Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 1. Gel permeation chromatograms and SDS-Page analysis of different rh-s-ACE2 products. A) rh-s-ACE2, B) rh-s-ACE2i and C) rh-s-ACE2i-biotin, as pre pared (full line) and after 24 h storage at 4 ◦C in PBS buffer (dashed lines). Rh-s-ACEi reacted with biotin-C6-NHS and rh-s-ACE2i-biotin were also analyzed after mixing with ANANAS (ACE2:NP = 30 mol:mole) (dash-dot lines). Only if biotinylated the protein binds to ANANAS and elutes together with the nanoparticle fraction with a retention volume of about 8 mL; D) SDS-PAGE (4–15 %) analysis of the different rh-s-ACE2 products after 24 h storage at 4 ◦C, conducted in reducing (R) and non-reducing (NR) conditions. The expected protein (MW ~ 69500 Da) displays a band at around 70 kDa. The high MW smeared signal in the NR rh-s-ACE2 sample corresponds to protein multimers, which are reverted to the expected monomeric protein by the reducing (R) treatment.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: SDS Page

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 2. Pre-formulation studies. A) Size of rh-s-ACE2i-biotin:ANANAS assemblies at ACE2:NP molar ratio in solution between 15 and 120; B) Assemblies Z-potential as a function of rh-s-ACE2i-biotin:NP molar ratio in solution mixture. Values were measured in PBS buffer, where core ANANAS exhibit a slightly negative value of −15.43 mV; C) Gel permeation chromatograms of rh-s-ACE2i-biotin (5 μg/run) as such or when mixed with ANANAS at molar ratios ACE2:NP equal to 30, 60 and 90. The peak at retention volume of about 17 mL corresponds to the free protein, the peak at 8 mL corresponds to the nanoassembly; D) Number of rh-s-ACE2i-biotin molecules linked onto the NPs as a function of the ratio of ACE2i-biotin:NP in solution mixtures. Values were calculated from the chromatograms of panel C by comparing the areas of the rh-s-ACE2i-biotin peak in the assembly mixtures with that of the protein in the absence of the nanoparticles.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Formulation

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 3. Affinity of rh-s-ACE2-biotin in solution or multimerized onto the NP surface. Representative curves of binding to RBD of rh-s-ACE2-biotin in free form, and multimerized onto the surface of nanoparticles at ACE2:NPs molar ratios between 15:1 and 60:1 measured by ELISA; A) Wuhan, B) Omicron variants, C) dissociation constants calculated from the ELISA curves; D) dissociation constants of the rh-s-ACE2i-biotin:RBD interaction for ACE2i differently formulated relative to the protein in solution. An R value > 1 indicates lower affinity. Kds were computed using the GraphPad-Prism® software and the one site specific binding equation. Data indicate mean ± SD of 2–4 independent experiments each performed in duplicate. Full data are also reported in the Supplementary Information (S.I.) file (Table S1).

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 4. Inhibition of SARS-CoV-2 infectivity by monomeric and nanoassembled rh-s-ACE2i-biotin. A) Inhibition of SARS-CoV-2 infection in Calu-3 by rh-s- ACE2i-biotin in monomeric form as a function of its concentration. Infection (Wuhan variant) was performed at MOI (multiplicity of infection) of 0.1. The virus was pre-incubated for 10 min with rh-s-ACE2i-biotin prior to Calu-3 cells infection; B) Efficacy of inhibition of SARS-CoV-2 infection (Wuhan variant) by rh-s-ACE2i- biotin at 2.5 μg/mL in monomeric or multimerized form on the ANANAS core at different ACE2:NP molar ratios. To keep the concentration of ACE2 in the assay constant at 2.5 μg/mL, the concentration in nanoparticles was different in the different samples (12.9, 25.7 and 51.5 μg/mL for ACE2:NP-R60, ACE2:NP-R30 and ACE2:NP-R15, respectively). Non-functionalized nanoparticles were therefore tested as control at these three concentrations, together with rh-s-ACE2i-biotin at 2.5 μg/mL in monomeric form; C/D) Efficiency in viral (Wuhan/Omicron Variants) cycle inhibition of increasing ACE2:NP-R30 concentrations (0–5 μg/ml). Bars indicate the mean of n = 2 biological replicates. Each condition was tested in triplicate per replicate. Individual data points are shown as dots.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Inhibition, Infection, Concentration Assay, Variant Assay, Virus, Incubation, Control

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 5. Transmission Electron Microscopy. Representative images of A) SARS-CoV-2, B) the ANANAS:rh-s-ACEi2-30 (ACE2:NP-R30) nanoassembly and C1/C2) the mixture of SARS-CoV-2:ACE2:NP-R30 generated at PFU:NP molar ratio = 1:100. Samples were negatively stained with 1 % uranyl acetate, followed by biotin- nanogold (5 nm). The gold nanoparticles appear ad black dots. More images can be found in the Supplementary information file (Fig. S3).

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Transmission Assay, Electron Microscopy, Generated, Staining

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 6. Ex vivo imaging (A) Ex vivo optical imaging of excised organs from animals sacrificed 15′, 2 h, 24 h and 72 h after vehicle, ANANAS, and ACE2:NP-R30 administration. Br. = brain, Lu. = lungs, Li. = liver, Sp. = spleen, Ki. = kidneys. (B) Quantification of ex vivo optical imaging signal. Data are reported as mean ± SE. The data were analyzed by One-way ANOVA using Dunnet’s test. *p < 0.1, **p ≤0.01.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Ex Vivo, Imaging, Optical Imaging

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 7. Nanoparticles localization in tissues. Representative images of the tissue distribution of ANANAS or ACE2:NP-R30 in lungs 15′, 2, 24 and 72 h after treatment. The blue signal refers to the nuclei (Hoechst 33258 staining), green corresponds to the lysosomal component of macrophages (CD68 Antibody), red is associated with the alexa633 dye linked to the NPs, yellow corresponds to co-localized red and green signals. Scale bar = 100 μm. In the lower panel, representative images of higher magnification (15X) of lungs for each timepoint (yellow arrows: nanoparticles co-localized with macrophages, red arrows: nanoparticles alone). Scale bar = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Staining

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 8. Histological evaluation by hematoxylin and eosin staining of lung tissue of mice treated with vehicle (CTR), ANANAS and ACE2:NP-R30 and sacrificed 15′, 2, 24, and 72 h after the treatment. Scale bar = 100 μm.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Staining

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 9. Cytokine analysis in serum of untreated and treated animals. (A) IL-10, (B) INF-γ, and (C) TNF-α concentrations were measured in serum samples from treated animals and controls 72h after ANANAS or ACE2:NP-R30 administration. LDL = low detection limit. Data are reported as mean±SD. Statistical analysis was performed by One-way ANOVA, followed by Tukey post-hoc test. No differences were found comparing ANANAS or ACE2:NP-R30 with CTR mice, or directly comparing the two nanoformulations. Overall, these data indicate that the intranasal administration of these formulations to healthy mice does not stimulate an inflammatory response at least over the period of our analysis (72 h), confirming the evidence of lack of toxicity.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques:

Journal: bioRxiv

Article Title: Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

doi: 10.1101/2021.12.21.473668

Figure Lengend Snippet: ( A ) RBD-bound ACE2 proteins were simulated. Newly formed polar interactions between ACE2.v2.4 (orange) and RBD (yellow) are indicated by broken red lines. ( B ) MSM-weighted distance distributions of newly formed polar interactions between RBD and ACE2.v2.4. ( C - D ) Distance distributions between the centers of mass of RBD loops 1 and 2 with respect to ACE2 residues (Cα atoms) 27 ( C ) and 330 ( D ), respectively. Loop centers of mass are calculated from Cα atoms. The means of each distribution are shown by vertical dashed lines. Distributions from simulations of RBD-bound wild type ACE2 are blue, RBD-bound ACE2.v2.4 are orange.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, Addgene No. 141185), 8his-tagged monomeric sACE2 (ACE2 a.a. 1–615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4–8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE2 2 (ACE2 a.a. 1–732; pcDNA3-sACE2-WT(732)-IgG1, Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.

Techniques:

Journal: bioRxiv

Article Title: Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

doi: 10.1101/2021.12.21.473668

Figure Lengend Snippet: ( A - C ) sACE2 2 .v2.4-IgG1 was IV administered to mice (N=6 per time point; 2.0 mg/kg). Serum was collected and analyzed by human IgG1 ELISA ( A ), by ACE2 ELISA ( B ), and for ACE2 catalytic activity ( C ). ( D ) Serum samples from representative male mice were separated on a non-reducing SDS electrophoretic gel and probed with anti-human IgG1 using 10 ng of purified sACE2 2 .v2.4-IgG1 as a standard. Predicted molecular weight (MW; excluding glycans) of dimer is 216 kD. ( E - F ) sACE2 2 .v2.4-IgG1 was incubated in vitro at 37 °C with normal mouse ( E ) and human ( F ) serum. Samples at the indicated time points were separated on a reducing SDS gel and immunoblotted with anti-human ACE2. MW of monomer (excluding glycans) is 108 kD. Shown are representative blots from two experiments. ( G - H ) Wild type sACE2 2 -IgG1 (white circles) and sACE2 2 .v2.4-IgG1 (black circles) were administered IT at 1.0 mg/kg. Lung tissues were collected, and proteins were extracted and analyzed by ( G ) human IgG1 ELISA and ( H ) ACE2 ELISA. N=3 males per time point. ( I ) Lung extracts from representative mice IT administered sACE2 2 .v2.4-IgG1 were analyzed under non-reducing conditions by anti-human IgG1 immunoblot. ( J - K ) Mice inhaled nebulized sACE2 2 .v2.4-IgG1. Extracts from lung tissue were analyzed by ( J ) ACE2 ELISA and ( K ) human IgG1 ELISA. N=3 males per time point. ( L ) Representative extracts from lung tissue of mice receiving nebulized sACE2 2 .v2.4-IgG1 were analyzed by anti-human IgG1 immunoblot. Data are presented as mean ± SEM.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, Addgene No. 141185), 8his-tagged monomeric sACE2 (ACE2 a.a. 1–615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4–8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE2 2 (ACE2 a.a. 1–732; pcDNA3-sACE2-WT(732)-IgG1, Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Purification, Molecular Weight, Incubation, In Vitro, SDS-Gel, Western Blot

Journal: bioRxiv

Article Title: Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

doi: 10.1101/2021.12.21.473668

Figure Lengend Snippet: (A) mRNA expression levels of ACE-2(NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells, A549 cells that stably express hACE2and hLMVECs (human lung microvascular endothelial cells) were analyzed by one-step RT-PCR (above) and real-time PCR (below). Relative expression was normalized to GAPDH expression levels. (B) Cultured hACE2- A549, A549, and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 or 25 μg/ml for 1 hour. SARS-CoV-2 pseudovirus (MOI=0.1) was added to the cells and the cells were harvested at 24 hours. Virus entry was evaluated by luciferase activity. N = 4 replicates. (C) 10 mg/kg sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1, or peptide buffer (PBS + 0.2% BSA) was intravenously administrated into K18 hACE2 transgenic mice for 30 minutes prior to SARS-CoV-2 pseudo-entry virus (10 6 pfu) i.p. injection. Tissue lysates were prepared at 24h and virus entry in the selected organs was evaluated by luciferase activity. Peptide buffer was applied as control group. N=4. ( D - G ) K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 isolate WA-1/2020 at 1×10 4 PFU. The mice received control PBS or sACE2 2 .v2.4-IgG1 10mg/kg via IV injection 12h before inoculation. Mice were observed for survival ( D ) and body weight ( E ), N=5. Quantification of EBA as a marker of pulmonary transendothelial permeability ( F ) and lung wet/dry ratio ( G ) as a measure of lung edema. Data are mean ± SEM, N=4. *P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 by one-way ANOVA. ns: not significant.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, Addgene No. 141185), 8his-tagged monomeric sACE2 (ACE2 a.a. 1–615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4–8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE2 2 (ACE2 a.a. 1–732; pcDNA3-sACE2-WT(732)-IgG1, Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.

Techniques: Expressing, Stable Transfection, One Step RT-PCR, Real-time Polymerase Chain Reaction, Cell Culture, Virus, Luciferase, Activity Assay, Transgenic Assay, Injection, Control, IV Injection, Marker, Permeability

Journal: bioRxiv

Article Title: Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

doi: 10.1101/2021.12.21.473668

Figure Lengend Snippet: ( A ) Experimental design to test the therapeutic efficacy of sACE2 2 .v2.4-IgG1. The K18 hACE2 transgenic mice were inoculated by SARS-CoV-2 isolate WA-1/2020 at 1×10 4 PFU. Group 1 received control PBS via IV injection 24 hours post viral inoculation. Group 2 (V2.4 12H) received sACE2 2 .v2.4-IgG1 10mg/kg via IV injection 12 hours post inoculation, and then daily subsequent injections at the same dose. Group 3 (V2.4 24H) received sACE2 2 .v2.4-IgG1 15mg/kg via IV injection 24 hours post inoculation and then daily subsequent injections at the same dose. ( B ) Survival curves and ( C ) Weights for N = 10 mice for each group. ( D-F ) Mouse lungs were harvested at Day 7 post-inoculation for assessment of lung transvascular albumin permeability. ( D ) Macroscopic images of lungs at baseline and day 7 post-viral inoculation in the three experimental groups without EBA (Evans Blue Albumin) on the left and with EBA injection on the right. ( E ) Quantification of EBA in all three experimental groups. ( F ) Quantification of lung edema by wet/dry ratio in all three experimental groups were shown at baseline and Day 7 post-inoculation. ( G-H ) Time course of lung vascular permeability of Group 2 (V2.4 12H) ( G ) and Group 3 (V2.4 24H) ( H ) as assessed by the EBA assay and by the lung wet/dry ratio. ( I ) Representative H&E staining of lung sections at baseline (1 st column), control PBS group at day 7 post-inoculation with the WA isolate (2 nd column), sACE2.V2.4-IgG 12H treatment group at day 7 (3 rd column), and sACE2.V2.4-IgG 24H treatment group at day 7 (4 th column) post-inoculation with the WA isolate. The images in the first row are low magnifications. Rectangle areas (Red) are shown in higher magnification in the second row. Data are presented as mean ± SEM. **: P<0.01, ***: P<0.001, ****: P<0.0001 by Two-way ANOVA for E & F ; one-way ANOVA for G & H .

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, Addgene No. 141185), 8his-tagged monomeric sACE2 (ACE2 a.a. 1–615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4–8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE2 2 (ACE2 a.a. 1–732; pcDNA3-sACE2-WT(732)-IgG1, Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.

Techniques: Drug discovery, Transgenic Assay, Control, IV Injection, Permeability, Injection, Staining

Journal: bioRxiv

Article Title: Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

doi: 10.1101/2021.12.21.473668

Figure Lengend Snippet: ( A ) Human Expi293F cells expressing myc-tagged S from four SARS-CoV-2 variants (Wuhan, B.1.1.7/alpha, P.1/gamma, and B1.351/beta) were incubated with monomeric sACE2–8h (black) or dimeric sACE2 2 -IgG1 (grey) and bound protein was detected by flow cytometry. N=2. ( B ) Binding of mAbs versus sACE2 2 .v2.4-IgG1 to S proteins of four VOCs (B.1.1.7/alpha, P.1/gamma, B1.351/beta, and B.1.617.2/delta) measured by flow cytometry. N=2.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, Addgene No. 141185), 8his-tagged monomeric sACE2 (ACE2 a.a. 1–615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4–8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE2 2 (ACE2 a.a. 1–732; pcDNA3-sACE2-WT(732)-IgG1, Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.

Techniques: Expressing, Incubation, Flow Cytometry, Binding Assay

Journal: bioRxiv

Article Title: Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

doi: 10.1101/2021.12.21.473668

Figure Lengend Snippet: The three groups of K18 hACE2 transgenic mice were inoculated by SARS-CoV-2 variant P.1 (Brazil) at 1×10 4 PFU. Group 1 (PBS): PBS was given by IV injection 24 hours post inoculation. Group 2 (V2.4 12H): sACE2 2 .v2.4-IgG1 10mg/kg were given by IV injection 12 hours post inoculation. Group 3 (V2.4 24H): sACE2 2 .v2.4-IgG1 15mg/kg were given by IV injection 24 hours post inoculation. The mice were injected once per day for 7 days. The survival probability was observed ( A ) and mouse weights were measured ( B ). N=10 for each group. ( C-D ) The mouse lungs were harvested at Day 6 post-inoculation to evaluate lung transvascular permeability – EBA assay ( C ) and lung wet/dry ratio ( D ) with baseline mouse lungs as control. ( E ) The viral loads of SARS-CoV-2 in the lungs harvested at baseline and Day 6 post-inoculation of SARS-CoV-2 variant P.1 (Brazil) were measured by real-time quantitative PCR for the mRNA expression level of SARS-CoV-2 Spike and SARS-CoV-2 NSPs. ( F ) Viral Plague Form Assay was performed to measure the viral loads of SARS-CoV-2 in the lungs harvested at baseline and Day 6 post-inoculation of SARS-CoV-2 variant P.1 (Brazil). ( G-H ) Time course of lung transvascular permeability of V2.4 12H treatment group. The EBA assay ( G ) and Wet/dry ratio ( H ) were measured at baseline, Day 6 and Day 14 post-inoculation. ( I ) Representative H&E staining of lung sections at baseline (1 st column), control PBS group at day 7 post-inoculation with the P.1 variant (2 nd column), sACE2.V2.4-IgG 12H treatment group at day 7 (3 rd column), and sACE2.V2.4-IgG 24H treatment group at day 7 (4 th column) post-inoculation with the P.1 variant. The images in the first row are low magnifications. Highlighted areas (Red) are shown in higher magnification in the second row. N=4 for C , D , E , F , G , and H . Data are presented by mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 by Two-way ANOVA for C & D ; one-way ANOVA for E , F , G & H .

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, Addgene No. 141185), 8his-tagged monomeric sACE2 (ACE2 a.a. 1–615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4–8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE2 2 (ACE2 a.a. 1–732; pcDNA3-sACE2-WT(732)-IgG1, Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.

Techniques: Transgenic Assay, Variant Assay, IV Injection, Injection, Permeability, Control, Real-time Polymerase Chain Reaction, Expressing, Staining