Optimization of Vero Cells Grown on a Polymer Fiber Carrier in a Disposable Bioreactor for Inactivated Coxsackievirus A16 Vaccine Development

Figures

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  Figure 1. Vero cell culture in a micro bioreactor and optimization of the virus yield. (A) The total cell count in a container (volume = 30 mL;) and the GCR (black square; grams of glucose consumed every 24 h per liter) in the culture container of the micro bioreactor. (B) GCR in the bioreactor using inocula at different cell densities (0.5 × 107 cells (black squares), 1.0 × 107 cells (white squares), 1.5 × 107 cells (black triangles), and 2.0 × 107 cells (white triangles). (C) Viral production (yield) at different MOIs. MOI = 0.01 (white triangle), 0.1 (black square), 0.5 (black triangle), and 1.0 (white square). (D) Viral production under different temperatures (31, 32, 33, 34, and 35 °C) in the mini bioreactor. (E) Plaque purification of clonal CVA16. (F) CPE of CVA16-infected Vero cells at 68 hpi; magnification: 200. (G) Immunofluorescence staining of CVA16-infected Vero cells at 68 hpi; magnification: 200. (H) Transmission electron microscopy of CVA16. Scale bar represents 50 nm. GCR, glucose consumption rate; MOI, multiplicity of infection; CVA16, Coxsackie virus A16; CPE, cytopathic effect; hpi, hours post-infection.

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  Figure 2. CVA16 virus production using different culture strategies and medium supplements. The virus titer of the supernatant was collected after growth using five different medium addition strategies: (A) 48 hpi, (B) 72 hpi, (C) 96 hpi, and (D) 120 hpi (group G1–G5). CVA16, Coxsackie virus A16; hpi, hours post-infection.

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  Figure 3. Culture in the NBS-Bioflo310 and ACPB bioreactors. (A) Distribution of cells in a 5 L perfusion column (5-L) containing 150 g of polymer fiber carriers in ACPB-0. (B) The viral titer and GCR in ACPB-1 (n = 3, 3 replicates in one batch) utilizing the G1 strategy. (C) The viral titer and GCR in ACPB-2, 3, and 4 (n = 9, 3 replicates × 3 batches) utilizing the G5 (5% LH) strategy. (D) The viral titer and GCR in NBS-1 (n = 3, 3 replicates in one batch) in the NBS-Bioflo310 system utilizing the G5 (5% LH) strategy. NBS-Bioflo310, New Brunswick Scientific Bioflo310 bioreactor; ACPB, AmProtein Current Perfusion Bioreactor; GCR, glucose consumption rate; LH, lactalbumin hydrolysate.

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  Figure 4. Total yield of virus in the cell lysates (intracellular) and supernatants (extracellular) from the ACPB and Bioflo310 systems using the G1 and G5 strategies. NBS-Bioflo310, New Brunswick Scientific Bioflo310 bioreactor; ACPB, AmProtein Current Perfusion Bioreactor. The determination of the residual BSA of the harvested supernatant from the ACPB and NBS-Bioflo310 bioreactors showed a substantial decrease when using the G5 (0.5% LH) strategy compared with using the G1 strategy (Table 1).

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  Figure 5. Neutralizing antibodies produced by mice immunized using the inactivated CVA16 vaccine. Titers of neutralizing antibodies (GMTs and 95% CI) in serum samples collected from mice immunized with the inactivated CVA16 vaccine or aluminum as a control (n = 8) at 14 days after the first immunization. The neutralization test was based on the highest dilution of the serum that stopped cells from undergoing a cytopathic effect. Data are presented as means, n = 8, * p < 0.05 compared with the ACPB-1/G1. CVA16, Coxsackie virus A16; GMT, geometric mean titer; CI; confidence interval.