Antiviral activity of an ACE2-Fc fusion protein against SARS-CoV-2 and its variants

Abstract

SARS-CoV-2 has continued spreading around the world in recent years since the initial outbreak in 2019, frequently developing into new variants with greater human infectious capacity. SARS-CoV-2 and its mutants use the angiotensin-converting enzyme 2 (ACE2) as a cellular entry receptor, which has triggered several therapeutic strategies against COVID-19 relying on the use of ACE2 recombinant proteins as decoy receptors. In this work, we propose an ACE2 silent Fc fusion protein (ACE2-hFcLALA) as a candidate therapy against COVID-19. This fusion protein was able to block the binding of SARS-CoV-2 RBD to ACE2 receptor as measured by ELISA and flow cytometry inhibition assays. Moreover, we used classical neutralization assays and a progeny neutralization assay to show that the ACE2-hFcLALA fusion protein is capable of neutralizing the authentic virus. Additionally, we found that this fusion protein was more effective in preventing in vitro infection with different variants of interest (alpha, beta, delta, and omicron) compared to the D614G strain. Our results suggest the potential of this molecule to be used in both therapeutic and preventive settings against current and emerging mutants that use ACE2 as a gateway to human cells.

Fig 1. Characterization of ACE2-hFcLALA fusion protein.

(A) Schematic representation of ACE2-hFcLALA fusion protein. Subunits of the homodimeric protein are formed by the extracellular domain of the human ACE2 fused to the Fc region of a human IgG1 with LALA mutations. (B) SDS-PAGE (7.5%) of purified ACE2-hFcLALA in reducing conditions. MWM, molecular weight marker (Bio-Rad, 161–0373). (C) Western blots analysis using antibodies specific for hACE2 (left) and human Fc fragment (right).

Fig 2. Binding of ACE2-hFcLALA fusion protein to SARS-CoV-2 RBD and Spike proteins.

(A) ELISA plates coated with RBD-mFc fusion protein (5μg/mL) were incubated with ACE2-hFcLALA at different concentrations, followed by human Fc specific PA-conjugated antibody. PDL1-hFc was used as an irrelevant fusion protein. (B) ELISA plate coated with ACE2-hFcLALA (5μg/ml) were incubated with RBD-His and Spike-His at different concentrations, followed by a mouse His specific HRP-conjugated antibody. The x-axis displays the concentration of soluble purified RBD (RBD-His) or RBD domain in the context of the Spike protein. Binding was detected with pNPP (A) and OPD (B) substrates.

Fig 3. Inhibition of RBD-ACE2 interaction in vitro by ACE2-hFcLALA.

ELISA plates coated with ACE2-hFcLALA (5ug/mL) were incubated with RBD-mFc (25ng/mL, A) or Spike (2ug/mL, B) premixed with ACE2-hFcLALA at different concentrations. The binding of RBD-mFc and Spike was detected with a mouse Fc specific PA-conjugated antibody/pNPP substrate and mouse HisTag specific HRP-conjugated antibody/OPD substrate, respectively. VeroE6 (C) and HEK293-ACE2 (D) cells were incubated with RBD-mFc (100ng/mL) premixed with ACE2-hFc at different concentrations. The binding of RBD-mFc was detected by flow cytometry using a mouse Fc specific FITC-conjugated antibody.

Fig 4. Neutralization of SARS-CoV-2 virus by ACE2-hFcLALA.

VeroE6 cells were infected with a mixture of the D614G SARS-CoV-2 virus and ACE2-hFcLALA at different concentrations. After 1h at 37°C of contact with the mixture, the cells were PBS washed and kept in culture for 72h at 37°C. (A) Images of VeroE6 cells alone, virus infected, and infected with a mixture of viruses and different concentrations of ACE2-hFc. Magnification 10X. Scale bar 100 μm (B) The infectivity inhibition was determined by measuring the cellular viability of the VeroE6 cells. The human 5G4 antibody was used as infectivity inhibition negative control. (C) Recognition of SARS-CoV-2 N proteins using two different epitopes specific monoclonal antibodies against this protein.

Fig 5. Neutralization of SARS-CoV-2 virus progeny by ACE2-hFcLALA.

VeroE6 cells were infected with a single-round infection of D614G SARS-CoV-2 virus during 1h at 37°C. Then, the cells were PBS washed and kept in culture with different concentrations of the recombinant protein ACE2-hFcLALA for 72h at 37°C. (A) Images of VeroE6 cells alone, virus infected and infected and kept in culture with different concentrations of ACE2-hFc. Magnification 10X. Scale bar 100 μm (B) The infectivity inhibition was determined by measuring the cellular viability of the VeroE6 cells. The human 5G4 antibody was used as infectivity inhibition negative control.

Fig 6. Neutralization of SARS-CoV-2 virus variants by ACE2-hFcLALA.

VeroE6 cells were infected with a mixture of each of the five mutated SARS-CoV-2 virus variants and different concentrations of the recombinant protein ACE2-hFcLALA. After 1h at 37°C in contact with the mixture, the cells were PBS washed and kept in culture for 72h at 37°C. The infectivity inhibition was determined by measuring the cellular viability of the VeroE6 cells. The human 5G4 antibody was used as infectivity inhibition negative control.