SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies

  1. Ceri Alan Fielding
  2. Pragati Sabberwal
  3. James C Williamson
  4. Edward JD Greenwood
  5. Thomas WM Crozier
  6. Wioleta Zelek
  7. Jeffrey Seow
  8. Carl Graham
  9. Isabella Huettner
  10. Jonathan D Edgeworth
  11. David A Price
  12. Paul B Morgan
  13. Kristin Ladell
  14. Matthias Eberl
  15. Ian R Humphreys
  16. Blair Merrick
  17. Katie Doores
  18. Sam J Wilson
  19. Paul J Lehner
  20. Eddie CY Wang
  21. Richard J Stanton

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Abstract

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

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  1. Version published to 10.7554/elife.74489 on eLife
  2. Version published to 10.1101/2021.04.06.438630v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.04.06.438630: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Use of healthy volunteer PBMC for this project, including those from WBS, was ethically approved by the Cardiff University School of Medicine Research Ethics Committee (SMREC) nos. 20/55 and 20/101.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies used were against HLA-ABC (W632; AbD Serotec), NCR3LG1/B7-H6 (MAB7144, Biotechne R&D Systems), Nectin 1 (R1.302; Biolegend), MICA (AMO1-100; BAMOMAB), MICB (BMO2-100; BAMOMAB), ULBP2 (BUMO1; BAMOMAB), Spike (1A9; Insight), Nucleocapsid (1C7; Stratech), anti-mouse IgG AF647 (Thermofisher).
    HLA-ABC
    suggested: (Leica Biosystems Cat# NCL-HLA-ABC, RRID:AB_563879)
    MAB7144
    suggested: (R and D Systems Cat# MAB7144, RRID:AB_2636810)
    BMO2-100
    suggested: None
    BAMOMAB
    suggested: (BAMoMAB Cat# BM02-100, RRID:AB_2636812)
    ULBP2
    suggested: None
    anti-mouse IgG
    suggested: (SouthernBiotech Cat# 1030-31, RRID:AB_2794301)
    Target cells were harvested using TrypLE Express (Gibco), preincubated for 30 min with the relevant antibody or serum preparations, then mixed with PBMCs at an effector:target (E:T) ratio of 10:1 in the presence of GolgiStop (0.7 μl/ml, BD Biosciences), Brefeldin-A (1:1000, Biolegend) and anti-CD107a–FITC (clone H4A3, BioLegend).
    Brefeldin-A
    suggested: None
    anti-CD107a–FITC
    suggested: None
    Primary antibody (anti-nucleocapsid 1C7, Stratech, 1:500 dilution) was added in PBST containing 1% non-fast milk and incubated for 1h at room temperature.
    anti-nucleocapsid
    suggested: None
    After washing in PBST, secondary antibody (anti-mouse IgG-HRP, Pierce, 1:3,000 dilution) was added in PBST containing 1% non-fat milk and incubated for 1h.
    anti-mouse IgG-HRP
    suggested: None
    Antibody depletions: Anti-spike antibody was depleted from sera using magnetic bead conjugated spike trimer protein (Acrobiosystems).
    Anti-spike
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: A549 were transduced with lentiviruses expressing human ACE2, and TMPRSS2 (AAT cells), as previously described 27.
    A549
    suggested: None
    The England2 strain of SARS-CoV2 was obtained from Public Health England (PHE), grown on VeroE6 cells, and titrated by plaque assay on both VeroE6 and AAT, as previously described27.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Plasmids were midiprepped (Nucleobond Xtra Midi; Machery-Nagel), and transfected into 293T cells using GeneJuice (Merck) according to manufacturers’ instructions.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    Samples were loaded onto a trapping column (300μm x 5mm PepMap cartridge trap (Thermo Fisher)) at 10μL/min for 5 minutes at 60 degrees.
    PepMap
    suggested: (BioWorks, RRID:SCR_014594)
    During the gradient elution, mass spectra were acquired with the parameters detailed in Fig S4 using Tune v3.3 and Xcalibur v4.3 (Thermo Fisher).
    Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository74 with the dataset identifier PXD025000 and 10.6019/PXD025000.
    PRIDE
    suggested: (Pride-asap, RRID:SCR_012052)
    Functional Annotation Clustering: Assessment of enriched gene annotation terms in temporal cluster one was carried out using the Functional Annotation Clustering tool at DAVID (david.ncifcrf.gov) v6.875, using the default clustering settings for medium stringency and the following libraries: Uniprot UP_Keyword, GOTERM:MF_ALL, BIOCARTA,, KEGG_PATHWAY and REACTOME_PATHWAY.
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    Plasmids and transfections: Lentivirus plasmids encoding each SARS-CoV2 ORF individually, with a C-terminal twin-strep tag, were obtained from Addgene76.
    Addgene76
    suggested: None
    Target cells were harvested using TrypLE Express (Gibco), preincubated for 30 min with the relevant antibody or serum preparations, then mixed with PBMCs at an effector:target (E:T) ratio of 10:1 in the presence of GolgiStop (0.7 μl/ml, BD Biosciences), Brefeldin-A (1:1000, Biolegend) and anti-CD107a–FITC (clone H4A3, BioLegend).
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Data were acquired using an AttuneNxT (Thermo Fisher) and analyzed with Attune NxT software or FlowJo software version 10 (Tree Star).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Where sera were tested at a range of dilutions, the area under the curve (AUC) was calculated using Graphpad Prism 9.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    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.

    About SciScore

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  4. Version published to 10.1101/2021.04.06.438630v1 on bioRxiv