Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display
Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display
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DOI：
10.1016/j.jmb.2006.09.084
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发表时间：
2007-01-05
影响因子：
5.6
通讯作者：
Marks, J. D.
中科院分区：
文献类型：
--
作者：
Levy, R.;Forsyth, C. M.;Marks, J. D.
肉毒杆菌神经毒素(BONT)是已知的最有毒的物质，会导致自然发生的人类疾病(肉毒杆菌中毒)，是排名前六的生物治疗剂之一。肉毒杆菌中毒的治疗是用马身上产生的多克隆抗体来治疗的，这种抗体与全身反应的发生率很高。人类单抗(MAbbs)正被开发为一种更安全的治疗方法。识别BoNTs上的中和表位是产生中和单抗的重要步骤，并对疫苗的开发具有重要意义。在这里，我们发现ONT血清型A(BoNT/A)的三个结构域可以展示在酵母的表面，并用于将六个单抗与它们结合的毒素结构域进行表位映射。酵母的使用消除了表达和纯化每个结构域的需要，并且应该可以展示其他BONT亚型和血清型的结构域以用于表位映射。利用酵母展示的BONT/A结合域(H-C)突变体文库，结合缺失筛选，鉴定了3株中和BONT/A单抗的优良表位。两个单抗结合H-C的C-末端亚区，一个结合在毒素唾液神经节苷脂结合部位附近。最有效的中和单抗结合H-C的N-末端亚区，这一区域以前并不被认为具有重要的功能。对表位进行建模显示了所有三种单抗是如何同时与BoNT/A结合的，这可能在一定程度上解释了当这些抗体结合时，在体内观察到的戏剧性中和毒素的协同作用。结果表明，酵母展示可用于构象BONT抗体表位的结构域水平和精细映射，映射结果识别出三个中和BONT/A表位。
Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast-displayed BoNT/A binding domain (H-C) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H-C, with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H-C, in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.