Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrickTM design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

BioRxiv : the Preprint Server for Biology
Shuchang LiuDarren N Nesbeth

Abstract

Vaccinia virus (VACV) is an established tool for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. As a step toward realising these benefits we analysed the Lister Vaccinia virus genome with respect to refactoring options and propose a VACV genome engineering BioBrickTM. We then used the CV-1 cell line to produce a conventional recombinant Lister strain VACV, VACVL-15 RFP in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h-1 and 0.044 h-1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h-1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. VACV production in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increase...Continue Reading

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