Abstract

BackgroundChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.MethodsWe used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine.ResultsThe expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells.ConclusionsOverall, the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.

Highlights

  • ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is in use across the globe

  • Virus and cells Human MRC-5 cells, A549 cells and HEK293 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC)

  • For A549 cells, there was a slight decline over time but notably there were approximately fivefold fewer reads as a proportion of human mapped transcripts mapping to the ChAdOx1 genome compared to MRC-5 cells

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Summary

Introduction

ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is in use across the globe. Replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome of approximately 36 kbp that efficiently deliver their DNA genome to the nuclei of host cells for viral genome replication. The E1 region contains early genes required to trigger a transcription cascade enabling viral replication; E1-deleted vectors need to be grown in E1 transcomplementing cell lines such as HEK293 cells [8]. HEK293 cells have a 4-kbp region of human adenovirus type 5 (HuAd5) integrated into the cellular genome that provides the E1 genes in trans enabling efficient virus vector replication and recombinant virus production. The transgene to be expressed is inserted into the virus genome in place of the E1 region under the control of a highly active promoter

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