Japanese encephalitis (JE) is a serious disease prevalent throughout Asia  and is transmitted to humans by mosquito bite . Pigs are an important amplifier host for the virus  Vaccination of swine, therefore, can help prevent disease in humans .
DNA vaccines against JEV have shown great potential as preventative agents for their ability to elicit potent humoral and cytotoxic cellular immune responses against the plasmid-encoded protein in a broad range of hosts . A previous study demonstrated that plasmids carrying the JEV prM and E genes can induce high NEUT antibodies and protective immunity in mice. Most importantly, the signal of prM is included in DNA vaccine, thus it is likely that viral antigens can be secreted from transfected cells and these DNA vaccines can induce high levels of immune responses . In this study, we assessed the immunogenicity of several prME mutants to evaluate the potential of these mutants as DNA vaccines against JEV in swine.
The prM and E proteins of JEV are both exposed structural proteins. E is a major immunogenic antigen, and the prM is JEV induce protective immune additional components . It has been reported that the prM proteins of flaviviruses can form natively folded structures independent of the E protein, form hetero-dimers with the E protein, and appear to act as folding chaperones for E protein . In addition, the prME expression plasmids contain the signal of prM coding sequences for translocation into endoplasmic reticulum (ER), and the signal of prM can make the respective proteins are glycosylated or transported by the secretory pathway as supposed . To investigate the effect of mutating putative N-glycosylation sites on the immunogenicity of prM and E, these proteins must maintain their native conformation.
Previous studies have demonstrated that N-linked glycans on the glycoproteins of many viruses play important roles in modulating the immune response. Removal of N-glycosylation sites in the simian immunodeficiency virus envelope protein and influenza virus hemagglutinin protein have been observed to limit the neutralizing antibody response , while mutation of N-linked glycans in human immunodeficiency virus type 1 (HIV-1) envelope protein appears to enhance the production of CTL , and deletion of glycans in hepatitis C virus E1 can enhance cellular or humoral immune responses . The effects of the putative N-linked glycosylation sites in prM and E on the immune response to JEV are not known, however.
The JEV PrM protein contains one putative N-linked glycosylation site at N15, and the E protein also contains one putative N-linked glycosylation site, at N154. We demonstrate that immunization with the mutants prME-M2 and prME-M3, both of which contain the N154A mutation, induced a significantly enhanced antibody response, elevated IL-4 secretion levels, and full protection to lethal challenge of JEV compared to immunization with native prME, indicating that these mutations could elicit a stronger humoral immune response than the wild-type prME. We also demonstrate that mutating the N15 site (prME-M1) induces a humoral immune response comparable to that observed upon immunization with wild-type prME. This strongly suggests that mutating N15 in the prM glycoprotein does not strongly perturb the immune response to prME, but mutating N154 of the E glycoprotein does affect the immune response to prME.
Though DNA vaccines generally induce a stronger Th1 immune response, producing elevated levels of IFN-γ and IgG2a, the immune responses induced by DNA vaccines need to be improved. Our results show that a single mutation, N154A, significantly enhances the humoral immune response. We propose, therefore, that this highly immunogenic mutant could serve as a swine vaccine against JEV and should be further optimized for this purpose.