Figure 3: Enhanced CD8 T cell responses by immunization with a mixture of HIV Env-DNA and SHIV 89.6 VLPs but not by simultaneous injection at separate sites. Groups of mice (6 per group) were immunized by intramuscular injection at weeks 0 and 4 with different vaccine preparations as shown in Figure 2. Two weeks after the second immunization, mouse splenocytes were prepared and stimulated by the peptide IGPGRAFYAR corresponding to the dominant CD8 epitope in the HIV Env for Balb/c mice. The percentages of IFN 𝛾 -producing CD8 T cells were analyzed by intracellular-cytokine staining and flow cytometry. (a) Representative results of FACS analysis for IFNγ-producing CD8 T cells from each immunization group stimulated with the peptide IGPGRAFYAR. Numbers in lower-right boxes represent percentages of IFNγ staining positive CD8 T cells. Background levels of IFNγ-producing CD8 T cells similar to Group 1 were obtained for all samples stimulated with an irrelevant peptide AMQMLKETI (data not shown). (b) Percentages of IFNγ staining positive CD8 T cells for each immunization group after stimulation with the peptide IGPGRAFYAR. Error bars represent standard deviations for each group. * indicates the groups with significantly higher levels of CD8 T cell responses than Group 2 that received the HIV Env-DNA vaccine only ( 𝑃 < . 0 5 ).