Figure 3.

T cell NS1-derived peptide identification in pcTPANS1-immunized BALB/c mice by IFN-γ ELISPOT assays. Immunizations, virus challenge and euthanasia were outlined in Figure 2A. Splenocytes isolated from pcTPANS1-immunized BALB/c mice (n = 3, pooled), challenged or not with DENV2, were stimulated with NS1-derived peptides for 18 h, and the number of cells producing IFN-γ was measured by ELISPOT assay. Peptides and cells were evaluated in pools (A) and positive peptides were confirmed by tests performed with cells from individual mice (n = 5) (B,C). ELISPOT assays were performed using splenocytes collected 15 days following DNA inoculation (gray and light blue bars/dots) or 21 (A) and 7 days post-infection (B,C) (dark blue bars/dots). Cells from naïve or pcTPA-inoculated mice were used as negative controls. The horizontal dotted lines represent the cut-off selection point (≥5 SFC/5 × 10⁵ cells) (B,C). Bars represent the mean plus standard deviation of triplicate data in (A) and the means of each group in (B,C). Symbols represent significant differences between groups, using non-parametric two-tailed Mann-Whitney statistical tests: * pcTPA- vs. pcTPANS1-inoculated mice; # naïve animals vs. pcTPANS1-immunized mice after DENV2 challenge; + pcTPANS1-immunized mice vs. pcTPANS1-immunized animals after DENV2 challenge; Φ naïve animals vs. DENV2 challenged mice; λ pcTPANS1-immunized mice vs. DENV2 challenged animals; Ψ pcTPANS1-immunized animals challenged with DENV2 vs. DENV2 challenged mice. One symbol: p < 0.05; two symbols: p < 0.001; three symbols: p < 0.0001.