A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

Abstract

Background

The emergence of the coronavirus disease 2019 (COVID-19) pandemic and the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) requires the continuous development of safe, effective, and affordable prevention and therapeutics. Nanobodies have demonstrated antiviral activity against a variety of viruses, providing a new candidate for the prevention and treatment of SARS-CoV-2 and its variants.

Methods

SARS-CoV-2 glycoprotein spike 1 subunit (S1) was selected as the target antigen for nanobody screening of a naïve phage display library. We obtained a nanobody, named Nb-H6, and then determined its affinity, inhibition, and stability by ELISA, Competitive ELISA, and Biolayer Interferometry (BLI). Infection assays of authentic and pseudotyped SARS-CoV-2 were performed to evaluate the neutralization of Nb-H6. The structure and mechanism of action were investigated by AlphaFold, docking, and residue mutation assays.

Results

We isolated and characterized a nanobody, Nb-H6, which exhibits a broad affinity for S1 and the receptor binding domain (RBD) of SARS-CoV-2, or Alpha (B.1.1.7), Delta (B.1.617.2), Lambda (C.37), and Omicron (BA.2 and BA.5), and blocks receptor angiotensin-converting enzyme 2 (ACE2) binding. Moreover, Nb-H6 can retain its binding capability after pH or thermal treatment and effectively neutralize both pseudotyped and authentic SARS-CoV-2, as well as VOC Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.2 and BA.5) pseudoviruses. We also confirmed that Nb-H6 binds two distinct amino acid residues of the RBD, preventing SARS-CoV-2 from interacting with the host receptor.

Conclusion

Our study highlights a novel nanobody, Nb-H6, that may be useful therapeutically in SARS-CoV-2 and VOC outbreaks and pandemics. These findings also provide a molecular foundation for further studies into how nanobodies neutralize SARS-CoV-2 and variants and imply potential therapeutic targets for the treatment of COVID-19.

Keywords:

• COVID-19
• antiviral medication
• single-domain antibody
• neutralizing antibody
• bio-screening

Data Sharing Statement

All data are available upon request by contact with the corresponding author.

Ethics Approval

Since IRB approval is not necessary for this study, approval from an ethics committee was not required.

Acknowledgments

This study was supported by grants from the Guangdong Basic and Applied Basic Research Foundation (2021A1515010917 and 2020A1515110410), the Guangdong Medical Science and Technology Research Foundation (A2021336), the Pearl River Talent Project of Guangdong Province (2021QN02Y426), the National Nature Science Foundation of China (32170937), the Shenzhen Science and Technology Program (RCBS20200714114958310 and 20200803131335002), the Shenzhen Peacock Plan Project (827/000655), and SZU Top Ranking Project (86000000210) to Liang Ye. We sincerely thank Dr. Huawei Wei at East Mab (Jiangsu, China) for providing the spike proteins of Alpha, Delta, and Lambda, and the hACE2 protein.

Disclosure

Liang Ye and Dandan Wu report a pending patent on the combination of Nb-H6 mentioned in the paper. Chinese patent application No. 202210681887.X. The authors declare that they have no other competing interests in this work.