Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb) kills more people in the world, than any single infectious disease. The only approved vaccine for TB, the Bacilli Calmette-Guerin (BCG) provides protection against miliary and meningeal TB in children, but is NOT effective against pulmonary TB in adults, the most transmissible and prevalent form of the disease. Therefore, BCG vaccination has not thwarted the global epidemic of TB, and developing an effective TB vaccine ranks at the top among the current global health priorities. Mtb is a complex and highly adapted slow-growing organism and protection against TB will likely require multiple layers of immune defense that counter the pathogen?s growth and spread at multiple steps in its life cycle and different phases of the disease. Accumulating evidence suggests that a critical number of strategically positioned(Tissue resident [TRM], effector [TEM] and central [TCM] memory) and functionally diverse (TC1/TH1 and TC17/TH17) subsets of memory CD4 and CD8 T cells will be required to collectively provide effective defense against pulmonary TB. However, several of the current vaccines that are in preclinical or clinical stages of development are skewed towards inducing only TEM/TCM TH1 cells, which might be necessary but not sufficient to provide effective protection against pulmonary TB. We have identified a mucosal vaccine combination adjuvant consisting of a carbomer-based adjuvant, Adjuplex (ADJ) and a TLR-4 agonist, glucopyranosyl lipid A (GLA) that elicits a multipronged immunity consisting of high numbers of mucosally imprinted (TRM) and circulating (TEM and TCM) subsets of memory CD4/CD8 T cells that produce IFN-g and/or IL-17 in lungs. These exciting preliminary data underpinned the central hypothesis that a subunit vaccine consisting of ADJ+GLA and immunogenic TB antigens (Ag85B [early antigen], ESAT-6 [replicating] and Rv2660c [latency antigen]) will induce broad T cell-mediated immunity that protects against pulmonary TB. The specific aims are to test the hypothesis that: (1) mucosal imprinting of TRM, differentiation of TCM and TEM and functional programming of TB-specific memory CD4 and CD8 T cells are dictated by the adjuvant and the route of vaccine administration and not by the antigen; (2) vaccines that elicit functionally diverse (TC1, TC17, TH1 and TH17) TRM, TEM and TCM will provide effective immunity to pulmonary TB. This exploratory R21 proposal exploits the complementary expertise of two PIs: Dr. Suresh (T cell memory) and Dr. Talaat (TB pathogenesis and vaccines). This project is highly significant because it may lead to the development of a safe and effective subunit vaccine against pulmonary TB. The innovation of this project stems from the preclinical development of a novel combination adjuvant that elicits a potent T cell response with unprecedented phenotypic and functional diversity in the lungs. Overall, this low risk-high pay off exploratory project might provide new insights into mechanisms of protective immunity to TB and such findings are of potentially high impact in the field of TB vaccinology and public health.