Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell line which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.
Cell Extracts/chemistry/immunology ; Cell Line ; Human ; Immunochemistry/*methods ; Oncogene Proteins, Viral/*analysis/immunology ; Papillomavirus, Human/chemistry/*immunology

OBJECTIVETo construct recombinant adenovirus containing codon-modified HPV16L1 gene, and evaluate systemic and mucosal immunological responses induced after immunization with the recombinant virus.

METHODSThe recombinant adenovirus rAd-mod.HPV16L1 was constructed by Admax kit. The C57 BL/6 mice were immunized by purified rAd-mod.HPV16L1 through different inoculation routes. The immunological effect was evaluated by testing the specific neutralizing antibodies in sera and vaginal secretions of immunized mice through indirect ELISA and neutralization assay based HPV pseudovirus.

RESULTSThe result showed that intramuscular immunization could induce good systemic immunity, but the mucosal immunity was too weak, and immunization via intranasal route could induce satisfactory immunity both in sera and vaginal secretions, while intravaginal immunization failed to induce any specific immunological responses either in sera or vaginal secretions.

CONCLUSIONThe recombinant adenovirus containing codon- modified HPV16L1 gene was successfully constructed. Immunization through intranasal route could induce satisfactory immunity both in sera and vaginal secretions, while intramuscular immunization could only induce high titer of neutralizing antibodies in sera.

Adenoviridae ; genetics ; Animals ; Antibodies, Viral ; analysis ; immunology ; Antibody Specificity ; Capsid Proteins ; genetics ; immunology ; Codon ; genetics ; DNA, Recombinant ; genetics ; Female ; Genetic Engineering ; Human papillomavirus 16 ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; Vaccination

OBJECTIVETo prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1.

METHODSPurified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1.

RESULTSAfter 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells.

CONCLUSIONHigh titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.

Animals ; Antibodies, Viral ; analysis ; immunology ; isolation & purification ; Antibody Specificity ; immunology ; CHO Cells ; Capsid Proteins ; genetics ; immunology ; metabolism ; Chickens ; Cricetinae ; Cricetulus ; Enzyme-Linked Immunosorbent Assay ; Green Fluorescent Proteins ; genetics ; immunology ; metabolism ; Humans ; Immunization ; methods ; Immunoglobulins ; immunology ; Immunohistochemistry ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transfection

Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide. Despite the encouraging development of the preventive vaccine for HPV, a vaccine for both prevention and therapy or pre-cancerous lesions remains in high priority. Thus far, most of the HPV therapeutic vaccines are focused on HPV E6 and E7 oncogene. However these vaccines could not completely eradicate the lesions. Recently, HPV E5, which is considered as an oncogene, is getting more and more attention. In this study, we predicted the epitopes of HPV16 E5 by bioinformatics as candidate peptide, then, evaluated the efficacy and chose an effective one to do the further test. To evaluate the effect of vaccine, rTC-1 (TC-1 cells infected by rAAV-HPV16E5) served as cell tumor model and rTC-1 loading mice as an ectopic tumor model. We prepared vaccine by muscle injection. The vaccine effects were determined by evaluating the function of tumor-specific T cells by cell proliferation assay and ELISPOT, calculating the tumor volume in mice and estimating the survival time of mice. Our in vitro and in vivo studies revealed that injection of E5 peptide+CpG resulted in strong cell-mediated immunity (CMI) and protected mice from tumor growth, meanwhile, prolonged the survival time after tumor cell loading. This study provides new insights into HPV16 E5 as a possible target on the therapeutic strategies about cervical cancer.
Adult ; Aged ; Amino Acid Sequence ; Animals ; Cancer Vaccines ; administration & dosage ; immunology ; Cell Line ; Cell Line, Tumor ; Dependovirus ; genetics ; Female ; Gene Expression Regulation, Viral ; immunology ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred C57BL ; Middle Aged ; Neoplasms, Experimental ; immunology ; prevention & control ; virology ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus Infections ; immunology ; prevention & control ; virology ; Papillomavirus Vaccines ; administration & dosage ; immunology ; Survival Analysis ; T-Lymphocytes ; immunology ; metabolism ; Tumor Burden ; immunology ; Uterine Cervical Neoplasms ; immunology ; prevention & control ; virology ; Vaccines, Subunit ; administration & dosage ; immunology

OBJECTIVETo investigate the effects of anti-HPV16E6-ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line.

METHODSHRz was designed by computer programs. HRz's activity was identified by cleavage experiments in vitro. HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot. Cell growth curves and soft agar forming ability were studied. The ability of each cell line to form tumors was assessed in nude mice. Apoptosis rates and expression of c-myc, bcl-2, p53 and Fas were detected by flow cytometry (FCM). Antigens of tumor cells, HLA-1, HLA-2, B7-1 and B7-2 were also detected. NK, LAK, and CD(3)AK cells were induced. Their cytotoxicities were detected in CaSKi-R, CaSKi-P, and CaSKi cells.

RESULTSIn vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site-specific manner. HRz could be expressed stably in transfected CaSKi cells. Northern blot analysis showed that E6 mRNA levels were lower in CaSKi-R than in CaSKi. The growth rate of CaSKi-R was slower than those of CaSKi and CaSKi-P. The soft agar-forming rate of CaSKi-R was lower compared with those of CaSKi and CaSKi-P cells. The ability of CaSKi-R to form tumors in nude mice was also poor. The apoptosis rate of CaSKi-R cells was much higher than those of CaSKi and CaSKi-P. HRz could reduce the expression of E6, c-myc and bcl-2 proteins, and increase the expression of p53 as well. HRz could increase the expression of HLA-2, B7-1 and B7-2 antigens. The cytotoxicity of NK, LAK and CD(3)AK cells was much higher in CaSKi-R than in CaSKi-P and CaSKi cells.

CONCLUSIONHRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.

Animals ; Apoptosis ; Cell Division ; Cytotoxicity, Immunologic ; Female ; Flow Cytometry ; Gene Expression ; Genetic Therapy ; Humans ; Killer Cells, Natural ; immunology ; Mice ; Mice, Nude ; Oncogene Proteins, Viral ; genetics ; Phenotype ; RNA, Catalytic ; therapeutic use ; RNA, Messenger ; analysis ; Repressor Proteins ; Tumor Cells, Cultured ; Uterine Cervical Neoplasms ; genetics ; pathology ; therapy