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

项目总结摘要 艰难梭状芽胞杆菌感染(CDI)在美国是一种非常常见的医疗保健和社区相关感染 无论是孩子还是成年人。CDI的范围从轻度腹泻到危及生命的结肠炎，与 严重的发病率、死亡率和超额的医疗费用。疾控中心已将艰难梭菌列为 “迫在眉睫的公共卫生威胁需要采取紧急和积极的行动。”免疫学药物是一种 前景看好的预防CDI的新策略；一种针对艰难梭菌的单抗最近被FDA 被批准用于预防CDI，几种毒素疫苗正在临床开发中。尽管如此 产品显示出预防CDI的希望，但许多患者未能通过这些治疗。这些产品是 根据艰难梭菌在动物中的免疫原性知识开发的，这可能不足以代表 艰难梭菌在人类中的免疫原性。为了解决这一知识鸿沟，我们建议对 人类自然CDI后的浆母细胞反应。浆母细胞是浆细胞前体细胞，它能产生 针对病原体的抗体；抗原特异的浆母细胞可以从外周血中分离出来 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识别 靶向抗原。我们的目标是将这种创新方法应用于CDI。首先，我们将开发一个小组 来自急性艰难梭菌感染的人的单抗，具体如下：(1a) 16例1-2周龄儿童和成人外周血单细胞浆母细胞的分离和鉴定 急性CDI的发病；以及(1b)从每个克隆内扩增的浆母细胞中制备单抗 受试者的浆母细胞池。接下来，使用这些抗体，我们将识别艰难梭菌的抗原靶标- 特定的人类单抗通过以下方式进行：(2)执行全基因组 从每个受试者分离的艰难梭菌的测序；(2b)通过酶-鉴定毒素A和B的特异性抗体- 联用免疫吸附试验(ELISA)，确定它们中和细胞培养中毒素的能力 检测，并通过肽微阵列鉴定特定的毒素表位；(2c)对于不识别的抗体 毒素A或B，对艰难梭菌的非毒素抗原鞭毛蛋白(Flc)和表面进行酶联免疫吸附试验 A层蛋白(SLpA)，并使用从每个患者的感染中制备的表面蛋白制剂(SPP 分离；以及(2d)通过ELISA与受试者的艰难梭菌SPP结合但其靶标仍然存在的抗体 未知，进行蛋白质印迹(线性表位)和免疫沉淀(构象表位) 从每个受试者身上分离并鉴定出我串联质谱仪中的特定蛋白质。通过这件事 克隆人成浆细胞对CDI的反应的创新方法，我们将鉴定一系列抗原 以急性CDI后的人类体液免疫反应为靶点，并开发出一组艰难梭菌- 针对各种毒素和非毒素抗原和表位的特定人类单抗。我们 从而确定将为未来的免疫疗法和疫苗战略提供信息的候选抗原。

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.