Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence

  1. Gemma E. Hartley
  2. Emily S.J. Edwards
  3. Pei M. Aui
  4. Nirupama Varese
  5. Stephanie Stojanovic
  6. James McMahon
  7. Anton Y. Peleg
  8. Irene Boo
  9. Heidi E. Drummer
  10. P. Mark Hogarth
  11. Robyn E. O’Hehir
  12. Menno C. van Zelm

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Abstract

Memory B cells specific for SARS-CoV-2 spike and nucleocapsid proteins persist in peripheral blood after recovery from COVID-19.

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  1. Version published to 10.1126/sciimmunol.abf8891
  2. ScreenIT

    SciScore for 10.1101/2020.11.17.20233544: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Participants: Individuals with a PCR-confirmed diagnosis of COVID-19 and uninfected controls were enrolled in research studies to examine their peripheral blood B- and T-cell subsets (projects: Alfred Health Human Research and Ethics Committee Numbers 182/20 and 202/20, Monash University 2016-0289 and 2020-26385).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, 50 μl of whole blood was added to a Trucount tube (BD Biosciences) together with a 20 μl antibody cocktail containing antibodies to CD3, CD4, CD8, CD16, CD19, CD56 and CD45 (Supplementary Tables 1 and 2) and incubated for 15 minutes at room temperature in the dark.
    CD4
    suggested: (BioLegend Cat# 391503, RRID:AB_2721611)
    CD8
    suggested: (BioLegend Cat# 391503, RRID:AB_2721611)
    CD16
    suggested: (RayBiotech Cat# CS-11-0019, RRID:AB_1227882)
    CD56
    suggested: None
    CD45
    suggested: None
    For detection of antigen-specific B cells, 12.5 million PBMC were incubated with fixable viability stain 700 (BD Biosciences), antibodies against CD3, CD19, CD21, CD27, CD38, CD71, IgA, IgD, IgG1, IgG2, IgG3, IgG4, (Supplementary Tables 1 and 2) and 5 μg/ml (total of 1.25 ug per 250 µl stain) each of [NCP]4-BUV395, [NCP]4-BUV737, [RBD]4-BV480 and [RBD]4-BV650 for 15 minutes at room temperature in a total volume of 250 μl FACS buffer (0.1% sodium azide, 0.2% BSA in PBS).
    CD19
    suggested: (Agilent Cat# TC67401, RRID:AB_579635)
    CD21
    suggested: None
    CD27
    suggested: None
    CD38
    suggested: (Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay Cat# INQ-T10, RRID:AB_2721895)
    CD71
    suggested: None
    IgG1
    suggested: (LSBio (LifeSpan Cat# LS-C86863-250, RRID:AB_1665439)
    IgG4
    suggested: (LSBio (LifeSpan Cat# LS-C86863-250, RRID:AB_1665439)
    In addition, 5 million PBMC were similarly incubated with fixable viability stain 700 (BD Biosciences), antibodies against CD3, CD19, CD27 and IgD, plus BUV395-, BUV737-, BV480- and BV650-conjugated streptavidin controls (
    CD3
    suggested: None
    IgD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Measurement of SARS-CoV-2 neutralizing antibodies in plasma: Measurement of neutralizing antibodies was performed using SARS-CoV-2 retroviral pseudotyped particles and a 293T-ACE2 cell line (Crawford et al., 2020) as described before (Jackson et al., 2020).
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Software and Algorithms
    SentencesResources
    Briefly, 200 µl was used for whole blood cell counts (Cell Dyn analyser; Abbott core laboratory, Abbott Park, IL) and Trucount analysis (see flow cytometry section).
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    The percentage entry was plotted against the reciprocal dilution of plasma in GraphPad Prism 8 Software (GraphPad Software, La Jolla, CA) and curves fitted with a one-site specific binding Hill plot.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data analysis and statistics: All flow cytometry data was analyzed with FlowJo v10 software (
    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: We detected the following sentences addressing limitations in the study:
    To further overcome this limitation, we used non-protein polymer fluorochromes, which exhibited minimal non-specific B-cell binding and increased the sensitivity of our assay (Hartley et al., 2020). Our study, for the first time shows the kinetics and longevity of SARS-CoV-2-specific Bmem cell numbers. Other studies have identified SARS-CoV-2-specific B cells in COVID-19 patients with particular focus on the RBD of the spike protein, mainly for the purpose of cloning neutralizing SARS-CoV-2-specific antibodies (Juno et al., 2020; Nguyen-Contant et al., 2020; Robbiani et al., 2020; Rogers et al., 2020; Wilson et al., 2020). Such studies have observed a predominant IgG+ B-cell response to SARS-CoV-2 with lower frequencies of cells expressing IgM and IgA (Brouwer et al., 2020; Juno et al., 2020; Rodda et al., 2020). We have expanded on this through detailed flow cytometry with the inclusion of absolute cell counts to show that SARS-CoV-2-specific Bmem cells predominantly expressed IgM or IgG1. This distinction enabled the discrimination between the initially large fraction of IgM+ Bmem cells that tended to decline beyond 150 days, while the IgG expressing fraction persisted and those specific for RBD correlated strongly with circulating Tfh cells. The Bmem cell populations directed against the two SARS-CoV-2 targets showed remarkable differences. The RBD-specific Bmem cells were nearly all CD27+ and strongly correlated with Tfh cells, while this was not observed for NCP-specific...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 34. 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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  3. Version published to 10.1101/2020.11.17.20233544v1 on medRxiv