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.

项目概要摘要 艰难梭菌感染 (CDI) 是美国一种非常常见的医疗保健和社区相关感染 儿童和成人的 CDI 范围从轻度腹泻到危及生命的结肠炎，与 疾病预防控制中心 (CDC) 将艰难梭菌列为一种严重的发病率、死亡率和过度医疗支出。 “需要采取紧急和积极行动的直接公共卫生威胁。” FDA 最近批准了一种有前途的 CDI 预防新策略；一种针对艰难梭菌的单克隆抗体 尽管如此，一些基于毒素的疫苗仍在临床开发中。 尽管这些产品显示出预防 CDI 的希望，但许多患者的治疗效果不佳。 基于对动物中艰难梭菌免疫原性的了解而开发，这可能不足以充分代表 为了解决这一知识差距，我们提出了艰难梭菌在人类中的免疫原性特征。 人类天然 CDI 后的浆母细胞反应 浆母细胞是产生浆细胞的前体。 针对病原体的抗体；可以从外周血中分离出抗原特异性浆母细胞1- 感染后2周，这些细胞编码的抗体可以在体外产生并用于鉴定。 我们的目标是将这种创新方法应用于 CDI，首先，我们将开发一组抗原。 通过具体执行以下操作，从患有急性艰难梭菌感染的人中获得单克隆抗体：(1a) 术后 1-2 周从 16 名儿童和成人中分离和表征单细胞外周浆母细胞 急性 CDI 发作；和 (1b) 从每个细胞内克隆扩增的浆母细胞制备单克隆抗体 接下来，使用这些抗体，我们将识别艰难梭菌的抗原靶标。 通过具体执行以下操作来制备特定的人单克隆抗体： (2a) 执行全基因组分析 对从每个受试者分离的艰难梭菌进行测序；(2b)通过酶-鉴定毒素A和B特异性抗体 连锁免疫吸附测定 (ELISA)，确定其中和细胞培养物中毒素的能力 测定，并通过肽微阵列识别特定的毒素表位（2c）对于不识别的抗体； 毒素 A 或 B，对已充分表征的艰难梭菌非毒素抗原鞭毛蛋白 (FliC) 和表面进行 ELISA 层蛋白 A (SlpA)，并使用从每位患者的感染细胞中制备的表面蛋白制剂 (SPP) (2d) 对于通过 ELISA 与受试者的艰难梭菌 SPP 结合但其目标仍然存在的抗体 未知，对 SPP 进行蛋白质印迹（线性表位）和免疫沉淀（构象表位） 通过串联质谱法从每个受试者的分离物中鉴定出特定的蛋白质。 克隆人类浆母细胞对 CDI 反应的创新方法，我们将鉴定一系列抗原 急性 CDI 后人类体液免疫反应的目标，并开发了一组艰难梭菌- 针对多种毒素和非毒素抗原和表位的特异性人单克隆抗体。 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。