6–8 week old BALB/c mice were primed with 1 × 105 ffu BNSP-Gag, BNSP-Gag-IFN(−) or BNSP-Gag-GM-CSF. PBS mice were included as a negative control. The spleens were isolated from mice on days 3, 7, 10 and 16 (a). Flow cytometry analysis of CD8+ T cell markers was gated as shown (b). The quantity of Gag-specific cells was measured with a tetramer stain against H2d restricted AMQMKLETI epitope (c). Activated Gag-specific CD8+ T cells were gated on CD62Llo cells (d). To further measure the functionality of the cells, an IFN-γ ELISpot assay was done (e–f). Statistical analysis was performed using one-way ANOVA * P <0.05, ** P<0.01.