Project 1: Mucosal toxin subunit immunization as a strategy for C. difficile vaccine development

项目 1 摘要 艰难梭菌是一种产芽孢厌氧细菌，是导致以下疾病的主要原因： 美国医院获得性胃肠道感染。 曾经去过的老年人 尽管有报道称，接受广谱抗生素治疗的感染风险最大 年轻人中的社区获得性感染正在增加。临床上，艰难梭菌感染（CDI） 表现为轻度至重度腹泻，并且经常会复发，结果恶化。症状 由艰难梭菌分泌的一种或多种大毒素的活性引起：TcdA、TcdB 和 CDT（二元毒素）。高血清水平的抗 TcdA 抗体与 无症状携带有机体，并且获得性 TcdA 或 TcdB 免疫反应降低 复发的风险。这些数据为开发基于毒素的艰难梭菌提供了理论依据 疫苗。虽然辉瑞公司最近完成的一项使用 TcdA/TcdB 类毒素的临床试验并没有 达到其主要终点，有一些有希望的次要指标表明了机会 以便在下一次迭代中进行改进。项目 1 将定义可引发强健粘膜的毒素抗原 人类和小鼠的免疫反应。我们的首要目标是定义表位 TcdA、TcdB 和 CDT 提供有效且广泛的中和 IgA、分泌型 IgA (sIgA)、 和人类的 IgG 反应（目标 1）， 纳入 TcdB 序列的当前信息 艰难梭菌菌株的多样性。 纯化的单克隆抗体将基于高 对人类临床样本中毒素特异性 B 细胞受体进行通量序列分析。的 将评估抗体的毒素结合和中和作用，以及与毒素相关的表位 广泛、有效的中和将通过冷冻电子显微镜（cryo-EM）来定义。这个目标 最终将产生毒素亚单位，并评估患者的 sIgA 反应 唾液样本。第二个目标将评估 TcdA、TcdB 和 CDT 的变体作为免疫原 以及特定毒素结构域对产生保护性免疫的影响。实验的 工作流程涉及评估直肠潜在益处的系统方法 免疫方法产生针对毒素的强大粘膜免疫力。它将 建立可用于评估确定的毒素亚基和 项目 2 中出现的新型非毒素抗原。最后，促进免疫的策略 持久保护将用于研究保护的相关性，特别是粘膜 T 促进强大而持久的免疫记忆的细胞群。

PROJECT 1 SUMMARY Clostridioides difficile is a spore-forming anaerobic bacterium that is the leading cause of hospital-acquired gastrointestinal infection in the United States. Elderly people who have been treated with broad spectrum antibiotics are at greatest risk for infection, although reports of community-acquired infections in young adults are increasing. Clinically, C. difficile infection (CDI) presents as mild to severe diarrhea and can often recur with worsening outcomes. The symptoms result from the activity of one or more of the large toxins secreted by C. difficile: TcdA, TcdB, and CDT (binary toxin). High serum levels of antibodies against TcdA have been linked to asymptomatic carriage of the organism, and an acquired TcdA or TcdB immune response reduces the risk of recurrence. These data provide the rationale for developing a toxin-based C. difficile vaccine. While a recently completed clinical trial from Pfizer using a TcdA/TcdB toxoid did not meet its primary endpoint, there were promising secondary indicators that suggest opportunities for improvement in the next iteration. Project 1 will define toxin antigens that elicit robust mucosal immune responses in humans and in mice. Our first objective will be to define the epitopes of TcdA, TcdB, and CDT that provide effective and broadly neutralizing IgA, secreted IgA (sIgA), and IgG responses in humans (Aim 1), incorporating current information on TcdB sequence diversity across C. difficile strains. Purified monoclonal antibodies will be produced based on high- throughput sequence analysis of toxin-specific B cell receptors from human clinical samples. The antibodies will be evaluated for toxin binding and neutralization, and the epitopes associated with broad, potent neutralization will be defined using cryo-electron microscopy (cryo-EM). This aim will culminate in the creation of toxin subunits that will be evaluated for sIgA responses in patient saliva samples. The second aim will evaluate variants of TcdA, TcdB, and CDT as immunogens and the impact of specific toxin domains in generating protective immunity. The experimental workflow involves a systematic approach to evaluating the potential benefits of a rectal immunization method in the generation of robust mucosal immunity against the toxins. It will establish an immunization regimen that can be used to evaluate defined toxin subunits and the novel non-toxin antigens that emerge from Project 2. Finally, immunization strategies that promote durable protection will be used to study the correlates of protection, specifically, the mucosal T cell populations that promote robust and durable immunological memory.