Research & Development Centre, Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500032, Andhra Pradesh, India.
Vaccine. 2011 Oct 6;29(43):7326-34. doi: 10.1016/j.vaccine.2011.07.071. Epub 2011 Jul 29.
The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.
人乳头瘤病毒（HPV）的主要衣壳蛋白（L1）在异源系统中表达会组装成病毒样颗粒（VLPs）。我们报告了在甲醇营养酵母毕赤酵母中克隆和表达两种高危 HPV 型 16 和 18 的 HPV L1 基因的情况。在毕赤酵母中产生的 VLPs 经过三步纯化方法，包括密度梯度离心和大小排阻层析。使用针对构象特异性的单克隆抗体进行 ELISA 分析和透射电子显微镜观察，对富集的 VLPs 进行了表征。用双价 HPV16 和 HPV18 VLPs 免疫的小鼠产生了针对两种 HPV 类型的高血清抗体滴度，并且在接种后 190 天仍持续存在。用 HPV-VLP 制剂免疫的小鼠的血清可在体外试验中中和同源假病毒。我们的结果表明，在毕赤酵母中表达的 L1 蛋白正确折叠，证据是组装成 VLPs，并在小鼠中诱导了针对特定类型的中和抗体反应。这项工作是朝着开发替代生产平台以生产负担得起的 HPV 疫苗以满足发展中国家需求迈出的一步。
