Moreover, the Rv2299c protein significantly increased BMDMs secretion of TNF-a,IL-6 and IL-1ß in a dose-dependent manner, and the concentration of 10 μg/ml stimulated the highest cytokines (Supplementary Figure S2).The DNA vaccines pVAX1Ag85A, pVAX1Rv2299c, and pVAX1Rv2299cAg85A were compared in Balb/c mice for immunogenicity and protection against challenge infection 6 weeks after the primary immunization (Figure 1A). Here, TNF is linked to infection.