SARS-CoV-2 variant B.1.617 is resistant to bamlanivimab and evades antibodies induced by infection and vaccination

  1. Markus Hoffmann
  2. Heike Hofmann-Winkler
  3. Nadine Krüger
  4. Amy Kempf
  5. Inga Nehlmeier
  6. Luise Graichen
  7. Prerna Arora
  8. Anzhalika Sidarovich
  9. Anna-Sophie Moldenhauer
  10. Martin S. Winkler
  11. Sebastian Schulz
  12. Hans-Martin Jäck
  13. Metodi V. Stankov
  14. Georg M.N. Behrens
  15. Stefan Pöhlmann

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  1. Rapid Reviews Infectious Diseases

    Erik Boehm

    Review 1: "SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination"

    This preprint claims that the B.1.617 variant displays resistance to Bamlanivimab and a higher degree of immune escape to antibodies induced by either vaccination or prior infection. Reviewers found it timely but in need of minor revisions to make it more specific to B.1.617.2.

  2. Rapid Reviews Infectious Diseases

    Pragya Yadav, Sanjay Kumar

    Review 2: "SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination"

    This preprint claims that the B.1.617 variant displays resistance to Bamlanivimab and a higher degree of immune escape to antibodies induced by either vaccination or prior infection. Reviewers found it timely but in need of minor revisions to make it more specific to B.1.617.2.

  4. Version published to 10.1016/j.celrep.2021.109415
  5. ScreenIT

    SciScore for 10.1101/2021.05.04.442663: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Convalescent plasma samples were collected from COVID-19 patients treated at the intensive care unit of the University Medicine Göttingen under approval given by the ethic committee of the University Medicine Göttingen (SeptImmun Study 25/4/19 Ü).
    Sex as a biological variableCell culture: 293T (human, female, kidney; ACC-635, DSMZ, RRID: CVCL_0063), Huh-7 (human, male, liver; JCRB0403, JCRB; RRID: CVCL_0336, kindly provided by Thomas Pietschmann, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Authentication of cell lines was performed by STR-typing, amplification and sequencing of a fragment of the cytochrome c oxidase gene, microscopic examination and/or according to their growth characteristics.
    Contamination: In addition, cell lines were routinely tested for contamination by mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    Thereafter, cells received culture medium containing anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no. CRL-2700; except for cells expressing VSV-G, which received only medium) and incubated for 16-18 h.
    anti-VSV-G
    suggested: (LSBio (LifeSpan Cat# LS-C132924-50, RRID:AB_10835621)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: 293T (human, female, kidney; ACC-635, DSMZ, RRID: CVCL_0063), Huh-7 (human, male, liver; JCRB0403, JCRB; RRID: CVCL_0336, kindly provided by Thomas Pietschmann, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany)
    human , female , kidney; ACC-635 , DSMZ ,
    detected: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Huh-7
    detected: (KCB Cat# KCB 200970YJ, RRID:CVCL_0336)
    , BHK-21 (Syrian hamster, male, kidney; ATCC Cat# CCL-10; RRID: CVCL_1915, kindly provided by Georg Herrler, University of Veterinary Medicine, Hannover, Germany) and Vero76 cells (African green monkey, female, kidney; CRL-1586, ATCC; RRID: CVCL_0574, kindly provided by Andrea Maisner, Institute of Virology, Philipps University Marburg, Marburg, Germany) were cultivated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FCS, Biochrom or GIBCO), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).
    BHK-21
    detected: (IZSLER Cat# BS CL 8, RRID:CVCL_1915)
    Vero76
    detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574)
    Caco-2 (human, male, intestine;
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    HTB-37, ATCC, RRID:CVCL_0025), Calu-3 (human, male, lung; HTB-55, ATCC; RRID: CVCL_0609, kindly provided by Stephan Ludwig, Institute of Virology, University of Münster, Germany) and Calu-3 cells stably overexpressing ACE2, Calu-3 (ACE2) (Hoffmann et al., 2021c), were cultivated in minimum essential medium supplemented with 10% FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 1 mM sodium pyruvate (Thermo Fisher Scientific).
    HTB-37
    suggested: None
    ATCC
    detected: (RCB Cat# RCB0988, RRID:CVCL_0025)
    Calu-3
    detected: (ATCC Cat# HTB-55, RRID:CVCL_0609)
    Calu-3
    suggested: None
    A549-ACE2 (Hoffmann et al., 2021a) and A549-ACE2/TMPRSS2 cells (Hoffmann et al., 2021a) were derived from parental A549 cells (human, male, lung; CRM-CCL-185, ATCC, RRID:CVCL_0023; kindly provided by Georg Herrler) and cultivated in DMEM/F-12 Medium (ThermoFisher Scientific) supplemented with 10% FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA), 1 mM sodium pyruvate (Thermo Fisher Scientific) and 0.5 μg/ml puromycin (Invivogen).
    A549-ACE2/TMPRSS2
    suggested: None
    A549
    detected: (BCRC Cat# 60074, RRID:CVCL_0023)
    First, 293T cells that expressed the respective S protein, VSV-G (or no viral protein, control) following transfection were inoculated with VSV*ΔG-FLuc at a multiplicity of infection of three and incubated for 1 h at 37 °C.
    293T
    suggested: None
    For experiments addressing cell tropism and entry efficiency, Vero, Caco-2, Calu-3, Calu-3 (ACE2), 293T, A549 (ACE2), A549 (ACE2+TMPRSS2) and Huh-7 target cells were inoculated with identical volumes of pseudotype preparations.
    A549
    suggested: None
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    BHK-21 cells were transfected 24 h prior to pseudotype inoculation with either empty plasmid or ACE2 expression plasmid (0.1 μg/well) using Lipofectamine LTX (Thermo Fisher Scientific).
    BHK-21
    suggested: None
    Pseudotype particle neutralization test: For neutralization experiments, S protein bearing pseudotype particles were pre-incubated for 30 min at 37 °C with different concentrations of monoclonal antibodies (Casirivimab, Imdevimab, Casirivimab+Imdevimab [1:1], Bamlanivimab, Esetevimab, Bamlanivimab+Etesevimab [1:1] or unrelated control IgG [2, 0.2, 0.02, 0.002, 0.0002, 0.00002 μg/ml]) or plasma samples obtained from convalescent COVID-19 patients or individuals vaccinated with the Pfizer/BioNTech vaccine BNT162b2/Comirnaty (1:25, 1:100, 1:400, 1:1,600, 1:6,400, 1:25,600), before the mixtures were inoculated onto Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Recombinant DNA
    SentencesResources
    The resulting open reading frame was further inserted into the pCG1 plasmid (kindly provided by Roberto Cattaneo, Mayo Clinic College of Medicine, Rochester, MN, USA), making use of the unique BamHI and XbaI restriction sites.
    pCG1
    suggested: None
    Preparation of vesicular stomatitis virus pseudotypes: For this study, we employed rhabdoviral pseudotype particles that are based on a replication-deficient VSV vector that lacks the genetic information for VSV-G and instead codes for two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV*ΔG-FLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) (
    VSV*ΔG-FLuc
    suggested: None
    In order to study the antiviral activity of Camostat mesylate, Caco-2 target cells, which express endogenous TMPRSS2, were pre-incubated for 1 h with medium containing different concentrations (100, 10, 1, 0.1 or 0.01 μM) of Camostat mesylate (prepared from a 100 mM stock; Tocris) or the solvent dimethyl sulfoxide (DMSO, 1:1,000; Sigma-Aldrich) as control before being inoculated with VSV bearing S protein, VSV-G or no glycoprotein.
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    Protein models were generated using the YASARA software (http://www.yasara.org/index.html) and are based on PDB: 6XDG (Hansen et al., 2020),PDB: 7L3N (Jones et al., 2020) or PDB: 7C01 (Shi et al., 2020), or a template that was constructed by modelling the SARS-2-S sequence on PDB: 6XR8 (Cai et al., 2020), using the SWISS-MODEL online tool (https://swissmodel.expasy.org/).
    YASARA
    suggested: (YASARA, RRID:SCR_017591)
    Data ware analyzed using Microsoft Excel (as part of the Microsoft Office software package, version 2019, Microsoft Corporation) and GraphPad Prism 8 version 8.4.3 (GraphPad Software).
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  6. Version published to 10.1101/2021.05.04.442663v1 on bioRxiv