Intramuscular immunization with DNA vaccines has been shown to induce a broad range of immune responses and protective immunity in many animal models, but it is less effective in primates. One reason for this may be the low expression of vector-encoded antigen in cells. Here we report that the use of vaccine vector (pCJ-3) containing two regulatory elements, a chimeric intron and a bovine growth hormone (BGH) polyadenylation signal, markedly increased antigen expression both in vitro and in vivo. A positive correlation was seen between the level of expression of Japanese encephalitis virus (JEV) envelope proteins and the levels of antibodies in C3H/HeN mice. Immunization of mice with pCJ-3/ME (pCJ-3 containing the entire membrane and envelope protein genes) with or without cardiotoxin pretreatment resulted in higher antibody titers than immunization with vector containing only envelope protein and conferred full protection against infection with JEV. Electron microscopy showed that pCJ-3/ME expression resulted in the production of virus-like particles of JEV in vitro. The particles enhanced the production of higher titers of neutralizing antibodies and thus provided immunity against JEV. Consequently, the efficacy of the newly developed DNA vaccines was validated. This should pave the way to clinical trials in man.