The spike protein of SARS-CoV-2 (GenBank accession number YP_009724390.1), the surface glycoprotein responsible for receptor binding and fusion/entry into the host cell, was codon optimised for expression in human cell lines and synthesised with the tissue plasminogen activator (tPA) leader sequence at the 5’ end by GeneArt Gene Synthesis (Thermo Fisher Scientific). The sequence, encoding SARS-CoV-2 amino acids 2–1273 and tPA leader, was cloned into a shuttle plasmid using InFusion cloning (Clontech). The shuttle plasmid encodes a modified human cytomegalovirus major immediate early promoter (IE CMV) with tetracycline operator (TetO) sites, poly adenylation signal from bovine growth hormone (BGH), between Gateway® recombination cloning sites. ChAdOx1 nCoV-19 was prepared using Gateway® recombination technology (Thermo Fisher Scientific) between the shuttle plasmid described and the ChAdOx1 destination DNA BAC vector described in5 (link) resulting in the insertion of the SARS-CoV-2 expression cassette at the E1 locus. The ChAdOx1 adenovirus genome was excised from the BAC using unique PmeI sites flanking the adenovirus genome sequence. The virus was rescued and propagated in T-Rex 293 HEK cells (Invitrogen) which repress antigen expression during virus propagation. Purification was by CsCl gradient ultracentrifugation. Virus titers were determined by hexon immunostaining assay and viral particles calculated based on spectrophotometry16 ,17 .