SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus membrane binds to the angiotensin converting enzyme 2) ACE2 (receptor on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells

“…Indeed, in our fusion protein, the RBD domain ends at amino acid 524, thus preventing the formation of the native disulphide bond C391-C525. Other recombinant Fc-tagged RBD proteins (amino acids 331-524), thus lacking C525, were previously described as retaining structural and biological properties of the native RBD domain, such as efficient binding to the human ACE2 receptor [ 14 , 15 , 16 ]. Here, we additionally demonstrated the antigenic property of RBDmfc, by showing its ability to be recognised by SARS-CoV-2-specific IgG from COVID-19 convalescent patients, in agreement with previous results based on the His-tagged version of recombinant RBD (amino acids 331-524) [ 13 ].…”

“…Notably, several studies reported the development and characterization of recombinant RBD proteins (amino acids 331-524 of the spike protein) fused to a C-terminal tag. These studies showed that recombinant RBD protein (amino acids 331-524) efficiently binds the angiotensin-converting enzyme 2 (ACE2) receptor, is efficiently recognised by neutralising antibodies from COVID-19 convalescent individuals, and elicits a strong neutralising antibody response when administered as an immunogen in BALB/c mice [ 13 , 14 , 15 , 16 ]. Of particular interest, Sun et al expressed SARS-CoV-2 RBD (amino acids 331-524) either fused to a histidine (His) tag as a monomeric protein or fused to a human IgG1 Fc fragment to produce a Y-shaped dimeric protein.…”

SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

“…293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ 55 ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes. Control cells were stained only with PE-conjugated goat anti-human IgG.…”

Anti-human ACE2 antibody neutralizes and inhibits virus production of SARS-CoV-2 variants of concern

“…We next tested whether the commercial anti-ACE2 antibody can block the binding of RBD to ACE2. For that, we used a fusion protein of SARS-CoV-2 wild-type receptor binding domain (RBD) which we have iScience Article generated previously and demonstrated that it binds ACE2 (Chaouat et al, 2021). We incubated 293T-ACE2 cells with or without the commercial antibody and then stained the cells with RBD-Ig.…”