Severe Acute Respiratory Syndrome Coronavirus 2 Seroassay Performance and Optimization in a Population With High Background Reactivity in Mali

  1. John Woodford
  2. Issaka Sagara
  3. Alassane Dicko
  4. Amatigue Zeguime
  5. M’Bouye Doucoure
  6. Jennifer Kwan
  7. Irfan Zaidi
  8. Justin Doritchamou
  9. Maryonne Snow-Smith
  10. Nada Alani
  11. Jonathan Renn
  12. Ivan Kosik
  13. Jaroslav Holly
  14. Jonathan Yewdell
  15. Dominic Esposito
  16. Kaitlyn Sadtler
  17. Patrick Duffy

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Abstract

Background

False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa.

Methods

From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs.

Results

Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6–89.8]; specificity, 99.4% [95% CI, 97.7–99.9]).

Conclusions

We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.

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  1. Version published to 10.1093/infdis/jiab498
  2. ScreenIT

    SciScore for 10.1101/2021.03.08.21252784: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All pre-pandemic samples were collected during NIH-sponsored studies that were approved by the Malian USTTB FMPOS human research ethics committee and the NIAID/NIH institutional review board and were conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableThe four geographically distinct contributing sites were Sotuba, an urban center study population of healthy adults in urban Bamako; Bancoumana, a rural study population of healthy adults located along a major road transport route between Mali and Guinea; Ouelessebougou, a rural study population of women in their childbearing years located along a major road transport route between Mali and two neighboring countries, Burkina Faso and Cote d’Ivoire; and Kalifabougou, a rural study population of all ages.
    Cell Line AuthenticationContamination: BHK-21-ACE2 cell production: The BHK-21 cell line was maintained in DMEM (Gibco, 10566016) supplemented with 10% FBS, grown at 37°C, 9% CO2 humid atmosphere and tested mycoplasma-negative using Universal Mycoplasma Detection Kit (ATCC 30-1012K).

    Table 2: Resources

    Antibodies
    SentencesResources
    Negative control samples were tested by ELISA at LMIV/NIAID, Bethesda, for antibodies to SARS-COV-2 spike protein, RBD and NCP, and the spike proteins of SARS-CoV-1, MERS-CoV, OC43 and HKU1 (Table 1).
    SARS-COV-2 spike protein, RBD
    suggested: None
    HKU1
    suggested: None
    One hundred (100) microliters of goat anti-Human IgG (H+L) Cross-Adsorbed secondary antibody, HRP (ThermooFisher) diluted at 1:4000 in blocking buffer was added to wells, incubated for 1 hour at room temperature, then washed three times with 300 µL of wash buffer.
    goat anti-Human IgG
    suggested: None
    anti-Human IgG
    suggested: None
    The surface expression of ACE2 was confirmed by flow cytometry using anti-human ACE2 AlexaFluor 647-conjugated antibody (R&D Systems, FAB9332R) (Supplementary Figure 1).
    anti-human ACE2
    suggested: None
    The expression was further confirmed by western blot using ACE2 Antibody (Cell Signaling Technology, #4355).
    ACE2
    suggested: (Cell Signaling Technology Cat# 4355, RRID:AB_2797606)
    To exclude the contribution of potentially recycled VSV-Gp, we compared the susceptibility to infection of wtBHK21 and BHK21-ACE2 cells and infectious titer in the presence and absence of the 1E9F11 and 8G5F11 anti-VSV-Gp antibody mixture (both 10 µg/mL).
    anti-VSV-Gp
    suggested: None
    This is a two-antigen assay to detect antibodies to SARS-CoV-2 spike protein and RBD, developed for use in US population serosurveillance studies [14].
    SARS-CoV-2 spike protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expi293 cells were transfected using 1.4 mL of ExpiFectamine (Thermo Fisher) and 0.5 mg of plasmid DNA per 0.5 L of cells.
    Expi293
    suggested: RRID:CVCL_D615)
    Forty thousand BHK21-ACE2 cells dissociated with 0.5% Trypsin EDTA, no-phenol red (Gibco), and diluted in DPBS (Gibco) were transferred to each well of a new non-tissue culture treated polypropylene 96 round-bottom well plate (Corning).
    BHK21-ACE2
    suggested: None
    BHK-21-ACE2 cell production: The BHK-21 cell line was maintained in DMEM (Gibco, 10566016) supplemented with 10% FBS, grown at 37°C, 9% CO2 humid atmosphere and tested mycoplasma-negative using Universal Mycoplasma Detection Kit (ATCC 30-1012K).
    BHK-21-ACE2
    suggested: None
    BHK-21
    suggested: None
    Briefly, a T75 tissue flask was seeded with five to seven million 293T cells (60-70% confluency) in DMEM (Gibco) supplemented with 8% FBS (Hyclone) and 10mM HEPES (Corning).
    293T
    suggested: None
    VSVdG-EGFP-VSV-Gp P3 pseudotyped virus was titrated on BHK21 cells.
    BHK21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    To generate VSVdG-EGFP-SARS2-Sgp, a T75 tissue flask was seeded with eight million BHK21-SARS2-Sgp-CTd16aa cells in DMEM (Gibco) supplemented with 8% FBS (Hyclone) and 10 mM HEPES (Corning).
    BHK21-SARS2-Sgp-CTd16aa
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    SARS-CoV-2 antigens and other betacoronavirus spike proteins: SARS-CoV-2 full-length spike protein (VRC-SARS-CoV-2 S-2P-3C-His8-Strep2×2 [10]) and RBD protein (Ragon-SARS-CoV-2 S-RBD(319-529)-3C-His8-SBP [11]) and the full-length spike proteins for the other betacoronaviruses SARS-CoV-1, MERS-CoV, OC43 and HKU1 [12] were produced as previously described.
    VRC-SARS-CoV-2 S-2P-3C-His8-Strep2×2
    suggested: None
    Ragon-SARS-CoV-2 S-RBD(319-529)-3C-His8-SBP
    suggested: None
    Software and Algorithms
    SentencesResources
    One hundred (100) microliters of goat anti-Human IgG (H+L) Cross-Adsorbed secondary antibody, HRP (ThermooFisher) diluted at 1:4000 in blocking buffer was added to wells, incubated for 1 hour at room temperature, then washed three times with 300 µL of wash buffer.
    ThermooFisher
    suggested: None
    We performed the analysis performed using FlowJo software (TreeStar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Data analysis: Data were analyzed using Microsoft Excel and GraphPad Prism7 software.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    GraphPad Prism7
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT02942277CompletedSafety and Immunogenicity of Pfs25M-EPA/AS01 and Pfs230D1M-E…
    NCT03952650RecruitingSanaria PfSPZ Challenge With Pyrimethamine or Chloroquine Ch…
    NCT03989102Active, not recruitingSafety, Immunogenicity, and Protective Efficacy of Radiation…
    NCT01322581RecruitingA Longitudinal Systems Biological Analysis of Naturally Acqu…
    NCT03083847CompletedDose Escalation PfSPZ-CVac

    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 11. 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.

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  3. Version published to 10.1101/2021.03.08.21252784v1 on medRxiv