Mice were immunized with experimental subunit vaccines based on secreted antigens from Mycobacterium tuberculosis in a series of adjuvants, comprising incomplete Freund's adjuvant (IFA), dimethyl dioctadecyl ammoniumbromide (DDA), RIBI adjuvant, Quil-A saponin, and aluminum hydroxide. Immune responses induced by these vaccines were characterized by in vitro culture of primed cells, PCR analysis for cytokine mRNA, detection of specific immunoglobulin G isotypes induced, and monitoring of protective immunity to tuberculosis (TB). The study demonstrated marked differences in the immune responses induced by the different adjuvants and identified both IFA and DDA as efficient adjuvants for a TB subunit vaccine. Aluminum hydroxide, on the other hand, induced a Th2 response which increased the susceptibility of the animals to a subsequent TB challenge. DDA was further coadjuvanted with either the Th1-stimulating polymer poly(I-C) or the cytokines gamma interferon, interleukin 2 (IL-2), and IL-12. The addition of IL-12 was found to amplify a Th1 response in a dose-dependent manner and promoted a protective immune response against a virulent challenge. However, if the initial priming in the presence of IL-12 was followed by two booster injections of vaccine without IL-12, no improvement in long-term efficacy was found. This demonstrates the efficacy of DDA to promote an efficient immune response and suggests that IL-12 may accelerate this development, but not change the final outcome of a full vaccination regime. MATERIALS AND METHODS Mice. C57BL/6J female mice 8 to 12 weeks old were bred at Bomholtegaard breeding center, Ry, Denmark. Bacteria. M. tuberculosis H37Rv cells were grown at 37ЊC on Löwenstein-Jensen medium or in suspension in Sauton medium enriched with 0.5% sodium pyruvate and 0.5% glucose.