Integrated immune networks in SARS-CoV-2 infected pregnant women reveal differential NK cell and unconventional T cell activation

  1. Jennifer R Habel
  2. Brendon Y Chua
  3. Lukasz Kedzierski
  4. Kevin J Selva
  5. Timon Damelang
  6. Ebene R Haycroft
  7. Thi HO Nguyen
  8. Hui-Fern Koay
  9. Suellen Nicholson
  10. Hayley McQuilten
  11. Xiaoxiao Jia
  12. Lilith F Allen
  13. Luca Hensen
  14. Wuji Zhang
  15. Carolien E van de Sandt
  16. Jessica A Neil
  17. Fatima Amanat
  18. Florian Krammer
  19. Kathleen Wragg
  20. Jennifer A Juno
  21. Adam K Wheatley
  22. Hyon-Xhi Tan
  23. Gabrielle Pell
  24. Jennifer Audsley
  25. Irani Thevarajan
  26. Justin Denholm
  27. Kanta Subbarao
  28. Dale I Godfrey
  29. Allen C Cheng
  30. Steven YC Tong
  31. Katherine Bond
  32. Deborah A Williamson
  33. Fiona James
  34. Natasha E Holmes
  35. Olivia C Smibert
  36. Jason A Trubiano
  37. Claire L Gordon
  38. Amy W Chung
  39. Clare L Whitehead
  40. Stephen J Kent
  41. Martha Lappas
  42. Louise C Rowntree
  43. Katherine Kedzierska

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Abstract

Although pregnancy poses a greater risk for severe COVID-19, the underlying immunological changes associated with SARS-CoV-2 infection during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in pregnant and non-pregnant women during acute and convalescent COVID-19 up to 258 days post symptom onset, quantifying 217 immunological parameters. Additionally, matched maternal and cord blood were collected from COVID-19 convalescent pregnancies. Although serological responses to SARS-CoV-2 were similar in pregnant and non-pregnant women, cellular immune analyses revealed marked differences in key NK cell and unconventional T cell responses during COVID-19 in pregnant women. While NK cells, γδ T cells and MAIT cells displayed pre-activated phenotypes in healthy pregnant women when compared to non-pregnant age-matched women, activation profiles of these pre-activated NK and unconventional T cells remained unchanged at acute and convalescent COVID-19 in pregnancy. Conversely, activation dynamics of NK and unconventional T cells were prototypical in non-pregnant women in COVID-19. In contrast, activation of αβ CD4 + and CD8 + T cells, T follicular helper cells and antibody-secreting cells was similar in pregnant and non-pregnant women with COVID-19. Elevated levels of IL-1β, IFN-γ, IL-8, IL-18 and IL-33 were also found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, our study provides the first comprehensive map of longitudinal immunological responses to SARS-CoV-2 infection in pregnant women, providing insights into patient management and education during COVID-19 pregnancy.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.21.21262399: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Written informed consents were obtained from all blood donors prior to the study.
    IRB: The study was approved by the Alfred Hospital (#280/14), Melbourne Health (HREC/66341/MH-2020 and HREC/17/MH/53), Austin Health (HREC/63201/Austin-2020), Monash Health (HREC/15/MonH/64), Mercy Health (R14/25 and R04/29), Australian Red Cross Lifeblood (2015#08), and the University of Melbourne (#1442952, #1749349, #2056901, #1443540, #2056761, #1955465, 2020-20782-12450-1, 2021-13973-14410-3 and 2021-13973-14410-3) Human Research Ethics Committees.
    Sex as a biological variablePregnant and non-pregnant women with acute or convalescent COVID-19 were recruited via the Mercy Hospital for Women, Royal Women’s Hospital, Royal Melbourne Hospital, Austin Hospital, Alfred Hospital or The University of Melbourne.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Four antibody panels were used to determine activation of (1) monocytes, T, B, NK and γδ T cells, (2) TFH and ASC cell activation, (3) cytotoxicity profiles of T cells and NK cells expressing intracellular granzymes A, B, K and M and perforin, and (4) activation and phenotypes of MAIT and γδ T cells (Supp Fig 6 and 7).
    ASC cell activation, (3
    suggested: None
    SARS-CoV-2 Receptor Binding Domain and Nucleocapsid ELISA: ELISA for the detection of RBD- or N-specific IgG, IgM and IgA antibodies were performed as previously described (Amanat et al., 2020; Koutsakos et al., 2021; Rowntree et al., 2021), using flat bottom Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific) for antigen coating (2µg/ml), blocking with PBS (with w/v 1% BSA) and serial dilutions in PBS (with v/v 0.05% Tween and w/v 0.5% BSA).
    N-specific
    suggested: None
    N-specific IgG
    suggested: None
    IgA
    suggested: None
    Bound antibodies were then detected using either HRP-conjugated anti-human IgG or anti-human IgM antibody as described previously.
    anti-human IgG
    suggested: None
    anti-human IgM
    suggested: None
    Colour intensity was inversely dependent on the titre of anti-SARS-CoV-2 neutralizing antibodies.
    anti-SARS-CoV-2 neutralizing antibodies.
    suggested: (Creative Diagnostics Cat# CABT-CS064, RRID:AB_2891088)
    Luminex bead-based multiplex assay: A custom multiplex assay was used to investigate the isotypes and subclasses of SARS-CoV-1 and -2 specific antibodies present in plasma samples (Selva et al., 2021).
    -2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Heat inactivated sera (56°C for 30 min) was serially diluted and serum/virus mixtures assessed for residual virus infectivity in quadruplicate wells of Vero cells incubated in serum-free media containing 1μg/ml of TPCK trypsin at 37°C and 5% CO2.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were acquired on a LSRII Fortessa (BD Biosciences) and analyzed using FlowJo v10 software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Endpoint titres were determined by interpolation from a sigmoidal curve fit (all R-squared values >0.95; GraphPad Prism 9) as the reciprocal dilution of plasma that produced >15% (for IgA and IgG) or >30% (for IgM) absorbance of the positive control at a 1:31.6 (IgG and IgM) or 1:10 dilution (IgA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Principal component analysis: PCA was performed in the Eigenvectors PLS toolbox (Eigenvector Research, Inc., Manson, USA) in MATLAB.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)
    Statistical analysis: Data and statistical analysis were performed in GraphPad Prism (version 9).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    PESTLE and SPICE software (version 6.1) were used for analysis of cytotoxic granzymes and perforin in NK cell and T cell subsets (Roederer et al., 2011).
    SPICE
    suggested: (SPICE, RRID:SCR_016603)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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

    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.

  2. Version published to 10.1101/2021.08.21.21262399 on medRxiv