Repeated delivery of Ad5-vectored SIV vaccines through AVIP elicited robust antigen-specific immune responses in Ad5-seropositive macaques. (A) Immunization and challenge procedure. After the first immunization (Fig. 6), the same three groups of Ad5-seropositive macaques received the second and third immunizations: group 1 with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines delivered through AVIP; group 2 with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines injected intramuscularly; and group 3 with Ad5-empty injected intramuscularly as the control. Vaccine-elicited specific cellular immune responses were measured by IFN-γ ELISPOT assays following stimulation with SIV Gag, Pol, and Env peptide pools. (B) Gag-specific cellular immune responses; (C) Pol-specific cellular immune responses; (D) Env-specific cellular immune responses. Data were analyzed by a nonparametric Mann-Whitney U test. The bars represent the standard error. *, P < 0.05; **, P < 0.01.