Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein

  1. Jillian M. DiMuzio
  2. Baron C. Heimbach
  3. Raymond J. Howanski
  4. John P. Dowling
  5. Nirja B. Patel
  6. Noeleya Henriquez
  7. Chris Nicolescu
  8. Mitchell Nath
  9. Antonio Polley
  10. Jamie L. Bingaman
  11. Todd Smith
  12. Benjamin C. Harman
  13. Matthew K. Robinson
  14. Michael J. Morin
  15. Pavel A. Nikitin

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Abstract

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of V L and V H gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

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    SciScore for 10.1101/2021.01.27.428534: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Donors gave written consent to have their blood drawn and authorized the unrestricted use of their blood samples by Immunome.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After that, cells were washed twice in PBS and stained with a goat anti-human ACE2 polyclonal antibody (R&D Systems, Cat # AF933) or isotype goat polyclonal isotype control (R&D Systems, Cat # AB-108-C) at 1:100 dilution for 30 minutes on ice.
    anti-human ACE2
    suggested: (R and D Systems Cat# AF933, RRID:AB_355722)
    Next, cells were washed twice and stained with Alexa Fluor 488-conjugated donkey anti-goat antibody (Jackson ImmunoResearch, Cat #705-545-147) at 1:200 dilution for 30 minutes on ice.
    anti-goat
    suggested: (Jackson ImmunoResearch Labs Cat# 705-545-147, RRID:AB_2336933)
    HTRF Screening Assays: A homogeneous time-resolved fluorescence (hTRF) assay [26] comprised of terbium-labeled anti-human IgG (H+L) (Cisbio, custom label) donor and AF488-labeled anti-HIS (Cell Signaling, Cat # 14930S) acceptor antibodies was used to screen patient-derived antibodies for their binding to recombinantly produced SARS-CoV-2 antigens.
    anti-human IgG
    suggested: None
    anti-HIS
    suggested: None
    Flow cytometry-based cellular screens for antiviral antibodies: SARS-CoV-2 antigen sequences were cloned into pcDNA3.4 plasmids and transfected into 293F cells utilizing the Expi293 Expression System (Life Technologies, Cat # A14635) per manufacturer’s instructions.
    antiviral antibodies: SARS-CoV-2 antigen sequences
    suggested: None
    antiviral antibodies: SARS-CoV-2
    suggested: None
    A cocktail of Fc-specific secondary antibodies consisting of AF647 goat-anti-human IgG (Jackson ImmunoResearch, Inc.)
    IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293 cells expressing human Angiotensin converting enzyme 2 (ACE2) (BPS Biosciences, Cat #79951) were cultured in EMEM containing 10% FBS and 5 mg/mL Puromycin to select for ACE2-expressing cells.
    HEK293
    suggested: None
    Immunoglobulin expression fragments were cloned into the pcDNA3.4-based vectors and expressed in 293F cells.
    293F
    suggested: RRID:CVCL_D615)
    100 mL of pseudovirus at various dilutions was added to ACE2-293T cells.
    ACE2-293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells: 293TN Producer cell line (System Biosciences, Cat #LV900A-1) was maintained in DMEM containing 10% FBS.
    Cells
    suggested: None
    Data were analyzed using FlowJo software (BD).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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 did not find any issues relating to the usage of bar graphs.

    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 10.1101/2021.01.27.428534 on bioRxiv