Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: ACE2 screening in human cells and the characterization of ACE2-NPs. (a) Western blotting detection of ACE2 in five cell lines. β-actin was used as the reference. (b) Immunofluorescence microscopy showing the location of ACE2 (red) in HEK-293T-ACE2 cells. Nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. (c) Immunofluorescence microscopy showing the location of ACE2 (red) and the adherence of S1 (green) on ACE2-NPs. The scale bar indicates 500 nm. (d) TEM image of ACE2-NPs. The scale bar indicates 200 nm. (e) Hydrodynamic diameters and surface charges of NPs. The results are shown as the means ± standard deviations (SDs). (f) ELISA results showing the ACE2 levels in NPs and cell lysates. ***, P < 0.001, relative to the total cell lysate group. (g) Comparison of the amounts of ACE2 antibody bound to ACE2-NPs and HEK-293T-ACE2 cells containing equal amounts of membrane content. n.s., not significant.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Inhibitory effect of ACE2-NPs on S1 recruitment. (a) Binding kinetics for NPs and SARS-CoV-2 RBD loaded on SA biosensors. (b) Western blotting detection of S1 and (e) D614G-S1 binding to HK-2 in the absence and presence of NPs. β-actin was used as the reference. (c) Immunofluorescence microscopy revealing the protective effect of NPs on cells exposed to S1 (green). The region of interest in the S1-treated group is magnified in the inset graph. Nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. (d) Binding kinetics for increasing concentrations of ACE2 and D614G-S1 loaded on SA biosensors. The fitted curves are colored red. The fitting coefficient ( R 2 ) is 0.96.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Binding Assay, Western Blot, Immunofluorescence, Microscopy, Staining

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Inhibitory effect of ACE2-NPs on S1-induced apoptosis. (a) Top nine enriched biological processes in GO analysis. (b) Protein bands of OPA1, cytochrome c, and cleaved caspase (c-caspase) 3. β-actin was used as the reference. (c) OPA1 and (d) cytochrome c mRNA expression relative to β-actin expression. The results are shown as as the means ± SDs. ***, P < 0.001. (e) Apoptosis of HK-2 cells exposed to S1 and D614G-S1. Representative flow cytometry scatter plots of cells stimulated by S1 in the absence and presence of 100 μg mL –1 ACE2-NPs are shown. The overall results are presented in a bar graph. ***, P < 0.001.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Expressing, Flow Cytometry

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Evaluation of the antiviral activity of ACE2-NPs. (a) Immunofluorescence microscopy showing the invasion of S pseudovirions into HK-2 cells. S pseudovirions were traced by S staining using a FITC-labeled antibody (green). Nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. (b) TEM images of S pseudovirions adsorbed onto NPs. The regions of interest are magnified in the inset graphs, in which pseudovirions and NPs are colored red and blue, respectively. The scale bar indicates 200 nm. (c) Dose-dependent antiviral activity of ACE2-NPs and (d) 293T-NPs. The fitted curves obtained by linear regression are in burgundy. The fitting coefficients of ACE2- and 293T-NPs are 0.98 and 0.99, respectively.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Activity Assay, Immunofluorescence, Microscopy, Staining, Labeling

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Inhibitory effect of ACE2-NPs on S pseudovirion infection in mice. (a) Diagram depicting the establishment of the pseudovirion-based mouse infection model. Adenovirus, Adv; pseudovirions, Pv. (b) Protein bands of Flag-tag and His-tag in mouse lungs. β-actin was used as the reference. (c) EGFP mRNA expression relative to β-actin expression in mouse livers, (d) lungs, and (e) kidneys. The results are shown as the means ± SDs. ***, P < 0.001. (f) Immunofluorescence microscopy revealing the inhibitory effect of ACE2-NPs on pseudovirion infection in mouse lungs. Flag-tag and His-tag are shown in red and green fluorescence, respectively. The scale bar indicates 20 μm.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Infection, FLAG-tag, Expressing, Immunofluorescence, Microscopy, Fluorescence

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Distribution and toxicity analysis of ACE2-NPs. (a) ELISA results showing the ACE2 content in mouse serum at 1, 3, 6, and 12 h post injection of ACE2-NPs. (b) In vitro imaging of DiO-labeled ACE2-NPs in mouse kidneys, lungs, spleens, livers, and hearts, at 0.5, 1, 3, 6, 12, and 24 h post intravenous administration. (c) Counts of RBCs, WBCs, and PLTs in mouse blood at 1, 3, and 5 days post injection of ACE2-NPs. The results are presented as the means ± SDs. (d) HE staining of the organs of mice treated with sterile PBS and ACE2-NPs. The scale bar indicates 200 μm.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, In Vitro, Imaging, Labeling, Staining

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: Neutralization of SARS-CoV-2 infection and RBD binding to ACE2 (A) Indicated hmAbs were incubated with live SARS-CoV-2 (100 PFU/well) 1 h before (pre-treatment) or 1 h after (post-treatment) addition to Vero E6 cells. hmAbs were tested in quadruplicate cultures and NT 50 and upper 95% confidence interval (CI) indicated. (B) Representative titration curve of 1212C2 hmAb presented, mean and standard error presented. (C) Binding of indicated hmAb to SARS-CoV-2 or mock-infected Vero E6 cells measured by immunofluorescence; scale bar, 100 μm. (D) Indicated hmAb was incubated as single replicate with recombinant biotinylated RBD protein before incubation with HEK293-ACE2 cells measured by flow cytometry. Plot gated on 7-aminoactinomycin (7AAD)-ACE2 + cells.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Neutralization, Infection, Binding Assay, Incubation, Titration, Immunofluorescence, Recombinant, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: SPR epitope mapping (A) Representative sensorgram from the SPR competition assays used to subset hmAbs into distinct RBD binding epitopes. For each assay, a series of hmAbs were sequentially injected over immobilized SARS-CoV-2 RBD. In this example, 1212C2 was injected first, followed by a second injection of 1212C2, 2 injections of 1206D1, and the last injection was CR3022. (B) Summary of all epitope mapping data, in which each block (first experiment from A in the red box) with a bold hmAb at the top represents a different experiment (10 experiments total). The bold hmAb is the “first” hmAb injected. The percentage of binding (100 = 100% binding and 0 = 0% binding) of subsequent hmAbs was recorded. mAbs were considered to have a different epitope (denoted by a distinct color) if they exhibited binding levels >30% in the presence of other mAbs. Thus, in the first experiment, CR3022 is defined as a new epitope (cyan), distinct from 1212C2. (C) Schematic diagram of NmAb RBD epitopes defined in the mapping experiment. Five major epitopes (A–E) were identified, where the E epitope overlaps with control mAb CR3022 (cyan, epitope F). Four of the 5 epitopes (A–D) are located within the ACE2 binding site (purple), and all of the NmAbs are blocked by the 1212C2 epitope A (yellow). NmAbs with epitopes similar to B (orange) and C (green) are defined as B’ (light orange) and C’ (light green), respectively. The 1212C2 epitope A (yellow) blocks the binding of all NmAbs, with the exception of 1215B11, which occupies epitope E. Epitopes B and C are also blocked by epitope A NmAbs, but exhibit limited competition with each other.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Binding Assay, Injection, Blocking Assay, Control

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet:

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Synthesized, Expressing, Virus, Recombinant, Plasmid Preparation, Binding Assay, Saline, cDNA Synthesis, Transfection, Gel Extraction, Lysis, Luciferase, Software

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Tuning VLP viral tropism by altering viral glycoprotein. ( A ) Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike, or MERS spike) and reporter genes (luciferase or EGFP). ( B ) One microgram of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: WT 293T (293T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3, and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only hACE2-expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, and Calu-3), with SARS exhibiting higher luminescence intensity compared with SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types. ( C ) 3P EGFP-PS9 VLPs were produced with 4 μg VSV-G, 1 μg SARS2 spike, or 1 μg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provide detailed steps for VLP production. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: These include anti-SARS2 N protein mAb 1035111 (IgG2b, Catalog #: MAB10474), anti-SARS2 spike subunit 2 (S2) mAb 1034617 (IgG2a, Catalog #: MAB10557), Alexa Fluor 647-conjugated anti-human ACE2 mAb 535919 (IgG2a, Catalog #: FAB9332R), and rabbit anti-mouse ACE2 mAb 2818I (IgG, Catalog #: FAB34372G), all from R&D Systems (Minneapolis, MN).

Techniques: Produced, Luciferase, Expressing, Infection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: VLPs deliver functional Cas9 mRNA into target cells to achieve gene editing. ( A ) 3P Cas9-P2A-dTo-T20 VLPs (dTo: dTomato) were used for gene editing studies, with surface glycoprotein encoding for either VSV-G or SARS2 spike. ( B ) General workflow of gene editing study performed in panels (C)–(E). sgRNAs were transfected into cells on day −1 using plasmids carrying BFP reporter. VLP-carrying spCas9 mRNA was introduced into cells on day 0. Tropism of the VLP depends on surface glycoprotein. Gene editing efficiency was quantified on day 6. Editing efficiency quantified percentage of BFP(+) population that either turned EGFP(−) (panels C and E) or lost ACE2 expression based on anti-hACE2 binding (panel D). ( C ) sgRNAs targeting EGFP were introduced into 293T-hACE2-EGFP and spCas9 mRNA was delivered using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( D ) sgRNAs against hACE2 were introduced to knockout the receptor in 293T-hACE2 cells using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( E ) sgRNAs targeting EGFP were introduced in 293T-EGFP cells, with genome editing being performed using 3P Cas9-P2A-dTo-T20 VLPs bearing either VSV-G or SARS2 spike. In all panels, the target gene ( EGFP or hACE2 ) was knocked out in 20%–35% of cells expressing sgRNA. Higher VLP amount resulted in greater editing. ( F ) 293T-hACE2 stably expressed sgRNAs against SLC35A1 were infected with 3P SARS2 Cas9-P2A-dTo-T20 VLPs (1.885 μg/μl N protein equivalent) or without SARS2 spike (1.385 μg/μl N protein equivalent). Gene editing efficiency was evaluated based on increase in fluorescent peanut agglutinin lectin (PNA) binding to cells. More than 70% gene editing was observed upon using 3P VLPs to edit endogenous genes. Volume of VLP used in each assay is specified in individual panels. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: These include anti-SARS2 N protein mAb 1035111 (IgG2b, Catalog #: MAB10474), anti-SARS2 spike subunit 2 (S2) mAb 1034617 (IgG2a, Catalog #: MAB10557), Alexa Fluor 647-conjugated anti-human ACE2 mAb 535919 (IgG2a, Catalog #: FAB9332R), and rabbit anti-mouse ACE2 mAb 2818I (IgG, Catalog #: FAB34372G), all from R&D Systems (Minneapolis, MN).

Techniques: Functional Assay, Transfection, Expressing, Binding Assay, Knock-Out, Stable Transfection, Infection

Journal: Virologica Sinica

Article Title: Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

doi: 10.1007/s12250-020-00253-y

Figure Lengend Snippet: Characterization of CR KPTECs. A Organoid cultures of KPTECs. KPTECs were cultured in Matrigel 3D organoid culture as described in Materials and Methods. Left: bright field image of organoids. Middle: beta-catenin and DAPI. Right: Expression of ACE2. Scale bar, 20 μm. B CR KPTECs did not form colonies in soft agar. CR KPTECs did not form colonies in soft agar, but HeLa cells did. Scale bar, 1000 μm. C CR KPTECs maintained ability to repair DNA damage. Phosphorylated H2AX foci and DNA were stained and images were captured using immunofluorensence microscopy. CR KPTECs were able to repair DNA damage induced by irradiation in 24 h. Scale bar, 10 μm.

Article Snippet: Cells were grown on sterile glass cover slips and treated with 5 Gy of gamma irradiation and fixed in 4% (w/v) paraformaldehyde at indicated time points, permeabilized with 0.1% saponin in PBS containing 0.2% gelatin, and labeled with the primary (mouse anti-p63, Santa Cruz, sc-863; Goat polyclonal ACE2 antibody, R&D systems, Abingdon, UK) and secondary (Alexa Fluor 488 donkey anti-mouse IgG, anti-goat Alexa Fluor 488, Molecular Probe) antibodies, according to the manufacturer’s protocol.

Techniques: Cell Culture, Expressing, Staining, Microscopy, Irradiation

Journal: Virologica Sinica

Article Title: Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

doi: 10.1007/s12250-020-00253-y

Figure Lengend Snippet: Expression of endogenous transporters and ACE2 in KPTECs. A Expression of endogenous SLC34A3 and cubilin. Expression of two transporters was measured using quantitative RT-PCR. β-Actin was used as an internal control. Both SLC34A3 and cubilin were expressed in CR KPTECs, but not in HeLa cells. Expression of endogenous ACE2 in CR KPTECs was measured using IF ( B ) and quantitative RT-PCR ( C ). Scale bar, 10 μm.

Article Snippet: Cells were grown on sterile glass cover slips and treated with 5 Gy of gamma irradiation and fixed in 4% (w/v) paraformaldehyde at indicated time points, permeabilized with 0.1% saponin in PBS containing 0.2% gelatin, and labeled with the primary (mouse anti-p63, Santa Cruz, sc-863; Goat polyclonal ACE2 antibody, R&D systems, Abingdon, UK) and secondary (Alexa Fluor 488 donkey anti-mouse IgG, anti-goat Alexa Fluor 488, Molecular Probe) antibodies, according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Pathogens and Immunity

Article Title: Characteristics and Functions of Infection-enhancing Antibodies to the N-terminal Domain of SARS-CoV-2

doi: 10.20411/pai.v9i2.679

Figure Lengend Snippet: Mechanism of infection-enhancement of SARS-CoV-2. (A) Infection-enhancing activity of IgG and purified Fab generated from a panel of NTD-binding, infection-enhancing mAbs. Enhancement was measured in VSV-SARS-CoV-2 S pseudovirus (PV) assays using PV bearing S from Wuhan, Delta, or Omicron variants. Mean values for percentage enhancement by IgG and Fab against each PV were compared using a 2-tailed Student's t test, *** P <0.0001, NS=not significant. (B) Flow cytometry of cell-surface ACE2 expression on 293T-ACE2, intestinal (Caco-2), and respiratory (Calu-3) epithelial cell lines as compared to an isotype matched IgG2A control antibody. MFI=Mean Fluorescent Intensity. (C) Infection of 293T-ACE2, Caco-2 and Calu-3 epithelial cell lines with a Wuhan-PV. Luciferase activity was measured in cell lysates 24 hours after infection and expressed as the median of relative light units (RLU) for 5 replicate wells. (D) Percentage enhancement of Wuhan-PV infection by NTD-binding mAbs in 293T-ACE2, Calu-3, and Caco-2 cells. Mean values for percentage enhancement of infection between 2 cell types were compared using a 2-tailed Student's t test, *** P <0.0001.

Article Snippet: Cells were then stained with either anti-ACE2-Alexa Fluor® 647-conjugated antibody (R&D Systems) or an isotope-matched control mouse IgG2A antibody (R&D systems) for 30 minutes at room temperature.

Techniques: Infection, Activity Assay, Purification, Generated, Binding Assay, Flow Cytometry, Expressing, Control, Luciferase

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: Immunofluorescence staining of ACE2, SARS-CoV-2 N, CD31 in non-COVID-19 and COVID-19 lungs. A Immunofluorescence of lung sections from human non-COVID-19 for ACE2 (magenta), SARS2-N (stain for SARS-CoV-2 nucleocapsid protein in red) and CD31 (stain for endothelium in green). DAPI serves as a nuclear DNA counterstain (blue). Bar = 20 µm. B SARS2-N shows Type I/II pneumocyte infection. In the top row, immunofluorescence of COVID-19 decedent lung sections from patient #3, stained for ACE2 (magenta), SARS2-N (stain for SARS-CoV-2 nucleocapsid protein in red) and CD31 (green). DAPI serves as a nuclear DNA counterstain (blue). Positive N staining shows acute phase of diffuse alveolar damage with sloughed alveolar Type I/II pneumocytes (200× magnification). In the middle row, immunofluorescence of COVID-19 decedent lung sections from patient #6 stained for ACE2, SARS2-N and CD31. Positive N staining shows type 1 cells with long processes along the air sacs (200× magnification). In the bottom row, zoomed insets showing the presence of viral infection in type 1 cells (630× magnification). Bar = 20 µm

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Immunofluorescence, Staining, Infection

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: Endotheliitis is seen in tissue sections of COVID-19 lung endothelium. Immunofluorescence of COVID-19 decedent lung sections from patient #3, stained for ACE2 (magenta), SARS2-N (stain for SARS-CoV-2 nucleocapsid protein in red) and CD31(endothelium in green). Zoomed insets showing the presence of viral infection in these larger vessels (diameter > 100 µm) was consistently accompanied by signs of endothelial damage and vasculopathy, including disruption of the endothelial lining, sloughing of ECs into the vascular lumen, and apoptotic ECs with distorted nuclear shape (630× magnification). Yellow arrowheads indicate positive ACE2 staining and white arrowheads indicate positive SARS2-N staining in the cytoplasm. ACE2, SARS2-N and CD31 are colocalized in an apoptotic EC. Bar = 20 µm

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Immunofluorescence, Staining, Infection, Disruption

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: Immunofluorescence staining of mouse ACE2 and SARS2-N in tissue sections of K18-hACE2 transgenic mice. vWF (stain for endothelium in green), mouse ACE2 (red), SMA (stain for smooth muscle cell layer in green), SARS2-N (red) and DAPI serves as a nuclear DNA counterstain (blue). A Immunofluorescence of lung sections from uninfected K18-hACE2 transgenic mice. White arrowheads indicate co-localization of vWF and mouse ACE2 staining. Bar = 20 µm. B Immunofluorescence of lung sections from SARS-CoV-2-infected K18-hACE2 transgenic mice, 3.81 × 10 3 TCID 50 at 4 dpi. Bar = 20 µm

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Immunofluorescence, Staining, Transgenic Assay, Infection

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: IFNα induces ACE2 in human primary endothelial cell cultures. A Relative mRNA expression of ACE2 in different types of human endothelial cells, including the pulmonary arterial (PA), three pulmonary microvasculature (PM -1, -2, -3), myocardium (Myocardial), aorta (Aortic), cardiac microvascular (Cardiac), white adipose(Adipose), brain microvasculature (Brain) and bone (Bone). Endothelial expression is relative to that from the 16HBE human bronchial epithelial cell line (16HBE). Means ± SEM are from two technical replicates. B Relative mRNA expression of ACE2 in human pulmonary arterial endothelial cells (PAECs) when incubated in cytokine-free media (ϕ); with a cytokine cocktail (CC—consisting of IFNα, IFNγ, TNFα, IL6 & CXCL10); or with IFNα alone for 6, 24 and 6–24 h (a total of 6 h simulation and then total RNA collect in 24 h). ACE2 expression level in cytokine-free media were used as control. Means ± SEM derived from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to untreated control, unpaired t test. C IFNα dose-dependent mRNA expression of ACE2 in human PAEC. Fold-change of ACE2 expression is relative to that in untreated cells. Means ± SEM are from two biological replicates. D Relative mRNA expression of ACE2 in human PMVEC after IFNα incubation for 1, 3, 6, 9, 24 and 48 h, in a time-dependent manner. Means ± SEM are from two biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to untreated control (0 h), unpaired t test. E Immunofluorescence for ACE2 (magenta) TMPRSS2 (red) and CD31 (green) in PAECs treated or not with IFNα (7.4 × 10 5 units/mL) for 6, 24 and 6–24 h. Zoomed insets showing ACE2 expression on the cell membrane (630X magnification). Bar = 20 µm

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Expressing, Incubation, Control, Derivative Assay, Immunofluorescence, Membrane

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: SARS-CoV-2 infection in human primary arterial endothelial cells. A Representative Western blot results of ACE2 and TMPRSS2 protein expression in PAECs treated with IFNα (7.4 × 10 5 units/ml) for 6, 12, 16 and 24 h. 60ug total protein was loaded in each lane. β-actin serves as a loading control. B Optical Density (OD) quantification of ACE2 protein levels from A. Means ± SEM are from 4 biological replicates. * p < 0.05, ** p < 0.01 compared to untreated, unpaired t test. C GFP expression in PAECs after d19/ R682Q modifications in Spike pseudoviral transduction (200× magnification). Left: PAECs only; middle: without IFNα stimulation but transduced with pseudovirus; right: with IFNα stimulation and transduced with pseudovirus. Bar = 20 µm D Quantification of GFP positive cells vs. total number of cells from C. *** p < 0.001 compared to its control, unpaired t test. E Immunofluorescence images of viral nucleoprotein (SARS2-N, red) of PAECs infected with SARS-CoV-2 (MOI = 1 or 5) at 1 day post infection (200X magnification). Left: no IFNα stimulation but infected at MOI 5; middle left: IFNα 24 h but no viral infection; middle right: IFNα 24 h, infected at MOI 1; right: IFNα 24 h, infected at MOI 5. Bar = 20 µm. F Quantification of the number of positive SARS2-N cells (cytoplasmic red staining along with a nuclear DAPI staining) versus the total number of cells (the number of positive nuclear DAPI staining). Means ± SEM are calculated on six images from random fields with two biological replicates. * p < 0.001, *** p < 0.001, **** p < 0.0001 compared to its control, unpaired t test

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Infection, Western Blot, Expressing, Control, Transduction, Immunofluorescence, Staining

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: SARS-CoV-2 or Ebola virus infection of human primary PAECs after IFNα or β stimulation. A Schematic representation of ACE2 and delta ACE2(dACE2) transcripts and the position of the three PCR probes to generate wildtype ACE2, delta(d)ACE2 and full length ACE2 amplicons. B Expression of total ACE2 (left) and expression of full length ACE2 (right) in three PMECs after treated with IFNα, β, γ, λ for 6 h. C Representative Western blot results of ACE2 and TMPRSS2 protein expression in PAECs treated with IFNα or β for 24 h. 60 μg total protein was loaded in each lane. GAPDH serves as a loading control. Optical Density (OD) quantification of ACE2 protein levels vs GAPDH. Means ± SEM are from three biological replicates. D Immunofluorescence images of viral nucleoprotein (SARS2-N, red) of PAECs infected with SARS-CoV-2 (MOI = 5) at 1 day post infection (200X magnification). Left: Mock; middle: no IFNβ; right: IFNβ 24 h. Bar = 20 µm. E Immunofluorescence images of viral nucleoprotein (SARS2-N, red) of PAECs infected with recombinant SARS-CoV-2-mNeonGreen (MOI = 5) at 1 day post infection (200× magnification). Left: Mock; middle left: no IFN; middle right: IFNα; right: IFNβ 24 h. Bar = 20 µm. F Immunofluorescence images of viral nucleoprotein (EBOV NP, green) of PAECs at 24 h post infection or at 48 h post infection ( G ). Cells were pretreated with IFNs for 24 h and infected with wildtype Ebola virus (EBOV) (MOI = 5) Left: Mock; middle left: no IFN; middle right: IFNα; right: IFNβ. Bar = 50 µm. Quantification (both D , E , F and G ) of the number of positive SARS2-N or EBOV NP (cytoplasmic positive staining along with a nuclear DAPI staining) versus the total number of cells (the number of positive nuclear DAPI staining). Means ± SEM are calculated on six images from random fields with two technical replicates. * p < 0.05, *** p < 0.001 and **** p < 0.0001 compared to its control, unpaired t test

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Virus, Infection, Expressing, Western Blot, Control, Immunofluorescence, Recombinant, Staining

Journal: Angiogenesis

Article Title: Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2

doi: 10.1007/s10456-021-09823-4

Figure Lengend Snippet: Sequences and amplicons info of qPCR probe sequences for total ACE2, dACE2 and full length ACE2

Article Snippet: Primary antibodies of interest, including rabbit anti-vWF (1:200; A008202-5, Agilent), rabbit anti- SARS-N protein (1:500; 200-401-A50 Rockland), rat anti-mouse ACE2 (1:50; MAB3437, R&D Systems) and SMA-Alexa 647(1:50, sc-32251, Santa Cruz).

Techniques: Sequencing, Amplification

Journal: Frontiers in Microbiology

Article Title: A novel hACE2 knock-in mouse model recapitulates pulmonary and intestinal SARS-CoV-2 infection

doi: 10.3389/fmicb.2023.1175188

Figure Lengend Snippet: Generation and genetic characterization of human angiotensin-converting enzyme 2 (hACE2) knock-in (KI) mice. (A) The strategy of generation of hACE2 KI mice. A pair of single guide RNAs (sgRNAs) cutting exons 2 and 19 of mouse ACE2, respectively, was used to replace the translated exons of mouse ACE2 with the human ACE2 coding sequence (CDS) without the sequence coding signal peptide, and the human ACE2 CDS with signal peptide was placed downstream the sequence coding mouse ACE2 signal peptide as a result. Black boxes: untranslated exonic sequences of mouse ACE2; white boxes: translated exonic sequences of mouse ACE2; green box: human ACE2 CDS without signal peptide; blue lines: introns of mouse ACE2; the arrow in dotted line: the transcription direction; F1, F2, R1, and R2 show the positions of primers used for genetic screening of hACE2 KI mice; red lines indicate the locations of probes for Southern blotting. (B,C) Transgenic founder mice were subjected to genetic screening by PCR using primer pairs F2/R2 (B) and F1/R1 (C) , which covered the 5′ and 3′ junction regions of hACE2 KI allele, respectively. The PCR products were sequenced and the sequence chromatography images are shown. (D) Southern blotting of homozygous hACE2 KI individuals. The genomic DNAs of the homozygous individuals were completely digested by the endonucleases Bam HI and Mfe I, and then subjected to Southern blotting using the 5′ and 3′ probes shown in A, respectively. The sizes of Southern blot bands with the 5′ probe were 4.54 kb in the wild-type (WT) allele and 3.00 kb in the hACE2 KI allele, and those with 3′ probe were 10.83 kb in the WT allele and 4.25 kb in the hACE2 KI allele. WT: wild-type mouse genomic DNA; ~1–3: homozygous KI individuals.

Article Snippet: The primary antibodies were human ACE2 antibody (Sino Biological, Beijing, China; 10,108-RP01) and mouse ACE2 antibody (R&D Systems, Minneapolis, MN, United States; MAB34372), and both were used at 1:50 dilution.

Techniques: Knock-In, Sequencing, Southern Blot, Transgenic Assay, Chromatography

Journal: Frontiers in Microbiology

Article Title: A novel hACE2 knock-in mouse model recapitulates pulmonary and intestinal SARS-CoV-2 infection

doi: 10.3389/fmicb.2023.1175188

Figure Lengend Snippet: Expression of hACE2 in hACE2-KI mouse model. (A) The expression pattern of hACE2 mRNA in different organs including brain, heart, intestines, kidneys, liver, lungs, spleen, stomach, and trachea was detected by real-time quantitative PCR (qPCR) assay. (B) Representative immunoblotting image shows the expression of the ACE2 protein in mouse intestine and lung tissues normalized to total protein. Human ACE2 was highly expressed in the lungs and intestines of hACE2-KI mice, while mouse ACE2 protein was detected in WT mice but not in hACE2-KI mice. (C) Immunochemistry results of the hACE2 expression in different organs. The intestines, lungs, and trachea sections are shown in high-power magnification; scale bar: 100 μm. Statistical analysis was performed using unpaired Student’s t -test or Wilcoxon rank test. NS: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The primary antibodies were human ACE2 antibody (Sino Biological, Beijing, China; 10,108-RP01) and mouse ACE2 antibody (R&D Systems, Minneapolis, MN, United States; MAB34372), and both were used at 1:50 dilution.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: medRxiv

Article Title: The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera

doi: 10.1101/2021.01.19.21249592

Figure Lengend Snippet: (A) Strategy: Vero-E6 cells were transduced with a lentiviral vector expressing mACE-2 and a puromycin resistance gene. Cells were selected for mACE-2 expression by puromycin selection. (B) Expression in the selected polyclonal population was confirmed by Western blot.

Article Snippet: Anti-Tubulin and anti-mACE-2 (R&D Systems, Cat# MAB3437) primary antibodies were used at dilution of 1:1000 while secondary HRP-conjugated antibodies were used at dilutions of 1:10000 in 3% BSA-containing TBST.

Techniques: Transduction, Plasmid Preparation, Expressing, Selection, Western Blot