| "High risk" genotypes of the human papillomavirus(HPV), most commonly HPV genotype 16, are the primary etiologic agents of cervical cancer. Indeed HPV DNA is detected in 99% of cervical carcinomas. Thus, cervical cancer and other HPV-assoeiated malignancies might be prevented or treated by the induction of appropriate viral-antigen-specific immune responses. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation, therapeutic vaccines targeting E6 and E7 may provide the best opportunity to control HPV-assoeiated malignancies. Various candidate therapeutic HPV vaccines are currently being test whereby E6 and/or E7 are administered in live vectors, as peptide or proteins, in nucleic acid form, as components of chimeric VLPs, or in cell-based vaccines. Encouraging results for experimental vaccination systems in animal models have led to several therapeutic vaccine clinical trials, some trials are still ongoing. But, the results of the trials are not as desirable as expected. So, seeking ways to enhance the immunogeneeity is the emphases of therapeutic vaccine research.This study is about HPV therapeutic vaccine. HPV 16 E7 gene was chosen as target antigen gene in this study and the vaccine was in the form of nucleic acid (DNA). We optimized HPV 16 E7 gene in multiple ways to enhance the expression of antigen protein and to eliminate transformation activity, and then fused the optimized HPV 16 E7 (SHE7) gene with Myeobaeterial tuberculosis heat shock protein 70 (MtHSP70) gene, and constructed the recombinant plasmid of HPV16 ShE7/MtHSP70 fusing gene for DNA vaccination. To further enhance the vaccine efficacy, human intedeukin 15 (hIL-15) gene was used as molecular adjuvant for co-administration. After animal vaccination, NK cytotoxieity assay was performed and possible mechanism for elieitation of immune responses were discussed. The experimental strategy and results are as follows:1. HPV 16 E7 gene was optimized in multiple ways. First, we shuffled the gene by cut it into 3 parts a, b and e, and rearranged it in the abbc sequence, HLA*A0201 epitopes were added to the shuffled gene by AAY linker. Then we optimized the ShE7 gene with codons most frequently used in mammalian cells. Rb sites and zinc finger motifs were also mutated. These strategies were aimed at enhancement of immunogeneeity and elimination of transformation activities. The optimized gene were synthesized artificially.2. VR1012 was chosen as the eukaryotic vector. We constructed recombinant plasmid VR1012-HPV 16ShE7/MtHSP70 for DNA vaccination and VR 1012-WE7, VR1012-ShE7 for comparison. VR1012-hlL-15 and VR1012-hlL-2S/hlL-15 were also constructed and one of them was chosen for co-administration with VR1012- ShE7/Mt HSP70. All the recombinant plasmids were transfected transiently into Cos 7 cells and expression assays were performed with western blot, immunofluorescence and ELISA.3. Mice were vaccinated with recombinant plasmids mentioned above.6-8 weeks old C57BL/6 mice were divided into 6 groups randomly: WE7 group, ShE7group, ShE7/MtHSP70group, ShE7/MtHSP70+hlL-15group, hlL- 15group and empty plasmid VR1012 group was used for control. For DNA inoculation, 100μg of each DNA construct of interest in normal saline was injected to bilateral quadriceps muscles of C57BL/6 mice. In vivo electroporation was performed immediately after DNA inoculation at the injected sites. The immunization was boosted at week 2 and 4.Assays of immunogenecity and in vivo antitumor activity are necessary for therapeutic vaccine research. This study is the basis for further immunogenecity and in vivo antitumor activity assay and better understanding of the underlying mechanism of immune response elicited by coadministration of IL-15 and HPV16 ShE7/MtHSP70, it also provides experimental data for further clinical trial.
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