HTNV N-specific cellular immune response following DNA vaccination. (A) HTNV N-specific IFN-γ-producing CD8+ T cells demonstrated by ELISPOT Assay in groups of mice following DNA immunization. 4 mice of each group were euthanized on day 7, day 14 and day 21 after the first immunization. Splenocytes at 106 cells/well were tested in the presence of 10 μg/200 μl CTL epitope peptides M6. Number of spot forming cells (SFCs)/106 spleenocytes 2 was shown as mean plus standard deviation bar. SFCs in group of mice receiving pcDNA3/eCTLA4-S+M plasmids plus CpG motif was significantly higher than other groups (*p < 0.01) on day 21 after 1st immunization. (B) Detection of CD8+ CTL response specific for the epitope of hantavirus N protein by intracellular cytokine staining assay. One week after final boost, mice were euthanized. Splenocytes were restimulated with 10 μg/ml peptides (M6) for 5 h, and then stained with FITC-conjugated anti-CD8 and PE-conjugated anti-mouse IFN-γ McAbs. Mean frequencies of IFN-γ+ CD8+ cells in each group were shown in the right upper quadrants. Mean frequency of IFN-γ+ CD8+ cells in group of mice receiving pcDNA3/eCTLA4-S+M plasmids plus CpG motif was significantly higher than other groups (p < 0.05).