PROJECT SUMMARY Tuberculosis (TB) is one of the world's most important diseases, and a safe and effective vaccine against the causative agent Mycobacterium tuberculosis (Mtb) that is more potent than the currently available only partially effective M. bovis strain Bacille Calmette-Guérin (BCG) vaccine is sorely needed. It is generally acknowledged that both an improved replacement vaccine for BCG and a potent heterologous booster vaccine are needed in the fight against TB. The purpose of this project is to optimize and conduct advanced proof-of-concept studies in small animals and non-human primates (NHP) of a second-generation heterologous multiantigenic recombinant attenuated Listeria monocytogenes-vectored vaccine against TB. Live attenuated recombinant Listeria monocytogenes (rLm) vaccines offer major advantages over other approaches to booster vaccines, including protein in adjuvant and virus-vectored vaccines, in terms of cost, ease of manufacture, immunogenicity and efficacy. In preliminary studies, we have identified an improved multi-deletional Listeria vector (Lm ΔactA ΔinlB prfA*) and demonstrated that rLm vaccines expressing four key immunoprotective Mtb proteins (rLmMtb4Ag) substantially augment protective immunity when used as a heterologous booster vaccine in a prime-boost vaccination strategy against Mtb aerosol challenge in mice and guinea pigs. Moreover, delivering the immunoprotective Mtb protein via a first generation rLm vector was more efficacious than delivering it via a recombinant viral vector or administering it with a potent adjuvant. The goal of this application is to optimize expression of an Lm-vectored vaccine expressing 4 Mtb antigens; expand its antigen repertoire to six antigens to increase its potency; and to evaluate the optimized final lead rLm vaccine candidate for safety, immunogenicity and efficacy as a standalone vaccine and as a heterologous booster vaccine to BCG-primed animals in mouse, guinea pig, and non-human primate (NHP) models of pulmonary TB. We shall accomplish this goal by: a) Optimizing the protein expression cassette of rLmMtb4Ag vaccine; systematically evaluating additional novel Mtb antigens for immunogenicity and efficacy in mice, selecting the top two antigens, and subsequently constructing a rLmMtb6Ag lead vaccine candidate; b) Conducting comprehensive proof-of-concept studies of the optimized rLmMtb6Ag lead vaccine candidate for safety, immunogenicity, and efficacy as standalone and heterologous booster vaccine in the mouse model of pulmonary TB; c) Conducting selected proof-of-concept studies of the lead rLmMtb6Ag vaccine as a standa- lone and heterologous booster vaccine for safety, immunogenicity and efficacy in a guinea pig model of pulmonary TB; and d) as Aeras requires proof-of-concept in NHP for a vaccine to enter preclinical develop- ment, evaluating the lead rLmMtb6Ag candidate as a standalone vaccine for safety, immunogenicity and efficacy in a NHP model of pulmonary TB in c...