spike protein peptide microarray  (Pepscan Inc)

Journal: Nature Communications

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

doi: 10.1038/s41467-021-21968-w

Figure Lengend Snippet: 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 IC 50 and IC 90 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 IC 50 and IC 90 values were shown. Source data are provided as a Source Data file. c ELISA-based receptor-binding inhibition assay. MERS-S ecto pre-incubated with serially diluted mAbs was added to ELISA plates coated with soluble human DPP4. The binding of MERS-S ecto to DPP4 was detected using an HRP-conjugated antibody recognizing the C-terminal Strep-tag on MERS-S ecto . 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.

Article Snippet: Spike protein peptide microarray analysis to map linear epitopes of antibodies in convalescent sera was performed by PEPSCAN (Lelystad, The Netherlands).

Techniques: Neutralization, Infection, Luciferase, Incubation, Transduction, Strep-tag, Control, Plaque Reduction Neutralization Test, Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition, Transfection, Plasmid Preparation, Expressing, Membrane, Fluorescence, Microscopy, Staining

Journal: Nature Communications

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

doi: 10.1038/s41467-021-21968-w

Figure Lengend Snippet: a Antibody binding competition analysed by biolayer interferometry. Immobilized MERS-S ecto 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-S ecto (non-denatured: ‘nd’) versus MERS-S ecto 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-S ecto (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.

Article Snippet: Spike protein peptide microarray analysis to map linear epitopes of antibodies in convalescent sera was performed by PEPSCAN (Lelystad, The Netherlands).

Techniques: Binding Assay, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay, Residue, Strep-tag

Journal: Nature Communications

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

doi: 10.1038/s41467-021-21968-w

Figure Lengend Snippet: 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.

Article Snippet: Spike protein peptide microarray analysis to map linear epitopes of antibodies in convalescent sera was performed by PEPSCAN (Lelystad, The Netherlands).

Techniques: Peptide Microarray, Synthesized, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing