OBJECTIVETo construct one recombinant vaccinia virus expressing the HPV18 E6 and E7 fusion proteins as HPV18 therapeutic vaccine candidate, and test its immunogenicity.

METHODSThe fusion E7E6 genes were synthesized and mutated to inactivate their oncogenic potential, and inserted into a vaccinia virus plasmid vector to construct one recombinant vaccinia virus. Finally its immunogenicity was characterized in immunized mice.

RESULTSOne recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was constructed. Sequencing results of PCR products and Western blot tests showed that the E7E6 fusion genes were correct and expressed in CEF cells infected with the recombinant vaccinia virus. The specific antibodies against E6 and E7 proteins were elicited, however no positive responses were detected by ELISPOT in immunized mice.

CONCLUSIONSOne recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was generated and elicited specific antibodies against E6 and E7 proteins, but detected no positive cellular immune responses in immunized mice, which will provide the basis to develop the different animal model for examining the cellular immune responses of HPV18E6 and E7 proteins.

Animals ; Antibodies, Viral ; blood ; DNA-Binding Proteins ; genetics ; immunology ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Human papillomavirus 18 ; genetics ; immunology ; Humans ; Immunization ; Male ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus Infections ; immunology ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccinia virus ; genetics ; metabolism

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

OBJECTIVETo enhance the expression level of human papillomavirus (HPV) 16 L2E7 in Escherichia coli (E. coli), in aim of providing high-level expression of HPV16 L2E7 strain for pre-clinical high-throughout production.

METHODSThe whole L2E7 gene was optimized by software of Synthetic Gene Designer, reflecting E. coli codon usage. Two parts of codon-optimized gene were cloned into pET9a vector step by step. The positive clone, which was sequenced to be corrected, was transfected to BL21 (DE3+) via isopropyl-beta-D-thiogalactoside (IPTG) induction. They produced the HPV16 L2E7 fusion protein, which was further detected by SDS-PAGE and Western blot. The induction temperature, induction time, and IPTG concentration were also optimized by a series of experiments. Further purification modes of this protein were also explored.

RESULTSCodon-optimized HPV16 L2E7 was highly expressed in E. coli. The target protein accounted for nearly 60% of the total cell extract.

CONCLUSIONHigh-level expression of HPV16 L2E7 was successfully constructed.

Codon ; Escherichia coli ; genetics ; metabolism ; Human papillomavirus 16 ; metabolism ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the anti-tumor immunity of the non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins.

METHODSC57BL/6 mice were immunized by non-replicating recombinant vaccinia virus (NTVJmE6E7), and then specific CTLs were determined. Immune protection effects were evaluated by challenges of different doses of TC-1 tumor cells. Immunotherapeutic effects in form of recurrence were evaluated on the tumor-removed mice.

RESULTSMice immunized by NTVJmE6E7 could generate TC-1 cell specific cytotoxic T lymphocyte (CTL). Mice boosted with NTVJmE6E7 could tolerate the challenge of 1 x 10(4) TC-1 cells. NTVJmE6E7 could effectively prevent the tumor recurrence in the tumor-removed mice.

CONCLUSIONNTVJmE6E7 can be taken as a candidate of therapeutic vaccine for HPV-associated tumors and their precursor lesions.

Animals ; Cancer Vaccines ; Cells, Cultured ; Female ; Genetic Vectors ; Immunotherapy ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Neoplasms, Experimental ; immunology ; therapy ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Recombination, Genetic ; Repressor Proteins ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccinia virus ; genetics ; Viral Vaccines

OBJECTIVETo express HPV31 and 52 L2 fusion protein and detect its immunogenicity.

METHODSAccording to the amino acid sequences of HPV31 and 52 L2 11-200AA published in the GenBank database, weartificially synthesized the HPV31 and 52 L2 fusion gene which was optimized according to Escherichia coli codon usage and encodes 11-200 amino acid of HPV31 and HPV52 L2, then cloned it into pET-9a vector. The HPV31 and 52 L2 fusion protein was expressed in Prokaryotic expression system and the mice were immunized with the fusion protein after purification. The immunogenicity was characterized in vaccinated mice.

RESULTSHPV31 and 52 L2 fusion protein was highly expressed in E. coli, the amount of fusion protein is nearly 20% of the total bacterial protein. The purified fusion protein with aluminum adjuvant could induce specific high titer of IgG antibodies detected by ELISA, and also induce the neutralizing antibodies against pseudovirus of HPV31 and HPV52 and cross-neutralizing antibodies against pseudovirus of HPV45, 58, 16, 18.

CONCLUSIONHPV31 and 52 L2 fusion protein could induce neutralizing and cross-neutralizing antibodies against HPV pseudovirus. It provides laboratory basis for development of HPV L2 protein vaccine.

Animals ; Antibodies, Viral ; blood ; Escherichia coli ; genetics ; Female ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; immunology ; Papillomavirus Vaccines ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification

OBJECTIVETo generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer.

METHODSHPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified.

RESULTSDNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus.

CONCLUSIONNTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.

Animals ; Blotting, Western ; Capsid Proteins ; genetics ; metabolism ; Cells, Cultured ; Chick Embryo ; Cloning, Molecular ; Female ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomaviridae ; genetics ; immunology ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; immunology ; prevention & control ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; therapeutic use ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Transfection ; Tumor Virus Infections ; immunology ; prevention & control ; virology ; Uterine Cervical Neoplasms ; immunology ; prevention & control ; virology ; Vaccinia virus ; genetics ; Virus Replication

OBJECTIVETo construct the Escherichia coli (E. coli) prokaryotic expression system pET9aHPV11L2E7, purify the fusion protein L2E7 and study the immunnogenicity of the protein.

METHODSThe HPV11 L2, E7 coding region was amplified from condyloma acuminata tissue specimen by PCR. The recombinant plazmid pET9aHPV11L2E7 was established and sequenced. Fusion protein L2E7 (553 amino acids) was expressed in host strain BL21 (DE3plus) by IPTG inducing and identified by using SDS-PAGE and Western blotting. Then L2E7 protein purified with CM column was inoculated to Balb/c mice and its cell-mediated and humoral immunnogenicity was assessed by IFN-gamma enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA).

RESULTSThe E. coli prokaryotic expression system pET9aHPV11L2E7 was established and the purified fusion protein L2E7 was obtained successfully. The mice in vivo experiment indicated that the purified protein L2E7 could induce HPV11E7 specific cell-mediated immune responses and high level HPV L2E7 antibody was detected in serum.

CONCLUSIONThe purified fusion protein L2E7 could induce specific cell-mediated and humoral immune responses. It can be used as a candidate of genital wart immune therapeutic vaccine.

Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Human papillomavirus 11 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins, Viral ; genetics ; immunology ; isolation & purification ; Papillomavirus Infections ; immunology ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; isolation & purification ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification

In order to fabricate the HLA-DQA1 genotyping chip and develop an integrated, parallel technical platform to type HLA system, a pair of primers and a set of probes were designed according to the sequences of HLA-DQA1 exon 2, where the polymorphism is concentrated. The oligonucleotide chip was made with the methods developed in our laboratory. The target DNA was asymmetrically amplified with the labeled sense primer. The signals were scanned and analyzed after the hybridization between microarray and PCR product. The allele types of the samples were identified. The result was verified by the standard DNA and DNA sequencing. The results showed that the genotyping was successfully carried out in 50 standard DNA samples and 50 clinical samples. Among them, results of the 50 standard DNA samples matched their templates. In the other 50 samples, results of the randomly selected 10 matched their sequencing results except that two of them got the incompletely result. In reproducible tests, the signal reappear rate was 95%. It is concluded that HLA-DQA1 genotyping by using our array system is simple and convenient with satisfied accuracy and reproducibility.
Genotype ; HLA-DQ Antigens ; genetics ; immunology ; HLA-DQ alpha-Chains ; Humans ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo identify specific T lymphocyte epitope on E6 protein of human papillomavirus type 18 in mice.

METHODSInfection with one recombinant vaccinia virus rVVJ18 E7, E6 respectively in C57 BL/6 and BALB/c mice, specific cellular immune responses were detected by ELISPOT or intracellular cytokine stainings by using a series of overlapping synthetic peptides covering full length of the amino acid sequence of E6 and E7 proteins or various truncated peptides.

RESULTSThe rVVJ18 E7, E6 generated significant E6 specific T-cell immune responses in vaccinated mice. Mapping of the epitope of E6 revealed that the peptides E6(67-75 ( KCIDFYSRI) and E6(60-68) (IPHAAGHKC) presented respectively by C57BL/6 and BALB/c mice were the optimal peptides to activate E6-specific CD8+ T lymphocytes. However no positive cellular immune responses stimulated with various E7 peptides were detected by ELISPOT in immunized mice.

CONCLUSIONSTwo specific T lymphocyte epitopes were identified on E6 protein in C57BL/6 and BALB/c mice, which will provide the basis to evaluate cellular immune response elicited by HPV18 E6 protein based vaccine.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; DNA-Binding Proteins ; immunology ; Epitopes, T-Lymphocyte ; immunology ; Human papillomavirus 18 ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; immunology ; Papillomavirus Vaccines ; immunology

OBJECTIVETo express HPV6bL2deltaN360E7E6 fusion protein in E. coli and preliminarily evaluate its immune effect.

METHODSThree HPV6b gene fragments, which were L2(1-360 bp), E7 and E6, were fused by overlapping PCR, then were inserted into a prokaryotic expression vector and expressed in E. coli. C57BL/6 mice were immunized with purified fusion protein plus Al (OH)3 and/or CpG adjuvants through intramuscular route, the cellular and humoral immune responses were detected by IFN-gamma ELISPOT and ELISA respectively.

RESULTSProtein plus CpG adjuvant could induce the strongest cellular immune response to E7 and E6, high antibody titer against L2 could be detected in all immunized groups but there were no significant difference among these groups.

CONCLUSIONSHPV6bL2deltaN360E7E6 gene was successfully cloned into pQE30 vector and expressed in E. coli, the fusion protein was also purified and proved that could induce strong cellular and humoral immune responses with appropriate adjuvant in C57BL/ 6 mice and could be used for future research.

Adjuvants, Immunologic ; Animals ; Condylomata Acuminata ; genetics ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; methods ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Immunity, Humoral ; Immunization ; Mice ; Mice, Inbred C57BL ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; chemistry ; genetics