Fig. 2

Immune responses and protection induced by sequential Ad-based vaccination. CB6F1 mice were immunized at two time points with 10⁹ vp of the indicated vaccines; immunizations were performed in a three-week interval, only one vaccine was used in each immunization. The GagL_85–93-specific CD8⁺ T cell response was analyzed by MHC I tetramer staining of peripheral blood cells two weeks after the second immunization (a). Three weeks after completion of the immunizations, mice were challenged with FV, and 10 days after FV infection GagL_85–93-specific CD8⁺ T cell responses (b) and Env_123–141-specific CD4⁺ T cells (c) were analyzed in peripheral blood cells by MHC I and MHC II tetramer staining, respectively. FV load in plasma was analyzed 10 days (d), viral load in spleens three weeks after FV infection (e). F-MuLV-binding antibody titers were analyzed in sera collected two weeks after completion of the immunizations (f). In another experiment, CB6F1 mice were immunized once with Ad.TxnGagL, with or without an immunization with Ad.pIXgp70 three months earlier. GagL_85–93-specific CD8⁺ T cell responses were analyzed in peripheral blood by MHC I tetramer staining two weeks after Ad.TxnGagL immunization (g). The data shown were obtained in two independent experiments (a, d, e) or one experiment (b, c, f, g). Each dot represents one mouse, lines indicate mean [a–c, g (grey line: mean value calculated with exclusion of outlier)] or median (d–f) values, dashed lines indicate detection limits. Data were analyzed by Kruskall–Wallis One Way Analysis of Variance on Ranks and Dunn’s multiple comparison procedure (a–e, g without exclusion of outlier from the calculation), One Way Analysis of Variance and Student–Newman–Keuls multiple comparison procedure (g with exclusion of outlier from the calculation) or Mann–Whitney Rank Sum Test (f). Significant differences (P < 0.05) compared to unvaccinated mice are indicated by *, significant differences compared to mice vaccinated with the respective single vaccine are indicated by # (a, b: compared to Ad.TxnGagL; F: compared to Ad.pIXgp70); black symbols indicate significant differences calculated including all values, grey symbols indicate significant differences after exclusion of the outlier (g). Sufficient statistical power was verified [SP = 0.99 (a), 0.99 (b), 0.81 (c), 0.99 (d), 0.99 (e), 1.0 (f), 0.99 (g)]