Protective mechanisms induced by a Japanese encephalitis virus DNA vaccine: requirement for antibody but not CD8(+) cytotoxic T-cell responses

Abstract

We have previously shown that a plasmid (pE) encoding the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection against a lethal viral challenge. In the present study, we used adoptive transfer experiments and gene knockout mice to demonstrate that the DNA-induced E-specific antibody alone can confer protection in the absence of cytotoxic T-lymphocyte (CTL) functions. Plasmid pE administered by either intramuscular or gene gun injection produced significant E-specific antibodies, helper T (Th)-cell proliferative responses, and CTL activities. Animals receiving suboptimal DNA vaccination produced low titers of anti-E antibodies and were only partially or not protected from viral challenge, indicating a strong correlation between anti-E antibodies and the protective capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to sublethally irradiated hosts conferred protection against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protective immunity in Igmu(-/-) and I-Abeta(-/-) mice, whereas in CD8alpha(-/-) mice the pE-induced antibody titers and protective rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4(+) T cells but independent of CD8(+) T cells.

Figures

FIG. 1

Effects of injection routes and numbers on JEV DNA vaccine-induced protective immunity. Groups of C3H/HeN mice (n = 5 to 7) were given intramuscular (i.m.) or gene gun injections of pE one, two, or three times at 3-week intervals. Mice receiving three doses of pcDNA3 by intramuscular or gene gun injection served as negative controls. All animals were challenged with 50 LD50 of JEV Beijing-1 8 weeks after the first immunization. Following challenge, mice were observed for 30 days, and the percentage of survivors was calculated. The data are representative of three independent experiments.

FIG. 2

Anti-E antibody induced by JEV DNA vaccine. Groups of C3H/HeN mice as described in the Fig. 1 legend were analyzed for the presence of JEV E-specific antibodies before challenge. The concentration of anti-E antibodies was calculated from the standard curve generated from serially diluted reference antibodies and expressed as units per milliliter. i.m., intramuscular.

FIG. 3

T-cell immunity generated by DNA or live viral vaccines. Groups of C3H/HeN mice (n = 3) were immunized three times at 3-week intervals with pE or pcDNA3 or sublethally immunized twice at 3-week intervals with 6.0 × 105 PFU of JEV Beijing-1 as described in Materials and Methods. One week after the last immunization, splenocytes were examined for JEV E-specific proliferative responses (A) and CTL activities (B) as described in Materials and Methods. The data are representative of three independent experiments. i.m., intramuscular.

FIG. 4

Anti-E antibodies in recipient animals to which was transferred immune serum (A) or splenocytes (B). C3H/HeN mice that were immunized with pE or pcDNA3 three times at 3-week intervals or sublethally immunized twice at 3-week intervals with live JEV Beijing-1 were used as donor animals. Immune splenocytes and sera were prepared as described in Materials and Methods. Naïve animals that were sublethally irradiated 24 h before were inoculated intravenously with 300 μl of sera or 5 × 107 splenocytes and challenged with 50 LD50 of JEV Beijing-1 12 h after transfer. Serum samples were collected 4 h, 4 days, or 14 days after transfer and analyzed for the presence of E-specific antibodies. i.m., intramuscular.

FIG. 5

DNA-induced anti-E antibody and protective immunity in gene knockout mice. Gene knockout mice and wild-type (WT) controls (C57BL/6) were given intramuscular (i.m.) or gene gun injections of pE three times at 3-week intervals. Two weeks after the last immunization, mice were challenged with 50 LD50 of JEV Beijing-1. The concentration of anti-E antibodies was determined as described in the Fig. 2 legend. The percentage of animals in each group that survived JEV challenge is indicated above each column.