Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet: Impact of BA.2.12.1 and BA.4/5 mutations on Spike-mediated infection (A) Infection kinetics of Caco2, A549-ACE2, and A549-ACE2-TMPRSS2 cells infected by VSVpp carrying the indicated mutant S proteins. Infected GFP+ cells were automatically quantified over a period of 22 h. Exemplary kinetic representing one out of four independent experiments (from B), where each dot indicates the mean of technical duplicates. (B) Automatic quantification of infection events by counting GFP positive cells of Caco2, A549-ACE2, and A549-ACE2-TMPRSS2 cells infected by VSVpp carrying SARS-CoV-2 Spike protein of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red). Bars represent the mean of four independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Automatic quantification of infection events of Caco2 (upper panel), A549-ACE2 (middle panel), and A549-ACE2-TMPRSS2 (lower panel) cells transduced with VSVΔG-GFP pseudotyped with SARS-CoV-2 S of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red) or indicated mutant S proteins. Graphs in the center show Hu-based S mutants, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Graphs on the right show BA.2-based S mutants and infection events are normalized on the BA.2 S (set at 1) and p values indicate difference to the BA.2 S. Bars represent the mean of four independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Correlation of the pseudotype infection events of the wild-type S or indicated mutant S proteins in A549-ACE2-TMPRSS2/A549-ACE2 (upper panel), A549-ACE2/Caco2 (middle panel), and A549-ACE2-TMPRSS2/Caco2 (lower panel) cells. Each symbol represents the average value derived from four infection experiments (shown in C). Numbers indicate the combined mutations of BA.4/5 S proteins whose infection events do not correlate between A549 and Caco2 cells. Coefficient of correlation (r), coefficient of determination (R 2 -values) and two tailed p values are provided (see also Figure S2 ).

Article Snippet: A549 (human lung carcinoma) cells (ATCC: # CCL-185; derived from a type II pneumocyte lung tumor of a white, 58-year-old male in 1972) stably expressing human ACE2 and/or human TMPRSS2 as well as HEK293T cells stably expressing human ACE2 were kindly provided by Markus Hoffmann and Stefan Pöhlmann (DPZ Göttingen, Germany).

Techniques: Infection, Mutagenesis, Transduction, Derivative Assay, Two Tailed Test

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet: Impact of BA.2.12.1 and BA.4/5 mutations on TMPRSS2 dependency (A) Automatic quantification of infection events HEK293T-ACE2 cells alone or transfected with TMPRSS2, cathepsin L or B, and infected with VSVpp carrying SARS-CoV-2 S of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red) or indicated mutant S proteins. The left part of the graphs shows Hu-based S mutants, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Graphs on the right show BA.2-based S mutants and infection events are normalized to BA.2 S (set to 1) and p values indicate difference to the BA.2 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (B) Correlation of VSVpp infection events of the wild-type S or indicated mutant S proteins in HEK293T overexpressing ACE2 alone or together with TMPRSS2 or cathepsins. Each dot represents the average value derived from three infection experiments (shown in A). Coefficient of correlation (r), coefficient of determination (R 2 -values), and two tailed p values are provided. (C) Infection kinetics (left panel) or infection events (right panel) of A549-ACE2 cells infected by VSVpp containing wild type or mutant Hu-based S proteins. The left panel shows an exemplary kinetic representing one out of three independent experiments (from right panel), where each dot indicates the mean of technical duplicates. In the bar diagram, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Infection kinetics (left panel) or infection events (right panel) of A549-ACE2-TMPRSS2 cells infected by VSVpp containing the wild type or the indicated Hu-based S proteins. The left panel shows an exemplary kinetic representing one out of three independent experiments (from right panel), where each dot indicates the mean of technical duplicates. In the bar diagram, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (E) Correlation of the pseudotype infection events of the wild-type S or indicated Hu-based mutant S proteins harboring mutations characteristic of BA.1 and/or BA.2 S in A549 overexpressing ACE2 alone/with TMPRSS2. Each dot represents the average value derived from three infection experiments (shown in C and 3D). Coefficient of correlation (r), coefficient of determination (R 2 -values) and two tailed p values are provided. (F) Automated quantification of GFP fluorescence of A549-ACE2-TMPRSS2 (left panel) or Caco2 (right panel) cells infected with VSVΔG-GFP pseudotyped with the indicated S variants. Cells were pre-treated (1 h, 37°C) with 20 μM of camostat or E64d in the highest concentration and diluted in a 1:5 titration row. Symbols represent the mean of three independent experiments (±SEM). (G) Quantification of viral N RNA levels in the supernatant of A549-ACE2-TMPRSS2 (left panel) and Caco2 (right panel) cells infected with the indicated SARS-CoV-2 variants (MOI = 0.05), collected at 48 h post-infection. Cells were pre-treated (1 h, 37°C) with 20 μM of camostat or E64d. The right graph of each panel shows the reduction of vRNA levels in the supernatants relative to the untreated control (set at 1). Bars represent the mean of three independent experiments (±SEM). Non-infected controls were below the quantification level (<1000). Statistical significance was tested by two-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3 .

Article Snippet: A549 (human lung carcinoma) cells (ATCC: # CCL-185; derived from a type II pneumocyte lung tumor of a white, 58-year-old male in 1972) stably expressing human ACE2 and/or human TMPRSS2 as well as HEK293T cells stably expressing human ACE2 were kindly provided by Markus Hoffmann and Stefan Pöhlmann (DPZ Göttingen, Germany).

Techniques: Infection, Transfection, Mutagenesis, Derivative Assay, Two Tailed Test, Fluorescence, Concentration Assay, Titration, Control

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet:

Article Snippet: A549 (human lung carcinoma) cells (ATCC: # CCL-185; derived from a type II pneumocyte lung tumor of a white, 58-year-old male in 1972) stably expressing human ACE2 and/or human TMPRSS2 as well as HEK293T cells stably expressing human ACE2 were kindly provided by Markus Hoffmann and Stefan Pöhlmann (DPZ Göttingen, Germany).

Techniques: Virus, Recombinant, Saline, Mutagenesis, Binding Assay, Expressing, Construct, Gene Expression, Plasmid Preparation, Software, Modification, Membrane, Semi Dry Blot, Transfection