SARS-CoV-2 Cysteine-like Protease Antibodies Can Be Detected in Serum and Saliva of COVID-19–Seropositive Individuals

  1. Pedro Martínez-Fleta
  2. Arantzazu Alfranca
  3. Isidoro González-Álvaro
  4. Jose M Casasnovas
  5. Daniel Fernández-Soto
  6. Gloria Esteso
  7. Yaiza Cáceres-Martell
  8. Sofía Gardeta
  9. Celia López-Sanz
  10. Salomé Prat
  11. Tamara Mateu-Albero
  12. Ligia Gabrie
  13. Eduardo López-Granados
  14. Francisco Sánchez-Madrid
  15. Hugh T Reyburn
  16. José M Rodríguez Frade
  17. Mar Valés-Gómez

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Abstract

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2–specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity.

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  1. Version published to 10.4049/jimmunol.2000842
  2. ScreenIT

    SciScore for 10.1101/2020.07.16.20155853: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Patient samples and Institutional Review Boards: This study used samples from the research project “Immune response dynamics as predictor of COVID-19 disease evolution.
    Consent: All included patients (or their representatives) were informed about the study and gave a written informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The plates were washed again and 100 μL/well of the indicated detection antibody [(AffiniPure Rabbit Anti-Human IgM, Fcµ fragment specific;
    Anti-Human IgM
    suggested: None
    Rabbit Anti-Human Serum IgA, α chain specific; AffiniPure Rabbit Anti-Human IgG, Fcγ fragment specific) from Jackson Labs, or anti-human (Fab)’2 HRPO-labelled antibody from Thermo Fisher Scientific] was added and incubated for 1 hour at room temperature.
    Anti-Human Serum IgA
    suggested: None
    Anti-Human IgG
    suggested: None
    anti-human
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The recombinant cDNA was cloned in a vector derived from the pEF-BOS (11) for transient expression in HEK293 cells, and in the pBJ5-GS vector for stable protein production in CHO cells following the glutamine synthetase system (12).
    HEK293
    suggested: None
    CHO
    suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Graphics and statistical analysis was performed with Graph Pad Prism 8 Software (GraphPad Software, USA, www.graphpad.com) and Stata 14.0 for Windows
    GraphPad
    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.
    • 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.

  3. Version published to 10.1101/2020.07.16.20155853v1 on medRxiv