The production and application of SARS-CoV-2 reverse genetic systems to facilitate vaccine development and biosafe drug discovery platforms.

The development of vaccines and antiviral drugs against SARS-CoV-2 is hampered by the requirement to grow the virus under high-containment, which is limited to few laboratories worldwide. We have recently established a rapid procedure to produce a DNA copy of the SARS-CoV-2 RNA genome, that can be used to genetically engineer new viruses via a process termed "reverse genetics". We now propose to engineer the SARS-CoV-2 genome to produce 1) a SARS-CoV-2 virus that expresses a green fluorescent protein (turboGFP) and 2) a disabled biosafe form of the virus (replicon) that expresses turboGFP instead of the structurally important spike protein. When introduced into suitable cells, the replicon reproduces continously but does not produce infectious virus particles. The two engineered forms of the virus can then be used to develop high-throughput fluorescence based assays to monitor the entry of the virus into cells and its replication. The first can be used to identify antibodies induced by vaccine candidates that neutralise virus infectivity, and also viral mutations that allow escape from the antibody response. The replicon can be used for rapid screening of antiviral compounds, at a lower containment level, which would allow many more laboratories to participate in drug discovery efforts.

The development of coronavirus reverse genetic systems has been slow and limited to few laboratories worldwide. We have established a rapid approach to generate a SARS-CoV-2 reverse genetic system, by targeted recombination of synthetic SARS-CoV-2 cDNA fragments in yeast, using a yeast artificial chromosome (YAC) vector. Nanopore minION sequencing was used to quickly identify correctly assembled YAC clones, that can be transcribed in vitro to produce RNA corresponding to the SARS-CoV-2 genome, which can be used to recover recombinant SARS-CoV-2 (Figure 1A). The entire process takes 2-3 weeks from the receipt of synthetic DNA. We now propose to use this reverse genetic system to develop:

1) recombinant SARS-CoV-2 reporter viruses expressing one or more reporter genes, in place of genes that are non-essential in cell culture: SARS-CoV-2 reporter viruses can be used to rapidly characterise viral infectivity and will be used to develop high-throughput virus antibody neutralisation and escape assays, to underpin viral vaccine development.
2) a biosafe SARS-CoV-2 "replicon" which lacks the viral structural genes but can replicate intracellularly. Human cell lines stably expressing SARS-CoV-2 replicons are powerful tools for antiviral screening and in contrast to assays with SARS-CoV-2, can be undertaken at containment level 2.

These constructs will allow academic and commercial laboratories to undertake high-throughput antiviral assays, removing a bottleneck in drug discovery efforts. Furthermore, combined transcriptomics and proteomics will be used to characterise the effects of the recombinant reporter viruses and replicons on the host cell, ensuring that the engineered viruses faithfully represent the natural virus.