Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity and efficacy in horses

A new vaccine, V3526, is a live-attenuated virus derived by site-directed mutagenesis from a virulent clone of the Venezuelan equine encephalitis virus (VEEV) IA/B Trinidad donkey (TrD) strain, intended for human use in protection against Venezuelan equine encephalitis (VEE). Two studies were conducted in horses to evaluate the safety, immunogenicity, ability to boost and protective efficacy of V3526 against challenges of TrD and VEEV IE 64A99. Horses were vaccinated subcutaneously (SC) with 10(7), 10(5), 10(3) or 10(2) plaque-forming units (pfu) of V3526. Control horses were sham immunized. In the first study, challenge viruses (TrD or 64A99) were administered SC 28 days post-vaccination (PV). No viremia and only mild fluctuation in white blood cell counts were observed PV. None of the V3526 vaccinated horses showed clinical signs of disease or pathology of VEE post-challenge (PC). In contrast, control horses challenged SC with 10(4)pfu TrD became viremic and showed classical signs of VEE beginning on Day 3 PC, including elevated body temperature, anorexia, leukopenia and malaise. Moderate to severe encephalitis was found in three of five control horses challenged with TrD. Control horses challenged with 64A99 failed to develop detectable viremia, but did exhibit a brief febrile episode at 1-3 days PC. None of the 10 immunized horses challenged with 64A99 became pyrexic. Twenty four of 25 horses immunized with V3526 in the first study developed serum neutralizing antibody to TrD and 64A99 within 14 days PV. Vaccinations with V3526, at doses as low as 10(2)pfu, were safe and efficacious in protecting horses against a virulent TrD virus challenge. The second study supported that repeat dosing resulted in an increase in serum neutralizing antibody to TrD.     

A recombinant envelope protein vaccine against West Nile virus

West Nile (WN) virus is a flavivirus that first appeared in North America in 1999. Since then, more than 600 human deaths and 22,000 equine infections have been attributed to the virus in the United States. We expressed a truncated form of WN virus envelope (E) protein in Drosophila S2 cells. This soluble recombinant E protein was recognized by antibodies from naturally infected horses, indicating that it contains native epitopes. Mice and horses produced high-titer antibodies when immunized with recombinant E protein combined with aluminum hydroxide. Immunized mice were resistant to challenge with a lethal viral dose. Sera from immunized horses, administered to naive mice, conferred resistance against a lethal WN viral challenge. In addition, sera of immunized horses neutralized West Nile virus in vitro, as demonstrated by plaque reduction assays. This recombinant form of E protein, combined with aluminum hydroxide, is a candidate vaccine that may protect humans and horses against WN virus infections.     

Evidence that use of an inactivated equine herpesvirus vaccine induces serum cytotoxicity affecting the equine arteritis virus neutralisation test

Several laboratories worldwide have recently experienced problems related to serum cytotoxicity with the equine arteritis virus (EAV) neutralisation test (VN) when using Office International des Epizooties (OIE) reference laboratory prescribed rabbit kidney (RK-13) indicator cells. Cytotoxicity can be mistaken for viral cytopathic effect and has led to increasing difficulties in test interpretation, consequently causing disruption to both equine breeding and disease surveillance. Results from experimental and field-derived data suggest that this serum cytotoxicity is associated with use of a tissue-culture-derived equine herpesvirus vaccine, probably manifested through a vaccine-induced anti-cellular antibody response directed against RK-13 cells. Two alternative EAV VN methods were shown to significantly reduce the effects of cytotoxicity (from 73 to <5% prevalence) among vaccinated horses but did not completely eliminate the problem. Use of ELISA-based tests, which are not affected by serum cytotoxicity but which are not currently recognised as international standards, should be evaluated as a useful backup in screening equine sera for EAV VN antibodies.     

Experiences with new generation vaccines against equine viral arteritis, West Nile disease and African horse sickness

Viral diseases constitute an ever growing threat to the horse industry worldwide because of the rapid movement of large numbers of horses for competition and breeding. A number of different types of vaccines are available for protective immunization of horses against viral diseases. Traditional inactivated and live-attenuated (modified live virus, MLV) virus vaccines remain popular and efficacious but recombinant vaccines are increasingly being developed and used, in part because of the perceived deficiencies of some existing products. New generation vaccines include MLVs with deletions and/or mutations of critical genes, subunit vaccines that incorporate immunogenic proteins (or portions thereof) or expression vectors that produce these proteins as immunogens, and DNA vaccines. New generation vaccines have been developed for several viral diseases of horses. We recently have developed an alphavirus replicon-vectored equine arteritis virus (EAV) vaccine, and evaluated a commercial canary pox virus-vectored vaccine for West Nile disease. The success of these new-generation vaccines has catalyzed efforts to develop improved vaccines for the prevention of African horse sickness, a disease of emerging global significance.     

Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.     

Response of ELA-A1 horses immunized with lipopeptide containing an equine infectious anemia virus ELA-A1-restricted CTL epitope to virus challenge

Lipopeptide containing an ELA-A1-restricted cytotoxic T lymphocyte (CTL) epitope from the envelope surface unit (SU) protein of the EIAV(WSU5) strain was used to immunize three horses having the ELA-A1 haplotype. Peptide-specific ELA-A1-restricted CTL were induced in all three horses, although these were present transiently in PBMC. These horses were further immunized with lipopeptide containing the corresponding CTL epitope from the EIAV(PV) strain. Then, the three immunized horses and three non-immunized horses were challenged by intravenous inoculation with 300 TCID(50) EIAV(PV). All horses developed cell free viremia, fever and thrombocytopenia. However, there was a statistically lower fever and thrombocytopenia severity score in the immunized group. Shorter duration of plasma viral load in two of the three immunized horses likely explains the less severe clinical disease in this group. Results indicate that lipopeptide immunization had a protective effect against development of clinical disease following virus challenge.     

Comparison of protective efficacies of plasmid DNAs encoding Japanese encephalitis virus proteins that induce neutralizing antibody or cytotoxic T lymphocytes in mice

Mice immunized with a plasmid DNA encoding the premembrane (prM) and envelope (E) proteins of Japanese encephalitis (JE) virus (designated pcJEME) produce neutralizing antibodies and are protected from JE. To determine the role of the immune response to other viral proteins in protection, we constructed plasmid DNAs encoding other JE virus proteins and made a direct comparison among these plasmids using a mouse model. Cytotoxic T lymphocytes (CTLs) were induced by plasmids encoding capsid (C) or nonstructural proteins, NS1, NS2A, NS2B, NS3 or NS5. However, these plasmids provided only a partial protection against intraperitoneal challenge with a lethal dose of JE virus, whereas mice immunized with pcJEME were fully protected. In mice inoculated with CTL-inducing plasmids, high virus titers were detected in plasma immediately (1h) following challenge and in brain on day 4 post-challenge, but no virus infectivity was detected in plasma and brain of pcJEME-immunized mice during the 5 days following challenge. These results indicate that protection provided by the prM/E-encoding DNA consists of neutralizing antibody that prevents virus dissemination from the peripheral site to the brain, and that this antibody-mediated mechanism of protection is more efficient than the immunity induced by plasmids that generate CTL responses capable of killing JE virus-infected cells.     

Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines

We previously reported that an experimental live-attenuated equine infectious anemia virus (EIAV) vaccine, containing a mutated S2 accessory gene, provided protection from disease and detectable infection after virulent virus (EIAV(PV)) challenge [Li F, Craigo JK, Howe L, Steckbeck JD, Cook S, Issel C, et al. A live-attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses. J Virol 2003;77(13):7244-53; Craigo JK, Li F, Steckbeck JD, Durkin S, Howe L, Cook SJ, et al. Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy. J Virol 2005;79(5):2666-77]. To determine if attenuated EIAV vaccines actually prevent persistent infection by challenge virus, we employed a 14-day dexamethasone treatment of vaccinated horses post-challenge to suppress host immunity and amplify replication levels of any infecting EIAV. At 2 months post-challenge the horses were all protected from virulent-virus challenge, evidenced by a lack of EIA signs and detectable challenge plasma viral RNA. Upon immune suppression, 6/12 horses displayed clinical EIA. Post-immune suppression characterizations demonstrated that the attenuated vaccine evidently prevented detectable challenge virus infection in 50% of horses. These data highlight the utility of post-challenge immune suppression for evaluating persistent viral vaccine protective efficacy.     

Alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in BALB/c mice and functional serum antibodies in rhesus macaques

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.     

Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens

West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA–WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA–WNV vaccines in other preclinical models and use them as candidate vaccine in humans.     

Immunogenicity of virus-like particles containing modified human immunodeficiency virus envelope proteins

Extensive glycosylation and variable loops of the HIV envelope protein (Env) are reported to shield some neutralizing epitopes. Here, we investigated the immunogenicity of mutated HIV Envs presented in virus-like particles (VLPs). We immunized mice with simian human immunodeficiency virus (SHIV) VLPs containing mutant HIV Env with reduced glycosylation (3G), variable loop-deleted mutations (dV1V2), or combinations of both types of mutations (3G-dV2-1G), and evaluated immune responses. Immune sera from mice that received VLPs with modified HIV Envs (3G or dV1V2) showed higher neutralizing activities against the homologous HIV 89.6 virus as well as heterologous viruses when compared with wild type SHIV VLP-immunized mice. Lymphocytes from immunized mice produced HIV Env-specific cytokines, with the 3G-dV2-1G mutant producing high levels of cytokines. Interestingly, both dendritic cells and B cells were found to interact with VLPs suggesting that VLPs are effective immunogens. Therefore, this study suggests that VLPs containing modified HIV Env have the potential to be developed as candidate vaccines capable of inducing cellular and humoral immune responses including neutralizing activities.     

A DNA vaccine expressing dengue type 2 virus premembrane and envelope genes induces neutralizing antibody and memory B cells in mice

A dengue DNA vaccine candidate was developed and evaluated for immunogenicity in mice. The vaccine, designated pcD2ME, is a pcDNA3-based plasmid encoding the signal sequence of premembrane (prM), prM and envelope (E) genes of the New Guinea C strain of dengue type 2 virus. CHO-K1 cells transfected with pcD2ME expressed prM and E as determined by immunochemical staining with monoclonal antibodies. BALB/c mice inoculated intramuscularly with 100 microg of pcD2ME two or three times at an interval of 2 weeks developed a low level of neutralizing antibody (1:10 at a 90% plaque reduction). Immunization twice with 10 microg or 1 microg of pcD2ME or three times with 100 microg of pcDNA3 did not induce detectable levels of neutralizing antibody. Mice immunized two or three times with 100 microg of pcD2ME raised neutralizing antibody titers to 1:40 or greater on days 4 and 8 after challenge with 3x10(5) plaque forming units (PFU) of the New Guinea C strain of dengue type 2 virus, showing strong anamnestic responses to the challenge. In contrast, mice immunized two or three times with 100 microg of pcDNA3 developed no detectable neutralizing antibody on days 4 and 8 after challenge. These results indicate that immunization with pcD2ME induces neutralizing antibody and dengue type 2 virus-responsive memory B cells in mice.     

Efficacy of an inactivated Senecavirus A vaccine in weaned pigs and mature sows

Senecavirus A (SVA), commonly known as Seneca Valley virus (SVV) is a causative agent for vesicular disease in swine. It has been found across the globe including the United States, Brazil, and China. Clinical disease caused by this virus is identical to foot-and-mouth disease virus (FMDV). Since FMDV has the potential to cause severe economic consequences in FMDV-free countries, those countries are on high alert for signs of vesicles in swine and an investigation is performed to rule out the presence of FMDV if observed. In countries where SVA cases have continued to occur, investigations and testing can cause a burden on personnel and resources. The objectives of this study were to test the efficacy of a whole-virus inactivated SVA vaccine against challenge in nursery-aged pigs, mature sows, and to assess the protection of passive maternal immunity generated by immunized dams. Animals were given two doses of the vaccine intramuscularly three weeks apart and challenged intranasally two weeks after the second dose. Non-vaccinated animals challenged with SVA developed clinical signs of disease, replicated virus, and developed a neutralizing antibody response. Vaccinated animals had robust neutralizing titers after two doses; and after challenge, did not develop vesicular disease and had limited rectal shedding. Piglets suckling immunized dams and challenged with SVA at 3–6 days-of-age had neutralizing titers prior to challenge and did not replicate or shed virus. An efficacious vaccine could improve swine welfare and reduce the economic consequences of continued foreign animal disease investigations.     

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.     

Adjuvant-enhanced antibody responses to recombinant proteins correlates with protection of mice and monkeys to orthopoxvirus challenges

Recombinant proteins are being evaluated as smallpox and monkeypox vaccines because of their perceived safety compared to live vaccinia virus. Previously, we demonstrated that three or more injections of a Ribi-type adjuvant with a combination of three proteins from the outer membranes of intracellular (L1 protein) and extracellular (A33 and B5 proteins) forms of vaccinia virus protected mice against a lethal intranasal challenge with vaccinia virus. Here, we compared several adjuvants and found that QS-21 and to a lesser extent alum+CpG oligodeoxynucleotides accelerated and enhanced neutralizing antibody responses to a mixture of L1 and A33 proteins, provided the highest ratio of IgG2a to IgG1 isotype response, and protected mice against disease and death after only two immunizations 3 weeks apart. In addition, monkeys immunized with recombinant vaccinia virus proteins and QS-21 developed neutralizing antibody to monkeypox virus and had reduced virus load, skin lesions, and morbidity compared to the non-immunized group following monkeypox virus 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

Dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. Despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. To explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes. MVA recombinants expressing the highly immunogenic C-terminally truncated dengue type 2 virus (DEN2) or dengue type 4 virus (DEN4) envelope protein (E), approx. 80% of the full-length, were evaluated for their protective immunity in animal models. Each of these recombinants elicited an elevated antibody response to DEN2 or DEN4 E in mice following the booster inoculation, as detected by radio-immunoprecipitation. Recombinant MVA-DEN2 80%E, but not MVA-DEN4 80%E, induced a neutralizing antibody response. The MVA-DEN2 80%E recombinant was chosen to further evaluate its ability to induce resistance to wild type DEN2 challenge in monkeys. Monkeys immunized twice with recombinant MVA-DEN2 80%E developed a low to moderate antibody response and were partially protected against DEN2 challenge, as determined by the viremia pattern. Importantly, the subsequent study showed that all four monkeys immunized with the recombinant in a three dose schedule developed an increased level of antibodies and were completely protected against DEN2 challenge. The potential efficacy of recombinant MVA-DEN2 80%E to protect primates against dengue infection suggests that construction and evaluation of MVA recombinants expressing other serotypes of dengue virus E for use in a tetravalent vaccine strategy might be warranted.     

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI).Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine.This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus.     

Pro-inflammatory and antiviral cytokine expression in vaccinated and unvaccinated horses exposed to equine influenza virus

Most studies of the cytokine response to influenza virus infection have been carried out in human, porcine and murine models, however the data available on equine cytokines is limited. An experimental challenge study was undertaken in unvaccinated na?ve horses and horses vaccinated with a commercial inactivated influenza vaccine. The humoral antibody response to vaccination and virus challenge was measured by single radial haemolysis (SRH) assay and clinical signs of influenza and viral shedding were monitored post-challenge. Levels of three equine pro-inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha and the antiviral cytokine interferon (IFN)-alpha were examined by quantitative RT-PCR of mRNA. Vaccination provided significant clinical and virological protection and resulted in a significant reduction of IFN-alpha and IL-6 expression on day 2 post-challenge. The patterns of cytokine expression observed in control animals suffering from influenza after challenge are comparable to those reported in studies of other species.