ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Casaccia Research Center, Rome, Italy.
PLoS One. 2013 Apr 23;8(4):e61473. doi: 10.1371/journal.pone.0061473. Print 2013.
The E7 protein of the Human Papillomavirus (HPV) type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP) plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines.
METHODOLOGY/PRINCIPAL FINDINGS: An expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG), under the control of the C. reinhardtii chloroplast psbD 5' UTR and the psbA 3' UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein.
The C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.
人乳头瘤病毒（HPV）16 型的 E7 蛋白参与恶性细胞转化，是开发针对 HPV 相关病变和癌症的治疗性疫苗的关键抗原。按照良好生产规范（GMP）以活性形式重组生产这种疫苗抗原对于开发有效疫苗至关重要。已证明在植物中生产的基于 E7 的治疗性疫苗在临床前模型中具有肿瘤消退和保护作用。然而，全植物疫苗生产的一些缺点促使我们探索在莱茵衣藻中生产基于 E7 的治疗性疫苗，莱茵衣藻是一种易于生长和转化的生物，完全符合 GMP 指南。
方法/主要发现：一个表达盒，编码 E7GGG，E7 癌蛋白的一种突变、减毒形式，单独或与亲和标签（His6 或 FLAG）融合，在莱茵衣藻叶绿体 psbD 5'UTR 和 psbA 3'UTR 的控制下，通过同源重组被引入莱茵衣藻叶绿体基因组。该蛋白主要以可溶形式存在，达到总可溶性蛋白的 0.12%。优化并进行了亲和纯化两种标记形式。用总衣藻提取物和亲和纯化的蛋白免疫 C57BL/6 小鼠，检测到针对 E7 的特异性 IgG 和 E7 特异性 T 细胞增殖。用表达 E7 蛋白的肿瘤细胞系进行攻毒后，实现了高水平的肿瘤保护。
莱茵衣藻叶绿体是生产可溶性免疫原性 E7GGG 蛋白的合适表达系统。在封闭和无菌条件下生产突出了微藻作为替代平台用于生产人类用途疫苗的潜力。
