3月3日，国际学术期刊pLOS pathogens 在线发表了中国科学院上海生命科学研究院生物化学与细胞生物学研究所国家蛋白质科学中心（上海）丛尧课题组与中国科学院上海巴斯德研究所黄忠课题组的合作研究论文Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody。该研究采用高分辨率冷冻电镜技术结合生化分析揭示了抗肠道病毒71（EV71）单克隆抗体D5结合以及抑制病毒的结构基础，为基于D5抗体的抗EV71药物开发提供了新思路。

手足口病（hand-foot and mouth disease, HFMD）每年都在亚太地区的婴幼儿中广泛流行。在中国，每年有上百万儿童感染手足口病，严重影响儿童健康。手足口病主要由肠道病毒感染导致，EV71是最主要的致病原，也是导致重症手足口病的罪魁祸首。目前，EV71灭活疫苗刚被批准上市，尚无用于治疗EV71感染的特异性药物。黄忠课题组前期自主研发的抗EV71单抗D5拥有强大体外抗病毒活性，可用于进一步开发抗EV71药物，然而对于该抗体的作用机制及其结构基础的了解尚不足。冷冻电镜技术近几年发展迅速，已成为超大分子复合体高分辨率结构研究的重要手段，但针对病毒－抗体复合物体系，由于其动态性及组成的不均一性，分辨率水平普遍在10 Å左右，只有近期几个冷冻电镜结构达到6-8.8 Å。

丛尧与黄忠密切合作，带领博士后叶晓华和樊晨等实验室成员，依托国家蛋白质科学中心（上海）的先进冷冻电镜设施，解析了系列不同EV71病毒颗粒与D5抗体形成复合体的高分辨率冷冻电镜三维结构（最高达4.8 Å），成为首例分辨率突破5 Å的病毒－抗体复合体冷冻电镜结构。结构分析显示，D5抗体可以跨越病毒的两重对称轴，以双价的方式结合在成熟病毒颗粒上，该种双价结合方式很可能具有稳固病毒颗粒，阻碍病毒构象变化的作用，从而抑制后续的病毒基因组释放。进一步分析表明，D5抗体的结合表位为病毒Vp1蛋白的GH loop，该表位在不同EV71亚型间极度保守，并且直接参与病毒与其脱衣壳受体SCARB2蛋白的相互作用。因此，抑制病毒与脱衣壳受体的相互作用是D5抗体的一个主要作用机制。同时，功能实验显示D5抗体不仅可以在体外中和多种不同基因型的EV71毒株，而且在体内完全保护小鼠免于致死剂量的EV71病毒感染。该项研究揭示了广谱保护性抗体D5结合EV71病毒并抑制其感染的结构基础，从而促进了基于D5的抗EV71单抗药物的开发。

该研究得到国家蛋白质科学中心（上海）冷冻电镜系统及数据库与计算分析系统的大力支持。参与该研究的还包括清华大学教授张林琦，及purdue大学教授Wen Jiang。该研究得到国家自然科学基金委、国家科技部、中国科学院战略性先导科技专项（B类）和上海市科委等的资助。

　

（A-D）不同EV71病毒颗粒与D5抗体的完整IgG或Fab片段形成复合物的冷冻电镜结构；（E）4.8 Å的F-particle-Fab的Vp1蛋白局部细节放大图；（F）F-particle-Fab密度图中D5抗体Fab片段与EV71相互作用区域放大图。

原文摘要：

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved Vp1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved Vp1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.