A human antibody that broadly neutralizes betacoronaviruses protects against SARS-CoV-2 by blocking the fusion machinery

  1. Dora Pinto
  2. Maximilian M. Sauer
  3. Nadine Czudnochowski
  4. Jun Siong Low
  5. M. Alejandra Tortorici
  6. Michael P. Housley
  7. Julia Noack
  8. Alexandra C. Walls
  9. John E. Bowen
  10. Barbara Guarino
  11. Laura E. Rosen
  12. Julia di Iulio
  13. Josipa Jerak
  14. Hannah Kaiser
  15. Saiful Islam
  16. Stefano Jaconi
  17. Nicole Sprugasci
  18. Katja Culap
  19. Rana Abdelnabi
  20. Caroline Foo
  21. Lotte Coelmont
  22. Istvan Bartha
  23. Siro Bianchi
  24. Chiara Silacci-Fregni
  25. Jessica Bassi
  26. Roberta Marzi
  27. Eneida Vetti
  28. Antonino Cassotta
  29. Alessandro Ceschi
  30. Paolo Ferrari
  31. Pietro E. Cippà
  32. Olivier Giannini
  33. Samuele Ceruti
  34. Christian Garzoni
  35. Agostino Riva
  36. Fabio Benigni
  37. Elisabetta Cameroni
  38. Luca Piccoli
  39. Matteo S. Pizzuto
  40. Megan Smithey
  41. David Hong
  42. Amalio Telenti
  43. Florian A. Lempp
  44. Johan Neyts
  45. Colin Havenar-Daughton
  46. Antonio Lanzavecchia
  47. Federica Sallusto
  48. Gyorgy Snell
  49. Herbert W. Virgin
  50. Martina Beltramello
  51. Davide Corti
  52. David Veesler

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Abstract

The repeated spillovers of β-coronaviruses in humans along with the rapid emergence of SARS-CoV-2 escape variants highlight the need to develop broad coronavirus therapeutics and vaccines. Five monoclonal antibodies (mAbs) were isolated from COVID-19 convalescent individuals and found to cross-react with multiple β-coronavirus spike (S) glycoproteins by targeting the stem helix. One of these mAbs, S2P6, cross-reacts with more than twenty human and animal β-coronavirus S glycoproteins and broadly neutralizes SARS-CoV-2 and pseudotyped viruses from the sarbecovirus, merbecovirus and embecovirus subgenera. Structural and functional studies delineate the molecular basis of S2P6 cross-reactivity and broad neutralization and indicate that this mAb blocks viral entry by inhibiting membrane fusion. S2P6 protects hamsters challenged with SARS-CoV-2 (including the B.1.351 variant of concern) through direct viral neutralization and Fc-mediated effector functions. Serological and B cell repertoire analyses indicate that antibodies targeting the stem helix are found in some convalescent donors and vaccinees but are predominantly of narrow specificity. Germline reversion of the identified cross-reactive mAbs revealed that their unmutated ancestors are specific for the endemic OC43 or HKU1 viruses and acquired enhanced affinity and breadth through somatic mutations. These data demonstrate that conserved epitopes in the coronavirus fusion machinery can be targeted by protective antibodies and provide a framework for structure-guided design of pan-β-coronavirus vaccines eliciting broad protection.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.09.442808: (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
    AMBRA (antigen-specific memory B cell repertoire analysis) of IgG antibodies: Replicate cultures of total unfractionated PBMC from SARS-CoV-2 infected or vaccinated individuals were seeded in 96 U-bottom plates (Corning) in RPMI1640 supplemented with 10% Hyclone, sodium pyruvate, MEM non-essential amino acid, stable glutamine and PenicillinStreptomycin.
    antigen-specific memory B cell repertoire analysis) of IgG
    suggested: None
    Antibody discovery and expression: Antigen specific IgG+ memory B cells were isolated and cloned from total PBMCs of convalescent individuals.
    Antigen specific IgG+
    suggested: None
    Flow cytometry of antibody on S Protein expressing ExpiCHO-S cells: For Expi-CHO cell transient transfection, S plasmids (21, 58) were diluted in cold OptiPRO SFM, mixed with ExpiFectamine CHO Reagent (Life Technologies, A29130) and added to the cells seeded at 6×106 cells/ml in a volume of 5 ml in a 50 ml bioreactor.
    A29130
    suggested: None
    Twelve-point 3-fold serial dilutions of S2P6 were prepared in DMEM and OC43 S VSV pseudoviruses were added 1:1 (v/v) to each dilution in the presence of anti-VSV-G antibody from I1-mouse hybridoma supernatant diluted 50 times (final volume: 50 μl).
    anti-VSV-G
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Cell lines used in this study were obtained from ATCC (HEK293T and Vero-E6) or ThermoFisher Scientific (ExpiCHO cells, FreeStyle™ 293-F cells and Expi293F™ cells).
    HEK293T
    suggested: None
    293-F
    suggested: None
    In a BSL3 facility, serial dilutions of mAbs (1:4) were incubated with 200 PFU (plaque forming units, corresponding to a multiplicity of infection of 0.01) of authentic SARS-CoV-2 (isolate USA-WA1/2020, passage 3, passaged in Vero-E6 cells) for 30 minutes at 37°C.
    Vero-E6
    suggested: None
    To perform pseudotype neutralization assays, VeroE6-TMPRSS2 cells were used for VSV-SARS-CoV-2, VSV-SARS-CoV-1, and VSV-GD19 and Huh7 cells were used for VSV-MERS.
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Briefly, HEK-293T cells at 70~80% confluency were transfected with the pCDNA3.1 expression vectors encoding full-length OC43 S harboring a truncation of the 17 C-terminal residues along with a fusion to Ha-tag and the bovine coronavirus hemagglutinin esterase protein Fc-tagged at molar ratios of 7:1.
    HEK-293T
    suggested: None
    Recombinant DNA
    SentencesResources
    SARS-CoV-2 S D614G, used for production of SARS-CoV-2 postfusion, contains a mu-phosphatase signal peptide beginning at 14Q, a mutated S1/S2 cleavage site (SGAR), and ends at residue K1211 followed by a TEV cleavage, foldon trimerization motif, and an 8X his tag in a pCMV vector.
    pCMV
    suggested: RRID:Addgene_20783)
    VSV pseudotype virus production and neutralization: Sarbecovirus spike cassettes with a C-terminal deletion of 19 amino acids (D19) were synthesized and cloned into mammalian expression constructs (pcDNA3.1(+) or pTwist-CMV) for the following Sarbecoviruses: SARS-CoV-2 (Accession QOU99296.1), SARS-CoV-1 (Accession AAP13441.1), hCoV-19/pangolin/Guangdong/1/2019 (GD19, Accession QLR06867.1), and Middle East respiratory syndrome-related coronavirus (MERS, Accession YP_009047204).
    pTwist-CMV
    suggested: None
    Briefly, HEK-293T cells at 70~80% confluency were transfected with the pCDNA3.1 expression vectors encoding full-length OC43 S harboring a truncation of the 17 C-terminal residues along with a fusion to Ha-tag and the bovine coronavirus hemagglutinin esterase protein Fc-tagged at molar ratios of 7:1.
    pCDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    UCA sequences of the VH and VL were constructed using IMGT/V-QUEST.
    IMGT/V-QUEST
    suggested: (IMGT/V-QUEST, RRID:SCR_010749)
    Conservation analysis: Conservation analysis was performed as described previously (Pinto et al 2020).
    Conservation
    suggested: (Conservation, RRID:SCR_016064)
    The multiple sequences alignment was performed using MAFFT (https://mafft.cbrc.jp/alignment/software/) with the spike amino acid sequences as input.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Plates were imaged with an automated microscope (Cytation5, Biotek), and nuclei and cells positive for the SARS-CoV-2 Nucleoprotein were quantified using the supplied Gen5 software.
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    Percent neutralization data were analyzed using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Several subsequent rounds of model building and refinement were performed using Coot (71), ISOLDE (72), Refmac5 (73), and MOE, to arrive at a final model for the complex.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Two rounds of reference-free 2D classification were performed using cryoSPARC (76) to select well-defined particle images.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

  2. Version published to 10.1101/2021.05.09.442808v1 on bioRxiv