Venezuelan equine encephalitis vaccine with rearranged genome resists reversion and protects non-human primates from viremia after aerosol challenge.

Live-attenuated V4020 vaccine for Venezuelan equine encephalitis virus (VEEV) containing attenuating rearrangement of the virus structural genes was evaluated in a non-human primate model for immunogenicity and protective efficacy against aerosol challenge with wild-type VEEV. The genomic RNA of V4020 vaccine virus was encoded in the pMG4020 plasmid under control of the CMV promoter and contained the capsid gene downstream from the glycoprotein genes. It also included attenuating mutations from the VEE TC83 vaccine, with E2-120Arg substitution genetically engineered to prevent reversion mutations. The population of V4020 vaccine virus derived from pMG4020-transfected Vero cells was characterized by next generation sequencing (NGS) and indicated no detectable genetic reversions. Cynomolgus macaques were vaccinated with V4020 vaccine virus. After one or two vaccinations including by intramuscular route, high levels of virus-neutralizing antibodies were confirmed with no viremia or apparent adverse reactions to vaccinations. The protective effect of vaccination was evaluated using an aerosol challenge with VEEV. After challenge, macaques had no detectable viremia, demonstrating a protective effect of vaccination with live V4020 VEEV vaccine.

Novel DNA-launched Venezuelan equine encephalitis virus vaccine with rearranged genome.

Novel live-attenuated V4020 vaccine was prepared for Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The genome of V4020 virus was rearranged, with the capsid gene expressed using a duplicate subgenomic promoter downstream from the glycoprotein genes. V4020 also included both attenuating mutations from the TC83 VEEV vaccine secured by mutagenesis to prevent reversion mutations. The full-length infectious RNA of V4020 vaccine virus was expressed from pMG4020 plasmid downstream from the CMV promoter and launched replication of live-attenuated V4020 in vitro or in vivo. BALB/c mice vaccinated with a single dose of V4020 virus or with pMG4020 plasmid had no adverse reactions to vaccinations and developed high titers of neutralizing antibodies. After challenge with the wild type VEEV, vaccinated mice survived with no morbidity, while all unvaccinated controls succumbed to lethal infection. Intracranial injections in mice showed attenuated replication of V4020 vaccine virus as compared to the TC83. We conclude that V4020 vaccine has safety advantage over TC83, while provides equivalent protection in a mouse VEEV challenge model.

Plasmid DNA launches live-attenuated Japanese encephalitis virus and elicits virus-neutralizing antibodies in BALB/c mice.

We describe novel plasmid DNA that encodes the full-length Japanese encephalitis virus (JEV) genomic cDNA and launches live-attenuated JEV vaccine in vitro and in vivo. The synthetic cDNA based on the sequence of JEV SA14-14-2 live-attenuated virus was placed under transcriptional control of the cytomegalovirus major immediate-early promoter. The stability and yields of the plasmid in E. coli were optimized by inserting three synthetic introns that disrupted JEV cDNA in the structural and nonstructural genes. Transfection of Vero cells with the resulting plasmid resulted in the replication of JEV vaccine virus with intron sequences removed from viral RNA. Furthermore, a single-dose vaccination of BALB/c mice with 0.5 - 5µg of plasmid resulted in successful seroconversion and elicitation of JEV virus-neutralizing serum antibodies. The results demonstrate the possibility of using DNA vaccination to launch live-attenuated JEV vaccine and support further development of DNA-launched live-attenuated vaccine for prevention of JEV infections.