Project 2: Discovery of novel C. difficile antigens using genetic and biochemical approaches

项目2：利用遗传和生化方法发现新的艰难梭菌抗原

• 批准号: 10625693
• 负责人: Eric P Skaar
• 金额: $ 35.71万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-03 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625693
• 关键词: Address; Adherence; Adult; Anaerobic Bacteria; Animals; Antibiotic Resistance; Antibiotics; Antibodies; Antigen Targeting; Antigens; Attenuated; B-Lymphocytes; Bacteria; Bacterial Vaccines; Binding; Biochemical; Biotin; Biotinylation; Clinical; Clostridium difficile; Collaborations; Colon; Communities; Diarrhea; Disease; Elderly; Enzyme-Linked Immunosorbent Assay; Epitopes; Evaluation; Genetic; Goals; Hospitals; Human; Immune response; Immune system; Immunization; Immunocompromised Host; Immunoglobulin A; Immunoglobulin G; Incidence; Individual; Infection; Label; Lead; Libraries; Ligase; Link; Location; Mass Spectrum Analysis; Mediating; Membrane Proteins; Memory B-Lymphocyte; Methods; Modeling; Mus; Organism; Output; Patients; Peripheral Blood Mononuclear Cell; Population; Process; Proteins; Pseudomembranous Colitis; Public Health; Recurrence; Recurrent disease; Reproduction spores; Role; Sampling; Serum; Severities; Signal Transduction; Sorting; Surface; Symptoms; Testing; Toxin; Toxoids; United States; Vaccinated; Vaccination; Vaccine Antigen; Vaccines; Validation; Virulent; Zoonoses; adaptive immune response; antigen binding; antitoxin; bactericide; candidate identification; cost; efficacy testing; enteric infection; experimental study; fitness test; gene product; genetic approach; genetic selection; gut colonization; health care settings; immunogenicity; in vivo; innovation; mouse model; mutant; novel; pathogen; pre-clinical; preclinical trial; prevent; programs; resistant strain; response; safety testing; saliva sample; stem; success; transposon sequencing; unvaccinated; vaccine candidate; vaccine discovery; vaccine safety; vaccine trial

PROJECT 2 SUMMARY Clostridioides difficile is a spore-forming anaerobic bacterium that is the number one cause of hospital- acquired diarrhea and pseudomembranous colitis in the United States. The incidence of C. difficile infection (CDI) has been rapidly rising since the 1990s and is linked to increased antibiotic use. The emergence of new highly virulent strains over the past two decades has contributed to CDI cases spreading from elderly and immunocompromised populations in healthcare settings, to community-acquired and zoonotic infections among healthy adults. Although CDI is a toxin-mediated disease, vaccine trials targeting toxoids have failed to produce effective vaccines.There is a concern that even the most effective anti-toxin strategy will not prevent the intestinal colonization of C. difficile. Additional bacterial vaccine targets that promote immune system mediated clearance of both vegetative bacteria and spores are needed. A strong IgA/IgG response to a conserved antigen found on the C. difficile surface will provide the necessary target to promote a bactericidal immune response and/or by blocking bacterial adherence and colonization within the colon. This proposal presents two aims to identify relevant antigens and a third aim to test the new antigens for protection in a mouse immunization model. The first approach is to use a genetic selection in a murine model to identify antigens that are selectively targeted by the adaptive immune response. The second approach will use two biochemical strategies that will both leverage human clinical samples from recovered CDI patients. The first of these biochemical approaches will use a recently developed promiscuous biotin ligase to enzymatically biotinylate C. difficile antigens bound to antibodies from the human sera. The second biochemical approach will sort patient B-cells against fluorescently labeled C. difficile to identify antibodies that target surface proteins on C. difficile. These mABs will be characterized for binding to C. difficile and used for antigen discovery. In both biochemical approaches we will prioritize antigens associated with IgA class antibodies. In the third aim of this project, antigens will be assessed and prioritized. The most promising antigens, based on multiple considerations including conservation across isolates, strength of the identifying signals, and predicted cellular location, will be expressed and purified by Core 2. These antigens will be evaluated for binding patient sera samples by ELISA and the most promising candidates will be evaluated (in collaboration with Core 4) for immunogenicity and their ability to induce a protective immune response in our pre-clinical murine vaccine mode.

项目2摘要 梭状芽胞杆菌艰难梭菌是一种形成孢子的厌氧菌细菌，是医院的第一名 在美国获得了腹泻和假膜结肠炎。艰难梭菌感染的发生率 （CDI）自1990年代以来一直在迅速上升，与抗生素使用的增加有关。新的出现 在过去的二十年中，高毒的菌株已导致CDI病例从老年人和 医疗保健环境中的免疫功能低下的人群，以社区的领域和人畜共患感染 健康的成年人。尽管CDI是毒素介导的疾病，但针对毒素的疫苗试验未能产生 有效的疫苗。担心即使是最有效的抗毒素策略也不会阻止肠道 艰难梭菌的定殖。促进免疫系统介导的清除率的其他细菌疫苗靶标 需要两种营养细菌和孢子。对在 艰难梭菌表面将提供必要的目标，以促进杀菌性免疫反应和/或 阻断结肠内细菌的依从性和定殖。该提案提出了两个旨在确定的目标 相关的抗原和第三个目的是测试新抗原在小鼠免疫模型中的保护。这 第一种方法是在鼠模型中使用遗传选择来识别由 适应性免疫反应。第二种方法将使用两种都将利用的生化策略 来自恢复的CDI患者的人类临床样本。这些生化方法中的第一种将使用 最近开发了滥交生物素连接酶，以酶促生物素基甲酯艰难梭菌抗原与抗体结合 来自人类血清。第二种生化方法将对荧光标记的患者B细胞分类 艰难梭菌以鉴定靶向表面蛋白在艰难梭菌上的抗体。这些mab将被描述为 与艰难梭菌结合并用于抗原发现。在两种生化方法中，我们都将优先考虑抗原 与IgA类抗体相关。在该项目的第三个目标中，将评估和优先级。 最有希望的抗原，基于多种考虑因素，包括跨分离株的保护，强度 识别信号和预测的细胞位置将通过核心2表示和纯化。这些抗原 将通过ELISA评估结合患者血清样品，将评估最有前途的候选人 （与Core 4合作）免疫原性及其在我们的中诱导保护性免疫反应的能力 临床前鼠疫苗模式。