SARS-CoV-2 variants with adaptive mutations have continued to emerge, causing fresh waves of infection even amongst vaccinated population. The development of broad-spectrum antivirals is thus urgently needed. We previously developed two hetero-bivalent nanobodies (Nbs), aRBD-2-5 and aRBD-2-7, with potent neutralization activity against the wild-type (WT) Wuhan isolated SARS-CoV-2, by fusing aRBD-2 with aRBD-5 and aRBD-7, respectively. Here, we resolved the crystal structures of these Nbs in complex with the receptor-binding domain (RBD) of the spike protein, and found that aRBD-2 contacts with highly-conserved RBD residues and retains binding to the RBD of the Alpha, Beta, Gamma, Delta, Delta plus, Kappa, Lambda, Omicron BA.1, and BA.2 variants. In contrast, aRBD-5 and aRBD-7 bind to less-conserved RBD epitopes non-overlapping with the epitope of aRBD-2, and do not show apparent binding to the RBD of some variants. However, when fused with aRBD-2, they effectively enhance the overall binding affinity. Consistently, aRBD-2-5-Fc and aRBD-2-7-Fc potently neutralized all of the tested authentic or pseudotyped viruses, including WT, Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.1.1 and BA.2. Furthermore, aRBD-2-5-Fc provided prophylactic protection against the WT and mouse-adapted SARS-CoV-2 in mice, and conferred protection against the Omicron BA.1 variant in hamsters prophylactically and therapeutically, indicating that aRBD-2-5-Fc could potentially benefit the prevention and treatment of COVID-19 caused by the emerging variants of concern. Our strategy provides new solutions in the development of broad-spectrum therapeutic antibodies for COVID-19.

具有适应性突变的 SARS-CoV-2 变体不断出现，甚至在接种疫苗的人群中也引起了新的感染浪潮。因此，迫切需要开发广谱抗病毒药物。我们之前开发了两种异二价纳米抗体 (Nbs)，aRBD-2-5 和 aRBD-2-7，通过将 aRBD-2 与分别为 aRBD-5 和 aRBD-7。在这里，我们解析了这些与刺突蛋白的受体结合域 (RBD) 复合的 Nb 的晶体结构，并发现 aRBD-2 与高度保守的 RBD 残基接触并保留与 Alpha、Beta 的 RBD 的结合、Gamma、Delta、Delta plus、Kappa、Lambda、Omicron BA.1 和 BA.2 变体。相比之下，aRBD-5 和 aRBD-7 结合不太保守的 RBD 表位，与 aRBD-2 的表位不重叠，并且不显示与某些变体的 RBD 的明显结合。然而，当与 aRBD-2 融合时，它们有效地增强了整体结合亲和力。aRBD-2-5-Fc 和 aRBD-2-7-Fc 始终有效地中和所有经过测试的真实或假型病毒，包括 WT、Alpha、Beta、Gamma、Delta 和 Omicron BA.1、BA.1.1 和 BA .2. 此外，aRBD-2-5-Fc 在小鼠中提供了针对 WT 和小鼠适应性 SARS-CoV-2 的预防性保护，并在预防和治疗方面对仓鼠提供了针对 Omicron BA.1 变体的保护，表明 aRBD-2-5 -Fc 可能有利于预防和治疗由新出现的令人担忧的变体引起的 COVID-19。