组装型表位展示人乳头瘤病毒颗粒的分子设计及功能研究

Therapeutic human papillomavirus (HPV) vaccines are the most effective way to reduce the incidence of cervical cancer. Virus-like particles (VLPs) assembled by the HPV L1capsid protein is the unique listed vaccine form currently, however, its clinical application is limited due to the defect of cellular immune response. Chimeric VLPs harboring cytotoxic T lymphocyte (CTL) epitopes show therapeutic potential against HPV, but multiple epitopes inserted may cause that VLPs couldn’t self-assemble in vitro or their efficiency of assembly decreased. Therefore, how to enhance the capacity and diversity of the epitopes becomes the key technology for the reconstruction of chimeric VLPs vaccines. In this study, optimal chimeric sites in surface-exposed loops of L1 will be analyzed by homology modeling and chimeric VLPs harboring the reverse chain of leucine zipper heterodimers will be prepared. Meanwhile, CTL epitopes of E7 that their function have be confirmed in our previous study will be modified by forward chain of leucine zipper heterodimers at N-terminal, so immunodominant CTL epitopes will be displayed on the surface of capsid by zipper pairing with a high density, assemble multi-CTL epitopes displayed virus-like particles. Then it will be evaluated in terms of immunogenicity and inhibitory rate of the tumor in HLA-A2 transgenic mice. The assembled epitopes displayed virus-like particles designed in this study not only overcome the defect of deficiency in the current therapeutic HPV vaccine immune target designed, but also effective response epitopes of the vaccinators would assemble with VLPs freely. HPV vaccine design for individualized immunotherapy will be explored.

治疗性HPV疫苗是未来降低宫颈癌发病率的最有效手段。目前，病毒样颗粒作为唯一的预防性HPV疫苗形式已成功上市，但因其诱发的细胞免疫应答水平低导致临床应用受限。以病毒样颗粒为模体，嵌入细胞毒性T淋巴细胞表位可加强其治疗作用。然而，多表位的嵌入会导致衣壳蛋白无法在体外组装成病毒样颗粒或组装效率降低，因此，如何提高表位的装载量和多样性成为嵌合病毒样颗粒疫苗改造的关键技术。本课题设计将亮氨酸拉链嵌合到L1的环形区组装亮氨酸拉链嵌合VLPs，通过拉链的异二聚化作用分别将前期已筛选验证得到的CTL优势表位修饰到VLPs表面，组装多CTL表位展示病毒颗粒，并利用转基因鼠对其免疫原性及肿瘤抑制作用进行评价，本课题设计的组装型表位展示病毒颗粒不仅弥补了目前治疗性HPV疫苗免疫靶点单一的缺陷，而且可根据接种者自身的免疫应答特点，选择有效的应答表位与VLPs进行自由组装，探索个体化免疫治疗的HPV疫苗设计。

治疗性HPV疫苗是未来降低宫颈癌发病率的最有效手段。目前，病毒样颗粒作为唯一的预防性HPV疫苗形式已成功上市，但因其诱发的细胞免疫应答水平低导致临床应用受限。以病毒样颗粒为模体，嵌入细胞毒性T淋巴细胞表位可加强其治疗作用。然而，多表位的嵌入会导致衣壳蛋白无法在体外组装成病毒样颗粒或组装效率降低，因此，如何提高表位的装载量和多样性成为嵌合病毒样颗粒疫苗改造的关键技术。本研究利用密码子扩充原理通过叠氮-烷烃的环化加成点击化学技术制备E749-57-E786-93表位肽修饰VLPs，结果表明表位肽可以通过共价键地固定于VLPs表面，且与野生型HPV VLPs相比，可以刺激小鼠产生相似的中和抗体和更强的细胞免疫应答。本项目制备的表位肽修饰VLPs既不影响天然VLPs的组装效率又增加了其对外源表位的装载量和多样性，还可根据接种者自身的免疫应答特点，选择有效的应答表位与VLPs进行自由组装，实现精准的个体化免疫治疗的HPV疫苗设计，有望突破传统治疗性HPV疫苗免疫靶点单一的技术瓶颈，实现防治结合和个体化免疫的多重目标，为未来HPV疫苗研究拓宽思路。