Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) Average force profiles of WT (red), Alpha (blue), Beta (orange), Gamma (sky blue), Epsilon (green), Kappa (pink), and Delta (gray) variants as a function of the distance between the centers of mass of RBD and ACE2. (B) Initial snapshot of WT. Residues subjected to each mutation are shown as solid sticks (N501, K417, E484, L452, and T478). RBD and ACE2 are, respectively, colored in light gray and yellow. All N-glycans, water, and ions are hidden for clarity. (C) Initial snapshot of WT with clockwise 90° rotation along the normal from (B). All N-glycans are depicted in different colors. Any other residues, water, and ions are not shown for clarity.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Two-dimensional contact maps at D = 53 Å. (A) Interacting residue pairs between RBD WT and ACE2. RBD residues subjected to mutation are shown in colored boxes at the bottom: (B) blue for Alpha, (C) orange for Beta, and (D) green for Epsilon. The contact frequency is numbered with colors from light blue to dark blue. Dark red and yellow colors on the map, respectively, represent increased and decreased interactions between RBD and ACE2 upon mutations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) The average number of contacts between RBD residue 501 and ACE2. (B, C) Representative snapshots at D = 53 Å of (B) Alpha variant and (C) WT. (D) Average number of contacts between RBD residue 417 and ACE2 and (E, F) their interacting residue pairs at D = 53 Å of (E) Beta and (F) Alpha variants. (G) Average number of contacts between RBD residue 478 and ACE2 and (H, I) key interaction pairs at D = 78 Å of (H) Delta and (I) Epsilon variants. The overall color scheme is the same as in Figure , and each mutated residue in each variant is shown using the same colors (i.e., red for WT, blue for Alpha, orange for Beta, green for Epsilon, and gray for Delta). Interacting residues are depicted as solid sticks, and residues losing their interactions are shown as transparent sticks. RBD and ACE2 are presented in light gray and yellow, respectively.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Variant Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Binding affinities between RBD variants and ACE2 and its comparison with the simulation results. K d is obtained from microscale thermophoresis experiments. F WT / F is a ratio, where F WT and F are the respective maximum pulling force of WT and of each variant obtained from the SMD simulations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Binding Assay, Microscale Thermophoresis, Variant Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Comparison between RBDCoV-ACE2 and a virus neutralization test (VNT). Serum samples (n=16) of pre-pandemic (n=4) and COVID-19 convalescent (n=12) individuals were measured using both assays and analyzed by linear regression. The equation of the dashed regression line is shown next to the graph. VNT results are depicted as half-maximal inhibiting serum dilutions (VNT 50 ), RBDCoV-ACE2 results are shown in percentage inhibition of ACE2 binding. Correlation analysis was performed after Spearman and the correlation coefficient r is shown.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Neutralization, Inhibition, Binding Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between SARS-CoV-2 NeutraLISA and VNT and comparison to RBDCoV-ACE2. (a) Correlation and linear regression between NeutraLISA and VNT results for pre-pandemic (n=4) and COVID-19 infected (n=12) samples. Correlation analyses were performed after Spearman and correlation coefficients r are shown. (b) Descriptive statistics of the (c) correlation between NeutraLISA and RBDCoV-ACE2. One sample from each individual (n=168) was measured using both assays correlation was calculated after Spearman. Samples were classified as being negative (non-neutralizing) if they had a value below 20% (red lines).

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Infection

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: ACE2 binding inhibition varies between RBD mutants. Violin plots showing ACE2 binding inhibition (%) of individual serum samples from 7 to 49 days post PCR (n=50, depicted as dots) against RBD mutants. Black horizontal lines represent medians. Fold-decrease of ACE2 binding inhibition in comparison to wild-type corresponds to the ratio between the medians of wild-type and the respective RBD mutant. VOC-RBDs are shown in blue. Mutations of each RBD mutant are shown in the box above the violin plot.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Mutagenesis

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between anti-RBD IgG MFI signals and ACE2 binding inhibition (%) of serum samples from COVID-19 patients for wild-type and 11 RBD mutants. Regression analysis comparing ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type and all RBD mutants included in the study. Each circle represents one sample (n=168). For longitudinal donors with more than one sample available, the sample closest to 20 days post positive PCR diagnosis was selected. The percentage next to the bracket indicates the proportion of samples with ACE2 binding inhibition ≤ 20% (in orange). Spearman’s correlation coefficient (r) is specified for every correlation.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Longitudinal analysis of ACE2 binding inhibition and anti-RBD IgG levels in Covid-19 patients. Mean ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type RBD against time post positive PCR test for samples (n=149) taken from 1 to 92 days post PCR are shown (a, b). Black dots indicate mean responses with standard deviation indicated by the error bars. The same analysis is then shown for longitudinal samples of selected donors (n=6) for wild-type (c, d) and RBD delta (e, f). For all RBD mutants, mean ACE2 binding inhibition (%) and mean IgG responses (MFI) 1 to 92 days post PCR included in the study is shown (g, h). Each variant is illustrated by a different color according to the figure key.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Variant Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation of anti-RBD IgG levels and ACE2 binding inhibition with SARS-CoV-2 disease severity. Bar charts showing mean ACE2 binding inhibitions (%) against wild-type and RBD delta are correlated with WHO grades for disease severity for samples 7-49 days post PCR (a, b) and ≥ 50 days post PCR (c, d). Mean anti-RBD WT IgG and anti-RBD delta IgG levels are shown for samples 7-49 days post PCR (e, f) and ≥ 50 days post PCR (g, h). Individual samples are displayed as colored dots, bars indicate the mean of the dataset with error bars representing standard deviation. Number of samples is given below the columns (n). If no samples for a group were available, the column is labeled with “n/a”. WHO grade 1 - ambulatory / no limitations of activities, 2 - ambulatory / limitation of activities, 3 - hospitalized, mild disease / no oxygen therapy, 4 - hospitalized, mild disease / mask or nasal prongs, 6 - hospitalized, severe disease / intubation + mechanical ventilation, 7 - hospitalized, severe disease / ventilation + additional organ support (pressors, RRT, ECMO), 8 – Death. The study did not contain samples of WHO grade 5.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Labeling

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characteristics of potential blocking antibodies

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Expressing, Binding Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 1 Schematic Illustration. a Preparation of rhACE2-loaded electrospun nanofiber patch by hyaluronan (HA) micro-sol electrospun and the mechanism of formation of core-shell structure. b Myocardial infarction mice model established by precise ligation of the left anterior descending (LAD) coronary artery along with the illustration of rhACE2 patch implantation. c In situ rhACE2 patch niche degrading angiotensin II (AngII) into a cardioprotective heptapeptide, Ang1–7, which counter the AngII/AT1R mediated effects by inhibiting cardiac fibrosis and cardiomyocyte apoptosis. PLLA poly(L-lactic acid), rhACE2 recombinant human angiotensin-converting enzyme 2, AT1R angiotensin II receptor type 1, PKC protein kinase C, MAPK mitogen-activated protein kinase, ECM extracellular matrix.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: Ligation, In Situ, Recombinant

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 2 Characterization of the rhACE2 patch. The general view, SEM and TEM micrographs demonstrating the core-shell structure (a), dynamic light scattering (b), water contact angle (c) and stress-strain curve (d) from PLLA-HA/ACE2 electrospun nanofibers. Scale bars represent 1 cm in general view, 20 μm in SEM and 1 μm in TEM.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques:

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 3 Biocompatibility tests in vitro. a Representative live/dead fluorescence stained by Calcein AM (green) and Propidium Iodide (red) at day 3. Scale bars represented 50 and 20 μm for zoomed pictures. b Live and dead cell count per HPF of control, PLLA, PLLA-HA/ACE2 groups. n = 4/group. c CCK-8 cell viability quantification results in different groups after 1, 2, or 3 days. n = 4/group. Data were represented as the mean ± SEM and analyzed for statistical significance using One-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: In Vitro, Staining, Cell Counting, Control, CCK-8 Assay, Comparison

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 5 rhACE2 patch sustained-release activity test. a In vitro releasing curve of PLLA-HA/ACE2 fibrous membranes. b Releasing buffer collected at specified time points during release study was added into culture media of NRCMs undergone 6 h hypoxia. Representative immunofluorescence image of TUNEL (red) and nuclear visualized by DAPI (blue). Scale bars represented 50 μm. c Statistical analysis of TUNEL- positive cell counts. n = 3/group. Data were represented as the mean ± SEM and analyzed for statistical significance using One-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference; **P < 0.01 compared to the control group.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: Activity Assay, In Vitro, TUNEL Assay, Comparison, Control

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 6 rhACE2 patch preserve left ventricular function after acute myocardial infarction in vivo. a The animal research timeline design with images represented the acute MI model and successful implantation of rhACE2 patch. Echo, echocardiography. b Representative M-mode parasternal long axis view of left ventricle echocardiographic images of different groups at day 28 after LAD coronary artery ligation. Statistical analysis of left ventricular ejection fraction (c), shortening fraction (d), heart weight/body weight ratio (e), LV end-diastolic diameter (f), LV end- systolic diameter (g) and heart weight/tibial length (h) determined by echocardiography obtained from PLLA, intramyocardial injection (IM) and PLLA-HA/ACE2 treatment group at day 7, day 14 and day 28 after operation. n = 5/group. Data were represented as the mean ± SEM and analyzed for statistical significance using two-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference, *P < 0.05, **P < 0.01.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: In Vivo, Ligation, Injection, Comparison