Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection

  1. Natalie S. Haddad
  2. Doan C. Nguyen
  3. Merin E. Kuruvilla
  4. Andrea Morrison-Porter
  5. Fabliha Anam
  6. Kevin S. Cashman
  7. Richard P. Ramonell
  8. Shuya Kyu
  9. Ankur Singh Saini
  10. Monica Cabrera-Mora
  11. Andrew Derrico
  12. David Alter
  13. John D. Roback
  14. Michael Horwath
  15. James B. O’Keefe
  16. Henry M. Wu
  17. An-Kwok Ian Wong
  18. Alexandra W. Dretler
  19. Ria Gripaldo
  20. Andrea N. Lane
  21. Hao Wu
  22. Saeyun Lee
  23. Mindy Hernandez
  24. Vanessa Engineer
  25. John Varghese
  26. Sang Le
  27. Iñaki Sanz
  28. John L. Daiss
  29. F. Eun-Hyung Lee

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Abstract

Background

SARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential.

Methods

We developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD).

Results

To diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values −0.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months.

Conclusion

This SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness.

One Sentence Summary

In contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.04.410589: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Institutional Approval for Patient Sample Collection: All blood and tissue samples were collected under the Emory University Institutional Review Board approved protocols.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Luminex immunoassays for measurement of anti-SARS-CoV-2 antibodies: Fifty μL coupled microsphere mix was added to each well of clear-bottomed, 96-well, black, polystyrene microplates (Greiner Bio-One North America Inc.,
    anti-SARS-CoV-2
    suggested: None
    Median Fluorescent Intensity (MFI) using combined or individual detection antibodies (anti-IgA/anti-IgG/anti-IgM) was measured using the Luminex xPONENT software.
    anti-IgA/anti-IgG/anti-IgM
    suggested: None
    Monoclonal antibodies CR3022 (human IgG1 anti-S1-RBD(39); InvivoGen) and 6G9 (human:mouse chimeric IgG anti-N; Amsbio) were used as calibrators for SARS-CoV-2 antibody concentrations.
    human IgG1 anti-S1-RBD(39)
    suggested: None
    human IgG1
    suggested: None
    human:mouse chimeric IgG anti-N; Amsbio
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The Nucleocapsid protein (N; Cat. No. Z03480; expressed in E. coli), the S1 domain (S1; amino acids 16-685; Cat. No. Z03485; expressed in HEK293 cells) of the Spike protein, and the S1-Receptor Binding Domain (S1-RBD; Cat.
    HEK293
    suggested: None
    Software and Algorithms
    SentencesResources
    1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; Thermo Fisher Scientific; Waltham, MA, USA), and 5 mg/mL Sulfo-NHS (N-hydroxysulfosuccinimide; Thermo Fisher Scientific; Waltham, MA, USA)
    Thermo Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    After 30 minutes of incubation at 800 rpm in the dark, wells were washed three times in 100 μL of assay buffer, resuspended in 100 μL of assay buffer, and analyzed using a Luminex FLEXMAP 3D® instrument (Luminex; Austin, TX, USA) running xPonent 4.3
    xPonent
    suggested: None
    Comparisons among and between groups were made using one-sided ANOVA or two-sided t-tests 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.
    • No funding statement was detected.
    • 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 10.1101/2020.12.04.410589v1 on bioRxiv