Humans ; Squamous Cell Carcinoma of Head and Neck ; RNA, Messenger ; Immunohistochemistry ; Papillomavirus Infections/diagnosis* ; Oncogene Proteins, Viral/genetics* ; Head and Neck Neoplasms ; Cyclin-Dependent Kinase Inhibitor p16 ; Papillomaviridae ; Papillomavirus E7 Proteins/genetics* ; DNA, Viral

OBJECTIVE: Persistent infection of HPV increases the chance of carcinoma in situ of cervix through stages of cervical intraepithelial neoplasia (CIN) 1, 2, and 3, and finally progresses into cervical cancer. We aimed to explore the safety and efficacy of BLS-M07 which is orally administered agent expressing human papillomavirus (HPV) 16 E7 antigen on the surface of Lactobacillus casei in patients with CIN 3. METHODS: Patients with CIN 3 were recruited in our clinical trial. Reid Colposcopic Index (RCI) grading and serum HPV16 E7 specific antibody production were used to evaluate efficacy of BLS-M07. In phase 1, BLS-M07 was administered orally, 5 times a week, on weeks 1, 2, 4, and 8 with dosages of 500 mg, 1,000 mg, and 1,500 mg. In phase 2a, patients were treated with 1,000 mg. The primary endpoints were the safety and the pathologic regression on colposcopic biopsy. RESULTS: Nineteen patients were enrolled in the CIN 3 cohort. In phase 1, no patients experienced dose limiting toxicity. No grade 3 or 4 treatment-related adverse events or deaths were observed. At 16 weeks after treatment, RCI grading was improved and serum HPV16 E7 specific antibody production increased (p<0.05). Six of 8 (75%) patients with CIN 3 were cured in phase 2a. CONCLUSIONS: Oral immunization with BLS-M07 increases production of serum HPV16 E7 specific antibody which induces protective humoral immunity. The safety of this oral vaccine was proved and could be a competitive non-surgical therapeutic agent of CIN 3. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02195089
Antibody Formation ; Biopsy ; Carcinoma in Situ ; Cervical Intraepithelial Neoplasia ; Cervix Uteri ; Cohort Studies ; Female ; Humans ; Immunity, Humoral ; Immunization ; Lactobacillus casei ; Papillomavirus E7 Proteins ; Papillomavirus Vaccines ; Uterine Cervical Neoplasms

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.
Animals ; Cancer Vaccines ; immunology ; Listeria monocytogenes ; Mice ; Mice, Inbred C57BL ; Papillomavirus E7 Proteins ; immunology ; Papillomavirus Infections ; prevention & control ; Plasmids ; RAW 264.7 Cells ; Recombinant Fusion Proteins ; immunology ; Vaccines, Attenuated ; immunology ; Viral Vaccines ; immunology

OBJECTIVETo investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.

METHODSBoth SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively.

RESULTSCyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells.

CONCLUSIONSquamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.

Carcinoma, Squamous Cell ; metabolism ; virology ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cyclin D1 ; genetics ; metabolism ; Female ; Human papillomavirus 16 ; Humans ; Immunohistochemistry ; Papillomavirus E7 Proteins ; genetics ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Uterine Cervical Neoplasms ; metabolism ; virology

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.
Animals ; Capsid Proteins ; biosynthesis ; Codon ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Human papillomavirus 16 ; Mice ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; biosynthesis ; Papillomavirus E7 Proteins ; biosynthesis ; Papillomavirus Vaccines ; therapeutic use ; Recombinant Fusion Proteins ; biosynthesis

OBJECTIVETo explore the in vitro inhibitive effect and underlying mechanisms of Brucea Javanica oil emulsion (BJOE) on human papilloma virus (HPV) type 16 infected cells.

METHODSThe HPV16 E61E7 immortalized human ectocervical Ect1/E6E7 cell line and the CaSki cell line were selected as the in vitro models of premalignant cervical lesion and cervical cancer respectively. After treated with BJOE at different concentrations (5, 10, 20, and 40 microg/mL) at the operation time points (24, 48, and 72 h), the effects of BJOE on proliferative activities were measured by MTT assay. The morphologic changes of cell apoptosis stained with Hochest 33,258 were observed by fluorescence microscope. The effect on the cell apoptosis rate was analyzed by Annexin V-FITC/PI double-labeled flow cytometry. The mRNA expressions of HPV16 E6 and E7 were determined by semi-quantitative RT-PCR. The protein expressions of HPV16 E6, E7 oncogene, and specifically interacted p53, Rb antioncogene were stained by immunocytochemical staining (Elivison two-step procedure).

RESULTS(1) The proliferative activities of the Ect1/E6E7 cell and the CaSki cell treated with BJOE at different concentrations (5, 10, 20, and 40 p g/mL) at the operation time points (24, 48, and 72 h) were obviously inhibited, showing dose- and time-dependent manners (P <0.05). (2) Typical changes of apoptosis were observed in both HPV 16 positive cell lines after treated with BJOE. The cell apoptosis rates increased markedly after being cultured with BJOE at different concentrations (5, 10, and 20 microg/mL) for 48 h (P < 0.05). (3) After treated with BJOE at different concentrations (5, 10, and 20 microg/mL) for 48 h, the HPV16 E6 and E7 mRNA relative expressions in both HPV 16 positive cell lines decreased significantly (P < 0.05). (4) After treated with BJOE at different concentrations (5, 10, and 20 microg/ mL), the expressions of HPV16 E6, E7, and mutant p53 protein decreased gradually (P < 0.05), while the Rb protein expression increased gradually (P < 0.05).

CONCLUSIONSBJOE showed obvious in vitro inhibitory effects on HPV type 16 infected cells. Its underlying mechanisms might be possibly associated with down-regulating expressions of HPV16 E6 and E7 oncogenes.

Apoptosis ; drug effects ; Brucea ; chemistry ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Female ; Human papillomavirus 16 ; drug effects ; pathogenicity ; Humans ; Oncogene Proteins, Viral ; metabolism ; Papillomavirus E7 Proteins ; metabolism ; Papillomavirus Infections ; Plant Oils ; pharmacology ; Repressor Proteins ; metabolism

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.
Cancer Vaccines ; biosynthesis ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Female ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Papillomavirus Vaccines ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Uterine Cervical Neoplasms ; prevention & control ; Vaccines, DNA ; biosynthesis

OBJECTIVETo develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity.

METHODSThe E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay.

RESULTSSequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group.

CONCLUSIONSThe fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.

Animals ; Cancer Vaccines ; immunology ; Capsid Proteins ; genetics ; immunology ; Cell Transformation, Neoplastic ; Cloning, Molecular ; Codon ; Female ; Immunoglobulin G ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; NIH 3T3 Cells ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; Papillomavirus E7 Proteins ; genetics ; immunology ; Papillomavirus Vaccines ; immunology ; Plasmids ; Point Mutation ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Transfection ; Vaccines, DNA ; immunology

OBJECTIVETo investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.

METHODSThe HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.

RESULTSThe codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.

CONCLUSIONSThe data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cancer Vaccines ; immunology ; therapeutic use ; Capsid Proteins ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Codon ; Escherichia coli ; immunology ; metabolism ; Female ; Humans ; Immunization ; methods ; Immunotherapy ; methods ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; immunology ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Papillomavirus E7 Proteins ; genetics ; immunology ; metabolism ; Papillomavirus Vaccines ; immunology ; therapeutic use ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; immunology ; metabolism

To investigate E6 and E7 gene variations of human papillomavirus type 16 in Yunnan Province, DNA was extracted from 2000 gynecological outpatient samples. For Human papillomavirus (HPV) genotyping, the genomic DNA was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers, then the PCR products were subjected to direct DNA sequencing. A total of 20 HPV-16 viral DNAs were identified. E6 and E7 genes of HPV-16 viral DNA were then amplified using E6 and E7 specific primers, the PCR products were purified and sequenced. The results showed that mutations were found at nucleotide position 178 of HPV-16 E6 gene in 10 cases,the mutation rate was 50%; For HPV-16 E7 gene, the mutations were found at nucleotide position 647 in 10 cases; the mutation rate was 50%. Phylogenetic analysis showed that Asian (As) variants of HPV-16 were predominated in Yunnan, China. None of African-1, African-2 variants of HPV-16 was found in this region.
Adult ; Base Sequence ; China ; Female ; Genetic Variation ; Human papillomavirus 16 ; classification ; genetics ; isolation & purification ; Humans ; Middle Aged ; Molecular Sequence Data ; Mutation ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; genetics ; Papillomavirus Infections ; virology ; Phylogeny ; Repressor Proteins ; genetics