Continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles

Abstract

Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.

Figure 1. Overview of two-stage bioreactor setup for continuous virus propagation.

AGE1.CR cells were cultivated in two bioreactors. At time of infection, the influenza strain A/Puerto Rico/8/34 was added to the virus bioreactor at a multiplicity of infection of 0.025. Subsequently, the cell concentration in the cell bioreactor was kept at approx. 4–5×10⁶ cells/mL and cells were constantly fed into the virus bioreactor (feeding rates are depicted). Trypsin was added either to the feed or directly into the virus bioreactor. All green components correspond to the cell bioreactor, all red components to the virus bioreactor. Both reactors are connected via the purple tubing.

Figure 2. Continuous propagation of influenza A virus.

(A) Concentrations of AGE1.CR cells in the cell and virus bioreactor. (B) Virus titers determined by HA and TCID₅₀ assay. (C) MOI in the virus bioreactor based on the ratio of TCID₅₀ to cell count at each sampling time point. Results of two independent cultivations are shown. During the first cultivation additional trypsin (+T), seed virus (+V) or both were added to the virus bioreactor at indicated time points as an attempt to counteract decreasing virus titers.

Figure 3. Segment-specific PCR for the detection of full-length and defective interfering genome segments.

Using eight primer pairs directed against the 5′ and 3′-end of each influenza virus genome segment, full-length (FL) as well as defective interfering (DI) RNAs (smaller products in the range between 500 and 700 bp) were amplified. (A) Segment-specific PCR for all eight segments of the used virus stock and two samples of the second continuous cultivation. (B) Time course of the three polymerase segments 1–3 (encoding PB2, PB1 and PA, respectively) and segment 5 encoding the nucleoprotein (NP) from the second continuous virus propagation in which samples were taken every 12 hours. The size (in bp) of important marker bands is shown.

Figure 4. Model of continuous infection in the absence of DIPs.

(A) Schematic representation of the model for continuous influenza A virus infection in the absence of DIPs (see Eq. (2)). The continuous harvest of cells and viruses was omitted for illustrative reasons. (B, C) Simulated virus titers for a dilution rate of the virus reactor D which is (B) lower than the specific growth rate µ and (C) higher than the specific growth rate µ. Parameters were chosen according to Table S1 except that the dilution rate in (B) was reduced to D = 10⁻⁸ 1/h.

Figure 5. Model of continuous infection in the presence of DIPs.

(A) Schematic representation of the model for continuous influenza A virus infection in the presence of DIPs (see Eq. (1)). Dashed arrows indicate apoptosis or virus degradation. The continuous harvest of cells and viruses was omitted for illustrative reasons. (B) Simulated virus titers for the parameters used in Table S1. (C, D) Log₁₀ HA units/100 µL over process time for (C) various ratios of initial DIPs (V_d0) to STVs (V_s0) neglecting de novo DIP generation (f = 0) and (D) different rates of de novo DIP generation by STV-infected cells (f denoting the fraction of DIP to STV production) without DIPs being initially present (V_d0 = 0).