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

鉴定针对艰难梭菌感染的克隆抗体反应的抗原靶点

• 批准号: 10742376
• 负责人: LARRY K KOCIOLEK
• 金额: $ 25.72万
• 依托单位: LURIE CHILDREN'S HOSPITAL OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-11 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10742376
• 关键词: Acute; Address; Adult; Affect; Animal Model; Animals; Antibodies; Antibody Response; Antigen Presentation; Antigen Targeting; Antigens; Bacterial Infections; Binding; Biological Assay; Cell Culture Techniques; Cells; Child; Childhood; Classification; Clinical; Clonal Expansion; Cloning; Clostridium difficile; Colitis; Communities; Data; Development; Diarrhea; Enzyme-Linked Immunosorbent Assay; Epitopes; FDA approved; Feces; Flagellin; Future; Goals; Health Expenditures; Healthcare; Human; Human Cloning; Immune; Immune response; Immunization; Immunocompetent; Immunologics; Immunoprecipitation; Immunotherapy; In Vitro; Incidence; Infection; Infection prevention; Investigation; Knowledge; Life; Membrane Proteins; Methods; Molecular Conformation; Monoclonal Antibodies; Morbidity - disease rate; Mucocutaneous Lymph Node Syndrome; Mus; Pathogenesis; Patients; Peptides; Peripheral; Plasma Cells; Plasmablast; Preparation; Prevention; Prevention strategy; Proteins; Public Health; Recovery; Recurrence; Resolution; Surface; Toxin; Transgenic Mice; Vaccination; Vaccines; Vibrio cholerae; Virus Diseases; Western Blotting; Work; acute infection; candidate identification; clinical development; cytotoxicity; experimental study; genome sequencing; healthcare-associated infections; human monoclonal antibodies; immunogenicity; in vivo; infection risk; innovation; mortality; novel; pathogen; peripheral blood; preclinical study; prevent; public health priorities; public health relevance; recurrent infection; response; tandem mass spectrometry; therapeutically effective; transmission process; vaccine strategy; whole genome

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.

项目摘要摘要 梭状芽胞杆菌艰难梭菌感染（CDI）是美国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威胁生命的结肠炎，与 大量的发病率，死亡率和过多的医疗支出。 CDC已将艰难梭菌归类为 “需要紧急行动的立即公共卫生威胁。”免疫学剂是 有望预防CDI的新兴策略；针对艰难梭菌的单克隆抗体最近是FDA 批准用于预防CDI，几种基于毒素的疫苗正在临床开发中。尽管如此 表现出预防CDI的希望的产品，许多患者失败了这些疗法。这些产品是 基于对动物艰难梭菌免疫原性知识的知识，这可能无法充分代表 人类艰难梭菌免疫原性。为了解决这个知识差距，我们提出了 人类天然CDI后的浆膜反应。浆质是产生的浆细胞前体 针对病原体的抗体；可以从外周血1-中分离抗原特异性浆体 感染后2周。这些细胞编码的抗体可以在体外产生，并用于识别 目标抗原。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 通过专门进行以下操作，来自急性艰难梭菌感染的人类的单克隆抗体：（1a） 分离和表征来自16名儿童和成人的单细胞外围浆体后1-2周 急性CDI的发作； （1B）从每个克隆膨胀的浆液中制备单克隆抗体 受试者的plasmablast池。接下来，使用这些抗体，我们将确定艰难梭菌的抗原靶标 特定的人类单克隆抗体通过专门执行以下操作：（2a）执行整个基因组 与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特异性抗体 连接的免疫吸附测定法（ELISA）确定它们在细胞培养细胞毒性中中和毒素中和毒素的能力 测定并通过Pepperide微阵列确定特定的毒素表位； （2C）对于不识别的抗体 毒素A或B，进行ELISA，用于艰难梭菌非毒素抗原鞭毛蛋白（FLIC）和表面的ELISA 层蛋白A（SLPA），并使用每个患者感染制备的表面蛋白制备（SPP） 隔离; （2d）对于与受试者的艰难梭菌属于ELISA结合但其目标仍然存在的抗体 未知，执行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株中，并识别特定的蛋白质串联质谱法。通过这个 克隆人血浆对CDI的响应的创新方法，我们将确定一系列抗原 急性CDI后由人类的体液免疫反应靶向，并开发了一组艰难梭菌 - 特定的人类单克隆抗体靶向各种毒素和非毒素抗原和表位。我们 因此，将确定将为未来的免疫疗法和疫苗策略提供信息的候选人。