Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Sequences of sense strands of double-stranded RNA used to ablate specific protein expression in HEK-ACE2 and Huh7 cells

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shedding of ACE2 involves loss of its cytoplasmic tail. HEK293 cells were stably transfected with an expression vector encoding full-length ACE2 as described under “Materials and Methods.” OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier was added to exponentially growing cells and collected after 1 h. Following sedimentation of cells, the media were concentrated 10-fold, and 40 μg of media proteins ( M ) were separated by SDS-PAGE (6% v/v) alongside 20 μg of corresponding detergent cell extract ( C ) and immunoblotted with an antibody raised to the ectodomain of ACE2 ( left panel , ectodomain) or the cytosolic tail of ACE2 ( right panel , cytosolic). Immunoreactive bands were visualized with enhanced chemiluminescence as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Sedimentation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shed ACE2 occurs as two distinct glycoforms. HEK-ACE2 cells were incubated in OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier for 1 h, and the media were collected and concentrated as described. Media proteins ( M ; 40 μg) or detergent cell extracts ( C ; 20 μg) were incubated at 37 °C for 16 h in the presence or absence of endoglycosidase H ( Endo H ) or PNGase F and subsequently separated by SDS-PAGE. Following electrotransfer, immunoblotting was carried out using an antibody to the ectodomain of ACE2 as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page, Electrotransfer, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shedding of ACE2 is inhibited by broad spectrum hydroxamate-based metalloprotease inhibitors. HEK-ACE2 cells were incubated in OptiMEM containing various concentrations of the secretase inhibitors TAPI-1 or GM6001 or an equal volume of carrier (Me 2 SO). After 15 min, PMA (0.1 μ m ) or an equal volume of Me 2 SO was added, and incubation was continued for 1 h. The medium was subsequently harvested and concentrated as described; cells were pelleted and detergent extracts were collected as described under “Materials and Methods.” A , media proteins ( upper panel , 40 μg) and cell lysates ( lower panel , 20 μg) were separated by SDS-PAGE and immunoblotted for ACE2. Immunoreactive bands were visualized by enhanced chemiluminescence. B , graphical representation of results of densitometric analysis of three such experiments, ± S.E. Black shading , –PMA; gray shading , +PMA.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: PMA-stimulated ACE2 shedding is sensitive to ADAM17 inhibition. HEK-ACE2 cells were incubated for 15 min in the presence of the ADAM10 inhibitor GI254023X or the mixed ADAM10/ADAM17 inhibitor GW280264X or an equal volume of Me 2 SO. Subsequently, incubation was continued in the presence of PMA (0.1 μ m ). Media were harvested and concentrated as described under “Materials and Methods.” A , media proteins (40 μg) were separated by SDS-PAGE and immunoblotted for ACE2. B , graphical representation of results of densitometric analysis of three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Inhibition, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Stimulated ACE2 shedding is inhibited by NTIMP3 but not by NTIMP1. HEK-ACE2 cells were incubated for 15 min in the presence of NTIMP1, NTIMP3, or an equal volume of phosphate-buffered saline. Subsequently, incubation was continued in the presence of PMA (0.1 μ m ). Media were harvested and concentrated as described. A , media proteins (40 μg) were separated by SDS-PAGE and immunoblotted for ACE2. B , graphical representation of results of densitometric analysis of the immunoblots of three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Ablation of ADAM17 expression by siRNA reduces stimulated ACE2 shedding. HEK-ACE2 cells were transiently transfected with a mixture of double-stranded RNA derived from the coding sequence of ADAM10, ADAM17, or the control sequence GL2 as described under “Materials and Methods.” Twenty-four hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 1 h. Media were concentrated as described, and cell lysates were prepared. A , media proteins (40 μg) and cell lysates (50 μg) were separated by SDS-PAGE and immunoblotted for ACE2, ADAM10, and ADAM17, as indicated. Mock , mock transfection. B , graphical representation of densitometric analysis of immunoblots of media ACE2 from three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Transfection, Derivative Assay, Sequencing, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Overexpression of ADAM17 increases PMA-stimulated ACE2 shedding. HEK-ACE2 cells were transiently transfected with an expression vector encoding ADAM9, ADAM10, or ADAM17, as described under “Materials and Methods.” Thirty-six hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 1 h. Media were concentrated as described, and detergent cell extracts were harvested. A , media proteins (40 μg) and detergent cell extracts (50 μg) were separated by SDS-PAGE and immunoblotted for ACE2 ( upper panel , media), ADAM9, ADAM10, or ADAM17 ( lower panels , lysates). Membranes were stripped and reprobed for β-actin as a loading control. B , graphical representation of densitometric analysis of immunoblots of media ACE2 from three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: PMA-stimulated endogenous ACE2 shedding is sensitive to ADAM17 inhibition. A , OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier was added to exponentially growing Huh7 cells and collected after 6 h. Following sedimentation of cells, the media were concentrated, and 100 μg of media proteins ( M ) was separated by SDS-PAGE (4–12% v/v) alongside 50 μg of corresponding detergent cell extract ( C ) and immunoblotted with an antibody raised to the ectodomain of ACE2 ( left panel , ectodomain) or the cytosolic tail of ACE2 ( right panel , cytosolic). Immunoreactive bands were visualized with enhanced chemiluminescence as described under “Materials and Methods.” B , Huh7 cells were incubated for 15 min in the presence of 50 μ m GM6001 or 1 μ m the ADAM10 inhibitor GI254023X or the mixed ADAM10/ADAM17 inhibitor GW280264X or an equal volume of Me 2 SO. Subsequently, incubation was continued for 4 h in the presence or absence of PMA (0.1 μ m ). Media were harvested and concentrated as described under “Materials and Methods.” Concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate, as described under “Materials and Methods.” Black shading , –PMA; gray shading , +PMA.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Inhibition, Sedimentation, SDS Page, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Ablation of ADAM17 expression by siRNA reduces regulated endogenous ACE2 shedding. Huh7 cells (50% confluent) were transfected with double-stranded RNA oligomers (as described under “Materials and Methods”) to ADAM10, ADAM17, or a negative control sequence. After 40 h, the media were aspirated and replaced with OptiMEM containing 100 n m PMA or an equal volume of Me 2 SO. After a further 5-h incubation, the media were concentrated, and cell lysates were prepared as described under “Materials and Methods.” Cell lysates (50 μg) were separated by SDS-PAGE and immunoblotted for ADAM10 ( A ) and ADAM17 ( B ). Concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Transfection, Negative Control, Sequencing, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Overexpression of ADAM17 increases PMA-stimulated endogenous ACE2 shedding. HEK-ACE2 cells were transiently transfected with an expression vector encoding ADAM9, ADAM10, or ADAM17, as described under “Materials and Methods.” Thirty-six hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 4 h. Media were concentrated as described, and detergent cell extracts were harvested. A , detergent cell extracts (50 μg) were separated by SDS-PAGE and immunoblotted for ADAM9, ADAM10, or ADAM17. Membranes were stripped and reprobed for β-actin as a loading control. B , concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-B) A549 cells were transduced to stably overexpress ACE2 (A549-A), DPP4 (A549-D) in the absence or presence of TMPRSS2 (A549-AT and A549-DT). ACE2 and DPP4 expression was confirmed by western blot (A) , while TMPRSS2 expression was confirmed using flow cytometry (B) . (C-D) A549-derived or Calu-3 cells were inoculated with lentiviral particles pseudotyped with the spike proteins of MERS-CoV (C) , VSV, SARS-CoV-1, WIV1-CoV, WIV16-CoV, SARS-CoV-2 Hu1, SARS-CoV-2 delta or SARS-CoV-2 omicron (D) or pseudoparticles lacking envelope protein (no env) (C-D) for 2 h, then incubated for an additional 72 h, at which point luciferase activity was measured to assess pseudoparticle entry. The data are expressed as fold change relative to the luciferase signal obtained with no envelope. Graphs show mean +/- SEM from three independent experiments performed in triplicate.

Article Snippet: For the generation of the A549-ACE2 and A549-DPP4 cell lines, lentiviral vectors encoding human ACE2 (Dr. Sonja Best, Addgene #154981) and DPP4 (Addgene #158452) were used, respectively.

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Derivative Assay, Incubation, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: BEAS-2B cells were transduced to stably overexpress ACE2 (BEAS-2B-ACE2). Expression of ACE2 and endogenous TMPRSS2 was confirmed by western blot (A). (B) BEAS-2B-ACE2 cells were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations, then infected with SARS-CoV-2pp for 2 h at 37°C. Inocula were removed and cells were incubated in complete media for 72 h, at which point luciferase activity was measured to assess viral entry. (C-D) BEAS-2B-ACE2 cells were pre-treated with NanH diluted to 50 μg/mL in serum-free media for 30 minutes at 37°C. Cells were then washed and processed for fluorescence microscopy (C) or inoculated with SARS-CoV-2 pseudoparticles (D) . NanH-treated cells were stained with SNA-FITC (binds sialic acid) or ECL-FITC (binds galactose) diluted to final concentration of 20 μg/mL in PBS, then washed with PBS and imaged by fluorescence microscopy (10X magnification; scale bar, 200 μm). Lectin staining confirmed removal of sialic acid by the NanH treatment. (E-F) BEAS-2B-ACE2 cells were treated with protease inhibitors or NanH as described (B-C) , then infected with replication-competent recombinant VSV-SARS-CoV-2-S expressing GFP for 2 h. After 7.5 h, cells were fixed and GFP fluorescence was assessed. Representative images are shown (20X magnification; scale bar, 50 μm). The percentage of infected cells in each condition was determined using ImageJ. Graphs show mean +/- SEM from three independent experiments performed in triplicate. Statistical significance was assessed by one-way or two-way ANOVA (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: For the generation of the A549-ACE2 and A549-DPP4 cell lines, lentiviral vectors encoding human ACE2 (Dr. Sonja Best, Addgene #154981) and DPP4 (Addgene #158452) were used, respectively.

Techniques: Stable Transfection, Expressing, Western Blot, Infection, Incubation, Luciferase, Activity Assay, Fluorescence, Microscopy, Staining, Concentration Assay, Recombinant

Journal: bioRxiv

Article Title: Novel ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV2

doi: 10.1101/2020.06.15.152157

Figure Lengend Snippet: Binding of RBD (A, C) or spike protein (B, D) and hACE2 by ELISA. Assays were performed at room temperature (A,B) or at 37° C (C, D). (E) Catalytic activity of WT or the MDR504 mutant in a fluorogenic ACE2 assay ( n = 3). Significant differences are designated using one-way ANOVA followed by Tukey’s multiple comparisons test (B, D) and unpaired t-test (E) based on the AUC. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For this study, we utilized the murine model of SARS-CoV2 were C57Bl/6 mice were first inoculated with adenovirus encoding human ACE2 (Vector Biolabs).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Novel ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV2

doi: 10.1101/2020.06.15.152157

Figure Lengend Snippet: Neutralization of pseudovirus were compared among the different constructs in (A) 89 nM and (B) 223 nM of various hACE2-IgG1 fusions. MDR504 mutant and MDR505 double mutant showed significantly higher neutralization. Similarly, SARS-CoV2 neutralization with WT and MDR503 mutant at 223 nM attested the result by plaque assay (C). IC 50 of the different ACE2-IgG1 fusions in the placque assay (D). Significant differences are designated using one-way ANOVA (A, B, C) and two-way ANOVA (D) followed by Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (E) Calculated IC 50 of each construct based on the placque assay data.

Article Snippet: For this study, we utilized the murine model of SARS-CoV2 were C57Bl/6 mice were first inoculated with adenovirus encoding human ACE2 (Vector Biolabs).

Techniques: Neutralization, Construct, Mutagenesis, Plaque Assay

Journal: bioRxiv

Article Title: Novel ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV2

doi: 10.1101/2020.06.15.152157

Figure Lengend Snippet: In vivo pharmacokinetics of the WT and MDR504 mutant ACE2-IgG1 was assayed after intravenous injection of 4 mg / kg body weight of protein assayed in in serum (A) and BAL fluid (B).

Article Snippet: For this study, we utilized the murine model of SARS-CoV2 were C57Bl/6 mice were first inoculated with adenovirus encoding human ACE2 (Vector Biolabs).

Techniques: In Vivo, Mutagenesis, Injection

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: (A) Sarbecovirus (Lineage B) phylogenetic tree classified based on RBD sequence conservation. (B) Left: Cartoon rendering of SARS-CoV-2 S trimer (PDB 6VYB) showing location of ‘up’ RBD (surface, orange and purple). Right: Amino acid sequence conservation of 12 RBDs calculated as described plotted on a surface representation of a SARS-CoV-2 RBD structure (PDB 7BZ5). Primary RBD epitopes for the indicated representatives from defined classes of RBD-binding antibodies (class 1-4) are indicated as yellow dotted lines (PDB 7K90, 6W41, 7JX3,7K8M). C022 epitope indicated as blue dotted line. (C) Comparison of binding of the indicated monoclonal IgGs to a panel of sarbecovirus RBDs from ELISA data shown as area under the curve (AUC) values. Data presented are mean AUC values from two independent experiments. IOMA IgG is an anti-HIV-1 antibody serving as a negative control . (D) Neutralization IC 50 values for the indicated IgGs against SARS-CoV-2 (D614G version of the original variant (GenBank: NC_045512 )), SARS-CoV-2 variants of concern, and other ACE2-tropic sarbecovirus pseudoviruses. Geomean = geometric mean IC 50 in which IC 50 values >50000ng/mL were entered as 50000 ng/mL for the calculation. SD = standard deviation. IC 50 values are means of 2-7 independent experiments.

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Sequencing, Binding Assay, Comparison, Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization, Variant Assay, Standard Deviation

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: (A) SARS-CoV-2 RBD surface representation (grey) with N343 glycan (teal). The ACE2 binding site is represented by a green dashed line. (B) SARS-CoV-2 RBD surface representation (grey) with overlaid bound models of V H V L of antibodies for class 1 (C102, green, PDB: 7K8M), class 2 (C144, purple, PDB: 7K90), Class 3 (S309, brown, 7JMX), and class 4 (CR3022, orange, PDB: 6W41). (C) SARS-CoV-2 surface representation with overlay of bound C118 Fab (blue), C022 Fab (red), and CR3022 Fab (orange, PDB: 6W41). (D) SARS-CoV-2 RBD surface representation (grey) at 45 ° angle from previous panels. Dotted outlines show epitopes of C102 (green), C022 (red), C118 (blue), and CR3022 (orange) mapped onto SARS-CoV-2 RBD.

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Glycoproteomics, Binding Assay

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: (A) Left: Cartoon model of SARS-CoV-2 RBD (RBD from C022-RBD structure) showing locations of point mutations as red spheres and the N343 RBD N -glycan as teal sticks. Right: Comparison of binding of the indicated monoclonal IgGs to RBD mutants from ELISA data shown as AUC values normalized to antibody binding to ‘wt’ SARS-CoV-2 RBD. Data presented are normalized mean AUC values from two independent experiments. (B-C) Normalized relative luminescence values for cell lysates of HT1080 ACE2 cells 48h after infection with SARS-CoV-2 pseudovirus carrying indicated spike variants in the presence of increasing concentrations of monoclonal IgGs C118 (B) and C022 (C). The mutants represented substitutions found in circulating SARS-CoV-2 sequences with frequencies >0.01% in GISAID . Mean and standard deviation of two experiments, each performed in duplicate (n=4), is shown. (D) Half-maximal inhibitory concentrations (IC 50 ) calculated from the neutralization curves in panels B and C for monoclonal IgGs C022 and C118 for neutralization of ‘wt’ (D614 S trimer) and the indicated mutant SARS-CoV-2 S pseudotyped viruses, as well as other sarbecovirus pseudoviruses. IC 50 values are means of 2 independent experiments. Colors indicate IC 50 ranges, as indicated. The E484K substitution was constructed in an R683G (furin cleavage site mutant) background to increase infectivity .

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Glycoproteomics, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection, Standard Deviation, Neutralization, Mutagenesis, Construct

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: (A,B) Cartoon renderings of crystal structures of (A) C0118 Fab complexed with SARS-CoV RBD, and (B) C022 Fab complexed with SARS-CoV-2 RBD. Dashed circle shows location of SARS-CoV N357 RBD residue, with the inset showing the N357 RBD asparagine and glycan modeled based on the SARS-CoV spike-S230 structure (PDB 6NB6). (C,D) CDR loops and RBD epitope residues of (C) C118 Fab and (D) C022 Fab overlaid on RBDs represented as gray surfaces with stick representations of epitope residues. Framework region residues, which account for some of the contacts for both antibodies, are not shown in right panels. (E) Comparison of Fab poses for binding to an RBD-ACE2 complex. C118 Fab (blue), C022 Fab (red), CR3022 Fab (PDB 6W41; orange), and EY6A Fab (PDB 6CZC pink) modeled onto an ACE2-RBD structure (PDB 6M0J; RBD shown as a gray surface and ACE2 shown as a green cartoon).

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Residue, Glycoproteomics, Comparison, Binding Assay

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: SARS-CoV-2 RBD was coupled to a biosensor chip using primary amine chemistry. An IgG was injected first (seconds 0-600). Seconds 600 - 730 represent the delay required to switch samples for a subsequent injection. From seconds 730 – 1030, soluble ACE2 was injected. Buffer was injected after 1030 seconds.

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Injection

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: (A) Epitopes for ACE2 and monoclonal antibodies calculated from analyses of structures of RBD or S trimer complexes (human antibodies isolated from COVID-19 patients are C118, C022, COVA1-16, EY6A, and S2A4). RBDs shown are derived from SARS-CoV-2 except for the C118 panel, which is SARS-CoV RBD. (B) Alignment of sequences for sarbecovirus RBDs (residue numbering for SARS-CoV-2 RBD). Secondary structure for SARS-CoV-2 RBD shown above alignment. Dots designate binding or neutralization for C118 (blue), C022 (red), or CR3022 (orange) for each strain. Diamonds designate RBD epitope residues for C118 binding to SARS-CoV (blue) and C022 (red) or CR3022 (orange) binding to SARS-CoV-2. Left boxes show binding by ELISA or neutralization of pseudovirus for each antibody for each strain; data for COVA1-16 from . Circles show binding or neutralization, blank spaces designate not tested, and dashes designate no binding or neutralization. Shadings in the sequence alignment indicate conserved portions of epitope (green). Colored boxes show differing portion of epitope covering the α4 helix and following loop (pink. (C) Cartoon representation of SARS-CoV-2 RBD (gray) showing overlapping antibody-interacting residues (green) as sticks in epitopes for C118, C022, COVA1-16, and CR3022 (corresponding to green shading in panel B). (D) Cartoon representation of SARS-CoV-2 RBD (gray) showing α4 helix and following (sticks, pink) that differ in their contacts with C118, C022, COVA1-16, and CR3022 (pink shading in panel B). (E) Cartoon representation of RBDs showing α4 region of RBD and C118 (left) or C022 (right) interacting loops with interacting Fab residues in light blue (C118) and light pink (C022).

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Bioprocessing, Isolation, Derivative Assay, Residue, Binding Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: bioRxiv

Article Title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

doi: 10.1101/2021.04.23.441195

Figure Lengend Snippet: (A) 3.4Å cryo-EM density for the C118 – S trimer complex (State 1). Side view (left panel) illustrates orientation with respect to the viral membrane (dashed line). Top view (right panel) shows symmetric binding at the trimer apex with C118 HC (blue) oriented in the interior. (B) 4.4Å cryo-EM density for the C118 – S trimer complex (State 2). Top view illustrates asymmetry of complex due to RBD rotation in one protomer. (C) Composite model of an open SARS-CoV-2 trimer bound by class 4 Fabs: C118 (this paper, blue), EY6A (PDB 6ZDH, pink), S2A4 (PDB 7JVC, brown), the class 4 anti-SARS antibody S304 (PDB 7JW0, green), and H014 (PDB 7CAK, yellow). (D) Comparison of S trimer openness by measurements of Cα distances for D428 RBD between adjacent ‘up’ RBDs in S trimers complexed with: the class 1 antibody S2E12 (PDB 7K43, gray), soluble ACE2 (PDB 7KMS, green) and the class 4 antibodies C118 (this study, blue) and EY6A (PDB 6ZDH, pink). (E) Prediction of potential intra-spike avidity effects by measurement of Cα distances between the C-termini of adjacent C H 1 domains for the mAb-S trimer complexes described in panel C. Measurements were used to evaluate the potential for intra-spike crosslinking by an IgG binding to a single spike trimer as described . For the H014-S complex, the C H 1-C L domains were rigid body fit into the cryo-EM density (EMD-30333) prior to measurements. (F) IC 50 values and molar neutralization ratios (MNRs) defined as: [IC 50 Fab or bispecific IgG (nM)/IC 50 IgG (nM)] for C118 and C022. IC 50 values shown for the IgGs are from . IC 50 values for all assays against SARS-CoV-2 and SARS-CoV are means of 2-7 independent experiments. Two MNRs are presented in the MNRs (bispecific/Fab) column: the MNR calculated using a bispecific IgG versus the bivalent IgG (left) and the MNR calculated using a Fab versus the bivalent IgG (right). Neutralization results with MNRs ≤5 are indicated as not demonstrating avidity effects (-), >10 are indicated as demonstrating minimal avidity (+), results with one MNR> 50 are indicated as moderate avidity (++), and MNRs demonstrating strong avidity effects (one MNR >700) are indicated as +++.

Article Snippet: A gene encoding a 6xHis-tagged soluble human ACE2 construct (residues 1-615) was purchased from Addgene (Catalog # 149268) and expressed and purified as described ( ).

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay, Comparison, Neutralization