FIG 5.

NS3 coexpressed with PRF is more immunogenic than NS3 alone. Splenocytes from vaccinated mice were stimulated with NS3 peptides, and cytokine production was analyzed using ICS and flow cytometry. Memory CD4⁺ and CD8⁺ T cells were gated to assess the frequency of IFN-γ-producing CD8⁺ T_EM cells (A), TNF-α-producing CD8⁺ T_EM cells (B), IL-2-producing CD8⁺ T_EM cells (C), IFN-γ/TNF-α-double-producing CD8⁺ T_EM cells (D), TNF-α/IL-2-double-producing CD8⁺ T_EM cells (E), IFN-γ/IL-2-double-producing CD8⁺ T_EM cells (F), IFN-γ/TNF-α/IL-2-triple-producing CD8⁺ T_EM cells (G), and the proliferation of CFSE-labeled CD3⁺ CD8⁺ T cells (H). Splenocytes were labeled with CFSE and restimulated for 5 days with peptides representing the C-terminal third of NS3; the proliferation of CD3⁺ CD8⁺ T cells is shown as a percentage of total CD3⁺ CD8⁺ T cells. (I) Frequency of IFN-γ-producing CD4⁺ T_EM cells after NS3 peptide stimulation. Each symbol represents an individual mouse, and the data show the means (n = 8 to 9) ± the SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney test).