Department of Infectious Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
J Transl Med. 2011 May 18;9:69. doi: 10.1186/1479-5876-9-69.
The HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated.
E7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months.
In western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant.
This report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines.
HPV16 E7 蛋白既是肿瘤特异性抗原，也是肿瘤排斥抗原，是开发用于治疗 HPV16 相关癌症及其前体病变的治疗性疫苗的理想靶点。E7 在病毒相关性致癌作用中起关键作用，包含 98 个氨基酸，具有两个手指型结构，可结合一个 Zn++离子。评估了组装成颗粒的大肠杆菌产生的 E7 制剂在 TC-1-C57BL/6 小鼠肿瘤模型中针对 HPV16 相关肿瘤的诱导保护性免疫的能力。
E7 在大肠杆菌中表达，通过一步变性方案纯化，并在天然缓冲液中透析后制成可溶性悬浮状态。通过非还原 SDS-PAGE 和负染色电子显微镜（EM）分析 E7 制剂中是否存在颗粒形式。通过质谱分析 Zn++离子含量。每只小鼠给予 10μg 蛋白，无佐剂一次、两次或三次。使用基于 E7 的 ELISA 在小鼠血清中监测 E7 特异性体液反应，并用淋巴细胞增殖和 IFN-γ ELISPOT 测定分析小鼠脾细胞中的细胞介导免疫反应。用 TC-1 肿瘤细胞对 E7 免疫的小鼠进行攻毒，并监测两个月的肿瘤生长情况。
在 Western blot 分析中，E7 以多聚体和高分子质量寡聚体的形式出现。EM 显微照片显示该蛋白呈不同形状和大小的聚集体分散。该蛋白以微聚集体、纳米聚集体和结构颗粒的形式聚集。用该蛋白制剂免疫的小鼠表现出明显的 E7 特异性体液和细胞介导免疫反应，为混合 Th1/Th2 型。在没有任何添加佐剂的情况下，用三剂 10μg 的 E7 疫苗接种，可完全保护小鼠免受肿瘤生长。
本报告表明，HPV16 E7 的颗粒形式无需佐剂即可在 C57BL/6 小鼠中诱导 E7 特异性肿瘤保护。保护性免疫由体液和细胞介导免疫反应共同维持。在体外组装成微纳米颗粒的大肠杆菌衍生的 HPV16 E7 不仅是抗原呈递细胞摄取和处理的良好底物，而且是生产新一代 HPV 亚单位疫苗的具有成本效益的手段。
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