SARS-CoV-2 Antigenemia as a Confounding Factor in Immunodiagnostic Assays: A Case Study

  1. Konstantinos Belogiannis
  2. Venetia A. Florou
  3. Paraskevi C. Fragkou
  4. Stefanos Ferous
  5. Loukas Chatzis
  6. Aikaterini Polyzou
  7. Nefeli Lagopati
  8. Demetrios Vassilakos
  9. Christos Kittas
  10. Athanasios G. Tzioufas
  11. Sotirios Tsiodras
  12. George Sourvinos
  13. Vassilis G. Gorgoulis

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Abstract

Humoral immunity has emerged as a vital immune component against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nevertheless, a subset of recovered Coronavirus Disease-2019 (COVID-19) paucisymptomatic/asymptomatic individuals do not generate an antibody response, constituting a paradox. We assumed that immunodiagnostic assays may operate under a competitive format within the context of antigenemia, potentially explaining this phenomenon. We present a case where persistent antigenemia/viremia was documented for at least 73 days post-symptom onset using ‘in-house’ methodology, and as it progressively declined, seroconversion took place late, around day 55, supporting our hypothesis. Thus, prolonged SARS-CoV-2 antigenemia/viremia could mask humoral responses, rendering, in certain cases, the phenomenon of ‘non-responders’ a misnomer.

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  1. Version published to 10.3390/v13061143
  2. ScreenIT

    SciScore for 10.1101/2021.01.26.21250561: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethical Statement: Written informed consent was obtained by the patients for the collection and processing of the samples as well as for the publication of this case report.
    IRB: This case report was conducted within the frame of ‘Emblematic action to handle SARS-CoV-2 infection: Epidemiological study in Greece via extensive testing for viral and antibody detection, sequencing of the virome and genetic analysis of the carriers’, which has been approved by the Ethics Committee of Medical School of National Kapodistrian University of Athens (Approval No. 317/12-06-2020).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Monoclonal antibody production: The monoclonal antibodies 480-S2 and 479-G2 used in the present immunoassays have been generated by immunizing mice against the receptor binding domain (RBD) region of the spike (S) protein of SARS-CoV-2 via a modification of the method described by Koehler and Milstein16.
    Milstein16
    suggested: None
    Immunohistochemistry: Immunohistochemistry was performed using the anti-SARS-CoV-2 monoclonal antibody (479-G2).
    anti-SARS-CoV-2
    suggested: None
    Software and Algorithms
    SentencesResources
    : Line graph representing the optical density (O.D) values of Viral/Antigen load and anti-SARS-CoV-2 antibody titers as a function of time, was constructed with GraphPad Prism version 7.00 (www.graphpad.com).
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    This emphasizes the importance of humoral immunity in viral clearance and points towards a different explanation, whilst raising an important question: does the phenomenon of non-responders reflect a true immunobiological event or a limitation of currently available diagnostic tools in precisely portraying the immune landscape? According to a study comparing different antibody detecting assays, this observation was reproducible by all methods, implying that absence of seroconversion represents a biological rather than a technical issue22. To the contrary, serum of presumably non-responders inhibited cell line infection upon culture with viable SARS-CoV-2, as assessed by a neutralization assay, the gold standard for antibody efficiency12,23. The latter supports that a technical limitation of the currently available diagnostic tools might be at play12. Our findings suggest that delayed and/or absent kinetics of the antibody reaction is most probably due to the prolonged and increased presence of the virus or its constituents (spike antigen in the present case) in the serum, saturating the antibody response, thus rendering antibody detection feasible only when antigenemia/viremia drops below a certain threshold (Suppl. Figure 4). Similarly to the prolonged shedding seen in nasopharyngeal and gastrointestinal tract secretions, it is likely that such an event could take place in the blood stream, as well26. A probable source of antigenemia/viremia could be PANoptosis (Pyroptosis, ...

    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 10.1101/2021.01.26.21250561v1 on medRxiv