Molecular mechanisms of botulinum neurotoxin neutralization

肉毒杆菌神经毒素中和的分子机制

• 批准号: 9918242
• 负责人: Rongsheng Jin
• 金额: $ 56.75万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-10 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918242
• 关键词: Affinity; Animals; Antidotes; Bacterial Toxins; Binding; Binding Sites; Bontoxilysin; Botulism; Cell surface; Centers for Disease Control and Prevention (U.S.); Cleaved cell; Clostridium botulinum; Communities; Complement; Complex; Crystallization; Dangerousness; Data; Development; Diagnostic; Disease; Endopeptidases; Enteral; Epitopes; Exposure to; GTP-Binding Protein alpha Subunits, Gs; Gene Therapy Agent; Goals; Health; Human; Immune Sera; Infection; Infectious Agent; Intoxication; Invaded; Knowledge; Length; Link; Lung; Mediating; Membrane; Methods; Microbe; Modeling; Molecular; Monoclonal Antibodies; Mutation; Neurons; Paralysed; Pathogenicity; Pathology; Peptide Hydrolases; Peptides; Prevention approach; Prevention strategy; Process; Property; Proteins; Reagent; Research; Risk; Role; SNAP receptor; Serotyping; Serum; Severities; Site; Specificity; Structural Models; Structure; Symptoms; Syndrome; Technology; Testing; Therapeutic; Time; Toxic effect; Toxin; Virus; alpha Toxin; antitoxin; base; biodefense; biothreat; design; flexibility; gene therapy; improved; inhibitor/antagonist; innovation; mouse model; neurotransmission; novel; novel strategies; pathogen; prevent; public health relevance; receptor; receptor binding; small molecule; treatment strategy

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.

项目摘要 肉毒杆菌病是由梭状芽胞杆菌产生的称为肉毒神经毒素（BONTS）的蛋白质毒素引起的 肉毒杆菌。 BONT是CDC Tier 1 Select Agent，目前不存在解毒剂。七个不同的BONT血清型 迄今已发现（BONT/A-G），许多人具有许多额外的BONT亚型。但是唯一的 目前可用的治疗是基于血清的抗毒素产物，这些抗毒素产物来自大型动物，只有在 爆发后不久就进行了管理。因以下事实加剧了开发BONT治疗药物的挑战 七个已知的BONT血清型都是不同的毒素，具有不同的受体特异性和蛋白酶，它们在 网罗蛋白上的不同位点破坏神经传播。由于风险的严重程度，治疗的缺乏 选择以及挑战的复杂性，预防和治疗胸部中毒的新方法是 显然需要。现在，我们在多个毒素模型中有大量证据，表明基于双特异性VHH 中和剂（VNA）由两种共价连接的毒素中和VHHs组成，是具有势力的抗毒素 通常超过当前单克隆和多克隆抗毒素剂的那样。此外，VNA提供大量 与血清和MAB抗毒素产品相比，它们具有经济性和用途广泛的优势；奉献 创新的毒素暴露和感染的新预防和治疗策略，可产生毒素的病原体 例如基因疗法以及直接递送到挑战的肠和肺部。在此提案中，我们测试了 假设将结构和机械信息整合到VNA设计中将导致更大的抗毒素 功效和多功能性。具体目的是（1）确定选定的BONT结合VHHS的晶体结构 与他们的目标主体复杂； （2）定义在AIM 1块BONT毒性中选择VHH的机制， 和; （3）通过利用结构/功能数据来设计和测试具有增强抗毒素特性的双特异性VNA 目标1和2。这将是中和表位的第一个全面的结构映射，这将是 补充了BONT功能和Bont-Host相互作用的机理研究。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一般理解 有效的VNA抗毒素剂，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治疗剂。

项目成果

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.

• DOI: 10.1038/s41598-022-05008-1
• 发表时间: 2022-02-02
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Nowakowska MB;Selby K;Przykopanski A;Krüger M;Krez N;Dorner BG;Dorner MB;Jin R;Minton NP;Rummel A;Lindström M
• 通讯作者: Lindström M

Intramuscular delivery of formulated RNA encoding six linked nanobodies is highly protective for exposures to three Botulinum neurotoxin serotypes.

• DOI: 10.1038/s41598-022-15876-2
• 发表时间: 2022-07-08
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Mukherjee, Jean;Ondeck, Celinia A.;Tremblay, Jacqueline M.;Archer, Jacob;Debatis, Michelle;Foss, Alexa;Awata, Junya;Erasmus, Jesse H.;McNutt, Patrick M.;Shoemaker, Charles B.
• 通讯作者: Shoemaker, Charles B.