Almeida Ana M, Tomás Joana, Pereira Patrícia, Queiroz João A, Sousa Fani, Sousa Ângela
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, Covilhã, 6200-506, Portugal.
Biotechnol Prog. 2018 Mar;34(2):546-551. doi: 10.1002/btpr.2603. Epub 2018 Jan 19.
DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV-16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:546-551, 2018.
在过去几十年中，DNA疫苗作为预防多种传染病的替代方法而出现，但它们也可用于治疗特定疾病，如由人乳头瘤病毒（HPV）引起的宫颈癌。这种病毒产生E6和E7癌蛋白，它们会改变细胞周期调控并可能干扰DNA修复系统。这些特征最终可导致HPV感染细胞后宫颈癌的进展。因此，开发针对这两种蛋白的DNA疫苗成为治疗这种疾病的一个有趣选择。尽管如此，在评估其治疗潜力之前，生物制药的纯度水平必须符合监管机构的规范。此前，我们的研究小组通过亲和色谱法成功纯化了重组HPV-16 E6/E7 DNA疫苗的超螺旋异构体，纯度接近100%。本研究旨在评估pDNA样品纯度水平对靶蛋白表达可能产生的影响。因此，进行了体外研究，以评估疫苗产生靶蛋白的能力，并比较通过亲和色谱法获得的pDNA样品（仅呈现超螺旋异构体且符合监管机构建议）与通过商业纯化试剂盒回收的相同DNA疫苗（包含不同的pDNA异构体）之间的表达效率。我们的研究结果表明，通过亲和色谱法纯化的E6/E7 DNA疫苗比用商业试剂盒纯化的DNA疫苗能促进更高水平的E6和E7 mRNA及蛋白表达。总体而言，这些结果强调了纯化策略在重组DNA疫苗治疗效果中的重要性，设想它们作为生物制药的进一步应用。© 2018美国化学工程师学会生物技术进展，34:546-551，2018。
