Productivity, apoptosis, and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants

In cell culture-based influenza vaccine production significant efforts are directed towards virus seed optimization for maximum yields. Typically, high growth reassortants (HGR) containing backbones of six gene segments of e.g. influenza A/PR/8, are generated from wild type strains. Often, however, HA and TCID₅₀ titres obtained do not meet expectations and further optimization measures are required.     

Glycan analysis in cell culture-based influenza vaccine production: Influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin

Mammalian cell culture processes are commonly used for production of recombinant glycoproteins, antibodies and viral vaccines. Since several years there is an increasing interest in cell culture-based influenza vaccine production to overcome limitations of egg-based production systems, to improve vaccine supply and to increase flexibility in vaccine manufacturing. With the switch of the production system several key questions concerning the possible impact of host cell lines on antigen quality, passage-dependent selection of certain viral phenotypes or changes in hemagglutinin (HA) conformation have to be addressed to guarantee safety and efficiency of vaccines. In contrast to the production of recombinant glycoproteins, comparatively little is known regarding glycosylation of HA, derived from mammalian cell cultures. Within this study, a capillary DNA-sequencer (based on CGE-LIF technology), was utilized for N-glycan analysis of three different influenza virus strains, which were replicated in six different cell lines. Detailed results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Strong host cell but also virus type and subtype dependence of HA N-glycosylation was found. Clear differences were already observed, by N-glycan fingerprint comparison. Further structural investigations of the N-glycan pools revealed that host cell dependence of HA N-glycosylation was mainly related to minor variations of the (monomeric) constitution of single N-glycans. To some extent, shifts in the N-glycan pool composition regarding the proportion of different N-glycan types were observed. In contrast to this, a principal switch of the N-glycan type attached to HA was observed when comparing different virus types (A and B) and subtypes (H1N1 and H3N2).     

Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻⁵, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log₁₀ HAU/100 μL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log₁₀ HAU/100 μL and TCID₅₀ of 1 × 10⁹ and 2.37 × 10⁹ infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.