Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

  1. Emma C. Thomson
  2. Laura E. Rosen
  3. James G. Shepherd
  4. Roberto Spreafico
  5. Ana da Silva Filipe
  6. Jason A. Wojcechowskyj
  7. Chris Davis
  8. Luca Piccoli
  9. David J. Pascall
  10. Josh Dillen
  11. Spyros Lytras
  12. Nadine Czudnochowski
  13. Rajiv Shah
  14. Marcel Meury
  15. Natasha Jesudason
  16. Anna De Marco
  17. Kathy Li
  18. Jessica Bassi
  19. Aine O’Toole
  20. Dora Pinto
  21. Rachel M. Colquhoun
  22. Katja Culap
  23. Ben Jackson
  24. Fabrizia Zatta
  25. Andrew Rambaut
  26. Stefano Jaconi
  27. Vattipally B. Sreenu
  28. Jay Nix
  29. Ivy Zhang
  30. Ruth F. Jarrett
  31. William G. Glass
  32. Martina Beltramello
  33. Kyriaki Nomikou
  34. Matteo Pizzuto
  35. Lily Tong
  36. Elisabetta Cameroni
  37. Tristan I. Croll
  38. Natasha Johnson
  39. Julia Di Iulio
  40. Arthur Wickenhagen
  41. Alessandro Ceschi
  42. Aoife M. Harbison
  43. Daniel Mair
  44. Paolo Ferrari
  45. Katherine Smollett
  46. Federica Sallusto
  47. Stephen Carmichael
  48. Christian Garzoni
  49. Jenna Nichols
  50. Massimo Galli
  51. Joseph Hughes
  52. Agostino Riva
  53. Antonia Ho
  54. Marco Schiuma
  55. Malcolm G. Semple
  56. Peter J.M. Openshaw
  57. Elisa Fadda
  58. J. Kenneth Baillie
  59. John D. Chodera
  60. Suzannah J. Rihn
  61. Samantha J. Lycett
  62. Herbert W. Virgin
  63. Amalio Telenti
  64. Davide Corti
  65. David L. Robertson
  66. Gyorgy Snell

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Abstract

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  1. ScreenIT

    SciScore for 10.1101/2020.11.04.355842: (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
    S protein was surface captured via anti-AviTag pAb covalently immobilized on a CM5 chip, RBD protein was surface captured via StrepTactin XT covalently immobilized on a CM5 chip, and ACE2-mFc was surface captured via covalent immobilization of the Cytiva Mouse antibody capture kit on a C1 chip.
    anti-AviTag
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For S binding measurements, recombinant ACE2 (residues 19-615 from Uniprot Q9BYF1 with a C-terminal thrombin cleavage site-TwinStrep-10xHis-GGG-tag, and N- terminal signal sequence) was expressed in Expi293 cells at 37°C and 8% CO2 in a humified incubator.
    Expi293
    suggested: RRID:CVCL_D615)
    Viral growth curve: VeroE6-ACE2 cells (VeroE6 cells induced to overexpress Ace2) either with or without TMPRSS2 overexpression (Rhin et al., 2020 under review) were seeded in a 12- well plate and inoculated with an MOI of 0.01 with either the GLA1 (N439/D614G) or GLA2 (N439K/D614G) virus isolates for 1hr before washing the cells three times in PBS and replacing with 2% DMEM.
    VeroE6-ACE2
    suggested: None
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Lenti-X™ 293T cells (Takara, 632180) were seeded in 10-cm dishes at a density of 1e5 cells/cm2 and the following day transfected with 5 μg of spike expression plasmid with TransIT-Lenti (Mirus, 6600) according to the manufacturer’s instructions.
    293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Reads were size filtered, demultiplexed and trimmed with Porechop (https://github.com/rrwick/Porechop), and mapped against reference strain Wuhan-Hu-1 (MN908947).
    Wuhan-Hu-1
    suggested: None
    Software and Algorithms
    SentencesResources
    RBD residues within 6.0A distance of any ACE2 atoms (determined using MOE) were determined for each of the two copies of the complex in the asymmetric unit, and then were combined to obtain the RBM. 6M0J was obtained from the Coronavirus Structural Task Force server (https://github.com/thorn-lab/coronavirus_structural_task_force) and was further refined (using Refmac5 v5.8.0258), manually fitted (using Coot v0.9) and prepared (using MOE, as described above) in multiple iterative cycles.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Generated amplicons were used to prepare either Oxford Nanopore or Illumina sequencing libraries.
    Oxford Nanopore
    suggested: (Oxford Nanopore Technologies, RRID:SCR_003756)
    Oxford Nanopore libraries were prepared as described in the link above and sequenced in a flow cell R9.4.1 (Oxford Nanopore Technologies, Part Number FLO- MIN106D), using MinKNOW version 19.12.6.
    MinKNOW
    suggested: None
    Reads were size filtered, demultiplexed and trimmed with Porechop (https://github.com/rrwick/Porechop), and mapped against reference strain Wuhan-Hu-1 (MN908947).
    Porechop
    suggested: (Porechop, RRID:SCR_016967)
    Variants were called using Nanopolish 0.11.3 and accepted if they had a log- likelihood score of greater than 200 and minimum read coverage of 20.
    Nanopolish
    suggested: (Nanopolish, RRID:SCR_016157)
    Reads were trimmed with trim_galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped with BWA (Li and Durbin, 2009)) to the Wuhan-Hu-1 (MN908947) reference sequence, followed by primer trimming and consensus calling with iVar (Grubaugh et al., 2019) and a minimum read coverage of 10.
    http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
    suggested: (Trim Galore, RRID:SCR_011847)
    BWA
    suggested: (BWA, RRID:SCR_010910)
    A maximum-likelihood phylogenetic tree was constructed using IQ-TREE with the the following parameters: -czb -blmin 0.0000000001 -m HKY --runs 5 and all other parameters set to default.
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)
    The tree was visualised with custom python code using the baltic library, https://github.com/evogytis/baltic.
    python
    suggested: (IPython, RRID:SCR_001658)
    Change in molecules bound to the biosensors caused a shift in the interference pattern that was recorded in real time and plotted using GraphPad Prism 8 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data were analyzed and visualized with Prism (Version 8.4.3).
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    Results from OddPub: Thank you for sharing your code.

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    ISRCTN66726260NANA

    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 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 10.1016/j.cell.2021.01.037
  3. ScreenIT

    SciScore for 10.1101/2020.11.04.355842: (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

    No key resources detected.

    Results from OddPub: Thank you for sharing your data.

    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.

    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.

  4. Version published to 10.1101/2020.11.04.355842v1 on bioRxiv