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: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: Cell Reports Medicine

Article Title: Cross-Sectional Evaluation of Humoral Responses against SARS-CoV-2 Spike

doi: 10.1016/j.xcrm.2020.100126

Figure Lengend Snippet: Anti-S Neutralizing Antibody Titers Decrease over Time Pseudoviral particles coding for the luciferase reporter gene and bearing the glycoproteins SARS-CoV-2 S (A, D, G, and H), SARS-CoV S (B, E, and I), or VSV-G (C and F) were used to infect 293T-ACE2 cells. Pseudoviruses were incubated with serial dilutions of samples from 10 COVID-19-negative or 106 COVID-19-positive patients (T1, T2, T3, T4, and convalescent) at 37°C for 1 h prior to infection of 293T-ACE2 cells. Infectivity at each dilution was assessed in duplicate and is shown as the percentage of infection without sera for each glycoprotein. Neutralization half maximal inhibitory serum dilution (ID 50 ) (G and I) and ID 80 (H) values were determined using a normalized non-linear regression using GraphPad Prism software. Undetectable measures are represented as white symbols. Neutralizer represent patients with an ID 50 over 100 (G and I) or an ID 80 (H). Statistical significance was tested using Mann-Whitney U tests (∗p < 0.05; ∗∗p < 0.01).

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: Luciferase, Incubation, Infection, Neutralization, Software, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Cross-Sectional Evaluation of Humoral Responses against SARS-CoV-2 Spike

doi: 10.1016/j.xcrm.2020.100126

Figure Lengend Snippet:

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: Recombinant, Modification, Saline, Western Blot, Lysis, Expressing, Transfection, Staining, Labeling, Plasmid Preparation, Software, Flow Cytometry

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) GDF15 levels; B) ACE2 levels ; C) ratio GDf15/ACE2 ; The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); GDF15 (Growth differentiation factor 15); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 is positive associated with age (A), while ACE2 is not correlated with age (B). Each dot represents an individual value. The solid blue line represents the regression line. The grey shade represents the confidence interval.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) SARS-CoV-2 cell entry assay strategy: Lentiviral-based replication-defective pseudovirus were generated in HEK293T cells from lentiviral parental genes, SARS-CoV-2 Spike and encapsulating a mCherry reporter. Since the entry steps of the SARS-CoV-2 pseudovirions are governed by the coronavirus Spike protein at their surface, they enter cells in a similar fashion to native counterparts. A549 airway cells were transfected with exogenous GFP-hACE2 enabling SARS-CoV-2 pseudovirus to effectively infect the cells with mCherry reporter. Double-positive GFP/mCherry cells were quantified by flow cytometry to assess viral infection capacity. B,C) Following the strategy described in A, A549 cells expressing GFP-hACE2 were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ****P < 0.0001; **P < 0.01; *P < 0.1 by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Generated, Transfection, Flow Cytometry, Infection, Expressing, Plasmid Preparation, Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A549 cells were transfected with either GFP-ACE2 WT, GFP-ACE2 polymorphisms or GFP alone. Then, ACE2 protein expression was analyzed by A) Western Blot with MA5-32307 antibody B) Immunocytochemistry (red) with either MA5-32307 antibody (left panel) or MAB933 antibody (right panel). Nuclei was stained with DAPI (blue). Transfected cells contain GFP (green). CT: secondary antibody control to detect unspecific binding. Images were acquired with Cell Observer-Zeiss. Scale bar: 50 μm

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Transfection, Expressing, Western Blot, Immunocytochemistry, Staining, Control, Binding Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Studying the effect of ACE2 SNPs. Non-synonymous ACE2 single nucleotide polymorphism were selected among those fulfilling the triple criteria of high allelic frequency (Allele freq > 1.00e-4; Allele count > 20); involved in ACE2-claw S-protein RBD-binding interface and previously associated to clinical outcome. B, C) Following the strategy described in , A549 cells expressing either GFP-ACE2 either WT or polymorphisms were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ***P < 0.001, **P < 0.01, *P < 0.1, ns P >0.1 to WT by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Variant Assay, Flow Cytometry

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Heatmap showing the distribution of ACE2 variants in the hospitalization severity groups. Coloured squares indicate de presence of the ACE2 variants. Red: Promoting; Green: Protective; Yellow: No-effect. B) Frequencies of ACE2 SNPs among hospitalization severity groups. Bars represent frequencies of the SNP in each group C) Frequencies of ACE2 SNPS among susceptibility groups.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels across ACE2 variants.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels among ICU and non-ICU COVID-19 patients and ACE2 genotypes.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Genotype 0 corresponding to subjects that did not carry any variant; Genotype 1, subjects that carry at least one promoting variant; Genotype 2, subjects that carry at least one protective variant; Genotype 3, subjects that carry at least one promoting and one protective variant ( ; ). We only found differences in the levels of the ACE2 mRNA ( P <0.05).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Then, PCR1, PCR2 and PCR3 were performed in order to amplify the regions comprising the studied SNPs. PCR products were sequenced and aligned against reference ACE2 (NM_021804). PCR1: residues from Ser3 to Met249 (743 bp), PCR2: residues Phe308 to Arg621 (944 bp), PCR3: residues from Val670 to Val752 (250 bp) A) Agarose gel electrophoresis with the PCR products of several patients was performed to confirm specificity. B) Representative image of the alignment of the sequenced (forward and reverse) PCR products against reference ACE2 using SnapGene® Software. In particular, the image corresponds to the PCR2 of the non-ICU patient 10 that presents the L351V variant.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Agarose Gel Electrophoresis, Software, Variant Assay