Cell Line Development by Systems Biology


Within an interdisciplinary project which combines the expertise of two industrial and five academic partners it is our aim to provide an innovative and highly productive cell culture-based influenza vaccine manufacturing platform. Therefore, we want to optimize influenza virus replication in well-defined human designer cells supplied by ProBioGen AG (Berlin) by manipulating the expression level of cellular factors involved in virus-host cell interactions. Based on results obtained by a genome-wide RNAi screen [1], different cell lines with specific genetic manipulations are being produced at the Helmholtz Centre for Infection Research (HZI, Braunschweig) and at the Max Planck Institute for Infection Biology (MPIIB, Berlin). All modified cell lines that show an improved virus production will be analyzed in more detail to understand underlying mechanisms. In addition, our cooperation partners at the Otto von Guericke University (OvGU, Magdeburg) are developing mathematical models on the single cell and on the cell population scale to quantitatively assess the impact of cell line modifications on virus yields in bioreactors. After validation and refinement of these models by experimental approaches, they shall enable us to develop strategies for the rational design of high-yield vaccine manufacturing processes.

Aim of the project

The main focus of this subproject is the thorough characterization of influenza virus propagation in genetically manipulated cell lines. Therefore, we will use a broad panel of recent influenza vaccine strains and state-of-the-art analytical tools, i.e. real-time RT-qPCR and imaging flow cytometry, to study how genetic modifications affect virus replication. Moreover, we analyze the impact of the innate immune response of human cells on viral titers, and we optimize process parameters such as cell density at time of infection, multiplicity of infection, and harvest time to improve the process yields. Combining these findings we hope to establish a novel high-productive influenza vaccine manufacturing platform based on genetically engineered human cells.

Figure 1: Influenza virus life cycle and potential host cell interaction partners. 
During the first step of influenza virus infection, the viral surface glycoprotein HA binds to host cell surface receptors and gets internalized through endocytosis. The low pH in late endosomes triggers a conformational change in the HA protein, leading to the fusion of the viral envelope and endosomal membranes, thereby releasing the viral ribonucleoprotein complexes (vRNPs) into the cytoplasm. vRNPs are subsequently transported into the nucleus, where mRNA transcription and vRNA replication occur. In the next step viral pre-mRNAs become spliced by host cell factors and translocated back into the cytoplasm for translation. After post-translational modifications of certain viral proteins, the structural components of the viral particle are transported to the plasma membrane where assembly and budding take place. To identify cellular key players in influenza virus replication, several siRNA screens where performed. The validations of some identified gene candidates could already confirm the strong dependency of influenza virus on host cell factors during most steps of the viral life cycle [2].


[1] Karlas, A., Machuy, N., Shin, Y., Pleissner, K. P., Artarini, A., Heuer, D., Becker, D., Khalil, H., Ogilvie, L. A., Hess, S., Maurer, A. P., Muller, E., Wolff, T., Rudel, T., Meyer, T. F. (2010). "Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication." Nature 463(7282): 818-822.

[2] Watanabe, T., Watanabe, S., Kawaoka, Y. (2010). "Cellular networks involved in the influenza virus life cycle." Cell Host Microbe 7(6): 427-439.

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