A potent human monoclonal antibody with pan-neutralizing activities directly dislocates S trimer of SARS-CoV-2 through binding both up and down forms of RBD

  1. Xiaofei Wang
  2. Ao Hu
  3. Xiangyu Chen
  4. Yixin Zhang
  5. Fei Yu
  6. Shuai Yue
  7. Arong Li
  8. Junsong Zhang
  9. Zhiwei Pan
  10. Yang Yang
  11. Yao Lin
  12. Leiqiong Gao
  13. Jing Zhou
  14. Jing Zhao
  15. Fang Li
  16. Yaling Shi
  17. Feng Huang
  18. Xiaofan Yang
  19. Yi Peng
  20. Luoyang Tu
  21. Huan Zhang
  22. Huanying Zheng
  23. Jun He
  24. Hui Zhang
  25. Lifan Xu
  26. Qizhao Huang
  27. Yongqun Zhu
  28. Kai Deng
  29. Lilin Ye

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel coronavirus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

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  1. Version published to 10.1038/s41392-022-00954-8
  2. Version published to 10.1101/2021.11.29.470356v4 on bioRxiv
  3. Version published to 10.1101/2021.11.29.470356v3 on bioRxiv
  4. Version published to 10.1101/2021.11.29.470356v2 on bioRxiv
  5. ScreenIT

    SciScore for 10.1101/2021.11.29.470356: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsConsent: Human samples: The COVID-19 patients enrolled in the study were admitted to Guangzhou Eighth People’s Hospital (January to March 2020) and provided written informed consent.
    IRB: Protection against SARS-CoV-2 in hACE2 mice: All animal experiments were carried out in strict accordance with the guidelines and regulations of Laboratory Monitoring Committee of Guangdong Province of China and were approved by Ethics Committee of Zhongshan School of Medicine of Sun Yat-sen University on Laboratory Animal Care (SYSU-IACUC-2021-00432)
    Sex as a biological variablenot detected.
    RandomizationThe hACE2 mice of the same sex were randomly assigned to each group.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    FITC-conjugated anti-CD19 antibody (Biolegend),
    anti-CD19
    suggested: None
    PE-conjugated anti-CD20 antibody (Biolegend),
    anti-CD20
    suggested: None
    APC-Cy7-conjugated anti-CD3 antibody (Biolegend),
    anti-CD3
    suggested: None
    anti-CD14 antibody (Biolegend),
    anti-CD14
    suggested: None
    anti-CD56 antibody (Biolegend) and APC-Cy7-conjugated LIVE/DEAD dye (Life Technologies) at 4 °C for 30 min.
    anti-CD56
    suggested: None
    After washing with PBST (PBS + 0.1% Tween 20), the bound antibodies were incubated with HRP-conjugated goat anti-human IgG antibody (Bioss Biotech) for 30 min, followed by washing with PBST and addition of TMB (Beyotime).
    anti-human IgG
    suggested: None
    Cells were then incubated with anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Sino Biological), followed by an HRP-labeled secondary antibody (Proteintech).
    anti-SARS-CoV-2 nucleocapsid protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: Vero E6 cells were obtained from ATCC and maintained in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher) and 1% penicillin-streptomycin (ThermoFisher) at 37 °C with 5% CO2.
    Vero E6
    suggested: None
    The S-2P and S-6P plasmids were transiently transfected into HEK293F cells using 25-kDa linear polyethyleneimine (PEI) (Polysciences) with the PEI:DNA mass ratio of 3:1 and 1 mg DNA for per liter of culture when the cell density reached 2 × 106 cells per mL.
    HEK293F
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    For infection, ICR-hACE2 mice were anesthetized with isoflurane and inoculated intranasally with 4×104 PFU SARS-CoV-2 virus (GISAID: EPI_ISL_402125).
    ICR-hACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    To generate a standard curve, the SARS-CoV-2 nucleocapsid (N) gene was cloned into a pcDNA3.1 expression plasmid and in vitro transcribed to obtain RNAs for standards.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    The S-2P and S-6P plasmids were transiently transfected into HEK293F cells using 25-kDa linear polyethyleneimine (PEI) (Polysciences) with the PEI:DNA mass ratio of 3:1 and 1 mg DNA for per liter of culture when the cell density reached 2 × 106 cells per mL.
    S-6P
    suggested: None
    The dose-weighted images were then imported into cryoSPARC51 for the following image processing, including CTF estimation, particle picking and extraction, 2D classification, ab initio 3D reconstruction, heterogeneous 3D refinement and non-uniform homogeneous refinement.
    cryoSPARC51
    suggested: None
    Software and Algorithms
    SentencesResources
    EC50 values were determined by using Prism 6.0 (GraphPad) software after log transformation of the mAb concentration using sigmoidal dose-response nonlinear regression analysis.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Local refinements of the RBD-35B5 Fab region were performed to improve the interface density in cryoSPARC.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Structural figures were generated using UCSF ChimeraX version 1.256. Statistics: In the mouse study assessing mAb protection against WT SARS-CoV-2, the comparisons of lung viral titers and lung cytokine/chemokine mRNA were performed using one-way ANOVA with Tukey’s post hoc test by Prism 6.0 (GraphPad).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56, 50 and 52. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  6. Version published to 10.1101/2021.11.29.470356v1 on bioRxiv