Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8 ⁺ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8 ⁺ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Abstract

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4 ؉ and CD8 ؉ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1-and E7-specific responses in CD4 ؉ and CD8 ؉ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8 ؉ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4 ؉ and CD8 ؉ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical cancer. Although more than 100 distinct HPV genotypes have been described, and at least 20 are associated with cervical cancer, HPV type 16 (HPV-16) is by far the most frequently detected in cervical neoplasia regardless of the geographical origin of the patients (4). In the last few years significant advances have been made in the development of candidate prophylac...

Self-Adjuvanting Polymer–Peptide Conjugates As Therapeutic Vaccine Candidates against Cervical Cancer

“…These limitations have prompted research into the development of therapeutic vaccines that treat HPV-related cancer. 2,[7][8][9][10][11] Therapeutic vaccine candidates against HPV-related cancers were usually designed to elicit a cytotoxic T-cell response by targeting E7 oncoprotein. This protein is constitutively expressed in cancer cells and is required for the induction and maintenance of tumor growth.…”

A randomized trial of immunotherapy for persistent genital warts

“…L1 protein is predominantly expressed in the superficial layers of the epidermis in the course of natural infection, where it would not be thought susceptible to cell mediated immune responses of the sort induced by VLP immunotherapy. 7 However, sufficient L1 protein for susceptibility to cell mediated immunity is presented by immature cells, 13,14 and protein from immature 14,15 epithelial cells can be cross presented from skin by vascular endothelial cells to enable epithelial rejection. 16 As local inflammation has been shown important for local function of immune effector cells induced by vaccination, 17 the current study was designed to use VLPs as immunotherapy following conventional destructive therapy which was expected to induce the necessary local inflammation.…”

Development of New Human Papillomavirus Vaccines

“…Taken together, these data demonstrate consistent expression of L1 in primary cervical tumours and the possibility of inducing effective L1/tumour-specific CD4+ and CD8+ Tlymphocyte responses in patients harbouring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harbouring established HPVinfected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations (Shedlock & Shen, 2003;Bellone et al, 2009). …”

“…These results may have potential implications for future clinical translation. Therefore, further work needs to be done to complete this study (Bellone et al, 2009;Peng et al, 2001;Shedlock & Shen, 2003;Silk & Finn, 2007).…”