Journal: Cell

Article Title: Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

doi: 10.1016/j.cell.2021.04.033

Figure Lengend Snippet: SARS-CoV-2 neutralization by receptor blocking antibodies. (A) Infection of CHO-ACE2 cells by SARS-CoV-2 pseudovirus were determined in the presence of receptor-blocking IgGs (left panel) or Fabs (right panel). Luciferase activities in the CHO-ACE2 cells were measured, and the percent neutralization was calculated. Data are presented as mean ± SEM in triplicates and are representative of two independent experiments. The IC 50 was calculated by a variable-slope four-parameter non-linear regression model using GraphPad Prism 7 software or the Quest Graph IC 50 Calculator from AAT Bioquest ( https://www.aatbio.com/tools/ic50-calculator ) with top and bottom constraints set at 100% and 0%, respectively. (B) Infection of Vero E6 C1008 cells by SARS-CoV-2 live virus (isolated from a nasopharyngeal swab of an individual in Singapore) were determined in the presence of receptor-blocking IgGs (left panel) or Fabs (right panel). Infection-induced cytopathic effect was determined by detecting the amount of ATP present in the uninfected live cells from which the percent neutralization was calculated. Data are presented as mean ± SEM in triplicates and are representative of two independent experiments. The IC 50 was calculated by a variable-slope four-parameter non-linear regression model in GraphPad Prism 7 software. Pseudovirus and live virus neutralization assays were not performed for 6F8 Fab because of 6F8 IgG’s low and similar potency to other clones. (C) Evaluation of antiviral activity of 5A6 in a model of reconstituted human airway epithelium (HAE). Viral genome quantification was performed using qRT-PCR, and results are expressed in relative virus production (intracellular or apical) compared with the control. Bars represent the mean ± SD in duplicates. ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 compared with the control (no Ab) by one-way ANOVA. The trans -epithelial electrical resistance (TEER in Ω/cm 2 ) was measures at 48 hpi (hours post-infection). (D) Correlation curve of affinity/avidity for RBD and live virus neutralization potency (IC 50 ) of receptor-blocking IgG antibodies (circles), Fab antibodies (triangles), and ACE2-Fc (black diamond). The IC 50 values were calculated using a four-parameter logistic regression model in the Quest Graph IC 50 Calculator from AAT Bioquest. (E) Binding of the IgG (solid lines, circles) and 5A6 Fab (dashed line, red triangle) to the purified SARS-CoV-2 pseudovirus. Data are presented as mean ± SD in duplicates and are representative of two independent experiments. See also Figure S2 and .

Article Snippet: EC 50 values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism and IC 50 values were calculated either using the [Inhibitor] versus response variable slope four parameter non-linear regression model of GraphPad Prism, or the four parameter logistic regression model in the Quest Graph IC 50 Calculator from AAT Bioquest, Inc ( https://www.aatbio.com/tools/ic50-calculator ).

Techniques: Neutralization, Blocking Assay, Infection, Luciferase, Software, Isolation, Clone Assay, Activity Assay, Quantitative RT-PCR, Binding Assay, Purification

Journal: Cell

Article Title: Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

doi: 10.1016/j.cell.2021.04.033

Figure Lengend Snippet: SARS-CoV-2 live virus neutralization and antibody binding to the Spike trimer measured by SPR, related to (A) The potency of 2H4, 3D11 and 5A6 IgG antibodies in neutralizing live SARS-CoV-2 virus assays determined by measuring the viral genome copy number (GCN). Infection of Vero E6 C1008 cells by SARS-CoV-2 live virus (isolated from a nasopharyngeal swab of a patient in Singapore) were determined in the presence of receptor blocking IgGs 2H4, 3D11 and 5A6. 48 hours post infection, culture supernatant was harvested and viral GCN was determined by RT-qPCR targeting the N gene and GCN values were determined by comparing Ct values against a logGCN standard curve. The GCN values were then converted to percent neutralization and plotted with a non-linear regression curve fit using PRISM. IC 50 was calculated by a variable slope four parameter non-linear regression model in Graphpad PRISM 7 without or ˆwith top and bottom constraints set at 100% and 0% respectively. Data are presented as mean ± SEM from 6 replicates. (B) Binding avidity of IgG clones 2H4, 3D11, 3F11, 5A6, and 6F8 for intact Spike trimer measured by surface plasmon resonance (SPR). A range of IgG concentrations from 12.5 nM to 0.39 nM (in 2-fold serial dilution) are shown, with sensorgrams in black and curve fits in red. (C) Binding affinity of Fab clones 1F4, 2H4, 3D11, 3D11, and 5A6 for intact Spike trimer measured by surface plasmon resonance (SPR). A range of Fab concentrations from 100 nM to 3.125 nM (in 2-fold serial dilution) are shown, with sensorgrams in black and curve fits in red. (D) Log-log scatterplot comparing antibody binding constants for Fc-RBD (x axis) to those for Spike trimer (y axis). Affinity or avidity of antibodies or Fab fragments for the flexible Fc-RBD construct represent binding without geometric constraints, while measurements using immobilized Spike trimers represent binding with the specific geometries afforded by the Spike:antibody complexes. For most species, SPR and BLI measurements are similar, however 3D11 IgG and ACE2-Fc bind significantly more weakly to relatively unrestricted Fc-RBD than to Spike trimer (note that the 3D11 IgG binds > 10x more tightly than 3D11 Fab in both sets of experiments, indicating avid binding). 5A6 IgG binds somewhat more tightly to Spike trimer than to Fc-RBD, perhaps indicating that RBDs within a Spike trimer have particularly favorable geometries for binding.

Article Snippet: EC 50 values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism and IC 50 values were calculated either using the [Inhibitor] versus response variable slope four parameter non-linear regression model of GraphPad Prism, or the four parameter logistic regression model in the Quest Graph IC 50 Calculator from AAT Bioquest, Inc ( https://www.aatbio.com/tools/ic50-calculator ).

Techniques: Neutralization, Binding Assay, Infection, Isolation, Blocking Assay, Quantitative RT-PCR, Clone Assay, SPR Assay, Serial Dilution, Construct

Journal: Cell

Article Title: Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

doi: 10.1016/j.cell.2021.04.033

Figure Lengend Snippet: Neutralization of the SARS-CoV-2 pseudovirus with Spike mutant D614G, related to (A) Neutralization by 5A6 of SARS-CoV-2 pseudovirus bearing either wild-type Spike protein (red), or Spike with the D614G mutation (black). The efficacy of 5A6 IgG is against D614G mutant Spike is improved over wild-type. (B) Neutralization by 3D11 of SARS-CoV-2 pseudovirus bearing either wild-type Spike protein (red), or Spike with the D614G mutation (black). 3D11 IgG suffers a severe loss of efficacy against the mutant pseudovirus (more than 5-fold weaker IC 50 ). Data are presented as mean ± SEM in triplicates and are representative of two independent experiments. IC 50 was calculated by variable slope four parameter non-linear regression model using Graphpad PRISM 7 Software without or ˆwith top and bottom constraints set at 100% and 0% respectively.

Article Snippet: EC 50 values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism and IC 50 values were calculated either using the [Inhibitor] versus response variable slope four parameter non-linear regression model of GraphPad Prism, or the four parameter logistic regression model in the Quest Graph IC 50 Calculator from AAT Bioquest, Inc ( https://www.aatbio.com/tools/ic50-calculator ).

Techniques: Neutralization, Mutagenesis, Software

Journal: Cell

Article Title: Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

doi: 10.1016/j.cell.2021.04.033

Figure Lengend Snippet:

Article Snippet: EC 50 values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism and IC 50 values were calculated either using the [Inhibitor] versus response variable slope four parameter non-linear regression model of GraphPad Prism, or the four parameter logistic regression model in the Quest Graph IC 50 Calculator from AAT Bioquest, Inc ( https://www.aatbio.com/tools/ic50-calculator ).

Techniques: Isolation, Recombinant, Transfection, Modification, Luciferase, Plasmid Preparation, Software