Manufacturing Process Study Of HIV-1 DNA Vaccine And Construction, Expression And Purification Of PTD-P24 Fusion Protein

DNA vaccine, with huge promising market potential, is a new generation of vaccine following the traditional vaccine and subunit vaccine. The large scale manufacturing process of plasmid DNA is of significant importance for the industrialization of DNA vaccine. In the first section of this thesis, we aimed to establish the pilot-scale manufacturing process of HIV-1 DNA vaccine based on the demand of national scientific research project and the prospect of biopharmaceutical industry. This task was successfully finished with the cooperation of people from the upstream and downstream teams. Based on a lab-scale process, the DNA vaccine manufacture process for clinical-grade plasmid was scaled up under GMP conditions. The fed-batch fermentation process of 75L fermentor was established. After 20 hours fermentation, the yield of E. coli cells reached 60g/L (wet weight) and plasmid yield was above 60mg/L. High yield of plasmid was achieved at relatively high cell density. By combining several purification strategies and optimizing the parameters, we then established a robust, simple, and easy-to-scale-up purification process of DNA vaccine. Most impurities in plasmid solution were removed by LiCl precipitation and spermine precipitation. These initial purification steps are of high-capacity with low cost. The money and time consuming chromatography process steps could be simplified based on the high efficiency of these primary purification steps. Whole purification flow-chart was successfully simplified. Stratagy of enhancing primary purification and simplify higher purification was involved in the process development of DNA vaccine. The quality of DNA vaccine reaches the specifications for clinical application. Total yield of plasmid was above 40%. When compared with previously described processes, the process reported here contains less high purification steps, therefore shortens purification period and lowers the cost. In addition, we established a complete product quality monitor system to monitor the stability of the manufacture process and the product quality. The quality of the final product has passed the quality testing by the National Institute for the Control of Pharmaceutical and Biological Products and been approved to be applied in the AIDS vaccine clinical trial.The major disadvantage of subunit protein vaccine is the inefficiency in eliciting antigen-specific cellular immune responses. In the second section of this thesis, we aimed to enhance the cellular immune responses initiated by protein vaccine via directly introducing the protein antigen into host cells through protein transduction domain (PTD). The cDNA coding PTD-P24 fusion protein was generated by fusing the PTD sequence from HIV Tat protein to the upstream of P24 gene of HIV-1 CN54 through PCR. Plasmid pThioHisA-PTD-P24 was constructed by inserting PTD-P24 coding sequence into the pThioHisA vector between the Nde I and EcoR I sites. Plasmid pThioHisA-PTD-P24 could also serve as a gene modification vector through replacing the P24 gene with other gene of interest by BamH I and EcoR I subcloning. High level of PTD-P24 expression was achieved, with the recombinant protein representing about 45% of the total bacterial protein. The recombinant protein is soluble after optimizing the expression conditions. Western blotting assay revealed that the recombinant PTD-P24 has excellent antigenicity. PTD-P24 fusion protein was purified through weak cation-exchange chromatography. The purity of PTD-P24 reached above 95%. The purification process is simple and easy-to-scale up. The construction, expression and purification of PTD-P24 have established a solid foundation for the preclinical study and pilot-scale production.