Immunoinformatics Design and In Vivo Immunogenicity Evaluation of a Conserved CTL Multi-Epitope Vaccine Targeting HPV16 E5, E6, and E7 Proteins.

Human papillomavirus type 16 (HPV16) infection is responsible for more than 50% of global cervical cancer cases. The development of a vaccine based on cytotoxic T-lymphocyte (CTL) epitopes is a promising strategy for eliminating pre-existing HPV infections and treating patients with cervical cancer. In this study, an immunoinformatics approach was used to predict HLA-I-restricted CTL epitopes in HPV16 E5, E6, and E7 proteins, and a set of conserved CTL epitopes co-restricted by human/murine MHCs was screened and characterized, with the set containing three E5, four E6, and four E7 epitopes. Subsequently, the immunogenicity of the epitope combination was assessed in mice, and the anti-tumor effects of the multi-epitope peptide vaccine E5E6E7pep11 and the recombinant protein vaccine CTB-Epi11E567 were evaluated in the TC-1 mouse tumor model. The results demonstrated that mixed epitope peptides could induce antigen-specific IFN-γ secretion in mice. Prophylactic immunization with E5E6E7pep11 and CTB-Epi11E567 was found to provide 100% protection against tumor growth in mice. Moreover, both types of the multi-epitope vaccine significantly inhibited tumor growth and prolonged mouse survival. In conclusion, in this study, a multi-epitope vaccine targeting HPV16 E5, E6, and E7 proteins was successfully designed and evaluated, demonstrating potential immunogenicity and anti-tumor effects and providing a promising strategy for immunotherapy against HPV-associated tumors.

EXPRESSION FEATURES OF HUMAN PAPILLOMA VIRUS TYPE 16 AND ANTI-EPSTEIN-BARR VIRUS IN PLEOMORPHIC ADENOMA, SURROUNDING AND INTACT SALIVARY GLAND.

OBJECTIVE: The aim is to reveal the expression features of MCA to human papilloma virus type 16 and anti-Epstein-Barr virus in the pleomorphic adenoma, surrounding and intact salivary gland. PATIENTS AND METHODS: Materials and methods: It was used surgical and biopsy material from 30 patients, represented by pleomorphic adenomas with surrounding to tumor tissue of the salivary gland and intact tissue of the salivary gland (the distance between the tumor and the intact salivary gland - 10 mm). Immunohistochemical study was performed using mouse monoclonal antibody (MCA) to human papilloma virus type 16 (clone CAMVIR-1, «Diagnostic BioSystems¼, USA) and anti-Epstein-Barr virus (LMP, clone CS. 1-4, «Dako¼, Denmark). Visualization was performed, using an EnVisionTM FLEX detection system (Dako, Denmark). Antigen unmasking was carried out in citrate buf f er pH 6.0 at 95°C. Primary antibodies were incubated at room temperature for 30 minutes, secondary antibodies - 20 minutes. Sections were counterstained with Gill hematoxylin. We assessed the immunohistochemical reaction by a semi-quantitative method by counting the percentage of positively stained cells in the fi eld of view of a microscope × 400. Microspecimens were studied and photoarchived on an Olympus BX-41 microscope (Japan). RESULTS: Results: In this study it was detected a positive immunohistochemical reaction with MCA to human papilloma virus type 16 and anti-Epstein-Barr virus, respectively, in 26 (86.7%) and 8 (26.7%) cases. Epithelial, mixed and mesenchymal variants of pleomorphic adenoma of the salivary glands are characterized, respectively, by the severely expressed, moderately expressed and minimally expressed of MCA to human papilloma virus type 16 and anti-Epstein-Barr virus. The parenchymal component of pleomorphic adenoma is characterized by more marked expression of these markers as compared to the stromal component. The epithelial cells of the salivary glands, surrounding the pleomorphic adenoma, as well as intact salivary glands, express MCA to human papilloma virus type 16 and anti-Epstein-Barr virus. The severity of the expression of these markers in the salivary gland is determined by the histological variant of the tumor (severely expressed in the epithelial variant, moderately expressed in the mixed variant, and minimally expressed in the mesenchymal variant). CONCLUSION: Conclusions: The immunohistochemical study has shown that the Epstein-Barr virus and, especially, human papilloma virus type 16 can act as exogenous trigger factors involved in the development of pleomorphic adenoma of the salivary glands. The revealed immunohistochemical features of MCA expression to human papilloma virus type 16 and anti-Epstein-Barr virus in the salivary gland surrounding the pleomorphic adenoma and in the intact tissue of the salivary gland make it possible to recommend the extracapsular dissection of the tumor with resection of the adjacent intact tissue of the salivary gland at a distance of 10 mm in patients with pleomorphic adenoma.

IMMUNOHISTOCHEMICAL FEATURES OF THE EXPRESSION OF HUMAN PAPILLOMA VIRUS TYPE 16 IN PLEOMORPHIC ADENOMAS OF SALIVARY GLAND.

OBJECTIVE: The aim is to reveal the immunohistochemical features of human papilloma virus type 16 expression in various histological variants of pleomorphic adenomas of the salivary gland. PATIENTS AND METHODS: Materials and methods: The material of the study was surgical and biopsy material from 30 patients with pleomorphic adenomas of the salivary glands, among which in 15 cases mesenchymal was detected, in 10 - mixed, in 5 cases - epithelial histological variant, respectively. Immunohistochemical study was performed, using mouse monoclonal antibody to human papilloma virus type 16. Visualization was performed, using an EnVisionTM FLEX detection system. Histological sections of grade III cervical intraepithelial neoplasia (CIN III) were used as a positive control; for a negative control, the procedure was performed without primary antibodies. The immunohistochemical reaction was assessed by a semi-quantitative method by counting the percentage of positively stained cells in the field of view of a microscope × 400. Microspecimens were studied, photoarchived on an Olympus BX-41 microscope. RESULTS: Results: Expression of human papilloma virus type 16 of varying severity was determined in 26 cases of pleomorphic adenomas of the salivary glands, which was 86.7%. The epithelial component of the pleomorphic adenoma of the salivary gland was characterized by a more pronounced expression of the monoclonal antibody to human papilloma virus type 16 compared to the mesenchymal component of the tumor. The severity of the immunohistochemical reaction with a monoclonal antibody to human papilloma virus type 16 depended on the histological variant of the pleomorphic adenoma of the salivary gland. Epithelial, mixed and mesenchymal variants of pleomorphic adenoma of the salivary gland were characterized, respectively, by the most pronounced, pronounced and moderately pronounced expression of a monoclonal antibody to human papilloma virus type 16. CONCLUSION: Conclusions: A comprehensive immunohistochemical study with a monoclonal antibody to human papilloma virus type 16 revealed the presence of a causal relationship between the infection of a patient with human papilloma virus type 16 and development of pleomorphic adenoma of the salivary gland in him.

Sustainable Tumor-Suppressive Effect of iPSC-Derived Rejuvenated T Cells Targeting Cervical Cancers.

Immunotherapy utilizing induced pluripotent stem cell (iPSC) technology has great potential. Functionally rejuvenated cytotoxic T lymphocytes (CTLs) can survive long-term as young memory T cells in vivo, with continuous tumor eradication. Banking of iPSCs as an unlimited "off-the-shelf" source of therapeutic T cells may be feasible. To generate safer iPSCs, we reprogrammed human papilloma virus type 16 (HPV16) E6-specific CTLs by Sendai virus vector without cotransduction of SV40 large T antigen. The iPSCs efficiently differentiated into HPV16-specific rejuvenated CTLs that demonstrated robust cytotoxicity against cervical cancer. The tumor-suppressive effect of rejuvenated CTLs was stronger and more persistent than that of original peripheral blood CTLs. These rejuvenated HPV16-specific CTLs provide a sustained tumor-suppressive effect even for epithelial cancers and constitute promising immunotherapy for cervical cancer.

Expression Difference and Its Mechanism Study of miR-27a-3p and HOXB8 Protein in Cervical Cancer Tis-sues of HPV16-positive and HPV16-negative Patients / 中国药房

OBJECTIVE:To investigate the expression difference and its mechanism of miR-27a-3p and HOXB8 protein in cer-vical cancer tissues of HPV16-positive and HPV16-negative patients. METHODS:A total of 120 patients with cervical cancer in our hospital during Jan. 2012-Jan. 2016 were divided into HPV16-positive group(60 cases)and HPV16-negative group(60 cases) according to HPV16 infection situation. The expression of miR-27a-3p mRNA and HOXB8 mRNA in cervical cancer tissue were de-tected by RT-qPCR. The expression of HOXB8 protein was detected by Western blotting assay. DNA methylation level of miR-27a-3p promoter region was detected by nested-down methylation specific PCR (nMS-PCR). Histone methylation level of miR-27a-3p promoter region was detected by chromatin immunoprecipitation PCR (CHIP-PCR). The expression of miR-27a-3p mRNA and HOXB8 mRNA were detected after transfecting miR-27a-3p mimic and inhibitor into Human cervical cancer cell line SiHa,respectively. RESULTS:The expression of miR-27a-3p in HPV16-positive group was significantly lower than HPV16-nega-tive group,while HOXB8 mRNA and protein expression,DNA and histone methylation levels of miR-27a-3p promoter region were significantly higher than HPV16-negative group,with statistical significance (P<0.05 or P<0.01). After transfecting miR-27a-3p mimic into SiHa cells,the expression of miR-27a-3p was increased significantly,while that of HOXB8 mRNA was de-creased significantly;after miR-27a-3p inhibitor transfection,the expression of miR-27a-3p was decreased significantly,while that of HOXB8 mRNA was increased significantly,with statistical significance(P<0.01). CONCLUSIONS:HPV16 may down-regu-late the expression of miR-27a-3p through DNA methylation and histone methylation of promoter region,so as to influence the gen-eration and development of cervical cancer. HOXB8 may be the target protein of miR-27a-3p.

Production of human papilloma virus type 16 e6 oncoprotein as a recombinant protein in eukaryotic cells.

BACKGROUND: Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm. METHODS: In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/T-E6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression vector. The pcDNA3-E6 vector was propagated in E.coli strain DH5α and transfected into CHO cells 72 hours post-transfection. RESULTS: The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody. CONCLUSION: HPV16-E6 target protein recognized by specific antibody could be an appropriate form of protein, which can be used for further studies. Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies.