Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain.
Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain.
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DOI：
10.1186/s12896-015-0206-0
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发表时间：
2015-09-17
影响因子：
3.5
通讯作者：
Pelat T
中科院分区：
文献类型：
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作者：
Avril A;Miethe S;Popoff MR;Mazuet C;Chahboun S;Rasetti-Escargueil C;Sesardic D;Thullier P;Hust M;Pelat T
肉毒杆菌中毒是一种自然发生的疾病，主要由摄入受肉毒神经毒素(BoNTs)污染的食物引起。肉毒杆菌神经毒素是最致命的。它们被疾病控制中心归类为六种主要的生物战剂。BoNTs作用于胆碱能运动神经元，在那里它们裂解与乙酰胆碱囊泡胞吐有关的蛋白质。这种胞吐抑制会导致迟缓性瘫痪，逐渐影响所有肌肉，通常会引起呼吸窘迫。BoNTs还被用于医学，主要用于治疗神经肌肉疾病，防止大规模接种疫苗。肉毒杆菌中毒的特殊治疗需要注射抗毒素，通常是马源性的，因此耐受性很差。因此，开发人类或类似人类的中和抗体是一个重要的兴趣，它是名为“AntiBotABE”的欧洲框架项目的主题。本研究从重组BoNT/A1重链免疫猕猴(BoNT/A1-HC)出发，构建了免疫抗体噬菌体展示文库，分离并鉴定了具有纳分子亲和力的抗体片段(单链可变型)。在小鼠膈神经-横隔膜实验中分析这些单链抗体的中和能力。经过三轮淘洗，共分离到24个亲和力大于10nM的抗体片段。在小鼠膈神经-横隔膜实验中，其中3个有效地中和了BONT/A1，2个交叉中和了BONT/A1和BONT/A2亚型。这是第一批交叉中和BONT/A1和BONT/A2的类似人类的单抗。筛选出了A1HC38抗体，可用于肉毒杆菌中毒的预防和治疗。本文的在线版本(doi：10.1186/s12896-0150206-0)包含补充材料，授权用户可以使用。
Botulism is a naturally occurring disease, mainly caused by the ingestion of food contaminated by the botulinum neurotoxins (BoNTs). Botulinum neurotoxins are the most lethal. They are classified among the six major biological warfare agents by the Centers for Disease Control. BoNTs act on the cholinergic motoneurons, where they cleave proteins implicated in acetylcholine vesicle exocytosis. This exocytosis inhibition induces a flaccid paralysis progressively affecting all the muscles and generally engendering a respiratory distress. BoNTs are also utilized in medicine, mainly for the treatment of neuromuscular disorders, preventing large scale vaccination. Botulism specific treatment requires injections of antitoxins, usually of equine origin and thus poorly tolerated. Therefore, development of human or human-like neutralizing antibodies is of a major interest, and it is the subject of the European framework project called “AntiBotABE”. In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1 (BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay. After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1 efficiently and two cross-neutralized BoNT/A1 and BoNT/A2 subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1 and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism. The online version of this article (doi:10.1186/s12896-015-0206-0) contains supplementary material, which is available to authorized users.