Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2

  1. Sarah A. Clark
  2. Lars E. Clark
  3. Junhua Pan
  4. Adrian Coscia
  5. Lindsay G.A. McKay
  6. Sundaresh Shankar
  7. Rebecca I. Johnson
  8. Anthony Griffiths
  9. Jonathan Abraham

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Abstract

The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.13.381533: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Donors: This study was approved by the Harvard Medical School Office of Human Research Administration Institutional Review Board (IRB20-0365) as was the use of healthy donor control blood (IRB19-0786).
    Consent: We received informed, written consent from a healthy adult male participant (C1) who recovered from confirmed SARS2-CoV-2 infection, with mild illness not requiring hospitalization, five weeks before blood donation.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableWe received informed, written consent from a healthy adult male participant (C1) who recovered from confirmed SARS2-CoV-2 infection, with mild illness not requiring hospitalization, five weeks before blood donation.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After washing three times and resuspending the cells, we added anti-IgG-APC antibody (BD Biosciences), anti-CD19-FITC antibody (BD Biosciences), and streptavidin-PE (Invitrogen).
    anti-IgG-APC
    suggested: (Miltenyi Biotec Cat# 130-093-194, RRID:AB_1036183)
    anti-CD19-FITC
    suggested: None
    We detected bound antibody with horseradish peroxidase (HRP)-coupled anti-human (Fc) antibody (Sigma Aldrich catalog number A0170).
    anti-human (Fc)
    suggested: (Sigma-Aldrich Cat# A0170, RRID:AB_257868)
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: We maintained HEK293T cells (ATCC CRL-11268) in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v
    HEK293T
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    A HEK293T-hACE2 stable cell line was a gift from Huihui Mou and Michael Farzan and an Expi293F-His6-tagged SARS-CoV-2 S2P stable cell line that expresses a gift from Bing Chen.
    HEK293T-hACE2
    suggested: None
    S2P
    suggested: RRID:CVCL_2H49)
    A T225 flask of VeroE6 cells was inoculated with 90 μl starting material in 15 ml DMEM containing 2% (v/v) of heat inactivated FBS (HI-FBS) and incubated in a humidified incubator at 37 °C with periodic rocking for 1 h.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    For crystallography, we produced the protein by PEI transfection of GnTI-/- HEK293S cells grown in suspension or HEK293T cells grown in suspension and also in presence of kifunensine (5 μM), purified by nickel affinity purification, and removed the His6-tag by TEV digestion followed by reverse nickel affinity purification.
    HEK293S
    suggested: RRID:CVCL_A784)
    Software and Algorithms
    SentencesResources
    After washing three times and resuspending the cells, we added anti-IgG-APC antibody (BD Biosciences), anti-CD19-FITC antibody (BD Biosciences), and streptavidin-PE (Invitrogen).
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    We used IMGT/V-QUEST31 (http://www.imgt.org) to analyze IgG gene usage and the extent of variable segment somatic hypermutation.
    http://www.imgt.org
    suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)
    Diffraction data were indexed and integrated using XDS (build 202 00131)36 and merged using AIMLESS (v0.5.32)37.
    AIMLESS
    suggested: (AIMLESS, RRID:SCR_015747)
    The structure of C1A-B12 Fab:RBD (space group P212121) was determined by molecular replacement using Phaser (v2.8.3)38, with coordinates for the B38 Fab variable domain, constant domain and RBD (PDB ID: 7BZ5) (Wu et al., 2020) used as search models.
    Phaser
    suggested: (Phaser, RRID:SCR_014219)
    We performed iterative model using O39 and refinement in Phenix (v1.18.2-3874)40 and Buster (v2.10.3)41, during which we also built alternative conformations where density was apparent.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    During refinements, we updated TLS groups calculated using Phenix40 and a python script, as well as occupancy restraints calculated in Buster.
    python
    suggested: (IPython, RRID:SCR_001658)
    Structural Analysis: We analyzed the structures and generated figures using PyMOL (Schrödinger)
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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: We did not find any issues relating to colormaps.

    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.

    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.

  2. Version published to 10.1101/2020.11.13.381533 on bioRxiv