Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

  1. Panpan Zhou
  2. Ge Song
  3. Hejun Liu
  4. Meng Yuan
  5. Wan-ting He
  6. Nathan Beutler
  7. Xueyong Zhu
  8. Longping V. Tse
  9. David R. Martinez
  10. Alexandra Schäfer
  11. Fabio Anzanello
  12. Peter Yong
  13. Linghang Peng
  14. Katharina Dueker
  15. Rami Musharrafieh
  16. Sean Callaghan
  17. Tazio Capozzola
  18. Oliver Limbo
  19. Mara Parren
  20. Elijah Garcia
  21. Stephen A. Rawlings
  22. Davey M. Smith
  23. David Nemazee
  24. Joseph G. Jardine
  25. Yana Safonova
  26. Bryan Briney
  27. Thomas F. Rogers
  28. Ian A. Wilson
  29. Ralph S. Baric
  30. Lisa E. Gralinski
  31. Dennis R. Burton
  32. Raiees Andrabi

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Abstract

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  1. Version published to 10.1016/j.immuni.2023.02.005
  2. ScreenIT

    SciScore for 10.1101/2022.03.04.479488: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: COVID-19 infected-vaccinated donors: Sera and PBMC samples from convalescent COVID-19 donors, vaccinated donors, and COVID-19-recovered vaccinated donors, were provided through the “Collection of Biospecimens from Persons Under Investigation for 2019-Novel Coronavirus Infection to Understand Viral Shedding and Immune Response Study” UCSD IRB# 200236 as reported earlier (35).
    Consent: All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the
    IACUC: S Animal Welfare Assurance Number: D16-00256 (A3410-01), under approved IACUC protocols.
    Euthanasia Agents: Immediately prior to infection, mice were anesthetized by injection of ketamine and xylazine intraperitoneally and weighed.
    Sex as a biological variable12-month-old female Balb/c mice (strain 047) were purchased from Envigo for Sarbecovirus challenge experiments (65, 85).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Fluorescently labeled antibodies specific for cell surface markers were prepared as 1:100 dilution as a master mix in FACS buffer, to stain the PBMC samples for CD3 (APC-Cy7, BD Pharmingen cat.#
    CD3
    suggested: None
    For recombinant protein ELISA, mouse anti-His antibody (Invitrogen cat.
    anti-His
    suggested: None
    After another washing, alkaline phosphatase-conjugated goat anti-human IgG Fc secondary antibody (Jackson ImmunoResearch cat.#
    anti-human IgG
    suggested: None
    902360) were used to determine the reactivity of monoclonal antibodies to human epithelial type 2 (HEp2) by indirect immunofluorescence.
    human epithelial type 2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: FreeStyle293-F cells (Thermo Fisher Scientific cat.#
    FreeStyle293-F
    suggested: RRID:CVCL_D603)
    R79007) were grown in FreeStyl 293 Expression Medium (Gibco cat.# 12338018), and Expi293F cells (Gibco cat.#
    Expi293F
    suggested: RRID:CVCL_D615)
    Adherent HEK293T cells and HeLa-ACE2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat-inactivated FBS, 4mM L-Glutamine and 1% P/S, maintaining in the incubator at 37°C, 5% CO2.
    HeLa-ACE2
    suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)
    The stable hACE2 or hDPP4-expressing HeLa cell line was generated using an ACE2 lentivirus protocol previously described (7).
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    ELISA using peptides or recombinant proteins: N-terminal biotinylated peptides corresponding to stem helix of SARS-CoV-1/2, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E and HCoV-NL63 were synthesized at A&A Labs (
    HCoV-229E
    suggested: JCRB Cat# JCRB1838, RRID:CVCL_B3M4)
    Pseudovirus production: HIV-based lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene #8455), pBOB-Luciferase (Addgene #170674) were co-transfected into HEK293T cells along with full-length or variously truncated SARS-CoV1, WIV1, SHC014, Pang17, SARS-COV2, SARS-CoV-2 variants of concern [(B.1.1.7(alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) and B.1.1.529 (Omicron)] and MERS-CoV spike using Lipofectamine 2000 (ThermoFisher Scientific cat.# 11668019) to produce single-round infection-competent pseudoviruses (
    HEK293T
    suggested: None
    HEp2 epithelial cell polyreactive assay: According to manufacturer’s instructions, HEp2 slides (Hemagen cat.#
    HEp2
    suggested: CLS Cat# 300397/p694_Hep-2, RRID:CVCL_1906)
    Mock-transfected 293T cells were used as a negative control.
    293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    Virus titration: SARS-CoV-2-MA10, SARS-CoV-1-MA15 and MERS-CoV-M35c4 were grown and titered using VeroE6 cells as previously described (87).
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    12-month-old female Balb/c mice (strain 047) were purchased from Envigo for Sarbecovirus challenge experiments (65, 85).
    Balb/c
    suggested: None
    C57Bl/6 288/330+/+ mice, which encode two human codons in the mouse dipeptidyl peptidase gene, were used for MERS-CoV mouse adapted challenge experiments (66).
    C57Bl/6 288/330+/+
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmid construction: To generate soluble S ectodomain proteins from SARS-CoV-1 (residues 1-1190; GenBank: AAP13567) ,SARS-CoV-2 (residues 1-1208; GenBank: MN908947) , HCoV-HKU1 (residue 1-1295; GenBank: YP_173238.1) , HCoV-OC43 (residues 1-1300; GenBank: AAX84792.1), MERS-CoV (residues 1-1291; GenBank: APB87319.1), HCoV-229E (residues 1-1110; GenBank: NP_073551.1) and HCoV-NL63 (residues 1-1291; GenBank: YP_003767.1) , we synthesized the DNA fragments from GeneArt (Life Technologies) and cloned them into the phCMV3 vector (Genlantis cat.#
    phCMV3
    suggested: RRID:Addgene_173431)
    Briefly, the pBOB-hACE2 or hDPP4 plasmid and lentiviral packaging plasmids (
    pBOB-hACE2
    suggested: None
    hDPP4
    suggested: None
    pMDL, pREV, and pVSV-G (Addgene #12251, #12253, #8454)) were co-transfected into HeLa cells using Lipofectamine 2000 reagent (ThermoFisher Scientific cat.# 11668019).
    pMDL
    suggested: None
    pREV
    suggested: None
    pVSV-G
    suggested: RRID:Addgene_138479)
    Pseudovirus production: HIV-based lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene #8455), pBOB-Luciferase (Addgene #170674) were co-transfected into HEK293T cells along with full-length or variously truncated SARS-CoV1, WIV1, SHC014, Pang17, SARS-COV2, SARS-CoV-2 variants of concern [(B.1.1.7(alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) and B.1.1.529 (Omicron)] and MERS-CoV spike using Lipofectamine 2000 (ThermoFisher Scientific cat.# 11668019) to produce single-round infection-competent pseudoviruses (
    pCMV-dR8.2
    suggested: RRID:Addgene_8455)
    pBOB-Luciferase
    suggested: None
    Software and Algorithms
    SentencesResources
    The protocol was approved by the UCSD Human Research Protection Program.
    UCSD Human Research Protection Program
    suggested: None
    08-771-23) and sorted for S protein-specific memory B cells using BD FACSMelody sorter.
    BD FACSMelody
    suggested: None
    Single colonies were picked for sequencing and analysis on IMGT V-Quest online tool (http://www.imgt.org) and downstream plasmid production.
    http://www.imgt.org
    suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)
    Fifty percent maximal response concentrations (EC50) were calculated using the Asymmetrical dose-response model of Richard’s version in GraphPad Prism 7 (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Following three washes with FACS buffer, the cells were resuspended and analyzed by flow cytometry (BD Lyrics cytometer), and the binding data were generated by calculating the Mean Fluorescence Intensity using FlowJo 10 software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

    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 10.1101/2022.03.04.479488v1 on bioRxiv