Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual

  1. Emilie Seydoux
  2. Leah J. Homad
  3. Anna J. MacCamy
  4. K. Rachael Parks
  5. Nicholas K. Hurlburt
  6. Madeleine F. Jennewein
  7. Nicholas R. Akins
  8. Andrew B. Stuart
  9. Yu-Hsin Wan
  10. Junli Feng
  11. Rachael E. Nelson
  12. Suruchi Singh
  13. Kristen W. Cohen
  14. M. Juliana McElrath
  15. Janet A. Englund
  16. Helen Y. Chu
  17. Marie Pancera
  18. Andrew T. McGuire
  19. Leonidas Stamatatos

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Abstract

B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.

IN BRIEF

SARS-CoV-2 infection leads to expansion of diverse B cells clones against the viral spike glycoprotein (S). The antibodies bind S with high affinity despite being minimally mutated. Thus, the development of neutralizing antibody responses by vaccination will require the activation of certain naïve B cells without requiring extensive somatic mutation.

Highlights

  • Analysis of early B cell response to SARS-CoV-2 spike protein

  • Most antibodies target non-neutralizing epitopes

  • Potent neutralizing mAb blocks the interaction of the S protein with ACE2

  • Neutralizing antibodies are minimally mutated

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  1. ScreenIT

    SciScore for 10.1101/2020.05.12.091298: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All participants signed informed consent, and the following institutional human subjects review committee approved the protocol prior to study initiation: University of Washington IRB (Seattle, Washington, USA)
    IACUC: All participants signed informed consent, and the following institutional human subjects review committee approved the protocol prior to study initiation: University of Washington IRB (Seattle, Washington, USA)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Plates were washed 4X in wash buffer and the secondary antibody Goat anti-Human Ig-HRP (Southern Biotech, Cat# 2010-05)
    anti-Human Ig-HRP
    suggested: (SouthernBiotech Cat# 2010-05, RRID:AB_2795564)
    The antibody concentration or plasma dilution that neutralized 50% of infectivity (IC50 or ID50, respectively) was interpolated from the neutralization curves determined using the log(inhibitor) vs. response -- Variable slope (four parameters) fit using automatic outlier detection in Graphpad Prism Software.
    IC50
    suggested: (GeneTex Cat# GTX21096, RRID:AB_384608)
    ID50
    suggested: (bNAber Cat# bNAberID_50, RRID:AB_2491067)
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, plasmids expressing the HIV-1 Gag and pol (pHDM-Hgpm2, BEI resources Cat# NR-52517), HIV-1Rev (pRC-CMV-rev1b, BEI resources Cat# NR-52519), HIV-1 Tat (pHDM-tat1b, BEI resources Cat# NR-52518), the SARS CoV2 spike (pHDM-SARS-CoV-2 Spike, BEI resources Cat# NR-52514) and a luciferase/GFP reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W, BEI resources Cat# NR-52516) were co-transfected into 293T cells at a 1:1:1:1.63:4.63 ratio using 293 Free transfection reagent (EMD Millipore Cat# 72181) according to the manufacturer’s instructions.
    293T
    suggested: None
    72 hours later the culture supernatant was harvested, clarified by centrifugation and frozen at −80°C. 293 cells stably expressing ACE2 (BEI resources Cat# NR-5251) were seeded at a density of 4 X103 cells/well in a 100µl volume in 96 well flat bottom tissue culture plates.
    293
    suggested: None
    The media was aspirated from 293T-ACE2 cells and 100µl of the virus-antibody mixture was added.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Software and Algorithms
    SentencesResources
    The RBD+ frequency of sorted B cells was analyzed post-sort using the index file data in Flow Jo version 9.9.4 (Becton, Dickinson and Company).
    Flow Jo
    suggested: (FlowJo, RRID:SCR_008520)
    To quantify the number of amino acid mutations, the sequence alignments were exported from Geneious and imported into R (Version 3.4.1) for analysis (R Core Team, 2017
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    This analysis uses the packages Biostrings (Pages H, 2018), seqinr (Charif D, 2007), and tidyverse (Wickham, 2017) in R andGraphPad Prism were used to create graphs.
    Biostrings
    suggested: (Biostrings, RRID:SCR_016949)
    The antibody concentration or plasma dilution that neutralized 50% of infectivity (IC50 or ID50, respectively) was interpolated from the neutralization curves determined using the log(inhibitor) vs. response -- Variable slope (four parameters) fit using automatic outlier detection in Graphpad Prism Software.
    Graphpad Prism
    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 33. 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.

    About SciScore

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  2. Version published to 10.1101/2020.05.12.091298v1 on bioRxiv