Materials & Methods
Cell line and culture
media
The suspension adapted Vero cell line was provided by the National
Research Council (NRC) of Canada, Montreal, Canada, and its adaptation
process has been described previously (Shen et al., 2019). For routine
passaging, the cells were harvested by centrifugation for 5 minutes at
500 g and the cell pellet was resuspended in fresh medium to a seeding
cell density of 2.5–5 × 105 cells/mL in 125 mL
polycarbonate shake flasks (TriForest Enterprises, USA) and maintained
at 37 °C, 135 rpm and 5% CO2 in an humified Multitron
orbital shaker (Infors HT, Switzerland). The cells were cultivated in 20
mL working volume of either IHM03 medium, provided by the NRC, or in
MDXK medium (Xell AG, Germany), supplemented with 4 mM GlutaMAX (Thermo
Fisher Scientific, USA).
Viruses
Origin and viral seed stock amplification of rVSV-ZEBOV and
rVSV-B6-A74Env(PN6)/SIVtm (hereafter referred to as rVSV-HIV) have been
described previously (Kiesslich et al., 2020) (Mangion et al., 2020).
The recombinant COVID-19 vaccine candidate
rVSVInd-msp -SF-Gtc was
constructed as follows: Codon-optimized full-length spike protein gene
of SARS-CoV-2 (GenBank: JX869059.2) was purchased from Genscript USA Inc
(Pscataway, NJ, USA), cloned into an avirulent Indiana serotype of
vesicular stomatitis virus [VSVInd(GML)] as has been
described previously (Kim et al., 2015). The honeybee melittin signal
peptide (msp ) was inserted at the NH2-terminus
and the transmembrane domain and cytoplasmic tail (Gtc) of the S protein
was substituted by the Gtc of VSVInd G protein at the
COOH-terminus. In addition, the VSV intergenic sequences were added in
front of the spike protein gene in order to provide the transcription
termination signal, polyadenylation signal, and the transcription
re-initiation signal. The modified SARS-CoV-2 spike protein gene was
inserted into the G and L gene junction of the
VSVInd(GML) at Pme I and Mlu I sites.
Recombinant
rVSVInd-msp -SF-Gtc virus
was recovered by VSV reverse genetics as has been described previously
(Kim et al., 2015). The recombinant virus was purified by three
consecutive plaque picking, and a stock virus was prepared by infecting
BHK21 cells.
Shake flask virus studies
For virus infection studies in shake flasks, suspension adapted Vero
cells were harvested and seeded at the indicated cell density in fresh
medium. Vero cells were infected with rVSV at the indicated MOI and the
temperature was shifted to either 34 °C (rVSV-ZEBOV, rVSV-HIV) or 31 °C
(rVSVInd-msp -SF-Gtc ).
Samples for virus titration were centrifuged for 5 minutes at 1200 × g
to remove cellular debris, aliquoted and stored at −80 °C.
Bioreactor cultures
Bioreactor cultures were performed in a 1 L bioreactor (Applikon
Biotechnology, The Netherlands) equipped with a marine impeller, pH
sensor, temperature sensor, and dissolved oxygen (DO) concentration
sensor. MDXK medium was supplemented 4 mM L‑glutamine (GE Healthcare,
USA) instead of GlutaMAX to enable monitoring of L‑glutamine
consumption. Vero cell seed cultures were grown in progressively larger
polycarbonate shake flasks (TriForest Enterprises, USA), harvested by
centrifugation and resuspended in fresh medium before inoculation. The
bioreactor was inoculated at a cell density of
2.5 × 105 cells/mL in 850 mL working volume. The
culture was agitated at 100 rpm and kept at 37 °C. The DO concentration
was kept at 50 % air-saturation by continuous surface aeration of 5
mL/min air and injection of pure oxygen through the sparger when
required. The pH was set to 7.2 and regulated by injection of
CO2 into the headspace or addition of
NaHCO3 (90 g/L) (Sigma, USA). Samples were taken once or
twice daily, depending on the progress of the culture, to subsequently
determine viable cell density, metabolite concentration and virus titer.
Samples for metabolite analysis and virus titration were centrifuged for
5 minutes at 1200 × g to remove cellular debris, aliquoted and stored at
−80 °C.
For virus production, Vero cells were infected with rVSV at an MOI of
0.01 once the targeted cell density was reached and the temperature was
shifted to 34 °C or 31 °C, respectively, during the virus production
phase. The glucose concentration was estimated once daily and if
required adjusted to 2 g/L by feeding glucose (Sigma) concentrate (180
g/L). In addition, L-glutamine was maintained at a minimum concentration
of 2 mM.
Analytical Methods
Vero cell concentration and viability were determined via the Vi-CELL XR
cell counter (Beckman Coulter, USA). The Median Tissue Culture
Infectious Dose (TCID50) assay and digital PCR assay
used in this work to quantify the infectious titer and the number of
viral genomes, respectively, has been described previously (Gélinas et
al., 2020; Kiesslich et al., 2020). For
rVSVInd-msp -SF-Gtc , the
TCID50 plates were incubated at 31 °C, due to the
temperature sensitivity of this construct.
Metabolite analysis
During cell culture cultivations, the glucose concentration was
estimated using the D-Fructose/D-Glucose Assay Kit (Megazyme, Ireland).
More extensive metabolite analysis was performed offline via Bioprofile
400 (Nova Biomedical, USA) from samples that were stored at −80 °C.