A potent and broad-spectrum neutralizing nanobody for SARS-CoV-2 viruses, including all major Omicron strains.

SARS-CoV-2 viruses are highly transmissible and immune evasive. It is critical to develop broad-spectrum prophylactic and therapeutic antibodies for potential future pandemics. Here, we used the phage display method to discover nanobodies (Nbs) for neutralizing SARS-CoV-2 viruses especially Omicron strains. The leading nanobody (Nb), namely, Nb4, with excellent physicochemical properties, can neutralize Delta and Omicron subtypes, including BA.1, BA.1.1 (BA.1 + R346K), BA.2, BA.5, BQ.1, and XBB.1. The crystal structure of Nb4 in complex with the receptor-binding domain (RBD) of BA.1 Spike protein reveals that Nb4 interacts with an epitope on the RBD overlapping with the receptor-binding motif, and thus competes with angiotensin-converting enzyme 2 (ACE2) binding. Nb4 is expected to be effective for neutralizing most recent Omicron variants, since the epitopes are evolutionarily conserved among them. Indeed, trivalent Nb4 interacts with the XBB1.5 Spike protein with low nM affinity and competes for ACE2 binding. Prophylactic and therapeutic experiments in mice indicated that Nb4 could reduce the Omicron virus loads in the lung. In particular, in prophylactic experiments, intranasal administration of multivalent Nb4 completely protected mice from Omicron infection. Taken together, these results demonstrated that Nb4 could serve as a potent and broad-spectrum prophylactic and therapeutic Nb for COVID-19.

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© 2023 The Authors. MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.

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H.Y. designed and performed the phage display screening assay, purified selected Nbs, and characterized them using BLI assay with assistance from Zhiying Z. and Y.Z. H.Y. generated all plasmids, purified Nbs, and some RBDs for all follow‐up mechanistic, crystallization, Cryo‐EM, and functional experiments. The crystal structure was also analyzed by H.Y. H.W. purified ACE2 and Nb4‐Fc proteins, performed binding kinetics and competition assays, crystallized and determined crystal structure of the RBD1–Nb4 complex, and tested Nb stabilities. Zhaoyong Z. and X.X. evaluated. J.Z. and Y.W. conceived and designed the protection capacity of Nbs in vitro and in vivo. Y.L. under supervision of Haitao Y. solved Cryo‐EM structures of BA.1/Nb4 and BA.5/Nb4 complexes. H.Y., Z.W., and W.X. drafted the manuscript with inputs and revisions from all co‐authors. All authors have read and approved the final manuscript.H.Y., W.X., H.W., Zhiying Z., Y.Z., Z.W., Y.L., and Haitao Y. filed patents on Nb4 nanobody and the application for COVID‐19 treatment and prevention and the PCT number is PCT/CN2022/084209. Zhaoyong Z., X.X., Y.W., and J.Z. declare that they have no conflicts of interest.