Salivary anti-SARS-CoV-2 IgA as an accessible biomarker of mucosal immunity against COVID-19

  1. Atul Varadhachary
  2. Dev Chatterjee
  3. Javier Garza
  4. R. Patrick Garr
  5. Christopher Foley
  6. Andrea Letkeman
  7. John Dean
  8. David Haug
  9. Juliet Breeze
  10. Robbyn Traylor
  11. Andrew Malek
  12. Rohan Nath
  13. Leo Linbeck

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Abstract

Background

Mucosal immunity, including secretory IgA (sIgA), plays an important role in early defenses against respiratory pathogens. Salivary testing, the most convenient way to measure sIgA, has been used to characterize mucosal immune responses to many viral infections including SARS, MERS, influenza, HIV, and RSV. However, its role has not yet been characterized in the COVID-19 pandemic. Here, we report development and validation of a rapid immunoassay for measuring salivary IgA against the SARS-CoV-2 virus, and report quantitative results in both pre-COVID-19 and muco-converted subjects.

Methods

We developed and refined a specific test for salivary IgA against SARS-CoV-2 on the Brevitest platform, a rapid immunoassay system designed for point-of-care use. A qualitative test was validated as per FDA guidelines with saliva obtained from subjects prior to the emergence of COVID-19, and from PCR-confirmed COVID-19 patients. We also generated a quantitative measure of anti-SARS-CoV-2 salivary IgA. Time taken for saliva self-collection was measured and its ease-of-use assessed.

Results

We successfully validated a qualitative salivary assay for SARS-CoV-2 IgA antibodies, with positive and negative predictive values of 92% and 97%, respectively, and no observable cross-reactivity with any of seven potential confounders. Pre-COVID-19 saliva samples showed an 8-fold range of IgA concentrations, suggesting a broad continuum of natural antibody resistance against the novel virus, though at levels lower than that observed in COVID-19 PCR-confirmed subjects. Samples from muco-positive subjects also shown a ~9-fold variation in salivary IgA levels, with elevated salivary IgA observed beyond three months after onset of symptoms. We observed a correlation (r=0.4405) between salivary IgA levels and COVID-19 disease severity. In anecdotal observations, we observed individuals who exhibited antibodies early in the course of their disease, contemporaneously with a positive PCR test, as well as individuals who muco-converted despite no known direct exposure to a COVID-19 patient, no symptoms, and negative molecular and/or serum antibody tests. Salivary collection took 5-10 minutes, and was reported as being easy (mean of 1.1 on a scale of 1 to 10).

Implications

Mucosal immunity, including secretory IgA, plays an important role in host defense against respiratory pathogens, and our early data suggest it may do so in COVID-19. Salivary IgA, an accessible marker of mucosal immunity, may be a useful indicator of several key parameters including individual and community immune response, disease severity, clinical risk, and herd immunity. The non-invasive nature and ease of saliva collection facilitates its potential use as a biomarker for ongoing patient assessment and management, as well as a community surveillance tool. By measuring mucosal immune responses directly and systemic immune responses indirectly, salivary IgA could be useful in developing and deploying a vaccine(s) against COVID-19. Quantitative IgA assessment could also potentially serve as a tool to segment the population into different risk categories and inform individual and collective decisions relating to appropriate activities and vaccine prioritization/delivery. These data reinforce the importance of further investigation into the role of mucosal immunity and IgA in host responses against COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.07.20170258: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: The clinical protocol for sample and data collection and the informed consent document were approved by IntegReview, an independent Institutional Review Board.
    IRB: The clinical protocol for sample and data collection and the informed consent document were approved by IntegReview, an independent Institutional Review Board.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    BRAVO is an enzyme-Linked Immunosorbent Assay (ELISA) capable of quantitative detection of IgA antibodies to SARS-CoV-2 in human saliva though the assay has only been validated for qualitative IgA detection.
    IgA
    suggested: None
    Secondary antibody: Goat Anti-Human IgA conjugated to HRP (Cat: PA1-74395, Thermo Fisher Scientific).
    Secondary antibody: Goat Anti-Human IgA
    suggested: None
    Anti-Human IgA
    suggested: (Thermo Fisher Scientific Cat# PA1-74395, RRID:AB_10985660)
    Assay Validation: The BRAVO assay was validated as described in the Results section, using true negative pre-COVID saliva samples (Lee Biosolutions) and tested for cross-reactivity using serum samples for anti-influenza B IgG, anti-respiratory syncytial virus IgG, antinuclear antibodies, IgG, rheumatoid factors (Biomex ZMBH), HIV, and Hemophilus influenza antibodies (Cantor Bioconnect).
    anti-influenza B IgG
    suggested: None
    anti-respiratory syncytial virus IgG, antinuclear antibodies, IgG
    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 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.
    • Thank you for including a protocol registration statement.

    About SciScore

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  2. Version published to 10.1101/2020.08.07.20170258v1 on medRxiv