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

Journal: Immunology and Cell Biology

Article Title: The vaccinia‐based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses

doi: 10.1111/imcb.12539

Figure Lengend Snippet: Spike‐specific antibody and SARS‐CoV‐2 neutralization responses following SCV‐S vaccination. (a) S1 and S2 subunit endpoint IgM and IgG ELISA titers determined from serum of female C57BL/6J mice ( n = 3) at the indicated times after a single vaccination with 10 7 PFU of SCV‐S, with (b) ratio of S1‐ and S2‐specific IgG2c to IgG1 endpoint ELISA titers determined 28 days after vaccination. (c) S1 IgG ELISA binding titers (left panel) and cPass neutralization titers (right panel) in outbred ARC(s) and inbred C57BL/6J female mice ( n = 5) 21 days after a single vaccination with 10 7 PFU of SCV‐S or vector control, with (d) durability of response in the outbred cohort shown by S1‐specific endpoint IgG ELISA titers (left panel) and neutralization titers (right panel) at the indicated times. (e) S1‐specific IgG ELISA (left panel) and neutralization titers (right panel) 50 days after a single‐dose (day 0) or prime‐boost (day 0 and 28) vaccination of female C57BL/6J mice ( n = 5) with 10 7 PFU SCV‐S or control vector, with (f) neutralizing activity in ACE2 and RBD blocking ELISA, and (g) neutralizing activity (IC 80 titers) against lenti‐SARS‐CoV‐2‐S pseudoviruses bearing spike protein from the Wuhan reference strain, the alpha or beta variant, shown for prime‐boost samples. (h) Neutralization titers in young (6–8 weeks old; n = 5) and middle‐aged (9–10 months old; n = 10) C57BL/6J mice at the indicated times after prime‐boost vaccination with 10 7 PFU SCV‐S. Results shown are representative of four independent experiments (indicated above) with binding and neutralizing antibody levels comparable at similar doses and time points across all experiments. Symbols represent individual mice and bars show the mean ± s.e.m. from independent experiments. Data were log transformed and statistical significance determined using Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ACE2, angiotensin‐converting enzyme‐2; IC 80 , 80% inhibitory concentration; Ig, immunoglobulin; ns, not significant; PFU, plaque‐forming units; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SCV‐S, Sementis Copenhagen Vector spike protein.

Article Snippet: ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Activity Assay, Blocking Assay, Variant Assay, Transformation Assay, Concentration Assay