Construction of an Adenovirus Vector with Laco-regulated Antigen Expression Using the SARS-Cov-2 Spike as a Transgene Model

Abstract

The adenovirus vector has been widely studied for vaccines and gene therapies. During the production of the adenovirus vector, a high virus titer is desired to obtain enough virus. The adenovirus vector has been widely studied for the vaccinations and gene therapies, where a high virus titer is desired to obtain sufficient quantities of the virus. For an adenovirus vector-based vaccine, suppression of antigen expression during production would improve the virus titer during production. This study aimed to construct an adenovirus vector with lacO-regulated antigen expression using the SARS-CoV-2 spike as a transgene model, which would improve the adenovirus titer during production. Methods: The lacO expression cassette was designed and prepared as a synthetic gene in pUC57. The lacO expression cassette was then subcloned into pShuttle-CMV. The SARSCoV- 2 spike gene was then inserted into the pShuttle-CMV harboring lacO to generate pShuttlelacO_ S and pShuttle-lacO-intron_S. Recombinant pShuttle was then used to generate a recombinant adenovirus genome using Escherichia coli BJ5183 pAdeasy-1. Transfection of the PacI-linearized adenovirus genome into AD293 and HEK293 cells was used to generate adenovirus primary stock for 14 days of incubation. Recombinant adenovirus genomes, pAdeasy-lacO_S and pAdeasy-lacO-intron_S, were successfully generated and characterized using PacI restriction and PCR. In the production of adenovirus primary stocks, the adenovirus titer produced in AD293 cells was higher than in HEK293 cells. The primary stock titer of AdV_lacO-intron-S was higher than AdV_lacO-S and AdV_S titers. Production of adenovirus with lacO and spike gene, either with or without intron, was successful with a higher titer as compared to AdV_S titer.