Journal: Nature chemical biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: Fig. 5 | ACE2 decoys carrying the v2.4 mutations bind the S protein from multiple highly transmissible SARS-CoV-2 VOCs and SARS-CoV-1. a–d, sACE2 carrying the v2.4 mutations has increased S binding compared with WT sACE2. Human Expi293F cells expressing myc-tagged S from 4 SARS-CoV-2 variants (Wuhan (a), B.1.1.7/Alpha (b), B1.351/Beta (c) and P.1/Gamma (d)) were incubated with monomeric sACE2-8 h (black) or dimeric sACE22-IgG1 (gray); bound protein was detected by flow cytometry. WT ACE2 proteins are shown as broken lines, v2.4 proteins are shown as solid lines. n = 3 independent replicates; data are shown as the mean ± s.e.m. e–i, Binding of sACE22.v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE22.v2.4-IgG1 to the S proteins of SARS-CoV-2 VOCs (B.1.1.7/Alpha (e), B1.351/Beta (f), B.1.617.2/Delta (g) and P.1/Gamma (h)) and S protein of SARS-CoV-1 (i), as measured by flow cytometry. n = 3 independent replicates; data are shown as the mean ± s.e.m.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4myc-ACE2, plasmid no. 141185; Addgene), 8 histidine-tagged monomeric sACE2 (ACE2 amino acids 1–615; pcDNA3-sACE2(WT)-8his, plasmid no. 149268 and pcDNA3-sACE2v2.4-8his, plasmid no. 149664; Addgene), SARS-CoV-1 S protein with C-terminal C9 tag (pcDNA3.1-SARS-spike, plasmid no. 145031; Addgene), 8his-tagged RBD (pcDNA3-SARS-CoV-2-S-RBD-8his, plasmid no. 145145; Addgene) and human IgG1-Fc fused dimeric sACE22 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

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: 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 K-18 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