Implementation of a fast and reliable assay for the high-throughput screening of antiviral molecules active against SARS-CoV-2 – Alpha-COV

This year, the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to an unprecedented pandemic termed Covid-19 (Coronavirus disease 2019). Even as human trials of candidate vaccines are already ongoing, there is still a doubt that people can develop protective immunity against SARS-CoV-2. There is therefore an urgent need to identify safe and effective drugs for treatment. The goal of this project is to implement an innovative assay to rapidly detect SARS-CoV-2 experimental infections to allow for the high-throughput screening (HTS) of antivirals to tackle Covid-19. We have established an assay, termed Alpha-Centauri, that allows the quantification, using a Nanoluciferase-based Protein Complementation Assay (PCA), of the nuclear translocation of innate immunity transcription factors by HTS. Since all viral infections trigger signalling pathways downstream of PRR to varying degrees, which all converge on the phosphorylation and nuclear translocation of IRF3, our system should provide a fast and reliable read-out for experimental SARS-CoV-2 infections.
Our project falls within axis 2 of the ANR Flash COVID-19 call: Development of animal and cell models & Therapeutic targets and models to assess candidate drugs. Our team has already started on the project, and has secured a proof-of-concept with Sendai Virus, a RNA virus that causes severe respiratory disease in mice
For this project, we aim to (i) translate our system to lung adenocarcinoma A549 cells, which are an appropriate cellular model for SARS-CoV-2; we will use both overexpressing cell lines and generate cell lines in which endogenous IRF3 is modified by CRISPR/Cas9-mediated genome editing to allow nuclear PCA of endogenous protein (Aims 1 and 2); and (ii) validate our system for HTS by miniaturisation of the protocol and by testing reported positives and negatives on SARS-CoV-2 infection (Aim 3).
The project will be carried out as a partnership between two teams from the "Institut de Recherche en Infectiologie de Montpellier" (IRIM): Team "Viral Trafficking, Restriction and Innate Signaling" (Sébastien Nisole) who will develop the cellular models, and Team "Membrane Dynamics & Viruses" (Raphaël Gaudin) who will perform all infections with SARS-CoV-2 in the biosafety level 3 (BSL3) laboratories at the "Centre d’études des Maladies Infectieuses et Pharmacologie Anti-Infectieuse" (CEMIPAI, Montpellier).
The deliverables of the project will be the tools and the engineered cell lines that will be made accessible to the academic and pharmaceutical scientific community to screen compound libraries for antivirals active against SARS-CoV-2. Our technology will allow to screen chemical libraries for antiviral molecules, validate candidate molecules, or optimise validated antivirals. Our assay will enable to identify antivirals that target SARS-CoV-2 directly, and also molecules that boost the host cell interferon response. The latter will therefore constitute a precious arsenal to target other viral infections, which like SARS-CoV-2, are highly sensitive to interferon.