Project 2: Discovery of novel C. difficile antigens using genetic and biochemical approaches

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PROJECT 2 SUMMARY Clostridioides difficile is a spore-forming anaerobic bacterium that is the number one cause of hospital- acquired diarrhea and pseudomembranous colitis in the United States. The incidence of C. difficile infection (CDI) has been rapidly rising since the 1990s and is linked to increased antibiotic use. The emergence of new highly virulent strains over the past two decades has contributed to CDI cases spreading from elderly and immunocompromised populations in healthcare settings, to community-acquired and zoonotic infections among healthy adults. Although CDI is a toxin-mediated disease, vaccine trials targeting toxoids have failed to produce effective vaccines.There is a concern that even the most effective anti-toxin strategy will not prevent the intestinal colonization of C. difficile. Additional bacterial vaccine targets that promote immune system mediated clearance of both vegetative bacteria and spores are needed. A strong IgA/IgG response to a conserved antigen found on the C. difficile surface will provide the necessary target to promote a bactericidal immune response and/or by blocking bacterial adherence and colonization within the colon. This proposal presents two aims to identify relevant antigens and a third aim to test the new antigens for protection in a mouse immunization model. The first approach is to use a genetic selection in a murine model to identify antigens that are selectively targeted by the adaptive immune response. The second approach will use two biochemical strategies that will both leverage human clinical samples from recovered CDI patients. The first of these biochemical approaches will use a recently developed promiscuous biotin ligase to enzymatically biotinylate C. difficile antigens bound to antibodies from the human sera. The second biochemical approach will sort patient B-cells against fluorescently labeled C. difficile to identify antibodies that target surface proteins on C. difficile. These mABs will be characterized for binding to C. difficile and used for antigen discovery. In both biochemical approaches we will prioritize antigens associated with IgA class antibodies. In the third aim of this project, antigens will be assessed and prioritized. The most promising antigens, based on multiple considerations including conservation across isolates, strength of the identifying signals, and predicted cellular location, will be expressed and purified by Core 2. These antigens will be evaluated for binding patient sera samples by ELISA and the most promising candidates will be evaluated (in collaboration with Core 4) for immunogenicity and their ability to induce a protective immune response in our pre-clinical murine vaccine mode.