An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies

  1. Yafei Liu
  2. Wai Tuck Soh
  3. Jun-ichi Kishikawa
  4. Mika Hirose
  5. Emi E. Nakayama
  6. Songling Li
  7. Miwa Sasai
  8. Tatsuya Suzuki
  9. Asa Tada
  10. Akemi Arakawa
  11. Sumiko Matsuoka
  12. Kanako Akamatsu
  13. Makoto Matsuda
  14. Chikako Ono
  15. Shiho Torii
  16. Kazuki Kishida
  17. Hui Jin
  18. Wataru Nakai
  19. Noriko Arase
  20. Atsushi Nakagawa
  21. Maki Matsumoto
  22. Yukoh Nakazaki
  23. Yasuhiro Shindo
  24. Masako Kohyama
  25. Keisuke Tomii
  26. Koichiro Ohmura
  27. Shiro Ohshima
  28. Toru Okamoto
  29. Masahiro Yamamoto
  30. Hironori Nakagami
  31. Yoshiharu Matsuura
  32. Atsushi Nakagawa
  33. Takayuki Kato
  34. Masato Okada
  35. Daron M. Standley
  36. Tatsuo Shioda
  37. Hisashi Arase

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Abstract

No abstract available

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  1. Version published to 10.1016/j.cell.2021.05.032
  2. ScreenIT

    SciScore for 10.1101/2020.12.18.423358: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consent was obtained from the participants according to the relevant guidelines of the institutional review board.
    IRB: Written informed consent was obtained from the participants according to the relevant guidelines of the institutional review board.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: The cells were routinely checked for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-spike antibodies from COVID-19 patients: The V regions of anti-SARS-CoV-2 spike mAb from COVID-19 patients were synthesized according to the published sequence (IDT)9-11,14.
    anti-SARS-CoV-2
    suggested: None
    IDT)9-11,14
    suggested: None
    The concentration of unpurified IgG in the cell culture supernatants used in Fig. 1a was measured using the protein A-coupled latex beads (Thermo A37304) and APC-labeled anti-human IgG F(ab’)2 antibodies (Jackson) against IgG standards of known concentration.
    anti-human IgG F(ab’)2
    suggested: None
    Protein A-purified 8D214 and C14410 antibodies were labeled using DyLight 650 amine-reactive dye according to the manufacturer’s protocol.
    C14410
    suggested: (LSBio (LifeSpan Cat# LS-C14410-50, RRID:AB_860017)
    Antibodies and recombinant proteins: Mouse anti-human ACE2 mAb (R&D Systems,
    anti-human ACE2
    suggested: None
    The flow cytometric analysis of antibodies: The plasmid expressing the full-length SARS-CoV-2 spike protein, Flag-NTD-PILR-TM, Flag-RBD-PILR-TM, Flag-S2-PILR-TM, or mutated spike proteins were co-transfected with the GFP vector into HEK293T cells.
    SARS-CoV-2 spike protein , Flag-NTD-PILR-TM , Flag-RBD-PILR-TM , Flag-S2-PILR-TM ,
    suggested: None
    C-terminal deleted spike protein and GFP were cotransfected into HEK293T cells and the transfectants were mixed with various concentrations of anti-spike antibodies at 4°C for 30 min, followed by incubation with a biotinylated-ACE2-Fc fusion protein at 1 μg / ml and 4°C for 30 min.
    GFP
    suggested: None
    anti-spike
    suggested: None
    The pseudotyped SARS-CoV-2 virus was pre-incubated with anti-NTD monoclonal antibodies for 30 minutes and mixed with the ACE2-expressing cells.
    anti-NTD
    suggested: None
    The non-enhancing anti-S2 antibody, 2147, was used as a control.
    anti-S2
    suggested: None
    Detection of enhancing antibody in uninfected individuals: The plasmids expressing the wild-type NTD-TM, which was recognized by the enhancing antibodies, and the NTD-TM mutants (W64A, H66A, K187A,V213A, and R214A) not recognized by all enhancing antibodieswere transfected into HEK293T cells with the GFP vector as a transfection marker.
    V213A
    suggested: None
    The transfectants were mixed with 1:100 diluted serum from uninfected individuals, and the bound antibodies were detected with the APC-labeled anti-human IgG Fc antibody.
    anti-human IgG
    suggested: None
    The 4A8 anti-NTD antibody14 was used as a standard to calculate the relative concentration of the antibody against NTD.
    antibody against NTD.
    suggested: None
    Similarly, the relative level of the anti-RBD antibody was measured using RBD-TM transfectants and C144 as a standard.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and cell culture: HEK293T cells (RIKEN Cell Bank), Huh7 cells (the National Institute of Infectious Diseases) and TMPRSS2-expressing VeroE6 cells (Japanese Collection of Research Bioresources Cell Bank, JCRB1819) were cultured in DMEM (GIBCO, 11995-073) supplemented with 10% FBS (Biological Industries, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Nacalai, Japan) and cultured at 37°C in 5% CO2.
    Huh7
    suggested: None
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    The Expi293 cells (Thermo) were cultured with the Expi293 medium.
    Expi293
    suggested: RRID:CVCL_D615)
    C-terminal deleted spike protein and GFP were cotransfected into HEK293T cells and the transfectants were mixed with various concentrations of anti-spike antibodies at 4°C for 30 min, followed by incubation with a biotinylated-ACE2-Fc fusion protein at 1 μg / ml and 4°C for 30 min.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    The primers for mutagenesis were designed on Agilent’s website (https://www.agilent.com/store/primerDesignProgram.jsp).
    Agilent’s
    suggested: None
    Transfection: A pME18S expression plasmid containing the full-length or subdomain spike protein was transiently transfected into HEK293T cells using PEI max (PolyScience); the pMx-GFP expression plasmids were used as the marker of transfected cells.
    PolyScience)
    suggested: None
    Then, the antibodies bound to the stained cells were analyzed using flow cytometers (Attune™, Thermo; FACSCalibur BD bioscience)
    FACSCalibur
    suggested: None
    Antibody binding to the GFP-positive cells were shown in the figures using a FlowJo software (BD bioscience).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The top-scoring docked poses were rendered on the full-length spike model using PyMOL.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Data and statistical analysis: FlowJo version 10.7 (BD Biosciences, USA) was used to analyze the flow cytometry data, and Graphpad Prism version 7.0e was used for graph generation and statistical analysis.
    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 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. ScreenIT

    SciScore for 10.1101/2020.12.18.423358: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementWritten informed consent was obtained from the participants according to the relevant guidelines of the institutional review board.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationThe cells were routinely checked for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-spike antibodies from COVID-19 patients The V regions of anti-SARS-CoV-2 spike mAb from COVID-19 patients were synthesized according to the published sequence (IDT)9-11,14.
    Anti-spike
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    anti-SARS-CoV-2
    suggested: None
    IDT)9-11,14
    suggested: None
    The concentration of unpurified IgG in the cell culture supernatants used in Fig. 1a was measured using the protein A-coupled latex beads (Thermo A37304) and APC-labeled anti-human IgG F(ab')2 antibodies (Jackson) against IgG standards of known concentration.
    anti-human IgG
    suggested: None
    )2
    suggested: None
    Protein A-purified 8D214 and C14410 antibodies were labeled using DyLight 650 amine-reactive dye according to the manufacturer's protocol.
    C14410
    suggested: (LifeSpan Cat# LS-C14410-50, RRID:AB_860017)
    Antibodies and recombinant proteins Mouse anti-human ACE2 mAb (R&D Systems, USA)
    anti-human ACE2
    suggested: None
    The flow cytometric analysis of antibodies The plasmid expressing the full-length SARS-CoV-2 spike protein, Flag-NTD-PILR-TM, FlagRBD-PILR-TM, Flag-S2-PILR-TM, or mutated spike proteins were co-transfected with the GFP vector into HEK293T cells.
    SARS-CoV-2 spike protein , Flag-NTD-PILR-TM , FlagRBD-PILR-TM , Flag-S2-PILR-TM ,
    suggested: None
    C-terminal deleted spike protein and GFP were cotransfected into HEK293T cells and the transfectants were mixed with various concentrations of anti-spike antibodies at 4°C for 30 min, followed by incubation with a biotinylated-ACE2-Fc fusion protein at 1 μg / ml and 4°C for 30 min.
    GFP
    suggested: None
    The non-enhancing anti-S2 antibody, 2147, was used as a control.
    anti-S2
    suggested: None
    Detection of enhancing antibody in uninfected individuals The plasmids expressing the wild-type NTD-TM, which was recognized by the enhancing antibodies, and the NTD-TM mutants (W64A, H66A, K187A,V213A, and R214A) not recognized by all enhancing antibodieswere transfected into HEK293T cells with the GFP vector as a transfection marker.
    V213A
    suggested: None
    The transfectants were mixed with 1:100 diluted serum from uninfected individuals, and the bound antibodies were detected with the APC-labeled antihuman IgG Fc antibody.
    antihuman IgG
    suggested: (GeneTex Cat# GTX28798, RRID:AB_374523)
    The 4A8 anti-NTD antibody14 was used as a standard to calculate the relative concentration of the antibody against NTD.
    anti-NTD
    suggested: None
    antibody against NTD.
    suggested: None
    Similarly, the relative level of the anti-RBD antibody was measured using RBDTM transfectants and C144 as a standard.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and cell culture HEK293T cells (RIKEN Cell Bank), Huh7 cells (the National Institute of Infectious Diseases ) and TMPRSS2-expressing VeroE6 cells ( Japanese Collection of Research Bioresources Cell Bank, JCRB1819) were cultured in DMEM (GIBCO, 11995-073) supplemented with 10% FBS (Biological Industries, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL) (Nacalai, Japan) and cultured at 37°C in 5% CO2.
    Huh7
    suggested: None
    The Expi293 cells (Thermo) were cultured with the Expi293 medium.
    Expi293
    suggested: RRID:CVCL_D615)
    Transfection A pME18S expression plasmid containing the full-length or subdomain spike protein was transiently transfected into HEK293T cells using PEI max (PolyScience); the pMx-GFP expression plasmids were used as the marker of transfected cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    The stock virus was amplified in TMPRSS2-expressing VeroE6 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    Transfection A pME18S expression plasmid containing the full-length or subdomain spike protein was transiently transfected into HEK293T cells using PEI max (PolyScience); the pMx-GFP expression plasmids were used as the marker of transfected cells.
    PolyScience)
    suggested: None
    Then, the antibodies bound to the stained cells were analyzed using flow cytometers (Attune™, Thermo; FACSCalibur BD bioscience)
    FACSCalibur
    suggested: None
    Antibody binding to the GFP-positive cells were shown in the figures using a FlowJo software (BD bioscience).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The topscoring docked poses were rendered on the full-length spike model using PyMOL.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Data and statistical analysis FlowJo version 10.7 (BD Biosciences, USA) was used to analyze the flow cytometry data, and Graphpad Prism version 7.0e was used for graph generation and statistical analysis.
    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 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 10.1101/2020.12.18.423358v1 on bioRxiv