Human neutralizing antibodies elicited by SARS-CoV-2 infection

  1. Bin Ju
  2. Qi Zhang
  3. Jiwan Ge
  4. Ruoke Wang
  5. Jing Sun
  6. Xiangyang Ge
  7. Jiazhen Yu
  8. Sisi Shan
  9. Bing Zhou
  10. Shuo Song
  11. Xian Tang
  12. Jinfang Yu
  13. Jun Lan
  14. Jing Yuan
  15. Haiyan Wang
  16. Juanjuan Zhao
  17. Shuye Zhang
  18. Youchun Wang
  19. Xuanling Shi
  20. Lei Liu
  21. Jincun Zhao
  22. Xinquan Wang
  23. Zheng Zhang
  24. Linqi Zhang

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Abstract

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    SciScore for 10.1101/2020.03.21.990770: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementThis study received approval from the Research Ethics Committee of Shenzhen Third People 's Hospital , China ( approval number: 2020-084) .Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Among a total of 69 antibodies from P#2 , the majority ( 59 % ) were scattered across various branches and the remaining ( 41 % ) were clonally expanded into three major clusters ( Figure 3A) .
    total of 69
    suggested: None
    Control antibodies from P#1 demonstrated even lower competing power with ACE2 .
    ACE2
    suggested: None
    We selected a total of six antibodies with ACE2 competitive capacities of at least 70 % and analyzed them in a pairwise competition fashion using SPR .
    SPR
    suggested: None
    The most potent antibody , P2C-1F11 , did not seem target the same epitope as the relatively moderate antibody P2C-1C10 .
    P2C-1F11
    suggested: None
    Finally , despite successfully isolating and characterizing a large of number mAbs against SARS-CoV-2 , we cannot draw any firm correlation between antibody response and disease status at this time.
    SARS-CoV-2
    suggested: None
    The third staining at 4 °C for 30min involved either: Streptavidin-APC ( eBioscience ) and/or Streptavidin-PE ( BD Biosciences ) to target the Strep tag of RBD , or antihis-APC and anti-his-PE antibodies ( Abcam ) to target the His tag of RBD .
    antihis-APC
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti-his-PE</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The IgG heavy and light chain variable genes were amplified by nested PCR and cloned into linear expression cassettes or expression vectors to produce full IgG1 antibodies as previously described 29,41 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>full IgG1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR products were purified and cloned into the backbone of antibody expression vectors containing the constant regions of human IgG1 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>human IgG1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV antibodies ( S230 and m396 ) previously isolated by others 42 were synthesized and sequences verified before expression in 293T cells and purification by protein A chromatography .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HIV-1 antibody VRC01 was a broadly neutralizing antibody directly isolated from a patient targeting the CD4 binding site of envelope glycoprotein 40 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CD4 binding site of envelope glycoprotein 40</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were then stained with PE labeled anti-human IgG Fc secondary antibody ( Biolegend ) at a 1:20 dilution in 50 μl staining buffer at room temperature for 30 minutes .</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">VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HIV-1 envelope glycoprotein.</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The recombinant RBD was labeled with either a Strep or His tag and used alone or in combination to identify and isolate RBD-specific single B cells through staining with the Streptavidin-APC and/or Streptavidin-PE, or anti-His- APC and anti-His-PE antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-His- APC</b></div>
        <div>suggested: None</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">SARS-CoV-2 , SARS-CoV and MERS-CoV pseudovirus were generated by cotransfection of human immunodeficiency virus backbones expressing firefly luciferase ( pNL43R-E-luciferase ) and pcDNA3.1 ( Invitrogen ) expression vectors encoding the respective S proteins into 293T cells ( ATCC ) 37,38,44,45</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Huh7 cells ( ATCC ) ( approximately 1.5 × 104 per well ) were added in duplicate to the virusantibody mixture.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Huh7</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The isolate was amplified in Vero cell lines to make working stocks of the virus ( 1 × 105 PFU/ml) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serial dilutions of mAbs were mixed separately with 100 PFU of SARS-CoV-2 , incubated at 37 °C for 1 h , and added to the monolayer of Vero E6 cells in duplicates .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <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">The genes encoding the heavy and light chains of isolated antibodies were separately cloned into expression vectors containing IgG1 constant regions and the vectors were transiently transfected into HEK293T or 293F cells using polyethylenimine ( PEI ) ( Sigma) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293F</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_D615">CVCL_D615</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK 293T</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">Bioinformatic and biologic characterization indicates that these antibodies are derived from broad and diverse families of antibody heavy and light chains .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Bioinformatic</b></div>
        <div>suggested: (QFAB Bioinformatics, <a href="https://scicrunch.org/resources/Any/search?q=SCR_012513">SCR_012513</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Finally , the cells were re-suspended and analyzed with FACS Calibur instrument ( BD Biosciences , USA ) and FlowJo 10 software ( FlowJo , USA)</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">Half-maximal inhibitory concentrations ( IC50 ) of the evaluated mAbs were determined by luciferase activity 48h after exposure to virusantibody mixture using GraphPad Prism 6 ( GraphPad Software Inc . ) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    
      <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">The IgG heavy and light chain variable genes were aligned using Clustal W in the BioEdit sequence analysis package ( https://bioedit.software.informer.com/7.2/).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BioEdit</b></div>
        <div>suggested: (BioEdit, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007361">SCR_007361</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phylogenetic analyses were performed by the Maximum Likelihood method using MEGA X ( Molecular Evolutionary Genetics Analysis across computing platforms) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MEGA X</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr></table>

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  2. Version published to 10.1038/s41586-020-2380-z
  3. Version published to 10.1101/2020.03.21.990770v2 on bioRxiv
  4. Version published to 10.1101/2020.03.21.990770v1 on bioRxiv