Partial nucleotide sequence of the Japanese encephalitis virus genome. for 21 days, whereas only one of the two remaining animals survived. No unimmunized animals exhibited a rise of neutralizing antibody or survived challenge. Levels of JE virus-specific immunoglobulin M class antibodies were elevated following challenge in half of the unimmunized mice and in the single pcDNA3JEME-immunized mouse that died. In the second experiment, JE virus-specific main cytotoxic T-lymphocyte (CTL) activity was detected in BALB/c mice immunized once with 100 g of pcDNA3JEME 4 days after challenge, indicating a strong postchallenge recall of CTLs. In the third experiment, evaluation of induction of CTLs and antibody activity by plasmids made up of portions of the prM/E cassette exhibited that induction of CTL responses alone were not sufficient to prevent death. Finally, we showed that antibody obtained from pcDNA3JEME-immunized mice 4 days following challenge could partially protect recipient mice from lethal challenge. Taken together, these results show that neutralizing antibody produced following challenge provides the crucial protective component in pcDNA3JEME-vaccinated mice. Japanese encephalitis (JE) is usually a mosquito-borne viral disease causing contamination of the central nervous system in humans and equines. It is generally believed AC-42 that JE computer virus present in mosquito saliva replicates at or near the bite site and is then transported via the bloodstream into the brain, where it may cause contamination and encephalitis. AC-42 Two major factors have been reported to be important for protection from encephalitis: neutralizing antibody and cytotoxic T lymphocytes (CTLs) specific for JE computer virus. Passive transfer of monoclonal antibodies to the envelope (E) protein (1, 5, 21), Mouse monoclonal to BRAF T cells obtained from infected mice (23, 25), and CTLs (26) can safeguard mice from a lethal challenge. High levels of neutralizing antibody (29) and JE virus-specific T lymphocytes (8) have been detected in JE patients in the convalescent phase. We have previously analyzed the immunogenicity of JE gene products in a mouse model using recombinant poxviruses expressing the transmission of the premembrane (prM), the prM gene, and the envelope (E) gene. Cells infected with these poxviruses produce subviral extracellular particles (EPs). These subviral particles are similar to the slowly sedimenting hemagglutinin particles produced by cells infected with JE computer virus, suggesting that this prM, membrane (M), and E proteins in these EPs are comparable to the authentic forms of these proteins (11, 13, 22). Mice immunized with poxvirus-based recombinants encoding the signal-prM-E gene cassette induced high levels of neutralizing antibody and memory CTLs and were guarded from lethal challenge (9, 12, 14). However, these mice were not protected from contamination by the challenge computer virus, since high levels of antibody to the nonstructural (NS) proteins were detected in mice surviving challenge (11). Recently, naked DNA plasmids encoding flavivirus genes have been reported to induce neutralizing antibody and/or protection in mice, using the NS1 gene of JE computer virus (19) and the prM/E gene of dengue type 2 (6, 30), AC-42 St. Louis encephalitis (27), and tick-borne encephalitis (31) viruses. We have exhibited that mice immunized with a plasmid encoding the JE computer virus signal-prM-E gene cassette (pcDNA3JEME) were also guarded from a lethal challenge (15). Interestingly, although mice immunized with this DNA produced CTLs that could be detected after in vitro activation, the levels of neutralizing antibody induced by these DNAs were low or undetectable. Therefore, this system provides a mouse model useful for studying the mechanism of protection against JE. Some other DNA vaccines also have been reported to protect in the absence of neutralizing antibody responses (19, 19a). In this study, we analyzed the postchallenge immune responses in pcDNA3JEME-immunized mice to elucidate the role of neutralizing antibody and CTLs in protection. MATERIALS AND METHODS Viruses. The prototype Nakayama strain of JE computer virus (20) AC-42 was utilized for construction of plasmids, neutralization assessments, and spleen cell activation, and the virulent Beijing P3 strain (22) was utilized for mouse challenge studies. Recombinant vaccinia viruses used for contamination of target cells in cytotoxicity assays were vP555, encoding the prM, E, and NS1 genes of the Nakayama strain; vP658, encoding E and NS1; vP829, encoding prM and E; and their parent computer AC-42 virus, vP410 (11, 22). vP829 and vP410 were also utilized for preparing.