MTBK_24820-specific immune responses in C56BL/6 mice immunized with adjuvant, M. bovis BCG, or MTBK_24820. (A) Three weeks after the last immunization with adjuvant alone, BCG, or MTBK_24820, the IgG antibodies specific to MTBK_24820 were measured in sera. OD, optical density; adjuvant, DDA and MPL. (B) MTBK_24820-specific recall responses induced by immunization were measured in spleen cell culture supernatants after stimulation with 0, 0.1, 1, or 10 μg/ml of MTBK_24820 prior to M. tuberculosis Beijing/K strain aerosol infection. Concentrations of IL-2, IL-6, IFN-γ, and IL-17 were determined using multiplex bead assays. Data are presented as means ± SD for duplicate determinations from three mice. Significant differences between groups were analyzed by one-way ANOVA (*, P < 0.05; **, P < 0.01).