Production of antiviral “OP7 chimera” defective interfering particles free of infectious virus

Figures

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  Fig. 1. Scheme of OP7 chimera DIPs and Seg 1 cDIPs. Reverse genetics was used to generate a mixture of a) OP7 chimera DIPs and b) Seg 1 cDIPs. Both together replicate without addition of infectious standard virus as the missing PB2 is provided by MDCK-PB2(sus) cells. Figure adapted from (Dogra et al. 2023). Created with BioRender.com

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  Fig. 2. Batch mode production of OP7 chimera DIPs in shake flasks with MD and temperature decrease to 32 °C. MDCK-PB2(sus) cells, cultivated in 250 mL shake flasks (100 mL VW) at 37 °C, were grown to about 4.0 × 106 cells/mL. Subsequently, the suspension culture was diluted (MD, 1:2) with fresh medium (100 mL VW), cells were infected at a MOI of 10–4 and temperature was reduced to 32 °C. For comparison, two cultivations were performed at 37 °C, one with MD at an optimal MOI of 10–3, one with CME at an optimal MOI of 10–4. a) VCC. b) HA titer. c) Fraction of OP7 chimera DIPs, calculated using the extracellular Seg 7-OP7 and Seg 7-WT vRNA concentrations, quantified by real-time RT-qPCR. a-c depict the results of one experiment. d) Interference assay. WT MDCK(adh) cells were infected with STV PR8 at a MOI of 10 (negative control, NC) or co-infected with 125 μL of indicated OP7 chimera DIP material. Infectious virus particle release is indicated by the plaque titer and total virus particle release by HA titer (16 hpi). Interference assay was performed in three independent experiments. Error bars indicate standard deviation (SD). The optimal harvest time point (37 °C CME: 44 hpi, 37 °C MD: 52…

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  Fig. 3. OP7 chimera DIP production in batch mode in a 1 L STR versus shake flask production and interfering efficacy of produced material after purification by SXC. MDCK-PB2(sus) cells were grown in a 1 L STR (400 mL VW, 37 °C). After growth to about 4.0 × 106 cells/mL, cells were diluted (1:2) with fresh medium (approx. 700 mL final VW), temperature was set to 32 °C, pH was set to 7.4, and cells were infected at a MOI of 10–4. In total, three independent productions were carried out in STR (STR 1–3) and compared to two independent productions in shake flasks (SF 1–2, 100 mL VW) for comparison (also shown in Fig. 2 and Fig. S2). a) VCC. b) HA titer. c) Fraction of OP7 chimera DIPs (see Fig. 1). d) Interference assay with MDCK(adh) cells was performed in seven independent experiments for NC, and three experiments for STRs and SFs. e) Interference assay with MDCK(adh) cells of purified (SXC) vs non-purified material. Dilutions of the tested materials are indicated. Interference assay was performed in three independent experiments. Error bars indicate the SD. The optimal harvest time point (STR 1: 52 hpi, STR 2: 46 hpi, STR 3: 58 hpi, SF 1: 44 hpi, SF 2: 44 hpi) was analyzed for c, d and e

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  Fig. 4. Interfering efficacy in human alveolar epithelial Calu-3 cells of OP7 chimera DIP material purified by SXC. Calu-3 cells were infected with STV PR8 alone at a MOI of 0.05 (NC) or co-infected with 125 μL of indicated OP7 chimera DIP material. a) Infectious virus particle release, shown by the plaque titer (24 hpi). b) Fold change in IFN-β gene expression, quantified by real-time RT-qPCR. Results of six independent experiments for NC, or three experiments for the co-infection are shown. Material produced in shake flasks at 37 °C with CME (37 °C CME, Fig. 2) was compared to material produced in a STR at 32 °C with MD and further SXC purification (32 °C MD Downstream Processing (DSP), Fig. 3e). Error bars indicate the SD

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  Fig. 5. Production of OP7 chimera DIPs in a 1 L STR (700 mL VW) in perfusion mode. Inoculation of MDCK-PB2(sus) cells at 1.2 × 106 cells/mL, perfusion start after 24 h of batch mode by using an alternating tangential flow filtration system (ATF2) with a hollow fiber membrane (0.2 μm pore size). Perfusion rate during cell growth controlled by capacitance probe measuring of VCC. Before infection at a MOI of 10–4, one RV was exchanged with fresh medium and temperature was reduced to 32 °C. a) VCC. b) Perfusion rate. c) Glucose, lactate, glutamine and ammonium concentration. d) HA titer. e) Fraction of OP7 chimera DIPs. a-e depict the results of one experiment. The optimal harvest time point (45 hpi) was analyzed for e and f. f) Interference assay with MDCK(adh) cells. For comparison, material produced in batch mode using medium dilution (MD) (STR 1) is shown (Fig. 3). Interference assay was performed in three independent experiments. Error bars indicate the SD