PROJECT SUMMARY AND ABSTRACT Guinea pigs are an exceptional mid-pipeline model for evaluation of vaccine candidates against tuberculosis (TB) disease. Guinea pigs develop the full range of pathological disease observed in humans, where mice do not, and are still fairly simple to house unlike nonhuman primates. Unfortunately, unlike mice or nonhuman primates the immunological reagents for guinea pig endpoints are widely underdeveloped or non-existent. There is a gap in resources and endpoints for guinea pig research in the TB vaccine pipeline which limits their use. Here we propose adopting and optimizing the peripheral blood monocyte cell (PBMC)-based mycobacterial growth inhibition assay (MGIA) for use with guinea pig samples. The MGIA has been used for humans, mice and nonhuman primates as a surrogate efficacy endpoint which evaluates the ability of PBMC from vaccinated subjects to control mycobacteria ex vivo. To-date, the assay has not been evaluated using guinea pig samples. Due to the relatively low number of cells required and elimination of the need for in vivo challenge this assay affords a more high throughput evaluation of vaccine efficacy and addresses the 3Rs of animal research: reduction, replacement, and refinement. PBMC and autologous serum samples have been collected from control and vaccinated guinea pigs in historical studies which are available for this project. Using these available samples allows a reduction in the need for live animals and helps make use of existing materials. The ex vivo challenge of the MGIA replaces the need for in vivo challenge of live animals while also refining the endpoint. Our colleagues at Oxford University have previously demonstrated that ex vivo efficacy correlates well with in vivo challenge and subsequent bacterial control in preclinical models. We hypothesize that PBMC and serum from bacillus Calmette–Guérin immunized guinea pigs will better control both bacillus Calmette–Guérin (BCG) and Mycobacterium tuberculosis challenge in an ex vivo co-culture than non-immunized cohorts. Furthermore, we expect that we will be able to identify the correct cell number and challenge dose to best resolve vaccine- mediated differences. If successful, this optimized protocol would help align the preclinical pipeline efficacy endpoints and provide a much needed tool for the guinea pig model. We will also make use of the co-culture supernatants to evaluate the secreted effector molecules produced and look for differences between samples that did or did not inhibit mycobacterial growth. Further mechanistic studies will explore the role of host cell glycolytic metabolism on subsequent control. Together, this proposal aims to develop a new core methodology for a highly translational animal model and begins to interrogate the mechanisms behind efficacy readouts.