J Virol 2019 Apr 17. Epub 2019 Apr 17.
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Enterovirus A71 (EV-A71) is the major pathogen which causes hand-foot-and-mouth disease (HFMD) which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon deoptimized VP1 capsid protein (VP1-CD) showed similar replication rate and antigenicity to wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques, but was still able to examined by TCID50 titers which were similar to the others, thus indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF) and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in mice infection model. After 14 days post-immunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates proof-of-concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, which may be a good candidate for next generation EV-A71 vaccine development. Enterovirus A71 (EV-A71) can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here we developed a novel vaccine strategy with the combination of codon-deoptimization (CD) and high-fidelity (HF) substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated that the simultaneous introduction of CD genome sequences and HF substitutions as a potentially new strategy to develop attenuated vaccine seed virus. Our work provided an insight to develop the low virulent candidate vaccine virus (CVV) through a series of genetic editing on virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next generation vaccine development of EV-A71.