Abstract
Vaccinia virus (VACV) is an established vector 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. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of 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%. Virus productivity 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 increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.
Highlights
Vaccinia virus (VACV) is an enveloped, brick-shaped particle typically 300×240×120 nm containing a double stranded DNA genome which, for the Lister strain (Garcel et al 2007), is 189.4 kilobase-pairs in size, encoding up to 201 open reading frames (ORFs)
Considerations for VACV genome refactoring and BioBrickTM-based VACV plasmid tools Industrial synthetic biology seeks to go beyond conventional bioprocessing and design host organisms (‘chassis’) with predictable process behaviour
We indicated the location of the EcoRI, XbaI, SpeI and PstI sites in the Lister strain VACV genome (Figure 1A) as these sites define the BioBrickTM DNA fragment assembly standard (Shetty et al 2008) widely used in the synthetic biology community and beyond
Summary
Vaccinia virus (VACV) is an enveloped, brick-shaped particle typically 300×240×120 nm containing a double stranded DNA genome which, for the Lister strain (Garcel et al 2007), is 189.4 kilobase-pairs (kb) in size, encoding up to 201 open reading frames (ORFs). After the final decanting of supernatant 0.2 mL aqueous crystal violet solution (0.1% w/v Crystal Violet, 0.1M citric acid, 0.1% v/v Triton X-100) was added to the microcarriers slurry and the mixed by pipetting up and down 25 times before the Eppendorf tube was transferred to an incubator set at 37°C / 5% CO2 for 1.5 hours. This treatment causes cells to lyse and release stained nuclei. Released nuclei were counted using an Improved Neubauer haemocytometer (1080346, Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany) as an indicator of cell numbers
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