Xu Jianliang, Xu Kai, Jung Seolkyoung, Conte Andrea, Lieberman Jenna, Muecksch Frauke, Cetrulo Lorenzi Julio Cesar, Park Solji, Wang Zijun, Tessarollo Lino, Bylund Tatsiana, Chuang Gwo-Yu, Olia Adam S, Stephens Tyler, Teng I-Ting, Tsybovsky Yaroslav, Zhou Tongqing, Hatziioannou Theodora, Bieniasz Paul D, Nussenzweig Michel C, Kwong Peter D, Casellas Rafael
bioRxiv. 2021 Mar 4:2021.03.04.433768. doi: 10.1101/2021.03.04.433768.
Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.
自2019冠状病毒病（COVID-19）大流行开始以来，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）已在全球范围内导致超过200万人死亡。迄今为止，多种疫苗已投入使用，但病毒受体结合域（RBD）的持续进化最近对其效力提出了挑战。特别是，源自英国（B.1.1.7）、南非（B.1.351）和纽约（B.1.526）的SARS-CoV-2变体降低了康复期血清的中和活性，并损害了获得紧急使用授权的抗体鸡尾酒疗法的效力。虽然疫苗可以定期更新以应对新出现的变体，但迫切需要补充策略来避免病毒逃逸。一种潜在的替代方法是使用骆驼科VHH（也称为纳米抗体），由于其尺寸小，它们可以识别传统抗体无法接近的蛋白质缝隙。在这里，我们从美洲驼以及我们工程改造以产生从羊驼、单峰骆驼和骆驼克隆的VHH的“纳米小鼠”中分离出抗RBD纳米抗体。通过结合试验和冷冻电子显微镜，我们鉴定出两组高度中和性的纳米抗体。第一组表达的VHH通过识别ACE2结合位点之外的一个区域来规避RBD抗原漂移，该区域在冠状病毒中保守，但通常不是单克隆抗体的靶向区域。第二组几乎完全聚焦于RBD-ACE2界面，无法中和携带E484K或N501Y替代突变的假病毒。然而，值得注意的是，当它们以同源三聚体形式表达时，确实能够中和RBD变体，与迄今为止针对SARS-CoV-2产生的最有效的抗体相当。这些发现表明，多价纳米抗体通过两种不同的机制克服SARS-CoV-2变体突变：增强对ACE2结合域的亲和力，以及识别人类抗体基本无法接近的保守表位。因此，虽然新出现的SARS-CoV-2突变体将继续出现，但当疫苗效力受损时，纳米抗体是预防COVID-19死亡的有前景的工具。
