Fig. 5

Influence of pre-existing immunity on Ad-based induction of Env-specific binding antibodies and CD4⁺ T cells. CB6F1 mice were immunized with Ad.Env without pre-treatment, three weeks after two pre-immunizations with 10⁹ vp Ad.empty in three-week intervals, or one day after transfer of 10⁷ CD4⁺ T cells, or 200 µl plasma, or both from Ad pre-immune mice. Binding antibodies were analyzed two weeks after immunization with Ad.Env (a). In another transfer experiment, mice received plasma or antibody-cleared plasma from Ad pre-immune mice, or the Ad-neutralizing antibody 9C12 before immunization with 10⁹ vp Ad.Env. Binding antibodies were analyzed two weeks after one (b) or two immunizations with Ad.Env (c). Two weeks after the first Ad.Env immunization, blood cells were stimulated with Env-derived peptides and cytokine production by CD4⁺ T cells was analyzed by flow cytometry (d). Three weeks after the second Ad.Env immunization, mice were infected with FV and three days later, Env_123–141-specific CD4⁺ T cells were analyzed by MHC II tetramer staining (e). The data shown were obtained in two or three (b: Ad.Env and Ad-plasma,Ad.Env) independent experiments with four mice per group. Each dot represents one mouse (a–c, e) or mean values (d), lines indicate median (a–c) or mean (e) values, dashed lines indicate the limits of detection. Axes of the radar charts all have the scale as indicated for the first axis; the order of data shown in all plots is as indicated for the first radar chart (d). Data were analyzed by Kruskall–Wallis One Way Analysis of Variance on Ranks and Dunn’s multiple comparison procedure. Significant differences (P < 0.05) compared to unvaccinated mice are indicated by *, significant differences compared to mice vaccinated with Ad.Env are indicated by #. Sufficient statistical power was verified [SP = 0.99 (a), 1.0 (b), 1.0 (c), 0.93 (d: IFNγ), 0.99 (e)]