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

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: Primer-specific RT-qPCR for relative mRNA expression of mouse ACE2 (mACE2) (A), human ACE2 (hACE2) (B), and mouse ACE1 (mACE1) (C) from lung tissue homogenates of wild-type control (C57BL/6J) mice, K-18 promoter-driven hACE2 overexpression ( K18-hACE2 ) mice, and humanized ACE2 ( hACE2 ) mice (**, P<0.0005; ***, P<0.0001; compared to C57BL/6J by ANOVA; n=8, 4 male and 4 female mice). (D) Representative photomicrographs of hACE2 (red) expression in lung airways marked for alpha-tubulin (green) with corresponding quantitative data (E). Hoechst, blue. Bar, 200 microns. Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Quantitative RT-PCR, Expressing, Control, Over Expression

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: (A) Kaplan-Meier plot for survival post intranasal infection of C57BL/6J, K18-hACE2 , and hACE2 mice with 1000 PFU/dose of SARS-CoV-2 or mock infection with vehicle control (***, P<0.0001 by log rank compared to mock or C57BL/6J controls; n=16, 8 male and 8 female, for SARS-CoV-2 infections; n=8, 4 male and 4 female, for vehicle control). (B) Percent body weight relative to pre-infection weight over time after SARS-CoV-2 infection carried out as in (A) (***, P<0.0001 compared to mock or C57BL/6J controls using ANOVA; n=16, 8 male and 8 female, for SARS-CoV-2 infections; n=8, 4 male and 4 female, for vehicle controls). Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Infection, Control

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: (A) Heat map depicting relative plasma inflammatory chemokine, cytokine, and growth factor expression patterns, as measured by Luminex® INTELLIFLEX, from C57BL/6J, K18-hACE2 , or hACE2 mice 6 days after intranasal infection with 1000 PFU/dose of SARS-CoV-2. Pattern 1 revealed elevations specific to K18-hACE2 mice and included CCL5 (B), CCL7 (C), IL-6 (D), LEPTIN (E), TNF-α (F), and CSF-3 (G) (**, P<0.001; ***, P<0.0001 compared to all others using ANOVA; n=12, 6 male and 6 female). Pattern 2 revealed elevations in K18-hACE2 and hACE2 mice and included CCL2 (H) and IL-10 (I) (*, P<0.05; ***, P<0.0001 compared to C57BL/6J using ANOVA; n=12, 6 male and 6 female). Pattern 3 revealed elevations specific to hACE2 mice and included IL-1α (J), IL-1β (K), IL-17α (L), and IL-22 (M) (**, P<0.001; ***, P<0.0001 compared to all others using ANOVA; n=12, 6 male and 6 female). Pattern 4 revealed decreased expression specific to K18-hACE2 mice and included VEGF-A (N) (***, P<0.0001 compared to all others using ANOVA; n=12, 6 male and 6 female). Pattern 5 revealed decreased expression in both the K18-hACE2 and hACE2 mice and included IL-33R (O) (***, P<0.0001 compared to C57BL/6J using ANOVA; n=12, 6 male and 6 female).

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Expressing, Luminex, Infection

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: (A) Representative immunofluorescent photomicrographs from lungs of C57BL/6J, K18-hACE2 , and hACE2 mice harvested on day 6 post intranasal infection with 1000 PFU/dose of SARS-CoV-2. Blue, Hoechst. Red, fluorescent in situ hybridization (FISH) for SARS-CoV-2 negative strand. Green, CD68. Bar, 200 microns. Quantitation of the number of CD68 + cells (B), number of FISH + cells (C), and number of CD68 + FISH + cells (D). (**, P≤0.008; ***, P<0.0001 compared to all others using ANOVA; ††† , P<0.0001 compared to C57BL/6J using ANOVA; n=8, 4 male and 4 female).

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Infection, In Situ Hybridization, Quantitation Assay

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: (A) BMDMs from control or myeloid IL-1β -deleted mice (mIL-1β KO) hACE2 mice were primed with or without LPS (10 ng/mL) and exposed to cholesterol crystals (0 or 1000 μg/mL) for 24 hours to induce inflammasome activation, followed by RT-qPCR for relative IL-1β mRNA expression (***, P<0.0001 compared to all others using ANOVA; n=6 mice total, 3 males and 3 females). (B) BMDMs treated as in (A), followed by ELISA on cell culture supernatants for secreted, mature IL-1β protein (***, P<0.0001 compared to all others using ANOVA; n=6 mice total, 3 males and 3 females). (C) BMDMs treated as in (A), followed by quantitative RT-qPCR for relative hACE2 mRNA expression (***, P<0.0001 compared to all others by ANOVA; n=6 mice total, 3 males and 3 females). (D) Quantitative PCR of the ACE2 promoter after ChIP, using antibody specific to p65 (RELA) relative to isotype control, of lysate from BMDMs stimulated with LPS+IFN-γ for 24 hours (***, P=0.0001 by t -test; n=6 mice total, 3 males and 3 females). (E) BMDMs from control or mIL-1β KO mice were treated with LPS+IFN-γ for 24 hours in the presence or absence of the NF-κB inhibitor, JSH23, at indicated concentrations, followed by RT-qPCR for relative hACE2 mRNA expression in cell lysates (***, P≤0.0001 compared to control at 0 concentration by ANOVA; n=6 mice total, 3 males and 3 females). (F) Volcano plot demonstrating differential RNA-seq transcriptome data from BMDMs cultured from hACE2 mice and stimulated with LPS+IFN-γ for 18 hours, followed by infection with SARS-CoV-2 for 24 hours (n=3 male mice). Dashed line, P adj =0.05. (G) Heatmap illustrating normalized counts for the top 20 differentially expressed genes derived from (F) with each column representing an individual mouse and each row representing mRNA from a single gene (n=3 male mice). Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Control, Activation Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Concentration Assay, RNA Sequencing Assay, Infection, Derivative Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-B) A549 cells were transduced to stably overexpress ACE2 (A549-A), DPP4 (A549-D) in the absence or presence of TMPRSS2 (A549-AT and A549-DT). ACE2 and DPP4 expression was confirmed by western blot (A) , while TMPRSS2 expression was confirmed using flow cytometry (B) . (C-D) A549-derived or Calu-3 cells were inoculated with lentiviral particles pseudotyped with the spike proteins of MERS-CoV (C) , VSV, SARS-CoV-1, WIV1-CoV, WIV16-CoV, SARS-CoV-2 Hu1, SARS-CoV-2 delta or SARS-CoV-2 omicron (D) or pseudoparticles lacking envelope protein (no env) (C-D) for 2 h, then incubated for an additional 72 h, at which point luciferase activity was measured to assess pseudoparticle entry. The data are expressed as fold change relative to the luciferase signal obtained with no envelope. Graphs show mean +/- SEM from three independent experiments performed in triplicate.

Article Snippet: For the detection of cell-surface ACE2 expression, washed cells were resuspended in 100 μL of FACS buffer with 20 μg/mL (1:50) of anti-human ACE2 polyclonal antibody (Invitrogen #PA5-116467) for 1 h at room temperature.

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Derivative Assay, Incubation, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: BEAS-2B cells were transduced to stably overexpress ACE2 (BEAS-2B-ACE2). Expression of ACE2 and endogenous TMPRSS2 was confirmed by western blot (A). (B) BEAS-2B-ACE2 cells were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations, then infected with SARS-CoV-2pp for 2 h at 37°C. Inocula were removed and cells were incubated in complete media for 72 h, at which point luciferase activity was measured to assess viral entry. (C-D) BEAS-2B-ACE2 cells were pre-treated with NanH diluted to 50 μg/mL in serum-free media for 30 minutes at 37°C. Cells were then washed and processed for fluorescence microscopy (C) or inoculated with SARS-CoV-2 pseudoparticles (D) . NanH-treated cells were stained with SNA-FITC (binds sialic acid) or ECL-FITC (binds galactose) diluted to final concentration of 20 μg/mL in PBS, then washed with PBS and imaged by fluorescence microscopy (10X magnification; scale bar, 200 μm). Lectin staining confirmed removal of sialic acid by the NanH treatment. (E-F) BEAS-2B-ACE2 cells were treated with protease inhibitors or NanH as described (B-C) , then infected with replication-competent recombinant VSV-SARS-CoV-2-S expressing GFP for 2 h. After 7.5 h, cells were fixed and GFP fluorescence was assessed. Representative images are shown (20X magnification; scale bar, 50 μm). The percentage of infected cells in each condition was determined using ImageJ. Graphs show mean +/- SEM from three independent experiments performed in triplicate. Statistical significance was assessed by one-way or two-way ANOVA (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: For the detection of cell-surface ACE2 expression, washed cells were resuspended in 100 μL of FACS buffer with 20 μg/mL (1:50) of anti-human ACE2 polyclonal antibody (Invitrogen #PA5-116467) for 1 h at room temperature.

Techniques: Stable Transfection, Expressing, Western Blot, Infection, Incubation, Luciferase, Activity Assay, Fluorescence, Microscopy, Staining, Concentration Assay, Recombinant

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a – c Modulation after transfection: Flow cytometry staining of THP-1 cells for HLA-A/B/C, CD80, CD275, and HLA-DR surface expression 48 h after transfection with expression plasmids for ORF6 ( a ), ORF8 ( b ), or Envelope ( c ) proteins, compared with control transfection. d – i Modulation after infection: d A549-ACE2-TMPRSS2 cells were infected with recombinant wild-type (rCoV2), E**fs, ΔE G 68, or XBB.1.5 SARS-CoV-2 virus (MOI = 0.1) for 24 h and stained for HLA-A/B/C, CD44 and CD275. e Median fluorescence intensity (MFI) of HLA-A/B/C and CD275. The same infection was conducted on HEK293T-ACE2 and their respective supernatant was then applied on THP-1 for 48 h before surface staining and analysis. f Histogram showing the expression of CD44, HLA-A/B/C, CD80, CD275, and HLA-DR on THP-1 after 48 h. g Median fluorescence intensity of CD44, HLA-A/B/C, CD80, and CD275 markers on THP-1 after 48 h incubation. h Comparison of wild-type or ΔE G 68 conditions for their expression of CD80 and HLA-A/B/C. The frequency of cells inside the gate in ( h ) is shown in ( i ). Median is shown for ( e ) and ( g ), mean and S.D. for ( i ). The gating strategy is shown in Supplementary Fig. .

Article Snippet: Cells were sorted for surface expression of ACE2 stained by Mouse anti-human ACE2 (R&D #FAB9332G).

Techniques: Transfection, Flow Cytometry, Staining, Expressing, Control, Infection, Recombinant, Virus, Fluorescence, Incubation, Comparison

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a General scheme of the experiment, monocytes from vaccinated donors are differentiated into macrophages while a 24-h infection with wild-type (rCoV2), ΔE G 68, or E** fs at an MOI of 0.1 is started in A549-ACE2-TMPRSS2 cells. Cellular supernatant is then put in contact with the macrophages for 24 h before matching PBMCs and viruses at an MOI of 0.1 are added. After 24 h, cells are analyzed by flow cytometry ( n = 6). Comparison of IFNγ geometric mean fluorescence intensity (gMFI) ( b ), frequency of CD69 + ( c ) CD137 + ( d ), and CD69 + /CD137 + ( e ) in the T cell memory compartment (CD3 + /CD45RA - ). Comparison of frequencies of IFNγ + ( f ), IFNγ gMFI ( g ), frequency of CD69 + ( h ) or CD69 + /CD137 + ( i ) in actively secreting memory T cells (CD3 + /CD45RA - /CD107a + ). Comparison of IFNγ ( j ), CD137 ( k ), CD69 ( l ), and CD3 gMFIs ( m ) in activated T cells (CD3 + /CD45RA - /CD69 + /CD137 + ). The gating strategy is shown in Supplementary Fig. . The median is shown for all graphs, Friedman test with Dunn’s multiple comparisons ( b , g , j – m ), or one-way ANOVA with Tukey’s multiple comparison tests ( c – f , h , i ) were performed, respectively. The Friedman test is based on sample ranking, and due to the limited number of samples compared here, identical p values may result. Illustrations in ( a ) were created with BioRender.com.

Article Snippet: Cells were sorted for surface expression of ACE2 stained by Mouse anti-human ACE2 (R&D #FAB9332G).

Techniques: Infection, Flow Cytometry, Comparison, Fluorescence