SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity

  1. Annachiara Rosa
  2. Valerie E. Pye
  3. Carl Graham
  4. Luke Muir
  5. Jeffrey Seow
  6. Kevin W. Ng
  7. Nicola J. Cook
  8. Chloe Rees-Spear
  9. Eleanor Parker
  10. Mariana Silva dos Santos
  11. Carolina Rosadas
  12. Alberto Susana
  13. Hefin Rhys
  14. Andrea Nans
  15. Laura Masino
  16. Chloe Roustan
  17. Evangelos Christodoulou
  18. Rachel Ulferts
  19. Antoni G. Wrobel
  20. Charlotte-Eve Short
  21. Michael Fertleman
  22. Rogier W. Sanders
  23. Judith Heaney
  24. Moira Spyer
  25. Svend Kjær
  26. Andy Riddell
  27. Michael H. Malim
  28. Rupert Beale
  29. James I. MacRae
  30. Graham P. Taylor
  31. Eleni Nastouli
  32. Marit J. van Gils
  33. Peter B. Rosenthal
  34. Massimo Pizzato
  35. Myra O. McClure
  36. Richard S. Tedder
  37. George Kassiotis
  38. Laura E. McCoy
  39. Katie J. Doores
  40. Peter Cherepanov

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Abstract

SARS-CoV-2 spike N-terminal domain harbors a potent epitope that can be modulated by binding of natural linear tetrapyrroles.

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  1. Version published to 10.1126/sciadv.abg7607
  2. ScreenIT

    SciScore for 10.1101/2021.01.21.21249203: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    U8 MaxiSorp plates (Fisher Scientific) were coated overnight at 4°C with AffiniPure rabbit anti-human IgG antibody (Stratech; product code #309-005-008) diluted to 5 μg/ml in coating buffer (Clintech; product code #643005).
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 309-005-008, RRID:AB_2339626)
    , APC anti-IgM (clone MHM-88, Biolegend) and PE anti-IgA (clone IS11-8E10, Miltenyi Biotech) for 30 min (all antibodies diluted 1:200 in FACS buffer).
    anti-IgM
    suggested: None
    anti-IgA
    suggested: None
    Antibody binding was detected with APC anti-IgG (Biolegend) diluted 1:200 in FWB buffer.
    anti-IgG
    suggested: None
    Monoclonal human antibodies: The following IgGs COVA1-26, COVA1-23, COVA2-38, COVA2-17, COVA1-20, COVA2-26, COVA1-22, COVA3-07, COVA2-03, COVA1-18,COVA1-12, COVA1-16, COVA2-01, COVA2-02, COVA2-04, COVA2-07, COVA2-11, COVA2-15, COVA2-29, COVA2-39, COVA2-44, COVA2-46, COVA2-10, COVA2-25, COVA2-30 have been reported (10).
    COVA2-30
    suggested: None
    The blocking solution was removed cells were incubated with 2 µg/ml SARS-CoV-2 nuclear protein-specific murinized-CR3009 antibody in PBS supplemented with 1% milk at room temperature for 45 min.
    SARS-CoV-2 nuclear protein-specific murinized-CR3009
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells, seeded one day earlier in 10-cm dishes in complete Dulbecco’s modified Eagle’s medium (DMEM, supplemented with 10% fetal bovine serum, FBS) were co-transfected with 17 µg SIVMAC239-GFP, an env- and nef-defective provirus construct expressing a GFP reporter (65), and 4 µg pcDNA-SARS-CoV-2-del19, encoding SARS-CoV-2 spike with or without mutations in the biliverdin binding pocket.
    HEK293T
    suggested: None
    Six 5-fold serially diluted virus stocks were inoculated in quadruplicate in 96-well plates onto Huh-7/ACE-2 and Vero/ACE2, modified to overexpress the receptor ACE-2 from a transduced lentiviral vector carrying the puromycin resistance gene, seeded one day before infection in 96-well plates; 48 h post-infection, fluorescent cells were counted using the Ensight plate reader (Perkin Elmer).
    Huh-7/ACE-2
    suggested: None
    Pseudotype neutralization assay: HIV-1 particles pseudotyped with SARS-Cov-2 spike were produced in a T75 flask seeded the day before with 3 million HEK293T/17 cells (American Type Culture Collection; catalogue code CRL-11268) in 10 ml complete DMEM, supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin.
    HEK293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    HeLa cells stably expressing ACE-2 (provided by J.E. Voss, Scripps Institute)(69) were then added to the assay (10,000 cells per 100 µl per well).
    HeLa
    suggested: None
    The infectious virus titre was determined by plaque assay in Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    The media was removed from the pre-plated Vero-E6 cells and the serum-virus mixtures were added to the Vero E6 cells and incubated at 37°C for 24 h.
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Data was recorded using Xcalibur 3.0.63 software and analysed using Free Style 1.6 and Tracefinder 4.1 software (Thermo Scientific) according to the manufacturer’s workflows.
    Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    Cryo-EM image processing and real-space refinement: Micrograph movies were aligned with dose weighting applied using MotionCor2 (40), and the contrast transfer function (CTF) parameters were estimated from the frame sums with Gctf (41).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    real_space_refine before another round of manual rebuilding in Coot and a final round of phenix.real_space_refine.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Biliverdin (BLA) ligand geometry definition file was generated by Grade (Global Phasing) and model quality was assessed using Molprobity (55).
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    SARS-CoV-2 spike NTD (residues 14-290; PDB ID 6ZGE) (13) was used as a model for molecular replacement and yielded a solution containing one NTD per asymmetric unit, with a log likelihood gain of 490 and translation function Z-score of 22.7, in space group C2221 using Phaser (59) within the Phenix package (53).
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Samples were run on a Ze5 analyzer (Bio-Rad) running Bio-Rad Everest software v2.4, and analyzed using FlowJo v10
    Bio-Rad Everest
    suggested: None
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Measurements were performed in duplicate and used to calculate 50% inhibitory concentrations (IC50) in GraphPad Prism software.
    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 48 and 55. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

  3. Version published to 10.1101/2021.01.21.21249203 on medRxiv