Preexisting and de novo humoral immunity to SARS-CoV-2 in humans

  1. Kevin W. Ng
  2. Nikhil Faulkner
  3. Georgina H. Cornish
  4. Annachiara Rosa
  5. Ruth Harvey
  6. Saira Hussain
  7. Rachel Ulferts
  8. Christopher Earl
  9. Antoni G. Wrobel
  10. Donald J. Benton
  11. Chloe Roustan
  12. William Bolland
  13. Rachael Thompson
  14. Ana Agua-Doce
  15. Philip Hobson
  16. Judith Heaney
  17. Hannah Rickman
  18. Stavroula Paraskevopoulou
  19. Catherine F. Houlihan
  20. Kirsty Thomson
  21. Emilie Sanchez
  22. Gee Yen Shin
  23. Moira J. Spyer
  24. Dhira Joshi
  25. Nicola O’Reilly
  26. Philip A. Walker
  27. Svend Kjaer
  28. Andrew Riddell
  29. Catherine Moore
  30. Bethany R. Jebson
  31. Meredyth Wilkinson
  32. Lucy R. Marshall
  33. Elizabeth C. Rosser
  34. Anna Radziszewska
  35. Hannah Peckham
  36. Coziana Ciurtin
  37. Lucy R. Wedderburn
  38. Rupert Beale
  39. Charles Swanton
  40. Sonia Gandhi
  41. Brigitta Stockinger
  42. John McCauley
  43. Steve J. Gamblin
  44. Laura E. McCoy
  45. Peter Cherepanov
  46. Eleni Nastouli
  47. George Kassiotis

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Abstract

Immunological memory after infection with seasonal human coronaviruses (hCoVs) may potentially contribute to cross-protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Ng et al. report that in a cohort of 350 SARS-CoV-2–uninfected individuals, a small proportion had circulating immunoglobulin G (IgG) antibodies that could cross-react with the S2 subunit of the SARS-CoV-2 spike protein (see the Perspective by Guthmiller and Wilson). By contrast, COVID-19 patients generated IgA, IgG, and IgM antibodies that recognized both the S1 and S2 subunits. The anti-S2 antibodies from SARS-CoV-2–uninfected patients showed specific neutralizing activity against both SARS-CoV-2 and SARS-CoV-2 S pseudotypes. A much higher percentage of SARS-CoV-2–uninfected children and adolescents were positive for these antibodies compared with adults. This pattern may be due to the fact that children and adolescents generally have higher hCoV infection rates and a more diverse antibody repertoire, which may explain the age distribution of COVID-19 susceptibility.

Science , this issue p. 1339 ; see also p. 1272

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  1. ScreenIT

    SciScore for 10.1101/2020.05.14.095414: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAll patient sera and sera remaining after antenatal screening of healthy pregnant women were from residual samples prior to discarding, in accordance with Royal College Pathologists guidelines and the UCLH Clinical Governance for assay development and GOSH and ICH regulations.
    Cell Line AuthenticationContamination: Cell lines and virus: HEK293T cells were obtained from the Cell Services facility at The Francis Crick Institute, verified as mycoplasma-free and validated by DNA fingerprinting.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were incubated with sera (diluted 1:50 in PBS) for 30 min, washed with FACS buffer (PBS, 5% BSA, 0.05% Tween 20, 0.05% sodium azide), and stained with BV421 anti-IgG (Biolegend), APC anti-IgM (Biolegend) and PE anti-IgA (Miltenyi Biotech) for 30 min (all antibodies diluted 1:200 in FACS buffer).
    anti-IgG
    suggested: None
    anti-IgM
    suggested: None
    anti-IgA
    suggested: None
    Transfection efficiencies were determined by staining with a fixed concentration of the S1-reactive CR3022 antibody (100 ng/ml) (Absolute Antibodies) and control convalescent sera (1:50 dilution), followed by BV421 anti-IgG antibody.
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    At 24 hours post-infection, cells were fixed in 4% paraformaldehyde and permeabilised with 0.2% Triton-X-100/PBS and virus plaques were visualised by immunostaining, as described previously for the neutralisation of influenza viruses53, except using a rabbit polyclonal anti-NSP8 antibody and anti-rabbit-HRP conjugate and detected by action of HRP on a tetra methyl benzidine (TMB) based substrate.
    anti-NSP8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    anti-rabbit-HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero-E6 cells were from the National Institute for Biological Standards and Control, UK.
    Vero-E6
    suggested: None
    HCoV-NL63 S (UniProt ID: APF29071.1)
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    Lentiviral particle production and neutralisation: Lentiviral particles pseudotyped with either SARS-CoV-2 S or Vesicular Stomatitis Virus glycoprotein (VSVg) were produced by co-transfection of HEK293T cells with plasmids encoding either of these glycoproteins together with a plasmid encoding the SIVmac Gag-Pol polyprotein and a plasmid expressing an HIV-2 backbone with a GFP encoding gene, using GeneJuice (EMD Millipore).
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    SARS-CoV-2 plaque reduction neutralisation test: Confluent monolayers of Vero E6 cells were incubated with 10-20 plaque-forming units (PFU) of SARS-CoV-2 strain hCoV-19/England/2/2020 and two-fold serial dilutions of human sera (previously heat-treated at 56°C for 30 min) starting at 1:40 dilution, for 3 hours at 37°C, 5% CO2, in triplicate per condition.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were run on a Ze5 (Bio-Rad) running Bio-Rad Everest software v2.4 or an LSR Fortessa with a high-throughput sampler (BD Biosciences) running BD FACSDiva software v8.0, and analysed using FlowJo v10
    Bio-Rad Everest
    suggested: None
    BD FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Virus plaques were quantified and IC50 for sera was calculated using LabView software as described previously53.
    LabView
    suggested: (LabView , RRID:SCR_014325)
    Data analysis: Data were analysed and plotted in GraphPad Prism v8 (GraphPad Software) or SigmaPlot v14.0 (Systat Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    SigmaPlot
    suggested: (SigmaPlot, RRID:SCR_003210)
    Sequence alignments were performed with Vector NTI v11.5 (Thermo Fisher Scientific).
    Vector NTI
    suggested: (Vector NTI, RRID:SCR_014265)

    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: 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

    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 10.1126/science.abe1107
  3. ScreenIT

    SciScore for 10.1101/2020.05.14.095414: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableTo validate these findings , we tested samples from an additional cohort of 50 SARS-CoV-2uninfected pregnant women , all of which were negative when considering the presence of IgG , IgM and IgA SARS-CoV-2 S-reactive antibodies ( Extended data Fig . 3) .Cell Line AuthenticationCell lines HEK293T cells were obtained from the Cell Services facility at The Francis Crick Institute , verified as mycoplasma-free and validated by DNA fingerprinting .

    Table 2: Resources

    Antibodies
    SentencesResources
    SARS-CoV-2 S-reactive antibodies , exclusively of the IgG class , were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals with recent HCoV infection and targeted the S2 subunit .
    SARS-CoV-2 S-reactive
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>S2 subunit .</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In contrast , SARS-CoV-2 infection induced higher titres of SARS-CoV-2 Sreactive IgG antibodies , as well as concomitant IgM and IgA antibodies throughout the observation period of 6 weeks since symptoms onset.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Sreactive IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S2 exhibits a higher degree of homology among coronaviruses than S1 ( Extended data Fig . 1 ) and it was likely to be the main target of cross-reactive antibodies6 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antibodies6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Indeed , whereas the addition of recombinant soluble S1 completely abolished binding of the S1-reactive CR3022 antibody8 to SARS-CoV-2 S-expressing cells , it did not affect the binding of HCoV patient sera (</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S1-reactive</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CR3022</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In contrast , IgG antibodies to SARS-CoV-2 S1 or RBD and IgG , IgM and IgA antibodies to S detected by FACS were exclusive to COVID-19 patients ( Fig . 2e) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgG , IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Of note , a sample from an 81-year-old COVID-19 patient collected on day 16 post mild COVID-19 symptoms exhibited only IgG reactivity to SARS-CoV-2 S , but not to S1 , which would be more characteristic of pre-existing antibody memory to HCoVs , than a de novo response to SARS-CoV-2 ( Extended data Fig . 2) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">However , at least 5 of these samples ( all collected in 2018 ) showed evidence for lower levels of SARS-CoV-2 S-reactive IgG antibodies only , which could not have been elicited by SARS-CoV-2 infection ( Extended data Fig . 3) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S-reactive IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The vast majority of these ( 128 of 135 ) had readily detectable IgG , IgM and IgA antibodies to SARS-CoV-2 S ( Extended data Fig . 4) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgA</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S-reactive class-switched antibodies were detected by FACS concurrently with IgM antibodies in our COVID-19 patients , kinetics typically associated with anamnestic responses .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S-reactive class-switched</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Numerous serology assays that are being developed have also detected SARS-CoV-2reactive antibodies in a considerable proportion of SARS-CoV-2-uninfected individuals , which has often been considered as unspecific binding , rather than HCoV-elicited specific antibodies9-20,22 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antibodies9-20,22</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Despite the low numbers , the correlation between neutralising antibodies in HCoV patient sera with SARS-CoV-2 S-binding IgG antibodies determined by FACS was even stronger ( Fig . 3c)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S-binding IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Running the assay in sensitive mode still allows the distinction between pre-existing and de novo antibody responses to SARS-CoV-2 , based on the levels of SARS-CoV-2 S-reactive IgG and parallel detection of IgM and IgA .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV-2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Notably , these samples were described to have low or no SARS-CoV-2 S-reactive IgM antibodies31 , a feature we would associate with immune memory of HCoVs .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S-reactive IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated with sera ( diluted 1:50 in PBS ) for 30 min , washed with FACS buffer ( PBS , 5 % BSA , 0.02 % Tween 20 , 0.05 % sodium azide) , and stained with BV421 anti-IgG ( Biolegend) , APC anti-IgM ( Biolegend ) and PE anti-IgA ( Miltenyi Biotech ) for 30 min ( all antibodies diluted 1:200 in FACS buffer) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-IgG</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sera from COVID-19 patients at University College London Hospitals ( UCLH ) ( Table S1) , contained high levels of IgG , IgM and IgA antibodies recognising the wild-type Spike ( S ) glycoprotein of SARS-CoV-2 expressed on the surface of HEK293T cells , whereas control sera did not ( Fig . 1a)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Moreover , infection with SARS-CoV-2 has recently been shown to increase IgG seroreactivity to HCoVs21 , supporting the existence of shared epitopes .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV-2</b></div>
        <div>suggested: (Sino Biological Cat# 40143-R019, <a href="https://scicrunch.org/resources/Any/search?q=AB_2827973">AB_2827973</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were run on a LSR Fortessa with a high-throughput sampler ( BD Biosciences ) running BD FACSDiva v8.0 , and analysed using FlowJo v10</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BD FACSDiva</b></div>
        <div>suggested: (BD FACSDiva Software, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001456">SCR_001456</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>FlowJo</b></div>
        <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analysis Data were analyses and plotted in GraphPad Prism 7 (GraphPad Software) or SigmaPlot 14.0.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>GraphPad</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>SigmaPlot</b></div>
        <div>suggested: (SigmaPlot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003210">SCR_003210</a>)</div>
      </div>
    </td></tr></table>

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

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.05.14.095414 on bioRxiv