Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors

Abstract

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine

“…It was then linearized with PacI and transfected into HEK293A cells (RRID:CVCL_6910). Our recombinant adenovirus was then amplified using SF-BMAd-R cells 61 in 3 batches (Ad(Spike) 1-3), combined, and purified by ultracentrifugation on CsCl gradients as described previously 65 , before titration using a TCID 50 assay. A second human adenovirus serotype 5 (ΔE1-, ΔE3-; 1 st generation), lacking a gene cassette, was used as a negative control.…”

BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine

“… 73 It was then linearized with PacI and transfected into HEK293A cells (RRID:CVCL_6910). Our recombinant adenovirus was then amplified using SF-BMAd-R cells 70 in 3 batches (Ad(Spike) 1–3), combined, and purified by ultracentrifugation on CsCl gradients as described previously, 74 before titration using a TCID 50 assay. A second human adenovirus serotype 5 (ΔE1-, ΔE3-; 1 st generation), lacking a gene cassette, was used as a negative control.…”