2 Methods

2.1 DDVax Production Process Summary

2.1.1 Uninfected Cell Culture

Serum-Free Vero cells (2 x 104cells/cm2) were grown in OptiPro Serum-Free Media (SFM) (Lifetech, A31343) with Glutamax (Gibco, 35050-061) at 37°C and 5% CO2. Cells were expanded into 3 x 10-Layer CellStacks (6360 cm2) (Corning, 3271) and 1 x 1-Layer CellStacks (636 cm2) (Corning, 3268). For each passage, cells were seeded at either 2.0 x 104cells/cm2 for 48 ± 8-hours or 1.5 x 104 cell/cm2 for 72 ± 8-hours. Cell harvest was performed using TrypLE Select (Life Tech, 12563092). Cells were centrifuged at 500 x g for 5 minutes at 18°C and resuspended in OptiPro SFM. Cell enumeration was performed using a Vi-Cell Cell Viability Analyzer.

2.1.2 Generation of DDVax Pilot Material

Upon achieving cell confluency within the 3 x 10-Layer or 1 x 1-Layer CellStacks, Vero cells were infected with the DDVax Research Virus Stock (RVS), Lot # N16-5-20-RV at a multiplicity of infection (MOI) of 0.0005 PFU/cell. Infection volume used for each 10-Layer CellStack was 1300ml and the infection volume for the 1-Layer CellStack was 130ml. Infected cultures were then incubated at 37°C and 5% CO2 for 72-hours. Following the 72-hour infection incubation, flasks were examined for cytopathic effects (CPE). DDVax virus was then harvested by pumping the supernatant from each CellStack into a 5L Flexboy bag. The harvested pool underwent Benzonase treatment to digest Host Cell DNA (HCD). A 500mM MgCl2 solution was added to the Flexboy bag to achieve a final concentration of 1.5mM MgCl2 for Benzonase treatment. Benzonase (EMD Millipore, 101679) was added at a concentration of 50,000 U/L. The Flexboy bag was thoroughly rocked to mix and then incubated for 60 minutes at 37°C and 5% CO2, with rocking at 10-minute intervals. The Benzonase-treated pool was divided into 500ml conical tubes and centrifuged at 3000 x g for 15 minutes at 18°C to remove large cell debris. Supernatant containing the DDVax, now the clarified pool, was pumped out of the centrifuge bottles into a new 5L Flexboy bag. Virus was then concentrated by a factor of 6 using ultrafiltration (UF) using the KMPi TFF system (Repligen) over a 500kD Hollow Fiber Membrane (Repligen, S02-E500-05-N, 500kD, 20cm length, 0.5mm fiber diameter). Concentration was performed at a target shear rate of 3000 s-1 and TMP setpoint of 5 psi. The concentrated UF pool was diafiltered (DF) into a buffer containing 0.2M NaCl, 10mM Sodium Phosphate, 4% Sucrose, 5mM Glutamax, pH 7.4 ± 0.1. Buffer exchange was performed for 10 diavolumes. Diafiltration was performed at a target shear rate of 3000 s-1

2.2 DDVax sequencing and analysis

Illumina shotgun sequencing libraries were prepared from total RNA using the Kapa RNA HyperPrep kit following the manufacturer’s protocol. Dual indexed libraries were sequenced on an Illumina NextSeq 500 sequencer to generate single-end 150 nt reads.
We used two complementary approaches to detect and quantify viral variants. First, we used the lofreq tool to identify single nucleotide variants and short insertions and deletions (Wilm et al., 2012). Second, we used DI-tector to identify structural variants including longer deletions and insertions and copy back defective viral genomes (DVGs)(Beauclair et al., 2018; Vignuzzi & Lopez, 2019). These tools were run as part of a reproducible Nextflow pipeline, available at https://github.com/stenglein-lab/viral_variant_caller/releases/tag/DDVax_paper_release. Software dependencies and reference sequences (DDVax) are captured in this version-controlled release and in the conda environment contained therein.
To quantify variants, adapter-derived and low-quality bases were trimmed using Cutadapt(Martin, 2011). Host cell-derived reads were removed using bowtie2 to align reads to the Chlorocebus sabeus genome, accession GCF_000409795.2(Langmead & Salzberg, 2012). Host- and quality-filtered reads were aligned to the S, M, and L segment RVFV/DDVax reference sequences using the BWA aligner(Langmead & Salzberg, 2012; Li & Durbin, 2009). The reference sequences consisted of the RVFV-derived portions of the DDVax plasmid sequences. To improve accuracy of structural variant (indel) calls, base quality scores were recalibrated using GATK (McKenna et al., 2010). Single nucleotide variants (SNVs) and structural variants were called using LoFreq(Wilm et al., 2012). The minimum depth of coverage to call a variant was set at 40x coverage. SnpEff and SnpSift were used to predict the functional impact of variants(Cingolani, Patel, et al., 2012; Cingolani, Platts, et al., 2012). Variant calling distinguished between variants that were not detected despite sufficient data and positions that lacked sufficient data to call variants. Defective viral genomes were identified using the DI-tector tool(Beauclair et al., 2018). Outputs of these analyses were tabulated, processed, and visualized in R. Variants with frequencies ≥ 3% were reported(Grubaugh et al., 2019).

2.3 Virus strains

Stocks of DDVax were produced as described above. The passage history of MP-12 strain is unknown, but DQ375404.1 (L segment), DQ380208.1 (M segment) and DQ380154.1 (S segment) sequences were confirmed by Sanger sequencing. ZH501 strain virus was obtained from R. Bowen. V1 (Vero) passage stock was passaged twice in Vero cells to obtain V3 stocks used for this study.