FIG. 2.

Adenoviral vector immunization by the tonsillar route. (A) Experimental outline. The scale indicates the number of weeks after the first immunization. The type of vaccine or challenge virus is given above the time line. Four of the 10 monkeys of the Ad-oral group received a third oral immunization at week 16. (B) Mean titer and standard deviation of antibodies binding p27 CA or gp130 SU in 10 (weeks 0 to 12) and 4 (weeks 16 to 48) macaques of the Ad-oral group. The vertical dotted line in panel B indicates the time of challenge. (C) ELISPOT responses to the indicated peptide pools in the Ad-oral group were determined 4 and 0 weeks prior to immunization (Pre), and 2 and 4 weeks after the first, second, and third oral immunization with the adenoviral vector. ELISPOTs of unstimulated control cultures from each time point were subtracted, and the means for all available time points were used as single Pre, first, second, and third values for each animal. The means and standard deviations of the Pre, first, second, and third values for all animals after stimulation with the indicated peptide pools are shown. To determine whether there is a statistically significant difference between the means of the Pre, first, and second, ELISPOT values for each peptide, one-way ANOVA was used, followed by pairwise multiple comparison using the Tukey test. Numbers above the horizontal bars give the respective P values if a significant difference between two time points was obtained by ANOVA. Columns marked with an arrow indicate ELISPOT responses that are significantly higher (P < 0.05, t test) than the mock ELISPOT response to the respective peptide pool of the Ad-control group (D). For the 4 of the 10 animals that received a third oral immunization, a paired t test (#) was used to determine significant increases in ELISPOT responses between the second and third immunizations. (D) IFN-γ ELISPOT responses in six control animals were determined weeks −4, 0 (Pre), and 8 (mock) as described in for panel C. The t test was used to determine statistically significant differences between the Pre and mock ELISPOT responses for each peptide. *, due to failure of the equal variance test, ANOVA on ranks followed by pairwise comparison using Dunn's method was performed. (E) RNA load after challenge with SIVmac239. The mean and standard deviation of the viral RNA loads in the four animals receiving three oral adenoviral vector immunizations (Ad-oral) and in eight control monkeys infected in parallel are shown.