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

Lead Research Organisation: University of Bristol
Department Name: Cellular and Molecular Medicine

Abstract

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.

Technical Summary

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.
 
Description G2P-UK; A National Virology Consortium to address phenotypic consequences of SARSCoV-2 genomic variation
Amount £2,497,170 (GBP)
Funding ID MR/W005611/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2021 
End 07/2022
 
Title SARS-CoV-2 reverse genetics, 
Description My laboratory has produced a reverse genetic system for SARS-CoV-2. We are using the system to produce viruses in which i) specific genes have been replaced with marker genes ii) viruses which contain specific mutations corresponding to SARS-CoV-2 variants of concern and iii) sub-genomic replicons. The recombinant viruses will be provided to other scientists in the research community. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact None as yet 
 
Description G2P-UK; A National Virology Consortium to address phenotypic consequences of SARSCoV-2 genomic variation 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution A major role of my laboratory is to use SARS-CoV-2 reverse genetic analysis to investigate mutations in the genome of SARS-CoV-2 variants. My laboratory is only one of two in the UK that has established a reverse genetic system for SARS-CoV-2.
Collaborator Contribution The collaborators are other basic virologists who have expertise in reverse genetic analysis; virus neutralisation assays using pseudotype viruses; virus replication assays using cell based models; virus transmission studies using animal models and organoid cultures.
Impact Collaboration only started in Feb, 2021.
Start Year 2021
 
Description Interview for national news 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Broadcast on BBC Newsnight 22nd Feb, 2021 describing role in the G2P consortium investigating SARS-CoV-2 variants.
Year(s) Of Engagement Activity 2021
 
Description Multiple interviews for BBC Points West 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact In Dec 2020 and January 2021 I was interviewed 3x by BBC points West. On one occasion I was in the studio and the other two interviews were done by Zoom interview.
Year(s) Of Engagement Activity 2021