Entwicklung eines Prozesses zur Herstellung rekombinanter virusähnlicher Partikel als Vakzinkandidat gegen das Denguefieber

authored by: Janet Hirsch
supervised by: Thomas Scheper
Abstract: Dengue fever control is a global challenge as almost half of the world's population is at risk of a dengue infection and morbidity is increasing steadily. Dengue fever is a viral infectious disease that causes severe pain and can be lethal. The main challenge in dengue vaccine develop-ment is the coexistence of four dengue virus serotypes, which need to be covered all at the same time. The presented project on the development of a vaccine candidate aims the combination of different dengue envelope proteins in virus-like particles (VLPs). An envelope protein of dengue virus serotype 1 was expressed in Escherichia coli and accumulated in inclusion bodies (IBs). After cell disruption by sonication IBs were pelleted by centrifugation and solubilized using guanidine. Chromatographical purification was performed with a Ni2+-NTA column resulting in a target protein purity of 90-99 %. During subsequent dialysis the protein renatured and self-assembled VLPs, the average diameter of which was dependent on renaturation conditions. In carbonate buffer at pH 10 VLPs were observed to be spherical particles with an average diameter of 50 nm using both an atomic force microscope and a fluorescence microscope. Although their appearance and diameter equalled the original dengue virus, VLPs were not capable of eliciting neutralizing antibodies and did therefore not provide immunological funcionality. VLPs were stable during storage in carbonate buffer for at least one month, whereas VLPs in ammonia buffer tended to aggregate gradually. L-Arginine was found to hinder aggregation effects during storage. A temperature increase led to aggregation of VLPs in carbonate buffer at 50-55 °C, hence VLP stability was comparable with natural virus stability. A second target protein with few amino acid sequence changes could not be produced successfully with the same procedure. This observation is probably caused by molecular biological characteristics of the producing E. coli strain. To verify the combination of two different envelope proteins in the same VLP (bivalency) a fluorescent microscopical analysis was developed. However, by now there was no bivalency detected with this method. The production of further viral envelope proteins and their combination into VLPs are currently under development. This work contributes to the development of a vaccine candidate and hence supports the global dengue fever control efforts.
Organisation(s): Institute of Technical Chemistry
Type: Doctoral thesis
No. of pages: 106
Publication date: 2019
Publication status: Published
Sustainable Development Goals: SDG 3 - Good Health and Well-being
Electronic version(s): https://doi.org/10.15488/5586 (Access: Open)