V4020 Venezuelan Equine Encephalitis Vaccine: Mitigating Neuroinvasion and Reversion Through Rational Design

There is a need for safe and effective vaccines against the Venezuelan equine encephalitis virus that infects both humans and equines. However, development of a live-attenuated vaccine using the TC-83 strain has been hampered by substantial reactogenicity and the potential for neuroinvasion. In this study, we demonstrate that V4020, a new TC-83-based investigational VEEV vaccine with redundant safety features preventing neuroinvasion and reversion, exhibited no neuroinvasion potential in a murine model. Following subcutaneous or intramuscular administration, a subset of mice that received the TC-83 vaccine succumbed to central nervous system infection, with replicating virus detected in the CNS, demonstrating a low, yet detectable neuroinvasion potential of the TC-83 vaccine in vivo. Sequencing analysis of the TC-83 virus recovered from the brains identified a pseudoreversion of E2 R120I, as E2 R120 is known to confer attenuation for TC-83. In contrast, V4020 showed no evidence of virus in the CNS, highlighting one of the V4020 features, a new synonymous codon to minimize reversion to the wild-type residue. Overall, our study establishes V4020 as a rationally designed, safe vaccine candidate for VEEV with significantly reduced neuroinvasion risk.