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

Abstract

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.

Keywords: VEE; Venezuelan equine encephalitis; live attenuated VEEV vaccine; neuroinvasion; pseudoreversion.

Figure 1

Clinical signs of activity (ACT), grimace (GRI), grooming GRO), and neurological (NEU) abnormality from mice infected with TC-83 and V4020. Clinical signs from mice infected with TC-83 (A,C) and V4020 (B,D) after SC (A,B) and IM (C,D) administration. The scores on y-axis represent the mean scores from 10 mice. Scores were presented with a scale of 0–4 (none to severe) in the stacked column format.

Figure 2

Blood cell type analysis after vaccination with VEEV TC-83 (A) and V4020 (B). Graphs show mean ± S.D. from 10 samples per group. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 by Dunnett’s multiple comparison test compared to the PBS control.

Figure 3

Detection of viral RNA in the brain tissues by RNAScope analysis from TC-83-vaccinated mouse. Brain tissues from mock (A), TC-83 IN (B), TC-83 SC (C), and TC-83 IM (D) administrations were subjected to RNAScope assay with a probe specific to VEEV (brown stain). OB, olfactory bulbs; CO, cerebral cortex; H.C., hippocampus; CB, cerebellum. Representative images from three samples per group. Scale bars represent 250 µm.

Figure 4

Detection of viral RNA in the brain tissues by RNAScope analysis from V4020-vaccinated mice. Brain tissues from V4020 SC (A) and V4020 IM (B) administrations were subjected to RNAScope assay with a probe specific to VEEV (brown stain). CO, cerebral cortex; H.C., hippocampus; CB, cerebellum. Representative images from three samples. Scale bars represent 250 µm.

Figure 5

Tissue weight analysis of TC-83 and V4020 vaccinated mice. Tissue weight analysis of SC (A) or IM (B) infection of VEEV TC83 and V4020 in the Balb/c mouse (n = 3) model at DPI 6. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 by Tukey’s multiple comparison test.

Figure 6

Pseudoreversion of TC-83 and the strategy implemented in V4020 to minimize the risk.