Journal: bioRxiv

Article Title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

doi: 10.1101/2020.11.05.369264

Figure Lengend Snippet: a, Cells transfected with ACE2, L-SIGN, DC-SIGN, and CD147 (red line) or mock (shaded gray) were stained with SCoV2-NTD-Fc and RBD-Fc fusion protein (10 μg/mL). Each transfectant was also stained with a specific monoclonal antibody. b, Effect of mannan and anti-CD209 (anti-DC-SIGN) antibody on SCoV2-NTD-Fc binding to DC- and L-SIGN transfectants or mock (shaded gray). The transfectants were preincubated with mannan (light blue line) or anti-CD209 antibody (dark blue), followed by the staining with NTD-Fc fusion protein. c, Staining of DC- and L-SIGN transfectants (red line) or mock (shaded gray) with SCoV2-NTD-Fc and SCoV-NTD-Fc fusion proteins (10 μg/mL). d, Cells transfected with flag-tagged spike proteins of SCoV2, SCoV, human coronavirus OC43, or HKU1 (red line) or mock (shaded gray) were stained with DC-, L-SIGN-Fc fusion proteins or anti-Flag-tag antibody. Proportions of the stained cells are shown. Data are representative of three independent experiments.

Article Snippet: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Transfection, Staining, Binding Assay, FLAG-tag

Journal: bioRxiv

Article Title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

doi: 10.1101/2020.11.05.369264

Figure Lengend Snippet: a, SCoV2-PV infection on DC-SIGN, L-SIGN, mock, or ACE2 transfectants and Vero E6 cells. SCoV2-PV carrying a luciferase gene was used for the infection and luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived from unpaired T-test; * P = 0.0018; ** P = 0.0003 b, DC-SIGN or L-SIGN transfectants were infected with recombinant SCoV2 with the NanoBiT luciferase gene. The luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived from unpaired T-test; * P = 0.001; ** P = 0.0006. c, Cell-cell fusion assay of SCoV2 spike transfectants and DC- or L-SIGN transfectants. The effector cells expressing spike and T7 polymerase were cocultured with target cells expressing DC-SIGN, L-SIGN, mock, and T7 promoter-driven luciferase. Luciferase activities were measured after 24 h. Asterisks indicate statistical significance derived from unpaired T-test *** P <0.0001. d, Cell-cell fusion assay of SCoV2 spike transfectants (red) and DC-SIGN transfectants (green). Representative images are shown. Scale bar represents 50 μm in length. Data are representative of three independent experiments.

Article Snippet: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Infection, Luciferase, Activity Assay, Derivative Assay, Recombinant, Cell-Cell Fusion Assay, Expressing

Journal: Nature Communications

Article Title: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

doi: 10.1038/s41467-021-22781-1

Figure Lengend Snippet: a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

Article Snippet: For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively.

Techniques: Expressing, Immunohistochemistry

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Antibodies used for immunofluorescence staining.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence, Staining

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Composition of antibodies used in the multiplexed immunofluorescence panels.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: ( A , B ) Multiplex imaging analysis depicting expression of ACE2, the major receptor for SARS-CoV-2, in different cellular compartments of lung tissues in a representative patient with COVID-19 (case #6). ( A ) Red arrows point to ACE2-expressing pneumocytes, defined by double-expression of ACE2 (yellow) and PANCK (white). ( B ) Cyan arrows highlight ACE2-expressing macrophages, defined by double-expression of ACE2 (yellow) and CD68 (red). All images were acquired on the Vectra Polaris microscope using a 20x objective magnification, and zoomed in areas are depicted for visualization of macrophages. ( C ) The was no difference in the relative frequencies (% of total imaged cells) of CD3+ T cells in lung control tissues (non-COVID-19 DAD, n = 5) and COVID-19 lung tissues ( n = 5), expressed as mean values with SD at the scatter dot plots (Mann–Whitney U test, p = 0.87). ( D ) There was no statistically significant difference of the CD4+ to CD8+ T lymphocyte ratio between the two cohorts (Mann–Whitney U test, p = 0.55).

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Multiplex Assay, Imaging, Expressing, Microscopy, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

doi: 10.3389/fimmu.2022.1014309

Figure Lengend Snippet: Overview of the SARS-CoV-2 neutralization multiplex assay by flow cytometry (nC19BA). (A) Beads with different fluorescence intensities are coated with recombinant RBD proteins from the indicated variants and incubated with a given sample, followed by incubation with rhACE2-mIgG1. The binding of rhACE2 to RBD is detected with an anti-mouse IgG1 monoclonal antibody labelled with BV421 fluorochrome. Schematic created using BioRender.com. (B) Schematic illustration showing the mutations present in the RBD variants included in this assay. (C) Representative dot plots showing rhACE2 binding to the indicated RBD variants. SP, Signal peptide; NTD, N-terminal domain; RBD, Receptor binding domain; S2, S2 subunit; TM, transmembrane domain; CT, cytoplasmic tail .

Article Snippet: Human recombinant ACE2 with murine IgG1 (rhACE2-mIgG1, recombinant fusion protein of human ACE2 ectodomain fused with murine IgG1 Fc produced in HEK293T eukaryotic cells, SinoBiological 10108-H05H) was added at 0.18 mg/mL (diluted in 100 µL of PBS containing 5% FBS) and incubated for 30 minutes at 4°C protected from light.

Techniques: Neutralization, Multiplex Assay, Flow Cytometry, Fluorescence, Recombinant, Incubation, Binding Assay

Journal: Frontiers in Immunology

Article Title: A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

doi: 10.3389/fimmu.2022.1014309

Figure Lengend Snippet: Comparison of the performance of nC19BA against a cell-based neutralization assay. (A) Representative dot plots showing the percentage of infected HEK293T (left), HEK293T-ACE2 (middle) and HEK293T-ACE2 cells in the presence of Imdevimab (right), after incubation with pseudotyped viral particles expressing the Spike of SARS-CoV-2, measured by flow cytometry. (B) Comparison of the blocking activity achieved by the indicated concentrations of Imdevimab measured by two independent methods: a cell-based pseudotyped lentiviral assay (black) or the bead-based nC19BA assay (red). (C) Pearson correlation corresponding to the blocking activity of Imdevimab measured by nC19BA and a cell-based pseudotyped lentiviral assay. Pearson correlation coefficient (r), 95% confidence interval and its corresponding p-values are shown. (D) Bland-Altman analysis showing the agreement between nC19BA and a cell-based pseudotyped lentiviral assay for the measurement of the blocking activity of Imdevimab. (E) Pearson correlation corresponding to the blocking activity of preCOVID (n=10) and seropositive (n=18) serum samples corresponding to the indicating cohorts, measured by nC19BA and a cell-based pseudotyped lentiviral assay. Pearson correlation coefficient (r), 95% confidence interval and its corresponding p-values are shown. (F) Bland-Altman analysis showing the agreement between nC19BA and a cell-based pseudotyped lentiviral assay for the measurement of the blocking activity of serum samples.

Article Snippet: Human recombinant ACE2 with murine IgG1 (rhACE2-mIgG1, recombinant fusion protein of human ACE2 ectodomain fused with murine IgG1 Fc produced in HEK293T eukaryotic cells, SinoBiological 10108-H05H) was added at 0.18 mg/mL (diluted in 100 µL of PBS containing 5% FBS) and incubated for 30 minutes at 4°C protected from light.

Techniques: Neutralization, Infection, Incubation, Expressing, Flow Cytometry, Blocking Assay, Activity Assay

Journal: PLoS ONE

Article Title: Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

doi: 10.1371/journal.pone.0001082

Figure Lengend Snippet: (A) Vero E6 cells stained with propidium iodide. Light microscopy shows a syncytium (white arrow) in the center of the field of view (top), viewed under propidium iodide fluorescence (middle), and an overlay of both images (bottom). (B) Immuno-EM with 10 nm gold confirmed the attachment of ACE2 to the spikes. (C) Cryo-EM field of view of SARS-CoV decorated with ACE2, and select two-dimensional class averages of the SARS spike-ACE2 complex (D). Cryo-EM reconstruction of the SARS spike (E), and SARS spike-ACE2 complex (F), viewed from side and end-on perspectives (left, right). Color scheme: ACE2, violet; spike, green; stalk, blue; envelope, beige; nucleocapsid, red. Scale bars: (A) 50 µm, (B,C) 1000 Å, (D–F) 100 Å.

Article Snippet: The human ACE2-Fc (human IgG1) recombinant protein (Adipogen) had an initial concentration of 1 mg/ml in PBS and was diluted 1∶10 in PBS for incubation with SARS-CoV, for the remainder of this document this is referred to as ACE2.

Techniques: Staining, Light Microscopy, Fluorescence, Cryo-EM Sample Prep

Journal: PLoS ONE

Article Title: Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

doi: 10.1371/journal.pone.0001082

Figure Lengend Snippet: The resolution was estimated by Fourier shell correlation (FSC) between two half reconstructions of the SARS spike-ACE2 complex. The resolution was estimated to be 18.5 Å using the 0.5 FSC criteria (red dotted red line).

Article Snippet: The human ACE2-Fc (human IgG1) recombinant protein (Adipogen) had an initial concentration of 1 mg/ml in PBS and was diluted 1∶10 in PBS for incubation with SARS-CoV, for the remainder of this document this is referred to as ACE2.

Techniques:

Journal: PLoS ONE

Article Title: Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

doi: 10.1371/journal.pone.0001082

Figure Lengend Snippet: The cryo-EM reconstruction of the SARS spike (A,F) was subtracted from the SARS spike-ACE2 complex (E,J). The positive component attributed to the SARS spike in (B,G) indicates a re-arrangement in the S2 core, and the positive component attributed the SARS-CoV–ACE2 complex shows the addition of ACE2 and an exterior re-arrangement in S1 (D,I). The net difference map is presented in (C,H). The structures are presented from a side perspective (A–E), and end-on perspective (F–J). The arrows in (F) and (J) illustrate the mass reorganization that occurs in the central axis of the spike where one small central blob splits into three nubs. In (A,F) the region on the spike adjacent to ACE2 which corresponds to the receptor binding domain, has been highlighted with a dotted line and is colored purple. The color scheme used for the cryo-EM structure is the same as in . Scale bar, 100 Å.

Article Snippet: The human ACE2-Fc (human IgG1) recombinant protein (Adipogen) had an initial concentration of 1 mg/ml in PBS and was diluted 1∶10 in PBS for incubation with SARS-CoV, for the remainder of this document this is referred to as ACE2.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: PLoS ONE

Article Title: Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

doi: 10.1371/journal.pone.0001082

Figure Lengend Snippet: The atomic resolution structures; PDB ID code, 2AJF (ACE2, blue; receptor-binding domain, red) and PDB ID code, 2FXP (yellow) were docked within the SARS spike-ACE2 complex using SITUS. The SARS spike-ACE2 complex is shown from (A) side and (B) end-on perspectives, the arrow points to the C-terminus of ACE2. The color scheme used for the cryo-EM structure is the same as in . Scale bar, 100 Å.

Article Snippet: The human ACE2-Fc (human IgG1) recombinant protein (Adipogen) had an initial concentration of 1 mg/ml in PBS and was diluted 1∶10 in PBS for incubation with SARS-CoV, for the remainder of this document this is referred to as ACE2.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) Bar graph showing the percentage of cells with detectable ACE2 in islets from pancreata of donors with (n=2,705 cells) and without type 2 diabetes (n=12,185 cells). (B) Violin plot showing the distribution of ACE2 normalized expression in islet cells from pancreata of non-diabetic donors. (C) Bar graph showing the percentage of cells with detectable TMPRSS2 in islets isolated from pancreata of donors with (n=2,705 cells) and without type 2 diabetes (n=12,185 cells); * P <0.05, paired t-test for indicated comparisons. (D) Violin plot showing the distribution of TMPRSS2 normalized expression in islets cells from pancreata of non-diabetic donors; ***adjusted P <0.001, Wilcoxon rank sum tests. (E) Representative images of smFISH for ACE2 and TMPRSS2 mRNA in human pancreatic tissue sections counter stained for insulin (magenta). Inset highlights mRNA distribution in pancreatic ducts; scale bar: 20µm. (F) Representative smFISH images showing the presence of ACE2 and TMPRSS2 in CD34 positive cells in human pancreatic tissue sections; scale bar: 10µm. (G) Representative images of smFISH for ACE2 and TMPRSS2 mRNA in human pancreatic tissue sections counter stained for insulin (magenta). Inset highlights distribution in the endocrine pancreas; scale bar: 20µm. See also Table S1, Fig. S1, Table S2 , and Fig. S2A .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Expressing, Isolation, Staining

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) ACE2 protein structure illustrating the location of respective antibody directed antigen sites: SP, signal peptide; TM, transmembrane domain; CD, cytoplasmic domain. (B) Immunoblot analysis of four commercially available ACE2 antibodies using total pancreas lysates from three control organ donors (p1-p3) with accompanying Actin labeling. (C) Representative IHC images of human pancreas tissue sections stained for ACE2 using four commercially available ACE2 antibodies. Scale bars: 200 µm. Abbreviations: i, islet; d, duct; mv, microvasculature. See also Table S2 and Fig. S2B-D .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Western Blot, Labeling, Staining

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) Scheme of the experimental setup illustrating human pancreatic tissue processing, whole stained slide imaging, and machine learning algorithm application for the generation of ACE2 protein expression data. (B) Quantification of ACE2 protein expression in the pancreas of control organ donors with ages ranging from birth to 72 years shows progressive developmental changes. Data are presented as mean±SD with analysis by one-way ANOVA and Tukey’s post hoc test for multiple comparisons. (C) Representative confocal images of ACE2 protein expression in pancreatic ducts of control donors across different age groups. Scale bars (left to right): 100µm, 200µm, 200µm, 300µm, 300µm, 300µm. (D) Representative immunofluorescence images showing lack of ACE2 protein expression in pancreatic blood vessels from control donors across different age groups. Scale bars (left to right): 100µm, 200µm, 200µm, 200µm, 200µm, 100µm. (E) Representative immunofluorescence images show ACE2 protein expression in pancreatic microvasculature from control donors across different age groups. Scale bars: 100µm. (F) Representative immunofluorescence images of pancreatic islets showing ACE2 protein expression restricted to the islet’s microvasculature in pancreata from control organ donors across different age groups. Scale bars: 50µm. See also Table S2 and Fig. S3 .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Staining, Imaging, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) Pancreas tissue section from COVID-19 Patient 1 stained for H&E. Inset highlights fibrotic center with residual acinar cells and islet surrounding ductules. Scale bars: 3mm, inset 200µm. (B) Pancreas tissue section from COVID-19 Patient 2 stained for H&E. Inset highlights microthrombus without adjacent hemorrhages. Scale bars: 4mm, inset 400µm. (C) Pancreas tissue section of COVID-19 Patient 3 stained for H&E. Inset highlights a large, irregularly shaped pancreatic islet surrounded by fibrotic tissue. Scale bars: 4 mm, inset 200µm. (D) Representative pancreas tissue sections from three COVID-19 patients stained for ACE2, insulin (INS) and glucagon (GCG). Scale bars: 200µm. (E) SARS-CoV-2 NP observed in intralobular ducts (d) near an islet in the pancreas of COVID-19 Patient 1. Scale bars: 10µm. (F) Representative image of multiple ducts showing SARS-CoV-2 NP positivity in the pancreas of COVID-19 Patient 1. Scale bar: 20µm. See also Table S3 and Fig. S4 .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Staining