Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 Omicron sublineages show comparable cell entry but differential neutralization by therapeutic antibodies

doi: 10.1016/j.chom.2022.04.017

Figure Lengend Snippet: S proteins of Omicron sublineages do not exhibit major differences in ACE2 usage or ability to drive cell-cell and virus-cell fusion (A) Schematic overview of the SARS-CoV-2 spike (S) protein domain structure (left) and summary of the mutations found in the different Omicron sublineages (right; numbering is according to the S protein of SARS-CoV-2 B.1). S protein residues that are identical between the S proteins of some Omicron sublineages and B.1 are marked in green, whereas mutated residues are highlighted in red (note: the BA.1 S protein harbors an insertion between amino acid residues 214 and 215). Further, mutations found in all Omicron sublineages are indicated by a circle. Abbreviations: NTD, N-terminal domain; RBD, receptor-binding domain; TD, transmembrane domain; S1/S2 and S2’, cleavage sites in the S protein. (B) S-protein-driven cell entry. We added particles bearing the indicated S proteins (or no S protein) to the indicated cell lines and analyzed cell entry by measuring the activity of virus-encoded firefly luciferase in cell lysates 16–18 h after inoculation. Presented are the average (mean) data from 6–12 biological replicates (each conducted with four technical replicates) in which cell entry was normalized against B.1 (set as 1). Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t tests (p > 0.05, not significant [ns]; p ≤ 0.05, ∗ ; p ≤ 0.01, ∗∗ ; p ≤ 0.001, ∗∗∗ ). Please also see Figure S1 . (C) ACE2 binding efficiency. 293T cells transiently expressing the indicated S proteins (or no S protein) where first incubated with soluble ACE2 fused to the Fc portion of human immunoglobulin G (solACE2-Fc) and subsequently incubated with an Fc-specific AlexaFluor-488-coupled secondary antibody; then, solACE2-Fc binding was analyzed by flow cytometry. Presented are the average (mean) data from six biological replicates (each conducted with single samples) in which ACE2 binding was normalized against B.1 (set as 1). Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t tests (p > 0.05, not significant [ns]; p ≤ 0.05, ∗ ; p ≤ 0.01, ∗∗ ). (D) Blockade of S-protein-driven cell entry by an anti-ACE2 antibody. Vero cells were preincubated with serial dilutions of anti-ACE2 antibody before particles bearing the indicated S proteins were added. S-protein-driven cell entry was analyzed and normalized to samples without anti-ACE2 antibody (set as 1). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-way analysis of variance with Dunnett’s post hoc tests (p > 0.05, not significant [ns]; p ≤ 0.05, ∗ ; p ≤ 0.01, ∗∗ ; p ≤ 0.001, ∗∗∗ ). Please also see Tables S1 and . (E) Blockade of S-protein-driven cell entry by solACE2-Fc. Particles bearing the indicated S proteins were preincubated with serial dilutions of solACE2-Fc (or no solACE2-Fc) before being added to Vero cells. S-protein-driven cell entry was analyzed and normalized to samples without solACE2-Fc (= 0% inhibition). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-way analysis of variance with Dunnett’s post hoc tests (p > 0.05, not significant [ns]) . (F) Usage of mouse and bat ACE2 by Omicron S proteins. BHK-21 cells transiently expressing human, mouse, or horseshoe bat ( Rhinolophus pearsonii ) ACE2 orthologs (or no ACE2) were inoculated with particles bearing the indicated S proteins or VSV glycoprotein (VSV-G). Cell entry of pseudovirus particles was analyzed and normalized to particles bearing no viral glycoprotein (set as 1; indicated by dashed line). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t tests (p > 0.05, not significant [ns]; p ≤ 0.05, ∗ ). (G) Qualitative fusion assay. A549-ACE2 cells transfected to express the indicated S proteins (or no S protein) were fixed 24 h after transfection and stained with May-Gruenwald and Giemsa solution before microscopic images were taken (scale bar, 500 μm). Arrowheads indicate small syncytia in cells expressing the S proteins of the different Omicron sublineages. (H) Quantitative fusion assay. 24 h after transfection, 293T cells transiently expressing the indicated S proteins (or no S protein) along with the beta-galactosidase alpha fragment were resuspended and seeded on top of A549-ACE2 cells transiently expressing the beta-galactosidase omega fragment. After an additional 24 h of incubation, beta-galactosidase substrate was added, and luminescence was recorded. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t tests (p ≤ 0.05, ∗ ).

Article Snippet: Recombinant anti-ACE2 Neutralizing Antibody , Sino Biological , Cat# 10108-MM37; RRID: N/A.

Techniques: Binding Assay, Activity Assay, Luciferase, Two Tailed Test, Expressing, Incubation, Flow Cytometry, Inhibition, Single Vesicle Fusion Assay, Transfection, Staining

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 Omicron sublineages show comparable cell entry but differential neutralization by therapeutic antibodies

doi: 10.1016/j.chom.2022.04.017

Figure Lengend Snippet: S proteins of Omicron sublineages differ in terms of their resistance to therapeutic antibodies (A) Schematic overview of the SARS-CoV-2 S protein RBD (numbering is according to the S protein of SARS-CoV-2 B.1). Mutations found in the RBDs of the Omicron sublineages (compared with the B.1 S protein) are highlighted in red, whereas identical amino acid residues are marked in green. RBD residues that make direct contact with ACE2 or that are recognized as epitopes for therapeutic antibodies are highlighted. (B) Particles bearing the indicated S proteins were preincubated with serial dilutions of individual monoclonal antibodies used for COVID-19 treatment or cocktails thereof or an irrelevant control antibody (hIgG) before being added to Vero cells. Of note, for antibody cocktails, we used each antibody at half concentration in order to keep total antibody concentrations constant. S-protein-driven cell entry was analyzed and normalized to samples without antibody (= 0% inhibition). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-way analysis of variance with Dunnett’s post hoc tests (p > 0.05, not significant [ns]; p ≤ 0.05, ∗ ; p ≤ 0.01, ∗∗ ; p ≤ 0.001, ∗∗∗ ). Please see also Tables S1 and .

Article Snippet: Recombinant anti-ACE2 Neutralizing Antibody , Sino Biological , Cat# 10108-MM37; RRID: N/A.

Techniques: Concentration Assay, Inhibition

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 Omicron sublineages show comparable cell entry but differential neutralization by therapeutic antibodies

doi: 10.1016/j.chom.2022.04.017

Figure Lengend Snippet:

Article Snippet: Recombinant anti-ACE2 Neutralizing Antibody , Sino Biological , Cat# 10108-MM37; RRID: N/A.

Techniques: Recombinant, Produced, Reporter Gene Assay, Plasmid Preparation, Software

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: Postulated mechanism underlying the IFNβ-ACE2 fusion protein. ( A ) A SARS-CoV-2- or NL63-infected individual is given the IFNβ-ACE2 via nebulization to the lungs. The sACE2 domain is postulated to bind the Spike protein and coat the virion with a surface array of IFN-β. Based on this strategy, the IFN-β domain will drive IFN-β signaling pathways and antiviral activity in the target cell before viral entry. IFNβ-ACE2 is postulated to provide a more concentrated and targeted approach to delivering IFN-β to the exact site and time of imminent viral infection . ( B ) Schematic diagram of the fusion protein construct consisting of an IFN-β domain and a sACE2(18-611) domain. ( C ) Schematic diagrams of the soluble control proteins: sACE2(18-611), sACE2(18-740), and IFN-β. ( D ) Schematic diagrams of the transmembrane proteins expressed in HEK cells for the ACE2-binding assay. SDS-PAGE gels showing purity of IFNβ-ACE2 ( E ), sACE2(18-740) ( F ), sACE2(18-611) ( G ), and IFN-β ( H ) proteins.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Infection, Protein-Protein interactions, Activity Assay, Construct, Control, Binding Assay, SDS Page

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: The IFN-β and sACE2 domains of IFNβ-ACE2 exhibited predicted bioactivities. ( A ) To assay the IFN-β domain, TF-1 cells were incubated with GM-CSF and either IFN-β, sACE2(18-611), or IFNβ-ACE2 then pulsed with [ 3 H]thymidine during the last 24 h of a 3-day culture. The y -axis represents counts per minute (CPM), and error bars represent the SD. Statistical significance was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test comparing the three control groups to the IFNβ-ACE2 treatment group at each concentration (ns nonsignificant, * p < 0.05, **** p < 0.0001). ( B – E ) To assay the ACE2 domain, HEK-Spike or HEK-control cells were incubated with designated concentrations of either sACE2(18-611) or IFNβ-ACE2 for 1 h at 4 °C. After washing, cells were stained with AF647-conjugated anti-human ACE2 antibody for 1 h at 4 °C. Cells were analyzed for ACE2 binding by flow cytometry. ( B ) Viable, single, live, and GFP + stably transfected cells (parental gate representing all cells in plot) were subgated to show the ACE2 + subset. Representative dot plots show binding of either sACE2(18-611) or IFNβ-ACE2 (2 μM each) to HEK-Spike or HEK-control cells. ( C ) Shown are percentages of ACE2 + HEK-Spike cells (ACE2 + gate/parental gate). ( D ) The MFIs of anti-ACE2 fluorescence are shown for the parental gate. ( E ) Bar graphs show mean percentages of HEK-Spike or HEK-control cells bound to ACE2 (ACE2 + gate/parental gate). Each data point represents the mean value (n = 2), and error bars represent SD. These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Incubation, Control, Concentration Assay, Staining, Binding Assay, Flow Cytometry, Stable Transfection, Transfection, Fluorescence

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: The sACE2 domain of IFNβ-ACE2 targeted IFN-β to the surface of NL63. NL63 was incubated at 4 °C with designated concentrations of IFNβ-ACE2, sACE2(18-611), recombinant IFN-β, or IFN-β (Peprotech). After a 1 h incubation, NL63 was washed of any unbound protein using 300kD centrifugal filters. NL63-protein complexes were then added to Vero E6-TMPRSS2-T2A-ACE2 cultures (100 μL) in a 96-well plate. The cells were harvested after a 2-day incubation at 33 °C, stained with LIVE/DEAD Fixable Blue Dead Cell Stain, and then surface-labeled with FITC-conjugated anti-human ACE2 and PE-conjugated anti-human TMPRSS2. After fixation and permeabilization, cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells were gated on viable, single, and live cells (parental gate) before subgating on nucleocapsid + , ACE2 high , or TMPRSS2 high cells. Shown are representative dot plots (( A , D , I ), x -axis = FSC-A as in ( A )) when NL63 was incubated with 1 nM IFNβ-ACE2 or controls. The IFNβ-ACE2 versus sACE2(18-611) groups were compared based on percentages of nucleocapsid + cells ( B ), percentages of ACE2 high cells ( E ), MFI of anti-ACE2 staining ( F ), percentages of TMPRSS2 high cells ( J ), and MFI of anti-TMRSS2 staining ( K ). The IFNβ-ACE2 versus IFN-β groups were compared based on percentages of nucleocapsid cells ( C ), percentages of ACE2 high cells ( G ), MFI of anti-ACE2 staining ( H ), percentages of TMPRSS2 high cells ( L ), and MFI of anti-TMPRSS2 staining ( M ). Cell percentages were calculated by dividing events in the positive subgate by the parental gate, and MFI values represent all events in the parental gate. Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance comparing IFN-β and ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical significance comparing each protein group at each concentration to blank was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (° p < 0.05, °° p < 0.01, °°° p < 0.001, °°°° p < 0.0001). These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Incubation, Recombinant, Staining, Labeling, Infection, Flow Cytometry, Concentration Assay

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: The covalent linkage of IFN-β and ACE2 was required for IFN-β targeting to NL63. NL63 was incubated at 4 °C with either IFNβ-ACE2 or the unlinked combination of sACE2(18-611) and IFN-β. After a 1 h incubation, NL63 was repeatedly washed with 300kD centrifugal filters to remove proteins that lacked binding to virions. The retentates, which included virions and virion-bound proteins, were added to Vero E6-TMPRSS2-T2A-ACE2 cells in a 96-well plate. Cells were harvested after a 2-day incubation at 33 °C and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. Cells were surface-stained with PE-conjugated mouse anti-human TMPRSS2 and FITC-conjugated mouse anti-human ACE2. After fixation and permeabilization, cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were analyzed for viral infection by flow cytometry. Viable, single, and live cells in the parental gate were subgated as the nucleocapsid + subset ( A ), the ACE2 high subset ( C ), and the TMPRSS2 high subset ( F ) as shown for the 1 nM concentration value. Shown are the percentages of nucleocapsid + , ACE2 high , and TMPRSS2 high subsets together with the respective MFI values ( B , D , E ), and ( G , H ), respectively. Cell percentages were calculated by dividing the events in the subset-positive/high subgate by those in the parental gate. MFI values were gated on all viable, single, and live cells (i.e., cells in the parental gate). Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance of the IFNβ-ACE2 versus the ‘IFN-β + sACE2’ treatment group at each concentration was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical significance was also assessed for treatment groups at each concentration compared to the ‘blank’ control via two-way ANOVA with Tukey’s multiple comparisons test (°° p < 0.01, °°° p < 0.001, °°°° p < 0.0001). These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Incubation, Binding Assay, Staining, Infection, Flow Cytometry, Concentration Assay, Control

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: In a non-washed in vitro infection system, IFNβ-ACE2 exhibited enhanced antiviral activity compared to IFN-β alone, ACE2 alone, or the unlinked combination. NL63 was incubated for 1 h at 4 °C with either IFNβ-ACE2, sACE2(18-611), sACE2(18-740), recombinant IFN-β, IFN-β (Peprotech), or the unlinked combination of sACE2(18-611) and IFN-β. In contrast to experiments shown in and , we omitted the virus-washing step. The NL63 + protein mixtures were added to Vero E6-TMPRSS2-T2A-ACE2 cells in a 96-well plate. The cells were harvested after a 2-day incubation at 33 °C and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. Cells were surface-stained with FITC-conjugated mouse anti-human ACE2, were fixed and permeabilized, and then were intracellularly stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells gated as viable, single, and live cells (parent gate) were subgated to define nucleocapsid + and ACE2 high subsets. Shown ( A , D ) are representative dot plots showing percentages of the nucleocapsid + subset at the 1 pM concentration. Shown ( B , F ) are the percentages of the nucleocapsid + subset for each group over concentrations ranging from 100 fM to 1 μM. Bar graph ( C ) shows mean percentage values of nucleocapsid + cells at the 1 pM concentration. Shown ( E ) are representative dot plots showing percentages of the ACE2 high subset at the 1 pM concentration. Shown ( G ) are the percentages of ACE2 high subset for each group over concentrations ranging from 100 fM to 1 μM. Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance was analyzed by ( C ) one-way ANOVA with the Dunnett multiple comparisons test or ( F , G ) two-way ANOVA with Tukey’s multiple comparisons test comparing the unlinked combination of IFN-β and ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: In Vitro, Infection, Activity Assay, Incubation, Recombinant, Virus, Staining, Flow Cytometry, Concentration Assay

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: IFNβ-ACE2 exhibited virus-specific targeting in accordance with viral host receptor specificity . The NL63 or 229E viruses were incubated at 4 °C with either IFNβ-ACE2, sACE2(18-611), IFN-β, or the unlinked combination of IFN-β and sACE2(18-611). After a 1 h incubation, the virus + protein mixtures were washed, and the retentate containing virion–protein complexes was used for infection of Vero E6-TMPRSS2-T2A-ACE2 or A549 cells, respectively, in a 96-well plate ( A , B ). After the washing step, IFNβ-ACE2 or the unlinked combination of IFN-β and sACE2(18-611) were directly added to designated groups ( B ). Alternatively, the virus + protein mixtures were not subjected to a virus-washing step and the mixtures were used for infection of the respective host cells ( C – E ). The cells were harvested after a 2-day incubation and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. After fixation and permeabilization, Vero E6-TMPRSS2-T2A-ACE2 cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody and A549 cells were stained with AF647-conjugated rabbit anti-229E nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells were gated on viable, single, and live cells before subgating on nucleocapsid + cells. Shown ( A ) are the percentages of nucleocapsid + cells for each treatment group normalized to the ‘no protein’ control group (1 nM concentrations). ( B ) Bar graph shows mean percentages of nucleocapsid + 229E-infected cells when proteins were or were not added after the washing step (1 nM concentrations). Shown ( C ) are representative dot plots including percentages of nucleocapsid + 229E-infected cells (1 nM concentrations). Shown ( D ) are the percentages of nucleocapsid + cells for each treatment group normalized to the ‘no protein’ group for each virus (1 μM concentrations). Shown ( E ) are the percentages of nucleocapsid + 229E-infected cells for each treatment group at designated concentrations (100 fM to 100 nM). Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test comparing the IFN-β and/or ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration (unless otherwise noted in the figure) (ns nonsignificant, **** p < 0.0001). Experiments shown are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Virus, Incubation, Infection, Staining, Flow Cytometry, Control, Concentration Assay