Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses
- Department of Molecular Biosciences, The University of Texas at Austin, United States
- Department of Chemical Engineering, The University of Texas at Austin, United States
- Department of Chemistry and Biochemistry, The University of Texas at Dallas, United States
- Division of Chemical Biology and Medicinal Chemistry, The University of Texas at Austin, United States
- Texas Biomedical Research Institute, United States
- Laboratory of Veterinary Zoonosis, College of Veterinary Medicine, Chonnam National University, Republic of Korea
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Biophysics Graduate Program, University of California, Berkeley, United States
- Retired, United States
- Department of Chemistry, University of California, Berkeley, United States
- LaMontagne Center for Infectious Diseases, The University of Texas at Austin, United States
Figures
Figure 1 with 6 supplements
The hinge epitope spans the HR1/CH helices at the S2 apex.
(a) Monomeric SARS-2 2P spike (PDBID: 6VSB chain B) colored according to the HDX difference in deuterium fractional uptake between SARS-2 HexaPro spike alone and with 3A3 IgG at 102 s exchange. …
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Figure 1—source data 1
Summary and complete HDX data for spike peptides.
- https://cdn.elifesciences.org/articles/83710/elife-83710-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Sequence conservation is higher in the S2 domain than the S1 domain across coronaviruses that infect humans.
Percent sequence identity and similarity between the spike (a and c, respectively) S1 subunits and (b and d, respectively) S2 subunits of the seven coronaviruses known to infect humans were analyzed …
Figure 1—figure supplement 2
Many cross-reactive scFv-phage target the foldon domain.
Most monoclonal phage tested by ELISA on SARS-2 spike or the unrelated RSV F foldon-coated plates had cross-reactive binding, indicating targeting of the shared foldon domain. After round 4 of …
Figure 1—figure supplement 3
Peptides monitored through all timepoints of deuteration for SARS-2 HexaPro spike alone and with 3A3 IgG or Fab.
A total of 192 peptides were monitored, covering 56.3% of the SARS-2 HexaPro spike sequence and averaging 3.34 redundancy per amino acid. All peptides were manually checked. SARS-2 spike features …
Figure 1—figure supplement 4
Deuterium uptake of SARS-2 HexaPro spike alone suggests the trimer is maintained under HDX conditions.
(a) Trimeric (i and ii) and monomeric (iii) SARS-2 2P spike (PDB: 6VSB) colored according to fractional deuterium uptake of the SARS-2 HexaPro spike alone after 103 s of exchange. The figure was …
Figure 1—figure supplement 5
HDX identified the apex of the S2 domain as the 3A3 epitope.
Volcano plots showing changes in deuterium uptake in SARS-2 HexaPro spike peptides upon addition of (a) 3A3 IgG or (b) Fab after 102 s exchange. Significance cutoffs are an average change in …
Figure 1—figure supplement 6
HDX identified the spike-binding paratope in 3A3 IgG.
Volcano plots showing changes in deuterium uptake in 3A3 IgG (a) heavy chain and (b) light chain upon addition of SARS-2 HexaPro spike after 102 s exchange. Significance cutoffs are an average …
Figure 2 with 1 supplement
The hinge epitope is accessible only in an RBD-up and S2-open spike conformation.
(a) Trimeric SARS-2 spike in various conformations colored according to difference in deuterium fractional uptake between SARS-2 HexaPro spike alone and with 3A3 IgG. The hinge epitope within S2 is …
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Figure 2—source data 1
BLI sensorgram data.
- https://cdn.elifesciences.org/articles/83710/elife-83710-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
HexaPro E1031R variant exposes the hinge epitope.
The kinetics of interconversion between the closed-S2 and open-S2 states of HexaPro and HexaPro E1031R were evaluated by HDX as previously described (Costello et al., 2022). The spike proteins were …
Figure 3 with 4 supplements
SARS-2 spike residues 985–988 are recognized by 3A3 and impair spike function upon substitution.
(a) Residues important for 3A3 binding were identified by single residue changes in HexaPro that increased or decreased binding to 3A3 relative to HexaPro. Each variant was tested with duplicate …
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Figure 3—source data 1
ELISA binding data and relative luminescence data for pseudovirus infection assays.
- https://cdn.elifesciences.org/articles/83710/elife-83710-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
HexaPro variants with reduced 3A3 binding retain trimer SEC profile.
The single-point mutants of HexaPro, D985L, E988I, D994A, and L1001A, had reduced binding to 3A3 relative to HexaPro (Figure 3a), but retained the overall size of unmodified HexaPro by SEC with …
Figure 3—figure supplement 2
The hinge epitope is nonlinear and inaccessible in aggregated or misfolded protein.
(a) 3E11 binds reduced, denatured SARS-2 HP, SARS-2, and MERS spike proteins by Western blot, but 3A3 does not. The ladder molecular weight is labeled in kDa on the left side. (b) SDS-PAGE analysis …
Figure 3—figure supplement 3
Original blot images for Figure 3—figure supplement 2a.
Figure 3—figure supplement 4
Adding a disulfide bond slightly improves 3A3 binding by ELISA to spike with P986 and P987 reverted to the native sequence.
Antibody 3A3 was coated on high binding plates and allowed to bind dilutions of SARS-2 HexaPro spike, 4P spike (HexaPro with P986K and P987V reversions), or 4P-DS spike (4P with an additional …
Figure 4 with 4 supplements
The hinge epitope is conserved across β-coronaviruses and variably accessible in authentic spike.
The hinge epitope recognized by 3A3 (SARS-2 amino acids 980–1006) is highly conserved across the spike (a) sequences and (b) structures of β-coronaviruses known to infect humans, including Alpha, …
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Figure 4—source data 1
ELISA data and flow cytometry mean fluorescence intensity data.
- https://cdn.elifesciences.org/articles/83710/elife-83710-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Antibody hu3A3 binds SARS-2 HexaPro similarly to 3A3 by ELISA.
SARS-2 HexaPro spike was coated on high binding plates and allowed to bind dilutions of 3A3 or hu3A3 purified antibody, then anti-human Fc-HRP for detection.
Figure 4—figure supplement 2
Antibody hu3A3 yeast display libraries were enriched for binding to 4P-DS.
Yeast surface display of irrelevant Fab or the hu3A3 Fab resulted in minimal binding to AF647 conjugated 4P-DS spike by flow cytometry. Site-directed (SD) or error-prone (EP) strategies to introduce …
Figure 4—figure supplement 3
Antibody RAY53 retains epitope specificity while exhibiting higher affinity than 3A3 for SARS-2 HexaPro spike with reverted 2P changes.
(a) ELISA data with spike 4P-DS or 4P coated on high binding plates, followed by the addition antibody 3A3 or RAY53 in a 1:5 dilution series and detection with anti-human Fc-HRP secondary antibody. …
Figure 4—figure supplement 4
Antibodies 3A3 and RAY53 have low-to-mid nanomolar affinities for stabilized SARS-2 spike variants.
Binding of 3A3 Fab to HexaPro S2 was measured by (a) BLI with the HexaPro S2 trimer immobilized on the biosensor and (b) SPR with Fab immobilized on the chip. SPR was also used to evaluate binding …
Figure 5 with 1 supplement
Targeting the hinge epitope recruits Fc effector functions.
(a) Neutralization was evaluated by pre-incubating antibody with pseudotyped HIV particles that were then added to HEK 293T cells stably expressing ACE2 (SARS-1 and SARS-2 pseudoviruses) or DPP4 …
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Figure 5—source data 1
Data reporting antibody effect on infection with pseudovirus, authentic virus, phagocytosis score, and cellular lysis.
- https://cdn.elifesciences.org/articles/83710/elife-83710-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Antibody 3A3 inhibits cellular fusion induced by the interaction of SARS-2 spike with human ACE2.
(a) HEK 293 cells stably expressing human ACE2 were stained with Cell Trace Far Red and incubated with a CHO-based cell line transiently expressing authentic SARS-2 spike and EGFP. The cultures were …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-NP 1C7C7 | Thomas Moran (The Icahn School of Medicine at Mount Sinai) | N/A | |
| Antibody | Anti-spike CR3022 | Constructed based on ter Meulen et al., 2006. | N/A | |
| Antibody | Anti-spike mAb 2–4 | Constructed based on Liu et al., 2020 . | N/A | |
| Antibody | Anti-spike S309 | Constructed based on Pinto et al., 2020. | N/A | |
| Antibody | Anti-StrepTagII Fab (clone C23.21) | Constructed based on patent WO2015067768A1 (Institut Pasteur) | N/A | |
| Antibody | Antibody variants: 3A3, hu3A3, RAY53, 3E11 | This study | N/A | Sequences can be found in Supplementary file 4. |
| Antibody | Goat anti human κ HRP | SouthernBiotech | Cat# 2060-05 | |
| Antibody | Goat anti human IgG Fc-AF647 | Jackson ImmunoResearch | Cat# 109-605-008 | |
| Antibody | Goat anti mouse Ig HRP | SouthernBiotech | Cat# 1010-05 | |
| Antibody | Goat anti-human IgG Fc-HRP (polyclonal) | SouthernBiotech | Cat# 2047-05 | |
| Antibody | Human Fab2 anti-strep-tag (clone C23.21) | Jason McLellan Lab | N/A | |
| Antibody | Mouse anti c-myc, clone 9E10 | BioXCell | Cat #MA1-980 | |
| Antibody | Mouse anti FLAG (M2) HRP | Sigma-Aldrich | Cat# A8592 | |
| Antibody | Mouse anti FLAG (M2) PE | BioLegend/Prozyme | Cat# 637309/ #PJ315 | |
| Antibody | Mouse anti-M13 pVIII-HRP, clone RL-pH1 | Santa Cruz Biotech | Cat# sc53004 | |
| Cell line (Cricetulus griseus) | CHO-T | Acyte BioTech | N/A | |
| Cell line (C. griseus) | CHOK-1 | ATCC | Cat# CCL-61 | |
| Cell line (C. griseus) | ExpiCHO | Thermo Fisher Scientific | Cat# A29133 | |
| Cell line (Homo sapiens) | Expi293 | Thermo Fisher Scientific | Cat# A41249 | |
| Cell line (H. sapiens) | Freestyle HEK293-F | Thermo Fisher Scientific | Cat# R79007 | |
| Cell line (H. sapiens) | HEK-293T-hACE2 | BEI Resources | Cat# NR-52511 | |
| Cell line (H. sapiens) | HEK293T | ATCC | Cat# CRL-3216 | |
| Cell line (H. sapiens) | NK-92 V158 | ATCC | Cat# PTA-8836 | |
| Cell line (H. sapiens) | THP-1 | ATCC | Cat# TIB-202 | |
| Cell line (H. sapiens) | Vero HL | Piepenbrink et al., 2022 | N/A | |
| Chemical compound, drug | Biotin | Sigma-Aldrich | Cat# B4501-10G | |
| Chemical compound, drug | Calcein AM | BD Pharmingen | Cat# 564061 | |
| Chemical compound, drug | Flash Red 1μ Beads | Bangs Laboratories | Cat# FSFR004 | |
| Chemical compound, drug | pHrodo iFL Green STP Ester | Thermo Fisher Scientific | Cat# P36013 | |
| Chemical compound, drug | TMB Substrate | Thermo Fisher Scientific | Cat# 34021 | |
| Commercial assay or kit | Alexa Fluor 647 Protein Labelling Kit | Fisher Scientific | Cat# A20173 | |
| Commercial assay or kit | ExpiFectamine 293 Transfection Kit | Thermo Fisher Scientific | Cat# A14524 | |
| Commercial assay or kit | ExpiFectamine CHO Transfection Kit | Thermo Fisher Scientific | Cat# A29129 | |
| Commercial assay or kit | HiTrap Protein A columns | Cytiva | Cat# 17-5498-54P | |
| Commercial assay or kit | IMAC Sepharose 6 Fast Flow resin | Cytiva | Cat# 17092107 | |
| Commercial assay or kit | Lipofectamine 2000 | Thermo Fisher Scientific | Cat# 11668019 | |
| Commercial assay or kit | Mycostrip test | Invivogen | Cat# rep-mys-10 | |
| Commercial assay or kit | Octet Anti-Human Fab-CH1 2nd Generation (FAB2G) Biosensors | Forte Bio | Cat# 18-5125 | |
| Commercial assay or kit | Octet Streptavidin (SA) Biosensor | Forte Bio | Cat# 18-5019 | |
| Commercial assay or kit | Protein Thermal Shift Dye Kit | Thermo Fisher Scientific | Cat# 4461146 | |
| Commercial assay or kit | Series S Sensor Chip CM5 | Cytiva | Cat# BR100530 | |
| Commercial assay or kit | Strep-Tactin XT Superflow high capacity cartridge | IBA | Cat# 2-4026-001 | |
| Commercial assay or kit | Superdex 200 Increase 10/300GL | Cytiva | Cat# 28-9909-44 | |
| Organisms (Mus musculus) | Balb/c mice | Charles River | Cat# 028 | |
| Other | HBS EP+buffer | Cytiva | Cat# BR100669 | |
| Peptide, recombinant protein | Avidin | Sigma-Aldrich | Cat# A9275-25MG | |
| Peptide, recombinant protein | Streptavidin AF647 | Jackson ImmunoResearch | Cat# 016600084 | |
| Peptide, recombinant protein | Streptavidin PE | BioLegend | Cat# 405204 | |
| Recombinant DNA reagent | AbVec hIgG1 | Smith et al., 2009 | N/A | |
| Recombinant DNA reagent | AbVec hIgKappa | Smith et al., 2009 | N/A | |
| Recombinant DNA reagent | HDM-IDTSpike-fixK | BEI Resources | Cat# NR-52514 | |
| Recombinant DNA reagent | M13KO7 helper phage (virus) | NEB | N0315S | |
| Recombinant DNA reagent | pcDNA3.1(-)- Wuhan-Hu-1 Spike | Walls et al., 2020 BEI Resources | Cat# NR-52420 | |
| Recombinant DNA reagent | pCMV-VSV-G | Cell Biolabs | Cat# RV-110 | |
| Recombinant DNA reagent | pCTCon-Fab | Wang et al., 2018 | N/A | |
| Recombinant DNA reagent | pHAGE-CMV-Luc2-IRS-ZsGreen-W | BEI Resources | Cat# NR-52516 | |
| Recombinant DNA reagent | pHAGE2-EF1aInt-ACE2-WT | BEI Resources | Cat# NR-52512 | |
| Recombinant DNA reagent | pLEX307-DPP4-G418 | Addgene | Cat# 158453 | |
| Recombinant DNA reagent | pMoPac24 | Hayhurst et al., 2003 | N/A | |
| Sequence-based reagent | Primers for cloning mouse variable regions | Krebber et al., 1997 | N/A | |
| Software, algorithm | Astra Software V6.1.2 | Wyatt Technology | RRID:SCR_016255 | |
| Software, algorithm | Biacore X100 Evaluation Software V2.0.1 | GE Healthcare | N/A | |
| Software, algorithm | cisTEM | Grant et al., 2018 | RRID:SCR_016502 | |
| Software, algorithm | cryoSPARC | Punjani et al., 2017 | RRID:SCR_016501 | |
| Software, algorithm | DynamX v3.0 | Waters | Part# 720005145en | |
| Software, algorithm | Excel 1808 | Microsoft | N/A | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | |
| Software, algorithm | FlowJo 10.7.1 | BD Biosciences | RRID:SCR_008520 | |
| Software, algorithm | GraphPad Prism, v9.2.0 | Motulsky and Brown, 2006 | RRID:SCR_002285 | |
| Software, algorithm | HD-eXplosion v 1.2 | Naifu Zhang and Sheena D’Arcy (The University of Texas at Dallas) | N/A | |
| Software, algorithm | Image J v1.53e | NIH | RRID:SCR_003070 | |
| Software, algorithm | Octet Data Analysis Software V11.1 | Forte Bio | N/A | |
| Software, algorithm | ViiA 7 Software | Thermo Fisher Scientific | N/A | |
| Strain, strain background (Escherichia coli) | DH5α electrocompetent cells | NEB | Cat# C2987H | |
| Strain, strain background (E. coli) | XL1-Blue | Agilent | Cat# 200228 | |
| Strain, strain background (Saccharomyces cerevisiae) | AYW101 | Wentz and Shusta, 2007 | N/A | |
| Strain, strain background (S. cerevisiae) | EBY100 yeast | ATCC | Cat# MYA-4941 |
Additional files
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Supplementary file 1
HDX summary table for antibody peptides.
- https://cdn.elifesciences.org/articles/83710/elife-83710-supp1-v1.docx
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Supplementary file 2
Complete HDX data for antibody peptides.
- https://cdn.elifesciences.org/articles/83710/elife-83710-supp2-v1.xlsx
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Supplementary file 3
Width of isotopic distributions for example spike peptides.
Peptides were selected from regions identified as having bimodal distributions in Costello et al. We had no coverage in residues 626–636 and 1146–1166. Peak width (PW) in Da was calculated in triplicate for each peptide in the non-deuterated sample (ND Control) and at each time point of exchange. The SD is reported. The change in peak width (ΔPW) was calculated by subtracting the PW of the control from the PW of the sample. Bimodality was assessed by taking the maximum peak width for a particular peptide (ΔPWmax) and assessing if it was greater than 2 Da. Peak width was calculated using a method similar to Weis et al., 2006. Peptides were centroided with the Apex3D algorithm using DynamX (Waters). Following manual curation, ion stick data were transferred into Excel as two columns of data, m/z values and intensities, and the maximum peak in the isotopic envelope was determined. The list was then searched in descending m/z order to identify the two lowest m/z peaks that straddled 20% of the maximum peak intensity. The m/z value at an envelope intensity of 20% of the maximum intensity was determined using linear interpolation between these two peaks. This process was repeated with a search in ascending m/z order. The relative peak width was determined by multiplying by z (the charge state). For peptide spectra without peaks straddling 20% of the maximum peak intensity on one side of the maximum, typical for lower m/z peaks for peptides exhibiting low deuteration, the farthest isotopic centroid peak on that side was used as the m/z limit for calculating peak width while the other m/z limit was determined using the previously described method.
- https://cdn.elifesciences.org/articles/83710/elife-83710-supp3-v1.docx
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Supplementary file 4
Antibody variable region sequences.
- https://cdn.elifesciences.org/articles/83710/elife-83710-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/83710/elife-83710-mdarchecklist1-v1.pdf
