Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods

  1. Berislav Bošnjak
  2. Saskia Catherina Stein
  3. Stefanie Willenzon
  4. Anne Katrin Cordes
  5. Wolfram Puppe
  6. Günter Bernhardt
  7. Inga Ravens
  8. Christiane Ritter
  9. Christian R. Schultze-Florey
  10. Nina Gödecke
  11. Jörg Martens
  12. Hannah Kleine-Weber
  13. Markus Hoffmann
  14. Anne Cossmann
  15. Mustafa Yilmaz
  16. Isabelle Pink
  17. Marius M. Hoeper
  18. Georg M. N. Behrens
  19. Stefan Pöhlmann
  20. Rainer Blasczyk
  21. Thomas F. Schulz
  22. Reinhold Förster

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Abstract

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

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  1. Version published to 10.1038/s41423-020-00573-9
  2. ScreenIT

    SciScore for 10.1101/2020.07.12.20151407: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consent was obtained from all participants.
    IRB: Studies investigating serum samples from healthy controls and COVID-19 patients were approved by the HMS institutional review board (#9001_BO_K2020 and #7901_BO_K2018).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Unbound SARS-CoV-2-S-RBD was removed by four PBST washes before anti-His peroxidase-labelled monoclonal antibody (mAb; Clone 3D5, prepared in house) in carrier buffer was added for 1h at 37°C.
    anti-His
    suggested: None
    To test for the presence of neutralizing anti-SARS-CoV-2-S serum antibodies, 6 ng of SARS-CoV-2 S RBD was pre-incubated with test sera at final dilutions between 1:20 to 1:540 as indicated on the graphs for 1 h at 37°C, before adding them to plates coated with 150 or 300 ng/well ACE2.
    anti-SARS-CoV-2-S
    suggested: None
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In brief, pseudotyped VSV particles were produced by calcium-phosphate transfecting HEK293T cells with expression plasmids for the respective glycoproteins, either pCAGGS-VSV-G (35) for expression of VSV-G of the control virus or pCG1-SARS-2-SΔ18 (36) for the SARS-CoV2 spike protein.
    HEK293T
    suggested: None
    This VSV*ΔG-FLuc stock virus was propagated in BHK-21 G43 cells (38).
    BHK-21 G43
    suggested: None
    Medium was removed from Vero76 cells and replaced in triplicate wells with the serum/pseudotype particle mixture.
    Vero76
    suggested: IZSLER Cat# BS CL 101, RRID:CVCL_0603)
    Software and Algorithms
    SentencesResources
    All statistical analyses were conducted using GraphPad Prism 8.4 (GraphPad Software, USA).
    GraphPad
    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 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: 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.

  3. Version published to 10.1101/2020.07.12.20151407v1 on medRxiv