ace2 goat anti hace2  (R&D Systems)

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

doi: 10.1016/j.ymthe.2023.09.002

Figure Lengend Snippet: Figure 1. Generation and characterization of anti-hACE2-secreting hybridoma (A) Schematic representation of immunization schedule. Balb/c mice were immunized with 3 1011 vg of AAV6/hACE2 and 1 1012 vg of AAV9/hACE2 via the intratracheal and intraperitoneal routes, respectively, followed by boosts of Alum-hACE2 protein (10 mg) administered via intramuscular injection at weeks 10, 12, and 14 post primary immunization. (B) The titration of anti-hACE2 antibody in the mouse sera at week 14 p.i. (mean ± SEM). (C) The neutralizing activity (NT50) of hACE2-immunized mice serum at week 18 p.i. evaluated by tissue culture infectious dose assay (mean ± SEM; n = 4 for each group). The p values were calculated by two-tailed unpaired Student’s t test. (D) The binding ability of hybridoma supernatants from clones 2H2 (middle panel, red line) and 2G8 (right panel, blue line) to hACE2 expressed on 293T/hACE2 stable cell line was assessed by flow cytometry analysis. The plain medium (left panel, black line) was included as a control. Gray-shaded histograms represent the background staining of 293T/ hACE2 cells. MFI, mean fluorescence intensity.

Article Snippet: For the detection of chimerized anti-hACE2 (ch2H2) antibodies, the plates were coated with goat anti-human IgG, A, and M (Jackson Immuno Research, PA, USA).

Techniques: Injection, Titration, Activity Assay, Two Tailed Test, Binding Assay, Clone Assay, Stable Transfection, Cytometry, Control, Staining

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

doi: 10.1016/j.ymthe.2023.09.002

Figure Lengend Snippet: Figure 2. Characterization of hACE2-blocking ability of hybridoma clone 2H2 (A) Competitive binding of hybridoma clones 2H2 and 2G8 supernatants to 293T/hACE2 cells with mouse Fc-containing recombinant S-RBD protein, measured by FACS analysis. Plain medium (left panel, black line) was included as a control. Gray-shaded histograms represent the background staining of 293T/hACE2 cells. (B) The treatment of hybridoma supernatant from clone 2H2, but not 2G8, inhibited the fusion between 293T/spike cells (green) and 293T/hACE2 cells (red). The serum of hACE2-immunized mouse and irrelevant IgG served as positive and negative controls, respectively. Scale bar, 200 mm.

Article Snippet: For the detection of chimerized anti-hACE2 (ch2H2) antibodies, the plates were coated with goat anti-human IgG, A, and M (Jackson Immuno Research, PA, USA).

Techniques: Blocking Assay, Binding Assay, Clone Assay, Recombinant, Control, Staining

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

doi: 10.1016/j.ymthe.2023.09.002

Figure Lengend Snippet: Figure 3. 2H2 epitope mapping by HDX-MS and cryo-EM analyses (A) Structural mapping of 2H2 epitope on hACE2 by the HDX difference in percentage (%). The HDX difference (DHDX) is defined by subtracting the deuterium uptake of hACE2/2H2-Fab complex from that of hACE2. Color-coded DHDX (scale indicated by the color bar) is superimposed on a crystal structure of hACE2/S-RBD (PDB: 6M0J). The S-RBD model is shown in gold, and the N-terminal residues 21–29 of hACE2, which has the highest DHDX, are highlighted by the red dashed boxed. (B) Deuterium uptakes of hACE2 N-terminal peptides as a function of HDX time. Data are shown as mean ± SEM for triplicates. (C) Superimposition of cryo-EM structure of hACE2/2H2-Fab complex and crystal structure of hACE2/S-RBD complex (PDB: 6M0J). The partial cryo-EM map of hACE2/2H2-Fab is displayed in white. As for the ribbon models of proteins, the heavy chain (HC) and light chain (LC) of 2H2-Fab are shown in cyan and pink, respectively, and hACE2 is in wheat color and S-RBD in yellow. The mapped epitope of 2H2-Fab (N-terminal amino acids 21–29 of hACE2) is colored in red. (D) Enlarged panel of the competitive binding region of 2H2-Fab and S-RBD on hACE2 (highlighted by orange box in C).

Article Snippet: For the detection of chimerized anti-hACE2 (ch2H2) antibodies, the plates were coated with goat anti-human IgG, A, and M (Jackson Immuno Research, PA, USA).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

doi: 10.1016/j.ymthe.2023.09.002

Figure Lengend Snippet: Figure 4. Chimeric 2H2 (ch2H2) mAb has potent neutralizing activity against diverse SARS-CoV-2 variants in vitro (A) The kinetics of mAb ch2H2 binding to immobilized hACE2, analyzed by biolayer interferometry (BLI). Actual curves are displayed as gray lines, and the best fit of the data to a 1:1 binding model is indicated by red lines. (B) Neutralization potency of mAb ch2H2 against various pseudotyped SARS-CoV-2 variants. Data are shown as mean ± SEM for triplicates. The antibody concentrations resulting in 50% inhibition of infectivity (IC50) are also presented. (C) Neutralization of authentic SARS-CoV-2 variants by mAb ch2H2. Data are representative of two independent experiments.

Article Snippet: For the detection of chimerized anti-hACE2 (ch2H2) antibodies, the plates were coated with goat anti-human IgG, A, and M (Jackson Immuno Research, PA, USA).

Techniques: Activity Assay, In Vitro, Binding Assay, Neutralization, Inhibition, Infection

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

doi: 10.1016/j.ymthe.2023.09.002

Figure Lengend Snippet: Figure 5. AAV6/ch2H2 prophylaxis protected K18-hACE2 transgenic mice from SARS-CoV-2 Omicron BA.5 challenge (A) AAV6/ch2H2 transduction can result in long-term expression of mAb ch2H2 in mice serum (mean ± SEM). (B) Schematic overview of challenge experiment design. K18- hACE2 transgenic mice (n = 4 for each group), which received a dose of 3 1011 vg for both AAV6/ch2H2 and AAV6/empty, were challenged with SARS-CoV-2 Omicron BA.5 variant at a dose of 5 104 PFU after 1 week of AAV transduction. Mice were sacrificed on day 3 post challenge, and the lungs were collected for subsequent analyses. (C) The numbers of viral genomic RNA in the lungs of infected K18-hACE2 mice transduced with AAV6/empty (black circle) or AAV6/ch2H2 (red circle) measured by RT- QPCR. The dashed line indicates the limit of detection (LOD, 102 copies/mL of total RNA). (D) The titer of infectious virions in the lungs of infected K18-hACE2 mice transduced with AAV6/empty (black circle) or AAV6/ch2H2 (red circle), measured by TCID50 assay. The dashed line indicates the LOD (102 TCID50/mL). (E) The pulmonary pathophysiology of AAV6/empty- and AAV6/ch2H2-transduced K18-hACE2 mice shown by the H&E images of mouse lungs. Scale bar, 100 mm. (F) Histopathological scores of the lung pathology in infected AAV6/empty- and AAV6/ch2H2-transduced K18-hACE2 mice. In all analyses, p values were calculated by two-tailed unpaired Student’s t test.

Article Snippet: For the detection of chimerized anti-hACE2 (ch2H2) antibodies, the plates were coated with goat anti-human IgG, A, and M (Jackson Immuno Research, PA, USA).

Techniques: Transgenic Assay, Transduction, Expressing, Variant Assay, Infection, Quantitative RT-PCR, TCID50 Assay, Two Tailed Test