The Study On Cellular Immune Function Induced By Combination Immune Of Japanese Encephalitis DNA Vaccine And ICAM-1 Coding Gene Recombinant In BALB/c Mouse

ObjectiveWe constructed fusion plasmid named pJME/ICAM-1 with Japanese encephalitis virus (Japanese encephalitis virus, JEV) prME protein and BALB/c mouse intercellular adhesion molecule(intracellular adhesion molecule-1, ICAM-1) encoding gene, and pure plasmid named pICAM-1 encoding ICAM-1. To compare the adjuvant effect of ICAM-1 encoding gene in different ways on cell-mediated immunity induced by JE DNA vaccine and explore ICAM-1 coding gene immune-enhancing effect mechanism.Materials and methodsMaterials1,Cells and animalsChinese hamster ovary (CHO) cells, P815 cells were purchased from Shanghai Institutes for Biological Sciences, China. CHO cells were used for the transfection experiment and P815 cells were used as target cells; Female,4-week-old BALB/c mice were obtained from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, and maintained in sterile cages under specific pathogen-free conditions. The mice were later used directly for the experiment on DNA immunization.2,Plasmids and strains The recombinant plsmid named pJME encoding JEV prME was provided in our laboratory; Prokaryotic expression vector pMD19-T simple and Eukaryotic expression vector pcDNA3.1(+) were purchased separately from Takara Biotechnology (Dalian) Company and the U.S. Invitrogen corporation for plasmid sequencing and construction; Escherichia coli JM109 and DH5a were purchased from Takara company and used for expansion of recombinant plasmids.3,Main reagentsPlasmid Abstraction Kits were purchased from QIAGEN company; Lipofectamine2000 was purchased from Invitrogen company and was used for transfection;Goat-Anti-Mouse ICAM-1 were purchased from Santa company, and Fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP) labeled rabbit anti-goat IgG were purchased from Beijing Zhong Shan company, and they were used for immunofluorescence and western-blot analysis; The CytoTox96 Non-Radioactive Cytotoxicity assay was purchased from Promega company and used to detect mouse splenocyte CTL activites; Anti-CD3-PerCP, CD4-APC, CD8-PE, CD54-PE, CDllc-APC, MHCⅡ-FITC, CD80-PE, CD86-FITC monoclonal antibody were purchased from BD company and used to detect phenotype of spleen dendritic cells(DC); Anti-CD4-APC, CD8-PE monoclonal antibody were purchased from BD company and used to detect spleen T-lymphocytes subsets; Carboxyfluorescein diacetate, succinimidyl ester(CFSE) and FITC-dextran were purchased from Molecular Probes company and respectively used to detect mixed lymphocyte reaction(MLR) and phagocytic function of DC; Lympholyte-M was purchased from Canada Cedarlane company. Mitomycin C was purchased from Sigma company. IL-2 was purchased from Peprotech company. G418 was purchased from GiBCO company and used for screening of resistant clone; Restriction endonuclease EcoRI,BamHI and NotI were all purchased from Takara company, which were used construction and identification of recombinant plasmid. IL-4, IL-10, IL-12, IFN-y ELISA kit were purchased from the Shanghai SenXiong Technology company. ECL luminescence kit purchased from the United States Pierce company.Methods1,Construction and identification of pICAM-1,pJME/ICAM-1The coding sequence of murine ICAM-1 (1614 bp) was amplified by nested RT-PCR from total RNA isolated from stimulated BALB/c murine splenocytes.A 1659-bp fragment of murine ICAM-1and Gly-linker was digested with restriction enzymes Eco R I and Not I from pMD19-T simple-ICAM-1 and subcloned into the pcDNA3.1(+) vector at the EcoRI/Not I site. The construct containing the murine ICAM-1 gene was designated pICAM-1. To construct a DNA vaccine encoding fusion protein of prME and ICAM-1 (pJME/ICAM-1), a fragment of 2,001 bp of JEV prME isolated from pJME by BamH I and EcoR I digestion was inserted into the BamH I/EcoR I site of p ICAM-1.pICAM-1 and pJME/ICAM-1 transforming competent Escherichia coli DH5αwere amplified and extracted, and then verfied by restriction enzyme digestion and DNA sequencing.Plasmid pICAM-1 and pJME/ICAM-1 was transfected into CHO cells by Lipofectamine TM2000 according to the manufacturer's instruction. After 72h of transfection, the cells were selected with G418 (800μg/mL) for one week and then cultured with G418 (400μg/mL) for four weeks for establishment of stable transfectants. CHO cells transfected with an empty pcDNA3.1(+) vector and normal CHO were used as a control, stable transfectants were detected for expression of ICAM-1 and fusion protein prME/ICAM-1 by immunofluorescence and Western blot analysis.2,Animals and immunization procedureWe used 5 groups of 4-week-old female BALB/c mice (n=8 per group) which were as follows:pJME, pJME/ICAM-1, pICAM-1. For the i.m. immunization, mice were injected with 100μg pJME/GM-CSF,100μg pJME with 100μg of plasmid pICAM-1 into the quadriceps muscle mass of the left hind leg. All the mice received 2 booster doses in the same muscle (containing the same amount of plasmid as in the primary dose) 3 and 5 weeks after the primary injection. For the positive control group, each mouse was immunized with inactivated vaccine, a formalin-inactivated mouse brain-derived JEV vaccine (Beijing-1 strain) obtained from Liaoning Province Center of Disease Control and Prevention, and each mouse of the inactivated vaccine group was given an injection of 100μl (1/5 of a recommended adult dose) of inactivated vaccine and boosted with the same dose 3 and 5 weeks after the first immunization. The pcDNA3.1(+) immunized group was used as the negative control. The volume of vaccine solution injected into each thigh was adjusted to 100μl per mouse with PBS.3,Mouse spleen T lymphocytes and dendritic cells SeparationMouse spleen cell suspension were enriched according to the published methods. Briefly, mouse spleens were disrupted and cut up into small pieces, and passed through a fine screen mesh. A clean suspension was adjusted the cell concentration to a maximum of 2 x 107 nucleated cells per mL. Preparation of splenic T lymphocytes were performed in strict accordance with the instructions within the reagent kit. Another part of the spleen cell suspension were centrifuged at 1300 rpm for 5 min, resuspended in RPMI 1640 medium supplemented with 10% heat-activated fetal calf serum,2 mmol/1 Lglutamine,1 mmol/1 pyruvate,50 mmol/1 mercaptoethanol,100 U/mL penicillin, and 100 mg/mL streptomycin, and then incubated for 2 h at 37℃in a 5% CO2 atmosphere in plastic cell cultures plates. Culture plates were then washed vigorously four times with supplemented RPMI 1640 medium, and the nonadherent cells were discarded. The residual adherent cells were maintained in the culture medium and incubated overnight at 37℃in a 5% CO2 atmosphere. After incubation, DCs(which exhibit adherence capacity in the first hours of culture) become nonadherent and float in the medium. The DCs were collected and immediately used in our assays.4,Measurement of phenotype and function of spleen DC Spleen DC were isolated and Flow cytometry detected DC surface molecules CD54, CD11c, MHCⅡ, CD80, CD86, DC uptake of FITC-Dextran capability and capacity of DC to stimulate T lymphocyte proliferation.5,Spleen DC T-lymphocyte subsets and CTL function detectionSingle-cell spleen suspensions were prepared, from which erythrocytes were removed. T-lymphocyte subsets of mouse spleen cells were assayed with flow cytometry technique, and CTL activity of mouse spleen cells was detected with lactic dehydrogenase release test method.6,Dectection of cytokines secreted by T cells and DCSteps were carried out by product specification. WellscanMK3 microplate reader (Lab-syste ms Dragon Company) detected immediately 492nm wavelength absorbance A value after color and IL-12, IFN-γ, IL-4, IL-10 levels were calculated according to the standard curve.Results1,Construction and identification of pICAM-1 and pJME/ICAM-1Upon restriction enzyme digestion, pICAM-1 was digested with EcoR I/Not I into 1 fragment, as predicted, at 1,614bp in size, and confirmed that the DNA fragment released from the recombinant plasmid corresponded to the ICAM-1 coding sequence from the published murine ICAM-1 gene sequence. Meanwhile, the pJME/ICAM-1 was cut with BamH I/ EcoRI and Bam HI/Not I into 2 fragments as predicted at 2,001 and 3,660 bp in size, and confirmed that 2,001 bp was the same as that of the JEV prME gene and 3,660 bp was the sum of the murine ICAM-1 gene and the JEV prME gene. With sequence analysis using the T7 promoter primer located in pcDNA3.1(+), it was indicated that there was a normal junction of BamH I site between vectors and the prME gene of JEV, and EcoR I site between vectors and the murine ICAM-1 gene.Immunofluorescence result showed that the green fluorescence mark was mainly distributed obviously in endochylema of CHO cells transfected with pICAM-1 and pJME/ICAM-1, and not much in membrane. There was no specific green fluorescence in CHO cells untransfected and transfected with pcDNA3.1(+).In Western blots, the specific ICAM-1 protein and fusion protein prME/ICAM-1 were clearly visible in CHO cells transfected with both pICAM-1 and pJME/ICAM-1, but almost undetectable specificity protein in cells transfected with pcDNA3.1(+). Anti-murine ICAM-1 reactive protein bands of about 60 and 130 kDa were respectively in correspondence with ICAM-1 protein expressed by pICAM-1 and prME/ICAM-1 fusion protein expressed by pJME/ICAM-1, and there was no a corresponding protein band by Western blot analysis of CHO transfected with pcDNA3.1(+).2,The impact of pICAM-1 and pJME/ICAM-1 on mouse spleen DC phenotype and functionDC of each group express high levels MHCⅡ, pJME/ICAM-1, pJME+ pICAM-1 and pJME group was higher than JE inactivated vaccine and empty vector vaccinated groups (P<0.05),there were no statistically significant difference among the three groups (P>0.05). DC surface CD80 expression in spleen from the pJME/ICAM-1 vaccinated group was the highest in all groups, although there was no significant difference between pJME/ICAM-land pJME+ pICAM-1 group (P>0.05). There was significant difference in the expression of DC surface CD86 between 2 random groups except that there was no significant difference between JE inactivated vaccine and pJME vaccinated groups. DC surface ICAM-1 expression in spleen from the pJME/ICAM-1 and pJME+pICAM-1 vaccinated group was higher than that in the other groups, and there was significant difference between the two groups (P<0.05); DC surface ICAM-1 expression in spleen from the pJME and JE inactivated vaccine vaccinated group was higher than that in the pcDNA3.1(+) vaccinated group, and there was no significant difference between the two groups(P>0.05).Phagocytic capacity of dendritic cells in the pJME/ICAM-1 and pJME+pICAM-1 vaccinated groups was obviously higher than those in other groups(P<0.05), and there was no significant difference between the two groups (P>0.05).In order to further analyze the antigen-presenting function of mouse spleen dendritic cell, we determined the stimulating ability of dendritic cells to T lymphocytes in spleens of DNA-immunized mice by flow cytometry. The percentage of CD4+T and CD8+T cells division in spleen cells was respectively (73.69±7.32)% and (45.40±2.57)% in the pJME/ICAM-1 vaccinated group, which significantly increased the division capacity of CD4+T cell compared with other groups (P<0.05). The percentage of CD4+T and CD8+T cells division in spleen cells was respectively (53.48 +5.23)% and (35.85±4.46)% in the pJME+pICAM-1 vaccinated group, obviously higher than those in the pJME, JE inactivated vaccine and pcDNA3.1(+) vaccinated groups (P< 0.05).3,Dectection of cellular immune responsesThe adjuvant effects of the ICAM-1 DNA on the generation of CD4+T cells or CD8+T cells from spleen were compared. The percentage of CD4+T cells in spleen cells was (36.1±6.45)% in the pJME/ICAM-1 vaccinated group, which significantly increased the relative frequency of CD4+T subpopulations compared with other groups (P<0.05). The percentage of CD4+T cells in the pcDNA3.1(+) vaccinated group was (20.87±4.45)%, and lower than in all other groups(P<0.05). The percentage of CD4+T cells in the pJME+pICAM-1 vaccinated group was (31.23±6.55)%, and higher than pJME and JE inactivated vaccine group(P<0.05). There was no significant difference in the levels of CD8+T cells between 2 random groups(P>0.05).Mice immunized with 100μg pJME/ICAM-1 had higher CTL activity than those immunized with the same amount of other naked DNA(P<0.05). The CTL activities of the spleens of BALB/c mice immunized 3 times with indicated immunogens were as follows:pJME/ICAM-1 (56.38±5.69)%; pJME+pICAM-1 (50.37±6.33)%; pJME (35.25±4.78)%; JE inactivated vaccine (26.72±3.78)%, and pcDNA3.1(+) (12.34±7.73)%. There was significant difference in CTL activity between 2 random groups(P<0.05). 4,Dectection of cytokines secreted by T cells and DCSecretion of T cells and DC in the pJME/ICAM-1 and pJME+pICAM-1 vaccinated group was obviously higher than those in the pJME, JE inactivated vaccine and pcDNA3.1(+) vaccinated groups(P<0.05), pJME/ICAM-1 group acquired higher levels of IL-12, IFN-γthan pJME+pICAM-1 vaccinated group did(P<0.05). There was no significant difference in the levels of IL-4 secreted by T lymphocytes between 2 random groups(P>0.05). JE. inactivated vaccine vaccinated group obtained higher levels of IL-10 secreted by T lymphocytes compare with other groups(P<0.05). JE inactivated vaccine vaccinated group showed a great increase in levels of IL-10 and IL-4 secreted by DC compared with other groups(P<0.05), and there was no significant difference between any other groups(P>0.05).Conclusions1,The constructed recombinant pJME/ICAM-1 and pICAM-1 were confirmed by restrict enzyme digestion and DNA sequencing, and could express effectively the target protein in eukaryotic cells;2,ICAM-1 coding gene can promote T-cell cytokine secretion, and induced CD4+T cells to Thl cell differentiation;3,ICAM-1 coding gene can enhance the cytotoxic effects of T cells to target cells;4,Dendritic cells can regulate Thl/Th2 immune balance through ICAM-1 coding gene;5,ICAM-1 coding gene can promote the maturation of spleen DC induced by JE DNA vaccine;6,ICAM-1 coding gene can amplify costimulatory signals that be independent of B7 molecules;7,The fusion plasmid immunization group induced a stronger cellular immune response relative to the combined immune group.