Cell Line Engineering for Influenza Vaccine Production

Motivation

Influenza A virus (IAV) spreads worldwide causing several million infections per year and vaccination is the most effective measure to prevent or control disease outbreaks. Seasonal and pandemic influenza outbreaks require large amounts of vaccine doses to be produced in a timely manner. In this regard, animal cell cultures provide one suitable platform for vaccine manufacturing at large-scale. Moreover, cell culture-based manufacturing systems allow the increase in vaccine yields through different optimization strategies [1]. An option to enhance the final process yields is to improve the host cell capacity to replicate the virus. As such, designed cell lines offer higher cell-specific yields, and their use can lead to remarkably high volumetric yields when combined with appropriate culture media or advanced cultivation strategies.

Aim of the project

In this project, we engineer host cell lines to increase IAV replication for high-yield vaccine production. For this, we have selected cellular genes that were previously identified as regulators of IAV replication in different genome-wide screenings or individual studies [2-6]. Additionally, based on the bioinformatics analysis, we are investigating the use of specific miRNAs to target molecular pathways involved in the virus replication. Since the genes, as well as molecular pathways differ between the species, and not every genome is known, we work with various cell lines (HEK 293T, MDCK, VERO) aiming not only to increase the influenza virus vaccine production, but also to understand the biology of influenza virus life cycle and infection.

In order to engineer mammalian cells to transiently or stably express genes of interests or specific miRNAs, we use molecular biological and virological tools, such as PCR, cloning, transfection and transduction with lentiviral systems. After quality control to evaluate the stability of constructs, expression of proteins, and catalytic activity of enzymes, the cells can be used for detailed virus infection studies. Every step of virus life cycle can be checked, using the attachment and entry assays with virus surrogate systems, fusion assays, real time qPCR, fluorescence microscopy, and imaging flow cytometry as well as basic virology methods. Engineered cell lines showing increased virus yields or accelerated viral replication are selected to analyze the mechanisms that favor virus replication, and to determine their potential for designing host cells for improved influenza vaccine production.

Figure 1: Scheme of cell line engineering. Human HEK293 cells are genetically modified using lentiviral vectors which integrate into the genome of the cell to achieve a stable expression of short hairpin RNAs (shRNAs). The shRNA is processed by the cellular enzyme Dicer and subsequently integrated into the RISC complex in which single-stranded RNAs lead to the binding and degradation of complementary mRNAs. After the knockdown of the expression of cellular genes, the genetically modified cells are infected with influenza A virus and virus titers are determined by hemagglutination (HA) assay. — **Figure 1: Scheme of cell line engineering.** Human HEK293 cells are genetically modified using lentiviral vectors which integrate into the genome of the cell to achieve a stable expression of short hairpin RNAs (shRNAs). The shRNA is processed by the cellular enzyme Dicer and subsequently integrated into the RISC complex in which single-stranded RNAs lead to the binding and degradation of complementary mRNAs. After the knockdown of the expression of cellular genes, the genetically modified cells are infected with influenza A virus and virus titers are determined by hemagglutination (HA) assay.

**Figure 1: Scheme of cell line engineering.** Human HEK293 cells are genetically modified using lentiviral vectors which integrate into the genome of the cell to achieve a stable expression of short hairpin RNAs (shRNAs). The shRNA is processed by the cellular enzyme Dicer and subsequently integrated into the RISC complex in which single-stranded RNAs lead to the binding and degradation of complementary mRNAs. After the knockdown of the expression of cellular genes, the genetically modified cells are infected with influenza A virus and virus titers are determined by hemagglutination (HA) assay.

References

Gallo Ramirez, L. E.; Nikolay, A.; Genzel, Y.; Reichl, U.: Bioreactor concepts for cell culture-based viral vaccine production. Expert Review of Vaccines 14 (9), pp. 1181 - 1191 (2015)

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DOI

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[2] Karlas A, Machuy N, Shin Y et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature, 463(7282), 818-822 (2010).

[3] Konig R, Stertz S, Zhou Y et al. Human host factors required for influenza virus replication. Nature, 463(7282), 813-817 (2010).

[4] Shapira SD, Gat-Viks I, Shum BO et al. A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell, 139(7), 1255-1267 (2009).

[5] Wang P, Song W, Mok BW et al. Nuclear factor 90 negatively regulates influenza virus replication by interacting with viral nucleoprotein. J Virol, 83(16), 7850-7861 (2009).

[6] Wang S, Li H, Chen Y et al. Transport of influenza virus neuraminidase (NA) to host cell surface is regulated by ARHGAP21 and Cdc42 proteins. The Journal of biological chemistry, 287(13), 9804-9816 (2012).