1,5-Iodonaphthyl azide-inactivated V3526 protects against aerosol challenge with virulent venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus. VEEV is highly infectious in aerosolized form and has been identified as a bio-terrorism agent. There is no licensed vaccine for prophylaxis against VEEV. The current IND vaccine is poorly immunogenic and does not protect against an aerosol challenge with virulent VEEV. We have previously shown that VEEV inactivated by 1,5-iodonaphthyl azide (INA) protects against footpad challenge with virulent VEEV. In this study, we inactivated an attenuated strain of VEEV, V3526, with INA and evaluated its protective efficacy against aerosol challenge with wild type VEEV. We demonstrated that among three routes of immunization, intramuscular immunization with INA-inactivate V3526 (INA-iV3526) provided complete protection against aerosol challenge with virulent VEEV. Our data suggests that INA-iV3526 can be explored further for development as an effective vaccine candidate against aerosol challenge of virulent VEEV.

Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing.

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.

Evaluation of formalin inactivated V3526 virus with adjuvant as a next generation vaccine candidate for Venezuelan equine encephalitis virus.

V3526, a genetically modified strain of Venezuelan equine encephalitis virus (VEEV), was formalin inactivated for evaluation as a next generation vaccine candidate for VEEV. In this study, we tested formalin-inactivated V3526 (fV3526) with and without adjuvant for immunogenicity and efficacy in BALB/c mice and results were compared to the existing inactivated VEEV vaccine, C84. Mice were vaccinated intramuscularly (IM) or subcutaneously (SC) with fV3526 formulations and challenged with VEEV IAB Trinidad donkey (VEEV TrD) strain by SC or aerosol exposure. Efficacy following SC or aerosol challenge was not significantly different between the fV3526 formulations or compared to C84 despite C84 being administered in more doses and higher concentration of viral protein per dose. These data support further evaluation of fV3526 formulations as a next generation VEEV vaccine.

Comparison of the immunological responses and efficacy of gamma-irradiated V3526 vaccine formulations against subcutaneous and aerosol challenge with Venezuelan equine encephalitis virus subtype IAB.

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel further augmented the immune response in mice and resulted in 100% protection against aerosol challenge.

Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine.

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.

Protective cytotoxic T-cell responses induced by venezuelan equine encephalitis virus replicons expressing Ebola virus proteins.

Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8(+) T-cell responses to six Ebola virus proteins were examined. CD8(+) T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8(+) cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8(+) cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.