After removing the medium, find protocol splenocytes from individual mice at a density of 105 cells/well were stimulated with a pool of CSp peptides at a concentration of 5 μg/well for 48 h at 37 °C 5% CO2. Following incubation, plates were washed five times with PBS and were then incubated with 1 μg/ml of biotinylated anti-mouse antibodies (Mabtech) in PBS containing 0.5% FCS for 2 h at room temperature. After washing five times with PBS to remove free biotinylated anti-mouse antibodies, plates were incubated for 2 h with detection antibodies conjugated to streptavidin–alkaline phosphatase

at 1:1000 dilutions in the same buffer as above. The enzyme reaction was developed with nitroblue tetrazolium bromo-4-chloro-3-indolyl-phosphate chromogen substrate (Mabtech). The spot-forming units (SFU) per 105 cells were counted using a dissection microscope (Carl Zeiss, Stemi 2000-C). Multiscreen HTS-IP Filter Plates (96-wells, Millipore) were pre-wetted with 70% ethanol for 2 min, washed five times with

PBS and coated with 5 μg/ml of CSp in PBS Rapamycin solubility dmso overnight at 4 °C. Plates were blocked for 2 h at room temperature with complete medium. BM cells (105 cells per well) from the immunized mice were seeded in duplicates and stimulated individually with the C-CSp, N-CSp or IDE-CSp. Plates were incubated for 12 h at 37 °C, 5% CO2 and 85% humidity. After the incubation period plates were washed five times with PBS and incubated for 2 h at room temperature with HRP-conjugated goat anti-mouse IgG (1:1000; Southern Biotech) in PBS, 5% FCS. After washing with PBS five times, the reaction was developed using a Vectastain 3-amino-9-ethylcarbazole (AEC) substrate kit (Vector laboratories, Burlingame, CA) according to manufacturer’s instructions. The reactions were stopped by washing plates with deionized water. Plates were dried in the dark and spots were counted using a dissection microscope (Carl Zeiss, Stemi 2000-C). Data were analyzed using GraphPad Prism Version 5 (Graphpad Software, Inc.,

San Diego, CA). The nonparametric Kruskal–Wallis test was used for the comparison of means in different groups. For all enough tests, p ≤ 0.05 was considered significant. The combination of Ad35-CS and BCG-CS in a heterologous prime-boost regimen resulted in high-levels of CSp-specific IgG responses (Fig. 1). Moreover, antibody responses exhibited higher IgG2a (Th1-type responses) when comparing heterologous prime-boost Ad35-CS/BCG-CS to homologous prime-boost BCG-CS/BCG-CS immunizations (Fig. 1). Among the three CSp peptides tested (C-CSp, N-CSp and CSp-IDE), the response to C-CSp was synergistic and induced stronger IgG2a response in the group primed with Ad35-CS and boosted with BCG-CS (Fig. 2).