soluble dimeric ace2  (Addgene inc)

Journal: bioRxiv

Article Title: Bat sarbecovirus WIV1-CoV bears an adaptive mutation that alters spike dynamics and enhances ACE2 binding

doi: 10.1101/2025.04.14.648681

Figure Lengend Snippet: (a) Chimeric scVSV constructs in the spike NTD, RBD, or CTD2 regions are switched between WIV1-CoV and SHC014-CoV. (b-c) DBT-9 cells overexpressing Ra ACE2 were infected with genome-normalized amounts of scVSV-WIV1-CoV S either WT, K85R, SHC014 RBD, or H623Y (b) , or scVSV-SHC014-CoV S WT or Y623H (c) . Infection was scored by eGFP expression 16-18 hours post-infection (average±SD, n=6 from 3 independent experiments). A range of 3.98×10 8 to 6.06×10 4 viral genomes-equivalents (GEQ) was used. Groups were compared against WT with two-way ANOVA with Tukey’s correction for multiple comparisons, ns p>0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Cells were incubated with 50nM soluble Hs ACE2 for 1h at 4°C and stained with Strep-Tactin XT PE (IBA Lifesciences, Cat# 6-5400-001) for 1h at 4°C.

Techniques: Construct, Infection, Expressing

Journal: bioRxiv

Article Title: Bat sarbecovirus WIV1-CoV bears an adaptive mutation that alters spike dynamics and enhances ACE2 binding

doi: 10.1101/2025.04.14.648681

Figure Lengend Snippet: (a) Schematic of generated chimeric scVSV constructs in which single amino acid substitutions at positions 623 and 1167 are switched between WIV1-CoV and Rs3367-CoV spikes. (b) DBT-9 cells overexpressing Ra ACE2 were infected with genome-normalized amounts of scVSV-WIV1-CoV, scVSV-Rs3367-CoV, scVSV-Rs3367-CoV Y623H, or scVSV-Rs3367-CoV N1167D. Infection was scored by eGFP expression 16-18 hours post-infection (average±SD, n=6-12 from 2-4 independent experiments). A range of 2.19×10 10 to 3.33×10 6 viral genomes-equivalents (GEQ) was used. Groups were compared against WT with two-way ANOVA with Tukey’s correction for multiple comparisons, ns p>0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Cells were incubated with 50nM soluble Hs ACE2 for 1h at 4°C and stained with Strep-Tactin XT PE (IBA Lifesciences, Cat# 6-5400-001) for 1h at 4°C.

Techniques: Generated, Construct, Infection, Expressing

Journal: bioRxiv

Article Title: Bat sarbecovirus WIV1-CoV bears an adaptive mutation that alters spike dynamics and enhances ACE2 binding

doi: 10.1101/2025.04.14.648681

Figure Lengend Snippet: (a) Genome-normalized amounts of scVSV particles bearing spikes of WIV1-CoV, Rs3367-CoV, Rs3367-CoV Y623H, or Rs3367-CoV N1167D were diluted with 3-fold dilutions onto ELISA plates precoated with soluble Hs ACE2, followed by a spike-specific mAb, and incubation with an anti-human HRP-conjugated secondary antibody (average±SD, n=6-8 from 3-4 independent experiments). A range of 2.4×10 7 to 9.88×10 4 viral GEQ was used. Groups were compared against WT with two-way ANOVA with Tukey’s correction for multiple comparisons, ns p>0.05; ** p<0.01; *** p<0.001; **** p<0.0001. (b) Pre-titrated amounts of scVSV-WIV1-CoV, scVSV-Rs3367-CoV, scVSV-Rs3367-CoV Y623H, or scVSV-Rs3367-CoV N1167D were incubated with serial 3-fold dilutions of ADG-2 mAb, starting at 2.5uM, for 1h at 37°C. Virus:ADG-2 mixtures were applied to monolayers of DBT-9 cells overexpressing Rs ACE2. 16-18 hours post-infection, cells were fixed, and infected cells were scored by eGFP expression (average±SD, n=9 from 3 independent experiments). Relative infectivity (%) was calculated by normalizing to the infectivity percentage with no mAb present for each relative virus. AUC values were calculated for each curve, and groups were compared by one-way ANOVA with Dunnett’s post hoc test, ns p>0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Only the statistically significant comparisons are shown. (c-d) 293T cells were transfected with plasmids expressing either WIV1-CoV or Rs3367-CoV spikes and harvested 24 hours post-transfection. (c) Cells were incubated with 50nM of soluble Hs ACE2 and bound protein was detected with Strep-Tactin XT PE. Binding was assessed by flow cytometry (average±SD, n=5 from 3 independent experiments). (d) Cells were immunostained for cell surface expression by ADG-2, followed by a fluorescent secondary antibody, and analyzed using flow cytometry (average±SD, n=5 from 3 independent experiments). WIV1-CoV vs. Rs3367-CoV were compared with unpaired t-test with Welch’s correction, ns p>0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Cells were incubated with 50nM soluble Hs ACE2 for 1h at 4°C and stained with Strep-Tactin XT PE (IBA Lifesciences, Cat# 6-5400-001) for 1h at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Virus, Infection, Expressing, Transfection, Binding Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Bat sarbecovirus WIV1-CoV bears an adaptive mutation that alters spike dynamics and enhances ACE2 binding

doi: 10.1101/2025.04.14.648681

Figure Lengend Snippet: (a) scVSV particles bearing spikes of WIV1-CoV or Rs3367-CoV were mixed with 200 ug/mL of trypsin and added onto Vero cells. 16-18 hours post-infection, infection levels were scored by eGFP expression (average±SD. n=6 from 3 independent experiments). Infectivity values were normalized to infection by particles inoculated on cells in the absence of trypsin. (b) 293T cells were transfected with plasmid expression vectors encoding either WIV1-CoV or Rs3367-CoV spike proteins. Spike-expressing cells were incubated with trypsin at 5ug/mL for 1h at 4C. Binding by spike was assessed by mixing with soluble Hs ACE2 for 1h at 4C, followed by incubation with Streptactin PE and analysis by flow cytometry. (average±SD. n=5-6 from 3 independent experiments). Groups (no trypsin vs. trypsin treatment) were compared with unpaired t-test with Welch’s correction, ns p>0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Cells were incubated with 50nM soluble Hs ACE2 for 1h at 4°C and stained with Strep-Tactin XT PE (IBA Lifesciences, Cat# 6-5400-001) for 1h at 4°C.

Techniques: Infection, Expressing, Transfection, Plasmid Preparation, Incubation, Binding Assay, Flow Cytometry

Journal: MedComm

Article Title: Novel Trispecific Neutralizing Antibodies With Enhanced Potency and Breadth Against Pan‐Sarbecoviruses

doi: 10.1002/mco2.70191

Figure Lengend Snippet: Binding properties of novel trispecific antibodies. (A) Recombinant human ACE2 was immobilized on HIS1K sensors, followed by the addition of a mixture containing the indicated antibodies and SARS‐CoV‐2 WT spike trimer. As a positive control, the antibody was instead by PBST buffer, and then was loaded onto the human ACE2 immobilized biosensor (gray). (B) The XBB trimer was captured onto the HIS1K biosensors. Then, PW5‐5, PW5‐535, and PW5‐570 were sequentially used to saturate the trimer binding sites, and the association of Tri‐1 or Tri‐2 was subsequently measured. (C) The XBB trimer was initially captured onto the HIS1K biosensors, following the indicated prototype antibody (PW5‐5, PW5‐535, or PW5‐570) was injected onto the RBD‐connected biosensors, which have already saturated with Tri‐1 (left) or Tri‐2 (right). (D and E) The XBB.1.16 RBD was captured onto the HIS1K biosensors at 40, 200, and 1000 ng/mL for 300 s, respectively. Summary of the data (D) or scatter plot (E) of the affinities ( K D ), association ( K on ), and dissociation ( K off ) of Tri‐1‐ and structural‐related mAbs and bsAbs to indicated concentrations RBD.

Article Snippet: The mammalian expression plasmid encoding recombinant soluble dimeric ACE2 was obtained from Addgene.

Techniques: Binding Assay, Recombinant, Positive Control, Injection