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

项目1总结 艰难梭状芽胞杆菌是一种形成芽胞的厌氧细菌，是引起 美国的医院获得性胃肠道感染。 曾经经历过的老年人 使用广谱抗生素治疗的感染风险最大，尽管有报道称 年轻人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，经常复发，结果恶化。症状 由艰难梭菌分泌的一种或多种大型毒素TcdA、TcdB和 CDT(二进制毒素)。血清中高水平的抗TcdA抗体与 无症状的生物体携带，获得性TcdA或TcdB免疫反应减少 复发的风险。这些数据为开发以毒素为基础的艰难梭菌提供了理论基础。 疫苗。而辉瑞最近完成的一项使用TcdA/TcdB类毒素的临床试验并没有 达到其主要终点时，有一些很有希望的次要指标表明了机会 在下一次迭代中进行改进。项目1将定义毒素抗原，以诱导强大的粘膜 人类和小鼠的免疫反应。我们的第一个目标将是定义 TcdA、TcdB和CDT，提供有效和广泛的中和IgA、分泌型IgA(SIgA)， 和人类的免疫球蛋白反应(目标1)， 合并有关Tcdb序列的当前信息 艰难梭菌菌株间的多样性。 纯化的单抗将基于高纯度的 人类临床样本中毒素特异性B细胞受体的吞噬序列分析。这个 将对抗体进行毒素结合和中和评估，以及与 广泛、有效的中和将使用低温电子显微镜(CRYO-EM)来定义。这一目标 将最终产生毒素亚基，用于评估患者的SIgA反应 唾液样本。第二个目标是评估TcdA、TcdB和CDT的变种作为免疫原 以及特定毒素结构域在产生保护性免疫中的影响。实验性的 工作流程涉及一种评估直肠手术潜在益处的系统方法 免疫方法在产生对毒素的强健黏膜免疫力。会的 建立可用于评估定义的毒素亚基和 从项目2中产生的新的无毒素抗原。最后，促进 持久保护将用于研究保护的相关性，特别是粘膜T 促进强健和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.