Molecular mechanisms of botulinum neurotoxin neutralization

项目总结 肉毒杆菌中毒是由于接触到由梭状芽胞杆菌产生的称为肉毒杆菌神经毒素(BoNTs)的蛋白质毒素引起的 肉毒杆菌。BoNTs是CDC Tier 1选择剂，目前还没有解毒剂。七种不同的BONT血清型 到目前为止已发现(BONT/A-G)，许多具有许多额外的BONT亚型。然而，唯一的 目前可用的治疗方法是从大型动物中提取的血清抗毒素产品，只有在以下情况下才有效 在邦特中毒后不久给药。开发BONT疗法的挑战因以下事实而加剧 已知的七种BONT血清型都是不同的毒素，具有不同的受体特异性和切割酶 SNARE蛋白上的不同位置扰乱了神经传递。由于风险的严重性，治疗的匮乏 各种选择和挑战的复杂性，预防和治疗BONT中毒的新方法是 显然是有必要的。我们现在有多种毒素模型的广泛证据表明，基于双特异性VHH的 中和剂(VNAs)由两个共价连接的毒素中和VHH组成，是一种具有效力的抗毒素 这往往超过目前的单克隆性和多克隆性抗毒素药物。此外，VNA提供了大量的 相对于血清和单抗抗毒素产品的优势，因为它们生产成本低，通用性强；提供 创新的毒素暴露和产生毒素病原体感染的预防和治疗策略 例如基因疗法和直接输送到肠道和肺部具有挑战性的部位。在本提案中，我们测试 假设将结构和机械信息整合到VNA设计中将导致更强的抗毒素 功效和多功能性。具体目的是：(1)确定所选择的BONT结合的VHH的晶体结构 与其靶标BoNT的络合物；(2)定义在Aim 1中选择的VHH阻断BONT毒性的机制， 以及；(3)通过利用以下结构/功能数据设计和测试具有增强抗毒素特性的双特异性VNAs 目标1和2。这将是第一次全面绘制BONT中和表位的结构图，这将是 辅以对BONT功能和BONT-宿主相互作用的机制研究。此外，这项研究将 提高对结构和机械信息如何为设计提供更多信息的总体理解 有效的VNA抗毒素药物，应允许商业化抗毒素治疗药物的快速发展 暴露于所有BONT血清型和其他毒素生物制剂。

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.