Molecular mechanisms of botulinum neurotoxin neutralization

项目摘要 肉毒杆菌中毒是由暴露于梭菌产生的称为肉毒杆菌神经毒素（BoNT）的蛋白质毒素引起的 肉毒杆菌。BoNT是CDC第1层选择代理，目前没有解毒剂。七种不同的BoNT血清型 迄今为止已经发现了BoNT/A-G，其中许多具有许多另外的BoNT亚型。然而，唯一 目前可用的治疗是来自大型动物的基于血清的抗毒素产品， 在肉毒杆菌毒素中毒后立即注射开发BoNT疗法的挑战因以下事实而加剧： 七种已知的BoNT血清型各自是具有不同受体特异性的不同毒素， SNARE蛋白上的不同位点来破坏神经传递。由于风险的严重性，治疗的缺乏 选择，以及挑战的复杂性，预防和治疗BoNT中毒的新方法是 显然需要。我们现在在多种毒素模型中有广泛的证据表明，基于VHH的双特异性免疫抑制剂可以抑制细胞内的毒素。 中和剂（VNA）是由两个共价连接的毒素中和VHH组成的具有效力的抗毒素 其通常超过目前单克隆和多克隆抗毒素剂。此外，VNA提供了大量 优于血清和mAb抗毒素产品，因为它们生产经济且用途广泛; 针对毒素暴露和产毒病原体感染的创新性新预防和治疗战略 例如基因治疗和直接递送到肠道和肺部攻击部位。在本提案中，我们测试 将结构和机械信息整合到VNA设计中将导致更大的抗毒素的假设 功效和多功能性。具体目的是（1）确定在细胞中选择的BoNT结合VHH的晶体结构。 与其靶向BoNT复合;（2）定义目标1中选择的VHH阻断BoNT毒性的机制， 和（3）通过利用来自以下的结构/功能数据，设计和测试具有增强的抗毒素性质的双特异性VNA： 目标1和2。这将是BoNT中和表位的第一个全面的结构图谱， 补充BoNT功能和BoNT-宿主相互作用的机制研究。此外，本研究将 提高对结构和机械信息如何为设计提供更多信息的一般理解 有效VNA抗毒素剂，并应允许快速开发商业抗毒素治疗剂， 暴露于所有BoNT血清型和其他毒素生物威胁剂。

Project summary Botulism is caused by exposure to protein toxins called botulinum neurotoxins (BoNTs) that are produced by Clostridium botulinum. BoNTs are CDC Tier 1 select agent for which no antidote currently exists. Seven different BoNT serotypes have been discovered to date (BoNT/A-G), many having numerous additional BoNT subtypes. However the only currently available treatments are serum based antitoxin products derived from large animals that are only effective if administered soon after BoNT intoxication. The challenge of developing BoNT therapeutics is exacerbated by the fact that the seven known BoNT serotypes are each distinct toxins with distinct receptor specificities and proteases that cleave at distinct sites on SNARE proteins to disrupt nerve transmission. Due to the severity of the risk, the paucity of treatment options, and the complexity of the challenge, novel approaches to the prevention and treatment of BoNT intoxication are clearly needed. We now have extensive evidence in multiple toxin models demonstrating that bispecific VHH-based neutralizing agents (VNAs), consisting of two covalently linked, toxin-neutralizing VHHs, are antitoxins with potencies that often exceed that of current monoclonal and polyclonal antitoxin agents. Furthermore, VNAs offer substantial advantages over serum and mAb antitoxin products as they are economical to produce and highly versatile; offering innovative new prevention and treatment strategies for toxin exposures and infections with toxin-producing pathogens such as gene therapies and direct delivery to enteric and pulmonary sites of challenge. In this proposal, we test the hypothesis that integrating structural and mechanistic information into VNA design will lead to even greater antitoxin efficacy and versatility. The Specific Aims are to (1) determine the crystal structures of selected BoNT-binding VHHs in complex with their target BoNTs; (2) define the mechanisms by which VHHs selected in Aim 1 block BoNT toxicity, and; (3) design and test bispecific VNAs with enhanced antitoxin properties by exploiting structure/function data from Aims 1 and 2. This will be the first comprehensive structural mapping of BoNT neutralizing epitopes, which will be complemented with mechanistic studies of BoNT function and BoNT-host interactions. Furthermore, this study will improve general understanding of how structural and mechanistic information can inform the design of even more effective VNA antitoxin agents and should permit rapid development of commercial antitoxin therapeutics to treat exposures to all BoNT serotypes and other toxin biothreat agents.