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

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.

SUBMITTER: Li T

PROVIDER: S-EPMC9201380 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Li Tingting T Zhou Bingjie B Luo Zhipu Z Lai Yanling Y Huang Suqiong S Zhou Yuanze Y Li Yaning Y Gautam Anupriya A Bourgeau Salome S Wang Shurui S Bao Juan J Tan Jingquan J Lavillette Dimitri D Li Dianfan D

Frontiers in microbiology 20220602

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 <i>K</i> <sub>D</sub> ...[more]

PMID: 35722331

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Project description:IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), had a major impact on both the global health and economy. Numerous virus-neutralizing antibodies were developed against the S1 subunit of SARS-CoV-2 spike (S) protein to block viral binding to host cells and were authorized for control of the COVID-19 pandemic. However, frequent mutations in the S1 subunit of SARS-CoV-2 enabled the emergence of immune evasive variants. To address these challenges, broadly neutralizing antibodies targeting the relatively conserved S2 subunit and its epitopes have been investigated as antibody therapeutics and universal vaccines.MethodsWe initiated this study by immunizing BALB/c mice with β-propiolactone-inactivated SARS-CoV-2 (IAV) to generate B-cell hybridomas. These hybridomas were subsequently screened using HEK293T cells expressing the S2-ECD domain. Hybridomas that produced anti-S2 antibodies were selected, and we conducted a comprehensive evaluation of the potential of these anti-S2 antibodies as antiviral agents and versatile tools for research and diagnostics.ResultsIn this study, we present a novel S2-specific antibody, 4A5, isolated from BALB/c mice immunized with inactivated SARS-CoV-2. 4A5 exhibited specific affinity to SARS-CoV-2 S2 subunits compared with those of other β-CoVs. 4A5 bound to epitope segment F1109-V1133 between the heptad-repeat1 (HR1) and the stem-helix (SH) region. The 4A5 epitope is highly conserved in SARS-CoV-2 variants, with a significant conformational feature in both pre- and postfusion S proteins. Notably, 4A5 exhibited broad neutralizing activity against variants and triggered Fc-enhanced antibody-dependent cellular phagocytosis.DiscussionThese findings offer a promising avenue for novel antibody therapeutics and insights for next-generation vaccine design. The identification of 4A5, with its unique binding properties and broad neutralizing capacity, offers a potential solution to the challenge posed by SARS-CoV-2 variants and highlights the importance of targeting the conserved S2 subunit in combating the COVID-19.

Dataset Information

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

Publications

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

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