PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) latently infects one-fourth of the world's population, causing pulmonary tuberculosis (TB) in ~10 million people and resulting in ~1.6 million deaths each year. The currently available TB vaccine, Mycobacterium bovis BCG (BCG), shows variable efficacy. In addition, multi-drug resistant (MDR) Mtb strains have recently emerged. Thus, there is a great need for new TB vaccines. Our recent work has demonstrated that T helper type 17 (Th17) cells which produce the cytokine interleukin-17 (IL-17), are a primary effector cell mediating vaccine-induced protection against Mtb. Additionally, mucosal vaccines induce better mucosal immunity and confer superior protection against TB, when compared to systemic routes of immunization. Despite the ability of experimental mucosal adjuvants to induce protective Th17 responses and confer vaccine-induced protection, development of Th17-inducing mucosal TB vaccines for human use is slow. Therefore, there is an urgent need to identify safe and effective mucosal TB vaccines that can induce lung mucosal Th17 responses and understand their mechanism(s) of action. Nanoemulsions (NE) are oil-in-water emulsions formulated with antigen. NE adjuvant was safe and well-tolerated in human volunteers when used as a flu vaccine, and elicited both systemic and mucosal immunity following a single mucosal vaccination. In new published data, we show that mucosal delivery of NE along with Mtb antigens (NE-TB vaccine) can confer Mtb control, and is associated with decreased TB disease in mice. The overall goal of this proposal is to characterize and optimize the protective immune responses induced by NE-TB vaccine thus enabling development of a novel, safe, effective, first-of-kind Th17-inducing TB mucosal vaccine. Thus, we propose the following Specific Aims. In Specific Aim 1, using mouse model of TB, we will identify the prime-boost strategy and the antigen combination that enhances immunogenicity, and improves vaccine-induced protection of NE-TB vaccines, to exceed protection afforded by BCG vaccination. In Specific Aim 2, using the mouse model of TB, we will evaluate the efficacy of the best performing prime-boost NE-TB vaccine strategy against different Mtb strains, in genetically diverse hosts and upon pre-exposure to environmental mycobacteria. In Specific Aim 3, we will carry out preclinical development and proof-of-concept testing of NE-TB vaccines in non-human primates (NHP) infected with Mtb and identify correlates of protection. The work proposed in this grant will determine the mechanism(s) of action, and validate a novel first-of- kind Th17-inducing mucosal NE-TB lead candidate vaccine for use in humans in the near future.