IL-2 and IFN-γ are biomarkers of SARS-CoV-2 specific cellular response in whole blood stimulation assays

  1. Begoña Pérez-Cabezas
  2. Ricardo Ribeiro
  3. Inês Costa
  4. Sofia Esteves
  5. Ana Rafaela Teixeira
  6. Teresa Reis
  7. Ricardo Monteiro
  8. Alexandre Afonso
  9. Vitor Pinheiro
  10. Maria Isabel Antunes
  11. Maria Lucília Araújo
  12. João Niza Ribeiro
  13. Anabela Cordeiro-da-Silva
  14. Nuno Santarém
  15. Joana Tavares

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Abstract

A proper description of the immune response to SARS-CoV-2 will be critical for the assessment of protection elicited after both infection and vaccination. Uncoupled T and B cell responses have been described in acute and convalescent patients and exposed individuals. We assessed the potential usefulness of whole blood stimulation assays to identify functional cellular immune responses to SARS-CoV-2. Blood from COVID-19 recovered individuals (5 months after infection) and negative subjects was stimulated for 24 hours with HLA predicted peptide “megapools” of the Spike and Nucleoprotein, or the mixture of them. After stimulation, cytokines were quantified using a beads-based multiplex assay. Interleukin-2 and IFN-γ were found to be specific biomarkers of SARS-CoV-2 cellular response. Using the Spike and Nucleoprotein mixture, 91.3% of COVID-19 recovered individuals presented an IL-2 stimulation index over the cut-off, while 82.6% showed IFN-γ. All the negative individuals presented an IL-2 response under the cut-off, while 5.3% of these subjects presented positive IFN-γ stimulation indexes. Moreover, IL-2 production correlated with IgG levels for Spike 1, RBD, and Nucleocapsid. In conclusion, we demonstrate the potential of whole blood stimulation assays and the quantification of IL-2 and IFN-γ for the analysis of SARS-CoV-2 functional cellular responses.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.04.20248897: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study Approval: This study was approved by the Ethics Committees of Centro Hospitalar e Universitário de Coimbra (CHUC-084-20).
    Consent: Participants signed an informed consent form, according to the Helsinki principles, the Oviedo Convention and according to the General Data Protection Regulation – Regulation (EU) 2016/679.
    Randomizationnot detected.
    BlindingThe investigators were blinded to sample allocation during experiments.
    Power Analysisnot detected.
    Sex as a biological variableAmong the negative subjects, the median cohort age was 37.5 years (21-60 years), 9/19 (47%) were male, and 10/19 (53%) were female.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After a washing step, 100 μL anti-human IgG antibody conjugated with horseradish peroxidase (Sigma-Aldrich) diluted at 1:5000 was added and the plates were incubated at 37 °C for 1 h.
    100 μL anti-human IgG antibody
    suggested: None
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Nucleocapsid-His recombinant Protein (YP_009724397.2(335Gly/Ala)) (Met1-Ala419) expressed in Baculovirus-Insect Cells with a polyhistidine tag at the C-terminus (Reference 40588-V08B); Spike protein S1 Subunit (YP_009724390.1) (Val16-Arg685) expressed in HEK293 Cells with a polyhistidine tag at the C-terminus (Reference 40591-V08H); Spike protein Receptor Binding Domain (RBD) (YP_009724390.1) (Arg319-Phe541) expressed in HEK293 Cells with a polyhistidine tag at the C-terminus.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Negative SARS-CoV-2 group (n=19) included subjects with a negative qRT-PCR performed following risk-contact tracking and monitored by IgG serology (Alinity i, Abbott).
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    Samples were acquired in a LSRFortessa™ flow cytometer (BD Biosciences CA, USA) using the FACSDiva™ software v8.0 (BD Biosciences).
    FACSDiva™
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Microsoft Excel v.14.1.0
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    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.
    • 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.

  2. Version published to 10.1101/2021.01.04.20248897v1 on medRxiv