VLP vaccines against HPV-induced tumors

人乳头瘤病毒（HPV）是宫颈癌的一个必要原因，宫颈癌是全球妇女中第三大常见癌症。HPV DNA几乎在所有宫颈癌中检测到，其中约50-60%由高危HPV 16型占。有效的HPV复制条件尚未在体外模拟，从而阻碍了经典疫苗的开发。然而，当病毒的主要病毒体蛋白（L1）在真核细胞中过表达时，它自组装成病毒样颗粒（VLP）。HPV VLP已成为疫苗试验中通过诱导中和抗体预防未来HPV病变的主要策略，但它们不太可能对数百万已感染的妇女产生影响，也不会对不再表达L1基因的现有宫颈癌产生任何影响。然而，它们确实表达早期基因产物E6和E7，这些产物在宫颈癌中选择性保留和表达，并且是细胞转化状态所必需的。因此，将E6和E7蛋白掺入嵌合VLP（cVLP）中可以赋予基于VLP的疫苗治疗潜力。VLP在诱导T细胞应答、激活DC、肿瘤预防性环境中以及在肿瘤治疗性环境和基因递送中的初步方式中显示出巨大的潜力。基于这些研究，我们现在提出探索以下新目标：1）优化HPV VLP作为抗原递送系统的使用，2）HPV VLP作为定位HPV 16 E6/E7蛋白的人内源性呈递抗原表位的工具的使用，3）HPV VLP用于探索HPV与朗格汉斯细胞的相互作用，以及4）HPV VLP作为基因递送系统的使用。为实现这些目标，将采用以下方法：1）通过不同cVLP或不同途径递送HPV 16 E6/E7蛋白，以避免针对cVLP的抗体活性，所述抗体活性将中和针对E6/E7蛋白的重复接种，2）用负载有表达E6/E7蛋白的cVLP的自体DC体外免疫人外周血T细胞，3）人和小鼠朗格汉斯细胞的HPV VLP相互作用和活化，和4）将新的潜在肿瘤抑制基因或细胞因子基因递送至宫颈癌细胞，并将抗原编码基因或细胞因子基因递送至树突细胞。这些组合的目标将增加HPV VLP作为HPV诱导的宫颈癌的治疗剂的潜力和多功能性。

Human papillomaviruses (HPV are a necessary cause for cervical carcinoma, the third most common cancer among women worldwide. HPV DNA is detected in virtually every cervical carcinoma and about 50-60% of these are accounted for by the high risk HPV type 16. Efficient HPV replication conditions have not yet been mimicked in vitro thereby hampering the development of classical vaccines. However, when the major virion protein of the virus (L1) is over- expressed in eukaryotic cells, it self-assembles into virus-like particles (VLP). HPV VLP have become the leading strategy in vaccine trials for prevention of future HPV lesions by inducing neutralizing antibodies, but they are unlikely to have effects in the millions of women that are already infected and will not have any effect on existing cervical cancers that do no longer expression the L1 genes. They do, however, express the early gene products E6 and E7 that are selectively retained and expressed in cervical carcinoma and necessary for the transformed state of the cells. Therefore, incorporation of the E6 and E7 protein into chimeric VLP (cVLP) could confer a therapeutic potential to a VLP based vaccine. The tremendous potential of VLP has been shown in inducing T cell responses, in activating DC, in tumor preventive setting and, in preliminary ways also in tumor therapeutic settings and gene delivery. Based on these studies we now propose to explore the following new aims: 1) optimizing the use of HPV VLP as an antigen delivery system, 2) the use of HPV VLP as a tool for mapping human endogenously presented antigenic epitopes of HPV16 E6/E7 proteins, 3) the use of HPV VLP to explore the interaction of HPV with Langerhans cells, and 4) the use of HPV VLP as a gene delivery system. To achieve these aims the following methodology will be used: 1) delivery the of the HPV16 E6/E7 protein by different cVLP or different routes in order to avoid antibody activity against the cVLP that will neutralize repeated vaccination against the E6/E7 proteins, 2) in vitro immunization of human peripheral blood T cells with autologous DC loaded with cVLP expressing E6/E7 protein, 3) HPV VLP interaction and activation of human and mouse Langerhans cells, and 4) delivery of a new potential tumor suppressor gene or cytokine gene to cervical cancer cells and an antigen encoding gene or cytokine gene to dendritic cells. These combined aims will increase the potential and versatility of HPV VLP as therapeutic agents for HPV induced cervical cancer.