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

项目摘要 在美国，艰难梭菌感染（CDI）是一种非常常见的医疗保健和社区相关感染 儿童和成人。CDI的范围从轻度腹泻到危及生命的结肠炎， 严重的发病率、死亡率和过度的医疗支出。疾控中心将其归类为C类。difficile作为一个 “需要采取紧急和积极行动的直接公共卫生威胁”。免疫制剂是一种 有希望的CDI预防新策略;抗C. FDA最近 已批准用于预防CDI，并且几种基于毒素的疫苗正在临床开发中。尽管有这些 虽然这些产品显示出预防CDI的前景，但许多患者在这些治疗中失败了。这些产品 基于C.在动物中的免疫原性，这可能不足以代表 C.免疫原性。为了解决这一知识差距，我们提出了表征 人中天然CDI后的浆母细胞应答。浆母细胞是浆细胞前体， 针对病原体的抗体;抗原特异性浆母细胞可以从外周血1- 感染后2周。由这些细胞编码的抗体可以在体外产生并用于鉴定免疫原性。 靶向抗原。我们的目标是将这种创新方法应用于CDI。首先，我们将建立一个小组， 从患有急性C.通过具体执行以下操作来治疗艰难感染：（1a） 分离和表征16名儿童和成人在1-2周后的单细胞外周浆母细胞 急性CDI的发作;和（1b）从每个CDI内的克隆扩增的浆母细胞制备单克隆抗体。 受试者的浆母细胞池。接下来，我们将利用这些抗体来鉴定C.艰难的，艰难的 通过特异性地进行以下操作来制备特异性人单克隆抗体：（2a）进行全基因组测序， 测序C.（2b）通过酶联免疫吸附试验鉴定毒素A和B特异性抗体; 连接免疫吸附试验（ELISA），测定其中和毒素的能力，在细胞培养中的细胞毒性 （2c）对于不能识别的抗体， 毒素A或B，对充分表征的C进行ELISA。艰难梭菌非毒素抗原鞭毛蛋白（FliC）和表面 层蛋白A（SlpA），并使用由每个患者的感染制备的表面蛋白制剂（SPP）， 分离的;和（2d）对于结合受试者的C.通过ELISA检测艰难梭菌SPP，但其靶标仍然存在 未知，进行SPP的蛋白质印迹（线性表位）和免疫沉淀（构象表位） 从每个受试者的分离物中提取并通过串联质谱法鉴定出特定的蛋白质。通过这个 创新的方法来克隆人浆母细胞对CDI的反应，我们将确定一系列抗原 在急性CDI后被人体液免疫应答靶向，并产生一组C.艰难的，艰难的 靶向多种毒素和非毒素抗原和表位的特异性人单克隆抗体。我们 从而确定候选抗原，为未来的免疫疗法和疫苗策略提供信息。

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.