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

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.

In vitro evaluation of therapeutic antibodies against a SARS-CoV-2 Omicron B.1.1.529 isolate

The emergence and rapid spread of the Omicron variant of SARS-CoV-2, which has more than 30 substitutions in the spike glycoprotein, compromises the efficacy of currently available vaccines and therapeutic antibodies. Using a clinical strain of the Omicron variant, we analyzed the neutralizing power of eight currently used monoclonal antibodies compared to the ancestral B.1 BavPat1 D614G strain. We observed that six of these antibodies have lost their ability to neutralize the Omicron variant. Of the antibodies still having neutralizing activity, Sotrovimab/Vir-7831 shows the smallest reduction in activity, with a factor change of 3.1. Cilgavimab/AZD1061 alone shows a reduction in efficacy of 15.8, resulting in a significant loss of activity for the Evusheld cocktail (42.6 fold reduction) in which the other antibody, Tixagevimab, does not retain significant activity against Omicron. Our results suggest that the clinical efficacy of the initially proposed doses should be rapidly evaluated and the possible need to modify doses or propose combination therapies should be considered.

Endonuclease-based genotyping of the RBM, a first-line method for the surveillance of emergence or evolution of SARS-CoV-2 Variants.

Since the beginning of the Covid-19 pandemics, variants have emerged. Whereas most of them have no to limited selective advantage, some display increased transmissibility and/or resistance to immune response. To date, most of the mutations involved in the functional adaptation are found in the Receptor Binding Module (RBM), close to the interface with the human receptor ACE2. In this study, we thus developed and validated a fast and simple molecular assay allowing the detection and partial identification of the mutations in the RBM coding sequence. After the amplification of the region of interest, the amplicon is heat-denatured and hybridized with an amplicon of reference. The presence of a mutation in the heteroduplex can be cleaved by a mismatch-specific endonuclease and the cleavage pattern is analysed by capillary electrophoresis. The approach was first validated on viral RNA purified different SARS-CoV-2 variants produced in the lab before being implemented for clinical samples. The results highlighted the performance of the assay for the detection of mutations in the RBM from clinical samples. The procedure can be easily set up for high throughput identification of the presence of mutations and serve as a first-line screening to select the samples for full genome sequencing.

Niclosamide shows strong antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency against the UK B.1.1.7 and SA B.1.351 variant

SARS-CoV-2 variants are emerging with potential increased transmissibility highlighting the great unmet medical need for new therapies. Niclosamide is a potent anti-SARS-CoV-2 agent that has advanced in clinical development. We validate the potent antiviral efficacy of niclosamide in a SARS-CoV-2 human airway model. Furthermore, niclosamide is effective against the D614G, B.1.1.7 and B.1.351 variants. Our data further support the potent anti-SARS-CoV-2 properties of niclosamide and highlights its great potential as a therapeutic agent for COVID-19.

SARS-CoV-2 20I/501Y.V1 variant in the hamster model

Late 2020, SARS-CoV-2 Alpha variant from lineage B.1.1.7 emerged in United Kingdom and gradually replaced the G614 strains initially involved in the global spread of the pandemic. In this study, we used a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeeded to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicated to almost the same level and induced a comparable disease and immune response. A slight fitness advantage was noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium.

Replicative fitness SARS-CoV-2 20I/501Y.V1 variant in a human reconstituted bronchial epithelium

Since its emergence in 2019, circulating populations of the new coronavirus continuously acquired genetic diversity. At the end of 2020, a variant named 20I/501Y.V1 (lineage B.1.1.7) emerged and replaced other circulating strains in several regions. This phenomenon has been poorly associated to biological evidence that this variant and original strain exhibit different phenotypic characteristics. Here, we analyse the replication ability of this new variant in different cellular models using for comparison an ancestral D614G European strain (lineage B1). Results from comparative replication kinetics experiments in vitro and in a human reconstituted bronchial epithelium showed no difference. However, when both viruses were put in competition in a human reconstituted bronchial epithelium, the 20I/501Y.V1 variant outcompeted the ancestral strain. Altogether, these findings demonstrate that this new variant replicates more efficiently and could contribute to better understand the progressive replacement of circulating strains by the SARS-CoV-2 20I/501Y.V1 variant. ImportanceThe emergence of several SARS-CoV-2 variants raised numerous questions concerning the future course of the pandemic. We are currently observing a replacement of the circulating viruses by the variant from the United Kingdom known as 20I/501Y.V1 from B.1.1.7 lineage but there is little biological evidence that this new variant exhibit a different phenotype. In the present study, we used different cellular models to assess the replication ability of the 20I/501Y.V1 variant. Our results showed that this variant replicate more efficiently in a human reconstituted bronchial epithelium, which may explain why it spreads so rapidly in human populations.