Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 1 In‐house and commercial ACE2 enzymatic immunoassay (EIA) results of pre‐COVID‐19 donor control sera, COVID‐19 convalescent patient, and vaccine recipient sera. (A, B) IgM EIA results of COVID‐19 convalescent sera classified based on severity. (C, D) IgG EIA results of COVID‐19 convalescent sera classified based on severity. (E, F) IgG EIA results of COVID‐19 vaccine recipients based on type of vaccine. Bars represent median and interquartile range. Intergroup comparisons of medians were performed using Dunn's multiple comparisons test. Ns: not significant; *p ≤0.05; ***p ≤0.001; ****p ≤0.0001. ACE2, angiotensin‐converting enzyme 2; COVID‐19, coronavirus disease 2019.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay, Control

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 2 Correlations between ACE2 IgG enzymatic immunoassay optical densities (OD) and surrogate neutralizing antibody levels of CoronaVac (A, B) and Comirnaty (C, D) cohorts using commercial and in‐house ACE2 peptides. Strength of correlation was assessed using Spearman's rank correlation. ACE2, angiotensin‐converting enzyme 2.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 3 Trends of ACE2 IgG optical densities (ODs) using in‐house (A) and commercial (B) peptides for vaccine recipients testing positive at Day 56 post‐first dose. Each line represents trend for individual recipients. SNV020, SNV027, and SNV058 are CoronaVac recipients. BNT007, BNT012, BNT032, BNT081, BNT090, and BNT092 are Comirnaty recipients. The second timepoint is either Day 21 (for Comirnaty recipients) or Day 28 (for CoronaVac recipients). ACE2, angiotensin‐converting enzyme 2.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques:

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: Science immunology

Article Title: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

doi: 10.1126/sciimmunol.abc3582

Figure Lengend Snippet: Fig. 1. VSV-SARS-CoV-2 infects human small intestinal enteroids. (A) Mouse small intestinal cells were analyzed by scRNA-seq and resolved into 20 clusters based on gene expression profiles (left). Transcript levels of Cd26, Epcam, Cd44, Cd45, and Ace2 were indicated for different intestinal cell subsets. Clusters 10 and 18: intraepithelial lymphocytes; clusters 1, 2, 5, 8, 9, 17, 19, and 20: enterocytes; cluster 3: goblet cells; cluster 4: enteroendocrine cells; cluster 7: Tuft cells; cluster 11: crypt stem cells; cluster 12: Paneth cells. Each dot represents a single cell. Note that Ace2high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 105 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 105 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 m. (E) Same as (C) except that virus titers were mea- sured using a TCID50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, 80 m. (G) Same as (D) except that human colon enteroids were used instead. Scale bars, 80 m. For all figures except (A), experiments were repeated at least three times with similar re- sults. (A) was performed once with small intestinal tissues pooled from three mice. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Samples were then stained with the following primary antibodies or fluorescent dyes: ACE2 (sc-390851 AF594, Santa Cruz Biotechnology), 4′,6-diamidino-2-phenylindole (DAPI) (P36962, Thermo Fisher Scientific), SARS-CoV-2 S [CR3022 human monoclonal antibody (58)], villin (sc-58897 AF488, Santa Cruz Biotechnology), and phalloidin (Alexa 647–conjugated, Thermo Fisher Scientific).

Techniques: Gene Expression, Cell Culture, Staining, Infection, Expressing, Quantitative RT-PCR, Virus, TCID50 Assay

Journal: Science immunology

Article Title: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

doi: 10.1126/sciimmunol.abc3582

Figure Lengend Snippet: Fig. 2. VSV-SARS-CoV-2 and wild-type SARS-CoV-2 replicate in ACE2+ human mature enterocytes. (A) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically or basolaterally infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (B) Supernatants in both apical and basal chambers were collected from (A) and were subjected to a TCID50 assay to measure the amount of infectious virus. (C) Differentiated duodenum enteroids in monolayer were apically infected with 2.5 × 105 PFU of infectious SARS-CoV-2 virus (MOI = 0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a TaqMan assay and normalized to that of GAPDH. (D) Differentiated ileum enteroids in monolayer were apically or basolaterally infected with 2.5 × 105 PFU of infectious SARS-CoV-2 virus (MOI = 0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a TaqMan assay and normalized to that of GAPDH. (E) Same as (C) except that enteroids were fixed and stained for SARS-CoV-2 S (green), ACE2 (red), and nucleus (DAPI, blue). Scale bars, 32 m. SARS-CoV-2–infected ACE2-positive cells are enlarged in the inset (yellow box). (F) SARS-CoV-2–infected duodenum monolayers were imaged along the Z stacks and sectioned for YZ planes (top) and reconstructed for 3D images (bottom). For all figures except (C) to (E), ex- periments were repeated at least three times with similar results. (C) to (E) were performed twice with technical duplicates in each experiment. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Samples were then stained with the following primary antibodies or fluorescent dyes: ACE2 (sc-390851 AF594, Santa Cruz Biotechnology), 4′,6-diamidino-2-phenylindole (DAPI) (P36962, Thermo Fisher Scientific), SARS-CoV-2 S [CR3022 human monoclonal antibody (58)], villin (sc-58897 AF488, Santa Cruz Biotechnology), and phalloidin (Alexa 647–conjugated, Thermo Fisher Scientific).

Techniques: Cell Culture, Infection, Expressing, Quantitative RT-PCR, TCID50 Assay, Virus, TaqMan Assay, Staining

Journal: Science immunology

Article Title: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

doi: 10.1126/sciimmunol.abc3582

Figure Lengend Snippet: Fig. 3. TMPRSS2 and TMPRSS4, but not ST14, mediate SARS-CoV-2 S–mediated entry. (A) Bulk RNA-seq results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, and HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left), or transfected with indicated plasmid combination for 24 hours (right), and infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The ex- pression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with SARS-CoV-2 S and TMPRSS2 or TMPRSS4 for 48 hours. Cells were treated with trypsin at 0.5 g/ml for 10 min. The levels of S and GAPDH were measured by Western blot. The intensity of bands was quantified by ImageJ and shown as percentage of the bottom band versus the top band in each lane. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8 × 105 PFU of VSV-SARS-CoV-2 on ice for 1 hour, washed with cold phosphate-buffered saline for three times, and shifted to 37°C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type (WT) or human ACE2-expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 for 24 hours. The red arrows highlight the formation of large syncytia. Scale bars, 100 m. For all figures except (A), experiments were repeated at least three times with similar results. RNA-seq in (A) was performed once with duplicate samples. Data are represented as mean ± SEM. Statistical sig- nificance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Samples were then stained with the following primary antibodies or fluorescent dyes: ACE2 (sc-390851 AF594, Santa Cruz Biotechnology), 4′,6-diamidino-2-phenylindole (DAPI) (P36962, Thermo Fisher Scientific), SARS-CoV-2 S [CR3022 human monoclonal antibody (58)], villin (sc-58897 AF488, Santa Cruz Biotechnology), and phalloidin (Alexa 647–conjugated, Thermo Fisher Scientific).

Techniques: RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Stable Transfection, Western Blot, Incubation, Saline