DNA IMMUNIZATION WITH JAPANESE ENCEPHALITIS-VIRUS NONSTRUCTURAL PROTEIN NS1 ELICITS PROTECTIVE IMMUNITY IN MICE

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a z oonotic pathogen that is prevalent in some Southeast Asian countries a nd causes acute encephalitis in humans. To evaluate the potential appl ication of gene immunization to JEV infection, we characterized the im mune responses from mice intramuscularly injected with plasmid DNA enc oding JEV glycoproteins, including the precursor membrane (prM) plus e nvelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice surv ived after a lethal JEV challenge, whereas when immunized with the pla smid expressing NS1, 90% of the mice survived after a lethal challenge . As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effe ct of the plasmid DNA. Despite having no detectable neutralizing activ ity, the NS1 immunization elicited a strong antibody response exhibiti ng cytolytic activity against JEV-infected cells in a complement-depen dent manner. By contrast, immunization with a construct expressing a l onger NS1 protein (NS1'), containing an extra 60-amino-acid portion fr om the N terminus of NS2A, failed to protect mice against a lethal cha llenge. Biochemical analyses revealed that when individually expressed , NS1 but not NS1' could be readily secreted as a homodimer in large q uantity and could also be efficiently expressed on the cell surface. I nterestingly, when NS1 and NS1' coexisted in cells, the level of NS1 c ell surface expression was much lower than that in cells expressing NS 1 alone, These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.