Epidemic outbreaks of yellow fever (YF) in the forests and savannas of South America and Africa cause mortality rates between 20 % and 50 % and, thus, 29,000 - 60,000 deaths per year. Although there is an active immunization vaccine available on the market, a way to treat infected patients does not exist. In order to respond quickly to YF epidemics, a passive immunization approach based on virus-like particles (VLPs) is a promising opportunity.<br/>We develop a transient YF-VLP production strategy on a bioreactor scale using HEK as host cells. Therefore, the impact of different cultivation media and the transient transfection efficiency of the HEK 293T was investigated in order to produce high yields of YF-VLPs in adherent and suspension cultures. We used the cationic polymer polyethylenimine (PEI), as it is cost-effective, especially with regard to a large-scale production. Different plasmid DNA/PEI-ratios were investigated considering pDNA/PEI polyplex-size, as too large pDNA/PEI polyplexes may have a cytotoxic effect on the cells. Using DNA fluorescence staining, the attachment and internalization kinetics in cells of the pDNA/PEI polyplex were determined to follow the transfection until pDNA enters the nucleus. The production and the secretion kinetics of YF-VLPs will be analyzed by the fluorescently active eGFP. Furthermore, the size and morphology of the YF-VLPs were analyzed using Transmission Electron Microscopy. A specific detection of the YF-VLP proteins was carried out using Western Blot analysis.<br/>In order to produce YF-VLPs on a large scale, further studies are needed with regard to the YF-VLP-stability, e.g. temperature, pH value or the salt concentration in the medium. Furthermore, the influence of hydrodynamics on the YF-VLP quality and quantity in the stirred reactor will be investigated. We think that transient YF-VLP production is a promising way to rapidly produce the required boost for a passive immunization approach against YF.

Transient transfection of HEK 293T cells to produce yellow fever VLPs

2019 Spring National Meeting

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