纳米抗体 2S-1244 广谱抗 SARS-CoV-2 的中 和活性及其与 S 蛋白复合体结构的研究

为应对 SARS-CoV-2 引起 COVID-19 大流行对人类健康造成的重大威胁，科 研人员开发出多种预防疫苗和中和抗体以应对病毒感染。然而，随着病毒在传播 过程中的不断变异，多种变异株相继出现。特别是 2021 年 Omicron 变异株的广 泛传播极大地增加了人体再次感染病毒的风险。与原始 SARS-CoV-2 株及其他变 异株相比，Omicron 变异株的刺突蛋白 (spike protein,S 蛋白) 产生多位点突变，这 些突变可显著抵抗 COVID-19 康复者、接种 mRNA 疫苗或灭活疫苗的个体所产生 抗体的中和能力。最近研究表明，包括 FDA 批准的单克隆抗体在内的大多数针对 SARS-CoV-2 特异性的单克隆抗体已经失去对 Omicron 变异株的功效。因此，迫切 需要研发新的疫苗或广谱中和抗体以应对 SARS-CoV-2 的变异。 纳米抗体 (Nanobody,Nbs) 是源自骆驼科动物抗体的新型单体抗原结合片段。 Nbs 的优点包括低免疫原性、高特异性、稳定性和亲和力。这些特性使其可以迅速 被筛选、廉价大规模生产、简便储存和运输。我们实验室筛选了一种针对 SARSCoV-2 的 2S-1244 纳米抗体，在此基础上，本项目开展了其与病毒结合的生物功能 学实验和结构生物学实验。本研究展示了该纳米抗体对多种变异株 (Alpha、Delta、 Omicron 等) 的高效中和活性，并利用冷冻电镜单颗粒重构技术对 2S-1244 与 S 蛋 白复合体的结构进行深入研究。 对 2S-1244 中和野生型及 Alpha、Beta、Gamma、Delta 及 Omicron 各亚型的 中和活性进行测定，结果表明 2S-1244 为靶向 SARS-CoV-2 刺突蛋白受体结构域 (receptor binding domain,RBD) 的广谱高效中和纳米抗体。为进一步探究该纳米抗 体中和 SARS-CoV-2 的机制，本研究利用冷冻电镜单颗粒重构技术，对 2S-1244 全长纳米抗体与 S 蛋白的复合物进行结构解析，得到整体分辨率为 3.94 Å 的 2S1244-S 蛋白复合体的结构。结构显示，S 蛋白的三个 RBD 区域在与 2S-1244 纳米 抗体结合后处于”up”状态，一个 2S-1244 抗体的两个 VH 部分分别与两个 S 蛋白 RBD 区域的三位点结合，最终形成三个 2S-1244 全长抗体的 6 个 VH 部分与两个 S 蛋白的 6 个 RBD 区域分别进行结合的复合物，该结合方式可能使得病毒聚集在 一起，遮挡 S 蛋白与宿主细胞表面受体血管紧张素转化酶 2 的结合位点，阻止了 S 蛋白与 ACE2 的识别，使病毒入侵宿主的第一步失败，从而阻断病毒感染。 综合以上结论，本文证明 2S-1244 是一种高效中和 SARS-CoV-2 的广谱纳米抗 体，并且解析了 2S-1244 中和 S 蛋白发挥作用的机制。

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