Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Structural elements in the ACE2-Fc constructs. a Schematic depiction of the main parts in an engineered ACE2-Fc molecule and their functional properties. b Design of the ACE2-Fc fusion protein; ACE2 parts in light and dark blue, IgG-Fc part in gray, spike (S) protein trimer in green and the receptor-binding domain (RBD) located at the tip of each spike protein in orange and dark green. The binding region as well as active site residues H374 and H378 important for the enzymatic activity of ACE2 are highlighted. Structures of the following Protein Data Bank (PBD) identifiers were used for modeling: 6M17, 6M0J, 6VSB, 5DK3. c Nomenclature and structural variations in the ACE2-Fc constructs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Construct, Functional Assay, Binding Assay, Activity Assay

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Structural and functional characteristics of the ACE2-Fc proteins. a Far-UV CD spectra (left) and Near-UV CD spectra (right) of ACE2-Fc constructs indicating that all proteins exhibit similar secondary and tertiary structures. b Chromatograms and molecular mass from size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) indicating that the ACE2-Fc molecules form homodimers. c Non-reducing (top) and reducing (bottom) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showing that intermolecular disulfide bonds in the homodimers are formed. d Comparison of fluorescence signals over time obtained in an assay testing the cleavage of a fluorescent peptidyl-4-methylcoumaryl-7-amide (MCA). Relative fluorescent units (RFU) are given. e Amount of MCA cleaved after 30 min of incubation with the ACE2-Fc constructs. Ref1 and Ref2 are two different commercially available ACE2-Fc proteins from Genscript and Acrobiosystems, respectively. Bars are mean values; error bars depict the 95% confidence interval of six independent experiments shown as circles.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Functional Assay, Circular Dichroism, Construct, Size-exclusion Chromatography, Multi-Angle Light Scattering, Polyacrylamide Gel Electrophoresis, SDS Page, Comparison, Fluorescence, Incubation

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Interaction of the ACE2-Fc constructs with the SARS-CoV-2 RBD. a Surface plasmon resonance (SPR) was performed to obtain binding curves of ACE2-Fc fusion constructs and an unrelated Fc fusion protein (aflibercept) to an immobilized RBD from SARS-CoV-2. An exemplary binding curve is shown (RU = Response Units). b Binding constants of the ACE2-Fc constructs towards the RBD of SARS-CoV-2 (Mean ± SD of triplicate measurements). c ACE2-Fc fusion proteins were pre-incubated with the SARS-CoV-2 spike S1 protein and tested in a competition ELISA for their ability to neutralize S1 binding to immobilized ACE2 protein. Potent inhibition of SARS-CoV-2 spike S1 protein by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right). Data are represented as means ± SD of at least two independent experiments.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Construct, SPR Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Binding affinities of ACE2-Fc constructs to Fc-receptors.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Binding Assay, Construct

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Effect of ACE2-Fc on SARS-CoV-2-GFP infection and inhibition of SARS-CoV-2 primary isolates. a ACE2-IgG4-Fc reduces SARS-CoV-2-GFP replication. Representative fluorescent images of Vero E6 cells infected with SARS-CoV-2-GFP (multiplicity of infection (MOI) = 0.6 IU/cell) pre-incubated with ACE2-IgG4-Fc fusion construct 1 (632 nM). b ACE2-Fc fusion proteins potently neutralize coronaviruses. Serial dilutions of ACE2-Fc fusion proteins were pre-incubated with different coronaviruses and tested for their ability to neutralize the virus before infection of Vero E6 cells. Neutralization of SARS-CoV (top), SARS-CoV-2-Jan (middle) and SARS-CoV-2-April (bottom) by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right) is shown. Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50) determined as well as the 95% confidence interval (CI 95%) are given for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Infection, Inhibition, Incubation, Construct, Virus, Neutralization

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Neutralization potency of ACE2-IgG4-Fc fusion proteins increases with evolution of pandemic SARS-CoV-2 variants. Serial dilutions of ACE2-IgG4-Fc fusion constructs 1 and 3 were pre-incubated with the indicated SARS-CoV-2 primary isolates or VoCs and tested for their ability to prevent cytotoxicity following infection of A549-hACE2 cells. Neutralization of SARS-CoV-2-Jan, SARS-CoV-2-April and SARS-CoV-2 VoCs alpha, beta and delta by enzymatically active ACE2-IgG4-Fc construct 1 (left) and enzymatically inactive ACE2-IgG4-Fc construct 3 (right). Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50 values) determined as well as the 95% confidence interval (CI 95%) are shown for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Neutralization, Construct, Incubation, Infection