Identificaiton of Novel Immunogenic Human Papillomavirus Type 16 E7-Specific Epitopes Restricted to HLA-A*33;03 for Cervical Cancer Immunotherapy

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.

Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7 / 生物工程学报

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD₅₀) of LM4△hly::E7 strain was 3.863×10⁹ CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

Expression of Aurora-A, MCM7 and HPV 16 E7 in uterine cervical squamous cell carcinoma and their relationship with clinicopathological factors / 临床与实验病理学杂志

Purpose To explore the expression of Aurora kinase A (Aurora-A), minichromosome maintenance protein 7 (MCM7) and human papillomavirus type 16 E7 protein (HPV 16 E7) in uterine cervical squamous cell carcinoma (CSCC) and to investigate their relationship with clinicopathological factors. Methods Immunohistochemical method was employed on 20 cases of low-grade cervical intraepithelial neoplasia (CIN1) , 30 cases of high-grade cervical intraepithelial neoplasia (CIN2+3), 40 cases of CSCC, and 20 ca-ses of chronic cervicitis. Results (1) Aurora-A localized in the cytoplasm and nucleus. MCM7 protein positive staining localized in the nucleus. In the nucleus, and (or) the cytoplasm appeared brown particles positive for HPV 16 E7. (2) The expression of Aurora-A, MCM7 and HPV 16 E7 were higher in the group of CIN2+3 and CSCC than that in the group of chronic cervicitis or CIN1 ( P0. 05). Conclusion Aurora-A, MCM7 and HPV 16 E7 expression are gradually increased with disease progres-sion, and closely related to the occurrence and development of cervical cancer, they are expected to be early diagnosis, early treatment of biological indicators of cervical cancer.

The expression and clinic significance of CIP2A and HPV16-E7 protein in cervical cancer / 实用肿瘤学杂志

Objective To explore the expression and correlation of CIP 2A and HPV16-E7 protein in cervical cancer.Methods The expressions of CIP2A and HPV16-E7 in 50 cases of cervical cancer ,34 cases of CINⅠ,40 cases of CINⅡ,45 cases of CINⅢand 12 cases of normal tissues were assessed by immunohistochemi -cal method.R esults The rate of expressions of CIP2A protein in cancer tissues and CINⅢ were 68%(34/50) and 13.3%(6/45)respectively;The rate of expressions of CIP2A protein in CINⅡ、CINⅠtissues and normal tis-sues were negative.The expressions of CIP2A protein in cancer tissues were stronger than in CIN tissues and nor-mal tissues(P0.05).The expression of CIP2A was positively associated with the expression of HPV16-E7 in cervical cancer(r=0.514,P<0.05).Conclu-sion The expressions of CIP2A and HPV16-E7 are very strong in cervical cancer;CIP2A and HPV16 may play important roles in the carcinogenesis and the development of cervical cancer .

Effects of photodynamic therapy on the proliferation and apoptosis of human papillomavirus type 16 E7 proteinexpressing HaCaT cells / 中华皮肤科杂志

Objective To explore the effects of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the proliferation and apoptosis of HaCaT cells stably expressing human papillomavirus type 16 E7 protein (HaCaT/HPVl6 E7 cells).Methods Cultured HaCaT/HPV16 E7 cells were divided into several groups: blank control group receiving no treatment, ALA group treated with ALA alone, irradiation group irradiated with 630-nm red laser (30 mW/cm2,12 J/cm2), ALA-PDT groups pretreated with ALA for 5 hours followed by 630-nm red laser radiation at 4, 8, 12 J/cm2 respectively.CCK8 assay was performed to determine the survival rate of cells at 24 hours after PDT, and flow cytometetry and confocal microscopy were conducted to detect cell apoptosis and observe cell morphology respectively at 3 hours.Results At 24 hours, the survival rate of cells was 68.98％ ± 1.03％, 46.03％ ± 2.96％ and 23.57％ ± 3.83％ in the 4-,8-and 12-J/cm2 ALA-PDT groups respectively, significantly lower than that in the blank control group, ALA group and irradiation group (99.15％ ± 0.64％, 98.13％ ± 0.83％ and 96.85％ ± 1.37％ respectively, all P ＜ 0.05).With the increase in radiation dose, cell apoptosis was accelerated with obvious morphological changes and shrinkage of cells in the ALA-PDT groups.Conclusion ALA-PDT can inhibit the proliferation, and promote the apoptosis of HPV-infected HaCaT cells in a dose-dependent manner within a certain range of radiation dose.

Protein expression of HPV16 and its early gene E6, E7 in human laryngeal squamous cell carcinoma and its significance / 第二军医大学学报

Objective: To investigate the protein expression of HPV16 and its early gene E6, E7 in human laryngeal squamous cell carcinoma tissues, and to analyze their relationship with the clinical stage and pathological classification of laryngeal squamous cell carcinoma. Methods: The expression of HPV16, HPV16 E6, and HPV16 E7 protein was detected in 147 specimens of different laryngeal lesions immunohistochemically. The specimens included 82 laryngeal carcinoma, 39 non-carcinoma tissues (including 27 specimens of vocal cord polyp and 12 specimens of normal laryngeal tissues taken from more than 1.0 cm adjacent to the carcinoma), and 26 precancerous lesions (leukoplakia) of the larynx. The relationship between the protein expression with the clinical stage and histopathological classification of laryngeal squamous cell carcinoma was analyzed. Results: The positive rates of HPV16, HPV16 E6, and HPV16 E7 protein in precancerous tissues (30.77%, 26. 92%, and 26. 92%, respectively) were significantly lower than those in laryngeal carcinoma lesions (45. 12%, 39. 02%, and 42. 68%, respectively; P<0. 05 or 0. 01), but were significantly higher than those in non-carcinoma tissues (23. 08%, 5. 13%, and 2.56%, respectively; P<0.05 or 0.01). In non-carcinoma tissues, the positive rate of HPV16 protein was significantly higher than that of E6 or E7 (P<0.05 or 0.01), while there was no difference between their positive rates in laryngeal carcinoma or precancerous lesions. We found that human laryngeal carcinoma tissues with different clinical stages and different pathological classifications also had different positive rate of HPV16 protein (P<0.05), but they had a similar positive rate of HPV16 E6 and E7. Conclusion: The expression of HPV16 E6, E7 proteins after the HPV16 infection might be one of the reasons for development of human laryngeal carcinoma. Inhibition of HPV16E7 expression by immunologic strategy may have a potential for treatment of laryngeal carcinoma.

Prokaryotic cloning and expression of HPV16 E7 gene / 重庆医科大学学报

Objective:To explore the cloning and expression of HPV16 E7 protein from laryngeal carcinoma. Methods: HPV16 E7 gene was amplified by PCR. The amplified fragment was inserted into the plasmid pET28a (+) digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET28/E7 was transformed into E.coli JM109 which was selected with ampicillin. HPV16 E7 recombinant protein expression in the E.coli BL21(DE3) was identified by SDS-PAGE and Western blot. Results: The prokaryotic recombinant plasmid pET28/E7 was successfully constructed. The BL21(DE3) transformed recombinant plasmid pET28/E7 had expressed HPV16 E7 recombinant protein effectively. Conclusion:The construction of the prokaryotic recombinant plasmid pET28/E7 and the successful expression of the recombinant protein HPV16 E7 pave way for the profound research of the biological properties and the transformational mechanism of the HPV16 E7 protein on the specific cells.

Immunization with Adenoviral Vectors Carrying Recombinant IL-12 and E7 Enhanced the Antitumor Immunity against Human Papillomavirus 16-associated Tumor / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Human papillomavirus (HPV) infection has a significant role in cervical carcinogenesis, and HPV oncoprotein E7 plays an important part in the formation and maintenance of cervical cancer. Interleukin-12 (IL-12) has been reported to induce a cellular immune response, and to suppress the tumor growth and the E7 production. Here we describe the use of adenoviral delivery of the HPV 16 E7 subunit (AdE7) along with adenoviral delivery of IL-12 (AdIL-12) in mice with HPV-associated tumors. MATERIALS AND METHODS: Mice were injected with TC-1 cells to establish TC-1 tumor, and then they were immunized with AdIL-12 and/or AdE7 intratumorally. The anti tumor effects induced by AdIL-12 and/or E7 were evaluated by measuring the size of the tumor. E7-specific antibody and INF-gamma production in sera, and the T-helper cell proliferative responses were then measured. Cytotoxic T-lymphocyte (CTL) and T cell subset depletion studies were also performed. RESULTS: Combined AdIL-12 and AdE7 infection at the tumor sites significantly enhanced the antitumor effects more than that of AdIL-12 or AdE7 single infection. This combined infection resulted in regression of the 9 mm sized tumors in 80% of animals as compare to the PBS group. E7-specific antibody and INF-gamma production in the sera, and the T-helper cell proliferative responses were significantly higher with coinfection of AdIL-12 and AdE7 than with AdIL-12 or AdE7 alone. CTL response induced by AdIL-12 and AdE7 in the coinjected group suggested that tumor suppression was mediated by mostly CD8+ and only a little by the CD4+ T cells. CONCLUSION: IL-12 and E7 application using adenovirus vector showed antitumor immunity effects against TC-1 tumor, and this system could be use in clinical applications for HPV-associated cancer.

Study on Expression of ANP63 and HPV16 E7 in Cervical Intraepithelial Neoplasia / 重庆医科大学学报

objective: To examine the potential of ANP63 and HPV16 E7 as one of biomarkers for cervical premalignant lesions in Thi-nPrep smears, and to search for the identification of specific biomarkers for cervical intraepithelial neoplasia (CIN) as a new examining method to evaluate evolution of CIN. Method; Streptavidin peroxidase (SP) method was used. Immunocytochemical analysis of ANP63 expression was performed in 71 ThinPrep smears. RT - PCR was used to detect the mRNA of HPV16 E7 in 71 ThinPrep liquid - based smears specimens. Result; Positive expression rate of ANP63 in CIN was higher than that in inflammation (P