Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants

Tingting Li; Bingjie Zhou; Zhipu Luo; Yanling Lai; Suqiong Huang; Yuanze Zhou; Yaning Li; Anupriya Gautam; Salome Bourgeau; Shurui Wang; Juan Bao; Jingquan Tan; Dimitri Lavillette; Dianfan Li

doi:10.3389/fmicb.2022.875840

Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants

Front Microbiol. 2022 Jun 2:13:875840. doi: 10.3389/fmicb.2022.875840. eCollection 2022.

Authors

Tingting Li¹, Bingjie Zhou^{2

3}, Zhipu Luo⁴, Yanling Lai^{1

2}, Suqiong Huang^{2

3

5}, Yuanze Zhou⁶, Yaning Li^{1

2}, Anupriya Gautam^{2

3}, Salome Bourgeau^{2

3

7}, Shurui Wang⁶, Juan Bao¹, Jingquan Tan⁶, Dimitri Lavillette^{3

8}, Dianfan Li¹

Affiliations

¹ State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.
² University of CAS, Beijing, China.
³ CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai CAS, Shanghai, China.
⁴ Institute of Molecular Enzymology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou, China.
⁵ College of Pharmacy, Chongqing Medical University, Chongqing, China.
⁶ Nanjing Crycision Biotech Co., Ltd., Nanjing, China.
⁷ Institut National de la Santé et de la Recherche Médicale, École des Hautes Etudes en Santé Publique, Institut de Recherche en Santé, Environnement et Travail, Université de Rennes, Rennes, France.
⁸ Pasteurien College, Soochow University, Suzhou, China.

Abstract

SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K _D of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC₅₀ of 0.41 μg mL^-1. Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC₅₀ values ranging from 0.35 to 1.66 μg mL^-1 for the Alpha/Beta/Gamma/Delta and an IC₅₀ of 0.66 μg mL^-1 for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a "conformation competition" mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.

Keywords: COVID-19; SARS-CoV-2; conformation competition; coronavirus; crystal structure; nanobody; receptor-binding domain.