Abstract Clostridioides difficile is a spore-forming and toxin-producing anaerobic bacterium. It is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of life-threatening pseudomembranous colitis. Current treatment options of C. difficile infection (CDI) with very few antibiotics are plagued by high recurrence rates (15-35%). CDI symptoms are mainly caused by toxins TcdA and TcdB. In addition, 5-30% of C. difficile isolates produce binary toxin (CDT), which is associated with increased morbidity and mortality rates. Active vaccination provides the attractive opportunity to prevent CDI and recurrence, but no vaccine against CDI is licensed. Vaccines should target all three toxins and C. difficile cells/spores that transmit the disease and cause recurrence. The goal of this project is to develop multivalent parenteral/mucosal vaccines that target three C. difficile toxins and colonization. Specifically: 1) We have demonstrated effective protection of animals from CDI with a potent immunogen Tcd169FI, which includes immunodominant regions of both TcdA and TcdB. 2) We found that parenteral immunizations with FliCD (a fusion containing C. difficile flagellins FliC and FliD) effectively protected mice against CDI and significantly decreased C. difficile spores and toxin levels in the feces after infection. 3) CDT consists of CDTa and CDTb. CDTa is the enzymatic component, and CDTb is the binding component. The receptor-binding domain 2 (RBD2) of CDTb is critical for host cell toxicity. We found that parenteral immunizations with RBD2 induced potent immune responses to CDTb and provided mice full protection against a lethal challenge of CDT. In this R01 project, we will investigate immune responses to immunizations with combined protein antigens (Tcd169Fl, FliCD, and RBD2) via intramuscular, sublingual, and intranasal routes or “Protective Immunity Enhanced Salmonella Vaccine (PIESV)” platform expressing these protein antigens via oral route. We will evaluate protection efficacy of these vaccine candidates in animal models of CDI and recurrence to select 2 best vaccine candidates for further clinical trial in the next funding period.