Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects

  1. Madeleine F. Jennewein
  2. Anna J. MacCamy
  3. Nicholas R. Akins
  4. Junli Feng
  5. Leah J. Homad
  6. Nicholas K. Hurlburt
  7. Emily Seydoux
  8. Yu-Hsin Wan
  9. Andrew B. Stuart
  10. Venkata Viswanadh Edara
  11. Katharine Floyd
  12. Abigail Vanderheiden
  13. John R. Mascola
  14. Nicole Doria-Rose
  15. Lingshu Wang
  16. Eun Sung Yang
  17. Helen Y. Chu
  18. Jonathan L. Torres
  19. Gabriel Ozorowski
  20. Andrew B. Ward
  21. Rachael E. Whaley
  22. Kristen W. Cohen
  23. Marie Pancera
  24. M. Juliana McElrath
  25. Janet A. Englund
  26. Andrés Finzi
  27. Mehul S. Suthar
  28. Andrew T. McGuire
  29. Leonidas Stamatatos

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Abstract

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

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    SciScore for 10.1101/2021.03.23.436684: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Human subjects: Blood and peripheral blood mononuclear cells (PBMCs) were isolated from COVID19+ patients using protocols approved by Institutional Review Boards at Fred Hutch Cancer Research Center, University of Washington and Seattle Children’s Research Institute.
    Consent: Informed consent was obtained from all participants and the University of Washington and/or Fred Hutchinson Cancer Research Center and CHUM Institutional Review Boards approved the entire study and procedures.
    IACUC: All experiments adhered to the guidelines approved by the Emory University Institutional Animal Care and Committee.
    Randomizationnot detected.
    BlindingPeripheral blood mononuclear cells (PBMCs) and serum from pre-pandemic controls were blindly selected at random from the study “Establishing Immunologic Assays for Determining HIV-1 Prevention and Control”, with no considerations made for age, or sex, participants were recruited at the Seattle Vaccine Trials Unit (Seattle, Washington, USA).
    Power Analysisnot detected.
    Sex as a biological variableCell Lines: All cell lines were incubated at 37°C in the presence of 5% CO2. 293-6E (human female, RRID:CVCL_HF20) and 293T cells (human female, RRID:CVCL_0063) cells were maintained in Freestyle 293 media with gentle shaking.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    We recently reported an initial characterization of the anti-S antibody responses generated by CV1 (Seydoux et al., 2020).
    anti-S
    suggested: None
    Some samples appear to show enhanced binding in the presence of ACE2, perhaps because ACE2 binding stabilizes and exposes their binding sites, these antibodies are considered not competitive with ACE2. mAb competition BLI: To measure competition between individual mAbs for binding to SARS-CoV-2 S-2P and RBD, S-2P and RBD were biotinylated using EZ-Link NHS-PEG4 Biotin at a molar ratio of 1:2/ Biotinylated protein was purified using a Zeba spin desalting column.
    ACE2
    suggested: None
    SARS-CoV-2
    suggested: None
    S-2P
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E S1+S2 ECTs (Cat#’s 40607-V08B, 40606-V08B, 40604-V08B, 40605-V08D), SARS-HCoV-2 S1 domain (CAT#: 40591-V08B1), SARS-CoV-2 S1 N-terminal domain (CAT#40591-V41H) SARS-HCoV-2 S2 extra-cellular domain (CAT#: 40590-V08B) and SARS-CoV2 RBD (CAT#: 40150-V05H) were purchased from SinoBiologicals.
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    Cell Lines: All cell lines were incubated at 37°C in the presence of 5% CO2. 293-6E (human female, RRID:CVCL_HF20) and 293T cells (human female, RRID:CVCL_0063) cells were maintained in Freestyle 293 media with gentle shaking.
    the
    detected: ( RRID:CVCL_HF20)
    293T
    detected: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Briefly, plasmids expressing the HIV-1 Gag and pol (pHDM-Hgpm2), HIV-1Rev (pRC-CMV-rev1b), HIV-1 Tat (pHDM-tat1b), the SARS CoV2 spike (pHDM-SARS-CoV-2 Spike) and a luciferase/GFP reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W) were co-transfected into 293T cells at a 1:1:1:1.6:4.6 ratio using 293 Free transfection reagent (EMD Millipore Cat #72181) according to the manufacturer’s instructions.
    293T
    suggested: None
    The culture supernatant was harvested after 72 hours at 32°C, clarified by centrifugation, filtered and frozen at −80C. 293 cells stably expressing ACE2 (HEK293T-hACE2) were seeded at a density of 4000 cells/well in a 100 µl volume in flat clear bottom, black walled, tissue culture 96-well plates.
    293
    suggested: None
    The media was aspirated from 293T-ACE2 cells and 100 µl of the virus-antibody mixture was added.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Monitoring RBD-binding to 293-ACE2 cells by flow cytometry: 8 pmol of biotinylated S-2P with strep tag peptide sequence on C terminus were mixed with 10 pmol of mAb and incubated for 10 min at RT in a round-bottom tissue culture 96-well plate. 200,000 HEK293T-hACE2 cells in 50 µL of cDMEM were then added to each well and the mixture of cells + RBD or S-2P + mAb was incubated for 20 min on ice.
    293-ACE2
    suggested: RRID:CVCL_DR94)
    Experimental Models: Organisms/Strains
    SentencesResources
    Infection of k18-hACE2 mice with SARS-CoV-2: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from Jackson Laboratories.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Software and Algorithms
    SentencesResources
    IMGT/V-QUEST was used to assign V, D, J gene identity, and CDRL3 length to the sequences (Brochet et al., 2008).
    IMGT/V-QUEST
    suggested: (IMGT/V-QUEST, RRID:SCR_010749)
    The V(D)J enriched library was sequenced on an Illumina HiSeq or MiSeq.
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Remaining model building was completed using COOT (Emsley and Cowtan, 2004) and refinement was performed in Phenix (Adams et al., 2004).
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Structural figures were made in Pymol.
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Particles were picked with DogPicker (Voss et al., 2009) and processed in RELION 3.0 (Scheres, 2012).
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Sequence analysis: Sequences were analyzed using Geneious software (Version 8.1.9)
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    Statistical Analysis: All graphs were completed using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 10.1101/2021.03.23.436684v1 on bioRxiv