PROJECT SUMMARY/ABSTRACT Enteroviruses, such as group B Coxsackieviruses, are common suspects in myocarditis and pancreatitis. However, no vaccines are currently available for these viruses, in part because multiple serotypes of Coxsackieviruses can induce similar diseases. Thus, exploration of alternative strategies to protect against them may have merit. The research team has made an unexpected observation that animals immunized with complete Freund’s adjuvant (CFA) were completely protected from both myocarditis and pancreatitis induced with Coxsackievirus B3 (CVB) pointed to a possibility that trained immunity may be an underlying mechanism of CFA-mediated effects, as demonstrated with the Bacillus Calmette-Guérin (BCG). The long-term goal of this research is to identify adjuvants that induce trained immunity to enhance anti-viral vaccine responses in the prevention of enteroviral infections. The objective of this application is to determine if macrophages primed with CFA mediate trained immunity. The central hypothesis is that the CFA contributes to the induction of anti-viral responses by trained immunity. This hypothesis will be tested by two specific aims: 1) characterize anti-viral responses in animals immunized with CFA and 2) determine the mechanisms of CFA- mediated protection in CVB infection. Methods and tools to be used include multiplex cytokine bead array, virus neutralization test, and major histocompatibility complex class II dextramers to assess antigen-specific immune responses and chromatin immunoprecipitation and metabolite assays to investigate the mechanisms of CFA-induced trained immunity. The project is innovative because it aims to determine whether trained immunity offers protection against CVB infections. The proposed studies are significant because they will: 1) mechanistically investigate whether trained immunity is sufficient to prevent enterovirus infections and 2) identify pathways that contribute to CFA-induced trained immunity. These outcomes may have a significant impact on strategies for preventing and/or treating viral infections using mycobacterial components that are known to possess immune-stimulating properties.