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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Rapid restoration of potent neutralization activity against the latest Omicron variant JN.1 via AI rational design and antibody engineering.

doi: 10.1073/pnas.2406659122

Figure Lengend Snippet: Fig. 2. Therapeutic efficacy of antibody 8G3 against SARS-CoV-2 infection in transgenic mice. (A) Human ACE2 transgenic mice were challenged with 300-fold TCID50 of WA1/2020 or 2.3 × 104-fold TCID50 of Omicron BA.1 viruses and then received two doses of 8G3 treatment at 4 h and 48 h postinfection. (B) Mouse survival number. (C and D) Percentage of Body weight of mice. Clinical manifestations were observed at least once daily. The data points for antibody groups are identical, resulting in complete overlap on the graph. Data with duplications are shown as means ± SD. (E and F) Clinical score of mice. The clinical well-being of mice was assessed based on a 1 to 4 grading system. Data with duplications are shown as means ± SD. (G and H) Viral titer in lung and brain tissues on day 4 and day 7; W represents WA1/2020, and O represents BA.1. Data are shown as mean ± SD. Statistical differences were determined by one-way ANOVA, in which we set the data of body weight of dead mice in (C) and (D) to 0, and the clinical scores of dead mice in (E) and (F) to 4 (the maximum value). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Permissive AC70 human ACE2 transgenic mice (Taconic Biosciences) were intranasally challenged with 300 half tissue culture infectious dose (TCID50) of WA1/2020 virus (US_WA- 1/2020) or 2.3 × 104 TCID50 of BA.1 virus (Strain: EHC_C19_2811c).

Techniques: Drug discovery, Infection, Transgenic Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Rapid restoration of potent neutralization activity against the latest Omicron variant JN.1 via AI rational design and antibody engineering.

doi: 10.1073/pnas.2406659122

Figure Lengend Snippet: Fig. 3. Cryo-EM structural analysis of the 8G3 antibody and Omicron BA.1 RBD complex. (A) Domain-colored cryoelectron microscopy structures of the SARS- CoV-2 S ectodomain trimer and 8G3 fragment complexes. (B) Cryo-EM structure of 8G3 and the complex with BA.1 RBD. (C) Amino acid residues of the RBD that interact with ACE2, the heavy chain of 8G3, and the light chain of 8G3. (D–I) Details of the binding of BA.1 RBD (cyan) to the heavy (D–H) and light (I) chains of 8G3. (F) Density map of residues 455 to 456 of RBD and the amino acids on the antibody heavy chain within 5 Å of these residues. The density map is displayed at a contour level of 0.6. In this study, the amino acid residues of the antibodies were numbered in sequence order starting from the variable region without considering the regional and structural characteristics of the antibodies.

Article Snippet: Permissive AC70 human ACE2 transgenic mice (Taconic Biosciences) were intranasally challenged with 300 half tissue culture infectious dose (TCID50) of WA1/2020 virus (US_WA- 1/2020) or 2.3 × 104 TCID50 of BA.1 virus (Strain: EHC_C19_2811c).

Techniques: Cryo-EM Sample Prep, Cryo-Electron Microscopy, Binding Assay, Sequencing

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