreaking the control program a tedious task. Until now, no natural EI origin infections have been reported explicitly in humans. Recent advances in the diagnostics have led to efficient surveillance and rapid detection of EIV infections at the onset of outbreaks. Incessant surveillance programs will aid in opting a better control strategy for this virus by updating the circulating vaccine strains. Recurrent vaccination failures against this virus due to antigenic drift and shift have been disappointing, however better understanding of the virus pathogenesis would make it easier to design effective vaccines predominantly targeting the conserved epitopes (HA glycoprotein). Additionally, the cold adapted and canarypox vectored vaccines are proving effective in ceasing the severity of disease. Furthermore, better understanding of its genetics and molecular biology will help in estimating the rate of evolution and occurrence of pandemics in future. Here, we highlight the advances occurred in understanding the etiology, epidemiology and pathobiology of EIV and a special focus is on designing and developing effective diagnostics, vaccines and control strategies for mitigating the emerging menace by EIV.

Four vaccines for feline leukemia virus (FeLV) are available in the United States. This study's purpose was to compare the efficacy of Nobivac feline 2-FeLV (an inactivated, adjuvanted whole-virus vaccine) and PureVax recombinant FeLV (a live, canarypox virus-vectored vaccine) following FeLV challenge. Cats were vaccinated at 9 and 12 weeks with Nobivac feline 2-FeLV (group A,n= 11) or PureVax recombinant FeLV (group B,n= 10). Group C (n= 11) comprised unvaccinated controls. At 3 months postvaccination, cats were immunosuppressed and challenged with FeLV-A/61E. The outcomes measured were persistent antigenemia at 12 weeks postchallenge (PC) and proviral DNA and viral RNA at 3 to 9 weeks PC. Persistent antigenemia was observed in 0 of 11 cats in group A, 5 of 10 cats in group B, and 10 of 11 cats in group C. Group A was significantly protected compared to those in groups B (P< 0.013) and C (P< 0.0001). No difference was found between groups B and C (P> 0.063). The preventable fraction was 100% for group A and 45% for group B. At 9 weeks PC, proviral DNA and viral RNA were detected 1 of 11 cats in group A, 6 of 10 cats in group B, and 9 of 11 cats in group C. Nucleic acid loads were significantly lower in group A than in group C (P< 0.01). Group A had significantly lower proviral DNA loads than group B at weeks 6 to 9 (P< 0.02). The viral RNA loads were significantly lower in group A than in group B at weeks 7 to 9 (P< 0.01). The results demonstrate that Nobivac feline 2-FeLV-vaccinated cats were fully protected against persistent antigenemia and had significantly smaller amounts of proviral DNA and plasma viral RNA loads than PureVax recombinant FeLV-vaccinated cats and unvaccinated controls.

The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1β) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination., Author summary Development of a safe and efficacious HIV vaccine is a critical global health priority. Recombinant viral vectors are an important platform for HIV vaccine delivery. Recent clinical trials testing candidate HIV vaccines based on Ad5 vectors failed and reported excess HIV infections in some vaccine recipients, underscoring the necessity to investigate HIV susceptibility of viral vector-specific CD4 T cells in HIV vaccination. By using PBMC samples from clinical trials that examined two important HIV vaccine vectors (canarypox viral vector ALVAC and human Ad5 vector), we here report that compared to Ad5 vector, the ALVAC-specific CD4 T cells are more resistant to HIV infection, providing evidence for distinct HIV susceptibility of CD4 T-cell populations induced by different HIV vaccine vectors. Our findings present new insights into our understanding of HIV vaccine-induced immunity and help improve the design and immune assessment of viral vectors for the development of HIV vaccines.

The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay.

Canarypox viruses (CNPV) are excellent candidates to develop recombinant vector vaccines due to both their capability to induce protective immune responses and their incompetence to replicate in mammalian cells (safety profile). In addition, CNPV and the derived recombinants can be manipulated under biosafety level 1 conditions. There is no commercially available system to obtain recombinant CNPV; however, the methodology and tools required to develop recombinant vaccinia virus (VV), prototype of the Poxviridae family, can be easily adapted. This chapter provides protocols for the generation, plaque isolation, molecular characterization, amplification and purification of recombinant CNPV.

Poxviruses cause many diseases in humans and animals worldwide, and there is a need for vaccines with improved safety and good efficacy. In addition, poxvirus vectors are widely used as recombinant vaccines for various infectious diseases and as recombinant and oncolytic vaccines for cancer. One concern with poxvirus vaccine vectors is that some poxviruses can infect a developing fetus and cause fetal loss or congenital disease. This can be an issue both for patients receiving a vaccine and for pregnant health care providers, including doctors, nurses, and veterinarians, who might receive accidental exposure to the poxvirus by injection or during patient care. We describe here a method for analyzing the safety of virus exposure in pregnant mammals using a mouse model testing vaccinia, canarypox, and raccoonpox virus vectors.

Captive Eld's deer (Rucervus eldi thamin) were evaluated for the presence of rabies virus–neutralizing antibodies using a rapid fluorescent focus inhibition after vaccination with either a live canarypox-vectored recombinant rabies vaccine or a killed monovalent rabies vaccine. Twelve deer were vaccinated with 1.0 ml of killed, adjuvanted, monovalent rabies vaccine at 5–33 mo of age then annually thereafter, and 14 deer were vaccinated with 1.0 ml nonadjuvanted, live canarypox-vectored rabies vaccine at 3–15 mo of age then annually thereafter. Banked serum was available or collected prospectively from deer at 6 mo and 1 yr after initial vaccination, then collected annually. Rabies virus–neutralizing antibodies considered adequate (>0.5 IU/ml) were present in 20/34 samples vaccinated with canarypox-vectored rabies vaccine and in 12/14 samples vaccinated with killed adjuvanted rabies vaccine. Poor seroconversion was noted in deer less than 6 mo of age vaccinated with the canarypox-vectored rabies ...

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

Red pandas (Ailurus fulgens) are susceptible to canine distemper, with a number of reported vaccine-induced canine distemper cases. Canarypox-vectored recombinant canine distemper vaccines (PureVax Ferret Distemper [PFD] and Recombitek CDV [rCDV]) provide protection without inoculating a live distemper virus, but there are currently no published data regarding these vaccines' safety and efficacy in red pandas. One hundred twenty-two serum samples were collected from 50 captive red pandas and analyzed for antibodies to canine distemper. All naive red pandas (n = 20) had negative titers. Naive pandas receiving two PFD vaccinations had either negative or intermediate titers (n = 4). In contrast, naive pandas receiving a series of two or three rCDV vaccinations (n = 14) had greater antibody responses. Red pandas vaccinated with PFD >12 mo since their last vaccination and a rCDV booster vaccination showed the highest titers observed. We recommend red pandas be administered a series of at least three recombinant vaccine (PDF or rDCV) vaccinations, followed by annual booster vaccinations.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo . The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo , that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

Previous studies in experimental ponies using interferon gamma (IFN-γ) as a marker for cell mediated immune (CMI) response demonstrated an increase in IFN-γ gene expression following vaccination with an ISCOM subunit, a canarypox recombinant and more recently, an inactivated whole virus vaccine. The objective of this study was to carry out an independent comparison of both humoral antibody and CMI responses elicited following vaccination with all these vaccine presentation systems. Antibody response of 44 Thoroughbred weanlings was monitored for three weeks following the second dose of primary vaccination (V2) by single radial haemolysis (SRH). The pattern of antibody response was similar for all vaccines. The antibody response of horses vaccinated with the inactivated whole virus vaccine (Duvaxyn IE-T Plus) was superior to that of the horses vaccinated with the ISCOM-matrix subunit (Equilis Prequenza Te) and canarypox recombinant (ProteqFlu-Te) vaccine. In this study 39% of weanlings failed to seroconvert following their first dose of primary vaccination (V1). Poor response to vaccination (H3N8) was observed among weanlings vaccinated with Equilis Prequenza Te and ProteqFlu-Te but not among those vaccinated with Duvaxyn IE-T Plus. PAXgene bloods were collected on days 0, 2, 7 and 14 following V1. Gene expression levels of IFN-γ, IL-1β (proinflammatory cytokine) and IL-4 (B cell stimulating cytokine) were measured using RT-PCR. Mean gene expression levels of IL-1β and IL-4 peaked on day 14 post vaccination. The increase in IL-4 gene expression by horses vaccinated with Equilis Prequenza Te was significantly greater to those vaccinated with the other two products. Vaccination with all three vaccines resulted in a significant increase in IFN-γ gene expression which peaked at 7 days post V1. Overall, there was no significant difference in IFN-γ gene expression by the horses vaccinated with the whole inactivated, the subunit and the canarypox recombinant vaccines included in this study.

Vaccination is highly effective to prevent, control, and limit the impact of equine influenza (EI), a major respiratory disease of horses. However, EI vaccines should contain relevant equine influenza virus (EIV) strains for optimal protection. The OIE expert surveillance panel annually reviews EIV evolution and, since 2010, the use of Florida clade 1 and 2 sub-lineages representative vaccine strains is recommended. This report summarises the development process of a fully- updated recombinant canarypox-based EI vaccine in order to meet the last OIE recommendations, including the vaccine mode of action, production steps and schedule. The EI vaccine ProteqFlu contains 2 recombinant canarypox viruses expressing the haemagglutinin of the A/equine/Ohio/03 and A/equine/Richmond/1/07 isolates (Florida clade 1 and 2 sub-lineages, respectively). The updated EI vaccine was tested for efficacy against the representative Florida clade 2 EIV strain A/equine/Richmond/1/07 in the Welsh mountain pony model. Protective antibody response, clinical signs of disease and virus shedding were compared with unvaccinated control ponies. Significant protection was measured in vaccinated ponies, which supports the vaccine registration. The recombinant canarypox-based EI vaccine was the first fully updated EI vaccine available in the EU, which will help to minimise the increasing risk of vaccine breakdown due to constant EIV evolution through antigenic drift.

Only four HIV-1 vaccine concepts have been tested in six efficacy trials with no product licensed to date. Several scientific and programmatic lessons can be learned from these studies generating new hypotheses and guiding future steps.RV144 [ALVAC-HIV (canarypox vector) and AIDSVAX B/E (bivalent gp120 HIV-1 subtype B and CRF01_AE)] remains the only efficacy trial that demonstrated a modest vaccine efficacy, which led to the identification of immune correlates of risk. Progress on subtype-specific, ALVAC (canarypox vector) and gp120 vaccine prime-boost approaches has been slow, but we are finally close to the launch of an efficacy study in Africa in 2016. The quest of a globally effective HIV-1 vaccine has led to the development of new approaches. Efficacy studies of combinations of Adenovirus type 26 (Ad26)/Modified Vaccinia Ankara (MVA)/gp140 vaccines with mosaic designs will enter efficacy studies mid-2017 and cytomegalovirus (CMV)-vectored vaccines begin Phase I studies at the same time. Future HIV-1 vaccine efficacy trials face practical challenges as effective nonvaccine prevention programs are projected to decrease HIV-1 incidence.An HIV-1 vaccine is urgently needed. Increased industry involvement, mobilization of resources, expansion of a robust pipeline of new concepts, and robust preclinical challenge studies will be essential to accelerate efficacy testing of next generation HIV-1 vaccine candidates.

Context Numerous equine influenza (EI) epizooties are reported worldwide. EI vaccination is the most efficient methods of prevention. However, not all horses develop protective immunity after immunisation, increasing the risk of infection and transmission. Objectives This field study aimed to understand the poor response to primary EI vaccination. Study design The EI antibody response was measured in 174 Thoroughbred foals set in 3 stud farms (SF#1 to SF#3) over a 2 years period. All foals were immunised with a commercial recombinant canarypox-based EI vaccine. Sera were tested by single radial haemolysis against the A/equine/Jouars/4/06 EIV strain (H3N8) at the time of the first vaccination (V1), 2 weeks and 3 months after the second immunisation (V2), 2 days and 3 months after the third immunisation (V3). Results The frequency of poor-responders (no detectable antibody titres) was surprisingly elevated after V2 (56.8%), increased to 81.7% at V2 + 3 months and reached 98.6% at V3. The frequency of poor-responder was still 19.2%, 3 months after V3. Two independent influential factors were identified. The short (V2 + 2 weeks) and mid-term (V2 + 3 months, V3 + 3 months) antibody levels were positively correlated to the age at V1 ( p -value = 0.003, 0.031 and 0.0038, respectively). Presence of maternally-derived antibodies (MDA) at V1 was negatively correlated with antibody levels after V3 only ( p -value = 0.0056). Given that SF#1 antibody response was below clinical protective levels at all-time points studied, the annual boost immunisation (V4) was brought forward by 7.0 ± 1.1 months. V1 was delayed by 7 weeks the following year, which significantly increased short- and mid-term antibody titres ( p -value = 9.9e−07 and 2.31e−07, respectively). Conclusion The age and MDA at first immunisation with the canarypox-based IE vaccine play an independent role in the establishment of antibody levels. This study also highlights the benefit provided by serological surveillance to evaluate herd immunity and to implement corrective management/vaccination measures.

In 2007, Australia experienced the most extensive equine influenza outbreak observed in recent years. Extraordinary measures were rapidly implemented in order to control and prevent the spread of this highly contagious disease. The control strategy involved stringent movement restriction and disease surveillance, seconded by emergency post-outbreak vaccination strategies. Sixteen months after the first case and 12 months following the last reported case, Australia regained its equine influenza-free OIE status. This systematic review reports and summarises information relating to the implementation of emergency vaccination during the 2007 Australian equine influenza outbreak, including the choice of vaccine and implementation strategies.

Avipoxvirus (APV) is a fairly common virus affecting birds that causes morbidity and mortality in wild and captive birds. We studied the prevalence of pox-like lesions and genetic diversity of APV in house sparrows (Passer domesticus) in natural, agricultural and urban areas in southern Spain in 2013 and 2014 and in central Spain for 8 months (2012¿2013). Overall, 3.2% of 2,341 house sparrows visually examined in southern Spain had cutaneous lesions consistent with avian pox. A similar prevalence (3%) was found in 338 birds from central Spain. Prevalence was higher in hatch-year birds than in adults. We did not detect any clear spatial or temporal patterns of APV distribution. Molecular analyses of poxvirus-like lesions revealed that 63% of the samples were positive. Molecular and phylogenetic analyses of 29 DNA sequences from the fpv167 gene, detected two strains belonging to the canarypox clade (subclades B1 and B2) previously found in Spain. One of them appears predominant in Iberia and North Africa and shares 70% similarity to fowlpox and canarypox virus. This APV strain has been identified in a limited number of species in the Iberian Peninsula, Morocco and Hungary. The second one has a global distribution and has been found in numerous wild bird species around the world. To our knowledge, this represents the largest study of avian poxvirus disease in the broadly distributed house sparrow and strongly supports the findings that Avipox prevalence in this species in South and central Spain is moderate and the genetic diversity low

Hendra virus (HeV) is an emerging zoonotic pathogen, which causes severe respiratory illness and encephalitis in humans and horses. Since its first appearance in 1994, spillovers of HeV from its natural reservoir fruit bats occur on almost an annual basis. The high mortality rate in both humans and horses and the wide-ranging reservoir distribution are making HeV a serious public health problem, especially for people exposed to sick horses. This study has aimed to develop an efficient low-cost HeV vaccine for horses based on Canarypox recombinant vector expressing HeV glycoproteins, attachment glycoprotein (G) and fusion protein (F). This vaccine was used to immunise hamsters and then challenged intraperitoneally with HeV 3 weeks later. The higher tested dose of the vaccine efficiently prevented oropharyngeal virus shedding and protected animals from clinical disease and virus-induced mortality. Vaccine induced generation of seroneutralising antibodies and prevented virus-induced histopathological changes and a production of viral RNA and antigens in animal tissues. Interestingly, some vaccinated animals, including those immunised at a lower dose, were protected in the absence of detectable specific antibodies, suggesting the induction of an efficient virus-specific cellular immunity. Finally, ponies immunised using the same vaccination protocol as hamsters developed strong seroneutralising titres against both HeV and closely related Nipah virus, indicating that this vaccine may have the ability to induce cross-protection against Henipavirus infection. These data suggest that Canarypox-based vectors encoding for HeV glycoproteins present very promising new vaccine candidate to prevent infection and shedding of the highly lethal HeV. French researchers have found a new potential vaccine against the rare, often-deadly Hendra virus that spreads from horses to humans. A team led by Branka Horvat at the International Center for Infectiology Research in the collaboration with Merial, developed new vaccine by combining a safe version of the Canarypox virus with Hendra virus (HeV) proteins that promote an immune response in the host. The researchers vaccinated hamsters and then administered a lethal dose of virus. The vaccine provided immunity from HeV by stimulating the production of neutralizing antibodies, inhibiting viral shedding and protecting animals from clinical disease. The immunogenicity of this vaccine was then confirmed in horses. The study puts forward a potential new route to stop the spread of HeV and protect communities that come into contact with horses, thereby protecting both animal and human health.

Despite the availability of efficacious vaccines for animals and humans, rabies is still a major zoonosis. Prevention of rabies in dogs and cats is key for reducing the risk of transmission of this deadly disease to humans. Most veterinary vaccines are adjuvanted inactivated vaccines and have been shown to provide one to four-year duration of immunity. In response to debates about the safety of adjuvanted vaccines in cats, a non-adjuvanted feline rabies vaccine with one-year duration of immunity claim was specifically developed using the canarypoxvirus vector technology. The objective of this study was to validate a vaccination program based on primary vaccination, revaccination one year later and boosters every three years. Seronegative cats were vaccinated at 12 weeks of age and received a booster vaccination one year later. This vaccination regimen induced a strong and sustained antibody response, and all vaccinated animals were protected against virulent rabies challenge carried out 3 years after vaccination. These results validated 3-year duration of immunity after a complete basic vaccination program consisting in primary vaccination from 12 weeks of age followed by revaccination one year later with a non-adjuvanted canarypox-vectored vaccine.

A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from

Highly attenuated poxviruses are promising vectors for protective and therapeutic vaccines. These vectors do not replicate in human cells and can therefore be safely given even to immunocompromised recipients. They can accommodate very large inserts and provide strong stimulation of the immune system against the vectored antigen. Disadvantages include that very high numbers of infectious units are required per dose for full efficacy. Because they are difficult to produce, improved cellular substrates and processes are urgently needed to facilitate programs intended to reach a large number of vaccinees. We have developed a fully scalable and very efficient chemically-defined production process for modified vaccinia Ankara (MVA), canarypox (CNPV, strain ALVAC) and fowlpox viruses (FPV) based on a continuous cell line.

A phase III clinical trial finished in Thailand recently showed that an ALVAC HIV-1 vaccine prime-gp120 protein boost vaccination regimen could modestly protect persons from HIV-1 infection, demonstrating that development of an effective and safe HIV-1 preventive vaccine is possible. ALVAC HIV-1 vaccines are candidate HIV-1 vaccines based on canarypox vectors. Previous clinical trials proved that ALVAC HIV-1 vaccines were safe but weak in immunogenicity when used in human subjects. We have been exploring to use tumor necrosis factor superfamily (TNFSF) members as adjuvants to enhance the immunogenicity of ALVAC HIV-1 vaccines. In this commentary, we will summarize our findings in using two TNFSF molecules, CD40L and OX40L, as adjuvants for an ALVAC HIV-1 vaccine in mouse model. We will also briefly discuss the challenges and prospects of using TNFSF molecules as adjuvants for HIV-1 vaccines in humans.

Fowlpox (FP)-vectored avian influenza (FP-AI) vaccines are used in 1-day-old chickens, but they have also recently been shown to be immunogenic in ducks. The objectives of this work were 1) to evaluate safety and to compare the immunogenicity in ducks of three poxvirus vectors (fowlpox, canarypox, and vaccinia) expressing the same hemagglutinin gene from an H5N1 isolate, 2) to study the effect of the dose of the FP-AI and the presence of an adjuvant in 1-day-old Pekin ducks on antibody response after a boost with inactivated vaccine given 3 wk later, and 3) to confirm the immunogenicity of such a heterologous prime-boost vaccination scheme in 1-day-old Muscovy ducks. Immunogenicity induced by the three poxvirus vectors was comparable, and the FP vector was selected for the other studies. As published previously, there was a strong dose effect of the FP-AI priming on the hemagglutination inhibition (HI) titers induced after the boost with an inactivated vaccine. In contrast, the two tested adjuvants did not significantly increase the activity of FP-AI priming. The heterologous prime-boost regimen given to both Muscovy and Pekin ducklings at 1 and 14 or 21 days of age, respectively, was shown to be at least as immunogenic as two administrations of inactivated vaccines given at 2 and 5 wk of age. However, HI antibody titers were of short duration for both vaccine schemes, and their persistence was heterogeneous among individual birds.

The immunogenicity of current human immunodeficiency virus-1 (HIV-1) canarypox vaccines is weak and needs to be improved. Ligation of OX40 (CD134), a member of tumor necrosis factor receptor superfamily (TNFRSF), by its ligand OX40L (CD252), a tumor necrosis factor superfamily (TNFSF) molecule, has been demonstrated to provide a pivotal costimulatory signal to enhance CD4(+) T cell help of humoral and cytotoxic T cell immune responses. The present study examined whether an OX40L-expressing vector could boost the immunogenicity of the HIV-1 canarypox vaccine, vCP1452, in mice. Co-immunization of mice with OX40L-expressing canarypox and vCP1452 augmented HIV-1 specific CD8(+) T cell responses in terms of frequency and cytokine expression. OX40L-expressing canarypox enhanced the frequency of antigen specific CD8(+) T cells with an effector (CD127(-)CD62L(-)) phenotype, which was associated with an ex vivo expansion of HIV-1 specific CD4(+) T cells. This was in contrast to our previous work in which a CD40L-expressing construct preferentially enhanced antigen specific memory responses [Liu J, Yu Q, Stone GW, Yue FY, Ngai N, Jones RB, et al. 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. Vaccine 2008;26(32):4062-72]. Surprisingly, OX40L did not enhance antibody responses elicited by the HIV-1 canarypox vaccine. We saw no added benefit by combining OX40L and CD40L vectors as an adjuvant strategy for vCP1452. Our results indicate that, similar to CD40L, canarypox vectors expressing OX40L can enhance the cellular but not humoral immunogenicity of HIV-1 canarypox vaccines. In summary, our findings show that OX40L can be used as a molecular adjuvant to enhance T cell immune responses.

This study investigates the safety, immunogenicity and efficacy of different pox-vector vaccines expressing the haemagglutinin of a highly pathogenic (HP) H5N1 avian influenza virus (AIV) (A/chicken/Indonesia/7/03) in pigs. Pigs were vaccinated twice, with a 4-week interval, with a fowlpox (TROVAC®), a canarypox (ALVAC®), or a vaccinia (NYVAC) vector vaccine combined with an oil-in-water adjuvant, with the unadjuvanted NYVAC, or left unvaccinated. Six weeks after the second vaccination, all pigs were challenged intra-tracheally with low pathogenic (LP) H5N2 AIV A/chicken/Belgium/150/99. Sera were examined in haemagglutination inhibition (HI) tests against the H5N1 AIV from which the vaccine haemagglutinin derived, the challenge virus and the human A/Vietnam/1194/04 HPAIV. After challenge pigs were compared for H5N2 virus replication in the trachea and 4 lung lobes at 24 or 72 h post-challenge. Vaccination was well tolerated by all animals. Antibody titres peaked 2 weeks after the second vaccination and were 2- to 4-fold higher against the vaccine virus than heterologous H5 viruses. The NYVAC and ALVAC adjuvanted vaccines consistently induced higher antibody titres than TROVAC or NYVAC without adjuvant. Following challenge, the H5N2 challenge virus was isolated from all unvaccinated pigs, while 19 out of 21 vaccinates showed complete virological protection. Pox-vector vaccines were safe, immunogenic and efficacious against challenge with a heterologous H5 AIV, offering an alternative to classical inactivated vaccines. It remains to be seen whether they would protect against a swine-adapted H5 virus, which may replicate 100–1000 times better than our challenge virus.

Human immunodeficiency virus type 1 (HIV-1) canarypox vaccines are safe but poorly immunogenic. CD40 ligand (CD40L), a member of the tumor necrosis factor superfamily (TNFSF), is a pivotal costimulatory molecule for immune responses. To explore whether CD40L can be used as an adjuvant for HIV-1 canarypox vaccine, we constructed recombinant canarypox viruses expressing CD40L. Co-immunization of mice with CD40L expressing canarypox and the canarypox vaccine expressing HIV-1 proteins, vCP1452, augmented HIV-1 specific cytotoxic T lymphocyte (CTL) responses in terms of frequency, polyfunctionality and interleukin (IL)-7 receptor alpha chain (IL-7Ralpha, CD127) expression. In addition, CD40L expressed from canarypox virus could significantly augment CD4+ T cell responses against HIV-1 in mice. CD40L expressed from canarypox virus matured human monocyte-derived dendritic cells (MDDCs) in a tumor necrosis factor-alpha (TNF-alpha) independent manner, which underwent less apoptosis, and could expand ex vivo Epstein-Barr virus (EBV)-specific CTL responses from healthy human individuals and ex vivo HIV-1-specific CTL responses from HIV-1-infected individuals in the presence or absence of CD4+ T cells. Taken together, our results suggest that CD40L incorporation into poxvirus vectors could be used as a strategy to enhance their immunogenicity.