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: a , mRNA expression of ACE2 (NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells. A549 cells stably expressing hACE2 and hLMVECs were analyzed by one-step RT–PCR (top) and real-time PCR (bottom). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase expression. b , Cultured hACE2-A549 cells, A549 cells and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 µg ml −1 or 25 µg ml −1 for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was added to the cells, which were collected at 24 h. Virus entry was evaluated by luciferase activity. n = 4 replicates. c , A dose of 10 mg kg −1 sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1 or buffer (PBS + 0.2% BSA) was administrated intravenously into K18-hACE2 transgenic mice for 30 min before SARS-CoV-2 pseudovirus (10 6 PFU) intraperitoneal injection. Tissue lysates were prepared at 24 h and virus entry in the selected organs was evaluated by luciferase activity. Buffer was applied as the control group, n = 4. d – g , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Mice received control PBS or sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h before inoculation. Mice were observed for survival ( d ) and body weight ( e ), n = 5. EBA as a marker of pulmonary endothelial permeability ( f ) and lung wet/dry ratio ( g ) as a measure of lung edema were quantified. Data are presented as the mean ± s.e.m., n = 4. b , c , f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

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

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: K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Group 1 received control PBS via intravenous injection 24 h postviral inoculation. Group 2 (v2.4 12 h) received sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h postinoculation and then daily subsequent injections at the same dose. Group 3 (v2.4 24 h) received sACE2 2 .v2.4-IgG1 15 mg kg −1 via intravenous injection 24 h postinoculation and then daily subsequent injections at the same dose. a , b , Survival curves ( a ) and weights ( b ) for n = 10 mice for each group. c – e , Mouse lungs were obtained on day 7 postinoculation for assessment of lung vascular albumin permeability. c , Macroscopic images of lungs at baseline and day 7 postviral inoculation in three experimental groups without EBA (left) and with EBA injection (right). d , Quantification of EBA lung vascular endothelial permeability in all three experimental groups. e , Quantification of lung edema by wet/dry ratio in all three experimental groups at baseline and day 7 postinoculation. f , g , Time course of lung vascular endothelial permeability and edema formation of groups 2 (v2.4 12 h) ( f ) and 3 (v2.4 24 h) ( g ) as assessed by EBA assay and lung wet/dry ratio. Data are presented as the mean ± s.e.m. d , e , P values were calculated by two-way ANOVA with Tukey post hoc test. f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Transgenic Assay, Control, Injection, Permeability

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: 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 sACE2 2 -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 sACE2 2 .v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE2 2 .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 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

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: The 3 groups of K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 variant P.1 (Brazil) at 1 × 10 4 PFU. Group 1 (PBS): PBS was given by intravenous injection 24 h postinoculation. Group 2 (v2.4 12 h): sACE2 2 .v2.4-IgG1 10 mg kg −1 was given by intravenous injection 12 h postinoculation. Group 3 (v2.4 24 h): sACE2 2 .v2.4-IgG1 15 mg kg −1 was given by intravenous injection 24 h postinoculation. a , b , Mice were injected once per day for 7 d. Survival probability was calculated ( a ) and mouse weights were measured ( b ). n = 10 mice for each group. c , d , Mouse lungs were examined on day 6 postinoculation to evaluate lung vascular permeability and lung edema using the EBA assay ( c ) and lung wet/dry ratio ( d ), with baseline mouse lungs as controls. e , Viral loads of SARS-CoV-2 in the lungs obtained at baseline and day 6 postinoculation of the SARS-CoV-2 variant P.1 (Brazil) were measured by real-time qPCR for mRNA expression of SARS-CoV-2 S protein and SARS-CoV-2 NSP. f , A viral plaque formation assay was performed to measure the viral loads of SARS-CoV-2 in lungs obtained at baseline and on day 6 postinoculation of the SARS-CoV-2 P.1 (Brazil) variant. c – e , f , n = 4. Data are presented as the mean ± s.e.m. c , d , P values were calculated by two-way ANOVA with Tukey post hoc test. e , f , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Transgenic Assay, Variant Assay, Injection, Permeability, Expressing, Plaque Formation Assay

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: a – e , K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 isolate WA-1/2020. f – k , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate P.1. K18-hACE2 transgenic mice not inoculated were used as controls, n = 10 mice for each group. a , b , Mice inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU and 1 × 10 5 PFU, respectively were observed for survival ( a ) and weight ( b ). Only live mice were measured for body weight. c – e , Cells from mice inoculated with or without SARS-CoV-2 WA-1/2020 at 1 × 10 4 PFU were collected on day 7. n = 4 mice. c , Macroscopic images of lungs after EBA tracer injection. d , e , EBA, as a marker of pulmonary endothelial permeability ( d ), and lung wet/dry ratio, as a measure of lung edema ( e ), were quantified. f , Survival curves of mice inoculated with WA-1/2020 or P.1 variant at 1 × 10 4 PFU. g , Weight loss of mice inoculated with P.1. h , i , Mice were examined on day 6 to evaluate lung vascular leak by EBA assay ( h ) and lung wet/dry ratio ( i ). n = 4 mice. RT–PCR was used to measure viral loads in the lungs of mice with the SARS-CoV-2 P.1 variant on day 6 postinoculation and WA-1/2020 strain on day 7 postinoculation. j , k , mRNA expression of SARS-CoV-2 S ( j ) and SARS-CoV-2 NSP ( k ) is shown. Data are presented as the mean ± s.e.m. PPIA, peptidylprolyl isomerase A. d , e , h , i , P values were calculated by two-sided Student’s t -test. j , k , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Transgenic Assay, Injection, Marker, Permeability, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

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: a – c , Based on simulations of RBD-bound ACE2, new polar interactions were identified between the ACE2.v2.4 (orange) and RBD (yellow) loops (red) ( a ). ACE2 mutations are presented as sticks colored cyan with nearby RBD residues as sticks colored yellow. New polar interactions between ACE2.v2.4 and RBD loops 1 ( b ) and 2 ( c ) are indicated by the dotted blue lines. d , MSM-weighted distance distributions of newly formed hydrogen bonds. For each interacting pair, the ACE2.v2.4 residue is listed first and the RBD residue is listed second. e , Probabilities for stable hydrogen bond interactions using a 4-Å distance criterion between accepter and donor. f , Root mean square fluctuation (RMSF) of RBD residues when bound to WT (cyan) or v2.4 (orange) ACE2 receptors. The RBD regions that interface with ACE2 are shaded gray. The absolute difference in RMSF between WT and v2.4 proteins is shown in black. Distance distributions, hydrogen bond probabilities and RMSF calculations were based on 40,000 frames from the simulations. Frames were selected based on the MSM stationary probability to represent the entire conformational ensemble. The error bars represent the 95% confidence intervals calculated from 20 bootstrapped samples.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Residue

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: a , mRNA expression of ACE2 (NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells. A549 cells stably expressing hACE2 and hLMVECs were analyzed by one-step RT–PCR (top) and real-time PCR (bottom). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase expression. b , Cultured hACE2-A549 cells, A549 cells and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 µg ml −1 or 25 µg ml −1 for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was added to the cells, which were collected at 24 h. Virus entry was evaluated by luciferase activity. n = 4 replicates. c , A dose of 10 mg kg −1 sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1 or buffer (PBS + 0.2% BSA) was administrated intravenously into K18-hACE2 transgenic mice for 30 min before SARS-CoV-2 pseudovirus (10 6 PFU) intraperitoneal injection. Tissue lysates were prepared at 24 h and virus entry in the selected organs was evaluated by luciferase activity. Buffer was applied as the control group, n = 4. d – g , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Mice received control PBS or sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h before inoculation. Mice were observed for survival ( d ) and body weight ( e ), n = 5. EBA as a marker of pulmonary endothelial permeability ( f ) and lung wet/dry ratio ( g ) as a measure of lung edema were quantified. Data are presented as the mean ± s.e.m., n = 4. b , c , f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

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

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: K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Group 1 received control PBS via intravenous injection 24 h postviral inoculation. Group 2 (v2.4 12 h) received sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h postinoculation and then daily subsequent injections at the same dose. Group 3 (v2.4 24 h) received sACE2 2 .v2.4-IgG1 15 mg kg −1 via intravenous injection 24 h postinoculation and then daily subsequent injections at the same dose. a , b , Survival curves ( a ) and weights ( b ) for n = 10 mice for each group. c – e , Mouse lungs were obtained on day 7 postinoculation for assessment of lung vascular albumin permeability. c , Macroscopic images of lungs at baseline and day 7 postviral inoculation in three experimental groups without EBA (left) and with EBA injection (right). d , Quantification of EBA lung vascular endothelial permeability in all three experimental groups. e , Quantification of lung edema by wet/dry ratio in all three experimental groups at baseline and day 7 postinoculation. f , g , Time course of lung vascular endothelial permeability and edema formation of groups 2 (v2.4 12 h) ( f ) and 3 (v2.4 24 h) ( g ) as assessed by EBA assay and lung wet/dry ratio. Data are presented as the mean ± s.e.m. d , e , P values were calculated by two-way ANOVA with Tukey post hoc test. f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Transgenic Assay, Control, Injection, Permeability

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: 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 sACE2 2 -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 sACE2 2 .v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE2 2 .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 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

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: The 3 groups of K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 variant P.1 (Brazil) at 1 × 10 4 PFU. Group 1 (PBS): PBS was given by intravenous injection 24 h postinoculation. Group 2 (v2.4 12 h): sACE2 2 .v2.4-IgG1 10 mg kg −1 was given by intravenous injection 12 h postinoculation. Group 3 (v2.4 24 h): sACE2 2 .v2.4-IgG1 15 mg kg −1 was given by intravenous injection 24 h postinoculation. a , b , Mice were injected once per day for 7 d. Survival probability was calculated ( a ) and mouse weights were measured ( b ). n = 10 mice for each group. c , d , Mouse lungs were examined on day 6 postinoculation to evaluate lung vascular permeability and lung edema using the EBA assay ( c ) and lung wet/dry ratio ( d ), with baseline mouse lungs as controls. e , Viral loads of SARS-CoV-2 in the lungs obtained at baseline and day 6 postinoculation of the SARS-CoV-2 variant P.1 (Brazil) were measured by real-time qPCR for mRNA expression of SARS-CoV-2 S protein and SARS-CoV-2 NSP. f , A viral plaque formation assay was performed to measure the viral loads of SARS-CoV-2 in lungs obtained at baseline and on day 6 postinoculation of the SARS-CoV-2 P.1 (Brazil) variant. c – e , f , n = 4. Data are presented as the mean ± s.e.m. c , d , P values were calculated by two-way ANOVA with Tukey post hoc test. e , f , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-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 sACE2 2 (ACE2 amino acids 1–732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously.

Techniques: Transgenic Assay, Variant Assay, Injection, Permeability, Expressing, Plaque Formation 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: ( 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