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Production of rVSV-ZEBOV in serum-free suspension culture of HEK 293SF cells

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Supplemental Methods

Cells. VeroE6 and HEK 293T (ATCC) stocks were thawed and cultured in T75 cell culture flasks (Thermo Fisher Scientific) with DMEM 4.5 g/L glucose with sodium pyruvate (Thermo Fisher Scientific) enriched with heat inactivated 10% FBS (Wisent, St-Bruno, QC, Canada) and 1% GlutaMAX (Life Technologies). The flasks were left in a 37C humidified incubator with 5% CO2 until a confluence around 80%. The

attached cells were washed with 37C warmed DPBS (Thermo Fisher Scientific) and detached at room temperature with Trypsin-EDTA (Wisent). After diluting the trypsinised cells with 37C fresh DMEM, the mix was centrifuged at 200 × g for 5 minutes. The supernatant was discarded, and the pelleted cells were resuspended in DMEM, 10% FBS and 1% GlutaMAX and split between 1:3 and 1:10 depending on the original confluency.

Plasmids. The original stock of virus was generated by viral rescue using the plasmids pATX.V2.B6.Full, pBS-N, pBS-L, pBS-P and pCAGGS T7. For the genome plasmid pATX.V2.B6.Full, the backbone pATX.V2 containing the full VSV genome except for the wild-type envelope protein was optimized and synthesized by GenScript (Piscataway, NJ, USA). The Zaire ebolavirus envelope glycoprotein B6 sequence was previously published [1] and this sequence was synthesized by GenScript. It was inserted into the pATX.V2 backbone using NheI and XhoI cut sites and the In-Fusion cloning protocol (Takara Bio, Mountain View, CA, USA). The pBS-P plasmid [2], containing the phosphoprotein of VSV, Indiana serotype, was also synthesized by GenScript. The two other helper genes, L (VSV polymerase) and N (VSV nucleoprotein) were synthesized separately and subsequently cloned into the pBS vector using In-Fusion and HindIII and EcoRI cut sites to generate pBS-L and pBD-N. The T7 RNA polymerase sequence was synthesized from GenScript and inserted into the pCAGGS [3] using EcoRI and BglII cut-sites and In-Fusion. All plasmids were amplified and purified using Mini, Midi and/or Maxipreps (Qiagen, Valencia, CA, USA) and the sequences of inserted genes were confirmed by sequencing.

Viral rescue. Viral rescue of rVSV-ZEBOV has been described previously [4] and was performed here with slight changes. The day before transfection, VeroE6 cells and HEK 293T cells were co-cultured in 6-well plates (Sarstedt) with DMEM with 3% FBS and 1% GlutaMAX (Thermo Fisher Scientific) to an estimated confluency of 50%. The following day, the cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific). Briefly, the culture medium was removed from the cells and replaced with 500 L/well DMEM without any supplement addition. The plate

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was left in the incubator at 37C for 30-60 minutes. Meanwhile, the pATX.V2.B6.Full plasmid along with the helper plasmids were mixed at different ratios (pATX.V2.B6.Full and pCAGGS T7 from 2 to 2.5 g/well, pBS-N from 0.5 to 0.625 g/well, pBS-L from 0.25 to 0.75 g/well and pBS-P from 1.25 to 1.56 g/well) and diluted in 90 L/well Opti-MEM (Thermo Fischer Scientific). Lipofectamine 2000 was diluted 14 L/well in 90 L Opti-MEM and all solutions were incubated for 5 minutes at room temperature. The tubes were then combined and incubated for a further 15 minutes at room temperature. Finally, 1 mL of Opti-MEM was added slowly to each tube. The cells were incubated at 37C for 24 hours with Opti-MEM mixes which was then replaced by DMEM with 2% FBS and 1% GlutaMAX. The cultures were followed daily. Rescues were done 48h, 72h, 96h and/or 120h post-transfection. Fresh VeroE6 cells were plated out on 6-well plates with DMEM containing 10% FBS and 1% GlutaMAX. Estimated confluency around 80-90%. The following day, the medium from each well was aspirated and replaced with 500 L from the transfected wells. The cells were left incubating for 30-60 min at 37 °C. After incubation, 1.5 mL of DMEM with 2% FBS and 1% GlutaMAX was added to each well and the plate was left at 37 °C. The cultures were monitored daily for signs of cytopathogenic effect. If a cytopathogenic effect was observed the virus was titrated and stored at -80 °C in aliquots. This original stock was then amplified over 72 hours on HEK 293A cells using an MOI of 0.001. The supernatant was harvested by centrifugation at 500 × g for 5 minutes to generate a larger stock.

References

[1] Urbanowicz RA, McClure CP, Sakuntabhai A, Sall AA, Kobinger G, Muller MA, et al. Human Adaptation of Ebola Virus during the West African Outbreak. Cell.

2016;167:1079-87 e5.

[2] Garbutt M, Liebscher R, Wahl-Jensen V, Jones S, Moller P, Wagner R, et al. Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses. J Virol. 2004;78:5458-65.

[3] Richardson JS, Yao MK, Tran KN, Croyle MA, Strong JE, Feldmann H, et al. Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine. PLoS One. 2009;4:e5308.

[4] Wong G, Qiu X. Designing Efficacious Vesicular Stomatitis Virus-Vectored Vaccines Against Ebola Virus. Vaccine Design2016. p. 245-57.

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