Detection of cross-reactive immunoglobulin A against the severe acute respiratory syndrome-coronavirus-2 spike 1 subunit in saliva

  1. Keiichi Tsukinoki
  2. Tatsuo Yamamoto
  3. Keisuke Handa
  4. Mariko Iwamiya
  5. Juri Saruta
  6. Satoshi Ino
  7. Takashi Sakurai

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Abstract

Abundant secretory immunoglobulin A (SIgA) in the mucus, breast milk, and saliva provides immunity against infection of mucosal surfaces. Pre-pandemic breast milk samples containing SIgA have been reported to cross-react with SARS-CoV-2; however, it remains unknown whether SIgA showing the cross-reaction with SARS-CoV-2 exists in saliva. We aimed to clarify whether SIgA in saliva cross-reacts with SARS-CoV-2 spike 1 subunit in individuals who have not been infected with this virus. The study involved 137 (men, n = 101; women, n = 36; mean age, 38.7; age range, 24–65 years) dentists and doctors from Kanagawa Dental University Hospital. Saliva and blood samples were analyzed by polymerase chain reaction (PCR) and immunochromatography for IgG and IgM, respectively. We then identified patients with saliva samples that were confirmed to be PCR-negative and IgM-negative for SARS-CoV-2. The cross-reactivity of IgA-positive saliva samples with SARS-CoV-2 was determined by enzyme-linked immunosorbent assay using a biotin-labeled spike recombinant protein (S1-mFc) covering the receptor-binding domain of SARS-CoV-2. The proportion of SARS-CoV-2 cross-reactive IgA-positive individuals was 46.7%, which correlated negatively with age (r = –0.218, p = 0.01). The proportion of IgA-positive individuals aged ≥50 years was significantly lower than that of patients aged ≤49 years (p = 0.008). SIgA was purified from the saliva of patients, which could partially suppress the binding of SARS-CoV-2 spike protein to the angiotensin converting enzyme-2 receptor. This study demonstrates the presence of SARS-CoV-2 cross-reactive SIgA in the saliva of individuals who had never been infected with the virus, suggesting that SIgA may help prevent SARS-CoV-2 infection.

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  1. Version published to 10.1371/journal.pone.0249979
  2. ScreenIT

    SciScore for 10.1101/2021.03.29.21253174: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: We analyzed samples from 137 (male, n = 101; female, n = 36; mean age, 38.7 [24–65] years) individuals, under the fully informed consent.
    IRB: This study was conducted under the approval of the Kanagawa Dental University Research Ethics Review Board (Approval number: #690).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableWe analyzed samples from 137 (male, n = 101; female, n = 36; mean age, 38.7 [24–65] years) individuals, under the fully informed consent.

    Table 2: Resources

    Antibodies
    SentencesResources
    IgA purification: We purified sIgA using IgA purification kits (#20395; Thermo Fisher Scientific K.K., Waltham, MA, USA) as described by the manufacturer, then confirmed its molecular weight by standard western blotting using primary anti-human IgA rabbit monoclonal antibodies (ab184863; Abcam Plc, Cambridge, UK) diluted 1:500 and secondary anti-rabbit polyclonal antibodies (#P0448; Dako A/S., Glostrup, Denmark; dilution, 1:1000).
    anti-human IgA
    suggested: (Antibodies-Online Cat# ABIN135040, RRID:AB_10775110)
    anti-rabbit polyclonal antibodies
    suggested: (Agilent Cat# P0448, RRID:AB_2617138)
    Software and Algorithms
    SentencesResources
    Spike 1 protein-bound IgA was determined at 450 nm using a microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    All data were statistically analyzed using SPSS version 26 (IBM Corp., Armonk, NY, USA).
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    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: We detected the following sentences addressing limitations in the study:
    One limitation of the present study was the low number of participants. Although vaccines stimulate the production of cross-reactive antibodies [7], we found no significant association between vaccines and cross-reactive antibodies. Future investigations should compare individuals who are not involved in medical care (without experience of vaccination).

    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

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  3. Version published to 10.1101/2021.03.29.21253174v2 on medRxiv
  4. Version published to 10.1101/2021.03.29.21253174v1 on medRxiv