An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice

  1. Guillaume Beaudoin-Bussières
  2. Yaozong Chen
  3. Irfan Ullah
  4. Jérémie Prévost
  5. William D. Tolbert
  6. Kelly Symmes
  7. Shilei Ding
  8. Mehdi Benlarbi
  9. Shang Yu Gong
  10. Alexandra Tauzin
  11. Romain Gasser
  12. Debashree Chatterjee
  13. Dani Vézina
  14. Guillaume Goyette
  15. Jonathan Richard
  16. Fei Zhou
  17. Leonidas Stamatatos
  18. Andrew T. McGuire
  19. Hughes Charest
  20. Michel Roger
  21. Edwin Pozharski
  22. Priti Kumar
  23. Walther Mothes
  24. Pradeep D. Uchil
  25. Marzena Pazgier
  26. Andrés Finzi

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Abstract

Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

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    SciScore for 10.1101/2021.09.08.459408: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: PBMCs from healthy individuals as a source of effector cells in our ADCC assay were obtained under CRCHUM institutional review board (protocol #19.381).
    Consent: All participants were adults and provided informed written consent prior to enrollment in accordance with Institutional Review Board approval.
    IACUC: Mouse Experiments: All experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of and Institutional Biosafety Committee of Yale University (IBSCYU).
    Euthanasia Agents: All mice were anesthetized via isoflurane inhalation (3 - 5 % isoflurane, oxygen flow rate of 1.5 L/min) prior and during BLI using the XGI-8 Gas Anesthesia System.
    Sex as a biological variableAntibodies: The human antibodies (CV3-1, CV3-25 and CV3-13) used in the work were isolated from blood of male convalescent donor CV3 recovered 41 days after symptoms onset using fluorescent recombinant stabilized Spike ectodomains (S2P) as probes to identify antigen-specific B cells as previously described (Lu et al., 2020; Seydoux et al., 2020).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    At 48 hours post transfection, 293T cells were stained with CR3022, CV3-1, CV3- 13 WT, CV3-13 LALA, CV3-13 GASDALIE and CV3-25 antibodies (5μg/mL) for 45 minutes at 37°C before being washed 2 times in PBS.
    CV3-25
    suggested: None
    A mouse anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody (Clone 1C7, Bioss Antibodies) solution was prepared at 1 μg/mL in PBS + 1% non-fat milk and added to all wells for one hour at room temperature.
    anti-SARS-CoV-2 nucleocapsid protein
    suggested: None
    Following extensive washing (3×) with PBS, an anti-mouse IgG HRP secondary antibody solution was formulated in PBS + 1% non-fat milk.
    anti-mouse IgG
    suggested: None
    After 48 hours, infected Vero E6 cells were detached with PBS-EDTA and were incubated with 5 μg/mL of indicated antibodies for 30 minutes at 37℃, followed by staining with anti-human –AF647 secondary antibody and 1:1000 dilution of viability dye AquaVivid (Thermo Fisher Scientific) for 20 minutes at room temperature.
    anti-human –AF647
    suggested: None
    Stained target cells were incubated with 100 µL of the titrated concentrations of CV3-1, CV3-13 WT, CV3-13 LALA and CV3-13 GASDALIE antibodies (0,625 µg/mL, 1,25 µg/mL, 2,5 µg/mL, 5 µg/mL and 10 µg/mL) for 1h at 37°C, followed by two washes with media.
    CV3-13 GASDALIE
    suggested: None
    Antibody-mediated phagocytosis was determined by flow cytometry, gating on THP-1 cells that were triple- positive for eFluor450 and eFluor670 cellular dyes and GFP.
    GFP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    EXPERIMENTAL MODEL AND SUBJECT DETAILS: Cell and Viruses: Vero E6 (CRL-1586, American Type Culture Collection (ATCC) or Vero E6-TMPRSS2 (Craig B. Wilen, Yale University), were cultured at 37°C in RPMI supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non- essential amino acids, and 100 U/ml of penicillin–streptomycin.
    Vero E6
    suggested: None
    CEM.NKr, CEM.NKr-Spike, THP-1 and peripheral blood mononuclear cells (PBMCs) were maintained at 37°C under 5% CO2 in RPMI media, supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. 293T (or HEK293T) and 293T-ACE2 cells were maintained at 37°C under 5% CO2 in DMEM media, supplemented with 5% FBS and 100 U/mL penicillin/streptomycin. CEM.NKr (NIH AIDS Reagent Program) is a T lymphocytic cell line resistant to NK cell-mediated lysis.
    HEK293T
    suggested: None
    293T-ACE2 cells were cultured in medium supplemented with 2 mg/mL of puromycin (Millipore Sigma).
    293T-ACE2
    suggested: None
    The expression plasmids encoding the heavy and light chains of CV3-1, CV3-25, CV3-13 WT, CV3-13 LALA and CV3-13 GASDALIE IgG were transfected into Freestyle 293F cells (Thermo Fisher Scientific) using ExpiFectamine 293 transfection reagent as per the manufacturer’s protocol (Thermo Fisher Scientific).
    293F
    suggested: None
    Briefly, 293T cells were transfected by the calcium phosphate method with the pNL4.3 R-E- Luc plasmid (NIH AIDS Reagent Program) and a plasmid encoding for SARS- CoV-2 Spike at a ratio of 10:1.
    293T
    suggested: None
    Cell-surface staining of SARS-CoV-2-infected cells: 8M Vero-E6 cells were plated in T-175 flask 24 hours before infection.
    Vero-E6
    suggested: None
    THP-1 cells were used as effector cells and were stained with another cellular dye (cell proliferation dye eFluor670).
    THP-1
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    METHOD DETAILS: SARS-CoV-2 infection and treatment conditions: For all in vivo experiments, the 6 to 8 weeks male and female hACE2-K18 mice were intranasally challenged with 1 x 105 PFU in 25-30 µl volume under anesthesia (0.5 - 5 % isoflurane delivered using precision Dräger vaporizer with oxygen flow rate of 1 L/min).
    hACE2-K18
    suggested: None
    Recombinant DNA
    SentencesResources
    Light and heavy chains were cloned into the pTT expression plasmid (Durocher et al., 2002)
    pTT
    suggested: RRID:Addgene_44006)
    Flow cytometry analysis of cell-surface Spike staining: 10 µg of the different expressors of the original SARS-CoV-2 Spike (Hoffmann et al., 2020) or the different mutants of the SARS-CoV-2 Spike (B.1.1.7, D614G, Δ69-70, Δ144, N501Y, A570D, P681H, T716I, S982A and D1118H) (Li et al., 2021b; Prevost et al., 2021; Tauzin et al., 2021) were co-transfected with 2.5 µg of a green fluorescent protein (GFP) expressor (pIRES2-eGFP) into 2 × 106 293T cells using the standard calcium phosphate method.
    pIRES2-eGFP
    suggested: RRID:Addgene_14998)
    Briefly, 293T cells were transfected by the calcium phosphate method with the pNL4.3 R-E- Luc plasmid (NIH AIDS Reagent Program) and a plasmid encoding for SARS- CoV-2 Spike at a ratio of 10:1.
    pNL4.3 R-E-
    suggested: None
    Software and Algorithms
    SentencesResources
    Bioluminescence Imaging (BLI) of SARS-CoV-2 infection: All standard operating procedures and protocols for IVIS imaging of SARS-CoV-2 infected animals under ABSL-3 conditions were approved by IACUC, IBSCYU and YARC.
    YARC
    suggested: None
    Image sequences were assembled and converted to videos using Image J.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Samples were acquired on a LSRII cytometer (BD Biosciences) and data analysis was performed using FlowJo v10.5.3 (Tree Star).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Cryo-EM data processing, model building and analysis: Motion correction, CTF estimation, particle picking, curation and extraction, 2D classification, ab initio model reconstruction, volume refinements and local resolution estimation were carried out in cryoSPARC (Punjani et al., 2017; Rubinstein and Brubaker, 2015).
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Automated and manual model refinements were iteratively carried out in ccpEM (Burnley et al., 2017), Phenix (Liebschner et al., 2019) (real-space refinement) and Coot (Emsley and Cowtan, 2004).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Geometry validation and structure quality evaluation were performed by EM-Ringer (Barad et al., 2015) and Molprobity (Chen et al., 2010).
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Model- to-map fitting cross correlation and figures generation were carried out in USCF Chimera, Chimera X (Pettersen et al., 2021) and PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Quantification and Statistical Analysis: Statistical significance was derived by applying parametric unpaired t-test or non- parametric Mann-Whitney U test (two-tailed) available in GraphPad Prism software (La Jolla, CA, USA) depending on the normality distribution of the data.
    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 72, 73, 74, 76 and 77. 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.
    • 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 10.1101/2021.09.08.459408v1 on bioRxiv