NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

  1. Teresa R. Wagner
  2. Philipp D. Kaiser
  3. Marius Gramlich
  4. Elena Ostertag
  5. Natalia Ruetalo
  6. Daniel Junker
  7. Julia Haering
  8. Bjoern Traenkle
  9. Matthias Becker
  10. Alex Dulovic
  11. Helen Schweizer
  12. Stefan Nueske
  13. Armin Scholz
  14. Anne Zeck
  15. Katja Schenke-Layland
  16. Annika Nelde
  17. Monika Strengert
  18. Juliane S. Walz
  19. Georg Zocher
  20. Thilo Stehle
  21. Michael Schindler
  22. Nicole Schneiderhan-Marra
  23. Ulrich Rothbauer

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Abstract

As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide.

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  1. Version published to 10.1101/2020.09.22.308338v4 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.09.22.308338: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethical consent was granted from the Ethics Commission of the University of Tuebingen under the votum 179/2020/BO2.
    IRB: Ethical consent was granted from the Ethics Commission of the University of Tuebingen under the votum 179/2020/BO2.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Following panning 492 individual clones of the second selection round were screened by standard Phage-ELISA procedures using a horseradish peroxidase-labeled anti-M13 monoclonal antibody (GE-Healthcare).
    anti-M13
    suggested: None
    For immunoblotting, proteins were transferred on nitrocellulose membrane (Bio-Rad Laboratories) and detection was performed using anti-His antibody (Penta-His Antibody, #34660, Qiagen) followed by donkey-anti-mouse antibody labeled with AlexaFluor647 (Invitrogen) using a Typhoon Trio scanner (GE-Healthcare, Freiburg, Germany; excitation 633 nm, emission filter settings 670 nm BP 30).
    anti-His
    suggested: None
    donkey-anti-mouse
    suggested: (Jackson ImmunoResearch Labs Cat# 715-065-151, RRID:AB_2340785)
    For the detection of neutralizing serum antibodies, the bead-mix containing beads coupled with purified RBD (receptor-binding domain), S1 (S1 domain), spike (homotrimeric spike), S2 (S2 domain) or N (nucleocapsid) of SARS-CoV-2 was incubated with bivNb NM1257 (concentrations ranging from 1 µM to 6.1 pM) and serum samples of convalescent SARS-CoV-2 patients and healthy donors at a 1:400 dilution.
    S1
    suggested: None
    S1 domain
    suggested: None
    S2
    suggested: None
    S2 domain
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For the expression of SARS-CoV-2 proteins (RBD, stabilized homotrimeric spike and S1 domain), Expi293 cells were used18 and the hetero-bivalent Nb NM1267was expressed using the ExpiCHO system.
    Expi293
    suggested: RRID:CVCL_D615)
    Viral infection assay: For neutralization experiments, 1 ×104 Caco-2 cells/well were seeded in 96-well plates the day before infection in media containing 5% FCS.
    Caco-2
    suggested: None
    Software and Algorithms
    SentencesResources
    For immunoblotting, proteins were transferred on nitrocellulose membrane (Bio-Rad Laboratories) and detection was performed using anti-His antibody (Penta-His Antibody, #34660, Qiagen) followed by donkey-anti-mouse antibody labeled with AlexaFluor647 (Invitrogen) using a Typhoon Trio scanner (GE-Healthcare, Freiburg, Germany; excitation 633 nm, emission filter settings 670 nm BP 30).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Global fits were determined using the BLItzPro software and the global dissociation constant (KD) was calculated.
    BLItzPro
    suggested: None
    For quantification of infection rates, images were taken with the Cytation3 (Biotek) and Hoechst+ and mNG+ cells were automatically counted by the Gen5 Software (Biotek).
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    Inhibitory concentration 50 (IC50) was calculated as the half-maximal inhibitory dose using 4-parameter nonlinear regression (GraphPad Prism).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Analyses and Statistics: Graph preparation and statistical analysis was performed using the GraphPad Prism Software (Version 9.0.0).
    GraphPad Prism
    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

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  3. Version published to 10.1101/2020.09.22.308338v3 on bioRxiv
  4. Version published to 10.1101/2020.09.22.308338v2 on bioRxiv
  5. Version published to 10.1101/2020.09.22.308338v1 on bioRxiv