Broadly neutralizing antibodies target the coronavirus fusion peptide

  1. Cherrelle Dacon
  2. Courtney Tucker
  3. Linghang Peng
  4. Chang-Chun D. Lee
  5. Ting-Hui Lin
  6. Meng Yuan
  7. Yu Cong
  8. Lingshu Wang
  9. Lauren Purser
  10. Jazmean K. Williams
  11. Chul-Woo Pyo
  12. Ivan Kosik
  13. Zhe Hu
  14. Ming Zhao
  15. Divya Mohan
  16. Andrew J. R. Cooper
  17. Mary Peterson
  18. Jeff Skinner
  19. Saurabh Dixit
  20. Erin Kollins
  21. Louis Huzella
  22. Donna Perry
  23. Russell Byrum
  24. Sanae Lembirik
  25. David Drawbaugh
  26. Brett Eaton
  27. Yi Zhang
  28. Eun Sung Yang
  29. Man Chen
  30. Kwanyee Leung
  31. Rona S. Weinberg
  32. Amarendra Pegu
  33. Daniel E. Geraghty
  34. Edgar Davidson
  35. Iyadh Douagi
  36. Susan Moir
  37. Jonathan W. Yewdell
  38. Connie Schmaljohn
  39. Peter D. Crompton
  40. Michael R. Holbrook
  41. David Nemazee
  42. John R. Mascola
  43. Ian A. Wilson
  44. Joshua Tan

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Abstract

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2′ cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain–specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2′ cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

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  1. Version published to 10.1126/science.abq3773
  2. ScreenIT

    SciScore for 10.1101/2022.04.11.487879: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All participants in the convalescent cohort provided informed consent for their blood products to be used for research purposes by signing the standard New York Blood Center (NYBC
    IRB: For participants who received the SARS-CoV-2 mRNA-1273 vaccine (Moderna), whole blood, plasma and serum samples were obtained at the NIH Clinical Research Center in Bethesda, MD under protocols approved by the NIH Institutional Review Board, ClinicalTrials
    IACUC: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals.
    Field Sample Permit: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals.
    Sex as a biological variableHamsterization of human monoclonal antibodies: Genomes corresponding to the mouse IgG2a heavy and light chains were aligned to the genome assembly MesAur1.0 (GCA_000349665.1) for a female Syrian golden hamster downloaded from Genbank.
    RandomizationNo randomization or blinding was applied to the analysis of participants’ plasma, serum or PBMC samples, but all samples were anonymized before being used in this study.
    BlindingNo randomization or blinding was applied to the analysis of participants’ plasma, serum or PBMC samples, but all samples were anonymized before being used in this study.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Binding of secreted antibody to the beads was detected in the CY5 or TRED channels by capturing images at 6 min intervals over a 30 min time course.
    CY5
    suggested: None
    Shotgun mutagenesis epitope mapping of antibodies by alanine scanning: Epitope mapping was performed essentially as previously described (43), using a SARS-CoV-2 (Wuhan Hu-1 strain) S2 subunit shotgun mutagenesis mutation library, made using a full-length expression construct for the SARS-CoV-2 spike glycoprotein.
    SARS-CoV-2 spike glycoprotein .
    suggested: None
    Vaccinee and convalescent plasma binding to peptides: Polyclonal IgG antibodies from plasma or sera of vaccinated, convalescent, or naïve donors were purified using the Pierce Protein G Spin Plate (Thermo Scientific).
    Polyclonal IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Baculoviruses were produced by transfection of bacmid DNA into Sf9 cells and used to infect High Five cells (Life Technologies) at high (5 to 10) multiplicity of infection (MOI).
    Sf9
    suggested: None
    Plasma IgG reactivity to human coronaviruses and donor selection: Multiplexed beads for SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63 spike proteins, as well as CD4 as a negative control, were incubated with donor plasma diluted at 1/50, 1/250 or 1/1250 for 30 min at room temperature, then washed and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-170).
    HCoV-229E
    suggested: JCRB Cat# JCRB1838, RRID:CVCL_B3M4)
    mAbs were also expressed in-house by transient transfection of Expi293 cells (Gibco, A14527) using the ExpiFectamine 293 Transfection Kit (Gibco, A14524) according to manufacturer’s instructions.
    Expi293
    suggested: RRID:CVCL_D615)
    To generate green fluorescent protein (GFP)-tagged receptor cell lines, HeLa-ACE2 cells were transduced with lentivirus encoding GFP and sorted to collect the GFPhigh/ACE2high population.
    HeLa-ACE2
    suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)
    mAbs were added to the wells at a final concentration of 200 μg/mL and cultures were further incubated at 37 °C for 1h. 8,000 GFP+/ACE2+ HeLa cells were then added to each well and the co-cultures were maintained overnight to allow for syncytia development.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    A plasmid encoding cDNA for each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 h.
    HEK-293T
    suggested: None
    For neutralization assays, 5 × 104 RD cells were inoculated at TCID75% OC43-GFP virus and incubated for 1h at 35°C. 4-fold serial dilutions (73 ng/mL - 300 μg/mL) of each mAb were incubated with TCID75 OC43-GFP virus for 1h at 35°C. 60 μL of mAb- virus mixture was used to inoculate each well containing 5 × 104 RD cells and cultures were incubated for 24 h at 35°C.
    RD
    suggested: None
    transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and full-length spike plasmids from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E into 293T cells using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Asheville, NC, L3000-001) (49). 293 flpin-TMPRSS2-ACE2 cells (provided by Dr. Adrian Creanga, VRC/NIH) were used for SARS-CoV-2, SARS-CoV and hCoV-NL63 while HuH7.5 cells were used for MERS-CoV and hCoV-229E neutralization assay.
    HCoV-NL63
    suggested: None
    293T
    suggested: None
    HuH7.5
    suggested: RRID:CVCL_7927)
    SARS: Addgene #170447; SARS2 #170442; MERS #170448; NL63 #172666; alpha strain #170451; beta #170449; gamma #170450; delta #172320; omicron 180375) were co-transfected in HEK293T with Lipofectamine 2000 (ThermoFisher Scientific, 11668019) to produce single-round infection-competent pseudoviruses.
    HEK293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    SARS: Addgene #170447; SARS2 #170442; MERS #170448; NL63 #172666; alpha strain #170451; beta #170449; gamma #170450; delta #172320; omicron 180375) were co-transfected in HEK293T with Lipofectamine 2000 (ThermoFisher Scientific, 11668019) to produce single-round infection-competent pseudoviruses.
    #172666
    suggested: None
    Recombinant DNA
    SentencesResources
    Pre-fusion stabilized constructs for CCoV HuPn-2018 (Accession # QVL91811.1, aa1-1384 with E1140P and E1141P mutations) and PdCoV0081-4 ( Accession # MW685622.1, aa1-1092 with E854P and V855P mutations) were synthesized and cloned into pCDNA3.1- vectors (Genscript) with the following C-terminal modifications: T4 fibritin trimerization motif, HRV3C protease cleavage site, poly-GS linker, Avi-tag, and 8× His tag.
    pCDNA3.1-
    suggested: RRID:Addgene_52535)
    Briefly, the SARS-CoV-2 NTD and RBD were cloned into an in-house pFastBac vector.
    pFastBac
    suggested: RRID:Addgene_1925)
    The spike S2 domain (699 to 1207 with F817P, A892P, A899P, A942P, K986P, V987P) was constructed into phCMV3 vector which contained an N-terminal secreting signal peptide, and C-terminal thrombin cleavage site and His6 tag.
    phCMV3
    suggested: RRID:Addgene_173431)
    SARS-CoV-2 RBD, SARS- CoV-2 NTD, SARS-CoV-1 spike and SARS-CoV-1 RBD, MERS-CoV spike, OC43-CoV spike, CCoV-HuPn-2018 spike, pPDCoV-0081-4 spike, HCoV-NL63 spike, HCoV-229E spike, HCoV- HKU1 spike, H1 HA and recombinant CD4 (gifted by Gavin Wright, (35)).
    pPDCoV-0081-4
    suggested: None
    Spike-containing lentiviral pseudovirions were produced by co-transfection of packaging plasmid pCMVdR8.2,
    pCMVdR8.2
    suggested: RRID:Addgene_8455)
    transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and full-length spike plasmids from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E into 293T cells using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Asheville, NC, L3000-001) (49). 293 flpin-TMPRSS2-ACE2 cells (provided by Dr. Adrian Creanga, VRC/NIH) were used for SARS-CoV-2, SARS-CoV and hCoV-NL63 while HuH7.5 cells were used for MERS-CoV and hCoV-229E neutralization assay.
    pHR’
    suggested: None
    TMPRSS2
    suggested: RRID:Addgene_53887)
    2.5μg 2nd generation lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene, 8455), 2μg pBOBI-FLuc (Addgene, 170674) and 1μg truncated coronavirus spike expressing plasmids (
    pCMV-dR8.2
    suggested: RRID:Addgene_8455)
    pBOBI-FLuc
    suggested: RRID:Addgene_170674)
    Hamster genes with the highest homology to the mouse IgG2a heavy chain, lambda and kappa light chains genes were cloned into a pCDNA3.4 vector (Genscript) and expressed in Expi293 cells as described above.
    pCDNA3.4
    suggested: None
    Software and Algorithms
    SentencesResources
    ) high-throughput flow cytometer and FACS data were analysed with FlowJo (Version 10.8.1
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Analyses of the VH and Vλ/Vκ genes, CDR3 sequences, and percentage of somatic mutations were carried out using Geneious Prime (Version 2021.0.3, https://www.geneious.com) and the International Immunogenetics Information System database (IMGT, http://www.imgt.org/) (40).
    https://www.geneious.com
    suggested: (Geneious, RRID:SCR_010519)
    http://www.imgt.org/
    suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)
    Recombinant IgG mAbs were purified using HiTrap Protein A columns (Cytiva/GE Healthcare Life Sciences, 17040303).
    Cytiva/GE Healthcare
    suggested: None
    Phylogenetic tree generation: Full-length amino acid sequences of SARS-CoV-2 (accession #NC_045512.2), SARS-CoV (accession # AY278741.1), MERS-CoV (accession # NC_019843) , HCoV-NL63 (accession #NC_005831.2), HCoV-229E (accession #NC_002645.1), CCoV HuPn-2018 (accession #MW591993.2) and PDCov-0081-4 (accession #MW685622) were aligned using the L-INS-i method of MAFFT version 7.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    The sequence alignment was used to generate a sequence logo plot using the Weblogo 3.0 server and to color conserved amino acid residues on a pre-fusion stabilized spike protein (PDB 6VSB).
    Weblogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    Images were acquired in A488, A568 and DAPI channels using a BZ-X fluorescence microscope (KEYENCE) and processed using Fiji ImageJ (42)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Iterative model building and refinement were carried out in Coot (46) and PHENIX (47), respectively.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    Authentic OC43-CoV-GFP virus propagation and neutralization assay: Rhabdomyosarcoma cells (RD, ATCC CCL-136) were maintained at 37°C and 5% CO2 in No-glucose DMEM (Gibco, 11966-025), supplemented with 10% HI-FBS, 4500 mg/mL glucose, 1 mM sodium pyruvate (Gibco, 11360-070), 1 mM HEPES (Gibco, 15630-080) and 50 μg/mL gentamycin (Quality Biological, 120-098-661)
    Quality Biological
    suggested: None
    50% neutralization titers (NT50) were calculated using the dose- response-inhibition model with 5-parameter Hill slope equation in GraphPad Prism 9.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Descriptive statistics (mean ± SEM or mean ± SD) and statistical analyses were performed using Prism version 9.3.1 (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT00001281RecruitingStudies of Blood and Reproductive Fluids in HIV-Infected and…
    NCT05078905RecruitingVaccine Responses to SARS-CoV-2 and Other Emerging Infectiou…

    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.
    • Thank you for including a protocol registration statement.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2022.04.11.487879v1 on bioRxiv