SARS-CoV-2 Spike Protein Stabilized in the Closed State Induces Potent Neutralizing Responses

  1. George W. Carnell
  2. Katarzyna A. Ciazynska
  3. David A. Wells
  4. Xiaoli Xiong
  5. Ernest T. Aguinam
  6. Stephen H. McLaughlin
  7. Donna Mallery
  8. Soraya Ebrahimi
  9. Lourdes Ceron-Gutierrez
  10. Benedikt Asbach
  11. Sebastian Einhauser
  12. Ralf Wagner
  13. Leo C. James
  14. Rainer Doffinger
  15. Jonathan L. Heeney
  16. John A. G. Briggs

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Abstract

Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals.

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  1. Version published to 10.1128/jvi.00203-21
  2. ScreenIT

    SciScore for 10.1101/2021.01.14.426695: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAnimal work: 8-10 week old BALB/c females (Charles River) were immunized subcutaneously with 10 μg of purified protein and 10 μg of MPLA in a total volume of 50 μl PBS.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, PVDF membranes were coated with IFN-γ capture antibody overnight and then washed with PBS. 100 μL of test peptide (at a final concentration of 1 μg/ml) PepMixTM SARS-CoV-2 S (JPT, PM-WCPV-S-1) or murine anti-CD3 antibody (positive control) (Invitrogen, 16-0031-82) or CTL-TestTM medium (negative control) were applied to the wells and plates were then placed in a 37°C humidified 5% CO2.
    anti-CD3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Replication deficient SARS CoV-2 pseudotyped HIV-1 virions were produced in HEK293T wild-type (293T-wt) cells by transfection with pCAGGS-S, pCRV and CSGW as described previously (Price et al. 2014)
    HEK293T
    suggested: None
    HEK293T-hACE2 cells were plated into 96 well plates at a density of 7.5×103 cells per well and allowed to attach overnight.
    HEK293T-hACE2
    suggested: None
    The ACE2-Fc fusion protein was expressed in Expi293 cell and purified from cell culture media using a protein A column followed by size-exclusion chromatography.
    Expi293
    suggested: RRID:CVCL_D615)
    Cells and viruses for neutralisation assays: Vero (ATCC CCL 81) and HEK293T/17 (ATCC CRL-11268) cells were cultured in Dulbecco’s modified Eagle Medium, supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) and 10% foetal bovine serum.
    Vero
    suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
    Supernatants were taken after 48 h, filtered at 0.45 μm and titrated on HEK293T/17 cells transiently expressing human ACE-2 and TMPRSS2.
    HEK293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animal work: 8-10 week old BALB/c females (Charles River) were immunized subcutaneously with 10 μg of purified protein and 10 μg of MPLA in a total volume of 50 μl PBS.
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    Resultant reference-subtracted data were fitted to single or double phase association and dissociation kinetics to determine kon, koff and Kd(kin) (the binding constant determined from the ratio of the individual rate constants) using Prism 8.4.3 (GraphPad Software)
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The IC50 was estimated using a 3 parameter log-logistic regression fit in Graphpad Prism.
    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: 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 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.

  3. Version published to 10.1101/2021.01.14.426695v1 on bioRxiv