A potently neutralizing anti-SARS-CoV-2 antibody inhibits variants of concern by binding a highly conserved epitope

  1. Laura A. VanBlargan
  2. Lucas J. Adams
  3. Zhuoming Liu
  4. Rita E. Chen
  5. Pavlo Gilchuk
  6. Saravanan Raju
  7. Brittany K. Smith
  8. Haiyan Zhao
  9. James Brett Case
  10. Emma S. Winkler
  11. Bradley M. Whitener
  12. Lindsay Droit
  13. Ishmael D. Aziati
  14. Pei-Yong Shi
  15. Adrian Creanga
  16. Amarendra Pegu
  17. Scott A. Handley
  18. David Wang
  19. Adrianus C.M. Boon
  20. James E. Crowe
  21. Sean P.J. Whelan
  22. Daved H. Fremont
  23. Michael S. Diamond

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Abstract

With the emergence of SARS-CoV-2 variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here we developed a panel of neutralizing anti-SARS-CoV-2 mAbs that bind the receptor binding domain of the spike protein at distinct epitopes and block virus attachment to cells and its receptor, human angiotensin converting enzyme-2 (hACE2). While several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by historical SARS-CoV-2 strains, others induced escape variants in vivo and lost activity against emerging strains. We identified one mAb, SARS2-38, that potently neutralizes all SARS-CoV-2 variants of concern tested and protects mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engages a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of inhibitory antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.26.441501: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Plates were washed and bound human ACE2 was detected using HRP-conjugated anti-Flag antibody (Sigma-Aldrich, A8592, 1:5,000 dilution) and TMB substrate.
    anti-Flag
    suggested: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)
    A8592
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Hybridomas producing antibodies that bound to SARS-CoV-2-infected permeabilized Vero CCL81 cells by flow cytometry and to SARS-CoV-2 recombinant spike protein by direct ELISA were cloned by limiting dilution.
    Vero CCL81
    suggested: None
    All hybridomas were screened initially with a single-endpoint neutralization assay using hybridoma supernatant diluted 1:3 and incubated with SARS-CoV-2 for 1 h at 37°C prior to addition to Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Briefly, plaque assays were performed to isolate escape mutants on Vero-TMPRSS2 cells with neutralizing mAb in the overlay.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Recombinant antibodies were expressed in Expi293 cells following co-transfection of heavy and light chain plasmids (1:1 ratio) using Expifectamine 293 (Thermo Fisher Scientific).
    Expi293
    suggested: RRID:CVCL_D615)
    For post-attachment assays, 2 x 102 FFU of SARS-CoV-2 was adsorbed onto a monolayer of Vero cells for 1 h at 4°C.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    The mixture then was added to pre-chilled Vero E6, Vero-TMPRSS2, Vero-TMPRSS2-ACE2, or Calu-3 cells at an MOI of 0.005 and incubated at 4°C for 1 h.
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Experimental Models: Organisms/Strains
    SentencesResources
    MAb generation: BALB/c mice were immunized with 10 µg of SARS-CoV-2 RBD adjuvanted with 50% AddaVax™ (InvivoGen), via intramuscular route (i.m.), followed by i.m. immunization two and four weeks later with SARS-CoV-2 spike protein (5 µg and 10 µg, respectively) supplemented with AddaVax™.
    BALB/c
    suggested: None
    Recombinant DNA
    SentencesResources
    For initial modeling of SARS2-38 Fv, pBLAST was used to identify pre-existing Fab structures with high sequence similarity (PDB 1KIQ for VH, and PDB 5XJM for VL).
    pBLAST
    suggested: None
    Software and Algorithms
    SentencesResources
    MAb generation: BALB/c mice were immunized with 10 µg of SARS-CoV-2 RBD adjuvanted with 50% AddaVax™ (InvivoGen), via intramuscular route (i.m.), followed by i.m. immunization two and four weeks later with SARS-CoV-2 spike protein (5 µg and 10 µg, respectively) supplemented with AddaVax™.
    AddaVax™
    suggested: None
    Cryo-EM data processing: Movies were motion corrected using MotionCor2 v1.3.1 (Zheng et al., 2017), and contrast transfer function parameters were estimated using GCTF v1.18 (Zhang, 2016).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    GCTF
    suggested: (GCTF, RRID:SCR_016500)
    2D classification was performed in Relion 3.1 (Scheres, 2012; Zivanov et al., 2018), and particles in good classes from grids with or without ultra-thin carbon were combined for further processing.
    Relion
    suggested: (RELION, RRID:SCR_016274)
    To improve map quality at the Fab/spike interface, a mask was generated encompassing only the Fv and RBD, and particles were subjected to local non-uniform refinement in cryoSPARC v3.1.0.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    This model was docked into the full-spike map then refined using Coot v0.9.5
    Coot
    suggested: (Coot, RRID:SCR_014222)
    , Isolde v1.1.0, and Phenix v1.19.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Antibody-dose response curves were analyzed using non-linear regression analysis (with a variable slope) (GraphPad Software).
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 55. 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

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  2. Version published to 10.1101/2021.04.26.441501 on bioRxiv