Rapid characterization of spike variants via mammalian cell surface display

  1. Kamyab Javanmardi
  2. Chia-Wei Chou
  3. Cynthia I. Terrace
  4. Ankur Annapareddy
  5. Tamer S. Kaoud
  6. Qingqing Guo
  7. Josh Lutgens
  8. Hayley Zorkic
  9. Andrew P. Horton
  10. Elizabeth C. Gardner
  11. Giaochau Nguyen
  12. Daniel R. Boutz
  13. Jule Goike
  14. William N. Voss
  15. Hung-Che Kuo
  16. Kevin N. Dalby
  17. Jimmy D. Gollihar
  18. Ilya J. Finkelstein

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

No abstract available

Article activity feed

  1. Version published to 10.1016/j.molcel.2021.11.024
  2. Version published to 10.1101/2021.03.30.437622v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.03.30.437622: (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
    Antibodies were purified as described below or purchased from the following manufacturers: Mouse anti-FLAG M2 (Sigma-Aldrich; F3165); Goat anti-Mouse IgG(H+L), Human ads-Alexa Fluor® 488 (SouthernBiotech;
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)
    anti-Mouse IgG(H+L
    suggested: None
    Anti-FLAG signal was also used as an internal normalization control to correct for changes in transfection efficiency and spike expression when measuring antibody or ACE2 binding.
    ACE2
    suggested: None
    Cells were then washed with PBS-BSA and stained with secondary antibodies (5 μM Alexa Fluor® 488 anti-mouse (SouthernBiotech; 1031-30) and 10 μM Alexa Fluor® 647 anti-human (SouthernBiotech; 2048-31) in PBS-BSA), shaking at 950 rpm for 1 hr at 4°C.
    anti-human
    suggested: (SouthernBiotech Cat# 2048-31, RRID:AB_2795692)
    Membranes were blocked in LICOR Odyssey Blocking Buffer (Neta Scientific) incubated with the following primary antibodies overnight: mouse anti-FLAG M2 antibody (1:10,000; Sigma-Aldrich; F3165) for spike protein or RBD detection and mouse anti-beta tubulin antibody (1:5000; Thermo Fisher; MA5-16308) as a loading control.
    anti-beta tubulin
    suggested: None
    The membrane was washed three times with PBST (1X PBS, 0.1% Tween 20) for 10 min and then incubated with the following secondary antibody for 2 hrs: goat anti-mouse IRDYe® 680 conjugated antibody (1:10,000; abcam; ab216776).
    anti-mouse
    suggested: None
    The assay went through the following steps: 1) baseline: 60 s with BLI buffer; 2) IgG immobilizing: 360 s with anti-FLAG IgG; 3) spikes loading: 360 s with diluted supernatants; 4) baseline: 300 s with BLI buffer; 5) association: 600 s with serial diluted analytes (antibodies or ACE2); 6) dissociation: 600 s with BLI buffer.
    anti-FLAG IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expi293 cells were cultured in Expi293 Expression Medium (Sigma-Aldrich; A1435101) and maintained in a humidified atmosphere of 8% CO2 and 37 °C while shaking continuously at 125 rpm.
    Expi293
    suggested: RRID:CVCL_D615)
    The HIV particles pseudotyped with SARS-CoV-2 spike variants D614G or B.1.1.7 were generated in HEK293T cells, following previously published protocols68.
    HEK293T
    suggested: None
    The mixture was then added to HEK-293T target cells, stably expressing human ACE2 in 96-well white plates with a clear bottom.
    HEK-293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Cells were transiently co-transfected with plasmids for (1) HIV virion formation proteins (HDM-Hgpm2, pRC-CMV-Rev1b, and HDM-tat1b; (2) one of the envelope proteins (2019-nCoV Spike-D614G mutant, B.1.1.7 variant or VSV-G) and (3) the lentiviral backbone expressing luciferase reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W).
    Spike-D614G
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analyzed using FlowJo v9.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Half-maximal inhibitory concentrations (IC50) were calculated using a 3-parameter logistic regression equation (GraphPad Prism v9.0).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    EDTA-free Protease Inhibitor Cocktail; Millipore Sigma).
    Cocktail; Millipore Sigma
    suggested: None
    Particle stacks were then imported into cryoSPARC v3.1.0 for 2D classification, ab initio 3D reconstruction, heterogeneous 3D refinement, and homogeneous 3D refinement70.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    We then used the Biopython Bio.
    Biopython
    suggested: (Biopython, RRID:SCR_007173)
    We then used the python parasail package (https://github.com/jeffdaily/parasail-python) to perform a semi-global alignment of each sequence to the reference sequence71.
    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.

    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 10.1101/2021.03.30.437622v1 on bioRxiv