Efficient inhibition of SARS-CoV-2 strains by a novel ACE2-IgG4-Fc fusion protein with a stabilized hinge region
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Abstract
The novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) enters its host cells after binding to the angiotensin-converting enzyme 2 (ACE2) via its spike glycoprotein. This interaction is critical for virus entry and virus-host membrane fusion. Soluble ACE2 ectodomains bind and neutralize the virus but the short in vivo half-lives of soluble ACE2 limits its therapeutic use. Fusion of the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain can prolong the in vivo half-life but bears the risk of unwanted Fc-receptor activation and antibody-dependent disease enhancement. Here, we describe optimized ACE2-Fc fusion constructs that avoid Fc-receptor binding by using IgG4-Fc as a fusion partner. The engineered ACE2-IgG4-Fc fusion proteins described herein exhibit promising pharmaceutical properties and a broad antiviral activity at single-digit nanomolar concentration. In addition, they allow to maintain the beneficial enzymatic activity of ACE2 and thus are very promising candidate antivirals broadly acting against coronaviruses.
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SciScore for 10.1101/2020.12.06.413443: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources SARS-CoV-2-April was propagated by the infection of Caco-2 cells followed by passaging in Vero E6 cells. Caco-2suggested: NoneBriefly, HepG2 or Vero E6 cells were plated in a 12-well plate at 5 x 10E5 cells/well in DMEM medium (Gibco) supplemented with 5% FCS, 1% P/S, 200 mmol/L L-glutamine, 1% MEM-NEAA, 1% sodium-pyruvate (all from Gibco) and incubated overnight at 37°C and 5% CO2. HepG2suggested: NoneVero E6suggested: None… SciScore for 10.1101/2020.12.06.413443: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources SARS-CoV-2-April was propagated by the infection of Caco-2 cells followed by passaging in Vero E6 cells. Caco-2suggested: NoneBriefly, HepG2 or Vero E6 cells were plated in a 12-well plate at 5 x 10E5 cells/well in DMEM medium (Gibco) supplemented with 5% FCS, 1% P/S, 200 mmol/L L-glutamine, 1% MEM-NEAA, 1% sodium-pyruvate (all from Gibco) and incubated overnight at 37°C and 5% CO2. HepG2suggested: NoneVero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Virus strains: SARS-CoV-2-GFP (kindly provided by Volker Thiel, University of Bern, Switzerland) is based on the original Wuhan SARS-CoV-2 isolate (GenBank accession MT108784) and was reconstituted from a synthetic construct derived from SARS-2 BetaCoV/Wuhan/IVDC-HB-01/2019 [39]. SARS-CoV-2-GFPsuggested: NoneSoftware and Algorithms Sentences Resources Protein expression: Using the FreeStyle 293 Expression System (ThermoFisher), the different ACE2-Fc fusion proteins were transiently expressed in 3 x 240 mL culture media. ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)The main peak was pooled, the protein concentration was determined by slope spectrometry [67] and adjusted to 1 mg/mL. slopesuggested: (SLOPE, RRID:SCR_001185)The chromatograms were evaluated with the Astra software. Astrasuggested: (ASTRA, RRID:SCR_016255)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
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.
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SciScore for 10.1101/2020.12.06.413443: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Subsequently, Vero E6 cells were incubated with a 1:500 dilution of an antidsRNA J2 antibody (Jena Bioscience) in PBS supplemented with 1% FCS at 4°C overnight with shaking. antidsRNA J2suggested: NoneNext, the plates were incubated with a 1:2,000 dilution of a goat anti-mouse IgG2aHRP antibody (Southern Biotech) in PBS supplemented with … SciScore for 10.1101/2020.12.06.413443: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Subsequently, Vero E6 cells were incubated with a 1:500 dilution of an antidsRNA J2 antibody (Jena Bioscience) in PBS supplemented with 1% FCS at 4°C overnight with shaking. antidsRNA J2suggested: NoneNext, the plates were incubated with a 1:2,000 dilution of a goat anti-mouse IgG2aHRP antibody (Southern Biotech) in PBS supplemented with 1% FCS and incubated with gently shaking at RT for one hour. anti-mouse IgG2aHRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, HepG2 or Vero E6 cells were plated in a 12-well plate at 5 x 10E5 cells/well in DMEM medium (Gibco) supplemented with 5% FCS, 1% P/S, 200 mmol/L L-glutamine, 1% MEM-non-essential amino acids, 1% sodium-pyruvate (all from Gibco) and incubated overnight at 37°C and 5% CO2. HepG2suggested: NoneSARS-CoV-2 virus neutralization assay Viral neutralization assay followed by in-cell ELISA Vero E6 cells were plated in a 96-well plate at 1.6 x 10E04 cells/well in DMEM medium (Gibco) supplemented with 5% FCS, 1% penicillin-streptomycin, 200 mmol/L L-glumatine, 1% MEM-nonessential amino acids, 1% sodium-pyruvate (all from Gibco) and incubated overnight at 37°C and 5% CO2. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Protein expression Using the FreeStyle 293 Expression System (ThermoFisher), the different ACE2-Fc fusion proteins were transiently expressed in 3 x 240 mL culture media. ThermoFishersuggested: NoneThe main peak was pooled, the protein concentration determined by slope spectrometry [63] and adjusted to 1 mg/mL. slopesuggested: (SLOPE, RRID:SCR_001185)The chromatograms were evaluated with the Astra software. Astrasuggested: (ASTRA, RRID:SCR_016255)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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