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: Cell Metabolism

Article Title: Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19

doi: 10.1016/j.cmet.2020.11.005

Figure Lengend Snippet:

Article Snippet: Thereafter, the membranes were incubated at 4°C overnight with one of four primary antibodies (rabbit monoclonal anti-ACE2 (1:1,000 dilution, Abcam, Cambridge, MA), rabbit polyclonal anti-ACE2 (1:500 dilution, Abcam), mouse monoclonal anti-ACE2 (1:1,000 dilution, R&D Systems), goat polyclonal anti-ACE2 (1:500 dilution, R&D Systems, Minneapolis, MN)) and mouse monoclonal anti-b-actin (1:10,000 dilution; Sigma-Aldrich, St. Louis, MO) in Intercept Antibody Diluent (LI-COR Biosciences).

Techniques: Plasmid Preparation, Control, Recombinant, Protease Inhibitor, Blocking Assay, Avidin-Biotin Assay, Staining, RNAscope, Multiplex Assay, Fluorescence, BIA-KA, Generated, Gene Expression, Software

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – c , Based on simulations of RBD-bound ACE2, new polar interactions were identified between the ACE2.v2.4 (orange) and RBD (yellow) loops (red) ( a ). ACE2 mutations are presented as sticks colored cyan with nearby RBD residues as sticks colored yellow. New polar interactions between ACE2.v2.4 and RBD loops 1 ( b ) and 2 ( c ) are indicated by the dotted blue lines. d , MSM-weighted distance distributions of newly formed hydrogen bonds. For each interacting pair, the ACE2.v2.4 residue is listed first and the RBD residue is listed second. e , Probabilities for stable hydrogen bond interactions using a 4-Å distance criterion between accepter and donor. f , Root mean square fluctuation (RMSF) of RBD residues when bound to WT (cyan) or v2.4 (orange) ACE2 receptors. The RBD regions that interface with ACE2 are shaded gray. The absolute difference in RMSF between WT and v2.4 proteins is shown in black. Distance distributions, hydrogen bond probabilities and RMSF calculations were based on 40,000 frames from the simulations. Frames were selected based on the MSM stationary probability to represent the entire conformational ensemble. The error bars represent the 95% confidence intervals calculated from 20 bootstrapped samples.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Residue

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a , mRNA expression of ACE2 (NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells. A549 cells stably expressing hACE2 and hLMVECs were analyzed by one-step RT–PCR (top) and real-time PCR (bottom). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase expression. b , Cultured hACE2-A549 cells, A549 cells and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 µg ml −1 or 25 µg ml −1 for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was added to the cells, which were collected at 24 h. Virus entry was evaluated by luciferase activity. n = 4 replicates. c , A dose of 10 mg kg −1 sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1 or buffer (PBS + 0.2% BSA) was administrated intravenously into K18-hACE2 transgenic mice for 30 min before SARS-CoV-2 pseudovirus (10 6 PFU) intraperitoneal injection. Tissue lysates were prepared at 24 h and virus entry in the selected organs was evaluated by luciferase activity. Buffer was applied as the control group, n = 4. d – g , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Mice received control PBS or sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h before inoculation. Mice were observed for survival ( d ) and body weight ( e ), n = 5. EBA as a marker of pulmonary endothelial permeability ( f ) and lung wet/dry ratio ( g ) as a measure of lung edema were quantified. Data are presented as the mean ± s.e.m., n = 4. b , c , f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Expressing, Stable Transfection, One Step RT-PCR, Real-time Polymerase Chain Reaction, Cell Culture, Virus, Luciferase, Activity Assay, Transgenic Assay, Injection, Control, Marker, Permeability

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – d , sACE2 carrying the v2.4 mutations has increased S binding compared with WT sACE2. Human Expi293F cells expressing myc-tagged S from 4 SARS-CoV-2 variants (Wuhan ( a ), B.1.1.7/Alpha ( b ), B1.351/Beta ( c ) and P.1/Gamma ( d )) were incubated with monomeric sACE2-8 h (black) or dimeric sACE2 2 -IgG1 (gray); bound protein was detected by flow cytometry. WT ACE2 proteins are shown as broken lines, v2.4 proteins are shown as solid lines. n = 3 independent replicates; data are shown as the mean ± s.e.m. e – i , Binding of sACE2 2 .v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE2 2 .v2.4-IgG1 to the S proteins of SARS-CoV-2 VOCs (B.1.1.7/Alpha ( e ), B1.351/Beta ( f ), B.1.617.2/Delta ( g ) and P.1/Gamma ( h )) and S protein of SARS-CoV-1 ( i ), as measured by flow cytometry. n = 3 independent replicates; data are shown as the mean ± s.e.m.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Phylogenetic tree of bat ACE2 protein sequences with mammalian or avian ACE2 sequences. Phylogenetic trees were constructed by aligning CVB and PB ACE2 protein sequence with previously studied mammalian or bat ACE2 sequences. Alignments were performed using the Jukes–Cantor genetic distance model, tree was built using neighbor-joining algorithm using Chicken-ACE2 as an outgroup since it does not bind to SC2 S protein. Consensus tree was generated by resampling with 500 bootstraps. Branch labels indicate substitutions per site. Scale bar is shown.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Construct, Sequencing, Generated

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: CVB-ACE2 and PB-ACE2 cells support infection of SC2 variants. Line graphs show log 10 TCID 50 titers/mL of WA1, Delta, Lambda, and Omicron lineage SC2 viruses in CVB-ACE2/hTMPRSS2 ( A ), PB-ACE2/hTMPRSS2 expressing DF-1 cells ( B ) and DF-1 cells expressing human ACE2 and TMPRSS2 ( C ). Data represent mean ± SD from three independent experiments for each time point. Statistical comparisons were conducted with 2-way ANOVA using repeated measures with Geisser–Greenhouse correction and Tukey multiple comparisons test with individual variances computed for each comparison. Lines with different lowercase letters indicate statistically significant differences ( p < 0.05). Titers are indicated on Y-axis and time points of infection are indicated on the X-axis. CVB = Common vampire bat (Desmodus rotundus ), PB = Pallid bat ( Antrozous pallidus ), Hs = Homo sapiens .

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Infection, Expressing, Comparison

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Immunofluorescence microscopy of SC2 infected CVB-ACE2 cells. Confluent (75%) monolayers of CVB-ACE2 expressing DF-1 cells on iBID chamber slides were infected with WA1 ( A ), Delta ( B ), Lambda ( C ) or Omicron ( D ) variant of SC2 for 48 h and then stained for SC2 S protein and counterstained for nuclei using DAPI as stated in materials and methods. Scale bars at the bottom right represent 10× magnification.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Variant Assay, Staining

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Immunofluorescence microscopy of SC2 infected PB-ACE2 cells. Confluent (75%) monolayers of PB-ACE2 expressing DF-1 cells on iBID chamber slides were infected with WA1 ( A ), Delta ( B ), Lambda ( C ) or Omicron ( D ) variant of SC2 for 48 h and then stained for SC2 S protein and counterstained for nuclei using DAPI as stated in materials and methods. Scale bars at the bottom right represent 10× magnification.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Variant Assay, Staining