Journal: eLife

Article Title: A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants

doi: 10.7554/eLife.78633

Figure Lengend Snippet: ( A ) Schematic illustration of the designed mos-tri-RBD. In mos-tri-RBD, three heterologous RBDs were connected end to end into a single chain and co-assembled into a trimeric structure. For the three RBDs, one was derived from the Omicron (BA.1) variant (green color), and the other two were artificially designed harboring the key immune-evasion-related mutations that emerged in SARS-CoV-2 variants, in which one contained the mutations of K417N, L452R, T478K, F490S, and N501Y (cyan color), and the other one contained K417T, S477N, and E484K (blue color). These mutations are highlighted in the red ball-and-stick model in the figure. Each RBD subunit in mos-tri-RBD was composed of the residues 319–537 from the spike protein. The dotted curves in the figure represent the direct connection between the C-terminus of the former RBD and the N-terminus of the latter RBD. The schematic structure of mos-tri-RBD was drawn by Chimera software based on the PDB file with accession number 6zgi. ( B ) SDS-PAGE analysis of the recombinant mos-tri-RBD. ( C ) Concentration-dependent binding ability of mos-tri-RBD with an RBD-specific monoclonal neutralizing antibody MM117 tested using ELISA. ( D ) Binding avidity of mos-tri-RBD with the receptor hACE2 measured using SPR assay. In this figure, different curves represent different concentrations of analyte (top to bottom: 263.70 ng/ml, 131.85 ng/ml, 65.93 ng/ml, 32.96 ng/ml, and 16.48 ng/ml). Both the original (color curves) and fitted (black curves) data are displayed. Figure 1—source data 1. The raw files of SDS-PAGE results. Figure 1—source data 2. Concentration-dependent binding ability of mos-tri-RBD with the antibody MM117.

Article Snippet: Peptide, recombinant protein , Recombinant hACE2 protein (mammalian cell-expressed) , Sino Biological Inc, China , Cat#10108-H08H , .

Techniques: Derivative Assay, Variant Assay, Software, SDS Page, Recombinant, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: eLife

Article Title: A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants

doi: 10.7554/eLife.78633

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant hACE2 protein (mammalian cell-expressed) , Sino Biological Inc, China , Cat#10108-H08H , .

Techniques: Virus, Control, Isolation, Enzyme-linked Immunosorbent Assay, Recombinant, Plasmid Preparation, Expressing, Purification, Adjuvant, Produced, Software

Journal: bioRxiv

Article Title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

doi: 10.1101/2022.03.22.485248

Figure Lengend Snippet: A: HMDM and THP-1 cells were analysed by qPCR for ACE2 mRNA expression, with each data point showing an independent donor or experiment (n=3). B: HMDM were stimulated with IFNβ (10 ng/ml) for 6 h and protein extracts were analysed by immunoblot, alongside extracts from THP-1 cells (WT, THP-1-ACE2, THP-1-mSc). C: BAL macrophages from 3 donors were adhered overnight and lysed. Expression of ACE2 in BAL macrophages was analysed by immunoblot, relative to a loading control (Calnexin). Lysate from A549-cells overexpressing ACE2 were used as a positive control. D-E: Cells were infected with SARS-CoV-2 at MOI 0.5 or MOI 5. After 1h the virus inoculum was removed, cells were washed and cells or supernatants harvested at the indicated times. Cellular viral mRNA was analysed by qPCR (D-E), and infectious virions released into cell supernatants were measured by plaque assay (F-G). Data show the mean + SEM of 3-5 independent experiments, with data points representing individual experiments. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (two-way ANOVA, Tukey’s multiple comparison test).

Article Snippet: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin ( ) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids ( ).

Techniques: Expressing, Western Blot, Control, Positive Control, Infection, Virus, Plaque Assay, Comparison

Journal: bioRxiv

Article Title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

doi: 10.1101/2022.03.22.485248

Figure Lengend Snippet: A: THP-1 cells were infected with SARS-CoV-2 (MOI 5), which was washed away after 1 h, and incubated for a further 72h, after which cell death was analysed by ATPlite assay. Data are presented as cell viability relative to mock, and are mean + SEM of 3 independent experiments. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (two-way ANOVA, Tukey’s multiple comparison test). B: Schematic of TBK1 (BX-795) inhibition. C-F: THP-1-ACE2 or Calu3 cells were stimulated with SARS-CoV-2 at MOI 5. After 1 h, the viral inoculum was removed and BX-795 added. Supernatants were harvested at 72 h and CXCL10 was analysed by ELISA (C) and viral titres were analysed by plaque assay (D,E). Data show mean + SEM of at least 3 independent experiments, with each individual data point representing a different experiment. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (C: one-way ANOVA, Tukey’s multiple comparison test; D,E: ratio-paired t-test).

Article Snippet: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin ( ) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids ( ).

Techniques: Infection, Incubation, Comparison, Inhibition, Enzyme-linked Immunosorbent Assay, Plaque Assay

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 6. Surface expression and soluble receptor binding of wild-type and mutant SARS-CoV S 19 proteins. (A) Cell surface expression of the wild-type or mutant SARS-CoV S 19 protein was measured by flow cytometry. (B and C) Binding of the soluble hACE2-Fc receptor protein by cells expressing wild-type or mutant SARS-CoV S 19 detached with trypsin-EDTA (B) or EDTA alone (C). (D to F) Representative flow cytometry overlays shown to the right of corresponding bar graphs. Mock-transfected cells are shown in dark gray, and SARS-CoV S 19 is shown by a black line; representative SARS-CoV S 19 mutant proteins are shown by color (SARS-CoV S 19 Y1188A, green; S 19 W1194A, red; S 19 Y1197A, orange; S 19 Y1188A/Y1191A, cyan; and S 19 W1194/W1199A, yellow, respectively). Error bars represent standard errors of percent wild-type levels between at least three independent experiments.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Binding Assay, Mutagenesis, Cytometry, Transfection

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 8. Surface expression of hACE2. HEK-293 cells stably ex- pressing high levels of hACE2 were selected by flow cytometry.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Stable Transfection, Cytometry

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 9. Alanine substitutions in the JMD of the SARS-CoV S 19 protein reduce receptor-dependent entry of pseudotyped viruses. Rel- ative transduction activity of pseudotyped viruses with wild-type or mutant SARS-CoV S 19 proteins is shown. Pseudotypes in filtered medium were used to transduce HEK-293/hACE2 cells. Beta-galacto- sidase expression was analyzed 2 days posttransduction. Transduction units per ml were calculated {TU/ml [(% cells expressing reporter/ 100) total number of cells per well]/ml inoculum}. Data are repre- sented as percent transduction activity of wild-type SARS-CoV S 19 proteins and are representative of three to six experiments. Error bars represent standard errors. Student’s t test was used to evaluate the significance of differences between the transducing activities of pseudotyped viruses with mutant or wild-type proteins. (*, P 0.01 to 0.05; **, P 0.01 to 0.001; ***, P 0.0001).

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Transduction, Activity Assay, Mutagenesis, Expressing

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 10. Trypsin enhances receptor-dependent cell-cell fusion induced by wild-type SARS-CoV S 19 protein. The time course of syncytium formation was determined after 30 min, 1 h, 2 h, or 3 h. HEK-293 cells transfected with cDNA encoding the wild-type SARS-CoV S 19 glycoprotein (green) were stained with Celltracker Green (Molecular Probes) and were detached from the monolayer with EDTA alone or trypsin-EDTA and added to a 50% confluent monolayer of HEK-293/hACE2 cells (blue) stained with Hoechst 33342 (Sigma) at a ratio of one S-expressing cell to three hACE2-expressing cells. Cells were fixed with paraformaldehyde 2 h postmixing. Pictures were taken with a 20 objective. Pictures are representative of two independent experiments..

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Transfection, Staining, Expressing

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 11. JMD mutations reduce receptor-dependent cell-cell fu- sion induced by SARS S 19 proteins. (A) Fusion of HEK-293 cells expressing wild-type or mutant SARS-CoV S 19 proteins or empty vector with HEK-293/hACE2 cells. Fusion was scored as the percent- age of nuclei present in syncytia of any size at 2 h after mixing cells expressing spike and receptor. Background levels of fusion up to 15% were seen in HEK-293 cells. Data are expressed as a percentage of wild-type SARS-CoV S 19 fusion with background levels of fusion subtracted. Data are representative of three independent experiments. P values are 0.02 for all mutant proteins relative to results for wild- type SARS-CoV S 19. (B and C) Analysis of syncytium size induced by tyrosine-to-alanine (B) or tryptophan-to-alanine (C) mutants of the SARS-CoV S 19 protein. Each individual syncytium was scored for size (number of nuclei). Results are plotted as the cumulative percent- age of cells containing a given number of nuclei or less.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Mutagenesis, Plasmid Preparation