Identification of the antigenic targets of the clonal antibody response to Clostridioides difficile infection

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PROJECT SUMMARY ABSTRACT Clostridioides difficile infection (CDI) is a very common healthcare- and community-associated infection in US children and adults. CDI, which ranges from mild diarrhea to life-threatening colitis, is associated with substantial morbidity, mortality, and excess healthcare expenditures. The CDC has classified C. difficile as an “immediate public health threat that requires urgent and aggressive action.” Immunological agents are a promising emerging strategy for CDI prevention; a monoclonal antibody against C. difficile is recently FDA approved for CDI prevention, and several toxin-based vaccines are in clinical development. Despite these products showing promise for CDI prevention, many patients have failed these therapies. These products were developed based on knowledge of C. difficile immunogenicity in animals, which may not adequately represent C. difficile immunogenicity in humans. To address this knowledge gap, we propose characterization of the plasmablast response following natural CDI in humans. Plasmablasts are plasma cell precursors that produce antibodies directed against a pathogen; antigen-specific plasmablasts can be isolated from peripheral blood 1- 2 weeks after infection. Antibodies encoded by these cells can be produced in vitro and used to identify the targeted antigens. We aim to apply this innovative approach to CDI. First, we will develop a panel of monoclonal antibodies from humans with acute C. difficile infection by specifically doing the following: (1a) Isolate and characterize single cell peripheral plasmablasts from 16 children and adults at 1-2 weeks after onset of acute CDI; and (1b) Prepare monoclonal antibodies from clonally expanded plasmablasts within each subject’s plasmablast pool. Next, using these antibodies, we will identify the antigenic targets of C. difficile- specific human monoclonal antibodies by specifically doing the following: (2a) Perform whole genome sequencing of C. difficile isolated from each subject; (2b) Identify toxin A and B-specific antibodies by enzyme- linked immunosorbent assay (ELISA), determine their ability to neutralize toxin in a cell culture cytotoxicity assay, and identify the specific toxin epitopes by peptide microarray; (2c) For antibodies that do not recognize toxins A or B, perform ELISA for well-characterized C. difficile non-toxin antigens flagellin (FliC) and surface layer protein A (SlpA), and using a surface protein preparation (SPP) prepared from each patient’s infecting isolate; and (2d) For antibodies that bind to the subject’s C. difficile SPP by ELISA but whose target remains unknown, perform western blot (linear epitopes) and immunoprecipitation (conformational epitopes) of a SPP from each subject’s isolate and identify the specific protein my tandem mass spectrometry. Through this innovative approach to clone the human plasmablast response to CDI, we will identify an array of antigens targeted by the human humoral immune response following acute CDI and develop a panel of C. difficile- specific human monoclonal antibodies that target a variety of toxin and non-toxin antigens and epitopes. We will thereby identify candidate antigens that will inform future immunotherapies and vaccine strategies.