Effective host immunity to Mycobacterium tuberculosis is dependent on T cell-mediated responses against specific antigens of the bacillus. Although more than 2 million people die from tuberculosis every year, approximately 90% of infected humans who mount a normal immune response to the bacterium never develop clinical disease. This demonstrates the importance of a potent immune response in limiting or Preventing disease caused by M. tuberculosis, and provides a rationale for the development of an effective vaccine to prevent disease. Recently, it has become clear that many virulence-promoting factors produced by M. tuberculosis are lipids, which can have profound effects on modulating the immune response of the host. Functional inactivation of two genetic loci involved in lipid biosynthesis in M. tuberculosis, pcaA and panCD, are associated with reduced virulence and enhanced clearance of the bacilli in mice. A third genetic locus called RD1 plays a major role in virulence of M. tuberculosis most likely by controlling the secretion of toxic proteins. We hypothesize that M. tuberculosis harboring functional deletions of these genes will generate enhanced T cell responses and more robust protective immunity in infected animals. In addition, we propose that the T cell response against M. tuberculosis-associated protein antigens can be further enhanced by using novel synthetic glycolipid adjuvants in the alpha-galacotosyl ceramide family that activate CD1-restricted NK T cells. The following three specific aims are proposed: 1) To assess the impact of deletion of pcaA, panCD and RD1 on T cell responses to MHC class I and class II presented epitopes; 2) To analyze the basis for adaptive immunity to attenuated vaccine strains of M. tuberculosis in CD4-deficient mice; and 3) to determine the effect of synthetic immunomodulatory lipids in the alpha-galactosyl ceramide family on the T cell response and protective immunity induced by attenuated M. tuberculosis vaccine strains. This project will be enabled through an extensive network of collaborations with other members of this program project and the use of all of its core facilities. Combining these approaches for enhancing immunogenicity may lead to the development of highly effective attenuated live M. tuberculosis vaccines. This would be a major tool for enhancing the currently inadequate efforts to reduce the global burden of tuberculosis and prevent the emergence of multidrug resistant strains.