Journal: iScience

Article Title: A micro-electroporation/electrophoresis-based vaccine screening system reveals the impact of vaccination orders on cross-protective immunity

doi: 10.1016/j.isci.2023.108086

Figure Lengend Snippet: Cross-neutralizing antibodies induced by sequential vaccination of multiple SARS-CoV-2 DNA vaccines Mice were i.d. immunized with 10 μg SARS-CoV-2 S6P DNA vaccine coding the S protein of WT virus (pWT) or Omicron BA.1 variant (pBA.1) with or without MEES on day 0 and boosted on day 14. Sera were collected on day 14 (prime) or day 21 (boost). (A) IgG titers against WT S protein were measured by ELISA. n = 8–9. (B) IgG titers against BA.1 S protein were measured by ELISA. n = 8–9. (C–I) Mice were i.d. immunized with 10 μg SARS-CoV-2 S6P DNA vaccine encoding S protein of WT strain, Delta variant, or BA.1 variant on days 0, 21, and 42 with or without MEES. Sera were collected on day 56. Neutralization assays were performed to measure NAb titers against pseudoviruses whose S protein was from WT (C), Delta (D), BA.1 (F) or BA.5 (H) viral strains. n = 6–9 mice. (E) p values of the pairwise comparison of (D). (G) p values of the pairwise comparison of (F). (I) p values of the pairwise comparison of (H). Vaccines, immunized with DNA vaccine alone. +MEES, immunized with DNA vaccine followed by MEES treatment. The results were presented as geometric mean ±95% Cl (A-D, F, H). Statistical analysis, two-tailed Mann-Whitney U -test. ∗p < 0.05, ∗∗∗∗p < 0.0001, ns, no significance.

Article Snippet: The pCMV plasmids expressing the optimized spike protein from Delta (VG40804-UT) or Omicron BA.5 (VG40930-UT) variants were purchased from Sino Biological (Beijing, China).

Techniques: Vaccines, Virus, Variant Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A micro-electroporation/electrophoresis-based vaccine screening system reveals the impact of vaccination orders on cross-protective immunity

doi: 10.1016/j.isci.2023.108086

Figure Lengend Snippet: Cellular immune responses induced by sequential vaccinations and analysis of epitopes (A) The geometric means (GM) of PVNT 50 against multiple viral stains after sequential vaccinations were summarized as a heatmap. (B) The proportions of mice with PVNT 50 above the median calculated from all mice were summarized as a heatmap. (C) Mutations of SARS-CoV-2 variants were labeled on the structure of WT S protein (PDB ID: 6VYB ) using Pymol 2.6. (D) Mutation sites and CD8 + T cell epitopes in the S1 subunit of Delta, BA.1, and BA.5 were summarized and compared. (E) Mutation sites and CD8 + T cell epitopes in the S2 subunit were summarized and compared. (F) Mice were sequentially immunized with 10 μg of WT-BA.1-Delta or BA.1-WT-Delta DNA vaccines on day 0, day 21, and day 42. (G and H) Splenocytes were isolated on day 56. CD8 + T cells producing IFNγ after the S538 epitope peptide (G) or peptide pool (H) stimulation were measured by flow cytometry. The Peptide pool was a mixture of S263, S471, S510, S538, and S820 epitopes. n = 3–4. The results were shown as mean ± SEM. Statistical analysis, two-tailed Student’s t test. ∗p < 0.05 and ∗∗p < 0.01.

Article Snippet: The pCMV plasmids expressing the optimized spike protein from Delta (VG40804-UT) or Omicron BA.5 (VG40930-UT) variants were purchased from Sino Biological (Beijing, China).

Techniques: Labeling, Mutagenesis, Vaccines, Isolation, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: A micro-electroporation/electrophoresis-based vaccine screening system reveals the impact of vaccination orders on cross-protective immunity

doi: 10.1016/j.isci.2023.108086

Figure Lengend Snippet:

Article Snippet: The pCMV plasmids expressing the optimized spike protein from Delta (VG40804-UT) or Omicron BA.5 (VG40930-UT) variants were purchased from Sino Biological (Beijing, China).

Techniques: Virus, Recombinant, Cream, Enzyme-linked Immunosorbent Assay, Luciferase, Lysis, Staining, Membrane, Western Blot, Software

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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 using LALIGN/PLALIGN local alignment. The default values of the gap penalties of open = –12 and gap = –2 were used for S2 alignments but reduced to open = –10 and gap = –1 to allow for variable domain lengths in S1. GenBank protein sequence identification numbers (protein_id) used in these alignments were: NP_073551.1 for 229E, AFD64754.1 for NL63, BBA20979.1 for OC43, BBA20986.1 for HKU1, YP_009047204.1 for MERS, AYV99817.1 for SARS-1–1, and YP_009724390.1 for SARS-2.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Sequencing

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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 panning, these data show 3A3 in black, a close relative of 3A3 (two amino acid changes) in green, two other unique antibodies in red and purple, and foldon binders with closely related CDRH3 sequences in blue.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 10 2 s exchange. Residues lacking coverage are indicated in black. The figure was prepared using DynamX per residue output without statistics and PyMOL. ( b ) Cryo-EM 2D class averages of the SARS-2 S2 subunit bound to the 3A3 Fab. ( c ) Cryo-EM 3D reconstruction of the S2–3A3 Fab complex showing each S2 protomer binding one 3A3 Fab molecule. The pink 3D volume was generated from a focused refinement of one S2 protomer and 3A3 Fab. ( d ) A structure of the S2 subunit and a predicted structure of the 3A3 Fab shown as ribbons and fit into the cryo-EM map. The 3A3 Fab light (LC, orange) and heavy (HC, red) chains sandwich the apex of the spike S2 hinge (cyan). Figure 1—source data 1. Summary and complete HDX data for spike peptides.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Cryo-EM Sample Prep, Binding Assay, Generated

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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 are indicated on the sequence, including glycosylation sites. Glycosylation was not included in the peptide search, so no peptides are recovered surrounding these sites.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Sequencing

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 10 3 s of exchange. The figure was prepared using DynamX per residue output and PyMOL. Residues lacking coverage are indicated in black. Structural features are labeled, including the central trimer interface that shows relatively low deuterium incorporation. ( b ) Spectra from a spike peptide spanning residues 991–1001, within the 3A3 epitope. The spectra show the width of isotopic distributions in the deuterated samples suggestive of conformational heterogeneity.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Labeling

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: Volcano plots showing changes in deuterium uptake in SARS-2 HexaPro spike peptides upon addition of ( a ) 3A3 IgG or ( b ) Fab after 10 2 s exchange. Significance cutoffs are an average change in deuterium uptake greater than 0.2 Da and a p-value less than 10 –2 in a Welch’s t -test (hatched box). Black dots indicate peptides with a significant decrease in deuterium uptake and their residue boundaries are labeled. ( c ) Uptake plots for these peptides are shown. Traces are SARS-2 HexaPro spike alone (black), with 3A3 IgG (blue), and with 3A3 Fab (orange). Error bars are ± 2σ from three or four technical replicates and sometimes too small to be visible. The y-axis is 70% of max deuterium uptake assuming the N-terminal residue undergoes complete back-exchange. Data have not been corrected for back-exchange. ( d ) The difference in deuterium fractional uptake between SARS-2 HexaPro spike alone and with 3A3 Fab at 10 2 s exchange is projected onto monomeric SARS-2 2P spike (PDB: 6VSB chain B). Image was prepared using DynamX per residue output without statistics and PyMOL. Residues lacking coverage are indicated in black. Structural features are labeled, including the 2P mutations at residues 986 and 987 (shown as sticks).

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Labeling

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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 10 2 s exchange. Significance cutoffs are an average change in deuterium uptake greater than 0.2 Da and a p-value less than 10 –2 in a Welch’s t-test (hatched box). Black dots indicate peptides with a significant decrease in deuterium uptake and their residue boundaries are labeled. ( c ) Uptake plots for these peptides are shown. Traces are 3A3 IgG alone (black) and with SARS-2 HexaPro spike (gray). Error bars are ± 2σ from three or four technical replicates and sometimes too small to be visible. The y-axis is 70% of max deuterium uptake assuming the N-terminal residue undergoes complete back-exchange. Data have not been corrected for back-exchange.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Labeling

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 colored dark blue in structures of wild-type SARS-2 spike in the (i) three RBDs down or closed conformation (PDB: 6XR8) and in structures of stabilized spike with (ii) one RBD up (PDB: 6VSB), (iii) two RBDs up (PDB: 7A93), or (iv) three RBDs up while bound to ACE2 (red) in top-view and (v) sideview (PDB: 7A98). Residues lacking coverage in the HDX experiment are indicated in gray. ( b ) Antibody 3A3 (bottom) or control mAb 2–4 (top) were coupled to anti-Fab BLI sensors and allowed to capture HexaPro or nothing (buffer, green line), then dipped into buffer (baseline), and finally dipped into ACE2-Fc (ACE2, red) or nothing (buffer, blue). ( c ) BLI binding of immobilized control mAb 2–4 (top) or antibody 3A3 (bottom) to 100 nM HexaPro (solid) or HexaPro locked into the ‘closed’ conformation (dashed). Vertical dashed lines indicate start of dissociation phase. ( d ) The network of hydrogen bonds formed by residues E1031 and R1039 across protomers deep in the S2 core is shown on intact HexaPro spike and in detail in a top view (PDB: 6XKL). ( e ) Antibody 3A3 was coupled to anti-Fc BLI sensors and allowed to bind HexaPro or E1031R HexaPro (E1031R) spike protein. All BLI data are representative of biological duplicates. Each experiment was repeated in technical duplicate except e, which was tested once at each concentration to allow all data to be collected simultaneously for direct comparison. ( f ) Model of the kinetic changes required for antibody binding to the hinge epitope, including conversion of the RBDs into the up position and some degree of opening of the S2 domain in addition to typical antibody association and dissociation kinetics (generated using PDB 6XV8 and 7A98). Figure 2—source data 1. BLI sensorgram data.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Binding Assay, Concentration Assay, Generated

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 technical replicates in 2–6 independent ELISA assays. Significance relative to unaltered HexaPro was determined by ANOVA with post-hoc Tukey–Kramer test with α = 0.01; data meeting this criterion indicated by *. ( b ) Location of the residue changes altering binding to 3A3 shown in the HexaPro spike (6XKL) monomer and in ( c ) intact spike (side view) and the S2 domain (top-down view). All epitope residues (980–1006) are shown in space-fill, with residues colored according to their effect on 3A3 binding: improved binding (orange), reduced binding (teal), no effect (black), and those not altered (gray). The 2P stabilizing mutations within the hinge epitope are displayed in purple. ( d ) The infectivity of lentivirus pseudotyped with unmodified D614G SARS-2 spike or variants with D985L (green), E988A (light blue), and E988Q (dark blue) substitutions was compared by luciferase activity. Data shown are the mean luminescence with standard deviation of three technical replicates. Figure 3—source data 1. ELISA binding data and relative luminescence data for pseudovirus infection assays.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Binding Assay, Variant Assay, Enzyme-linked Immunosorbent Assay, Infection, Luciferase, Activity Assay, Standard Deviation

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 of fresh and stressed SARS-2 and SARS-2 HexaPro spikes shows a substantial aggregation of the stressed SARS-2 spike. For each spike, 8 μg of protein was analyzed by SDS-PAGE under nonreducing conditions. Reduced band intensity of stressed SARS-2 spike (bottom arrow) and appearance of a band just under the loading wells (top arrow) indicates aggregation products not apparent in the other samples. Densitometry analysis (ImageJ) of these bands indicates that the stressed SARS-2 spike’s main peak is diminished by ~20% relative to the fresh SARS-2 spike intensity. Ladder molecular weights in kDa are indicated to the left of the gel image. Binding of 3A3 Fab to HexaPro S2 measured ( c ) ELISA capture of fresh (red circles) or stressed (pink diamonds) SARS-2 spike on 3A3 coated plates. ( d ) Antibodies coated on ELISA plates captured fresh (dark blue or red) or stressed (pink or light blue) SARS-2 HP (blue) or SARS-2 (red) spike proteins. Duplicate dilutions of spike over ~5 log in concentration were used to calculate EC 50 values. For dilution series in which no binding was observed, EC 50 was assumed to be >1000 nM. Open symbols are replicate data and filled rectangles are average data.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Western Blot, Molecular Weight, Labeling, SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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 stabilizing disulfide). For detection, streptactin-HRP was allowed to bind the twin strep tag sequence at the C-terminus of any captured spike proteins.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Binding Assay, Strep-tag, Sequencing

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Binding Assay, Purification

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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 of SARS-2 4P-DS to immobilized ( c ) IgG RAY53, ( d ) Fab RAY53 or ( e ) control IgG22. For BLI, K d values were obtained using a 1:1 global fit model using the Octet instrument software. The SPR data were double-reference subtracted and fit to a 1:1 binding model using BIAevaluation software. ( f ) Binding of 3A3 and RAY53 IgG to additional spike variants was compared by incubating full-length IgGs immobilized on anti-Fc BLI sensors with serially diluted spike variants for an association step followed by a dissociation step in buffer only. Since the off-rates determined for this 3:1 spike protomer:antibody binding arm format using kinetic-based K d were extremely slow due to rebinding (~10 –12 s –1 ) and differential epitope accessibility is expected to influence the observed on-rate , on-rates ( k on ) were compared. Vertical lines indicate the start of the dissociation phase; data shown in solid lines with fits (1:1 association then dissociation fit using GraphPad Prism software) shown in red lines.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Binding Assay, Software

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: 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, Omicron BA.1, and other variants of concern (VOC; Beta, Gamma, Delta, Epsilon, Omicron BA.2 through BA.5). In ( a ), identical residues are indicated by a dot and similar residues are highlighted in gray. Residues conserved across all listed β-coronaviruses are in bold. Residues that lost binding to 3A3 when altered as shown in are in teal highlight and those whose disruption improved binding are orange. The location of the two proline mutations introduced to 2P variants are shown below the alignment. In ( b ), the structure of each epitope is displayed as follows: SARS-2 (6VSB) – red, SARS-1 (6CRV, RMSD = 0.8 Å) – magenta, MERS (5X5C, RMSD = 3.1 Å) – blue, HKU1 (5I08, RMSD = 0.5 Å) – teal, OC43 (6OHW, RMSD = 0.6 Å) – green. ( c ) Binding of full-length antibody 3A3 (black circles) and RAY53 (blue diamonds) to ancestral SARS-2, SARS-2 HexaPro (SARS-2 HP), SARS-2 4P, SARS-2 4P-DS, SARS-2 HexaPro Delta (SARS-2 HP Delta), SARS-2 HexaPro Omicron BA.1 (SARS-2 HP Omicron BA.1), MERS, SARS-1, HKU1, or milk (no coat) proteins by ELISA. Data are representative of duplicate biological replicates, each with duplicate technical replicates. The data midpoint is indicated with a bar. ( d ) Plasmids encoding full-length unstabilized spike proteins from SARS-2, SARS-2 Omicron BA.1, MERS, or SARS-1 were transiently transfected to Expi293 cells. The spike (blue or magenta histograms) or mock (grey histograms) transfected cells were stained with 100 nM RAY53 (top panels) or 10 nM control antibody S309 (bottom panels), followed by goat-anti-human Fc-PE secondary antibody, and flow cytometry scanning of 10,000 cells. The data shown are representative of triplicate experiments, with each condition repeated in technical duplicate. ( e ) Expi293 cells were transiently transfected with plasmids encoding SARS-2 spike and EGFP or EGFP only, then incubated with 3A3 (black circles) or RAY53 (blue diamonds) antibody (~1–300 nM) and anti-human Fc-PE before flow cytometric determination of the geometric mean fluorescence intensity (GMFI) in the PE channel for all green fluorescent cells. The GMFI of cells transfected with EGFP only was subtracted from the GMFI of cells expressing spike at each concentration, and the data fit to a three-parameter logistic curve to determine the effective K d (K d,eff ) for antibody binding. The data shown are representative of triplicate experiments; ND, not detected. Figure 4—source data 1. ELISA data and flow cytometry mean fluorescence intensity data.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Staining, Flow Cytometry, Incubation, Fluorescence, Expressing, Concentration Assay

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 imaged after 24 hr of incubation for EGFP (green) or Cell Trace Far Red (red) and the level of colocalization (yellow) was evaluated. ( b ) CHO cells not expressing SARS-2 spike and ( c ) HEK 293 cells not expressing ACE2 exhibited minimal fusion. ( d ) When the cultures were preincubated with an irrelevant isotype control antibody, extensive fusion and syncytia formation equivalent to no antibody was apparent. Incubation at ( e ) 6.7, ( f ) 67, and ( g ) 670 nM 3A3 reduced fusion in a dose-dependent manner with significance reached at 67 nM. Scale bar, 100 μm. ( h ) The percentage of HEK-ACE2 pixels (red) colocalizing with spike expressing CHO pixels (green) was analyzed with the JACoP plugin for ImageJ. ( i ) The same images were analyzed for the average cell size of fused HEK-ACE2 with ImageJ as a second statistical method to test the cell fusion level. Shown are the mean and standard deviation of at least 160 cells per condition from 8 to 9 independent images. The statistical analysis of average cell sizes under different conditions were performed with ANOVA followed by Tukey’s HSD test. Results shown are representative of four independent experiments; ****p<0.0001.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Stable Transfection, Expressing, Staining, Incubation, Standard Deviation

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet: ( 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 (MERS pseudovirus), with viral entry detected by luciferase luminescence. The entry efficiency of pseudoviruses without any treatment was considered 100%. ( b ) Neutralization of authentic SARS-2 wild-type virus was assessed by incubating viral particles with antibody before adding to Vero HF cells. Viral infection was assessed by ELISPOT 24 hr after infection by immunostaining with the anti-SARS-2 nucleocapsid antibody 1C7C7. ( c ) ADCP was performed by co-incubating undifferentiated THP-1 cells, antibodies and pHrodo-Green/APC-polystyrene beads coated with HexaPro or 4P-DS. The phagocytosis score was calculated as the percent of positive APC/FITC cells multiplied by the GMFI for APC. Data were collected from two separate experiments with the average and standard deviation shown. ( d ) ADCC was assessed by incubating NK-92 V/V cells, HEK-293T cells transfected to express either wild-type SARS-2 spike (spike) or nothing (mock) and antibody. For each panel, data shown are representative of three biological replicates. Duplicate technical replicates with the midpoint of each condition are shown. Figure 5—source data 1. Data reporting antibody effect on infection with pseudovirus, authentic virus, phagocytosis score, and cellular lysis.

Article Snippet: SARS-2 Omicron BA.1 (B.1.1.529) Spike Gene ORF cDNA was purchased from SinoBiological Inc SARS-2 Omicron BA.1, SARS-1 and MERS spike sequences were cloned into the pWT-SARS-2-spike plasmid for pseudovirus production.

Techniques: Neutralization, Stable Transfection, Expressing, Luciferase, Infection, Enzyme-linked Immunospot, Immunostaining, Standard Deviation, Transfection, Lysis