Developing broad-spectrum therapeutics against C. difficile toxins

项目摘要 艰难梭菌（C.艰难梭菌）是一种主要的机会致病菌，当正常肠道菌群 微生物群被破坏。大蛋白毒素TcdB是导致疾病的主要毒力因子 与C.艰难梭菌感染（CDI）。然而，开发中和性单克隆抗体的先前努力 并且针对TcdB的疫苗产生了意想不到的低效力或甚至失败。我们相信一把钥匙 这些先前研究的弱点可能是临床上所见毒素变异的复杂性。虽然单个 来自参考菌株的毒素序列已广泛用于所有先前的治疗开发中， 近年来，对临床分离株中的TcdB进行测序已经揭示了越来越多的C.艰难菌株也是如此 毒素序列的变异这可能是唯一的FDA- 批准的单克隆抗体bezlotoxumab，针对一些TcdB变体，例如由 高毒力菌株（核糖体型027）。序列变异和毒素的大尺寸（~270 kDa）也构成了 使用传统的类毒素方法开发有效的疫苗面临着巨大的挑战。基于我们最近 本文综述了近年来在毒素受体鉴定和TcdB结构与功能研究方面的进展， 建议开发基于受体诱饵的治疗性蛋白质作为广谱抗毒素和一种新的 以表位为重点的片段为基础的疫苗，可以提供有效的保护， 已知的TcdB变体。卷曲蛋白（FZD）和CSPG 4是TcdB的两种主要宿主受体， 我们先前已经揭示了TcdB识别FZD的机制。该项目的第一个目标是 建立TcdB与CSPG 4结合的结构理解。我们的第二个目标将集中在设计和 一个双特异性受体诱饵蛋白家族的表征，该家族由优化的TcdB- CSPG 4和FZD的结合片段。在第三个目标中，我们将利用我们对 TcdB全毒素、TcdB-抗体复合物和TcdB-受体复合物的结构，以设计候选 基于所选择的高度保守和功能关键的TcdB片段的疫苗。该项目是建立在 Jin实验室和Dong实验室之间长期的富有成效的合作，结合他们的高度 在结构生物学和蛋白质工程（Jin实验室）和TcdB受体/CDI方面的互补专业知识 发病机制/动物模型（Dong实验室）。该项目的成功完成将提供 用于免疫预防性治疗的抗毒素和提供预防和 持久的保护。

Project Summary Clostridioides difficile (C. difficile) is a major opportunistic pathogen that colonizes the colon when normal gut microbiota is disrupted. The large protein toxin TcdB is a major virulence factor responsible for diseases associated with C. difficile infection (CDI). However, prior efforts to develop neutralizing monoclonal antibodies and vaccines against TcdB have yielded unexpectedly low efficacy or even failure. We believe that a key weakness of these previous studies might be the complexity of toxin variations seen clinically. While a single toxin sequence from a reference strain has been widely used in all previous therapeutic development, sequencing TcdB in clinical isolates in recent years has revealed a growing number of C. difficile strains as well as variations in toxin sequences. This may account for the reduced neutralization efficacy of the only FDA- approved monoclonal antibody, bezlotoxumab, against some TcdB variants such as the one produced by a hypervirulent strain (ribotype 027). The sequence variation and the toxin’s large size (~270 kDa) also pose daunting challenges to develop effective vaccines using the traditional toxoid approach. Building on our recent progress in identification of toxin receptors and understanding the structure and function of TcdB, here we propose to develop receptor-decoy-based therapeutic proteins as broad-spectrum antitoxins and a new generation of epitope-focused fragment-based vaccines, which could provide effective protection against most of the known TcdB variants. Frizzled proteins (FZDs) and CSPG4 are two major host receptors for TcdB, and we previously have revealed the mechanism by which TcdB recognizes FZDs. The first aim in this project is to establish a structural understanding of TcdB binding to CSPG4. Our second aim will focus on design and characterization of a family of bi-specific receptor-decoy proteins, which are composed of the optimized TcdB- binding fragments of CSPG4 and FZDs. In the third aim, we will take advantage of our knowledge of the structures of TcdB holotoxin, TcdB–antibody complexes, and TcdB–receptor complexes to design candidate vaccines based on the selected highly conserved and functionally critical TcdB fragments. This project is built on long-standing productive collaborations between the Jin lab and the Dong lab, combining their highly complementary expertise in structural biology and protein engineering (Jin lab) and TcdB receptors/CDI pathogenesis/animal models (Dong lab). Successful completion of this project will provide prototypes of antitoxins for immunoprophylactic therapy and broad-spectrum candidate vaccines that offer prophylactic and long-lasting protection.