Figure 1. rHRV-DNA vaccination elicits robust CMI in the spleen.

Mice (n = 7) received either 2 doses (containing 5 × 10⁶ TCID₅₀ per dose per animal) of wild-type (wt)-HRV-A1 followed by 50 μg pVAX (vaccination control group) or 2 doses of rHRV-Gag/Tat (containing 5 × 10⁶ TCID₅₀ per dose per animal) followed by a single ID dose of 50 μg of a DNA cocktail (pVAX-Gag-Tat) containing equimolar concentrations of pVAX-sTat-IMX313 and pVAX-Gag-PRF. This vaccination regimen is referred to as rHRV-DNA prime-boost vaccination. Other mice were vaccinated with 3 ID doses (50 μg/dose per animal) of pVAX-Gag-PRF/pVAX-sTat-IMX313 and referred to as 3X pVAX-Gag-Tat vaccination. Splenocytes were collected 14 days after the final dose and restimulated in duplicate with overlapping peptides representing the entire Gag protein. (A) Gag peptide pool 1, (B) Gag peptide pool 2, (C) Gag peptide pool 3 and (D) Gag peptide pool 4 in an IFN-γ ELIspot. (E) Total Gag responses depicted in pools 1–4. (F) Tat-specific responses from cells stimulated with the complete Tat peptide pool. Splenocytes were also stained with the H-2K^d-Gag_197–205 tetramer for 1 h at room temperature and the number of tetramer-positive CD8⁺ T cells was analyzed by flow cytometry (G). The data are representative of 2 independent experiments (n = 7) and are plotted as mean SFU per 10⁶ splenocytes (±SEM). Each symbol represents an individual mouse. *p < 0.05, **p < 0.01 and ***p < 0.001 (Mann–Whitney U test).