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

Abstract

Dandan Wu,1,* Junxiao Cong,1,* Jiali Wei,1 Jing Hu,1 Wenhao Sun,1 Wei Ran,2 Chenghui Liao,1 Housheng Zheng,1 Liang Ye1 1Department of Immunology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, People’s Republic of China; 2State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Liang Ye, Department of Immunology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, 518055, People’s Republic of China, Tel [phone], Fax [phone], Email [email protected]: 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

Keywords

• covid-19
• antiviral medication
• single-domain antibody
• neutralizing antibody
• bio-screening.