Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: Neutralization of SARS-CoV-2 infection and RBD binding to ACE2 (A) Indicated hmAbs were incubated with live SARS-CoV-2 (100 PFU/well) 1 h before (pre-treatment) or 1 h after (post-treatment) addition to Vero E6 cells. hmAbs were tested in quadruplicate cultures and NT 50 and upper 95% confidence interval (CI) indicated. (B) Representative titration curve of 1212C2 hmAb presented, mean and standard error presented. (C) Binding of indicated hmAb to SARS-CoV-2 or mock-infected Vero E6 cells measured by immunofluorescence; scale bar, 100 μm. (D) Indicated hmAb was incubated as single replicate with recombinant biotinylated RBD protein before incubation with HEK293-ACE2 cells measured by flow cytometry. Plot gated on 7-aminoactinomycin (7AAD)-ACE2 + cells.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Neutralization, Infection, Binding Assay, Incubation, Titration, Immunofluorescence, Recombinant, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: SPR epitope mapping (A) Representative sensorgram from the SPR competition assays used to subset hmAbs into distinct RBD binding epitopes. For each assay, a series of hmAbs were sequentially injected over immobilized SARS-CoV-2 RBD. In this example, 1212C2 was injected first, followed by a second injection of 1212C2, 2 injections of 1206D1, and the last injection was CR3022. (B) Summary of all epitope mapping data, in which each block (first experiment from A in the red box) with a bold hmAb at the top represents a different experiment (10 experiments total). The bold hmAb is the “first” hmAb injected. The percentage of binding (100 = 100% binding and 0 = 0% binding) of subsequent hmAbs was recorded. mAbs were considered to have a different epitope (denoted by a distinct color) if they exhibited binding levels >30% in the presence of other mAbs. Thus, in the first experiment, CR3022 is defined as a new epitope (cyan), distinct from 1212C2. (C) Schematic diagram of NmAb RBD epitopes defined in the mapping experiment. Five major epitopes (A–E) were identified, where the E epitope overlaps with control mAb CR3022 (cyan, epitope F). Four of the 5 epitopes (A–D) are located within the ACE2 binding site (purple), and all of the NmAbs are blocked by the 1212C2 epitope A (yellow). NmAbs with epitopes similar to B (orange) and C (green) are defined as B’ (light orange) and C’ (light green), respectively. The 1212C2 epitope A (yellow) blocks the binding of all NmAbs, with the exception of 1215B11, which occupies epitope E. Epitopes B and C are also blocked by epitope A NmAbs, but exhibit limited competition with each other.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Binding Assay, Injection, Blocking Assay, Control

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet:

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Synthesized, Expressing, Virus, Recombinant, Plasmid Preparation, Binding Assay, Saline, cDNA Synthesis, Transfection, Gel Extraction, Lysis, Luciferase, Software

Journal: Biomedicines

Article Title: Pan-Cancer Analysis Identifies SNORA12 as a Prognostic Biomarker and Demonstrates Its Role in Upregulating TIGIT in Osteosarcoma

doi: 10.3390/biomedicines14030723

Figure Lengend Snippet: SNORA12 drives upregulation of the immune checkpoint TIGIT in osteosarcoma. ( A – D ) Representative Western blot analyses of TIGIT protein expression in the indicated cell lines under different treatments. SW1353 ( A ) and U2OS ( B ) osteosarcoma cells, as well as primary NK cells ( C ), were subjected to SNORA12 overexpression (OE-SNORA12), with respective controls (Control and OE-NC). NK92 cells ( D ), which endogenously express high levels of SNORA12, were subjected to SNORA12 knockdown (sh-SNORA12), with controls (Control and sh-NC). β-actin served as the loading control. ( E – H ) Quantitative analysis of TIGIT protein levels normalized to β-actin from ( A – D ), respectively. Data are presented as mean ± SD. Statistical significance was determined by Student’s t -test (* p < 0.05, ** p < 0.01, ns = not significant).

Article Snippet: After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with primary antibodies against TIGIT (1:1000, Proteintech, Cat# 66699-1-Ig, Rosemont, IL, USA) and β-actin (1:5000, serving as loading control).

Techniques: Western Blot, Expressing, Over Expression, Control, Knockdown

Journal: PLOS ONE

Article Title: Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

doi: 10.1371/journal.pone.0312402

Figure Lengend Snippet: (A) Schematic representation of ACE2-hFcLALA fusion protein. Subunits of the homodimeric protein are formed by the extracellular domain of the human ACE2 fused to the Fc region of a human IgG1 with LALA mutations. (B) SDS-PAGE (7.5%) of purified ACE2-hFcLALA in reducing conditions. MWM, molecular weight marker (Bio-Rad, 161–0373). (C) Western blots analysis using antibodies specific for hACE2 (left) and human Fc fragment (right).

Article Snippet: The blot was probed with a commercial ACE2 specific antibody (Novus Biological, AC18F) followed by a goat anti-mouse IgG-HRP (Cell Signaling Technologies, #7076).

Techniques: SDS Page, Purification, Molecular Weight, Marker, Western Blot

Journal: PLOS ONE

Article Title: Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

doi: 10.1371/journal.pone.0312402

Figure Lengend Snippet: (A) ELISA plates coated with RBD-mFc fusion protein (5μg/mL) were incubated with ACE2-hFcLALA at different concentrations, followed by human Fc specific PA-conjugated antibody. PDL1-hFc was used as an irrelevant fusion protein. (B) ELISA plate coated with ACE2-hFcLALA (5μg/ml) were incubated with RBD-His and Spike-His at different concentrations, followed by a mouse His specific HRP-conjugated antibody. The x-axis displays the concentration of soluble purified RBD (RBD-His) or RBD domain in the context of the Spike protein. Binding was detected with pNPP (A) and OPD (B) substrates.

Article Snippet: The blot was probed with a commercial ACE2 specific antibody (Novus Biological, AC18F) followed by a goat anti-mouse IgG-HRP (Cell Signaling Technologies, #7076).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Purification, Protein Binding

Journal: PLOS ONE

Article Title: Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

doi: 10.1371/journal.pone.0312402

Figure Lengend Snippet: ELISA plates coated with ACE2-hFcLALA (5ug/mL) were incubated with RBD-mFc (25ng/mL, A ) or Spike (2ug/mL, B ) premixed with ACE2-hFcLALA at different concentrations. The binding of RBD-mFc and Spike was detected with a mouse Fc specific PA-conjugated antibody/pNPP substrate and mouse HisTag specific HRP-conjugated antibody/OPD substrate, respectively. VeroE6 ( C ) and HEK293-ACE2 ( D ) cells were incubated with RBD-mFc (100ng/mL) premixed with ACE2-hFc at different concentrations. The binding of RBD-mFc was detected by flow cytometry using a mouse Fc specific FITC-conjugated antibody.

Article Snippet: The blot was probed with a commercial ACE2 specific antibody (Novus Biological, AC18F) followed by a goat anti-mouse IgG-HRP (Cell Signaling Technologies, #7076).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Flow Cytometry

Journal: PLOS ONE

Article Title: Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

doi: 10.1371/journal.pone.0312402

Figure Lengend Snippet: VeroE6 cells were infected with a mixture of the D614G SARS-CoV-2 virus and ACE2-hFcLALA at different concentrations. After 1h at 37°C of contact with the mixture, the cells were PBS washed and kept in culture for 72h at 37°C. ( A ) Images of VeroE6 cells alone, virus infected, and infected with a mixture of viruses and different concentrations of ACE2-hFc. Magnification 10X. Scale bar 100 μm ( B ) The infectivity inhibition was determined by measuring the cellular viability of the VeroE6 cells. The human 5G4 antibody was used as infectivity inhibition negative control. ( C ) Recognition of SARS-CoV-2 N proteins using two different epitopes specific monoclonal antibodies against this protein.

Article Snippet: The blot was probed with a commercial ACE2 specific antibody (Novus Biological, AC18F) followed by a goat anti-mouse IgG-HRP (Cell Signaling Technologies, #7076).

Techniques: Infection, Virus, Inhibition, Negative Control, Bioprocessing

Journal: PLOS ONE

Article Title: Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

doi: 10.1371/journal.pone.0312402

Figure Lengend Snippet: VeroE6 cells were infected with a single-round infection of D614G SARS-CoV-2 virus during 1h at 37°C. Then, the cells were PBS washed and kept in culture with different concentrations of the recombinant protein ACE2-hFcLALA for 72h at 37°C. ( A ) Images of VeroE6 cells alone, virus infected and infected and kept in culture with different concentrations of ACE2-hFc. Magnification 10X. Scale bar 100 μm ( B ) The infectivity inhibition was determined by measuring the cellular viability of the VeroE6 cells. The human 5G4 antibody was used as infectivity inhibition negative control.

Article Snippet: The blot was probed with a commercial ACE2 specific antibody (Novus Biological, AC18F) followed by a goat anti-mouse IgG-HRP (Cell Signaling Technologies, #7076).

Techniques: Infection, Virus, Recombinant, Inhibition, Negative Control

Journal: PLOS ONE

Article Title: Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

doi: 10.1371/journal.pone.0312402

Figure Lengend Snippet: VeroE6 cells were infected with a mixture of each of the five mutated SARS-CoV-2 virus variants and different concentrations of the recombinant protein ACE2-hFcLALA. After 1h at 37°C in contact with the mixture, the cells were PBS washed and kept in culture for 72h at 37°C. The infectivity inhibition was determined by measuring the cellular viability of the VeroE6 cells. The human 5G4 antibody was used as infectivity inhibition negative control.

Article Snippet: The blot was probed with a commercial ACE2 specific antibody (Novus Biological, AC18F) followed by a goat anti-mouse IgG-HRP (Cell Signaling Technologies, #7076).

Techniques: Infection, Virus, Recombinant, Inhibition, Negative Control