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. 2021 Mar 17;12(1):1715.
doi: 10.1038/s41467-021-21968-w.

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies

Chunyan Wang  1 , Rien van Haperen  2   3 , Javier Gutiérrez-Álvarez  4 , Wentao Li  1 , Nisreen M A Okba  5 , Irina Albulescu  1 , Ivy Widjaja  1   6 , Brenda van Dieren  1 , Raul Fernandez-Delgado  4 , Isabel Sola  4 , Daniel L Hurdiss  1 , Olalekan Daramola  7 , Frank Grosveld  2   3 , Frank J M van Kuppeveld  1 , Bart L Haagmans  5 , Luis Enjuanes  4 , Dubravka Drabek  2   3 , Berend-Jan Bosch  8
Affiliations

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies

Chunyan Wang et al. Nat Commun. .

Abstract

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.

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Conflict of interest statement

C.W., R.v.H., W.L., I.W., B.v.D., N.M.A.O., F.G., F.J.M.v.K., B.L.H., D.D., and B.J.B. are inventors on a patent application on monoclonal antibodies targeting MERS-CoV (patent publication no.: WO/2020/169755). F.G., D.D. and R.H. are non-substantial interest shareholders in Harbour Biomed and were part of the team that generated the mice. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Cross-reactivity of human monoclonal antibodies 28D9 and 1.6C7 to spike proteins of viruses in the Betacoronavirus genus.
a ELISA binding curves (upper panels) and corresponding ELISA-based half-maximal effective concentrations (EC50) titers (lower panels) of mAbs 28D9 and 1.6C7 to Strep-tagged spike ectodomains (Secto) and S2 ectodomains (S2ecto) of betacoronaviruses from different subgenera including MERS-CoV (subgenus Merbecovirus), SARS-CoV and SARS-CoV-2 (subgenus Sarbecovirus), HCoV-OC43, HCoV-HKU1 and MHV (subgenus Embecovirus), coated at equimolar concentrations. Anti-strep mAb targeting the Strep-tagged antigens was used to corroborate equimolar plate coating. n.b., no binding. Graph bars represent the average ± SD. Hollow circles represent individual data points for n = 2 biological replicates and 2 technical replicates. Source data are provided as a Source Data file. b Binding of mAbs 1.6C7 and 28D9 to HEK-293T cells expressing GFP-tagged membrane-anchored full-length spike proteins of MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-HKU1 and MHV detected by immunofluorescence assay. Cell nuclei in the overlay images were visualized by DAPI. The fluorescence images were recorded using a Leica SpeII confocal microscope. Representative images from n = 2 biological replicates. Scale bars are 100 μm.
Fig. 2
Fig. 2. Cross-neutralization capacity of mAbs 28D9 and 1.6C7 and mechanism of action.
a Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of MERS-CoV, SARS-CoV, SARS-CoV-2 and HCoV-OC43. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations were used to infect VeroCCL81 cells (MERS-S pseudotyped VSV), VeroE6 cells (SARS-S and SARS2-S pseudotyped VSV) or HRT-18 cells (OC43-S pseudotyped VSV) and luciferase activities in cell lysates were determined at 20 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD (n ≥ 6) from at least two independent experiments performed is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody was used as an antibody isotype control. The IC50 and IC90 values were shown. Source data are provided as a Source Data file. b Antibody-mediated neutralization of MERS-CoV, SARS-CoV and SARS-CoV-2 infection. Neutralization of authentic viruses was performed using a plaque reduction neutralization test (PRNT) on VeroCCL81 cells (MERS-CoV) or VeroE6 (SARS-CoV and SARS-CoV-2) as described earlier,. The experiment was performed with triplicate samples, the average ± SD is shown. The IC50 and IC90 values were shown. Source data are provided as a Source Data file. c ELISA-based receptor-binding inhibition assay. MERS-Secto pre-incubated with serially diluted mAbs was added to ELISA plates coated with soluble human DPP4. The binding of MERS-Secto to DPP4 was detected using an HRP-conjugated antibody recognizing the C-terminal Strep-tag on MERS-Secto. Data points represent the average ± SD, for n = 3 replicates from two independent experiments. Source data are provided as a Source Data file. d Cell-cell fusion inhibition assay. Huh-7 cells—transfected with plasmid expressing (GFP-tagged) MERS-CoV S were pre-incubated in the presence or absence of 1.6C7 and 28D9, or an irrelevant iso-type control antibody (Iso-CTRL), and then treated with trypsin to activate the membrane fusion function of the MERS-CoV S protein. The formation of MERS-S mediated syncytia was visualized by fluorescence microscopy. Merged images of MERS-S-GFP expressing cells (green) and DAPI-stained cell nuclei (blue) are shown. The experiment was performed twice, data from a representative experiment is shown. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. mAbs 1.6C7 and 28D9 target a linear epitope located in the stem region of S2 fusion subunit.
a Antibody binding competition analysed by biolayer interferometry. Immobilized MERS-Secto antigen was saturated in binding with a given mAb (step 1) and then exposed to binding by a second mAb (step 2). Additional binding of the second antibody indicates the presence of an unoccupied epitope, whereas lack of binding indicates epitope blocking by mAb1. As a control, the first mAb was also included in the second step to check for self-competition. The competitive binding was tested for the S2-targeting 1.6C7 and 28D9 antibodies and a MERS-S1 antibody control (7.7G6). Source data are provided as a Source Data file. b 1.6C7 and 28D9 recognize a linear epitope. ELISA binding curves of 1.6C7 and 28D9 to untreated MERS-Secto (non-denatured: ‘nd’) versus MERS-Secto that was heat-denatured in the presence of SDS and DTT (denatured: ‘d’). Two antibodies targeting the MERS-S1 domain (7.7G6) and the 8-residue long linear Strep-tag epitope (anti-strep) were used as controls. Source data are provided as a Source Data file. c 1.6C7/28D9 epitope maps to a 15-aa long stem region upstream of HR2 in MERS-S. ELISA-reactivity of 1.6C7 and 28D9 to a peptide library of 30-amino acid long peptides (with 15-a.a. overlap) covering the conserved C-terminal part of the MERS-Secto (residues 869-1,288). Both antibodies reacted with two peptides (blue and orange bars), and with a peptide corresponding to their 15-a.a. long overlap (green bar; MERS-S residues 1,229–1,243). The position of the epitope containing region is indicated in the MERS-S protein schematic with the spike subunits (S1 and S2), S1 domains (A through D), fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2) and transmembrane domain (TM) annotated. Source data are provided as a Source Data file. d The 1.6C7/28D9 epitope maps to a 8-aa long peptide ‘DELDEFFK’ detected by ELISA. ELISA binding curves of 1.6C7 and 28D9 to N- and C-terminally truncated versions of the 15-mer peptide fragment of MERS-S. Data points represent the average from n = 2 technical replicates. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Fine mapping the 1.6C7 and 28D9 antibody binding sites on the spike protein by mutagenesis.
a ELISA-based epitope alanine mutagenesis on the 15-mer spike peptide fragment comprising the linear 1.6C7 and 28D9 epitope, shown by half-maximum effective concentration (EC50) titers (μg/ml). The average from two independent experiments performed is shown. Source data are provided as a Source Data file. b Sequence alignment of 1.6C7/28D9 epitope region of MERS-CoV, HCoV-OC43, SARS-CoV, SARS-CoV-2, MHV and HCoV-HKU1. c Spike protein ectodomain single site mutagenesis to delineate 1.6C7 and 28D9 antibody binding sites. ELISA-based EC50 titers (μg/ml) of 1.6C7/28D9 binding to MERS-Secto mutants containing single amino acid substitutions in the core epitope region are indicated on the left. Anti-MERS-S1 control antibody 7.7G6 was used to control the expression level of all mutants. n.b., no binding. The average from n = 2 biological replicates is shown. Source data are provided as a Source Data file. d Binding of 28D9 and 1.6C7 antibodies to cell surface expressing (GFP-tagged) MERS-S mutants containing single amino acid substitutions in the core epitope region detected by flow cytometry. Antibody binding was detected using AlexaFluor 594 conjugated secondary antibody. The relative surface binding was determined by calculating the percentage of GFP+/Alexa Fluor 594+ cells over GFP+ cells. Anti-MERS S1 antibody 7.7G6 was used to control the cell surface expression levels of all single-site mutants. The asterisk indicates reduced cell surface expression of the L1235A mutant. n.b., no binding. The average from n = 2 biological replicates is shown. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. 1.6C7 and 28D9 bind the membrane-proximal stem helix of the coronavirus spike protein.
a Sequence alignment of spike protein region of alpha- and betacoronaviruses encompassing the 1.6C7/28D9 epitope region using EMBL-EBI Clustal Omega programme (https://www.ebi.ac.uk/Tools/msa/clustalo/). The 28D9 and 1.6C7 core epitope region is outlined by a rectangle box and residues critical for antibody binding are annotated by asterisks. A conserved glycosylation sequon (NxS/T) found in betacoronavirus spike proteins—one amino acid downstream of the core epitope—is underlined and annotated (ψ). The stem helix, heptad repeat region 2 (HR2) and the start of the transmembrane domain (TM) are indicated. Secondary structural elements of the SARS-CoV-2 prefusion spike (PDB: 6XR8) and postfusion S2 (PDB: 6XRA) structures are visualized using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/). b Structures of the SARS-CoV-2 spike (PBD: 6XR8) and S2 (PDB: 6XRA) in pre- and postfusion conformation, respectively. Structures are indicated as a grey cartoon with transparent surface presentation, and the segment corresponding to the stem helix epitope coloured in orange. Insets: zoom-in sections of the epitope region in both structures in two different orientations with the conserved N-glycan highlighted in red.
Fig. 6
Fig. 6. Antibodies towards the stem helix epitope are elicited during natural infection.
a Schematic representation of the MERS-CoV spike protein. The spike subunits (S1 and S2), S1 domains (A through D), fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2) and transmembrane domain (TM) are annotated. b Spike protein peptide microarray analysis using MERS-positive human and dromedary camel sera. 905 overlapping peptides covering the entire MERS-CoV S ectodomain (residues 1–1296) were synthesized with an offset of one or two residues. The binding of five convalescent MERS-positive human (H1–H5) and four dromedary camel (D1–D4) sera to the peptide library, as well as a MERS-negative serum from human (H-CTRL) or camel (D-CTRL) was assessed in a PEPSCAN-based ELISA (Lelystad, The Netherlands). A cumulative heatmap of signal intensities for individual peptides are shown. Signal intensities increase from light reddish to red, whereas white corresponds to background signal. Source data are provided as a Source Data file. c Reactivity of the human and dromedary sera to peptides covering the 1.6C7/28D9 epitope region (epitope core sequence highlighted in red). Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Antibody-mediated protection of mice against lethal MERS-CoV/SARS-CoV challenge.
a The in vivo prophylactic and therapeutic activity of the 1.6C7 mAb against lethal dose MERS-CoV challenge was tested in the K18 transgenic mouse model expressing human DPP4. A potent neutralizing MERS-S1 antibody (7.7G6) or an irrelevant IgG1 control antibody was taken along. Eight 20–30-week-old mice were injected with 50 μg of antibody (equivalent to 1.8 mg mAb/kg body weight) by intraperitoneal injection 24 h before (pre-) or 24 h after (post-) intranasal infection with a lethal dose of MERS-CoV. Survival rates (left) and weight loss (right, expressed as a percentage of the initial weight) were monitored daily until 10 days post-inoculation. Data points represent mean body weight relative to the initial weight ± SD, n = 8 mice. Source data are provided as a Source Data file. b Prophylactic efficacy of 28D9 against MERS-CoV infection. Five 20-week-old K18 mice were mock-infected or injected with 50 or 200 μg of 28D9 (equivalent to 1.8/7.2 mg mAb/kg body weight) or isotype control antibody by intraperitoneal injection 24 h before intranasal infection with a lethal dose of MERS-CoV. Data points represent mean body weight relative to the initial weight ± SD, n = 5 mice. Source data are provided as a Source Data file. c Prophylactic efficacy of 28D9 against SARS-CoV infection. Five 16-week-old Balb-C mice were mock-infected or administered with the 50 or 200 μg of 28D9 or isotype control antibody via intraperitoneal injection 24 h before intranasal infection with a lethal dose of mouse-adapted SARS-CoV. Survival rates (left) and weight loss (right, expressed as a percentage of the initial weight) were monitored daily until 13 days post-inoculation. Data points represent mean body weight relative to the initial weight ± SD, n = 5 mice. Source data are provided as a Source Data file.
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References

    1. Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–273. doi: 10.1038/s41586-020-2012-7. - DOI - PMC - PubMed
    1. Gorbalenya A, et al. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 2020;2020:03–04. - PMC - PubMed
    1. Peiris J, Guan Y, Yuen K. Severe acute respiratory syndrome. Nat. Med. 2004;10:S88–S97. doi: 10.1038/nm1143. - DOI - PMC - PubMed
    1. World Health Organization. MERS situation update, January 2020. http://www.emro.who.int/health-topics/mers-cov/mersoutbreaks.html (2020).
    1. Gaunt ER, Hardie A, Claas EC, Simmonds P, Templeton KE. Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method. J. Clin. Microbiol. 2010;48:2940–2947. doi: 10.1128/JCM.00636-10. - DOI - PMC - PubMed

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