SUMMARY: PROJECT 3 - IMMUNOLOGY The quality of the host immune response to Clostridioides difficile infection is one of the strongest predictors of disease severity. Despite the protective capacity of the host immune response, the immune parameters that promote immunity remain poorly understood. Approximately 25-35% of patients that recover from primary C. difficile infection will experience a recurrence episode indicating the host often fails to develop natural immunity following primary infection. Further, multiple vaccine trails have not met primary endpoint of reducing occurrence of infection despite the vaccine candidates eliciting robust antibody responses against C. difficile toxins, the primary virulence factors driving disease. A limited mechanistic understanding of why the natural immune response to infection often does not promote immunity represents a critical roadblock toward the goal of developing a vaccine that will elicit lasting protective immunity in high-risk populations. This project will systematically evaluate the natural immune response to C. difficile infection using both patient sample and a murine infection system. In aim 1 we will compare the capacity of the systemic and intestinal mucosal antibodies elicited following infection to detect and bind to C difficile residing in the intestinal lumen. Successful generation of an antibody response that targets C. difficile in the intestinal tract is dependent on a coordinated C. difficile-specific CD4+ T and B cell response in the intestine and associated draining lymph nodes and is the focus of studies proposed in aim 2. Last, in aim 3 we will investigate the in vivo biogeography and transcriptome of C. difficile in the presence of adaptive immune pressure to identify immune evasion mechanisms employed by C. difficile to promote persistence and transmission. The result of all three aims will feedback into Project 1 (Vaccine Development) to inform mRNA vaccine studies by providing a template how vaccine-induced immunity can be shaped to limit disease, prevent colonization, and recurrence of C. difficile. 1