FIG. 3.

Immunogenicity of rVSV-gag vectors following i.n. inoculation. Mice (n = 5) were primed by i.n. inoculation of rVSV_IN vectors (1 × 10⁷ PFU) and boosted 8 weeks later by i.n. inoculation with the corresponding rVSV_NJ vectors. T-cell responses were assessed in splenocytes at 7 days postprime and both 5 days and 4 weeks postboost. Humoral responses were assessed in serum at 4 weeks postboost. (A) HIV-1 Gag tetramer responses. Isolated splenocytes were stained with Gag tetramer (H-2K^d) (AMQMLKETI) and assessed by flow cytometry for the percentage of CD3⁺ CD8⁺ T cells that were Gag tetramer positive. (B) HIV-1 Gag IFN-γ ELISPOT responses. Isolated splenocytes were cultured overnight with the H-2K^d Gag immunodominant peptide (AMQMLKETI), and IFN-γ secretion was determined by ELISPOT analysis. The data are normalized to 10⁶ spleen cells. (C) HIV-1 Gag ex vivo CTL responses at 5 days postboost. Isolated splenocytes were used in 3-h ex vivo CTL assays to lyse Eu-labeled P815 target cells loaded with the H-2K^d Gag immunodominant peptide. (D) HIV-1 Gag in vivo CTL responses at 4 weeks postboost. Gag-specific in vivo cytolytic activity was assessed by 18-h in vivo CTL assays in immunized mice. (E) HIV-1 Gag serum antibody responses at 4 weeks postboost. Serum was collected from immunized mice and assessed for Gag p24-specific total IgG by ELISA. Data are presented as the geometric mean titers. (F) VSV N IFN-γ ELISPOT responses. Vector-specific responses were assessed by measuring the T-cell response to VSV nucleoprotein (VSV N). Isolated splenocytes were cultured overnight with the H-2L^d VSV N immunodominant peptide (MPYLIDFGL), and IFN-γ secretion was determined by ELISPOT analysis. Immune responses induced by attenuated rVSVs were compared to immune responses induced by rVSV-gag5. *, P < 0.05; **, P < 0.001.