| Human papillomaviruses (HPVs) infect people's epithelial cells in the skin and mucosa. They are closely linked with the benign and malignant hyperplasia of epithelial cells. Some subsets of HPVs cause benign hyperplasia of epithelial cells such as skin warts, condyloma acuminata and so on. The other subsets also cause genital cancers, such as cervical cancer, cancers of skin and the oral cavity. Due to no specific therapeutic methods, HPV infection is highly detrimental to human's health. Therefore, there is a great need to develop a safe and effective vaccine to prevent and treat HPV-induced diseases. Since we are not able to cultivate HPV in large quantities in the vitro at present, it is difficult to develop a traditional vaccine. Genetic immunization with naked DNA has emerged as an important strategy for vaccine development. DNA vaccines have a lot of advantages. For example, they are easy to prepare, safe and stable and capable of inducing persistent immune responses. The E7 oncoprotein of HPV16 is potentially an ideal target of immune therapy. Study showed that the HPV16 E7 vaccines can induce persistent cell-mediated immunity and humoral immunity, protect the mice from the attacks of HPV16 positive cancer cells, suppress the growth of the existing tumor or even clear it. However, when the vaccine is used on primates, its immunogenicity decreases, and when it is used on humans, the immune responses it induces are even weaker. As the vaccine will ultimately be used on humans, its immunogenicity must be enhanced. Research shows that immune response can be enhanced by co-administering some cytokines. Interleukin-15 (IL-15) is a recently identified cytokine which shares many biological properties of interleukin-2 (IL-2) such as activation of T cell, particularly CD8+CTLs. Unlike IL-2, it also plays a central role in the development and function of NK cells. IL-15 has been used in some experiments as an adjuvant and has significantly enhanced the induction of Ag-specific immune responses. But so far, there have been no reports concerning its effects on the immunity of HPV16 E7. This experiment subcloned IL-15 and constructed its eucaryon plasmids, which were injected along with the HPV16 E7 vaccine into BALB/c mice. Then, we investigated its effects on the Ag-specific immune responses of the mice. Dendritic cells (DCs) are the most effective antigen-presenting cells (APC) known that express a high amount of MHCâ… and â…¡molecules as well as costimulatory molecules. Presently, DC-based tumour immune therapy has been demonstrated to be effective for activating antigen specific T-cell responses against tumour cells.In HPV –associated tumour studies,application of antigen,peptide,virus-like particle and viral gene delivery has been tested for its ability to prime DC and then induce antitumour effects.Recent clinical trials using DC suggested a possible efficacy for treatment of HPV-associated cervical cancer.These findings support potential applications of DC for efficient promotion of antitumour immunity as an anticancer therapeutic modality.In order to increase further of immunotherapy efficacy we stimulated DC with HPV16E7 gene and IL-15 and evaluated the levels of immune responses induced by DC stimulated with E7+IL-15. New there have been no reports. 1 Construction of a DNA vaccine of HPV16 E7 gene and a IL-15 recombinant plasmid Objective:HPV16 E7 gene is persistently expressed in cervical cancer cells and therefore is considered a suitable tag for therapy of HPV-related cancers. Experimental studies have demonstrated that E7-specific T lymphocytes can prevent growth of tumour cells.This experiment co-immunized mice with pE7 as immunogen and pIL-15 as adjuvant in order to explore cell-mediated immune responses in vivo and in vitro. Methods: (1) According to DNA sequence of HPV16E7 in Genbank, thespecial primer of HPV16E7 were designed with Primer5. (2) The DNA fragment was amplified by PCR with HPV16 DNA template. (3)The amplified product was digested with EcoRI and BamHI which had 300bp and gel recovered. (4) pcDNA3.1 was digested with EcoRI and BamHI as well. Then the E7 DNA fragment and pcDNA3.1 were linked with T4 DNA ligase. (5) pGEM-T-IL-15 was digested with EcoRI and BamHI, the IL-15 gene fragments which had 500bp was recovered by gel.Then the IL-15 gene fragment and pcDNA3.1 were linked with DNA ligase. (6) The linked products were transfected into bacillus coli DH-5αcells and be cultured all night in LB medium. The DNA was amplified and purified .The integrity of plasmid DNA and the absence of Escherichia coli DNA was checked in each preparation using 1% agarose gel electrophoresis.DNA concentration was determined by the absorbance measured at 260 nm and 280 nm.The presence of inserted E7 fragment was confirmed by restriction enzyme digested with EcoRI and BamH. The amplified and purified was run in the same ways. (7) All plasmids were diluted with N.S. to the concentration needed and conserved in -20℃. Results: (1) The 300bp gene fragment which received from pcDNA 3.1-E7+ recombinant plasmid were equaled to intented gene fragment. (2) The 500bp gene fragment which received from pcDNA3.1-IL-15 were equaled to gene fragment which received from pGEM-T-IL-15. 2 Enhancement of HPV16 E7 DNA Vaccine-induced Specific Immune Response by Coimmunization with Il-15 Recombinat plasmid. Objective:IL-15 is a new T cell cytokine and can produce a kinds of biological effect. IL-15 can enhance cytotoxic T lymphocyte (CTL) activity of NK cells and stimulate cytokines induced NK cells. IL-15 has been used in some experiments as an adjuvant especially in HIV gene vaccine experiments and were confirmed that is potentially effective cytokine adjuvant. But so far, there have been no reports concerning its effects on the immunity of HPV16E7. Therefore this experiment subcloned IL-15 and constructed its eucaryon plasmids, which were injected along with the HPV16 E7 vaccineinto BALB/c mice. Then, we investigated its effects on the Ag-specific immune responses of the mice. Methods: Female BALB/c mice, which were divided randomly into seven groups, were immunized at the 6th week of age. All animals were pretreated with 100μl of 25% cane sugar each in tibialis anterio muscle 30min before the DNA immunization. One group of mice were then injected with a mixture of pE7+pIL-15, 100μg per mice, boostered with the same DNA vaccine after two weeks for three times. Mice were immunized respectively with pE7+pc,pIL-15+pc,pE7,pIL-15,pc and N.S. with the same method as control groups. After the last immunization, mice were killed, and the concentration of serum IFN-γwere determined by specific enzyme-linked immunosorbent assay (ELISA). Single-cell suspensions of splenocytes were prepared from mice of every group, and were restimulated with HPV16 E7 peptide or mediam only. Then, the T cell proliferation assay was measured by the MTT method. The disparity vale of OD570 represents the degree of T cell proliferation. Analysis of variance(ANOVA) were employed to test for differences in group means, A P value of less than 0.01 was considered to be significant. T cells subset in splenocytes and peripheral blood were detected by FCM. Results: (1) The concentration of serum IFN-γin the immunized mice with pE7+pIL-15,pE7,pE7+pc,pIL-15,pIL-15+pc,pc is 414.1 pg/ml, 152.12 pg/ml,153.24 pg/ml, 51.06 pg/ml,50.41 pg/ml, 16.32 pg/ml respectively. No IFN-γwas detected in mice administered with N.S. The concentration of IFN-γin the group of pE7+pIL-15 was raised to 414.1 pg/ml, which were significantly higher than that others (p<0.01). The concentration of IFN-γin the group of pE7 and the group of pE7+pc were induced to the higher levels.(2) The IFN-γproduction in splenocyes was significantly induced by E7 peptide in the group of pE7+pIL-15,in the group of pE7 and pE7+pc (p<0.05),as compared with the group.(3) The mice immunization with pE7 can induce a high level of specific T cell proliferation (The disparity vale of OD570 is 0.644), which is statistically significant with that of mice injected with pc and N.S. (P<0.01). Coadministration pIL-15 with pE7 can enhance specific T cellproliferation (The disparity vale of OD570 is 1.313), it is statistically significant with that of any control group of mice (P<0.01). (4) The detected results by FCM showed the percentages of CD4+ T cells and CD8+ T cells in splenocytes were increased in the group of pE7+pIL-15, especially CD8+T cells more significantly.(5) the percentages of CD4+ T cells and CD8+ T cells in peripheral blood in the group of pE7+pIL-15 were enhanced a bit higher than others (p<0.05). 3 The effects on the surface molecules and immune function of murine bone marrow-derived dendritic cells modified by HPV16E7 DNA vaccine and IL-15 Objective:DCs are an unique set of highly potent APCs that are very important players in inducetion and maintenance of immunity against a variety of pathogens,including viruses as well as tumors. Now It was not seen report that the relationship of HPV16E7+IL-15 and DCs.Therefore, DC-co-stimulate with E7+IL-15 were detected on surface marker, IL-12 and IFN-γto expore their immune activity. Methods: (1) Bone marrow DC were obtained from BALB/C mice and treated further with GM-CSF and IL-4. (2) DCs were treated in vitro with pE7+pIL-15 (50 μg/ml and 100 μg/ml),pE7 (50 μg/ml and 100 μg/ml),pIL-15 (50μg/ml and 100μg/ml),pc (50μg/ml and 100μg/ml) and control group, respectively. (3) Each sample was analysed using flow cytometry. The tested cells expressed surface marker ,such as CD40,CD80 and CD86 co-stimulatory molecule.These data confirm that the cells are bone marrow-derived DC in their phenotype. Moreove, the T cell subsets activied by DC were detected by FCM. (4) The mixed lymphproliferation response were detected by MTT. (5) Production of IL-12 from DC and the level of IFN-γin splenocytes suppernatans were detected by ELISA . Results: (1) The expression of co-stimulatory molecules ( CD40,CD80 and CD86) on surface of DCs detected by FCM were found a up-regulation.The expression of co-stimulatory molecules in the groups of pE7 and pIL-15 were significantly increased,as CD40(87.14%,90.44%), CD80 (74.36%,62.93%), CD86(83.17%,68.37%). But the expression ofDC-pE7+pIL-15 were downregulated (CD40 50.82%, CD80 27.87%, CD86 31.36%). (2) In MLR, the higher vaule of proliferation activity in the group of DC-pE7+pIL-15 were demonstrated than others (p<0.01). The vaule of proliferation activity is 0.32±0.08(1:1) and 0.42±0.14(1:10) in the group of DC-pE7+pIL-15,0.13 ±0.04(1:1) and 0.29 ±0.09(1:10) in the group of DC-pE7,0.17±0.07(1:1) and 0.27±0.08(1:10) in DC-pIL-15,0.04±0.02(1:1) and 0.07±0.04(1:10) in DC-pc,0.08±0.03(1:1) and 0.12±0.03(1:10) in N.S.(3) The detected results by FCM showed the percentages of CD4+ T cells and CD8+ T cells in splenocytes were increased in the group of DC-pE7+pIL-15(36.78 ±3.051,28.44 ±2.792) and DC-pIL-15(28.34 ±2.084,20.28 ±2.983), especially CD8+T cells more significantly. The percentage of CD4+ T cells in splenocytes also were increased in the group of DC-pE7.(4) IL-12 p70 production from DCs was more significant when DC were treated with pE7+pIL-15, as compared the other groups (p<0.01).The contents of IL-12p70 in groups were control(18.0 ±3.80),pc(27.3 ±3.54),pE7(40.1 ±2.1),pIL-15(38.5 ±4.7),pE7+pIL-15(78.4 ±6.6). (5) The IFN-γproduction induced by DCs in lymphocyte suppernatans were 1168.0±58.4(pg/ml)( in DC-pE7+pIL-15), 610.0±42.7(pg/ml) ( in DC-pE7), 968.0±25.5(pg/ml) ( in DC-pIL-15), 228.0±15.9(pg/ml) ( in DC-pc), 135.8(pg/ml) (in control), respectively. The IFN-γproduction in the group of DC-E7+IL-15 were significantly increased (p<0.01). CONCLUSION 1 The pcDNA3.1-HPV16E7 DNA vaccine and pcDNA3.1-IL-15 recombinant plasmid were contructed in this study. 2 The pcDNA3.1-E7 can induce special T lymphopoiesis response, the contents of IFN-γin serum detected showed that pcDNA3.1-E7 has immunogenicity and induced cell mediated immunity in mice. 3 It can enhance special T lymphopoiesis and increase IFN-γin serum by injecting pIL-15 and pE7 altogether. pIL-15 can increase the...
|
PDF Full Text Request