Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

  1. Natalia G. Herrera
  2. Nicholas C. Morano
  3. Alev Celikgil
  4. George I. Georgiev
  5. Ryan J. Malonis
  6. James H. Lee
  7. Karen Tong
  8. Olivia Vergnolle
  9. Aldo B. Massimi
  10. Laura Y. Yen
  11. Alex J. Noble
  12. Mykhailo Kopylov
  13. Jeffrey B. Bonanno
  14. Sarah C. Garrett-Thomson
  15. David B. Hayes
  16. Robert H. Bortz
  17. Ariel S. Wirchnianski
  18. Catalina Florez
  19. Ethan Laudermilch
  20. Denise Haslwanter
  21. J. Maximilian Fels
  22. M. Eugenia Dieterle
  23. Rohit K. Jangra
  24. Jason Barnhill
  25. Amanda Mengotto
  26. Duncan Kimmel
  27. Johanna P. Daily
  28. Liise-anne Pirofski
  29. Kartik Chandran
  30. Michael Brenowitz
  31. Scott J. Garforth
  32. Edward T. Eng
  33. Jonathan R. Lai
  34. Steven C. Almo

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Abstract

No abstract available

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  1. Version published to 10.1021/acsomega.0c03512
  2. ScreenIT

    SciScore for 10.1101/2020.06.14.150607: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: After obtaining informed consent, serum was obtained by venipuncture (BD Vacutainer, serum), centrifuged, aliquoted and stored at -80°C prior to use.
    IRB: Protocol approval was obtained by the Institutional Review Board (protocol IRB# 2016-6137) of the Albert Einstein College of Medicine.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    COVID-19 convalescence sera samples (2, 3, 5, 6, 7) were previously validated to be negative with RT-PCR for SARS-CoV-2 and positive ELISA for antibodies against SARS-CoV-2.
    SARS-CoV-2
    suggested: None
    The arrays were rinsed with 5% milk-PBST briefly and incubated for 1 hour at room temperature with a fluorescently labeled secondary antibody (Alexa Fluor 647 labeled goat anti-human IgG (H+L) cross-adsorbed secondary antibody, Thermo Fisher Scientific, Cat # A21445) diluted 1:150 in 5% milk-PBST.
    anti-human IgG
    suggested: (Molecular Probes Cat# A-21445, RRID:AB_2535862)
    Cells were then incubated with a PE-labeled anti-6x His tag antibody (Abcam Cat # ab72467), to detect Spike protein binding.
    anti-6x
    suggested: (Abcam Cat# ab72467, RRID:AB_1267596)
    After 45 minutes, cells were pelleted and washed twice, and then incubated with an anti-HIS PE antibody, washed twice, and analyzed on a SONY Spectral Analyzer.
    anti-HIS PE
    suggested: None
    Expression of hACE2, mACE2, CD147, CD26, Siglec9, Siglec10, Ceacam1, and Ceacam5 were confirmed by antibody staining of transfected HEK293F cells.
    CD147
    suggested: None
    Siglec10
    suggested: None
    Ceacam1
    suggested: None
    Ceacam5
    suggested: None
    Antibodies used: hACE2 and mACE2 (RND Cat # AF933-SP), CD147 (Biolegend Clone HIM6), CD26 (Biolegend Clone BA5b), Siglec9 (Biolegend Clone K8), Siglec10 (Biolegend Clone FG6), Ceacam1 and Ceacam5 (Biolegend Clone ASL-32).
    CD26
    suggested: None
    Siglec9
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293F Transient Transfections and Culture: HEK 293 suspension cells were cultured in HEK Freestyle Media (Invitrogen) at 37 C in a humidified shaking platform incubator (Kuhner) with 5% CO2.
    HEK 293
    suggested: None
    High-throughput Screening of Human Plasma Membrane Protein Library: The human plasma membrane protein library was transfected into HEK293F cells in 48-well format.
    HEK293F
    suggested: None
    Software and Algorithms
    SentencesResources
    Nickel eluates were concentrated by centrifugation in 100K concentrator (ThermoFisher Cat # 88533) by spinning at 1100 g for intervals of 8 minutes (if necessary) and further purified by gel filtration on a HiLoad™ 16/600 Superdex™ 200 column (GE) equilibrated with 50 mM TRIS, 100 mM ArgCl, 150 mM NaCl, 10% Glycerol, pH 8.0.
    ThermoFisher Cat
    suggested: None
    Protein concentration was determined using an extinction coefficient (1468500 M-1cm-1) estimated from amino acid sequence by Expasy online ProtParam tool[20]
    ProtParam
    suggested: (ProtParam Tool, RRID:SCR_018087)
    Protein concentration was determined using an extinction coefficient (33350 M-1cm-1), estimated from amino acid sequence by Expasy online ProtParam, and was further analyzed by SDS-PAGE.
    Expasy online ProtParam
    suggested: None
    The resulting supernatant was purified on an AKTA FPLC (GE Biosciences).
    GE Biosciences
    suggested: None
    Data was exported in Excel, and then analyzed and graphed by GraphPad Prism.
    Excel
    suggested: None
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The program Prism v8 was used to generate the AUC figures shown in the text and supplement.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Absorption was measured at OD450 using a Synergy4 plate reader (Biotek), and data was analyzed using GraphPad Prism7.0 to calculate IC50 values.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    It is important to note the limitations of the current platform. In particular, we stress that at present this is a qualitative approach, as a number of variables can impact the ability to detect antibody reactivity, including patient titers for specific antigens and the relative affinities of an antigen-specific pool. Furthermore, it should be appreciated that the current platform is programmed to detect the capture of IgG antibodies; thus, the resultant signal (or lack thereof) for a particular antigen could be the consequence of prevalences between different isotypes (IgA, IgD, IgE, IgG, IgM), which are known to evolve during the course of infection and subsequent resolution. Although currently focused on three antigens, this platform can be readily expanded to study differential antibody responses to different SARS-CoV-2 antigens and subdomains of those antigens amongst individuals, which are actively being investigated by others [52-55]. We are currently working to include not only other antigens from SARS-CoV-2 (E protein, M protein, etc.), but also antigens from other coronaviruses that may be cross reactive with SARS-CoV-2 antibodies. The analysis of antibody reactivity to multiple SARS-CoV-2 and related antigens will provide broad insight into the humoral immune response to SARS-CoV-2. Collectively, we provide standards and metrics for high quality protein reagents that can yield comparable clinical, biological and structural data as we continue to combat the global ...

    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.

  3. Version published to 10.1101/2020.06.14.150607v2 on bioRxiv
  4. Version published to 10.1101/2020.06.14.150607v1 on bioRxiv