Characterization of a Low-Fidelity Live-Attenuated Vaccine for Venezuelan Equine Encephalitis Virus

During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing 3 mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared to the parent TC-83. The TC-83 3x mutant was selected for further study due its decreased replication fidelity. While infection of live mosquitoes with TC-83 and TC-83 3x proved difficult, in vitro mosquito cell infection suggested increased attenuation of the TC-83 3x mutant in this host. TC-83 3x genetic stability was high, with no reversion of the 3 RdRp mutations during passaging in vitro and in vivo. Passaging of the TC-83 3x mutant using a reporter construct showed this mutant to have increased levels of recombination, as well as lowered fidelity. During serial passaging in a highly permissive mouse model, TC-83 3x increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine attenuation strategy.