Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals

  1. Davide F. Robbiani
  2. Christian Gaebler
  3. Frauke Muecksch
  4. Julio C. C. Lorenzi
  5. Zijun Wang
  6. Alice Cho
  7. Marianna Agudelo
  8. Christopher O. Barnes
  9. Anna Gazumyan
  10. Shlomo Finkin
  11. Thomas Hagglof
  12. Thiago Y. Oliveira
  13. Charlotte Viant
  14. Arlene Hurley
  15. Hans-Heinrich Hoffmann
  16. Katrina G. Millard
  17. Rhonda G. Kost
  18. Melissa Cipolla
  19. Kristie Gordon
  20. Filippo Bianchini
  21. Spencer T. Chen
  22. Victor Ramos
  23. Roshni Patel
  24. Juan Dizon
  25. Irina Shimeliovich
  26. Pilar Mendoza
  27. Harald Hartweger
  28. Lilian Nogueira
  29. Maggi Pack
  30. Jill Horowitz
  31. Fabian Schmidt
  32. Yiska Weisblum
  33. Eleftherios Michailidis
  34. Alison W. Ashbrook
  35. Eric Waltari
  36. John E. Pak
  37. Kathryn E. Huey-Tubman
  38. Nicholas Koranda
  39. Pauline R. Hoffman
  40. Anthony P. West
  41. Charles M. Rice
  42. Theodora Hatziioannou
  43. Pamela J. Bjorkman
  44. Paul D. Bieniasz
  45. Marina Caskey
  46. Michel C. Nussenzweig

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Abstract

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2 1–5 . Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC 50 s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.13.092619: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All participants provided written informed consent before participation in the study and the study was conducted in accordance with Good Clinical Practice.
    RandomizationThe GRAVY scores from all 533 IGH CDR3 amino acid sequences from this study (sequence COV047_P4_IgG_51-P1369 lacks CDR3 amino acid sequence) were used to perform the test and 5000 GRAVY scores of the sequences from the public database were randomly selected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableExclusion criteria included presence of symptoms suggestive of active SARS-CoV-2 infection, or hemoglobin < 12 g/dL for males and < 11 g/dL for females.
    Cell Line AuthenticationContamination: All cell lines have been tested negative for contamination with mycoplasma and were obtained from the ATCC (with the exception for Huh-7.5).

    Table 2: Resources

    Antibodies
    SentencesResources
    Plates were washed 6 times with washing buffer and then incubated with anti-human IgG or IgM secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research 109-036-088 and 109-035-129) in blocking buffer at a 1:5000 dilution.
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-036-088, RRID:AB_2337594)
    IgM
    suggested: None
    The antibody-virus-mix was then directly applied to VeroE6 cells (MOI of ~0.1 PFU/cell).
    antibody-virus-mix
    suggested: None
    A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (catalog no. GTX135357; GeneTex) was added to the cells at 1:500 dilution in blocking solution and incubated at 4°C overnight.
    anti-SARS-CoV-2
    suggested: None
    A goat anti-rabbit AlexaFluor 594 (catalog no. A-11012; Life Technologies) at a dilution of 1:2,000 was used as a secondary antibody.
    anti-rabbit
    suggested: None
    The enriched B cells were incubated in FACS buffer (1 X Phosphate-buffered Saline (PBS), 2% calf serum, 1 mM EDTA) with the following anti-human antibodies: anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41)
    anti-human antibodies: anti-CD20-PECy7 ( BD Biosciences , 335793) , anti-CD3-APC-eFluro 780
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The SARS-CoV-2 S 2P ectodomain and RBD constructs were produced by transient transfection of 500 mL of Expi293 cells (Thermo Fisher) and purified from clarified transfected cell supernatants four days post-transfection using Ni2+-NTA affinity chromatography (GE Life Sciences).
    Expi293
    suggested: RRID:CVCL_D615)
    To generate pseudotyped viral stocks, 293T cells were transfected with pNL4-3ΔEnv-nanoluc and pSARS-CoV2-Strunc or pSARS-CoV-S using polyethyleneimine.
    293T
    suggested: None
    Infectivity of virions was determined by titration on 293TACE2 cells.
    293TACE2
    suggested: RRID:CVCL_YZ65)
    SARS-CoV-2, strain USA-WA1/2020, was obtained from BEI Resources and amplified in VeroE6 cells at 33°C.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Viral titers were measured on Huh-7.5 cells by standard plaque assay (PA).
    Huh-7.5
    suggested: RRID:CVCL_7927)
    Software and Algorithms
    SentencesResources
    The average of its signal was used for normalization of all the other values on the same plate with Excel software prior to calculating the area under the curve using Prism 8 (GraphPad).
    Excel
    suggested: None
    The half-maximal inhibitory concentration for plasma (NT50) or monoclonal antibodies (IC50) was determined using 4-parameter nonlinear regression (GraphPad Prism)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All statistical analyses were done using Prism 8 software (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    For somatic hypermutations, IGHV and IGLV nucleotide sequences were aligned against their closest germlines using IgBlast and the number of differences were considered nucleotide mutations.
    IgBlast
    suggested: (IgBLAST, RRID:SCR_002873)
    Hydrophobicity scale based on free energy of transfer (kcal/mole)36 implemented by the R package Peptides available in the Comprehensive R Archive Network repository (https://journal.r-project.org/archive/2015/RJ-2015-001/RJ-2015-001.pdf).
    Comprehensive R Archive
    suggested: None
    Micrographs were recorded on a Thermo Fisher Talos Arctica transmission electron microscope operating at 200 keV using a K3 direct electron detector (Gatan, Inc) and SerialEM automated image acquisition software38.
    Thermo Fisher Talos
    suggested: None
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Reference-free 2D class averages and ab initio volumes were generated in cryoSPARC, and subsequently 3D-classified to identify classes of S-Fab complexes, that were then homogenously refined.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)

    Results from OddPub: Thank you for sharing your code and 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. ScreenIT

    SciScore for 10.1101/2020.05.13.092619: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll participants provided written informed consent before participation in the study and the study was conducted in accordance with Good Clinical Practice.RandomizationThe GRAVY scores from all 533 IGH CDR3 amino acid sequences from this study ( sequence COV047_P4_IgG_51-P1369 lacks CDR3 amino acid sequence ) were used to perform the test and 5000 GRAVY scores of the sequences from the public database were randomly selected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableThere was no age difference between females and males in our cohort ( Extended Data Fig . 1)Cell Line AuthenticationAll cell lines have been tested negative for contamination with mycoplasma and were obtained from the ATCC ( with the exception for Huh-7.5) .

    Table 2: Resources

    Antibodies
    SentencesResources
    However, little is known about the human antibody response to SARS-CoV-21-5.
    SARS-CoV-21-5
    suggested: None
    Plasma samples were tested for binding to the SARS-CoV-2 RBD and trimeric spike ( S ) proteins by ELISA using anti-IgG or -IgM secondary antibodies for detection ( Fig . 1 , Extended Data Table 1 and Extended Data Figs .
    anti-IgG
    suggested: None
    Notably , levels of anti-RBD- and -S IgG antibodies correlated strongly with NT50 ( Fig . 2 c , d)
    anti-RBD-
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>-S IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The average number of V genes nucleotide mutations for IGH and IGL was 4.2 and 2.8 , respectively ( Extended Data Fig . 8) , which is lower than in antibodies cloned from individuals suffering from chronic infections such as Hepatitis B or HIV-1 , and similar to antibodies derived from primary malaria infection or non-antigen-enriched circulating IgG memory cells6-8 ( Wang et al , in press , https://www.biorxiv.org/content/10.1101/2020.03.04.976159v1.full).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HIV-1</b></div>
        <div>suggested: (ImmunoDiagnostics Cat# 1101, <a href="https://scicrunch.org/resources/Any/search?q=AB_1305157">AB_1305157</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine whether human anti-SARS-CoV-2 monoclonal antibodies with neutralizing activity can bind to distinct domains on the RBD , we performed bilayer interferometry experiments in which a preformed antibody-RBD immune complex was exposed to a second monoclonal.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antibody-RBD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To further define the binding characteristics of Groups 1 and 2 antibodies , we imaged SARS-CoV2 S–Fab complexes by negative stain electron microscopy ( nsEM ) using C002 ( Group 1 , an IGHV3-30/IGKV1-39 antibody , which is clonally expanded in 2 donors) , C119 and C121 ( both in Group 2 ) Fabs ( Fig . 4 f-r and Extended Data Fig . 10) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IGHV3-30/IGKV1-39</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies are essential elements of most vaccines and will likely be crucial component of an effective vaccine against SARS-CoV-220 ( PMID:32434945; PMID:32434946) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV-220</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The observation that plasma neutralizing activity is low in most convalescent individuals , but that recurrent antiSARS-CoV-2 RBD antibodies with potent neutralizing activity can be found in individuals with unexceptional plasma neutralizing activity suggests that humans are intrinsically capable of generating anti-RBD antibodies that potently neutralize SARS-CoV-2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antiSARS-CoV-2 RBD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Asterisks indicate donors from which antibody sequences were derived. c , AUC for anti-RBD IgG ELISA ( X axis ) plotted against NT50 ( Y axis ) r=0.6432 , p=<0.0001 . d , AUC for anti-S IgG ELISA ( X axis ) plotted against NT50 ( Y axis ) r=0.6721 , p=<0.0001 . e , NT50 for outpatients and hospitalized individuals p=0.0495 . f , NT50 for all males and females in the cohort p=0.0031 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-RBD IgG</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>NT50</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-S IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interconnecting lines indicate the relationship between antibodies that share V and J gene segment sequences at both IGH and IGL .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IGL</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">C121 , C135 C144 and isotype control in red , green , purple , and black respectively , in all panels . b , Graph shows normalized relative luminescence values ( RLU , Y axis ) in cell lysates of 293TACE2 cells 48 hours after infection with SARS-CoV-2 pseudovirus in the presence of increasing concentrations of monoclonal antibodies ( X axis) . c , RLU for SARS-CoV-2 pseudovirus assay ( Y axis ) vs. titration of monoclonal antibodies C121 , CoV-2 real virus neutralization assay .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>C135</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>C144</b></div>
        <div>suggested: (Leinco Technologies Cat# C144, <a href="https://scicrunch.org/resources/Any/search?q=AB_2828501">AB_2828501</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">e , IC50s for antibodies assayed in b and d. f , Diagrammatic representation of biolayer interferometry experiment . g , Graph shows binding of C144 , C101 , C121 , C009 , C135 , and CR302210,11 to RBD . h-m ,</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>C101</b></div>
        <div>suggested: (Leinco Technologies Cat# C101, <a href="https://scicrunch.org/resources/Any/search?q=AB_2737447">AB_2737447</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Values are normalized by the subtraction of the autologous antibody control . o-q , Representative 2D-class averages and 3D reconstructed volumes for SARS-CoV-S 2P trimers complexed with C002 , C119 , and C121 Fabs .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>C002</b></div>
        <div>suggested: (GenWay Biotech Inc. Cat# GWB-C002D4, <a href="https://scicrunch.org/resources/Any/search?q=AB_10269310">AB_10269310</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>C119</b></div>
        <div>suggested: (Leinco Technologies Cat# C119, <a href="https://scicrunch.org/resources/Any/search?q=AB_2828355">AB_2828355</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>C121</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed 6 times with washing buffer and then incubated with anti-human IgG or IgM secondary antibody conjugated to horseradish peroxidase ( HRP ) ( Jackson Immuno Research 109-036-088 and 109-035-129 ) in blocking buffer at a 1:5000 dilution .</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: (Jackson ImmunoResearch Labs Cat# 109-036-088, <a href="https://scicrunch.org/resources/Any/search?q=AB_2337594">AB_2337594</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus-mix was then directly applied to VeroE6 cells ( MOI of ~0.1 PFU/cell) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antibody-virus-mix</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A rabbit polyclonal antiSARS-CoV-2 nucleocapsid antibody ( catalog no. GTX135357; GeneTex ) was added to the cells at 1:500 dilution in blocking solution and incubated at 4°C overnight .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antiSARS-CoV-2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A goat anti-rabbit AlexaFluor 594 ( catalog no. A-11012; Life Technologies ) at a dilution of 1:2,000 was used as a secondary antibody .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-rabbit</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The frequency distributions of human V genes in anti-SARS-CoV-2 antibodies from this study was compared to Sequence Read Archive SRP01097034 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-SARS-CoV-2</b></div>
        <div>suggested: (Abcam Cat# ab272854, <a href="https://scicrunch.org/resources/Any/search?q=AB_2847844">AB_2847844</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Clinical correlates of plasma antibody titers . a , AUC for IgG antiRBD ( Y axis ) for all cases and contacts in the cohort p=0.0107 . b , AUC for IgM anti-RBD ( Y axis ) for all cases and contacts in the cohort p=0.5371 . c , AUC for IgG anti-S ( Y axis ) for all cases and contacts in the cohort p=0.0135 . d , AUC for IgM anti-S ( Y axis ) for all cases and contacts in the cohort p=0.7838 . e , AUC for IgM anti-RBD ( Y axis ) for all males and females in the cohort p=0.9597 . f , AUC for IgG anti-S ( Y axis ) for all males and females in the cohort p=0.0275 . g , AUC for IgM anti-S ( Y axis ) for all males and females in the cohort p=0.5363 . h , AUC for IgM anti-RBD ( Y axis ) for all outpatient and hospitalized participants in the cohort p=0.0059 . i , AUC for IgG anti-S ( Y axis ) for all outpatient and hospitalized participants in the cohort p=0.0623 . j , AUC for IgM anti-S ( Y axis ) for all outpatient and hospitalized participants in the cohort p=0.2976 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antiRBD</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-RBD</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-S</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Additional clinical correlates of plasma antibody titers . a , Time between symptom onset and plasma collection in days ( X axis ) plotted against AUC for IgG antiRBD ( Y axis ) r=-0.0261 p=0.7533 . b , Time between symptom onset and plasma collection in days ( X axis ) plotted against AUC for IgG anti-S ( Y axis ) r=-0.1495 p=0.0697 . c , Time between symptom onset and plasma collection in days ( X axis ) plotted against AUC for IgM anti-S ( Y axis ) r=0.1496 p=0.0695 . d , Age ( X axis ) plotted against AUC for IgM anti-RBD ( Y axis ) r=0.0172 p=0.8355 . e , Age ( X axis ) plotted against AUC for IgG anti-S ( Y axis ) r=0.1523 p=0.0638 . f ,</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>plotted against AUC for IgG</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>plotted against AUC for IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">showing binding of antibodies C144, C101, C002, C121, C009, C019 (see also main text Fig. 4).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>C009</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>C019</b></div>
        <div>suggested: (GenWay Biotech Inc. Cat# GWB-C019A0, <a href="https://scicrunch.org/resources/Any/search?q=AB_10278338">AB_10278338</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The table displays the shift in nanometers after second antibody (Ab2) binding to the antigen in the presence of the first antibody (Ab1).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Ab1</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">The SARS-CoV-2 S 2P ectodomain and RBD constructs were produced by transient transfection of 500 mL of Expi293 cells ( Thermo Fisher ) and purified from clarified transfected cell supernatants four days post-transfection using Ni2+-NTA affinity chromatography ( GE Life Sciences) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Expi293</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">For constitutive expression of ACE2 in 293T cells , a cDNA encoding ACE2 , carrying two inactivating mutations in the catalytic site ( H374N & H378N) , was inserted into CSIB 3’ to the SFFV promoter25 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infectivity of virions was determined by titration on 293TACE2 cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293TACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6 kidney epithelial cells ( Chlorocebus sabaeus ) and Huh-7.5 hepatoma cells ( H . sapiens ) were cultured in Dulbecco’s Modified Eagle Medium ( DMEM ) supplemented with 1 % nonessential amino acids ( NEAA ) and 10 % fetal bovine serum ( FBS ) at 37oC and 5 % CO2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Huh-7.5</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_7927">CVCL_7927</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The day prior to infection VeroE6 cells were seeded at 12,500 cells/well into 96-well plates .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>VeroE6</b></div>
        <div>suggested: JCRB Cat# JCRB1819, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_YQ49">CVCL_YQ49</a></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">Red , blue , orange and yellow pie slices indicate clones that share the same IGHV and IGLV genes . c , Circos plot shows sequences from all 6 individuals with clonal relationships depicted as in b .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Circos</b></div>
        <div>suggested: (Circos, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011798">SCR_011798</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The average of its signal was used for normalization of all the other values on the same plate with Excel software prior to calculating the area under the curve using Prism 8 ( GraphPad) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Excel</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were done using Prism 8 software ( GraphPad) .</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>
    
      <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">For somatic hypermutations , IGHV and IGLV nucleotide sequences were aligned against their closest germlines using IgBlast and the number of differences were considered nucleotide mutations .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgBlast</b></div>
        <div>suggested: (IgBLAST, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002873">SCR_002873</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hydrophobicity scale based on free energy of transfer ( kcal/mole)36 implemented by the R package Peptides available in the Comprehensive R Archive Network repository ( https://journal.r-project.org/archive/2015/RJ-2015-001/RJ-2015001.pdf).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Comprehensive R Archive</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Micrographs were recorded on a Thermo Fisher Talos Arctica transmission electron microscope operating at 200 keV using a K3 direct electron detector ( Gatan , Inc ) and SerialEM automated image acquisition software38 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Thermo Fisher Talos</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>SerialEM</b></div>
        <div>suggested: (SerialEM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017293">SCR_017293</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reference-free 2D class averages and ab initio volumes were generated in cryoSPARC , and subsequently 3D-classified to identify classes of S-Fab complexes , that were then homogenously refined.</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></table>

    Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

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  3. Version published to 10.1101/2020.05.13.092619 on bioRxiv