SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

  1. Kizzmekia S. Corbett
  2. Darin Edwards
  3. Sarah R. Leist
  4. Olubukola M. Abiona
  5. Seyhan Boyoglu-Barnum
  6. Rebecca A. Gillespie
  7. Sunny Himansu
  8. Alexandra Schäfer
  9. Cynthia T. Ziwawo
  10. Anthony T. DiPiazza
  11. Kenneth H. Dinnon
  12. Sayda M. Elbashir
  13. Christine A. Shaw
  14. Angela Woods
  15. Ethan J. Fritch
  16. David R. Martinez
  17. Kevin W. Bock
  18. Mahnaz Minai
  19. Bianca M. Nagata
  20. Geoffrey B. Hutchinson
  21. Kapil Bahl
  22. Dario Garcia-Dominguez
  23. LingZhi Ma
  24. Isabella Renzi
  25. Wing-Pui Kong
  26. Stephen D. Schmidt
  27. Lingshu Wang
  28. Yi Zhang
  29. Laura J. Stevens
  30. Emily Phung
  31. Lauren A. Chang
  32. Rebecca J. Loomis
  33. Nedim Emil Altaras
  34. Elisabeth Narayanan
  35. Mihir Metkar
  36. Vlad Presnyak
  37. Catherine Liu
  38. Mark K. Louder
  39. Wei Shi
  40. Kwanyee Leung
  41. Eun Sung Yang
  42. Ande West
  43. Kendra L. Gully
  44. Nianshuang Wang
  45. Daniel Wrapp
  46. Nicole A. Doria-Rose
  47. Guillaume Stewart-Jones
  48. Hamilton Bennett
  49. Martha C. Nason
  50. Tracy J. Ruckwardt
  51. Jason S. McLellan
  52. Mark R. Denison
  53. James D. Chappell
  54. Ian N. Moore
  55. Kaitlyn M. Morabito
  56. John R. Mascola
  57. Ralph S. Baric
  58. Andrea Carfi
  59. Barney S. Graham

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Abstract

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.11.145920: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: ) Mouse Models: Animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and approval from the Animal Care and Use Committee of the Vaccine Research Center,
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableFor immunogenicity studies, 6-8-week-old female BALB/c (Charles River), BALB/cJ, C57BL/6J, or B6C3F1/J mice (Jackson Laboratory) were used.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN3) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: I-A/I-E (M5/114.15.2) PE, CD8a (53-6.7) BUV805, CD44 (IM7) BUV395, CD62L (MEL-14) BV605, and CD4 (RM4-5) BV480 in brilliant stain buffer (BD).
    CD8a
    suggested: (Meridian Life Science Cat# MAL75-211, RRID:AB_816933)
    CD44
    suggested: (BD Biosciences Cat# 740297, RRID:AB_2740036)
    CD62L
    suggested: (BD Biosciences Cat# 566111, RRID:AB_2739513)
    CD4
    suggested: None
    Cells were washed in perm/wash solution and stained with Fc Block (5 min at RT), followed by intracellular staining (30 min at 4°C) using a cocktail of the following antibodies purchased from BD, Biolegend, or eBioscience: CD3e (17A2) BUV737, IFN-γ (XMG1.2) BV650,
    CD3e
    suggested: (BD Biosciences Cat# 564618, RRID:AB_2738868)
    IFN-γ
    suggested: (BD Biosciences Cat# 563416, RRID:AB_2738193)
    Experimental Models: Cell Lines
    SentencesResources
    Cell Lines: HEK293T/17 (ATCC #CRL-11268), Vero E6 (ATCC), Huh7.5 cells (provided by Deborah R.
    HEK293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    Huh7.5
    suggested: RRID:CVCL_7927)
    Taylor, US Food and Drug Administration), and ACE-2-expressing 293T cells (provided by Michael Farzan, Scripps Research Institute) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, and 1% penicillin/streptomycin at 37°C and 5% CO2.
    293T
    suggested: None
    Vero E6 cells used in PRNT assays were cultured in DMEM supplemented with 10% Fetal Clone II and amphotericin B [0.25 μg/ml] at 37C and 5% CO2.
    Vero E6
    suggested: None
    Expi293 cells were maintained in manufacturer’s suggested media.
    Expi293
    suggested: RRID:CVCL_D615)
    In vitro mRNA Expression: HEK293T cells were transiently transfected with mRNA encoding SARS-CoV-2 WT S or S-2P protein using a TranIT mRNA transfection kit (Mirus).
    HEK293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    For immunogenicity studies, 6-8-week-old female BALB/c (Charles River), BALB/cJ, C57BL/6J, or B6C3F1/J mice (Jackson Laboratory) were used.
    BALB/c
    suggested: None
    C57BL/6J
    suggested: None
    B6C3F1/J
    suggested: RRID:IMSR_JAX:100010)
    For challenge studies to evaluate SARS-CoV-2 vaccines, BALB/cJ mice were challenged with 105 PFU of mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA).
    BALB/cJ
    suggested: None
    Software and Algorithms
    SentencesResources
    IC50 titers were determined using a log (agonist) vs. normalized response (variable slope) nonlinear function in Prism v8 (GraphPad)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    After 15 min, cells were washed with FC buffer then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to manufacturer instructions.
    BD Cytofix/Cytoperm
    suggested: None
    Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in Extended Data Figure 9
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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: 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 10.1101/2020.06.11.145920: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementTree Star , Inc . ) Mouse Models Animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and approval from the Animal Care and Use Committee of the Vaccine Research Center ,Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableImmunogenicity was assessed in six-week old female BALB/cJ , C57BL/6J , and B6C3F1/J mice by immunizing intramuscularly ( IM ) twice with 0.01 , 0.1 , or 1 µg of mRNA-1273 at a 3-week interval.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The spike ( S ) protein , a class I fusion glycoprotein analogous to influenza hemagglutinin ( HA) , respiratory syncytial virus ( RSV ) fusion glycoprotein ( F) , and human immunodeficiency virus ( HIV ) gp160 ( Env) , is the major surface protein on the CoV virion and the primary target for neutralizing antibodies .
    influenza hemagglutinin ( HA
    suggested: None
    mRNA-1273 induced dose-dependent S-specific binding antibodies after prime and boost in all mouse strains ( Fig . 2a-c)
    2a-c
    suggested: None
    Both immunogens elicited IgG2a and IgG1 subclass S-binding antibodies , indicating a balanced Th1/Th2 response ( Fig . 3a-c; Extended Data Fig . 6) .
    IgG1 subclass S-binding
    suggested: None
    Cells were then washed in FC buffer ( PBS supplemented with 2 % HI-FBS and 0.05 % NaN3 ) and resuspended in BD Fc Block ( clone 2.4G2 ) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: I-A/I-E ( M5/114.15.2 ) PE , CD8a ( 53-6.7 ) BUV805 , CD44 ( IM7 ) BUV395 , CD62L ( MEL-14 ) BV605 , and CD4 ( RM4-5 ) BV480 in brilliant stain buffer ( BD) .
    CD8a
    suggested: (BD Biosciences Cat# 564920, AB_2716856)
          <div style="margin-bottom:8px">
        <div><b>CD44</b></div>
        <div>suggested: (BD Biosciences Cat# 740297, <a href="https://scicrunch.org/resources/Any/search?q=AB_2740036">AB_2740036</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CD62L</b></div>
        <div>suggested: (BD Biosciences Cat# 566111, <a href="https://scicrunch.org/resources/Any/search?q=AB_2739513">AB_2739513</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CD4</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed in perm/wash solution and stained with Fc Block ( 5 min at RT) , followed by intracellular staining ( 30 min at 4°C ) using a cocktail of the following antibodies purchased from BD , Biolegend , or eBioscience: CD3e ( 17A2 ) BUV737 , IFN-γ ( XMG1.2 ) BV650 ,</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CD3e</b></div>
        <div>suggested: (Thermo Fisher Scientific Cat# A25978, <a href="https://scicrunch.org/resources/Any/search?q=AB_2536039">AB_2536039</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>IFN-γ</b></div>
        <div>suggested: (BD Biosciences Cat# 740498, <a href="https://scicrunch.org/resources/Any/search?q=AB_2740221">AB_2740221</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">Cell Lines HEK293T/17 ( ATCC #CRL-11268) , Vero E6 ( ATCC) , Huh7.5 cells ( provided by Deborah R .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T/17</b></div>
        <div>suggested: ATCC Cat# CRL-11268, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_1926">CVCL_1926</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Huh7.5</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">Expi293 cells were maintained in manufacturer’s suggested media .</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">In vitro mRNA Expression HEK293T cells were transiently transfected with mRNA encoding SARS-CoV-2 WT S or S-2P protein using a TranIT mRNA transfection kit ( Mirus) .</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">Briefly , HI serum was mixed with pseudoviruses , incubated , and then added to Huh7.5 cells or ACE-2expressing 293T cells , for MERS-CoV and SARS-CoV-2 respectively .</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">Virus/serum mixtures were incubated for 20 min at 37 ºC , followed by adsorption of 0.1 mL to each of two confluent Vero E6 cell monolayers ( in 10-cm2 wells ) for 30 min at 37°C .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</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">We thank members of the NIH NIAID VRC Translational Research Program for technical assistance with mouse experiments .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>VRC Translational Research Program</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MERS mRNA mouse challenge studies were funded under NIH Contract HHSN272201700036I Task Rrder No. 75N93019F00132 Requisition No. 5494549 ( to R . B . ) . K.S.C . ’s research fellowship was partially funded by the Undergraduate Scholarship Program , Office of Intramural Training and Education , Office of the Director , NIH . D.R . M. was funded by NIH NIAID grant T32-AI007151 and a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award. Author Contributions K.S.C . , D.K . E . , S.R . L . , O.M.A , S.B . B . , R.A . G . , S.H . , A . S. , C . Z . , A.T</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Undergraduate Scholarship Program</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data acquisition was performed on a BD LSRII Fortessa instrument ( BD Biosciences ) and analyzed by FlowJo software v10</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">IC50 titers were determined using a log ( agonist ) vs. normalized response ( variable slope ) nonlinear function in Prism v8 ( GraphPad)</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">After 15 min , cells were washed with FC buffer then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to manufacturer instructions .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BD Cytofix/Cytoperm</b></div>
        <div>suggested: None</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).

    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, including references cited, please follow this link.

  3. Version published to 10.1101/2020.06.11.145920 on bioRxiv