PROJECT SUMMARY/ABSTRACT As evident in the ongoing “tripledemic” of severe acute respiratory syndrome-Coronavirus-2, Respiratory Syncytial virus, and Influenza virus, as well as re-emergence of enterovirus D68, virus infection is an important health threat and a huge economic burden. Multivalent RNA vaccination is an effective measure for counteracting virus infections and cancerous threats. However, multivalent vaccines need to inject relatively larger doses of RNA than monovalent vaccines, which can trigger a potent innate immune response in vivo, causing reduced transgene expression and unwanted reactogenicity. We have recently found that high dose RNA vaccination can be safely achieved in mice by delivering self-amplifying replicon RNA (repRNA) with a family of muscle-localizing cationic nanocarriers, LIONTM. In vivo delivery of repRNA by LION restricted the formulation to the muscle and local lymph nodes without inducing unwanted systemic cytokine responses. Systemic blockade of type I IFN signaling increased transgene expression and the subsequent antibody and T cell responses. Notably, we also found that CD11b+ cells [mostly monocyte dendritic cells (DC)] expand over time after intramuscular injection of repRNA/LION. However, the role of the muscle-infiltrating cells in immunogenicity is unknown. Consequently, many questions remain about how repRNA/LION vaccines induce adaptive immune responses and how transgene expression and adaptive immune responses are inhibited by the type I IFN system. So, Aim 1 looks at skeletal muscle DC (SMDC) and characterizes their immunological function both in vitro and in vivo. Aim 2 focuses on type I IFN signaling and seeks to identify how type I IFN signaling limits transgene expression and adaptive immune responses to repRNA vaccines. Aim 3 spotlights the inflammatory responses to muscle damage and their role in inducing adaptive immune responses to repRNA/LION vaccines. The proposed studies will test the following hypotheses that (1) the muscle plays a critical role in eliciting the immune response to intramuscular-based RNA vaccines, and (2) muscle-restricted activation of the immune system is a novel strategy for RNA vaccines to achieve high efficacy with reduced reactogenicity.