Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy

Abstract

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 vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three 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 with the parent TC-83. During serial passaging in a highly permissive model, the mutants 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 strategy.

RNA Virus Fidelity Mutants: A Useful Tool for Evolutionary Biology or a Complex Challenge?

“…In the years since the identification of high-fidelity PV, a large number of fidelity variants have been identified from a diverse group of RNA virus families, including Picornaviridae [ 24 , 25 , 27 , 28 , 33 , 34 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ], Togaviridae [ 44 , 45 , 46 , 47 ], Flaviviridae [ 29 , 48 ], Caliciviridae [ 49 ], Arteriviridae [ 50 ], Orthomyxoviridae [ 31 , 51 ], and Coronaviridae [ 52 , 53 ]. The first fidelity mutants were identified similarly to the high-fidelity PV mutant by passaging virus multiple times in the presence of a nucleoside analog, usually Ribavirin.…”

“…Low-fidelity mutants generated by mutating residues important for high-fidelity are more sensitive to nucleoside analog treatment, regardless of the analog. However, low-fidelity mutants identified during nucleoside analog passaging are resistant to the nucleoside analog used for the identification, but are not necessarily resistant to other analogs [ 28 , 29 , 31 , 47 ].…”

Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes

“…While the time was too short (2 DPI) to consider selective pressure and adaptive immunity involvement, it is interesting to note that E3 has been identified as a key target of humoral responses which are protective against VEEV-TrD [58]. It has been reported that TC-83 strains with lower-fidelity RdRP are more attenuated and immunogenic [59]. nsP4, the viral RdRP [10], had a higher SMR in TC-83 than V4020, however we did not detect differences in mutation frequency across the genome ( Figure S3d), suggesting that no real difference in RdRP fidelity arose during passaging.…”

Tyr82 Amino Acid Mutation in PB1 Polymerase Induces an Influenza Virus Mutator Phenotype

“…The low-fidelity coronavirus vaccine strain was stable and did not revert to virulence during long-term persistent infection. Likewise, a live attenuated vaccine strain for Venezuelan equine encephalitis virus based on low-fidelity RdRp has been reported (36).…”

“…Biochemical experiments revealed that purified mutant viral polymerase with M296I tended to show a lower capacity to use ribavirin triphosphate as a substrate than the wild-type polymerase. Further, low-fidelity variants of poliovirus (32), coxsackievirus B3 (33), chikungunya virus (34), Sindbis virus (35), West Nile virus (19), and Venezuelan equine encephalitis virus (36) have been isolated by mutagen screening or introducing an amino acid change in RdRp.…”