Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

  1. Dora Pinto
  2. Young-Jun Park
  3. Martina Beltramello
  4. Alexandra C. Walls
  5. M. Alejandra Tortorici
  6. Siro Bianchi
  7. Stefano Jaconi
  8. Katja Culap
  9. Fabrizia Zatta
  10. Anna De Marco
  11. Alessia Peter
  12. Barbara Guarino
  13. Roberto Spreafico
  14. Elisabetta Cameroni
  15. James Brett Case
  16. Rita E. Chen
  17. Colin Havenar-Daughton
  18. Gyorgy Snell
  19. Amalio Telenti
  20. Herbert W. Virgin
  21. Antonio Lanzavecchia
  22. Michael S. Diamond
  23. Katja Fink
  24. David Veesler
  25. Davide Corti

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Abstract

No abstract available

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  1. ScreenIT

    SciScore for 10.1101/2020.04.07.023903: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics statement: Donors provided written informed consent for the use of blood and blood components (such as sera), following approval by the Canton Ticino Ethics Committee, Switzerland.
    IRB: Ethics statement: Donors provided written informed consent for the use of blood and blood components (such as sera), following approval by the Canton Ticino Ethics Committee, Switzerland.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Abs S303, S304, S306, S309, S310 and S315 were expressed as rIgG-LS antibodies.
    S304
    suggested: (Abcam Cat# ab63554, RRID:AB_1141794)
    S306
    suggested: None
    S309
    suggested: None
    An Alexa647-labelled secondary antibody anti-human IgG Fc was used for detection.
    anti-human IgG
    suggested: None
    KD determination of full-length antibodies compared to Fab: His-tagged RBD of SARS-CoV or SARS-CoV-2 were loaded at 3 µg/ml in KB for 15 minutes onto anti-HIS (HIS2) biosensors (Molecular Devices, ForteBio).
    anti-HIS
    suggested: None
    HIS2
    suggested: None
    After an overnight incubation at 37°C, cells were stained with anti-human CD14-APC antibody (BD Pharmingen, Cat. Nr.: 561708, Clone M5E2) to stain monocytes.
    anti-human CD14-APC
    suggested: (SouthernBiotech Cat# 9561-11S, RRID:AB_2796938)
    Plates were washed and sequentially incubated with 1 µg/mL of CR302246 anti-S antibody and HRP-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA.
    anti-S
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    ExpiCHO cells then were incubated with Jurkat cells expressing FcγRIIIa receptor or FcγRIIa on their surface and stably transfected with NFAT-driven luciferase gene (Promega, Cat. Nr.:
    Jurkat
    suggested: TKG Cat# TKG 0209, RRID:CVCL_0065)
    HEK293T cells were co-transfected with a SARS-CoV, SARS-CoV-2, CUHK, GZ02, or WiV1 S encoding-plasmid, an MLV Gag-Pol packaging construct and the MLV transfer vector encoding aluciferase reporter using the Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer’s instructions.
    HEK293T
    suggested: None
    VeroE6 cells or DBT cells transfected with human ACE2 were cultured in DMEM containing 10% FBS, 1% PenStrep and plated into 96 well plates for 16-24 hours.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Virus was passaged once in Vero CCL81 cells (ATCC) and titrated by focus-forming assay on Vero E6 cells.
    Vero CCL81
    suggested: None
    Recombinant Spike ectodomain production: The SARS-CoV-2 2P S (Genbank: YP_009724390.1) ectodomain was produced in 500mL cultures of HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37°C in a humidified 8% CO2 incubator rotating at 130 r.p.m, as previously reported6.
    HEK293F
    suggested: None
    Software and Algorithms
    SentencesResources
    Binding of mAbs to transfected cells was analyzed by flow-cytometry using a ZE5 Cell Analyzer (Biorard) and FlowJo software (TreeStar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Measurements were done in duplicate and relative luciferase units (RLU) were converted to percent neutralization and plotted with a non-linear regression curve fit in PRISM.
    PRISM
    suggested: (PRISM, RRID:SCR_005375)
    Data were processed using Prism software (GraphPad Prism 8.0).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data were acquired using the Leginon software 47 to control an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV.
    Leginon
    suggested: (Leginon, RRID:SCR_016731)
    For each data set two rounds of reference-free 2D classification were performed using cryoSPARC 50 to select well-defined particle images.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    CryoEM model building and analysis: UCSF Chimera 54 and Coot were used to fit atomic models (PDB 6VXX and PDB 6VYB) into the cryoEM maps.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Models were analyzed using MolProbity 59, EMringer 60, Phenix 61 and privateer 62 to validate the stereochemistry of both the protein and glycan components.
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    The spike ORF was localized by performing reference protein (YP_009724390.1)-genome alignments with GeneWise2.
    GeneWise2
    suggested: None
    Multiple sequence alignment was performed using MAFFT.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Variants were determined by comparison of aligned sequences (n=2,229) to the reference sequence using the R/Bioconductor package Biostrings.
    R/Bioconductor
    suggested: None
    Biostrings
    suggested: (Biostrings, RRID:SCR_016949)
    A similar strategy was used to extract and translate spike protein sequences from SARS-CoV genomes sourced from ViPR (search criteria: SARS-related coronavirus, full-length genomes, human host, deposited before December 2019 to exclude SARS-CoV-2, n=53).
    ViPR
    suggested: (vipR, RRID:SCR_010685)

    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

    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 10.1038/s41586-020-2349-y
  3. ScreenIT

    SciScore for 10.1101/2020.04.07.023903: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementMATERIALS AND METHODS Ethics statement Donors provided written informed consent for the use of blood and blood components ( such as sera) , following approval by the Canton Ticino Ethics Committee ,Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Fc-dependent effector mechanisms , such as NK-mediated antibody-dependent cell cytotoxicity ( ADCC ) and antibody-dependent cellular phagocytosis ( ADCP ) can contribute to viral control in infected individuals .
    antibody-dependent cellular phagocytosis ( ADCP
    suggested: None
    Collectively , these results demonstrate that in addition to potent in vitro neutralization , S309 may leverage additional protective mechanisms in vivo , as previously shown for other antiviral antibodies41,42 .
    antibodies41,42
    suggested: None
    Abs S303 , S304 , S306 , S309 , S310 and S315 were expressed as rIgG-LS antibodies .
    S304
    suggested: (Abcam Cat# ab63554, AB_1141794)
          <div style="margin-bottom:8px">
        <div><b>S306</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>S309</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">An Alexa647-labelled secondary antibody anti-human IgG Fc was used for detection .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-human IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">KD determination of full-length antibodies compared to Fab: His-tagged RBD of SARSCoV or SARS-CoV-2 were loaded at 3 µg/ml in KB for 15 minutes onto anti-HIS ( HIS2 ) biosensors ( Molecular Devices , ForteBio) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-HIS</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>HIS2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After an overnight incubation at 37°C , cells were stained with anti-human CD14-APC antibody ( BD Pharmingen , Cat. Nr . : 561708 , Clone M5E2 ) to stain monocytes .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-human CD14-APC</b></div>
        <div>suggested: (SouthernBiotech Cat# 9561-11S, <a href="https://scicrunch.org/resources/Any/search?q=AB_2796938">AB_2796938</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed and sequentially incubated with 1 µg/mL of CR302246 anti-S antibody and HRP-conjugated goat antihuman IgG in PBS supplemented with 0.1 % saponin and 0.1 % BSA .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-S</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>antihuman IgG</b></div>
        <div>suggested: (GeneTex Cat# GTX28798, <a href="https://scicrunch.org/resources/Any/search?q=AB_374523">AB_374523</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Eight out of the twenty five mAbs bound to SARS-CoV-2 S and SARS-CoV S transfected CHO cells with EC50 values ranging between 1.4 and 6,100 ng/ml, and 0.8 and 254 ng/ml, respectively (Fig. 1ab).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CHO</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ExpiCHO cells then were incubated with Jurkat cells expressing FcgRIIIa receptor or FcgRIIa on their surface and stably transfected with NFAT-driven luciferase gene ( Promega , Cat. Nr . : G9798 and G7018 ) at an effector to target ratio of 6:1 for FcgRIIIa and 5:1 for FcgRIIa .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Jurkat</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were co-transfected with a SARS-CoV , SARS-CoV-2 , CUHK , GZ02 , or WiV1 S encoding-plasmid , an MLV Gag-Pol packaging construct and the MLV transfer vector encoding aluciferase reporter using the Lipofectamine 2000 transfection reagent ( Life Technologies ) according to the manufacturer’s instructions .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus was passaged once in Vero CCL81 cells ( ATCC ) and titrated by focus-forming assay on Vero E6 cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero CCL81</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant Spike ectodomain production The SARS-CoV-2 2P S ( Genbank: YP_009724390.1 ) ectodomain was produced in 500mL cultures of HEK293F cells grown in suspension using FreeStyle 293 expression medium ( Life technologies ) at 37°C in a humidified 8 % CO2 incubator rotating at 130 r . p . m , as previously reported6 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293F</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding of mAbs to transfected cells was analyzed by flow-cytometry using a ZE5 Cell Analyzer ( Biorard ) and FlowJo software ( TreeStar)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>FlowJo</b></div>
        <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Measurements were done in duplicate and relative luciferase units ( RLU ) were converted to percent neutralization and plotted with a non-linear regression curve fit in PRISM.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PRISM</b></div>
        <div>suggested: (PRISM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005375">SCR_005375</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were processed using Prism software ( GraphPad Prism 8.0)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using the Leginon software 47 to control an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter , operated in zero-loss mode with a slit width of 20 eV .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Leginon</b></div>
        <div>suggested: (Leginon, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016731">SCR_016731</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For each data set two rounds of reference-free 2D classification were performed using cryoSPARC 50 to select well-defined particle images .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>cryoSPARC</b></div>
        <div>suggested: (cryoSPARC, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016501">SCR_016501</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">UCSF Chimera 54 and Coot were used to fit atomic models ( PDB 6VXX and PDB 6VYB ) into the cryoEM maps .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Coot</b></div>
        <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Models were analyzed using MolProbity EMringer 60 , Phenix 61 and privateer 62 59 , to validate the stereochemistry of both the protein and glycan components .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MolProbity</b></div>
        <div>suggested: (MolProbity, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014226">SCR_014226</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The spike ORF was localized by performing reference protein (YP_009724390.1)-genome alignments with GeneWise2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GeneWise2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Multiple sequence alignment was performed using MAFFT.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MAFFT</b></div>
        <div>suggested: (MAFFT, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011811">SCR_011811</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Variants were determined by comparison of aligned sequences (n=2,229) to the reference sequence using the R/Bioconductor package Biostrings.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>R/Bioconductor</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Biostrings</b></div>
        <div>suggested: (Biostrings, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016949">SCR_016949</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A similar strategy was used to extract and translate spike protein sequences from SARS-CoV genomes sourced from ViPR (search criteria: SARSrelated coronavirus, full-length genomes, human host, deposited before December 2019 to exclude SARS-CoV-2, n=53).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ViPR</b></div>
        <div>suggested: (vipR, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010685">SCR_010685</a>)</div>
      </div>
    </td></tr></table>

    Results from OddPub: Thank you for sharing your data.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, please follow this link.

  4. Version published to 10.1101/2020.04.07.023903v3 on bioRxiv
  5. Version published to 10.1101/2020.04.07.023903v1 on bioRxiv
  6. Version published to 10.1101/2020.04.07.023903v2 on bioRxiv