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概要 艰难梭菌是一种能形成孢子的厌氧菌，是导致住院的头号原因。 获得性腹泻和伪膜性结肠炎。C.菌感染 (CDI)自20世纪90年代以来一直在迅速上升，并与抗生素使用的增加有关。出现新 在过去的二十年里，高毒力菌株导致了CDI病例从老年人和 免疫功能低下的人群在医疗机构中，社区获得性和人畜共患感染， 健康成人虽然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 人们担心，即使是最有效的抗毒素策略也不能预防肠道疾病。 梭菌建群很难促进免疫系统介导清除的其他细菌疫苗靶标 细菌和孢子的繁殖体。一种强伊加/IgG反应的保守抗原发现， 梭艰难梭菌表面将提供必要的靶标以促进杀菌免疫应答和/或 阻止结肠内的细菌粘附和定植。该提案提出了两个目标，以确定 第三个目的是测试新抗原在小鼠免疫模型中的保护作用。的 第一种方法是在鼠模型中使用遗传选择来鉴定被选择性靶向的抗原， 适应性免疫反应第二种方法将使用两种生化策略， 来自恢复的CDI患者的人临床样品。这些生化方法中的第一种将使用 最近开发了混杂生物素连接酶以酶促生物素化C。与抗体结合的艰难抗原 从人类血清中分离出来第二种生化方法将根据荧光标记的 C.很难识别靶向C.很难这些mAB将被表征为 与C. difficile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 与伊加类抗体有关。在本项目的第三个目标中，将对抗原进行评估和优先排序。 最有前途的抗原，基于多种考虑，包括跨分离株的保守性，强度 的识别信号，和预测的细胞位置，将表达和核心2纯化。这些抗原 将通过ELISA评价与患者血清样品的结合，并评价最有希望的候选物 (in与核心4）的免疫原性和他们的能力，以诱导保护性免疫反应，在我们的 临床前鼠疫苗模式。