Figure 3: Immunogenicity comparison among groups of mice immunized with different epitope-based DNA vaccines. 100 μg of purified plasmid DNA solved in 100 μL sterile PBS was inoculated intramuscularly into tibialis anterior for three times at weeks 0, 2, and 4. 2 weeks after the final vaccination, the mice were sacrificed and splenocytes were freshly collected for intracellular cytokine staining (ICS) assay. (a) Representative flow cytometric analysis of IFN-γ and IL-2 secretion after stimulating mice splenocytes with Env2 peptide. The cytokines secretion profiles shown were gated on CD3+CD8+ T cells. (b) pSV-Env, pSV-triEnv, and pSV-sextEnv could elicit appreciable IFN-γ secretion in CD8+ T cells and the frequencies of IFN-γ + CD8+ T cells ranked as pSV-Env < pSV-triEnv < pSV-sextEnv (). No significant IL-2 response was observed in any of the groups. (c) Compared with pSV-Env(), the mean fluorescence intensity of IFN-γ in IFN-γ +CD8+ T cells elicited by pSV-triEnv () and pSV-sextEnv () was significantly lower. (d), the integrated mean fluorescence intensity (iMFI) of mice immunized with pSV-sextEnv was significantly higher than mice immunized with either pSV-triEnv or pSV-Env. The iMFI was calculated as multiplying the frequency of IFN-γ + CD8+ T cells by the corresponding mean fluorescence intensity of IFN-γ.