Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses

  1. Eric D. Laing
  2. Spencer L. Sterling
  3. Stephanie A. Richard
  4. Nusrat J. Epsi
  5. Si’Ana Coggins
  6. Emily C. Samuels
  7. Shreshta Phogat
  8. Lianying Yan
  9. Nicole Moreno
  10. Christian L. Coles
  11. Matthew Drew
  12. Jennifer Mehalko
  13. Caroline E. English
  14. Scott Merritt
  15. Katrin Mende
  16. Vincent J. Munster
  17. Emmie de Wit
  18. Kevin K. Chung
  19. Eugene V. Millar
  20. David R. Tribble
  21. Mark P. Simons
  22. Simon D. Pollett
  23. Brian K. Agan
  24. Dominic Esposito
  25. Charlotte Lanteri
  26. G. Travis Clifton
  27. Edward Mitre
  28. Timothy H. Burgess
  29. Christopher C. Broder

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Abstract

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 sero-surveillance. With the rollout of SARS-CoV-2 vaccines, such assays must be able to distinguish vaccine from natural immunity to SARS-CoV-2 and related human coronaviruses. Here, we developed and implemented multiplex microsphere-based immunoassay strategies for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2 and the endemic seasonal human coronaviruses (HCoV), enabling high throughout measurement of pre-existing cross-reactive antibodies. We varied SARS-CoV-2 antigen compositions within the multiplex assay, allowing direct comparisons of the effects of spike protein, receptor-binding domain protein (RBD) and nucleocapsid protein (NP) based SARS-CoV-2 antibody detection. Multiplex immunoassay performance characteristics are antigen-dependent, and sensitivities and specificities range 92-99% and 94-100%, respectively, for human subject samples collected as early as 7-10 days from symptom onset. SARS-CoV-2 spike and RBD had a strong correlative relationship for the detection of IgG. Correlation between detectable IgG reactive with spike and NP also had strong relationship, however, several PCR-positive and spike IgG-positive serum samples were NP IgG-negative. This spike and NP multiplex immunoassay has the potential to be useful for differentiation between vaccination and natural infection induced antibody responses. We also assessed the induction of de novo SARS-CoV-2 IgG cross reactions with SARS-CoV and MERS-CoV spike proteins. Furthermore, multiplex immunoassays that incorporate spike proteins of SARS-CoV-2 and HCoVs will permit investigations into the influence of HCoV antibodies on COVID-19 clinical outcomes and SARS-CoV-2 antibody durability.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.10.21251518: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Participant enrollment and sera collection: SARS-CoV-2 negative human serum specimens utilized were from sera collected from 2012 – 2018 in the ARIC Natural History Study (IDCRP-045) (32).
    IRB: ARIC (IDCRP-045), EPICC (IDCRP-085) and CAMP-NYC protocols were approved by the Uniformed Services University Institutional Review Board.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Secondary antibody (goat anti-human IgG cross-absorbed biotin-conjugated or goat anti-human IgM cross-absorbed biotin-conjugated; Thermo Fisher Scientific, Waltham, MA) was diluted 1:5000 in 1XPBS + 0.05% Tween20 (PBST) and 100 µL of each secondary was added to each well and incubated for 45 minutes with agitation, and plates were washed three times.
    anti-human IgG
    suggested: (LSBio (LifeSpan Cat# LS-C22913-30, RRID:AB_899274)
    anti-human IgM
    suggested: (LSBio (LifeSpan Cat# LS-C5000-1000, RRID:AB_858362)
    We established a cut-off of three standard deviations above the mean (99.7% probability) MFI of archival HCoV PCR-confirmed convalescent serum samples (n= 43) to establish a positivity threshold for detection of SARS-CoV-2 spike protein reactive IgG and IgM antibodies.
    SARS-CoV-2 spike protein reactive IgG
    suggested: None
    IgM
    suggested: None
    SARS-CoV-2 IgG antibody seroconversion was determined as a 4-fold increase in MFI compared to the baseline sera collection.
    SARS-CoV-2 IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The alphacoronavirus (α-CoV) HCoV-229E and HCoV-NL63 spike proteins were similarly constructed and prepared (LakePharma, Inc.).
    HCoV-229E
    suggested: JCRB Cat# JCRB1838, RRID:CVCL_B3M4)
    A mock antigen, consisting of cell culture supernatant from non-transfected HEK cells was collected via centrifugation then filtered through a 0.22 µM PES filter to remove debris.
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Figures were generated and statistical analyses were performed in GraphPad Prism version 7.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The positive predictive value and negative predictive value were calculated with MedCalc statistical software.
    MedCalc
    suggested: (MedCalc, RRID:SCR_015044)

    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:
    Whereas establishing the SARS-CoV-2 spike protein and NP MMIA will have utility for the future differentiation between antibody responses to SARS-CoV-2 vaccination and natural infection induced antibody responses, with some inherent limitations to sensitivity and specificity. Of additional importance, the MMIA approach demonstrated de novo IgG cross-reactivity with SARS-CoV and MERS-CoV in the SARS-CoV-2 PCR+/IgG+ EPICC and JMS cohorts compared with archival sera (ARIC). The EPICC geometric mean IgG levels reactive with SARS-CoV spike were near the indeterminate range of positivity, suggesting that significant rises in IgG levels after SARS-CoV-2 are driven by a subset of subjects that develop specific B cell repertoires that can be cross-reactive with SARS-CoV. A higher geometric mean IgG level to SARS-CoV and MERS-CoV were detected in JMS sera, which includes all hospitalized patients and suggests that induction of cross-reactivity is associated with COVID-19 severity. Conserved cross-neutralizing epitopes between SARS-CoV and SARS-CoV-2 S glycoproteins have been identified (52, 53), whether SARS-CoV-2 induced de novo IgG antibody responses to SARS-CoV and MERS-CoV spike proteins detected with this MMIA strategy are retained after affinity maturation, or are cross-neutralizing requires further investigation. The induction of cross-reactive SARS-CoV, SARS-CoV-2 and MERS-CoV antibodies further demonstrates that shared spike proteins epitopes exist and that rational-vaccine de...

    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.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.02.10.21251518 on medRxiv