Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases

  1. Javier Castillo-Olivares
  2. David A. Wells
  3. Matteo Ferrari
  4. Andrew C. Y. Chan
  5. Peter Smith
  6. Angalee Nadesalingam
  7. Minna Paloniemi
  8. George W. Carnell
  9. Luis Ohlendorf
  10. Diego Cantoni
  11. Martin Mayora-Neto
  12. Phil Palmer
  13. Paul Tonks
  14. Nigel J. Temperton
  15. David Peterhoff
  16. Patrick Neckermann
  17. Ralf Wagner
  18. Rainer Doffinger
  19. Sarah Kempster
  20. Ashley D. Otter
  21. Amanda Semper
  22. Tim Brooks
  23. Anna Albecka
  24. Leo C. James
  25. Mark Page
  26. Wilhelm Schwaeble
  27. Helen Baxendale
  28. Jonathan L. Heeney

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Abstract

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

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  1. Version published to 10.3389/fimmu.2021.748291
  2. ScreenIT

    SciScore for 10.1101/2021.05.21.21257572: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: COVID-19 patients hospitalised during the first wave and as well as NHS healthcare workers working at the Royal Papworth Hospital in Cambridge, UK served as the exposed HCW cohort (Study approved by Research Ethics Committee Wales, IRAS: 96194 12/WA/0148.
    Consent: All participants provided written, informed consent prior before enrolment in the study.
    Sex as a biological variablenot detected.
    RandomizationTo minimise noise between experimental runs the gradient, minimum, and maximum parameters were estimated based on a random subset of 200 samples and fixed for all other samples.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ) Seropositive HCW (n=24 samples); and c) Seronegative HCW (n=39) (Table 2). 2.2. Internal and External Calibration Sera: The reference antisera used as external, or primary, calibrants in our assays included: a) the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (NIBSC 20/136); b) the Anti-SARS-CoV-2 Antibody Diagnostic Calibrant (NIBSC 20/162; and c) the Research Reagent for anti-SARS-CoV-2 Ab (NIBSC 20/130).
    anti-SARS-CoV-2 immunoglobulin
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    Anti-nucleocapsid protein antibodies were detected using the qualitative Roche Elecsys® Anti-SARS-CoV-2 (ACOV2) ECLIA (Product code: 09203079190), whilst anti-RBD antibodies were detected using the quantitative Roche Elecsys® Anti-SARS-CoV-2 S (ACOV2 S) ECLIA (Product code 092892751902), as previously described 29,30.
    Anti-nucleocapsid protein
    suggested: None
    anti-RBD
    suggested: None
    Anti-spike results are expressed as units per ml (U/ml), with results of ≥ 0.8 U/ml interpreted as positive and a quantitative range of 0.4 to 2,500 U/ml. 2.6. Detection of SARS-CoV-2-S, -RBD and -N specific antibodies using a multiplex bead flow cytometry platform, Luminex™ platform: Detection of serum IgG reactive to SARS-CoV-2 N, S and RBD (receptor binding domain) antigens was done using a Luminex based assay following the methods previously described 31,32.
    RBD ( receptor binding domain )
    suggested: None
    Plasma IgG antibodies reactive against the SARS-CoV-2 Spike and Nucleocapsid proteins were analysed by immuno-blotting using the ‘Jess’ fully automated system (ProteinSimple; Bio-Techne) and the SARS-CoV-2 Multi-Antigen Serology Module (ProteinSimple; Bio-Techne, SA-001), following the manufacturer’s instructions.
    Plasma IgG
    suggested: None
    For the secondary antibody, ready to use HRP-conjugated goat anti-human IgG, IgA or IgM antibody was used.
    anti-human IgG
    suggested: None
    IgM
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells were maintained in DMEM containing 10% foetal bovine serum and 1% penicillin/streptomycin, at 37° C and 5% CO2.
    HEK293T
    suggested: None
    Vero ACE2/TMPRSS2 cells 35 were washed with PBS and resuspended in Buffer R (Thermo Fisher) at a concentration of 1 × 106 cells ml-1.
    Vero ACE2/TMPRSS2
    suggested: None
    Recombinant DNA
    SentencesResources
    Cells were transfected with 1000 ng of pcDNA-SARS-CoV-2 Spike plasmid, 1000ng of HIV 8.91 gag/pol plasmid and 1500ng of pCSFLW luciferase plasmid, using FuGENE HD (Promega, UK,) at a 1:3 ratio (plasmid:FuGENE HD).
    pcDNA-SARS-CoV-2
    suggested: None
    pCSFLW
    suggested: None

    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.
    • Thank you for including a protocol registration statement.

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

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  3. Version published to 10.1101/2021.05.21.21257572v1 on medRxiv