Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

Article activity feed

1. Version published to 10.1038/s41589-021-00965-6

   Jan 19, 2022
2. 

   ScreenIT

   Dec 24, 2021

   SciScore for 10.1101/2021.12.21.473668: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Toxicology: Animal experimental procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee (
   Sex as a biological variable	Toxicity of each peptide was assessed in 5 female and 5 male CD-1 IGS mice (8 weeks old).
   Randomization	Animals with sex- and age-matched littermates were randomly included in experiments.
   Blinding	Animal experiments were carried out in a blinded fashion whenever feasible.
   Power Analysis	For the number of animals needed to achieve statistically significant results, we conducted a priori power analysis.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After this, cells were stained with …

   SciScore for 10.1101/2021.12.21.473668: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Toxicology: Animal experimental procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee (
   Sex as a biological variable	Toxicity of each peptide was assessed in 5 female and 5 male CD-1 IGS mice (8 weeks old).
   Randomization	Animals with sex- and age-matched littermates were randomly included in experiments.
   Blinding	Animal experiments were carried out in a blinded fashion whenever feasible.
   Power Analysis	For the number of animals needed to achieve statistically significant results, we conducted a priori power analysis.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After this, cells were stained with antibodies including CD45-EF450 (1:2000, eBioscience #48-0451-82) and CD31-APC (1:100, eBioscience #17-0311-82) for 45 minutes at 4°C.	CD45-EF450 suggested: None CD31-APC suggested: None
   Anti-SARS-CoV-2 Monoclonal Antibodies: Sequences for monoclonal antibodies that had received Emergency Use Authorization from the U.S. Food and Drug Administration were pulled from the KEGG database (Accession No. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936).	Anti-SARS-CoV-2 suggested: None D12014; LY-CoV555, D11936 suggested: None LY-CoV555 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The SARS-CoV-2 were propagated in Vero E6 cells (CRL-1586; American Type Culture Collection).	Vero E6 suggested: None
   HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine®3000 (Invitrogen).	HEK293T suggested: None
   Cell culture: The 2019n-CoV/USA_WA1/2019 isolate as well as the P.1 variant of SARS-CoV-2 and ACE-2 expressing A549 cell lines were obtained from BEI Resources.	ACE-2 suggested: None A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Vero E6 (CRL-1586; American Type Culture Collection) Vero CCL81 (American Type Culture Collection),	Vero CCL81 suggested: None
   Assessment of ACE2 mutants: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described 22 and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA.	Expi293F suggested: RRID:CVCL_D615)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Hemizygous K18-hACE mice with c57BL/6J background (strain#034860: B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from The Jackson Laboratory.	K18-hACE suggested: None B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
   For in vivo studies, 10 mg/kg sACE22.v2.4-IgG1 (wt), sACE22.v2.4-IgG1 and control peptide buffer were intravenously administrated into K-18 hACE-2 mice for 30 minutes prior to SARS-CoV-2 pseudo-entry virus (106 pfu) i.p. injection for 1 dpi.	hACE-2 suggested: None
   Pharmacokinetics: 8-week-old CD-1 IGS mice (3 female and 3 male per time point) were IV administered protein solutions.	CD-1 IGS suggested: RRID:MGI:5461217)
   To characterize different routes side-by-side, peptides were administered to C57BL/6 mice, 3 males per time point.	C57BL/6 suggested: None
   Recombinant DNA
   Sentences	Resources
   HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine®3000 (Invitrogen).	pCMV3-SARS-CoV-2-spike suggested: None
   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 sACE22 (ACE2 a.a. 1-732; pcDNA3-sACE2-WT(732)-IgG1,	pcDNA3-sACE2 suggested: None pcDNA3-sACE2v2.4-8his suggested: RRID:Addgene_149664) pcDNA3-sACE2-WT(732)-IgG1 suggested: RRID:Addgene_154104)
   Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.	pcDNA3-sACE2v2.4 suggested: None
   Human codon-optimized S (GenBank No. YP_009724390.1) was subcloned into pCEP4 (Invitrogen) from pUC57-2019-nCoV-S(Human) (distributed by Molecular Cloud on behalf of Haisheng Yu, Chinese Academy of Medical Sciences) with a N-terminal HA leader (MKTIIALSYIFCLVFA), myc-tag (EQKLISEEDL), and linker (GSPGGA) upstream of the mature polypeptide (a.a. V16-T1273).	pCEP4 suggested: RRID:Addgene_16479) pUC57-2019-nCoV-S(Human suggested: None
   Assessment of ACE2 mutants: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described 22 and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA.	pCEP4-myc-ACE2 suggested: RRID:Addgene_141185)
   In vitro binding assays to S variants: Expi293F cells were transfected with pCEP4-myc-S plasmids as described above.	pCEP4-myc-S suggested: None
   H and L chains were cloned with CD5 leader sequences into pcDNA3.1(+) and expressed in Expi293F cells.	pcDNA3.1 suggested: RRID:Addgene_79663)
   Software and Algorithms
   Sentences	Resources
   Images were taken with a Zeiss microscope and analyzed by Zen software (Zeiss)	Zen suggested: None
   For sACE22.v2.4 without any tags or fusion partner (corresponding to ACE2 residues 19-732, protein provided by Orthogonal Biologics, Inc.), mice were IV administered (tail vein, 0.5 mg/kg) protein twice daily for 5 consecutive days (days 0-4) and sacrificed day 7.	Orthogonal Biologics suggested: None
   Mutations were introduced using PyMOL (https://pymol.org/2/) and the systems were solvated using TIP3P water and 150 mM NaCl using PACKMOL 71.	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   Protein residues and glycosylation sites were parameterized using AMBER ff14SB 72 and GLYCAM06 73 force fields.	AMBER suggested: (AMBER, RRID:SCR_016151)
   Anti-SARS-CoV-2 Monoclonal Antibodies: Sequences for monoclonal antibodies that had received Emergency Use Authorization from the U.S. Food and Drug Administration were pulled from the KEGG database (Accession No. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936).	KEGG suggested: (KEGG, RRID:SCR_012773)

   ---

   Results from OddPub: Thank you for sharing your code and data.

   ---

   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Another limitation is our focus on intravenous delivery, whereas intratracheal or inhalation delivery may be more readily applied in patients, especially in the outpatient setting. It would be useful in future studies to use varying doses and delivery route combinations for defined disease stages to identify the optimal combination prior to initiating human efficacy trials. Finally, we chose to fuse the ACE2 peptide to an unmodified Fc from IgG1 (isoallotype nG1m1), whereas others have considered fusions to Fc mutants or IgG4 to dampen interactions with FcγR subtypes that might contribute to inflammation24, 68. We instead reasoned that IgG1 effector functions will be necessary for optimum in vivo protection. In summary, we show for the first time that an engineered decoy ACE2 peptide demonstrating much higher affinity for the SARS-CoV-2 Spike protein is efficacious in vivo against multiple SARS-CoV-2 variants. This peptide prevented viral entry into cells and the peptide’s efficacy against a variant of concern prevented lung endothelial injury and ARDS and significantly reduced mortality. The results show the potential of this engineered peptide to treat COVID-19 patients and others with inadequate antibody titer to protect against emerging, more virulent SARS-CoV-2 variants.

   ---

   Results from TrialIdentifier: No clinical trial numbers were referenced.

   ---

   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

   ---

   Results from JetFighter: We did not find any issues relating to colormaps.

   ---

   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

   ---

   Results from scite Reference Check: We found no unreliable references.

   ---

   Read the original source
3. 

   Version published to 10.1101/2021.12.21.473668v1 on bioRxiv

   Dec 22, 2021