No abstract available.
Adolescent ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Burkitt Lymphoma/*pathology ; Child, Preschool ; Cyclophosphamide/therapeutic use ; Doxorubicin/therapeutic use ; Female ; Gene Rearrangement ; Herpesvirus 4, Human/metabolism ; Humans ; Immunohistochemistry ; Lymphoma, B-Cell/*diagnosis/drug therapy ; Lymphoma, Large B-Cell, Diffuse/*pathology ; Male ; Prednisone/therapeutic use ; Proto-Oncogene Proteins c-myc/genetics ; Tomography, X-Ray Computed ; Vincristine/therapeutic use ; Viral Matrix Proteins/immunology/metabolism

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

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 express and prepare polyclonal antibody of Human papillomavirus type 6b (HPV6b) E7 protein. a prokaryotic expression vector pGEX-4T-2/HPV6b E7 was constructed and GST-HPV6b E7 fusion protein was expressed as a soluble protein in E. coli. The expressed fusion protein was purified via Glutathione-Sepharose 4B column and thrombin cleavage in order to obtain HPV6b E7 protein. Polyclonal IgG antibody was prepared by immunizing New-Zealand rabbits with HPV6b E7 protein. Western-Blot and immunofluorescence analysis showed that the polyclonal IgG antibody could specifically recognize HPV6b E7 protein and its titer was identified. SDS-PAGE analysis demonstrated that large amounts of soluble GST-HPV6b E7 fusion protein was expressed in E. coli after 3.0-6.0 hours of IPTG induction. Polyclonal IgG antibody successfully prepared from immunized rabbits showed high titer and high specificity as confirmed by Western-Blot and immunofluorescence. The preparation of anti-HPV6b E7 polyclonal antibody will facilitate further research on the biological and immunological functions of HPV6b E7 protein.
Animals ; Antibodies ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression Regulation, Bacterial ; HEK293 Cells ; Humans ; Immunoglobulin G ; immunology ; metabolism ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.
Animals ; Antibodies, Viral ; immunology ; ultrastructure ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; Goats ; Human papillomavirus 16 ; genetics ; immunology ; ultrastructure ; Humans ; Male ; Oncogene Proteins, Viral ; genetics ; immunology ; isolation & purification ; Papillomavirus Infections ; immunology ; virology ; Rabbits ; Recombinant Proteins ; genetics ; immunology ; Vaccination ; Virion ; genetics ; immunology

Here, we presented a method to bacterially express the major structural protein L1 of Human Papillomavirus type 18 (HPV18) as soluble form. We found that the purified L1 could self-assemble to virus-like particles (VLPs). Further, we investigated the immunogenicity and the induced level of neutralizing antibody using these VLPs. First, the genome of HPV18 was cloned from a patient in Xiamen. It was used as template for PCR amplification of HPV18 L1 gene. The resultant DNA fragment was inserted into expression vector pTrxFus and expressed in Escherichia coli GI724. Second, L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography; and the purified L1 was subjected to self-assembly to form VLPs with the removal of premixed reductant DTT. Finally, the size and morphology of these VLPs was investigated by Dynamic Light Scattering and Transmission Electronic Microscopy as 29.34 nm in hydrated radius and globular particles similar with native HPV18. The half effective dosage (ED50) and maximum level of neutralizing antibody elicitation were measured by vaccinations on mice, rabbit and goat using pseudovirus neutralization cell model. The results showed that the ED50 of HPV18 VLPs is 0.006 microg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 10(7). As a conclusion, we can provide HPV18 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV18.
Animals ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Goats ; Human papillomavirus 18 ; immunology ; isolation & purification ; Mice ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Virion ; genetics ; 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