Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemia that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the virus RNA ratio allowing SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and S. Furthermore, monochrome, dual-color fluorescent or photoconvertible VLPs were produced. Thanks to live fluorescence and super-resolution microscopy, we quantified VLPs size and concentration. It shows a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a minimum concentration of 10e12 VLP/ml. SARS-CoV-2 VLPs could tolerate the integration of fluorescent N and M tagged proteins without impairing particle assembly. In this condition, we were able to establish incorporation of the mature Spike in fluorescent VLPs. The Spike functionality was then shown by monitoring fluorescent MNES-VLPs docking and endocytosis in human pulmonary cells expressing the receptor hACE2. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

Low selectivity index of ivermectin and macrocyclic lactones on SARS-CoV2 replication in vitro argues against their therapeutic use for COVID-19.

There are very limited antiviral therapeutic options for coronavirus infections, therefore global drug re-purposing efforts are paramount to identify available compounds that could provide clinical benefits to patients with COVID-19. Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin demonstrated anti-SARS-CoV-2 activity in vitro in human pulmonary cells in comparison to VeroE6 (with EC50 of 1-3 M). Similarly, the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin reduced SARS-CoV-2 replication in vitro (with EC50 of 2-5 M). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index <10 for all compounds. In conclusion, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se, due to low selectivity index. This is discussed in regards to recent clinical COVID studies on ivermectin.