Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization^1,2,3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies⁴. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD–ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and—to our knowledge—rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

自 COVID-19 大流行开始以来，SARS-CoV-2 已导致全球数百万人死亡。尽管已经部署了多种疫苗，但病毒受体结合域（RBD）的不断进化对其功效提出了挑战。特别是，新出现的变种 B.1.1.7、B.1.351 和 P.1（分别在英国、南非和巴西首次检测到）损害了从 COVID-19 中恢复的患者血清和免疫疗法的功效已获得紧急使用授权^1,2,3 。避免病毒逃逸的一种潜在替代方法是使用骆驼科动物 VHH（重链抗体的可变重链结构域（也称为纳米抗体）），它可以识别传统抗体通常无法接近的表位⁴ 。在这里，我们从美洲驼和小鼠中分离出抗 RBD 纳米抗体，这些纳米抗体是通过基因改造产生从羊驼、单峰骆驼和双峰驼克隆的 VHH 的。我们鉴定了两组高度中和的纳米抗体。第 1 组通过识别在冠状病毒中高度保守但很少被人类抗体靶向的 RBD 区域来规避抗原漂移。第 2 组几乎完全专注于 RBD-ACE2 界面，并且不会中和携带 E484K 或 N501Y 取代的 SARS-CoV-2 变体。然而，当以同源三聚体表达时，第 2 组中的纳米抗体保留了针对这些变体的完全中和活性，并且据我们所知，可以与迄今为止生产的最有效的针对 SARS-CoV-2 的抗体相媲美。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 突变：增强 ACE2 结合域的亲合力和识别人类抗体基本上无法接近的保守表位。 因此，尽管新的 SARS-CoV-2 突变体将不断出现，但纳米抗体代表了在疫苗受到损害时预防 COVID-19 死亡的有前途的工具。