Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

  1. Yiska Weisblum
  2. Fabian Schmidt
  3. Fengwen Zhang
  4. Justin DaSilva
  5. Daniel Poston
  6. Julio CC Lorenzi
  7. Frauke Muecksch
  8. Magdalena Rutkowska
  9. Hans-Heinrich Hoffmann
  10. Eleftherios Michailidis
  11. Christian Gaebler
  12. Marianna Agudelo
  13. Alice Cho
  14. Zijun Wang
  15. Anna Gazumyan
  16. Melissa Cipolla
  17. Larry Luchsinger
  18. Christopher D Hillyer
  19. Marina Caskey
  20. Davide F Robbiani
  21. Charles M Rice
  22. Michel C Nussenzweig
  23. Theodora Hatziioannou
  24. Paul D Bieniasz

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Abstract

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.21.214759: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All plasma samples were obtained under protocols approved by Institutional Review Boards at both institutions.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: All cell lines have been tested negative for contamination with mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody binding and ACE2 binding inhibition assay: A conformationally stabilized (6P) version of the SARS-CoV-2 S protein(25), appended at its C-terminus with a trimerization domain, a GGSGGn spacer sequence, NanoLuc luciferase, Strep-tag, HRV 3C protease cleavage site and 8XHis (S-6P-NanoLuc) was expressed and purified from the supernatant of 293T Expi cells.
    ACE2
    suggested: None
    Mutants thereof were also expressed and purifies following substitution of sequences encoding the RBD that originated from the unmodified S-expression plasmids For antibody binding assays, 20ng, 40ng, or 80ng S-6P-NanoLuc (or mutants thereof) were mixed with 100ng of antibodies, C121, C135, or C144, \ diluted in LI-COR Intercept blocking buffer, in a total volume of 60μl/well in 96-well plate.
    C144
    suggested: (Leinco Technologies Cat# C144, RRID:AB_2828501)
    For ACE2-binding inhibition assays, 20ng of S-6P-NanoLuc was mixed with 100ng of antibodies, C121, C135 or C144, diluted in 3% goat serum/PBS, in a total volume of 50μl.
    C121
    suggested: (Leinco Technologies Cat# C121, RRID:AB_2828361)
    C135
    suggested: None
    The half maximal inhibitory concentrations for plasma (NT50), and monoclonal antibodies (IC50) was calculated using 4-parameter nonlinear regression curve fit to raw or normalized infectivity data (GraphPad Prism).
    NT50
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: HEK-293T cells and derivatives were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2.
    HEK-293T
    suggested: None
    Briefly, 293T cells were transfected with pHIVNLGagPol, pCCNanoLuc2AEGFP and a WT or mutant SARS-CoV-2 expression plasmid (pSARS-CoV-2Δ19) using polyethyleneimine.
    293T
    suggested: None
    After 30 minutes incubation, the mixture was incubated with 1×105 293T cells, or 293T/ACE2cl.22 cells for 2 hours at 4°C.
    293T/ACE2cl.22
    suggested: None
    Software and Algorithms
    SentencesResources
    The PCR products were gel-purified and sequenced either using Sanger-sequencing or NGS as previously described (31).
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    For analysis of NGS data, the raw paired-end reads were pre-processed to remove adapter sequences and trim low-quality reads (Phred quality score <20) using BBDuk.
    Phred
    suggested: (Phred, RRID:SCR_001017)
    Information regarding RBD-specific variant frequencies, their corresponding P-values, and read depth were compiled using the Python programming language (version 3.7) running pandas (1.0.5), numpy (1.18.5), and matplotlib (3.2.2).
    Python
    suggested: (IPython, RRID:SCR_001658)
    matplotlib
    suggested: (MatPlotLib, RRID:SCR_008624)
    The half maximal inhibitory concentrations for plasma (NT50), and monoclonal antibodies (IC50) was calculated using 4-parameter nonlinear regression curve fit to raw or normalized infectivity data (GraphPad Prism).
    GraphPad
    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: 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 10.7554/elife.61312 on eLife
  3. ScreenIT

    SciScore for 10.1101/2020.07.21.214759: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll plasma samples were obtained under protocols approved by Institutional Review Boards at both institutions.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationAll cell lines have been tested negative for contamination with mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    Abstract Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2.
    SARS-CoV-2
    suggested: None
    Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARSCoV-2 agents.
    anti-SARSCoV-2
    suggested: None
    Results Selection of SARS-CoV-2 S variants using a replication competent VSV/SARS-CoV-2 chimeric virus To select SARS-CoV-2 S variants that escape neutralization by antibodies, we used a recently described replication-competent chimeric virus based on vesicular stomatitis virus that encodes the SARS-CoV-2 spike (S) protein and green fluorescent protein (rVSV/SARS-CoV2/GFP) (
    rVSV/SARS-CoV2/GFP
    suggested: None
    Antibodies that constitute at last part of the neutralizing activity evident in COV-NY plasma appear to recognize an epitope that includes and K444 and V445.
    V445
    suggested: None
    Antibody binding and ACE2 binding inhibition assay A conformationally stabilized (6P) version of the SARS-CoV-2 S protein(25), appended at its C-terminus with a trimerization domain, a GGSGGn spacer sequence, NanoLuc luciferase, Strep-tag, HRV 3C protease cleavage site and 8XHis (S-6P-NanoLuc) was expressed and purified from the supernatant of 293T Expi cells.
    ACE2
    suggested: None
    Mutants thereof were also expressed and purifies following substitution of sequences encoding the RBD that originated from the unmodified Sexpression plasmids For antibody binding assays, 20ng, 40ng, or 80ng S-6P-NanoLuc (or mutants thereof) were mixed with 100ng of antibodies, C121, C135, or C144, \ diluted in LI-COR Intercept blocking buffer, in a total volume of 60μl/well in 96-well plate.
    C121
    suggested: (Leinco Technologies Cat# C121, AB_2828361)
    Patent applications submitted by Rockefeller University are pending for anti SARS-CoV-2 antibodies (MCN, DR, inventors) and VSV/SARS-CoV-2 chimeric virus (PDB, TH FS and YW, inventors) A 10μg/ml C121, C135, C144 or plasma dilution 10μg/ml C121, C135, C144 or plasma dilution 1x106 IU rVSV/SARS-CoV-2/GFP p1 B p2 No Antibody Sequence, Isolate mutants by limiting dilution Plasma [5x initial] Sequence, Isolate mutants by limiting dilution Sequence Plasma [5x initial] p3 p4 10μg/ml C121 C Figure 1.
    anti SARS-CoV-2
    suggested: (Abcam Cat# ab273074, AB_2847846)
          <div style="margin-bottom:8px">
        <div><b>C135</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>p3</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Selection of SARS-CoV-2 S mutations that confer antibody resistance. A. Outline of serial passage experiments with replication competent VSV derivatives encoding the SARS-CoV-2 S envelope glycoprotein and a GFP reporter (rVSV/SARS-CoV-2/GFP) in 293T/ACE2(B) cells in the presence of neutralizing antibodies or plasma. B. Representative images of 293T/ACE2(B) cells infected with 1x106 PFU of rVSV/SARS-CoV2/GFP in the presence or absence of 10μg/ml of the monoclonal antibody C121. C.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV-2 S 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">C144 passaged rVSV/SARS-CoV-2/GFP Populations Mutant purification by limiting dilution rVSV/SARS-CoV-2/GFP (E484K) rVSV/SARS-CoV-2/GFP (Q493R) Figure 3 - supplement 1 Example of plaque purification of individual viral mutants from populations passaged in the presence of antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>rVSV/SARS-CoV-2/GFP</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acids whose substitution confers partial or complete (IC50 >10μg/ml) resistance to each monoclonal antibody in the HIV-pseudotype assays are indicated for C121 (red) C135 (green) and C144 (purple). E. Binding of S-NanoLuc fusion protein in relative light units (RLU) to 293T or 293T/ACE2cl.22 cells after preincubation in the absence or presence of C121, C135 and C144 monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <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;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 HEK-293T cells and derivatives were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 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>HEK-293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were transfected with pHIVNLGagPol, pCCNanoLuc2AEGFP and a WT or mutant SARS-CoV-2 expression plasmid (pSARS-CoV2Δ19) using polyethyleneimine.</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">Amino acids whose substitution confers partial or complete (IC50 >10μg/ml) resistance to each monoclonal antibody in the HIV-pseudotype assays are indicated for C121 (red) C135 (green) and C144 (purple). E. Binding of S-NanoLuc fusion protein in relative light units (RLU) to 293T or 293T/ACE2cl.22 cells after preincubation in the absence or presence of C121, C135 and C144 monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T/ACE2cl.22</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">The PCR products were gel-purified and sequenced either using Sanger-sequencing or NGS as previously described (31).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>NGS</b></div>
        <div>suggested: (NGSadmix, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003208">SCR_003208</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For analysis of NGS data, the raw paired-end reads were pre-processed to remove adapter sequences and trim low-quality reads (Phred quality score <20) using BBDuk.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Phred</b></div>
        <div>suggested: (Phred, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001017">SCR_001017</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Information regarding RBD-specific variant frequencies, their corresponding P-values, and read depth were compiled using the Python programming language (version 3.7) running pandas (1.0.5), numpy (1.18.5), and matplotlib (3.2.2).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Python</b></div>
        <div>suggested: (IPython, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001658">SCR_001658</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>matplotlib</b></div>
        <div>suggested: (MatPlotLib, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008624">SCR_008624</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The half maximal inhibitory concentrations for plasma (NT50), and monoclonal antibodies (IC50) was calculated using 4-parameter nonlinear regression curve fit to raw or normalized infectivity data (GraphPad Prism).</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></table>

    Data from additional tools added to each annotation on a weekly basis.

    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.

  4. Version published to 10.1101/2020.07.21.214759v1 on bioRxiv