Cell surface SARS-CoV-2 nucleocapsid protein modulates innate and adaptive immunity

  1. Alberto Domingo López-Muñoz
  2. Ivan Kosik
  3. Jaroslav Holly
  4. Jonathan W. Yewdell

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Abstract

SARS-CoV-2 nucleocapsid protein (N) induces strong antibody (Ab) and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2–infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12β, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS-CoV. Anti-N Abs bound to the surface of N-expressing cells activate Fc receptor–expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc-expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.

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  1. Version published to 10.1126/sciadv.abp9770
  2. ScreenIT

    SciScore for 10.1101/2021.12.10.472169: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: PBMCs were obtained from a healthy donor with informed consent, at the Department of Transfusion Medicine (NIH).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: Previously published Ab VH and VL amino acid sequences against SARS-CoV-2 N (# N1833) and SARS-CoV-2 S (# H434) were commercially synthesized, cloned into a human IgG1 vector backbone, produced and purified (Synbio Technologies).
    human IgG1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero cells (# CCL-81), BHK-21 (# CCL-10), Caco-2 (# HTB-37), Calu-3 (# HTB-55), CHO-K1 (# CCL-61), CHO-pgsA-745 (# CRL-2242), CHO-pgsB-618 (# CRL-2241), CHO-pgsD-677 (# CRL-2244), CHO-pgsE-606 (# CRL-2246), HEK293-FT (# CRL-11268), A549 (# CCL-185) and MOLT-4 (# CRL-1582) cells were from the American Type Culture Collection (ATCC).
    A549
    suggested: None
    Vero, BHK-21, Caco-2, Calu-3 and HEK293-FT cells were grown in DMEM with GlutaMAX (Thermo Fisher # 10566016).
    BHK-21
    suggested: None
    Caco-2
    suggested: None
    Calu-3
    suggested: None
    CHO-K1, CHO-pgsA-745, CHO-pgsB-618, CHO-pgsD-677, CHO-pgsE-606 and A549 cells were grown in F-12K medium (Thermo Fisher # 21127022).
    CHO-K1
    suggested: None
    CHO-pgsE-606
    suggested: None
    PBMCs, MOLT-4 and MonoMac-1 cells were grown in RPMI 1640 (Thermo Fisher # 11875119).
    MonoMac-1
    suggested: DSMZ Cat# ACC-252, RRID:CVCL_1425)
    BHK-21_hACE2, CHO-K1_hACE2, HEK293-FT_hACE2 and A549_hACE2 cells were grown in their correspondent medium with 250-500 μg/ml of blasticidin (Invivogen # ant-bl-1).
    A549_hACE2
    suggested: None
    The TCID50 and PFU of virus in clarified culture medium was determined on Vero cells after staining with crystal violet.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Abs and serum were titrated and specificity was tested, by flow cytometry on HEK293-FT cells transiently expressing SARS-CoV-2 N (Addgene # 141391), S (BEI # NR-52310) or Sst. Immunofluorescence: For confocal microscopy imaging, 2.5 x 104 cells were seeded on 12 mm glass coverslips in 24-well plates in indicated medium with gentamycin (25 μg/ml) overnight.
    HEK293-FT
    suggested: RRID:CVCL_6911)
    Then, 1 x 105 CHO-K1_hACE2 cells (SARS-CoV-2-infectable) were homogeneously mixed and co-seeded with indicated non-infectable cell type, being co-cultured overnight.
    CHO-K1_hACE2
    suggested: None
    PBMCs, MonoMac-1 and MOLT-4 cells (1.25 x 105) were placed on the upper compartment and separated from the lower chamber by a 3 or 5 μm pore size filter.
    MOLT-4
    suggested: None
    At 24 hpi, infected target cells were washed with DPBS and the medium was replaced with 50 μl of RPMI 1640 with 4% low IgG serum (Promega # G711A) containing 5 x 104 Jurkat effector cells (Promega # G701A) and serial dilutions of indicated human mAbs.
    Jurkat
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse polyclonal anti-SARS-CoV-2 Sst serum was produced as followed: 8-to-12-week C57B6 mice (Taconic Farms Inc) were immunized with 4 μg of Sst diluted in DPBS, adjuvanted by TiterMax® Gold (MilliporeSigma # T2684) (2:1) in 50 μl volume via intramuscular injections.
    C57B6
    suggested: None
    Recombinant DNA
    SentencesResources
    In brief, a semi-confluent 60 mm plate was seeded with each cell line and co-transfected with 0.5 μg of pCMV(CAT)T7-SB100 (Transposase vector, Addgene # 34879) and 5 μg of pSBbi-Bla hACE2 (Transposon vector), using TransIT-LT1 Transfection Reagent (Mirus Bio), following manufacturer instructions.
    pCMV(CAT)T7-SB100
    suggested: RRID:Addgene_34879)
    pSBbi-Bla hACE2
    suggested: None
    The open reading frame of hACE2 (kindly provided by Sonja Best from NIAID/NIH) was cloned into pSBbi-Bla vector (Addgene # 60526) as described 31.
    hACE2
    suggested: RRID:Addgene_1786)
    pSBbi-Bla
    suggested: RRID:Addgene_60526)
    Software and Algorithms
    SentencesResources
    Maximum intensity projections (MIPs) were processed from z-stacks (at least 15 0.3 μm z-steps per image); and for background correction (Gaussian filter) and color processing, using Imaris (Bitplane).
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    Animations (gifs) were generated with Photoshop 2022 (Adobe).
    Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    Data were analyzed with FlowJo (Tree Star) and plotted with Prism v9.1.1 software (GraphPad).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical analysis: Statistical analyses were performed using GraphPad Prism software.
    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: 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

    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 10.1101/2021.12.10.472169v1 on bioRxiv