Project 1: Mucosal toxin subunit immunization as a strategy for C. difficile vaccine development

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PROJECT 1 SUMMARY Clostridioides difficile is a spore-forming anaerobic bacterium that is the leading cause of hospital-acquired gastrointestinal infection in the United States. Elderly people who have been treated with broad spectrum antibiotics are at greatest risk for infection, although reports of community-acquired infections in young adults are increasing. Clinically, C. difficile infection (CDI) presents as mild to severe diarrhea and can often recur with worsening outcomes. The symptoms result from the activity of one or more of the large toxins secreted by C. difficile: TcdA, TcdB, and CDT (binary toxin). High serum levels of antibodies against TcdA have been linked to asymptomatic carriage of the organism, and an acquired TcdA or TcdB immune response reduces the risk of recurrence. These data provide the rationale for developing a toxin-based C. difficile vaccine. While a recently completed clinical trial from Pfizer using a TcdA/TcdB toxoid did not meet its primary endpoint, there were promising secondary indicators that suggest opportunities for improvement in the next iteration. Project 1 will define toxin antigens that elicit robust mucosal immune responses in humans and in mice. Our first objective will be to define the epitopes of TcdA, TcdB, and CDT that provide effective and broadly neutralizing IgA, secreted IgA (sIgA), and IgG responses in humans (Aim 1), incorporating current information on TcdB sequence diversity across C. difficile strains. Purified monoclonal antibodies will be produced based on high- throughput sequence analysis of toxin-specific B cell receptors from human clinical samples. The antibodies will be evaluated for toxin binding and neutralization, and the epitopes associated with broad, potent neutralization will be defined using cryo-electron microscopy (cryo-EM). This aim will culminate in the creation of toxin subunits that will be evaluated for sIgA responses in patient saliva samples. The second aim will evaluate variants of TcdA, TcdB, and CDT as immunogens and the impact of specific toxin domains in generating protective immunity. The experimental workflow involves a systematic approach to evaluating the potential benefits of a rectal immunization method in the generation of robust mucosal immunity against the toxins. It will establish an immunization regimen that can be used to evaluate defined toxin subunits and the novel non-toxin antigens that emerge from Project 2. Finally, immunization strategies that promote durable protection will be used to study the correlates of protection, specifically, the mucosal T cell populations that promote robust and durable immunological memory.