Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

  1. Florian A. Lempp
  2. Leah Soriaga
  3. Martin Montiel-Ruiz
  4. Fabio Benigni
  5. Julia Noack
  6. Young-Jun Park
  7. Siro Bianchi
  8. Alexandra C. Walls
  9. John E. Bowen
  10. Jiayi Zhou
  11. Hannah Kaiser
  12. Maria Agostini
  13. Marcel Meury
  14. Exequiel Dellota
  15. Stefano Jaconi
  16. Elisabetta Cameroni
  17. Herbert W. Virgin
  18. Antonio Lanzavecchia
  19. David Veesler
  20. Lisa Purcell
  21. Amalio Telenti
  22. Davide Corti

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Abstract

Investigating the mechanisms of SARS-CoV-2 cellular infection is key to better understand COVID-19 immunity and pathogenesis. Infection, which involves both cell attachment and membrane fusion, relies on the ACE2 receptor that is paradoxically found at low levels in the respiratory tract, suggesting that additional mechanisms facilitating infection may exist. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding Ig-like lectin 1 (SIGLEC1) function as auxiliary receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the N-terminal domain (NTD) or to the conserved proteoglycan site at the base of the Receptor Binding Domain (RBD), while poorly neutralizing infection of ACE2 over-expressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the Receptor Binding Motif (RBM), while potently neutralizing infection of ACE2 over-expressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.03.438258: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.
    SARS-CoV-2 nucleocapsid protein
    suggested: (Bioss Cat# bsm-41414M, RRID:AB_2848129)
    SARS-CoV-2 pseudotyped VSV was diluted 1:30 in media in the presence of 100 ng/mL anti-VSV-G antibody (clone 8G5F11, Absolute Antibody) and added 1:1 to each antibody dilution.
    anti-VSV-G
    suggested: (Absolute Antibody Cat# Ab01401-2.0, RRID:AB_2883992)
    For antibody-mediated inhibition of trans-infection, cells were pre-incubated with 10 ug/mL anti-SIGLEC1 antibody (Biolegend, clone 7-239) for 30 min.
    anti-SIGLEC1
    suggested: None
    Cells were incubated with primary antibodies anti-DC-SIGN/L-SIGN (Biolegend, cat. 845002, 1:500
    anti-DC-SIGN/L-SIGN
    suggested: None
    The cells were resuspended in 100 μL of FACS buffer prepared with 0.5% BSA (Sigma-Aldrich) in PBS containing the primary antibodies at a 1:100 dilution: mouse anti-DC/L-SIGN (Biolegend, cat. 845002), rabbit anti-DC-SIGN (Cell Signaling, cat. 13193), mouse anti-SIGLEC1 (Biologend, cat. 346002) or goat anti-ACE2 (R&D Systems, cat. AF933).
    anti-DC/L-SIGN
    suggested: None
    anti-DC-SIGN
    suggested: None
    anti-ACE2
    suggested: None
    After 1 h incubation on ice, the cells were washed two times and resuspended in FACS buffer containing the Alexa Fluor-488-labeled secondary antibodies at a 1:200 dilution: goat anti-mouse (Invitrogen cat.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Takara), Vero E6 (ATCC), MRC5 (Sigma-Aldrich), A549 (ATCC) or HeLa (ATCC) cells were transduced in the presence of 6 ug/mL polybrene (Millipore) for 24 h.
    A549
    suggested: None
    Single cell clones were derived from the A549-ACE2-TMPRSS2 cell line, all other cell lines represent cell pools.
    A549-ACE2-TMPRSS2
    suggested: None
    Serial 1:4 dilutions of the monoclonal antibodies were incubated with 200 pfu of SARS-CoV-2 (isolate USA-WA1/2020, passage 3, passaged in Vero E6 cells) for 30 min at 37°C in a BSL-3 facility.
    Vero E6
    suggested: None
    For viral neutralization, cells were seeded into black-walled, clear-bottom 96-well plates at 20,000 cells/well (293T cells were seeded into poly-L-lysine-coated wells at 35,000 cells/well) and cultured overnight at 37°C.
    293T
    suggested: None
    Trans-infection: Parental HeLa cells or HeLa cells stably expressing DC-SIGN, L-SIGN or SIGLEC1 were seeded at 5,000 cells per well in black-walled clear-bottom 96-well plates.
    HeLa
    suggested: None
    After 2 h inoculation, cells were washed four times with complete medium and 10,000 VeroE6-TMPRSS2 cells per well were added and incubated 17-20 h at 37°C for trans-infection.
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Cell-cell fusion of CHO-S cells: CHO cells stably expressing SARS-CoV-2 S-glycoprotein were seeded in 96 well plates for microscopy (Thermo Fisher Scientific) at 12’500 cells/well and the following day, different concentrations of mAbs and nuclei marker Hoechst (final dilution 1:1000) were added to the cells and incubated for additional 24h hours.
    CHO
    suggested: RRID:CVCL_Y503)
    Flow cytometry analysis for DC-SIGN, L-SIGN, SIGLEC1 and ACE-2: HEK293T cells expressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were resuspended at 4×106 cells/mL and 100 μL per well were seeded onto V-bottom 96-well plates (Corning, 3894).
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Micrographs were recorded using the Leginon software48 on a 100kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k x 4k CCD camera at 67,000 nominal magnification.
    Leginon
    suggested: (Leginon, RRID:SCR_016731)
    Selected particle images were subjected to the Bayesian polishing procedure implemented in Relion3.054 before performing another round of non-uniform refinement in CryoSPARC followed by per-particle defocus refinement and again non-uniform refinement.
    CryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 31. 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 rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  2. Version published to 10.1101/2021.04.03.438258v1 on bioRxiv