Developing broad-spectrum therapeutics against C. difficile toxins

开发针对艰难梭菌毒素的广谱疗法

• 批准号: 10548826
• 负责人: Rongsheng Jin
• 金额: $ 77.29万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-11 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10548826
• 关键词: Address; Affinity; Animal Model; Animals; Antibiotic Therapy; Antibiotics; Antibodies; Antigens; Binding; Binding Sites; Biology; CSPG4 gene; Cells; Clinical; Clostridium difficile; Collaborations; Colon; Complex; Cryoelectron Microscopy; Developed Countries; Disease; Environment; Epitopes; Escape Mutant; Extracellular Domain; FDA approved; Face; Failure; Family; Formaldehyde; Generations; Goals; Healthcare; Hospitalization; Human; Immune response; Immunize; In Vitro; Infection; Knowledge; Mediating; Membrane; Molecular; Monoclonal Antibodies; Mus; North America; Passive Immunotherapy; Pathogenesis; Patients; Phase; Phase I/II Trial; Prevention; Productivity; Protein Engineering; Proteins; Recovery; Recurrence; Recurrent disease; Relapse; Reproduction spores; Resistance; Ribotypes; Site; Speed; Structural Protein; Structure; Therapeutic; Toxin; Toxoids; Vaccines; Variant; Virulence Factors; antibody mimetics; antitoxin; clinical sequencing; design; gastrointestinal infection; gut microbiota; holotoxins; in vivo; infection risk; neutralizing antibody; neutralizing monoclonal antibodies; neutralizing vaccine; novel strategies; novel vaccines; opportunistic pathogen; pathogenic bacteria; patient population; prevent; prophylactic; protein complex; prototype; rational design; receptor; receptor binding; seal; standard of care; structural biology; therapeutic development; therapeutic protein; therapeutically effective; vaccine candidate; vaccine development; vaccine strategy

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.

项目概要 艰难梭菌 (C. difficile) 是一种主要的机会性病原体，当肠道正常时，它会在结肠中定殖。 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。 然而，之前开发中和单克隆抗体的努力。 我们认为，针对 TcdB 的疫苗疗效出乎意料地低，甚至失败。 先前这些研究的弱点可能是临床上看到的毒素变异的复杂性。 来自参考菌株的毒素序列已广泛用于之前的所有治疗开发中， 近年来，对临床分离株中的 TcdB 进行测序也发现了越来越多的艰难梭菌菌株 由于毒素序列的变化，这可能是 FDA 唯一的中和功效降低的原因。 批准的单克隆抗体 bezlotoxumab，针对某些 TcdB 变体，例如由 高毒力菌株（核糖体型 027）也构成了序列变异和毒素的大尺寸（~270 kDa）。 在我们最近的基础上，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。 毒素受体鉴定和TcdB结构与功能了解的进展，在这里我们 提议开发基于受体诱饵的治疗蛋白作为广谱抗毒素和新的 产生基于表位的片段疫苗，可以针对大多数疾病提供有效的保护 已知的 TcdB 变体（FZD）和 CSPG4 是 TcdB 的两个主要宿主受体，并且 我们之前已经揭示了 TcdB 识别 FZD 的机制。该项目的首要目标是： 建立 TcdB 与 CSPG4 结合的结构理解我们的第二个目标将侧重于设计和 双特异性受体诱饵蛋白家族的表征，该家族由优化的 TcdB- 组成 CSPG4 和 FZD 的结合片段 在第三个目标中，我们将利用我们的知识。 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物 该项目是基于选定的高度保守且功能关键的 TcdB 片段构建的疫苗。 金实验室和董实验室之间长期富有成效的合作，结合了他们的高度 结构生物学和蛋白质工程（金实验室）和 TcdB 受体/CDI 方面的互补专业知识 发病机制/动物模型（董实验室）的成功完成将提供原型。 用于免疫预防治疗的抗毒素和提供预防和预防作用的广谱候选疫苗 持久的保护。