Generation and evaluation of protease inhibitor-resistant SARS-CoV-2 strains (preprint)

Since the start of the SARS-CoV-2 pandemic, the search for antiviral therapies has been at the forefront of medical research. To date, the 3CLpro inhibitor nirmatrelvir (Paxlovid(R)) has shown the best results in clinical trials and the greatest robustness against variants. A second SARS-CoV-2 protease inhibitor, ensitrelvir (Xocova(R)), has been developed. Ensitrelvir, currently in Phase 3, was approved in Japan under the emergency regulatory approval procedure in November 2022, and is available since March 31, 2023. One of the limitations for the use of antiviral monotherapies is the emergence of resistance mutations. Here, we experimentally generated mutants resistant to nirmatrelvir and ensitrelvir in vitro following repeating passages of SARS-CoV-2 in the presence of both antivirals. For both molecules, we demonstrated a loss of sensitivity for resistance mutants in vitro. Using a Syrian golden hamster infection model, we showed that the ensitrelvir M49L mutation confers a high level of in vivo resistance. Finally, we identified a recent increase in the prevalence of M49L-carrying sequences, which appears to be associated with multiple repeated emergence events in Japan and may be related to the use of Xocova(R) in the country since November 2022. These results highlight the strategic importance of genetic monitoring of circulating SARS-CoV-2 strains to ensure that treatments administered retain their full effectiveness. HighlightsO_LISARS-CoV-2 resistant strains to clinical stage protease inhibitors have been generated after 16 passages in vitro C_LIO_LIThe ensitrelvir resistance mutation M49L induces a strong resistance to ensitrelvir in vitro in three different isolates C_LIO_LIM49L mutation alone renders ensitrelvir treatment ineffective in vivo C_LIO_LIThe prevalence of naturally occurring M49L-mutants has increased over recent months C_LI

Sotrovimab retains activity against SARS-CoV-2 Omicron variant BQ.1.1 in a non-human primate model (preprint)

The SARS-CoV2 Omicron variants have acquired new Spike mutations leading to escape from the most of the currently available monoclonal antibody treatments reducing the options for patients suffering from severe Covid-19. Recently, both in vitro and in vivo data have suggested that Sotrovimab could retain partial activity against recent omicron sub-lineage such as BA.5 variants, including BQ.1.1. Here we report full efficacy of Sotrovimab against BQ.1.1 viral replication as measure by RT-qPCR in a non-human primate challenge model.

In vivo activity of Sotrovimab against BQ.1.1 Omicron sublineage (preprint)

The successive emergence of SARS-CoV-2 Omicron variants has completely changed the modalities of use of therapeutic monoclonal antibodies. Recent in vitro studies indicated that only Sotrovimab has maintained partial activity against BQ.1.1, a sub-variant of BA.5 that is spreading in the USA and Europe. In the present study, we used the hamster model to determine whether Sotrovimab retains antiviral activity against BQ.1.1 in vivo. Our results show that at exposures consistent with those observed in humans, Sotrovimab remains active against BQ.1.1 variant, although at a lower level than that observed against the first globally dominant BA.1 and BA.2 Omicron sublineages.

Enhanced neutralization escape to therapeutic monoclonal antibodies by SARS-CoV-2 Omicron sub-lineages (preprint)

The landscape of SARS-CoV-2 variants dramatically diversified with the simultaneous appearance of multiple sub-variants originating from BA.2, BA.4 and BA.5 Omicron sub-lineages. They harbor a specific set of mutations in the spike that can make them more evasive to therapeutic monoclonal antibodies. In this study, we compared the neutralizing potential of monoclonal antibodies against the Omicron BA.2.75.2, BQ.1, BQ.1.1 and XBB variants, with a pre-Omicron Delta variant as a reference. Sotrovimab retains some activity against BA.2.75.2, BQ.1 and XBB as it did against BA.2/BA.5, but is less active against BQ.1.1. Within the Evusheld/AZD7442 cocktail, Cilgavimab lost all activity against all subvariants studied, resulting in loss of Evusheld activity. Finally, Bebtelovimab, while still active against BA.2.75, also lost all neutralizing activity against BQ.1, BQ.1.1 and XBB variants.

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

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 alphaReps), 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 alphaRep 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, alphaReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

Rapid characterization of a Delta-Omicron SARS-CoV-2 recombinant detected in Europe (preprint)

Recombination is a crucial process in the evolution of many organisms. Although the evolutionary reasons behind its occurrence in RNA viruses are debated, this phenomenon has been associated with major epidemiological events such as virus host range expansion, antigenic shift or variation in virulence 1,2, and this process occurs frequently in positive strand RNA viruses such as coronaviruses. The SARS-CoV-2 pandemic has been associated with the repeated emergence of variants of concern presenting increased transmissibility, severity or immune escape 3. The recent extensive circulation of Delta worldwide and its subsequent replacement by viruses of the Omicron lineage 4 (BA.1 then BA.2), have created conditions for genetic exchanges between viruses with both genetic diversity and phenotypic specificities 5-7. Here we report the identification and in vitro and in vivo characterization of a Delta-Omicron recombinant in Europe. This recombinant exhibits immune escape properties similar to Omicron, while its behavior in mice expressing the human ACE2 receptor is more similar to Delta. This recombinant provides a unique and natural opportunity to better understand the genotype to phenotype links in SARS-CoV-2.

In vitro activity of therapeutic antibodies against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5In vitro activity of therapeutic antibodies against SARS-CoV-2 Omicron BA.1 and BA.2 (preprint)

The replacement of the Omicron BA.1 variant of SARS-CoV-2 by the BA.2 and the rapid growth of the BA.5 sub lineage, which have both different sets of mutations in the spike glycoprotein, alters the spectrum of activity of therapeutic antibodies currently licensed in the European Union. Using clinical strains of the Omicron BA.2 and BA.5 variants, we compared the neutralising power of monoclonal antibodies against the Omicron BA.1, BA.2 and BA.5 variants, using an ancestral strain (lineage B.1, D614G) and a Delta variant strain as reference. Sotrovimab/Vir-7831 is less active against BA.2 than against BA.1 (fold change reduction ~1,4) and even less active against BA.5 (fold change reduction ~2.7). Within the Evusheld /AZD7442 cocktail, Cilgavimab/AZD1061 is more active against BA.2 and BA.5 than against BA.1 (fold change increase ~32), whilst the very low activity of Tixagevimab/AZD8895 against BA.1 is not enhanced against BA.2 nor BA.5. In total, compared to BA.1, the activity of the Evusheld/AZD7442 is significantly improved against BA.2 and that against BA.5 is intermediate but closer to that against BA.2.

Activity of Tixagevimab/Cilgavimab against the Omicron variant of SARS-CoV-2 in a hamster model (preprint)

The efficacy of pre-exposure prophylaxis by the Tixagevimab/Cilgavimab cocktail (AZD7442) was evaluated in hamsters against a clinical BA.1 strain of SARS-CoV-2 variant Omicron. AZD7442 retains inhibitory activity against Omicron despite a substantial loss of efficacy. We estimate that Omicron virus requires about 20-times more antibodies in plasma than the ancestral B.1 strain (G614) virus to achieve a similar drug efficacy in reducing lung infectious titers.

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

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.

Pre-clinical evaluation of antiviral activity of nitazoxanide against Sars-CoV-2 (preprint)

To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy. In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2. First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50. These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19.

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 (preprint)

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 (preprint)

Late 2020, SARS-CoV-2 20I/501Y.V1 variant from lineage B.1.1.7 emerged in United Kingdom and gradually replaced the D614G 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 20I/501Y.V1 variant with an ancestral D614G strain. The 20I/501Y.V1 variant succeeded to infect animals and to induce a pathology that mimics COVID-19. However, both strains induced replicated to almost the same level and induced a comparable disease and immune response. A slight fitness advantage was noted for the D614G strain during competition and transmission experiments. These data do not corroborate the current epidemiological situation observed in humans nor recent reports that showed a more rapid replication of the 20I/501Y.V1 variant in human reconstituted bronchial epithelium.

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

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.

Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2 (preprint)

Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of Imatinib in multiple models. Our results indicated that Imatinib and another TKI, the Masitinib, exhibit an antiviral activity in VeroE6 cells. However, Imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, Imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of Imatinib and similar TKIs as antivirals in the treatment of Covid-19.

A simple reverse genetics method to generate recombinant coronaviruses (preprint)

Engineering recombinant viruses is capital for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the large size of coronaviruses genome makes reverse genetics methods challenging.Here we describe a simple method based on “infectious subgenomic amplicons” (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstructing a full genomic cDNA. The method was applied to the SARS-CoV-2 and the feline enteric coronavirus, and allowed to rescue wild-type viruses with biological characteristics closely similar to original strains. Mutations and fluorescent red reporter gene were rapidly incorporated into the SARS-CoV-2 genome allowing the generation of a genomic variant and a fluorescent reporter strains which were studied during in vivo experiments, serological diagnosis and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies and to explore biological properties of SARS-CoV-2 variants or accelerating the development of therapeutic measures. 

Viral dynamic modeling of SARS-CoV-2 in non-human primates (preprint)

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques infected with 106 pfu of SARS-CoV-2 for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large daily viral production (>104 virus) and a within-host reproductive basic number of 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly cleared with a half-life of 9 hours, with no significant association between cytokine elevation and clearance. Translating our model to the context of human-to-human infection, human mild infection may be characterized by a peak occurring 4 days after infection, a viral shedding of ~11 days and a generation time of 4 days. These results improve the understanding of SARS-CoV-2 viral replication and better understand the infection to SARS-CoV-2 in humans.

Favipiravir and severe acute respiratory syndrome coronavirus 2 in hamster model (preprint)

Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we used a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment was initiated before or simultaneously to infection, favipiravir had a strong dose effect, leading to dramatic reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlated with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. The antiviral efficacy observed in this study was achieved with plasma drug exposure comparable with those previously found during human clinical trials and was associated with weight losses in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase (preprint)

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.