FIG. 1.

IFN-γ responses after vaccination. (A) Kinetics of ex vivo, direct responses after stimulation of PBMC (2 × 10⁵/well) with rAg85A (5 μg/ml). Results are depicted as mean net SFC (SFC with stimulant minus SFC of medium controls) ± standard errors of the means (SEM) for five cattle per group. *, P < 0.05 compared to the other two groups (one-way analysis of variance followed by the Tukey-Kramer multiple-comparison postanalysis test). Circles, BCG-rAd85A (n = 5); squares, rAd85A-BCG (n = 5); triangles, rAd85A-rAd85A (n = 5). Priming vaccination was done at week 0. B1, rAd85A boost of rAd85A-rAd85A group; B2, BCG or rAd85A boost of BCG-rAd85A or rAd85A-BCG group, respectively. Positive responses were recorded when the SFC with stimulant minus the SFC of medium controls was >50 SFC/10⁶ PBMC and more than the prevaccination values. (B) Cultured ELISPOT responses established 8 weeks after booster injections. PBMC were stimulated with rAg85A and IL-2 for 13 days, and IFN-γ ELISPOT assays were performed by stimulating cultured cells with rAg85A (5 μg/ml) or a peptide cocktail derived from Ag85A (6 μg/ml of each peptide). Cultures were performed in the presence of autologous macrophages as a source of antigen-presenting cells. Medium control values were subtracted, and data are presented as means of groups of five animals ± SEM. Statistical differences between groups were evaluated using unpaired, two-tailed t tests. Differences between the BCG-rAd85A and rAd85A-rAd85A groups did not reach statistical significance. The horizontal line shows peptide-stimulated cultured ELISPOT results for PBMC from a group of six unvaccinated control animals (SFC/10⁶ cells, 480 ± 480).