PROJECT SUMMARY/ABSTRACT The lack of effective vaccines against most infectious diseases is largely a result of our fundamental lack of understanding of mechanisms involved in protective immunity. Adjuvants incorporated into vaccine formulations have a major impact on vaccine efficacy via modulating and prolonging host immune responses; however, our understanding of their underlying mechanism(s) of action in driving specific immune parameters is incomplete. While vaccines are the most effective way to prevent and control infectious diseases, many pathogens that significantly impact human health remain without an effective vaccine. For example, one-fourth of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB)1, the leading infectious disease killer in the world. It is likely that for TB, and other major infectious diseases (e.g. AIDS and malaria), new adjuvants or adjuvant combinations will be essential for instructing a protective immune response. We and others have shown that targeting both the type 1 T helper (Th1) cells and type 17 T helper (Th17) cells enhance vaccine-induced immunity for TB5-8. Additionally, we have recently demonstrated that live vaccines (e.g. BCG) and adjuvants (e.g. β-glucan) generate innate memory response, termed trained immunity, via epigenetic reprogramming of monocytes/macrophages, thereby conferring protection against Mtb infection9,10. These data together suggest that combination adjuvants targeting both innate trained immunity and adaptive Th1/Th17 cellular responses can enhance protective immunity against pathogens. Thus, defining the mechanisms of action of combination adjuvants that generate potent trained immunity and protective Th1/Th17 axis, are the overall goals of this proposal. In the current proposal, we hypothesize that combinations of adjuvants that drive Th1 responses (AS01 or UM-1007, a novel TLR7/8 agonist) and Th17 responses (β- glucan, nanoemulsion, or UM-1098, a novel Mincle agonist) will result in Th1/Th17 adaptive responses and/or enhance trained immunity. We will achieve these overall goals through the following four Specific Aims. Specific Aim 1: To determine the mechanisms by which combination adjuvants elicit Th1/Th17 immune responses. Specific Aim 2. To determine the impact of combination adjuvants on hematopoietic stem cells and trained immunity. Specific Aim 3. To determine whether use of combination adjuvants improves recall Th1/Th17 responses and trained immunity upon challenge. Specific Aim 4. Determine the mechanism of action of combination adjuvants in a pre-clinical human-like rhesus macaque model. Together, the aims of this study will map out the pathways induced by combination of adjuvants that effectively drive Th1/Th17 responses and trained immunity. Through Mtb challenge studies, we will demonstrate whether the mechanisms by which Th1/Th17 and trained immunity are elicited are involved in protection again...