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

项目1概要 艰难梭菌是一种形成孢子的厌氧细菌， 医院获得性胃肠道感染在美国。 的老年人 使用广谱抗生素治疗的患者感染的风险最大，尽管有报告称， 年轻人的社区获得性感染正在增加。临床上，C。艰难梭菌感染 表现为轻度至重度腹泻，经常复发，结果恶化。症状 由C.分泌的一种或多种大毒素的活性引起。艰难梭菌：TcdA、TcdB和 CDT（二元毒素）。高血清水平的抗TcdA抗体与 无症状携带生物体，并且获得性TcdA或TcdB免疫应答降低 复发的风险。这些数据为开发基于毒素的C.艰难 疫苗虽然辉瑞最近完成的一项使用TcdA/TcdB类毒素的临床试验没有 在达到主要终点后，有一些有希望的次要指标表明， 在下一次迭代中进行改进。项目1将确定毒素抗原， 在人类和小鼠中的免疫反应。我们的第一个目标是确定 TcdA、TcdB和CDT，其提供有效和广泛中和的IgA、分泌型IgA（sIgA）， 和人的IgG应答（目标1）， 结合TcdB序列的当前信息 C的多样性。艰难菌株 纯化的单克隆抗体将基于高- 来自人临床样品的毒素特异性B细胞受体的通量序列分析。的 将评估抗体的毒素结合和中和，以及与 将使用冷冻电子显微镜（cryo-EM）来定义广泛、有效的中和作用。这一目标 将最终产生毒素亚单位，这些亚单位将用于评估患者中的sIgA应答。 唾液样本第二个目标是评估TcdA、TcdB和CDT的变体作为免疫原 以及特定毒素结构域在产生保护性免疫中的影响。实验 工作流程涉及一种系统的方法来评估直肠癌的潜在益处， 免疫方法在产生针对毒素的稳健粘膜免疫中的应用。它将 建立可用于评估确定的毒素亚单位的免疫方案， 项目2中出现的新型非毒素抗原。最后，免疫战略， 持久保护将用于研究保护的相关性，特别是粘膜T 细胞群，促进强大和持久的免疫记忆。

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.