Obtaining a plasmid DNA vaccine against HPV

• Autor(a) principal: Figueiredo, Vanessa Andrade
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6000
• topic: Cromatografia de Afinidade; Isoforma Superenrolada; Monolito de Arginina; Plasmídeo Hpv16 E6/E7mut; Transfeção; Vacinas de Dna; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

The Human papillomavirus (HPV) infection is one of the most common sexually transmitted virus and is responsible for various human epithelial lesions, including cervical cancer. The initial step for cancer progression consists in immortalization of normal epithelial cells. The carcinogenicity of HPV16 infections is associated with E6 and E7 proteins, which deregulate the host cell cycle by inhibiting tumor suppressor protein (p53) and retinoblastoma protein (pRb), respectively. DNA vaccines induce an immune response which allows the prevention or treatment of infections by viruses, bacteria and parasites. One of the best strategies used for this purpose is the plasmid DNA (pDNA). Our research group has been studying a way to effectively develop the production and purification of sc pDNA vaccine HPV16 E6/E7 through the use of a monolith modified with arginine ligands. However, it is necessary to ensure that these proteins are unable to induce cell immortalization. Thus, the E6 and E7 protein oncogenic potential was removed by introducing point mutations, which inhibit interactions of these proteins with p53 and pRb. Afterwards, affinity chromatography was used to purify sc HPV16 E6/E7MUT pDNA, taking advantage of a purification strategy with arginine monolith, previously explored by our research group for the non-mutated plasmid. A purity degree > 99% and a recovery yield of 82% was achieved, which were comprised within the values obtained in our group. After optimization of the purification strategy, impurities such as genomic DNA (gDNA), RNA, endotoxin and proteins were quantified. A significant decrease in impurities of purified sc pDNA sample was observed when compared to initial lysate sample. Finally, in vitro transfection studies were performed to evaluate the fibroblast cell transfection efficiency and the subsequent expression of the target proteins, encoded by the genes contained in the pDNA vaccine. These results allowed us to conclude that the transfection process was successful, leading to increased E6 and E7 protein expression compared to non-transfected cells.