Auto-immunoproteomics analysis of COVID-19 ICU patients revealed increased levels of autoantibodies related to the male reproductive system

  1. Frank Schmidt
  2. Houari B. Abdesselem
  3. Karsten Suhre
  4. Nishant N. Vaikath
  5. Muhammad U. Sohail
  6. Maryam Al-Nesf
  7. Ilham Bensmail
  8. Fathima Mashod
  9. Hina Sarwath
  10. Joerg Bernhardt
  11. Stephanie Schaefer-Ramadan
  12. Ti-Myen Tan
  13. Priscilla E. Morris
  14. Edward J. Schenck
  15. David Price
  16. Vidya Mohamed-Ali
  17. Mohammed Al-Maadheed
  18. Abdelilah Arredouani
  19. Julie Decock
  20. Jonathan M. Blackburn
  21. Augustine M. K. Choi
  22. Omar M. El-Agnaf

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Abstract

Background: Coronavirus disease (COVID-19) manifests many clinical symptoms, including an exacerbated immune response and cytokine storm. Autoantibodies in COVID-19 may have severe prodromal effects that are poorly understood. The interaction between these autoantibodies and self-antigens can result in systemic inflammation and organ dysfunction. However, the role of autoantibodies in COVID-19 complications has yet to be fully understood.

Methods: The current investigation screened two independent cohorts of 97 COVID-19 patients [discovery (Disc) cohort from Qatar (case = 49 vs. control = 48) and replication (Rep) cohort from New York (case = 48 vs. control = 28)] utilizing high-throughput KoRectly Expressed (KREX) Immunome protein-array technology. Total IgG autoantibody responses were evaluated against 1,318 correctly folded and full-length human proteins. Samples were randomly applied on the precoated microarray slides for 2 h. Cy3-labeled secondary antibodies were used to detect IgG autoantibody response. Slides were scanned at a fixed gain setting using the Agilent fluorescence microarray scanner, generating a 16-bit TIFF file. Group comparisons were performed using a linear model and Fisher’s exact test. Differentially expressed proteins were used for KEGG and WIKIpathway annotation to determine pathways in which the proteins of interest were significantly over-represented.

Results and conclusion: Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls ( p ≤ 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients who served as controls. Both cohorts showed substantial similarities ( r 2 = 0.73) and exhibited higher autoantibody responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. The sequences for SPANXN4 and STK25 were cross-validated using sequence alignment tools. ELISA and Western blot further verified the autoantigen–autoantibody response of SPANXN4. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19-associated male reproductive tract complications, and warrants further research.

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  1. Version published to 10.3389/fphys.2023.1203723
  2. ScreenIT

    SciScore for 10.1101/2022.02.09.479669: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical approval for this cohort was obtained from the Hamad Medical Corporation Institutional Review Board Research Ethics Committee (reference MRC-05-003), and Qatar Biomedical Research Institute-Institutional Review Board (Reference QBRI-IRB 2020-06-19).
    Consent: All participants (patients and controls) provided written informed consent prior to enrolment in the study.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Samples including controls were randomized and applied to the microarray slides for 2 hours and samples’ IgGs were then detected by secondary Cy3-labeled IgG antibodies.
    Cy3-labeled IgG
    suggested: (SeraCare KPL Cat# 5230-0359, RRID:AB_2892090)
    Biotinylated human IgG (detected by fluorescently labelled secondary antibody) and biotinylated human anti-IgG (detected only when plasma or serum is added to the slide) were used as positive controls to assess assay integrity.
    anti-IgG
    suggested: None
    To check this latter possibility, we selected two proteins (SPANXN4 and STK25) that showed the highest autoantibody alterations to perform their sequence alignment and antigen specificity analysis.
    SPANXN4
    suggested: None
    STK25
    suggested: None
    antigen specificity analysis.
    suggested: None
    Software and Algorithms
    SentencesResources
    Uniprot BLASTP program was used to compare proteins sequences.
    BLASTP
    suggested: (BLASTP, RRID:SCR_001010)
    Protein pathways prediction: The assingment of KREX array proteins to functional KEGG categories and their hierarchical organisation was displayed by using Paver, a software for the visualization of Voronoi Treemaps28.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    This analysis was performed on R 3.6.2 using clusterProfiler 3.14.3.
    clusterProfiler
    suggested: (clusterProfiler, RRID:SCR_016884)

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2022.02.09.479669v1 on bioRxiv