LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants

  1. Kathryn Westendorf
  2. Stefanie Žentelis
  3. Lingshu Wang
  4. Denisa Foster
  5. Peter Vaillancourt
  6. Matthew Wiggin
  7. Erica Lovett
  8. Robin van der Lee
  9. Jörg Hendle
  10. Anna Pustilnik
  11. J. Michael Sauder
  12. Lucas Kraft
  13. Yuri Hwang
  14. Robert W. Siegel
  15. Jinbiao Chen
  16. Beverly A. Heinz
  17. Richard E. Higgs
  18. Nicole L. Kallewaard
  19. Kevin Jepson
  20. Rodrigo Goya
  21. Maia A. Smith
  22. David W. Collins
  23. Davide Pellacani
  24. Ping Xiang
  25. Valentine de Puyraimond
  26. Marketa Ricicova
  27. Lindsay Devorkin
  28. Caitlin Pritchard
  29. Aoise O’Neill
  30. Kush Dalal
  31. Pankaj Panwar
  32. Harveer Dhupar
  33. Fabian A. Garces
  34. Courtney A. Cohen
  35. John M. Dye
  36. Kathleen E. Huie
  37. Catherine V. Badger
  38. Darwyn Kobasa
  39. Jonathan Audet
  40. Joshua J. Freitas
  41. Saleema Hassanali
  42. Ina Hughes
  43. Luis Munoz
  44. Holly C. Palma
  45. Bharathi Ramamurthy
  46. Robert W. Cross
  47. Thomas W. Geisbert
  48. Vineet Menachery
  49. Kumari Lokugamage
  50. Viktoriya Borisevich
  51. Iliana Lanz
  52. Lisa Anderson
  53. Payal Sipahimalani
  54. Kizzmekia S. Corbett
  55. Eun Sung Yang
  56. Yi Zhang
  57. Wei Shi
  58. Tongqing Zhou
  59. Misook Choe
  60. John Misasi
  61. Peter D. Kwong
  62. Nancy J. Sullivan
  63. Barney S. Graham
  64. Tara L. Fernandez
  65. Carl L. Hansen
  66. Ester Falconer
  67. John R. Mascola
  68. Bryan E. Jones
  69. Bryan C. Barnhart

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Abstract

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  1. Version published to 10.1016/j.celrep.2022.110812
  2. Version published to 10.1101/2021.04.30.442182v6 on bioRxiv
  3. Version published to 10.1101/2021.04.30.442182v5 on bioRxiv
  4. Version published to 10.1101/2021.04.30.442182v4 on bioRxiv
  5. Version published to 10.1101/2021.04.30.442182v3 on bioRxiv
  6. ScreenIT

    SciScore for 10.1101/2021.04.30.442182: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Single cells secreting target-specific antibodies were identified and isolated using three assay types (55): a multiplexed bead assay using multiple optically-encoded beads, each conjugated to the soluble pre-fusion stabilized S protein of either SARS-CoV-2 or WIV1 S with T4-foldon domain, 3C protease cleavage site, 6x His-tags, and twin-strep tags (34), the SARS-CoV-2 S1 subunit or negative controls (bovine serum albumin [BSA] His-tag and T4 FoldOn trimerization domain), and a live cell assay using passively dyed suspension-adapted Chinese hamster ovary (CHO) cells transiently transfected to surface-express full-length SARS-CoV-2 S protein (GenBank ID MN908947.3) with a green fluorescent protein (GFP) reporter, and non-transfected cells as a negative control.
    GFP
    suggested: None
    Antibodies were recombinantly produced by transient transfection in either human-embryonic kidney (HEK293) or CHO cells as described in Supplemental Methods.
    HEK293
    suggested: None
    Samples were prepared by mixing each antibody in 10-fold molar excess with antigen (1:1 freshly prepared mix of 400 nM antibody and 40 nM antigen, both diluted in 1X HBSTE + 0.05% BSA running buffer).
    antigen
    suggested: (W. Sieghart, Center for Brain Research, Medical University of Vienna; Vienna; Austria Cat# Beta1 (375-400, RRID:AB_2827800)
    To test the antibodies’ ability to block ACE2, antibodies coupled to the HC-30M chip as described above were exposed to SARS-CoV-2 S protein:ACE2 complex.
    ACE2
    suggested: None
    Multi-cycle kinetics on Biacore: The capture molecule, an anti-human IgG (Fc) antibody, was immobilized on a Biacore CM5 chip by direct coupling.
    anti-human IgG
    suggested: None
    CHO cells were washed, and binding was detected by using a fluorescently labeled anti-human secondary antibody.
    anti-human secondary antibody .
    suggested: None
    Median fluorescence intensity of each antibody was normalized over the median fluorescence intensity of the human isotype control for respective antigens.
    human isotype control for respective antigens .
    suggested: None
    Infectious titers were determined for a subset of virus preparations by infection of VeroE6 cells (ATCC CRL-1586) with serially diluted virus followed by staining with an anti-luciferase antibody (Novus Cat # NB600-307PEATT594) and analysis by fluorescence-activated cell sorting using a Becton Dickinson LSRFortessaTM X-20
    anti-luciferase
    suggested: None
    NB600-307PEATT594
    suggested: None
    Infected cells were detected using a primary detection antibody recognizing SARS-CoV-2 nucleocapsid protein (Sino Biological) following staining with secondary detection antibody (goat α-rabbit) conjugated to AlexaFluor 488.
    SARS-CoV-2 nucleocapsid protein
    suggested: (Bioss Cat# bsm-41414M, RRID:AB_2848129)
    α-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The extracellular domain (ECD, residues 18 to 618) of ACE2 was expressed in CHO cells as an Fc-fusion protein containing a TEV-protease recognition site.
    CHO
    suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
    Single cells secreting target-specific antibodies were identified and isolated using three assay types (55): a multiplexed bead assay using multiple optically-encoded beads, each conjugated to the soluble pre-fusion stabilized S protein of either SARS-CoV-2 or WIV1 S with T4-foldon domain, 3C protease cleavage site, 6x His-tags, and twin-strep tags (34), the SARS-CoV-2 S1 subunit or negative controls (bovine serum albumin [BSA] His-tag and T4 FoldOn trimerization domain), and a live cell assay using passively dyed suspension-adapted Chinese hamster ovary (CHO) cells transiently transfected to surface-express full-length SARS-CoV-2 S protein (GenBank ID MN908947.3) with a green fluorescent protein (GFP) reporter, and non-transfected cells as a negative control.
    Chinese hamster ovary ( CHO )
    suggested: None
    Briefly, 293T cells were transfected with individual mutant spike expression plasmids, and 16 to 20 hours later, transfected cells were infected with VSV-G-pseudotyped ΔGluciferase rVSV.
    293T
    suggested: None
    Infectious titers were determined for a subset of virus preparations by infection of VeroE6 cells (ATCC CRL-1586) with serially diluted virus followed by staining with an anti-luciferase antibody (Novus Cat # NB600-307PEATT594) and analysis by fluorescence-activated cell sorting using a Becton Dickinson LSRFortessaTM X-20
    VeroE6
    suggested: None
    For neutralization assay serial dilutions (2 dilutions at 10 and 1 μg/ml for the initial screen assay or 8 dilutions for the full curve at 10-0.0006 μg/ml) of monoclonal antibodies were mixed with titrated pseudovirus, incubated for 45 minutes at 37 °C and added to pre-seeded 293T-ACE2 cells (provided by Dr. Michael Farzan) in triplicate in 96-well white/black Isoplates (Perkin Elmer).
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Vero-76 cells were inoculated with SARS-CoV-2 (GenBank MT020880.1) at a MOI = 0.01 and incubated at 37°C with 5% CO2 and 80% humidity.
    Vero-76
    suggested: None
    The antibody-virus mixture was applied to monolayers of Vero-E6 cells in a 96-well plate and incubated for 1 hour at 37°C in a humidified incubator.
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Single-cell sequencing, bioinformatic analysis, and cloning: Single cell polymerase chain reaction (PCR) and custom molecular biology protocols generated NGS sequencing libraries (MiSeq, Illumina) using automated workstations (Bravo, Agilent).
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Plasmids were verified by Sanger sequencing to confirm the original sequence previously identified by NGS.
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    Neutralization IC50, IC80 and IC90 titers were calculated using GraphPad Prism 8.0.2.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Model building was performed with Coot (CCP4) and final structure validation with MolProbity (Chen et al., 2010) and CCP4 validation tools.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    , 2019.0101; Chemical Computing Group ULC), and detailed contact analysis with CCP4 CONTACT(Winn et al., 2011) and custom shell/Perl scripts.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    We aligned the genomes to the SARS-CoV-2 reference genome (Genbank file MN908947.3) using BWA-MEM (Danecek et al., 2021), and performed variant calling and annotation using SAMTools.
    BWA-MEM
    suggested: (Sniffles, RRID:SCR_017619)
    SAMTools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    All further analysis (i.e. slicing by time or geographic region) was performed using custom-written Python scripts.
    Python
    suggested: (IPython, RRID:SCR_001658)

    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.

  7. Version published to 10.1101/2021.04.30.442182v2 on bioRxiv
  8. Version published to 10.1101/2021.04.30.442182v1 on bioRxiv