Clostridioides difficile infection (CDI) is the leading cause of healthcare- and antibiotic-associated infection and has a 30% recurrence rate1–5. Previous vaccine strategies against CDI failed to reduce pathogen burden, a prerequisite for preventing C. difficile transmission and recurrence6–11. These vaccines were administered parenterally, which induced a systemic immune response, rather than a mucosal response in the colon, the site of infection. Here we compare protection and colonization burden between mucosal (rectal) and parenteral (intraperitoneal) administration routes of a multivalent, adjuvanted vaccine combining inactivated C. difficile toxins and novel surface antigens. We found that mucosal immunization, but not parenteral, clears C. difficile from the host. Unique correlates of decolonization included faecal IgG responses to vegetative surface antigens and a colonic, T helper type 17 (TH17)-skewed tissue-resident memory T cell response against spore antigen. Importantly, mucosal vaccination protected against morbidity, mortality, tissue damage and recurrence. Our results demarcate notable differences in correlates of protection and pathogen clearance between vaccine administration routes and highlight a mucosal immunization regimen that elicits sterilizing immunity against CDI.
