Journal: PLoS Pathogens

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1371/journal.ppat.1010175

Figure Lengend Snippet: (A) Schematic representation of our proposed treatments. (Left panel) SARS-CoV-2 spike protein binds to the ACE2 receptor on host cells to mediate virus entry. (Middle panel) Binding of ACE2-Ig to SARS-CoV-2 Spike protein may prevent its binding to the ACE2 receptor thus blocking infection. (Right panel) Binding of RBD-Ig to the receptor ACE2 on host cells may prevent the binding of the spike protein thus blocking infection. This figure was created by us using BioRender. (B) Staining of 293T-ACE2, 293T-NKp46 and 293T parental cells using an anti-Flag antibody (left panel). Quantification of mean fluorescence intensity (MFI) of 3 repetitions (right panel). (C) Staining of 293T-ACE2, 293T-NKp46 and 293T parental cells with RBD-Ig (left panel). Quantification of MFI of 3 repetitions (right panel). (D) Spike protein surface expression on 293T cells co-transfected with either SARS-CoV-2 Spike envelope plasmid (293T-Spike cells) Vesicular stomatitis virus (VSV) G envelope plasmid (293T-VSV-G cells). 293T parental cells (untransfected) were also stained as a control (left panel). Quantification of MFI of 3 repetitions (right panel). (E) Staining of 293T-Spike/ 293T-VSV-G/ 293T parental cells with ACE2-Ig. Quantification of MFI of 3 repetitions (right panel). In all histograms except from those made for 293T parental cells, we gated on GFP positive cells. *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. Data are mean ± SEM of three independent experiments.

Article Snippet: The ACE2-Ig and RBD-Ig fusion proteins secreted to the medium were purified on HiTrap Protein G High Performance column (Cat#GE17-0405-01, GE Healthcare).

Techniques: Binding Assay, Blocking Assay, Infection, Staining, Fluorescence, Expressing, Transfection, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1371/journal.ppat.1010175

Figure Lengend Snippet: (A) ACE2 enzymatic activity assay. Recombinant human ACE2 and ACE2-Ig were incubated with and without an ACE2 inhibitor, then MCA based peptide substrate was added and plate was immediately inserted in the fluorescent plate reader. *p<0.005, **p<0.0005, ***p<0.00005, Student’s t-test as compared to same treatment with inhibitor. (B) Staining of 293T-Spike cells with ACE2-Ig which was previously incubated for 15 minutes with or without an ACE2 inhibitor. (C) Plaque reduction neutralization test. Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ACE2-Ig or RBD-Ig. IC50 was calculated from the mean of the three experiments represented. Not significant (NS); Student’s t-test. Data are mean ± SEM of three independent experiments.

Article Snippet: The ACE2-Ig and RBD-Ig fusion proteins secreted to the medium were purified on HiTrap Protein G High Performance column (Cat#GE17-0405-01, GE Healthcare).

Techniques: Enzyme Activity Assay, Recombinant, Incubation, Staining, Plaque Reduction Neutralization Test, Infection

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1371/journal.ppat.1010175

Figure Lengend Snippet: RBD-Ig decreases disease severity of SARS-CoV-2 infected mice . (A) Homozygous female K18-hACE2 transgenic mice were infected with SARS-CoV-2 (day 0) and treated with 75 μg/mouse of either Control-Ig, RBD-Ig or ACE2-Ig. % of initial body weight was calculated from mice which were weighed daily. *P < 0.05; Student’s t-test as compared to Infected + Control-Ig. (B) Survival percentages of SARS-CoV-2 infected mice treated as described in A. *P < 0.05; Mantel-Cox test as compared to Infected + Control-Ig. (A-B) n = 7–8 , data are mean ± SEM of two independent experiments. (C-D) Homozygous female K18-hACE2 transgenic mice were infected with SARS-CoV-2 (day 0) and treated at 1, 2, 3 DPI with 2nmol/mouse of RBD-Ig ( n = 10 ) or ACE2-Ig ( n = 10 ), at 5 DPI mice were sacrificed, sera was collected and lung harvested. *P < 0.05; *** P < 0.0005; Student’s t-test. Data are mean ± SEM. (C) ELISA was performed from collected sera to detect fusion protein presence. (D) PFU in the harvested lungs of mice treated with ACE2-Ig or RBD-Ig.

Article Snippet: The ACE2-Ig and RBD-Ig fusion proteins secreted to the medium were purified on HiTrap Protein G High Performance column (Cat#GE17-0405-01, GE Healthcare).

Techniques: Infection, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1371/journal.ppat.1010175

Figure Lengend Snippet: (A) Staining of 293T-Parental cells and 293T-ACE2 cells with the mAb anti-ACE2 01 we generated. (B) Staining of 293T-Parental cells and 293T-ACE2 cells with RBD-Ig. Cells were incubated with or without anti-ACE2 01 for 1 hour at 4°C, washed and then staining was performed. (C) Staining of infected (MOI 0.5) and uninfected VERO E6 cells with either an anti-Spike antibody to verify infection (left panel) or with our anti-ACE2 01 antibody (right panel) at 16,24,48 hours PI. (D) Staining with anti-ACE 01 of 293T-ACE2 cells which were incubated with 1 μg of either Control-Ig or RBD-Ig for 1, 2, 6 and 24 hours. (A-D) All histograms were gated on GFP positive cells. (E) ACE2 enzymatic activity assay. Recombinant human ACE2 and 293T-ACE2 cells lysate (10 μg) were incubated with either Control-Ig (1 μg) or RBD-Ig (0.1 μg or 1 μg), then MCA based peptide substrate was added and plate was immediately read in the fluorescent plate reader. Not significant (NS), Student’s t-test as compared with Control-Ig. (F) Plaque reduction neutralization test. Vero E6 cells were infected with increasing SARS-CoV-2 titers and treated with 0.1 nmol/well of ACE2-Ig or RBD-Ig, picture of the plaques can be seen on the left panel and average number of plaques in the table on the right panel. Too many plaques to count (tm). Figures shows one representative experiment out of three performed.

Article Snippet: The ACE2-Ig and RBD-Ig fusion proteins secreted to the medium were purified on HiTrap Protein G High Performance column (Cat#GE17-0405-01, GE Healthcare).

Techniques: Staining, Generated, Incubation, Infection, Enzyme Activity Assay, Recombinant, Plaque Reduction Neutralization Test

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Schematic representation of our proposed treatments. SARS-CoV-2 infects ACE2 expressing cells (left panel). Binding of ACE2-Ig to SARS-CoV-2 Spike protein (middle panel) or binding of RBD-Ig to the ACE2 receptor (right panel) may prevent infection. (B) Staining of cells transfected to express ACE2 with an N-terminal Flag-tag (293T-ACE2 cells) and their parental cells that do not express a tag. This staining was performed using an anti-Flag antibody. (C) Staining of 293T-ACE2 cells with RBD-Ig. (D) Left panel: Spike protein surface expression on 293T cells co-transfected with either SARS-CoV-2 Spike envelope plasmid (293T-Spike cells) or Vesicular stomatitis virus (VSV) G envelope plasmid (293T-VSV-G cells). Right panel: Staining of 293T-Spike cells with ACE2-Ig. All histograms except from those made for 293T-Parental cells, were made from GFP positive gated cells. Figures shows one representative experiment out of 3 performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Expressing, Binding Assay, Infection, Staining, Transfection, FLAG-tag, Plasmid Preparation

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) ACE2 enzymatic activity assay. Recombinant human ACE2 and ACE2-Ig were incubated with and without an ACE2 inhibitor, then MCA based peptide substrate was added and plate was immediately inserted in the fluorescent plate reader. *p<0.005, **p<0.0005, ***p<0.00005, Student’s t-test as compared to same treatment with inhibitor. (B) Staining of 293T-Spike cells with ACE2-Ig which was previously incubated for 15 minutes with or without an ACE2 inhibitor. (C-D) Plaque reduction neutralization test. Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of either Control-Ig, ACE2-Ig (C) or RBD-Ig (D). % Neutralization was calculated as the percent of the decrease in plaque numbers, as compared with the background control. *P < 0.05; **P< 0.01; ***P <0.001; Student’s t-test as compared to Control-Ig. Figures shows one representative experiment out of 3 performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Enzyme Activity Assay, Recombinant, Incubation, Staining, Plaque Reduction Neutralization Test, Infection, Neutralization

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Homozygous female K18-hACE2 transgenic mice were infected with SARS-CoV-2 (day 0) and treated with 75ug/mouse of either Control-Ig, RBD-Ig or ACE2-Ig. % of initial body weight was calculated from mice which were weighed daily. (B) Survival percentages of SARS-CoV-2 infected mice treated as described in A. *P < 0.05; Mantel-Cox test as compared to Infected + Control-Ig. Figure shows the combined results of two independent experiments.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Transgenic Assay, Infection

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Anti-Spike IgG antibodies generated by mice following infection with SARS-CoV-2. Sera were taken 15 dpi from all mice groups and from naïve mice and diluted as indicated (upper right). Sera was incubated either with 293T-Spike cells (upper histograms) or with 293T-ACE2 cells (lower histograms) as a primary antibody then cells were stained with Alexa fluor 647 anti-mouse IgG secondary antibody. (B) ACE2-Ig staining of 293T-Spike cells in the presence or absence of sera from the various groups. Sera from all indicated groups were incubated with 293T-Spike cells for 1 hour at 4°C followed by staining with ACE2-Ig. All histograms were gated on GFP positive cells. Figure shows one representative experiment out of 2 performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Generated, Infection, Incubation, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

doi: 10.1101/2021.04.18.440302

Figure Lengend Snippet: (A) Staining of 293T-Parental cells and 293T-ACE2 cells with the mAb anti-ACE2 01 we generated. (B) Staining of 293T-Parental cells and 293T-ACE2 cells with RBD-Ig. Cells were incubated with or without anti-ACE2 01 for 1 hour at 4°C, washed and then staining was performed. (C) Staining of infected (MOI 0.5) and uninfected VERO E6 cells with either an anti-Spike antibody to verify infection (left panel) or with our anti-ACE2 01 antibody (right panel) at 16,24,48 hours PI. (D) Staining with anti-ACE 01 of 293T-ACE2 cells which were incubated with 1 ug of either Control-Ig or RBD-Ig for 1,2,6 and 24 hours. (A-D) All histograms were gated on GFP positive cells. (E) ACE2 enzymatic activity assay. Recombinant human ACE2 and 293T-ACE2 cells lysate (10 ug) were incubated with either Control-Ig (1 ug) or RBD-Ig (0.1 ug or 1ug), then MCA based peptide substrate was added and plate was immediately read in the fluorescent plate reader. Not significant (NS), Student’s t-test as compared with Control-Ig. (F) Plaque reduction neutralization test. Vero E6 cells were infected with increasing SARS-CoV-2 titers and treated with 20 ug/well of either Control-Ig, ACE2-Ig or RBD-Ig. % Neutralization was calculated as the percent of the decrease in plaque numbers, as compared with cells treated with Control-Ig. *P < 0.01; **P< 0.005; ***P <0.00001; Student’s t-test. Figures shows one representative experiment out of 3 (A-E) or 2 (F) performed.

Article Snippet: The enzymatic activity of the ACE2-Ig fusion protein was evaluated using the ACE2 Activity Assay Kit (Fluorometric) (Cat#BN01071, Assay Genie) according to the manufacturer instructions.

Techniques: Staining, Generated, Incubation, Infection, Enzyme Activity Assay, Recombinant, Plaque Reduction Neutralization Test, Neutralization