Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody

  1. Guillermo Valenzuela Nieto
  2. Ronald Jara
  3. Daniel Watterson
  4. Naphak Modhiran
  5. Alberto A Amarilla
  6. Johanna Himelreichs
  7. Alexander A. Khromykh
  8. Constanza Salinas
  9. Teresa Pinto
  10. Yorka Cheuquemilla
  11. Yago Margolles
  12. Natalia López González del Rey
  13. Zaray Miranda-Chacon
  14. Alexei Cuevas
  15. Anne Berking
  16. Camila Deride
  17. Sebastián González-Moraga
  18. Héctor Mancilla
  19. Daniel Maturana
  20. Andreas Langer
  21. Juan Pablo Toledo
  22. Ananda Müller
  23. Benjamín Uberti
  24. Paola Krall
  25. Pamela Ehrenfeld
  26. Javier Blesa
  27. Pedro Chana-Cuevas
  28. German Rehren
  29. David Schwefel
  30. Luis Ángel Fernandez
  31. Alejandro Rojas-Fernandez

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Abstract

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.09.137935: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Immunization and VHH library construction: The alpaca immunization process followed the guidelines established by the Bioethics Committee of the Austral University of Chile (certifications 338/2019 and 388/2020).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAdjuvant, GERBU FAMA) diluted 1:1 in sterile water and injected subcutaneously into a male alpaca (Vicugna pacos).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After this, the membrane was incubated with a Goat anti-mouse IgG antibody coupled to HRP (Invitrogen) (1:5000) in PBS-T containing 5% BSA, for 1h at room temperature, followed by 3 x 5 min washes with PBS-T and visualized using the ECL reagent (Pierce).
    anti-mouse IgG
    suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)
    After washing another 3 times with PBS, a mouse anti-myc antibody (Cell Signaling) was used at 1:3000 and incubated during 45 minutes at 37°C.
    anti-myc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    High content microscopy: Spike-GFP transfected HeLa cells were grown on a 96-well optical plate (Themofisher), washed with PBS 3 times and fixed with 4% paraformaldehyde at room temperature for 30 min.
    HeLa
    suggested: None
    Viruses were passaged three times in Vero E6 cells and titrated by focus-forming assay on Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)

    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 10.1101/2020.06.09.137935v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.09.137935: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementMaterial and Methods Immunization and VHH library construction The alpaca immunization process followed the protocol the guidelines established by the Bioethics Committee of the Austral University of Chile certifications 338/2019 and 388/2020.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableAdjuvant , GERBU FAMA ) diluted 1:1 in sterile water and injected subcutaneously into a male alpaca ( Vicugna pacos) .Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Nanobodies were myc tagged after incubation with a mouse anti-myc antibody and an anti-mouse Alexa647 secondary antibody was used for immunofluorescence assays.
    anti-myc
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti-mouse</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After this , the membrane was incubated with a Goat anti mouse IgG antibody coupled to HRP ( Invitrogen ) 1:5000 in PBST containing 5 % BSA , for 1h at room temperature , followed by 3 x 5 min wash with PBS-T and visualized using the ECL reagent ( Pierce) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti mouse IgG</b></div>
        <div>suggested: (Acris Antibodies GmbH Cat# SM5000, <a href="https://scicrunch.org/resources/Any/search?q=AB_1008244">AB_1008244</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image shows the reaction to decreasing amounts of Spike-1 and Bovine serum albumin ( negative control ) using a pre-immunization control , and after one immunization ( 1 week) , or two immunizations ( 3 weeks ) with full-length SARS-CoV-2 Spike , using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody . D ) ELISA assay before and after the second immunization ( 3 weeks) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-camelid IgG-HRP</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">B ) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody , followed by Mouse anti-Myc 1:3000 and anti-Mouse-Alexa 647 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-Mouse-Alexa</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">HeLa cells had a transfection efficiency of ~20%.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLa</b></div>
        <div>suggested: CLS Cat# 300194/p772_HeLa, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0030">CVCL_0030</a></div>
      </div>
    </td></tr></table>

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

  4. Version published to 10.1101/2020.06.09.137935v1 on bioRxiv