Antibody escape and cryptic cross-domain stabilization in the SARS-CoV-2 Omicron spike protein

  1. Kamyab Javanmardi
  2. Thomas H. Segall-Shapiro
  3. Chia-Wei Chou
  4. Daniel R. Boutz
  5. Randall J. Olsen
  6. Xuping Xie
  7. Hongjie Xia
  8. Pei-Yong Shi
  9. Charlie D. Johnson
  10. Ankur Annapareddy
  11. Scott Weaver
  12. James M. Musser
  13. Andrew D. Ellington
  14. Ilya J. Finkelstein
  15. Jimmy D. Gollihar

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Abstract

No abstract available

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  1. Version published to 10.1016/j.chom.2022.07.016
  2. ScreenIT

    SciScore for 10.1101/2022.04.18.488614: (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
    To each well, we added 500 μL total volume of 5 μM Alexa Fluor® 488 anti-mouse secondary (SouthernBiotech 1031-30) and 10 μM Alexa Fluor® 647 anti-human secondary (SouthernBiotech 2048-31) antibodies in PBS-BSA.
    anti-human secondary ( SouthernBiotech 2048-31 )
    suggested: None
    We used the following equation to calculate the expression of spike variant (x) relative to WT (6P-D614G): To correct for changes in transfection efficiency or spike expression in antibody or ACE2 binding measurements, we also included anti-FLAG signal as an internal normalization control.
    ACE2
    suggested: None
    anti-FLAG
    suggested: None
    We hydrated anti-mouse Fc capture (AMC) biosensors (FortéBio 18-5088) in BLI buffer for 10 min in an Octet RED96e (FortéBio) system and then immobilized mouse anti-FLAG M2 (Sigma-Aldrich F3165) antibodies to the AMC sensor tips.
    anti-mouse
    suggested: None
    For each assay, we performed the following steps: 1) baseline: 60 s with BLI buffer; 2) IgG immobilization: 360 s with anti-FLAG IgG; 3) spike loading: 360 s with diluted supernatants; 4) baseline: 300 s with BLI buffer; 5) association: 600 s with serially diluted analytes (antibodies or ACE2); 6) dissociation: 600 s with BLI buffer.
    anti-FLAG IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expression and purification of neutralizing anti-spike monoclonal antibodies: We cultured Expi293 cells in Expi293 Expression Medium (Sigma-Aldrich A1435101) and used a humidified cell culture incubator to maintain cells at 37°C and 8% CO2 with continuous shaking at 125 rpm.
    Expi293
    suggested: RRID:CVCL_D615)
    Briefly, we transfected the ACE2-Fc expression vector into Expi293T cells (Sigma-Aldrich).
    Expi293T
    suggested: None
    We added predetermined concentrations of primary antibody or chimeric cell receptor (ACE2-Fc) diluted in PBS-BSA and 50 μL (1.5 × 105) of HEK293T cells to each well.
    HEK293T
    suggested: RRID:CVCL_HA71)
    We incubated diluted serum or antibody with 100-150 fluorescent focus units (FFU) of mNG SARS-CoV-2 at 37°C for 1 h before loading the serum-virus mixtures into 96-well plates pre-seeded with Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    We used the following equation to calculate normalized binding measurements of spike variant (x) expression relative to WT (6P-D614G): We used FlowJo v9 for all flow cytometry data analyses.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    We plotted curves of relative infectivity versus serum dilution using Prism 9 (GraphPad).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All data were visualized in GraphPad Prism 9.
    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: We detected the following sentences addressing limitations in the study:
    Our study has several limitations. First, we used a prefusion stabilized spike protein that does not precisely mimic the dynamics of the native Omicron spike protein26. Second, our binding assays use a set of potent neutralizing mAbs which only serve as proxies for the antibodies found in patient antibody repertoires after immunization or natural infection. Third, our work only touches on antibody recognition and hACE2 binding; T-cell immunity plays a critical role in protecting against SARS-CoV-2 disease. Additional studies focused on the perturbations of spike variants on T-cell response will continue to bridge the gap in the understanding of immune escape between humoral and cell-mediated immunity. In the aggregate, the data presented here add critical new information about key features of Omicron spike protein mutations and how these mutations synergize to create spike variants that successfully evade antibodies while maintaining high affinity hACE2 binding. Our binding maps largely complement prior structure-based studies of binding escape, but now provide new insights into the role of compensatory substitutions in the NTD that impact both expression/stability and conformation. We conclude that the continuing accumulation of NTD mutations will further alter the conformational equilibrium and stability of the spike protein to allow for the accumulation of new, more virulent mutations in the RBD. Further, our study also highlights the importance of rapidly analyzing novel ...

    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.

  3. Version published to 10.1101/2022.04.18.488614v1 on bioRxiv