Strengthen The Human Papillomavirus Type 16 Of Me7/hsp70 Experimental Study Of The Dna Vaccine

Studies have suggested that the primary reason of cervical cancer are correlated directly with the persistent infection with high-risk HPV (Human papillomavirus), in which HPV 16 causes 56% cases of cervical cancer all over the world. At present, there are no effective therapeutic vaccines of cervical cancer on market. China is the high prevalence region of cervical cancer, it is very important to study and develop therapeutic vaccines targeted HPV 16.Ideal therapeutic vaccines are those vaccines that could activate tumor specific immunity, especially specific cellular immunity, so scientists attach great importance to versatile immunostimulating molecules in the therapeutic vaccines of tumor and virus, it is very significant in the use of DNA and protein vaccines especially. Comparing with live recombinant microbial vaccines or inactivated vaccines, although these vaccines possess single and definite ingredients and good safety, they are deficient in the ability to produce "danger signal" and not enough in the ability to activate innate immunity and antigen presenting cells, so go against the effective activation of specific immunity. Study has suggested that HSP originated from different species is a strong molecular adjuvant, which could induce APC to secret proinflammatory cytokines, up regulate the expression of MHC (Major histocompatibility complex) and costimulating molecules on DC (Dendritic cell), prompt APC to uptake antigen, play a role of molecule chaprone in the process of antigen processing and presenting and cross present the exogenous antigen peptide to specific CD8+T cell, facilitate the complete activation of immune response, especially the activation of CD8+T cell.HSP70 is one of the members of heat shock protein family with very strong imunostimulating activity; quantities of studies have suggested that the vaccines based on HSP70/peptide compound are able to enhance peptide specific anti-tumor immunity, these kinds of vaccines may be categorized into two groups, one is regular HSP70 fusion antigen vaccine, another is HSP70 fusion antigen vaccine preceded with signal peptide, such as the tumor cell vaccine transfected with the HSP70/peptide compound gene expression plasmid with signal peptide gene and HPV16E7/HSP70 DNA vaccine preceded with a signal peptide sequence. Studies suggested that these HSP/peptide compound vaccines with signal peptide are able to enhance specific anti-tumor immune as well as regular HSP/peptide compound vaccines, till today we haven't found the comparative study report about immune activity of these two forms of HSP/peptide compound vaccines.Safety and immunogenicity are the two key factors should be considered when developing effective vaccine. Through introducing HSP70, a kind of immune adjuvant, to enhance the immunogenicity of DNA vaccine will possibly acquire tumor therapeutic DNA vaccine with an enhanced immunogenicity, we also should consider the possible potential risk of adjuvant molecule used in human body, besides consider the safety of regular DNA vaccine. Study in the mice suggested that mycobacterium tuberculosis derived HSP70 could induce body to produce autoimmune-mediated intestine inflammation, further study suggested the reason of autoimmune-mediated intestine inflammation is the production of autologous HSP70-reactive T lymphocytes.The comparative study of immune activity between human derived HSP70/peptide compound vaccines and mycobacterium tuberculosis derived HSP70 could provide reference basis for the selection of corresponding vaccine used in human body.During the previous work of our lab, we have acquired the mE7 gene (Optimized and modified E7 gene) with deleted transformation activity and enhanced immunogenicity by combining with the methods of gene shuffling, point mutation and codon modification. The HPV16 therapeutic vaccines using mE7 gene as target antigen gene own the value of further development, so we apply for and acquire the Chinese invention patent. In order to prompt the further study of HPV16 therapeutic vaccines based on mE7 and develop the HPV16 therapeutic DNA vaccines possessing self-owned intellectual property rights as soon as possible, this study will construct pVR1012-SigmE7/MtHSP70 DNA vaccine with signal peptide gene and pVR1012-SigmE7/HuHSP70 DNA vaccine with signal peptide gene based on the regular pVR1012-mE7/MtHSP70, after that, compare with the difference of immune activity between regular pVR1012-mE7/MtHSP70 DNA vaccine and pVR1012-SigmE7/ MtHSP70 DNA vaccine with signal peptide gene, at the same time, compare with the difference between human derived pVR1012-SigmE7/HuHSP70 and mycobacterium tuberculosis derived pVR1012-SigmE7/MtHSP70 HSP70/peptide compound DNA vaccine.The methods and results are following:1.The construction of pVR1012-SigmE7/MtHSP70 and pVR1012-SigmE7/ HuHSP70:Using pVR1012-mE7 as template, acquired the SigmE7 gene with PCR and fused SigmE7 with MtHSP70 in C terminal and human CD33 signal peptide gene in N terminal together by enzyme cutting and linkage method, constructed the pVR1012-SigmE7/MtHSP70;using pMSHsp70 including human HSP70 gene as template, acquired the HuHSP70 gene with PCR and took place of the MtHSP70 in pVR1012-SigmE7/MtHSP70 with HuHSP70 and constructed pVR1012-SigmE7/ HuHSP70, identification of digestion with restriction enzyme and DNA sequencing results are correct.2. The expression identification of recombination plasmids:Extracted and purified plasmids DNA using methods of alkaline lysis and polyethylene glycol (PEG), transfected COS-7 cells with Lipofectamine 2000, collected cell lysates and supernatants after 48 hours, quantitified the total proteins of cell lysates and supernatants with BCA Protein Assay Kit, loaded sample of 1/10 volume cell lysates and supernatants respectively and identified the expression level of fusion protein in cell lysates and supernatants.Results suggested that corresponding fusion proteins were expressed in cell lysates and supernatants of pVR1012-mE7/MtHSP70, pVR1012-SigmE7/MtHSP70 and pVR1012-SigmE7/HuHSP70, their relative molecular weights were about 85,86 and 100kDa. The expression level of fusion proteins in cell lysates were higher than the expression level in supernatants, there were no significant difference of three fusion proteins in cell lysates and no significant difference of three fusion proteins in supernatants, all above results suggested that the recombination plsamid without signal peptide also expressed in extracellular matrix, there were no diffirence of extracellular expression level between recombination plasmids with signal peptide gene and without signal peptide gene, three fusion proteins were expressed in cytoplasm mainly. 3. Animal immunization and in vitro immune activity assay of vaccine:Female C57BL/6 mice of 6-8 weeks were divided into four groups randomly, with 7 mice in each group, named pVR1012-mE7/MtHSP70 (mE7/MtHSP70), pVR1012-SigmE7/MtHSP70 (SigmE7/Mt HSP70), pVR1012-SigmE7/HuHSP70 (SigmE7/HuHSP70) and Normal saline (NS) control group respectively. Each mouse was injected i.m. into the M. quadriceps with 50 ug on each side, these injections were repeated after 1 week, at 10 days after the second vaccination, harvested the splenocytes from the mice and restimulated with E749-57 and E730-67 at 2μg/ml respectively and testified the quantity of E7 specific CD8+T cells secreting IFN-γwith ELISPOT assay and the quantity of E7 specific CD8+T cells secreting IFN-y, CD4+/IFN-γ+Th1 cells and CD4+/IL-4+Th2 cells with flow cytometry, and collected the mice serum and identified the E7 specific antibody with ELISA. The results showed that the spot numbers of spleen cells secreting IFN-y in the groups of mE7/MtHSP70, SigmE7/MtHSP70, SigmE7/HuHSP70 and NS were 503,100,651 and 5 respectively in ELISPOT assay. The numbers of CD8+/IFN-γ+ double positive T cells were 497,389,686 and 280, the numbers of CD4+/IFN-γ+double positive T cells were 385,386,389 and 383 and the the numbers of CD4+/IL-4+double positive T cells were 165,168,158 and 150. The results indicated that mE7/MtHSP70 immunized mouse generated much higher CD8+T cell response than SigmE7/MtHSP70 with signal peptide immunized mouse, human derived SigmE7/HuHSP70 generated much higher CD8+T cell response than mycobacterium tuberculosis derived SigmE7/MtHSP70, there were no significant CD4+T cell response mediated by CD4+/IFN-γ+-Th1 cells or CD4+/IL-4+-Th2 cells in three DNA vaccines and no significant enhanced E7 specific antibody level in three DNA vaccines.4. In vivo anti-tumor activity experiments:In the prevention assay, immunized DNA vaccine intramuscularly in advance, on the 7 days after the last DNA vaccination, the female C57BL/6 mice were injected s.c.in the right flank with 7.5×104 TC-1 cells expressing HPV16E6/E7, on the 12 days after TC-1 were injected, one mouse in SigmE7/MtHSP70 group formed the tumor, the mice of mE7/MtHSP70 and SigmE7/HuHSP70 didn't form the tumor and the mice of NS group all formed the tumor; on the 55 days after TC-1 were injected, the rate of tumor-free mices of mE7/MtHSP70, SigmE7/MtHSP70 and SigmE7/HuHSP70 were 100%,86%and 71%.In the treatment assay, the female C57BL/6 mice were injected s.c. in the right flank with 7.5×104 TC-1 cells in advance, on the days after TC-1 injection, immunized DNA vaccine intramuscularly according to above methods.On the 14 days after TC-1 were injected, one mouse in SigmE7/MtHSP70 group formed the tumor, the mice of mE7/MtHSP70 and SigmE7/HuHSP70 didn't form the tumor and the mice of NS group all formed the tumor; on the 20 days after TC-1 were injected, one mouse in mE7/MtHSP70 group formed the tumor and another mouse in SigmE7/MtHSP70 group formed the tumor, there were no mice formed the tumor in SigmE7/HuHSP70 group; on the 60 days after TC-1 were injected, the rate of tumor-free mices of mE7/MtHSP70, SigmE7/MtHSP70 and SigmE7/HuHSP70 were 86%,71% and 100%. The results indicated that regular mE7/MtHSP70 immunized mouse generated much higher anti-tumor effector than SigmE7/MtHSP70 with signal peptide immunized mouse, human derived SigmE7/ HuHSP70 generated higher anti-tumor effector than mycobacterium tuberculosis derived SigmE7/MtHSP70, these results were consistent with the results of ELISPOT assay and flow cytometry.In summary, the above results suggested that comparing with the SigmE7/MtHSP70, regular mE7/MtHSP70 owned the stronger E7 specific CD8+T cell response and stronger anti-tumor activity. Human derived HSP70 played a higher immunostimulating activity than mycobacterium tuberculosis derived HSP70.The anti-tumor activities of the three kinds of mE7/HSP70 DNA vaccine were induced by antivited CD8+T cell. This study also suggested that the regular mE7/HuHSP70 DNA vaccine was eager to generate the stronger specific anti-tumor immunity, this study built a base for the experimental study of tumor therapeutic DNA vaccine of cervical cancer associated with HPV16 and other tumors known to have tumor specifi antigens.