Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

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1. Version published to 10.1126/sciadv.abn4188

   Jul 15, 2022
2. 

   ScreenIT

   Nov 30, 2021

   SciScore for 10.1101/2021.11.24.469776: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All animal experiments were performed according to Institute of Laboratory Animal Resources guidelines and the protocol was approved by the National Cancer Institute Animal Care and Use Committee. Euthanasia Agents: All mice were anesthetized via isoflurane inhalation (3 - 5 % isoflurane, oxygen flow rate of 1.5 L/min) prior and during BLI using the XGI-8 Gas Anesthesia System.
   Sex as a biological variable	6–8-week-old male and female mice were used for all the experiments.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Plates were washed three times and incubated with the goat …

   SciScore for 10.1101/2021.11.24.469776: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All animal experiments were performed according to Institute of Laboratory Animal Resources guidelines and the protocol was approved by the National Cancer Institute Animal Care and Use Committee. Euthanasia Agents: All mice were anesthetized via isoflurane inhalation (3 - 5 % isoflurane, oxygen flow rate of 1.5 L/min) prior and during BLI using the XGI-8 Gas Anesthesia System.
   Sex as a biological variable	6–8-week-old male and female mice were used for all the experiments.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Plates were washed three times and incubated with the goat anti-human-IgG Fc secondary antibody conjugated with alkaline phosphatase (AP, Southern Biotech) at a 1:1000 dilution in blocking buffer for 1 h at room temperature.	anti-human-IgG suggested: None
   Antibody-dependent cellular phagocytosis was determined by flow cytometry, gating on THP-1 cells that were triple-positive for GFP, efluor450 and efluor670 cellular dyes.	GFP suggested: None
   Samples were washed, sonicated, and incubated with goat anti-guinea pig C3 antibody conjugated with biotin (Immunology Consultants Laboratory) at RT for 1 h followed by incubation with streptavidin R-Phycoerythrin (PE,	anti-guinea pig C3 suggested: None
   Before and after injection, serum samples were collected at 0 min, 10min, 1 h, 6 h, 24 h and 48 h and the ACE2-Fc serum concentration was estimated by indirect ELISA in which SARS-CoV-2 RBDwt (200 ng/well) were used as capturing molecule and the goat-anti-human IgG conjugated with AP (1:1000 dilution) were used as secondary antibody.	IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   IgG1 Fc tag and 8xHis tag) (45), plasmids encoding the respective genes were transfected to 293F cells with the same protocol as described above.	293F suggested: RRID:CVCL_D615)
   To determine viral titers, hACE2-expressing 293T cells (gift from Dr. Allison Malloy, USUHS) were infected with serial PsV dilutions.	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Antibody-dependent cellular phagocytosis was determined by flow cytometry, gating on THP-1 cells that were triple-positive for GFP, efluor450 and efluor670 cellular dyes.	THP-1 suggested: None
   Dilutions from infected cell homogenates were applied on Vero E6 monolayer. 24 hour post infection, infected Vero E6 cells were washed with PBS, lysed with Passive lysis buffer and transferred into a 96-well solid white plate (Costar Inc) and nanoluciferase activity was measured using Tristar multiwell Luminometer (Berthold Technology) for 2.5 seconds by adding 20 µl of Nano-Glo® substrate in nanoluc assay buffer (Promega Inc).	Vero E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   96-well Nunc Maxisorp plates (Sigma) were coated with SARS-CoV-2 RBDwt (residue 319-541) (50ng), RBDB.1.351 (50 ng), S-2P (75 ng), SB.1.1.7 (75 ng), SB.1.351 (75ng), SP.1 (75 ng), SB.1.526 (75 ng) and SARS-CoV RBD (50ng) per well in Tris-buffered saline (TBS) at 4 °C overnight.	SB.1.1.7 suggested: None
   For in vivo PsV-based inhibition assays, 6-8-week-old K18-hACE2 mice were intranasally (i.n) treated with Synagis (control IgG, 25 µg), M27 or M81 (5 or 25 µg) one hour before challenge by SARS-CoV-2 PsVD614G or PsVB.1.617.2 (i.n., ∼108 RLU)	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   For PK studies, C57BL/6J mice were intravenously (i.v.) injected with 100 μg (5 mg/kg) of two engineered ACE2-Fc M81 or M86.	C57BL/6J suggested: None
   Recombinant DNA
   Sentences	Resources
   To generate SARS-CoV-2 RBDwt (residue 319-541 or residue 319-537, for crystallization), RBDB.1.1.7 (residue 329-527, N501Y) and RBDB.1.351 (residue 329-527, K417N/E384K/N501Y), the respective codon optimized DNA segments fused with an N-terminal secretion peptide and a C-terminal 6xHis tag were cloned into the pACP-tag (m)-2 vector using either EcoRI/NotI for RBDwt (319–541), RBD B.1.1.7 and RBDB.1.351 or BamHI/XhoI for RBDwt (319–537) as restriction enzymes.	pACP-tag suggested: RRID:Addgene_101126)
   Monomeric ACE2wt and engineered ACE2LFMYQY2HA plasmids encoding ACE2 (residue 1-615) with C-terminal HRV-3C-cleavable 8xHis tag (45) were transfected to FreeStyle 293F cells and the resulting protein was purified over Ni-NTA columns (Cytiva).	ACE2LFMYQY2HA suggested: None
   Software and Algorithms
   Sentences	Resources
   GraphPad Prism was used to display the mean and SEM for all groups and used to calculate the area under the curve (AUC) within the concentration range of 0.05-2.5 nM using 5% binding as baseline (Fig. 2D & S2)	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Iterative cycles of model building and refinement were done in Coot (79) and Phenix (80).	Coot suggested: (Coot, RRID:SCR_014222)
   Structural analysis and Fig. generation were performed in PyMOL (81)	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   All samples were acquired on an LSRII cytometer (BD Biosciences) and data analysis performed using FlowJo v10 (Tree Star)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Bioluminescence Imaging (BLI) of SARS-CoV-2 infection: All standard operating procedures and protocols for IVIS imaging of SARS-CoV-2 infected animals under ABSL-3 conditions were approved by IACUC, IBSCYU and YARC.	YARC suggested: None
   Images were acquired and analyzed with Living Image v4.7.3 in vivo software package (Perkin Elmer Inc).	Living Image suggested: (Living Image software, RRID:SCR_014247)
   The data were processed and plotted using GraphPad Prism 8 v8.4.3.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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

   Thus far, the in vivo protective potential of an ACE2-Fc therapeutic has been tested only once in a Syrian hamster model (28) which has several limitations due to its inability to fully recapitulate SARS-CoV-2 pathogenesis and severity. To better test our lead ACE2-Fc variant we utilized a well characterized K18-hACE2 mouse model (67). Due to the constitutive high endogenous human ACE2 expression, this model is highly susceptible to SARS-CoV-2 infection and the disease progression partially recapitulates the severe pathological features of SARS-CoV-2 infection in humans. The model has also been used extensively for evaluating contributions from direct neutralization and Fc-effector activities mediated by nAbs (68) and a non-neutralizing Ab (69). However, a high basal level of hACE2 on target cells in this model, particularly in the brain, poses a significant obstacle for soluble ACE2-based antivirals such as our engineered ACE2-Fc to surmount and achieve protection. Despite these limitations, we detected a strong benefit to the administration of ACE2740 LFMYQY2HA–Fc GASDALIE variant both prophylactically and therapeutically in K18-hACE2 mice. In both settings, ACE2-Fc treatments were associated with markedly improved in vivo efficacy, e.g., a reduction in virus-induced body weight loss, pro-inflammatory cytokine responses and mortality, particularly in the therapeutic context. Given the human Fc-mouse FcγR mismatch may compromise Fc-effector functionality of ACE-Fcs in K18-hA...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   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).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56, 51 and 60. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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

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3. 

   Version published to 10.1101/2021.11.24.469776v1 on bioRxiv

   Nov 24, 2021