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

项目2总结 艰难梭状芽胞杆菌是一种形成孢子的厌氧细菌，是医院的头号原因。 美国的获得性腹泻和伪膜性结肠炎。艰难梭菌感染的发生率 自20世纪90年代以来，CDI一直在迅速上升，并与抗生素使用的增加有关。新事物的出现 在过去20年中，高毒力菌株导致CDI病例从老年人和 到社区获得性和人畜共患传染病 健康的成年人。尽管CDI是一种毒素介导的疾病，但针对类毒素的疫苗试验未能产生 有效的疫苗。人们担心，即使是最有效的抗毒素策略也不能预防肠道 艰难梭菌的定植。促进免疫系统介导清除的额外细菌疫苗靶点 营养细菌和孢子都是必需的。对发现的一种保守抗原产生强烈的IgA/IgG反应 艰难梭菌表面将提供必要的靶点以促进杀菌免疫反应和/或通过 阻止细菌在结肠内的黏附和定植。这项提案提出了两个目标，以确定 第三个目标是测试新的抗原，以便在小鼠免疫模型中进行保护。这个 第一种方法是在小鼠模型中使用基因选择来识别选择性靶向的抗原 适应性免疫反应。第二种方法将使用两种生化策略，这两种策略都将利用 CDI康复者的临床样本。这些生化方法中的第一种将使用 新近开发的混杂生物素连接酶，用于酶促艰难梭菌抗原与抗体结合 从人类血清中分离出来。第二种生化方法将对患者B细胞进行荧光标记 艰难梭菌识别针对艰难梭菌表面蛋白的抗体。这些单抗的特征将是 与艰难梭菌结合并用于抗原发现。在这两种生化方法中，我们将优先考虑抗原 与IgA类抗体有关。在该项目的第三个目标中，将对抗原进行评估并确定优先顺序。 最有希望的抗原，基于多种考虑，包括跨分离株的保守性，强度 识别信号和预测的细胞位置，将由核心2表达和纯化。这些抗原 将通过酶联免疫吸附试验评估结合患者血清样本，并将评估最有希望的候选人 (与Core 4合作)的免疫原性及其诱导保护性免疫反应的能力 临床前小鼠疫苗模式。

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.