Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals

Abstract

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named Reps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent Rep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, Reps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment. Author SummaryThe entry of SARS-CoV-2 in permissive cells is mediated by the binding of its spike to angiotensin-converting enzyme 2 (ACE2) on the cell surface. To select ligands able to block this interaction, we screened a library of phages encoding artificial proteins (named Reps) for binding to its receptor binding domain (RBD). Two of them were able to bind the RBD with high affinity and block efficiently the virus entry in cultured cells. Assembled Reps through covalent or non-covalent linkages blocked virus entry at lower concentration than their precursors (with around 20-fold activity increase for a trimeric Rep). These Reps derivates neutralize efficiently SARS-CoV-2 {beta}, {gamma}, {delta} and Omicron virus variants. Instillation of an Rep dimer in the nasal cavity effectively reduced virus replication in the hamster model of SARS-CoV-2 and pathogenicity.