Vance David J, Tremblay Jacqueline M, Rong Yinghui, Angalakurthi Siva Krishna, Volkin David B, Middaugh C Russell, Weis David D, Shoemaker Charles B, Mantis Nicholas J
Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, USA
Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA.
Clin Vaccine Immunol. 2017 Dec 5;24(12). doi: 10.1128/CVI.00236-17. Print 2017 Dec.
We previously produced a heavy-chain-only antibody (Ab) VH domain (VH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538-36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VH-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHs grouped into more than 20 different competition bins. The RTA-specific VHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development.
我们之前用来自两只羊驼的仅重链抗体（Ab）VH结构域（VH）构建了噬菌体展示文库，这两只羊驼已用蓖麻毒素类毒素以及酶促蓖麻毒素A亚基（RTA）和结合性蓖麻毒素B亚基（RTB）的无毒混合物进行了免疫（D. J. 万斯、J. M. 特伦布莱、N. J. 曼蒂斯和C. B. 休梅克，《生物化学杂志》288:36538 - 36547，2013年，https://doi.org/10.1074/jbc.M113.519207）。通过直接酶联免疫吸附测定（ELISA）对该文库进行初次及后续筛选，得到了二十多个独特的RTA和RTB特异性VH，其中10个的结构随后解析为与RTA形成复合物的形式。为了生成更完整的蓖麻毒素抗原图谱并确定与毒素中和活性相关的表位，我们让该VH展示噬菌体文库在受体结合型蓖麻毒素和抗体捕获型蓖麻毒素上进行额外的“淘选”。我们现在报告68个独特VH的全长DNA序列、结合亲和力和中和活性：31个针对RTA，33个针对RTB，4个针对蓖麻毒素全毒素。通过用一组单克隆抗体（MAb）进行交叉竞争ELISA实现表位定位，并在某些情况下用氢 - 氘交换质谱法进行验证。这68个VH分为20多个不同的竞争组。具有强毒素中和活性的RTA特异性VH局限于与两个先前确定的中和热点（称为簇I和簇II）重叠的组中。四个具有强毒素中和活性的RTB特异性VH聚集在簇II附近RTA - RTB界面处的三个相邻组内。这些结果为蓖麻毒素表面表位的相互关系提供了重要见解，并描绘了可用于疫苗和治疗开发的脆弱区域。
