Nanobodies against C. difficile TcdA and TcdB reveal unexpected neutralizing epitopes and provide a toolkit for toxin quantitation in vivo

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo. Author SummaryC. difficile (C. diff) is a leading cause of diarrhea and is recognized as an urgent threat by the Centers for Disease Control. Disease symptoms are caused by two large, similar, protein toxins, TcdA and TcdB. These toxins are drug targets and are also important for diagnosis. Despite their clear importance, the understanding of how to neutralize toxin activity is incomplete, and there are no freely available tools to quantify toxin concentration in research studies. To address these issues, we have developed nanobodies that bind and neutralize TcdA and TcdB and have also used these nanobodies to develop quantitative assays for TcdA and TcdB detection. Neutralization studies led us to discover that many of the potent neutralizers of TcdA bind epitopes within the delivery domain. This finding suggests either a role for the delivery domain in receptor binding or that the nanobodies block pore-formation and thereby inhibit delivery of the toxin enzyme domains to the cytosol. The availability of nanobody assays that can differentiate the quantities of TcdA from TcdB should permit a better understanding of toxin-specific effects and how toxin levels change over the course of infection.