Directed Molecular Evolution Improves the Immunogenicity and Protective Efficacy of a Venezuelan Equine Encephalitis Virus DNA Vaccine

摘要

We employed a directed molecular evolution approach consisting of in vitro multigene DNA recombination and screening technologies in an attempt to improve the cross-reactivity and immunogenicity of Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins E1 and E2. DNA encoding E1 and E2 from three VEEV subtypes (IA/B, IIIA, IE) and one strain each of eastern (PE- 6) and western (CBA87) equine encephalitis viruses (EEEV and WEEV) were used to create libraries of recombined variants. Five different libraries were generated by recombining one or both parent glycoprotein genes. Several hundred of the recombined variants were used as naked plasmid DNA to vaccinate mice, and their serum samples were screened for the presence of antibodies in a virus- specific ELISA. Selected sera were further assayed for the presence of neutralizing antibodies by plaque reduction neutralization tests (PRNT). We found that approximately 10% of the recombined variants elicited greater levels of cross-reactive antibodies against all five viral antigens as compared to DNA vaccines expressing the parental genes. In addition, representative variants from a library in which only E2 genes were recombined elicited significantly increased neutralizing VEEV antibody titers as compared to the parent DNA vaccine and provided improved protection against aerosol VEEV challenge. These data demonstrate that in vitro, multigene DNA recombination and associated screening can lead to the selection of antigens that exhibit improved cross- reactivity, immunogenicity, and protective efficacy as compared to the wild- type proteins and suggest a broad utility for these technologies in optimizing vaccine antigens.