Neutralizing antibodies, which conferring protective immunity induced by DNA vaccine plasmids against hantavirus were evaluated by microneutralization assays. Pre-immune sera from all the groups exhibited no neutralizing activity. In contrast, immune sera collected 21 days after the first immunization with 100 μg of HTNV DNA vaccine plasmids in the presence or absence of CpG motifs showed neutralizing antibody titers of 8 to 32 (reciprocal of the highest dilution exhibiting 50% neutralization) against HTNV strain 84Fli. Immunization with pcDNA3 vector didn’t elicit any neutralizing antibody. Groups of mice receiving pcDNA3/eCTLA4-S+M plus CpG motifs, pcDNA3/eCTLA4-S+M alone, or pcDNA3/S+M plus CpG motifs, all achieved MN titers of ≧ 16. Only three of mice receiving pcDNA3/S+M alone could achieve MN titers of 16. The mean MN titer in mice vaccinated by pcDNA3/eCTLA4-S+M plus CpG motifs or pcDNA3/eCTLA4-S+M alone was significantly higher than that of mice immunized with pcDNA3/S+M alone (p < 0.05). These results indicated that eCTLA4 fusion strategy combine with CpG motif could induce better magnitude of neutralizing antibodies in mice against HTNV infection. eCTLA4 fusion strategy enhances CD8 T-cell responses
CD8+ T-cells play a vital role in protection against hantavirus infection by cell-mediated mechanisms. In order to evaluate the CD8+ T-cell response to vaccination, the splenocytes from mice vaccinated with DNA vaccine plasmids 1 week after each immunization were restimulated with HTNV N protein-specific peptides and analyzed by ELISPOT. The splenocytes from mice 1 week after third immunization were restimulated and analyzed by Intracellular Cytokine Staining assay. Number of CD8+IFN-γ-secreting splenocytes was significantly higher than other groups (p < 0.01) at 21 days after 1st immunization in mice receiving CpG+peCTLA4-M+S vaccine (Figure 3A). Consistently, mice vaccinated with pcDNA3/eCTLA4-S+M plasmids plus CpG1826 motif demonstrated higher frequencies of CD8+IFN+ T-cells to HTNV N protein-specific peptides compared with all the other groups in flow cytometery analysis. These results indicate that eCTLA4 fusion strategy could enhance the Th1-type cellular immune response. Discussion
DNA immunization with plasmids expressing hantavirus N protein and glycoprotein by intramuscular vaccination induced specific immune responses to the corresponding viral antigens in mice. In this study, we demonstrated that, a better magnitude of humoral and cellular immune responses could be generated in mice by DNA vaccine plasmids encoding HTNV N and GP fused to eCTLA4, a bioactive factor targeting to antigen presenting cells (APCs).