Abstract Title: Novel multivalent viral vectored tuberculosis vaccines targeting lung immunity Tuberculosis (TB) persists as the deadliest bacterial infection, with more than 10 million new cases of active TB diagnosed and 1.5 million deaths attributed to TB worldwide each year, because there is no highly effective preventative vaccine. Mycobacterium tuberculosis (Mtb) also causes asymptomatic latent infections in ~25% of the world’s population. Latently infected individuals have a 10% lifetime risk of developing active TB disease. Vaccines that prevent pulmonary Mtb infection, limit reactivation from latency and/or therapeutically treat active TB disease are urgently needed. The objective of this R21 proposal is to explore the unique immunological features of an innovative Pichinde virus (PICV)-based vaccine platform combined with novel Mtb antigens to develop next generation TB vaccines. PICV is a non-pathogenic arenavirus with a bi-segmented RNA genome. The proposal exploits a recombinant PICV engineered with three RNA segments, rP18tri, which can encode two additional open-reading frames (ORFs) to express antigens. The rP18tri platform is safe, versatile and induces balanced antibody and T cell responses. Moreover, the rP18tri platform is simple to modify to produce a variety of multivalent antigens, which enables rapid analysis of candidates to identify those antigens that induce the greatest protection. The proposed research will test the hypothesis that optimized multivalent antigens delivered intranasally via the rP18tri viral vector platform will induce robust protective immunity against pulmonary Mtb infection. Preliminary data from proof-of-concept studies establish that rP18tri-based TB vaccines can be efficiently generated, induce strong antigen-specific T cell immunity and protect against pulmonary Mtb infection in a mouse aerosol challenge model. In this R21 proposal, we will generate additional rP18tri vector-based multivalent TB vaccine candidates with novel immunogens (Aim 1), evaluate antibody as well as systemic and lung tissue-resident T cell responses induced by these vaccines in mice (Aim 2), and assess the efficacy of these vaccines for prevention of Mtb infection in a mouse model (Aim 3). The study is significant because it is expected to produce at least one viral vectored multivalent TB vaccine candidate with demonstrated safety and efficacy in mice to be advanced to the next phases of preclinical and clinical evaluations. The study is also expected to generate new knowledge on protective immunity induced by novel Mtb antigens, which will guide the design of next generation TB vaccines, and to advance development of the PICV vector platform, which will expand the toolbox for fighting infectious diseases.