ACE2 amino acid sequence modifications were designed by computer-aided modelling. ACE2 ectodomains of different length, Q18-G732 and Q18-S740, with or without mutation of the catalytic site (wild type or H374N/H378N mutant)65 were combined with the Fc fragment of IgG4 bearing a stabilizing S228P mutation in the hinge region.33 For comparison, the same ACE2 sequence variants were fused to the Fc fragment of IgG1 with a truncated hinge region (DKTHTCPPCPA).
Generation of expression plasmids
Plasmids encoding the Fc fusion proteins were generated at ThermoFisher. Genes of interest were subcloned into pcDNA3.1 Zeocin expression plasmids (Invitrogen V860-20) with an elongated CMV promoter using HindIII/XhoI restriction sites. Following amplification in Escherichia coli, expression plasmids were isolated and analyzed by restriction analysis as well as DNA sequencing.
Using the FreeStyle 293 Expression System (ThermoFisher), the different ACE2-Fc fusion proteins were transiently expressed in 3 x 240 mL culture media. On day six, samples were analyzed for cell viability as well as cell density and supernatants were harvested by centrifugation followed by sterile filtration.66 The material was either stored at -80°C until purification or subjected directly to purification. Small samples were taken from the pools to determine expression yields by bio-layer interferometry (BLI).
Purification of the fusion proteins secreted into the culture medium was performed by protein A column chromatography followed by preparative Size Exclusion Chromatography (SEC). For protein A purification, after loading the sample, the Amsphere A3 column (JSR Life Sciences) was washed and the ACE2-Fc fusion proteins were eluted using 40 mM NaAc, pH 3.0. Following elution, samples were first neutralized to pH 7.5 using 1 M Tris, pH 9.0, subsequently diluted 1:1 with 50 mM Tris, pH 7.5, 300 mM NaCl and concentrated to 10 mg/mL using spin filters. Concentrated proteins were further purified with a Superdex 200 increase (GE Healthcare) column equilibrated with 50 mM Tris, pH 7.5, 150 mM NaCl. The main peak was pooled, the protein concentration was determined by slope spectrometry67 and adjusted to 1 mg/mL. The protein solution was passed through a sterilizing filter and stored at 4°C until further usage.
Size exclusion chromatography with multi-angle light scattering (SEC-MALS)
A Shimadzu HPLC system with two concentration detectors (UV and refractive index) and a HELEOS II MALS detector were used for the measurements. The flow rate was 1 mL/min and the running buffer was 50 mM Tris, pH 7.5 and 150 mM NaCl. 50 µg of protein was injected on a Superdex 200 Increase 10/300 GL column (Cytiva). The chromatograms were evaluated with the Astra software.
Circular dichroism (CD)
All CD measurements were performed with a Jasco J-1500 spectropolarimeter at 20°C. The sample buffer consisted of 50 mM Tris, pH 7.5 and 150 mM NaCl. The Far-UV CD spectra were obtained in a 1 mm quartz cuvette using a protein concentration of 0.1 mg/mL. The Near-UV CD spectra were measured in a 5 mm quartz cuvette using a protein concentration of 1 mg/mL.
ACE2 activity assay
An ACE2 activity assay kit from Abcam (Cat.No. ab273297) was used to measure the enzymatic activity of the constructs. The assay was performed according to the manufacturer’s manual and is based on a synthetic peptidyl-4-methylcoumaryl-7-amide (MCA) that is cleaved by the ACE2 enzyme. Upon cleavage, free MCA is detected fluorometrically (Ex320nm/Em420nm) and quantified with a standard curve obtained with MCA standard solutions with known concentrations. Two commercially available ACE2-Fc proteins obtained from Genscript (Cat.No. Z03484-1) and Acrobiosystems (Cat.No. AC2-H5257) were used as reference.
Determination of binding affinity to spike protein RBD using surface plasmon resonance
The measurements were performed with a Biacore X-100 system and the Biotin CAPture kit (Cytiva). The running buffer was HBS-EP+ pH 7.4 (Cytiva). The ligand SARS-CoV-2 RBD with an AviTag (Acrobiosystems) was captured on the streptavidin chip to around 100 RU. Increasing concentrations of the analyte ACE2-Fc (0.32, 1.6, 8, 40 and 200 nM) were injected over the immobilized ligand in a single-cycle kinetic mode. The obtained sensorgrams were evaluated with the Biacore X-100 software to obtain a binding constant (KD).
Determination of binding affinity to Fc-receptors using surface plasmon resonance
A Biacore T200 was used for the Fc-receptor binding studies. For the FcgRI and the FcgRIIIa experiments, the his-tagged FcgRI and FcgRIIIa with a concentration of 1.5 nM were captured by injection of the solutions for 90 s with a flow rate of 5 µL/min over a covalently immobilized anti-his tag antibody on a CM5 chip. The running buffer was HBS-EP+ pH 7.4 (Cytiva). Five different concentrations of the ACE2-Fcs were injected in a single-cycle kinetic mode (3.7-300 nM for the experiment with FcgRI and 25-2000 nM for FcgRIIIa). The FcgRI-binding data was fit to a heterogeneous ligand model and the first binding constant was reported. The FcgRIIIa-binding data was fit to a two-state reaction model to derive the binding constant. For the FcRn experiments, the FcRn was covalently immobilized on a CM5 chip to around 50 RU. The sample buffer was HBS-EP+ pH 6.0 (Cytiva). The ACE2-Fcs were injected in five different concentrations from 205 to 8000 nM in a single-cycle kinetic mode. The FcRn binding was evaluated with a steady-state affinity fit.
SARS-CoV-2 Spike S1 Inhibition ELISA
Inhibition of binding of SARS-CoV-2 spike S1 protein to ACE2 was tested using the ACE2:SARS-CoV-2 Spike S1 Inhibitor Screening Assay Kit (BPS Bioscience; Cat.No. 79945) according to the manufacturer’s instructions with an adapted neutralization procedure. Briefly, biotinylated SARS-CoV-2 Spike S1 protein (25 nM) was incubated with serial dilutions of the ACE2-Fc fusion proteins in a 96-well neutralization plate at room temperature (RT) for one hour with slow shaking (= neutralization mix).
ACE2 protein was added to a nickel-coated 96-well plate at a concentration of 1 μg/mL and incubated at RT for one hour with slow shaking. Following a washing step to remove unbound ACE2, the plates were blocked at RT for 10 min with slow shaking. Subsequently, the neutralization mix was transferred to the ACE2 coated plate and the plate was incubated at RT for one hour with slow shaking. Following a 10 min blocking step, the plate was incubated with Streptavidin-HRP at RT for one hour with slow shaking. Following a washing and a 10 min blocking step, the HRP substrate was added and the plate was analyzed on a chemiluminescence reader.
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-Jan (SARS-CoV-2-Munich-TUM-1; EPI_ISL_582134), SARS-CoV-2-April (SARS-CoV-2 D614G; EPI_ISL_466888), SARS-CoV-2-B.1.1.7 (EPI_ISL_755639) and SARS-CoV (AY291315.1) were isolated from patient material in Germany. Briefly, SARS-CoV-2-Jan was isolated from a COVID-19 infected patient who was infected during the earliest documented COVID-19 outbreak in Germany at the end of January 2020 with a virus imported from Wuhan via a single contact in Shanghai.43 SARS-CoV-2-April was isolated during the first eminent wave of the pandemic in Europe in April 2020 from a patient in Munich, Germany. Both virus isolates as well as a control isolate from the early “Webasto” cluster outbreak68 contain the S1 D614G mutation showing significantly higher infectious titers in vitro.59
SARS-CoV-2-Jan, SARS-CoV-2-B.1.1.7, SARS-CoV and SARS-CoV-2-GFP39 were propagated and passaged in Vero E6 cells (derived from African green monkey kidney epithelial cells). SARS-CoV-2-April was isolated on Caco-2 cells followed by passaging in Vero E6 cells. All strains were cultured in DMEM medium (5% fetal calf serum (FCS), 1% penicillin/streptomycin (P/S), 200 mmol/L L-glutamine, 1% MEM-Non-Essential Amino Acids (NEAA), 1% sodium pyruvate (all from Gibco). Viral titer was determined by Plaque Assay.69
Viral titers were determined as described by Baer et al. 69 with some modifications. Briefly, HepG2 or Vero E6 cells were plated in a 12-well plate at 5E05 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. Cells were infected with serial dilution of virus sample in cell culture medium at 37°C for one hour. After discarding the supernatant, 1 mL of 5% carboxymethylcellulose (Sigma) diluted in Minimum Essential Media (Gibco) was added per well and the plate was incubated at 37°C until obvious plaques appeared. After removing the supernatant, cells were fixed with 10% paraformaldehyde (ChemCruz) at RT for 30 min. Next, a washing step with PBS was performed, followed by the addition of 1% crystal violet (Sigma; diluted in 20% methanol and water). Following an incubation time of 15 min at RT, the solution was washed away with PBS and the plate was dried. The viral titer (PFU/mL) of the sample was determined by counting the average number of plaques for a dilution and the inverse of the total dilution factor.
In vivo infection assay using SARS-CoV-2-GFP
Vero-E6 cells were plated in a 96-well plate at 1.4E04 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. Serial dilutions of ACE2-Fc fusion proteins and SARS-CoV-2-GFP were mixed in fresh media and pre-incubated at 37°C for one hour. Afterwards, Vero E6 cells were infected with the neutralized virus solution at a multiplicity of infection (MOI) of 0.6 infectious viruses per cell at 37°C. After one hour, the neutralization mix was replaced by cell culture medium. Plates were placed in the IncuCyte S3 Live-Cell Analysis System (Essen BioScience, Newark, UK) and real-time images of uninfected mock cells (Phase channel) and infected (GFP and Phase channel) cells were captured every four hours for 72 hours. Virus control cells were infected with the same virus stock but without prior incubation with the ACE2-Fc fusion constructs using the identical protocol.
Viral neutralization assay
Vero E6 cells were plated in a 96-well plate at 1.6E04 cells/well in DMEM medium (Gibco) supplemented with 5% FCS, 1% P/S, 200 mmol/L L-glumatine, 1% MEM-NEAA, 1% sodium-pyruvate (all from Gibco) and incubated overnight at 37°C and 5% CO2. Serial dilutions of the ACE2-Fc fusion proteins were mixed with virus in fresh media and pre-incubated at 37°C for one hour. The Vero E6 cells were infected at a multiplicity of infection (MOI) of 0.06 with the neutralized virus solution at 37°C for one hour. Next, the neutralization mix was removed, culture medium was added, and cells were incubated at 37°C for 24 hours. Mock cells represent uninfected Vero E6 cells, incubated with culture medium. After 24 hours, cells were washed once with PBS and fixed with 4% paraformaldehyde (ChemCruz) at RT for 10 min. Following a washing step with PBS, fixed Vero E6 cells were permeabilized with 0.5% saponin (Roth) in PBS at RT for 10 min to allow an in-cell ELISA. For this, cells were blocked with a mixture of 0.1% saponin and 10% goat serum (Sigma) in PBS with gentle shaking at RT for one hour. Subsequently, Vero E6 cells were incubated with a 1:500 dilution of an anti-dsRNA J2 antibody (Jena Bioscience) in PBS supplemented with 1% FCS at 4 °C overnight with shaking. Following four washing steps with wash buffer (PBS supplemented with 0.05% Tween-20 (Roth)). Next, the plates were incubated with a 1:2,000 dilution of a goat anti-mouse IgG2a-HRP antibody (Southern Biotech) in PBS supplemented with 1% FCS and incubated with gently shaking at RT for one hour. Following four washing steps, 3,3′,5,5′-Tetramethylbenzidin (TMB) substrate (Invitrogen) was added to the wells and incubated in the dark for 10 min. Colorimetric detection on a Tecan infinite F200 pro plate reader (Tecan) at 450 nm and at 560 nm was performed after stopping the color reaction by the addition of 2N H2SO4 (Roth).
Cell viability assay using SARS-CoV-2 variants of concern (VoC)
24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37°C and 5% CO2. A serial dilution of ACE2-IgG4-Fc construct 1 and 3 was performed and mixed with a defined volume of virus stocks of the indicated SARS-CoV-2 clinical isolates resulting in 80% cytotoxicity. After 1 h of pre-incubation at 37°C, the neutralization mix of ACE2-IgG4-Fc construct and the respective SARS-CoV-2 isolates was added to A549-hACE2 cells. Virus-mediated killing of target cells was determined using a luminometric readout of virus-induced cytotoxicity. In brief, 72 h after infection cells were treated according to manufacturer’s instructions: 15 µl CellTiter-Glo 2.0 reagent (Promega, Wisconsin, USA) were added to each well, incubated for 10 min in the dark at RT and luminescence was recorded (0.5 seconds integration time, no filter) using the Infinite F200 microplate reader (Tecan). Viability of cells and the corresponding infectious titer for each virus isolate was calculated by normalization of infected cells to untreated control cells (set to 100%).
The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files are available from the authors upon request. Source data are provided with this paper.