Mutation of putative N-Linked Glycosylation Sites in Japanese encephalitis Virus Premembrane and Envelope proteins enhances humoral immunity in BALB/C mice after DNA vaccination

Swine are an important host of Japanese encephalitis virus (JEV). The two membrane glycoproteins of JEV, prM and E, each contain a potential N-linked glycosylation site, at positions N15 and N154, respectively. We constructed plasmids that contain the genes encoding wild-type prME (contain the signal of the prM, the prM, and the E coding regions) and three mutant prME proteins, in which the putative N-linked glycosylation sites are mutated individually or in combination, by site-directed mutagenesis. The recombinant plasmids were used as DNA vaccines in mice. Our results indicate that immunizing mice with DNA vaccines that contain the N154A mutation results in elevated levels of interleukin-4 secretion, induces the IgG1 antibody isotype, generates greater titers of anti-JEV antibodies, and shows complete protection against JEV challenge. We conclude that mutation of the putative N-glycosylation site N154 in the E protein of JEV significantly enhances the induced humoral immune response and suggest that this mutant should be further investigated as a potential DNA vaccine against JEV.