Journal: Journal of Medical Virology

Article Title: Elevated Soluble ACE2 Activity in Children and Adults After SARS‐CoV‐2 Exposure Irrespective of Laboratory‐Confirmed Infection

doi: 10.1002/jmv.70098

Figure Lengend Snippet: Soluble ACE2 (sACE2) activity in sera from unexposed pre‐pandemic controls ( n = 154) and (A) participants 3 months ( n = 1192) and 1 year ( n = 345) after SARS‐CoV‐2 exposure, (B) categorized by participants with ( n = 594) and without ( n = 598) evidence of infection 3 months after exposure, and (D) categorized by participants with ( n = 236) and without ( n = 109) evidence of infection 12 months after exposure. (C) Longitudinal sACE2 activity from 345 participants 3−12 months after SARS‐CoV‐2 exposure. Individual data for each participant are depicted in gray. (A, B, D) Mean and 1 ± times standard deviation and (C) longitudinal mean are shown in black. p values were calculated using (A, B, D) one‐way ANOVA (A: F [2, 1688] = 184.4, p < 0.0001; B: F [2, 1343] = 61.9, p < 0.0001; D: F (2, 496) = 0.33, p = 0.72) with Tukey correction for multiple comparisons and (C) paired t ‐test.

Article Snippet: Plasma sACE2 enzyme activity was determined using the commercially available SensoLyte 390 ACE2 activity assay kit (72086; AnaSpec) according to the manufacturer's protocol.

Techniques: Activity Assay, Infection, Standard Deviation

Journal: Journal of Medical Virology

Article Title: Elevated Soluble ACE2 Activity in Children and Adults After SARS‐CoV‐2 Exposure Irrespective of Laboratory‐Confirmed Infection

doi: 10.1002/jmv.70098

Figure Lengend Snippet: Soluble ACE2 (sACE2) in sera of (A) 1192 participants 3 months and (B) 345 participants 1 year after SARS‐CoV‐2 exposure stratified by sex. Mean and 1 ± times standard deviation are shown in black and individual data for each participant are depicted in gray. p values were calculated using unpaired t ‐tests.

Article Snippet: Plasma sACE2 enzyme activity was determined using the commercially available SensoLyte 390 ACE2 activity assay kit (72086; AnaSpec) according to the manufacturer's protocol.

Techniques: Standard Deviation

Journal: Journal of Medical Virology

Article Title: Elevated Soluble ACE2 Activity in Children and Adults After SARS‐CoV‐2 Exposure Irrespective of Laboratory‐Confirmed Infection

doi: 10.1002/jmv.70098

Figure Lengend Snippet: Soluble ACE2 (sACE2) activity stratified by age group in sera of (A) 1192 participants 3 months and (B) 345 participants 1 year after SARS‐CoV‐2 exposure and participants (C) without and (D) with evidence of infection 3 months after SARS‐CoV‐2 exposure. Mean and 1 ± times standard deviation are shown in black and individual data for each participant are depicted in gray. p values were calculated using unpaired t ‐tests.

Article Snippet: Plasma sACE2 enzyme activity was determined using the commercially available SensoLyte 390 ACE2 activity assay kit (72086; AnaSpec) according to the manufacturer's protocol.

Techniques: Activity Assay, Infection, Standard Deviation

Journal: Journal of Medical Virology

Article Title: Elevated Soluble ACE2 Activity in Children and Adults After SARS‐CoV‐2 Exposure Irrespective of Laboratory‐Confirmed Infection

doi: 10.1002/jmv.70098

Figure Lengend Snippet: (A) Soluble ACE2 (sACE2) activity in sera of 67 exposed participants without prior evidence of SARS‐CoV‐2 infection (seronegative and no positive PCR) 3 months after SARS‐CoV‐2 exposure stratified by SARS‐CoV‐2 specific T‐cell response. (B) Longitudinal sACE2 activity from 22 T‐cell negative participants 3−12 months after SARS‐CoV‐2 exposure. Individual data for each participant are shown in gray. (A) Mean and 1 ± times standard deviation and (B) longitudinal mean are shown in black. p value was calculated using (A) unpaired and (B) paired t ‐test.

Article Snippet: Plasma sACE2 enzyme activity was determined using the commercially available SensoLyte 390 ACE2 activity assay kit (72086; AnaSpec) according to the manufacturer's protocol.

Techniques: Activity Assay, Infection, Standard Deviation

Journal: Journal of Medical Virology

Article Title: Elevated Soluble ACE2 Activity in Children and Adults After SARS‐CoV‐2 Exposure Irrespective of Laboratory‐Confirmed Infection

doi: 10.1002/jmv.70098

Figure Lengend Snippet: (A) Schematic illustration of in vitro experiments to determine shedding of soluble ACE2 (sACE2) upon exposure to pseudovirus particles bearing a SARS‐CoV‐2 spike protein (SCoV‐2 spike) (created with BioRender.com ). (B) Absolute soluble ACE2 (sACE2) activity and the change in sACE2 (∆sACE2) activity in the supernatant of Calu‐3 (human lung) cells after 16 h of exposure to pseudovirus with (yes) versus without (no) SARS‐CoV‐2 spike. Individual data points for each biologically independent experiment are depicted with black dots connected by lines. For ∆sACE2 activity, the mean and confidence intervals between the experiments are shown. The p ‐value was calculated using a paired t ‐test.

Article Snippet: Plasma sACE2 enzyme activity was determined using the commercially available SensoLyte 390 ACE2 activity assay kit (72086; AnaSpec) according to the manufacturer's protocol.

Techniques: In Vitro, Activity Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Choice of cell line for sACE2 2 .v2.4-IgG1 production impacts pharmacokinetics and glycosylation (A and B) sACE2 2 .v2.4-IgG1 was expressed and purified from human Expi293F (gray) or nonhuman ExpiCHO-S (black) cultures that were both transiently transfected. A 10 mg/kg amount of sACE2 2 .v2.4-IgG1 was injected into the tail vein of human FcRn transgenic mice. (A) ACE2 catalytic activity and (B) protein concentrations based on ELISA were measured in plasma. Data are mean ± SEM, n = 3 mice per time point. (C) N-glycan types from glycomics analysis of sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S vs. Expi293F cells. (D) Abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures. (E and F) O-Glycan analysis of sACE2 2 .v2.4-IgG1 produced in (E) ExpiCHO-S and (F) Expi293F cells. After PNGaseF treatment of protein, O-glycans were released, purified, permethylated, and analyzed by MALDI-TOF-MS. O-glycan structures were assigned using Glycoworkbench software based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x axis by their m/z ratio. GlcNAc, blue squares; GalNAc, yellow squares; Gal, yellow circles; Neu5Ac, purple diamonds.

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Purification, Transfection, Injection, Transgenic Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Produced, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: An engineered derivative of sACE2 2 .v2.4-IgG1 and its glycosylation (A) Left, the structure (PDB: 6M17 ) of dimeric ACE2 (chains “A” and “B” in dark and light green) bound to RBD (gray ribbons). Glycans are shown as orange sticks. PD, protease domain; CLD, collectrin-like dimerization domain. Center and right, residues mutated to fill cavities (blue spheres), introduce disulfides (yellow spheres), or add N-glycosylation motifs (purple spheres) are shown on a single ACE2 subunit. Lead candidate sACE2 2 .S19-IgG1 has mutations V491I, M662T, N720S. (B) N-glycan types on sACE2 2 .S19-IgG1 produced in Expi293F cells. (C) Abundance of sialylated and fucosylated N-glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells. (D) O-Glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells following O-glycan release and MALDI-TOF-MS analysis. (E) Occupancy of the N-glycosylation sites based on glycopeptidomics analysis of sACE2 2 .S19-IgG1 from Expi293F (green), sACE2 2 .v2.4-IgG1 from Expi293F (pale gray), and sACE2 2 .v2.4-IgG1 from ExpiCHO-S (dark gray). sACE2 2 .S19-IgG1 has added glycosylation sites at positions 660 and 718. (F) Percent of the glycoforms at each N-glycosylation site that have at least one sialic acid.

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Introduce, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: High sialylation and YTE mutations in the Fc region enhance the pharmacokinetics of sACE2 2 .S19-IgG1 (A) Intravenous administration of proteins at 10 mg/kg into human FcRn mice. Blood was collected into heparin via retroorbital route at the plotted time points. Plasma levels of the indicated proteins were measured by ELISA. (B and C) Proteins were injected s.c. in the flank of human FcRn mice at a dose of 10 mg/kg (solid lines) or 100 mg/kg (broken line). (B) ACE2 catalytic activity in plasma and (C) protein concentrations based on ELISA. (D) sACE2 2 .S19-IgG1(YTE) was treated with PNGase F or neuraminidase. Proteins (20 μg) were analyzed without further purification by SDS-PAGE under non-reducing conditions and stained with Coomassie. The calculated molecular weight (MW) of the mature polypeptide (excluding glycans) is 218 kD for the dimer. PNGase F has an MW of 36 kD. A. ureafaciens neuraminidase is a mixture of isoenzymes. (E) Proteins (10 μg) were analyzed by IEF gel electrophoresis. Glycosidase-treated proteins were analyzed without (−) and with (+) purification by gel filtration following treatment. (F) Purified proteins were injected i.v. at 10 mg/kg into human FcRN mice and concentrations in plasma were measured by ELISA for 5 days. For PK studies in this figure, data are mean ± SEM for n = 3 per group and proteins were purified from transiently transfected Expi293F.

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Activity Assay, Purification, SDS Page, Staining, Molecular Weight, Nucleic Acid Electrophoresis, Filtration, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: The v2.4 and S19 mutations in ACE2 are predicted to have low immunogenicity (A) Computational analysis of calculated affinity of peptides to HLA-II allotypes. The wild-type sACE2 2 -IgG1 sequence (top row) is scanned for peptides predicted to be displayed on a common set of 14 HLA-II allotypes. In this analysis, the minimum threshold for a peptide to be considered an antigen is predicted affinity for four HLA-II allotypes (Nhits ≥4) from a set of 14 common alleles. For sACE2 2 -IgG1 derivatives, the sequence is grayed out except for the regions where mutations are introduced to highlight whether a mutation is within a predicted epitope and/or changes the probability of presentation. Peptides that were analyzed experimentally by yeast display are indicated below with dark red bars. (B) Peptides were displayed on yeast and binding to HLA-II following an HLA-DM-dependent peptide loading reaction was measured by flow cytometry. The correlation plot shows the agreement between two independent replicates measuring mean fluorescence units (MFU) for bound HLA-II. Polyserine negative control reactions are blue, positive control peptide/HLA-II pairs are orange, and ACE2 peptides are gray. (C) Yeast display measurements of HLA-II binding to ACE2 peptides is plotted from no signal (dark blue) to high binding signal (orange).

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Sequencing, Mutagenesis, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Positive Control