Vaccine production: upstream processing with adherent or suspension cell lines.

The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered.

Impact of cultivation conditions on N-glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture.

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor-binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N-glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) based glycoanalysis (N-glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N-glycosylation pattern of Madin-Darby canine kidney (MDCK) cell-derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum-free growth has only a minor impact on the HA N-glycosylation pattern. Only relative abundances of N-glycan structures are affected. In contrast, host cell adaptation to serum-free suspension growth resulted in significant changes in the HA N-glycosylation pattern regarding the presence of specific N-glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of ß-propiolactone inactivation resulted-at best-only in minor changes in the relative N-glycan structure abundances of the HA N-glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N-glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum-free suspension growth significantly influenced HA N-glycosylation regarding both, the type of attached glycan structures as well as their abundances.

CAP, a new human suspension cell line for influenza virus production.

Forced by major drawbacks of egg-based influenza virus production, several studies focused on the establishment and optimization of cell-based production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high virus titers. From primary virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza virus production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log(10) HA units/100 µL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log(10) HA units/100 µL and maximum cell-specific virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza virus vaccine production.

Toward animal cell culture-based influenza vaccine design: viral hemagglutinin N-glycosylation markedly impacts immunogenicity.

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin-Darby canine kidney cell-derived and Vero cell-derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c(+) dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line-based influenza vaccine design.

Comparison of the human immunodeficiency virus (HIV) type 1-specific immunoglobulin G capture enzyme-linked immunosorbent assay and the avidity index method for identification of recent HIV infections.

The sensitivity and specificity of the human immunodeficiency virus (HIV) type 1-specific immunoglobulin G capture enzyme-linked immunosorbent assay (BED-CEIA) were compared with those of the avidity index method to identify recent HIV infection using a panel of 148 samples (81 patients) representing durations of infection ranging from 0 to 222 weeks. The results from the two tests were similar (sensitivity of 80% versus 74% [P = 0.53]; specificity of 86% versus 82% [P = 0.67]).