| Objective: Coxsackievirus group B(CVB), is the major pathogen of human viral myocarditis, which is responsible for over 50% of the cases and some of them develop into chronic myocaditis or dilated cardiomyopathy. CVB3 is the most common cause. So far, there is no specific strategy available to prevent CVB3 infection. VP1 is a major capsid protein of CVB3. It had been shown that the plasmid-based CVB3 VP1 DNA vaccine could induce the production of virus specific antibody in mice, but the titers of the antibody induced was too low to profect the mice from a lethal challenge of CVB3.In order to enhance the immunological effect of vaccines, some researchers constructed targeting DNA vaccines by recombining the genes of dendritic cells chemokines with target genes. These vaccines showed some advantages in uptaking, processing and presenting of antigen, and could promote the expression of co-stimulatory molecules by dendritic cells, which enhanced the immunogenicity of tumor antigens and virus antigens. Macrophage-derived chemokine (MDC), a CC type chemokine, has chemotactic effect on macrophage, dendritic cells and T lymphocyte. In our previous research, a fusion gene DNA vaccine pcDNA3/MDC-VP1 was constructed by recombining a MDC gene fused with a signal peptide gene with VP1 gene. A better protective effect was obserbed in mice immunized with this vaccine. But it could not still protect all the mice from a lethal challenge of CVB3.Compared with plasmid, adenovirus could enter a wider range of host cells, improve the expression of co-stimulatory molecules, induce the production of cytokine or chemokine and make a high expression of foreign genes. At the same time, Ad vector vaccines could not be integrated into the host's chromosomes and have no inserted mutagenicity. The Ad vector exhibited lots of superiorities as a vaccine vector. Using adenovirus as gene expressing vector, some researchs have shown satisfactory immune effects.In this study, AdEasy adenovirus system was used to construct recombinant adenovirus-based vaccine Ad/MDC-VP1, and the immune effects of the vaccine or the vaccine combined with DNA vaccine pcDNA3/MDC-VP1 were tested.Methods: 1 Construction of the recombinant adenoviral transfer plasmid pAdTrack-CMV/MDC-VP1: The fragment of MDC-VP1 was puriried from pcDNA3/MDC-VP1 cut with Hind III and XbaI and separated on agarose gel. The fragment obtaine was cloned into the transfer vector pAdTrack-CMV cut by the same endonucleases to construct the recombinant adenoviral transfer plasmid pAdTrack-CMV/MDC-VP1. The plasmid was transformed into competent bacterial cells which were selected by Kanamycin, the identificetion of the recombined plasmids was done by endonuclease analyses.2 Preparation of recombinant adenoviral plasmids: The recombinant plasmid was linearized with Pme I and co-transformed into E. coli strain BJ5183 with pAdEasy-1, the viral DNA plasmid. Recombinant was selected with kanamycin and identified by restriction enzyme analysis. The recombinant adenoviral plasmid was then transformed to competent E.coli DH5αfor large preparation and the plasmid obtained was purified by PEG.3 Packaging and amplification of the recombinant adenovirus particle: Linearized by PacI digestion, the recombinant adenoviral plasmid was transferred into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000 as a transfection reagent according to the manufacturer's guidelines, to produce viral particles. The expression of green fluorescent protein(GFP), which is a tag of the recombined virus, was monitored as a indictor of successful packging. The supernatant of the cell lysate was collected and the recombinant viruse was urther amplified in HEK 293 cells for 2 to 4 passages. the passage 4 virus was purified using the adenovirus purification kit.4 Viral titration : Cell-free virus stocks were diluted serially in serum-free medium at ten times, and titrated by plaque forming units (PFU) methods according to Ad-Easy vector system application manual。5 Amplify and purify the Ad packaged in the past year using the same method, and then the titrers of the virus was teseted.6 the HEK 293 cells were lysed by freezing /thawing 48 h post infection and the viral protein expressied was detected by Western blotting.7 Large preparations of pcDNA3/MDC-VP1 were performed from transformed E.coli DH5α, and then purified by PEG8000.8 BALB/c mice aged 6-8 weeks were divided into six groups: Ad/MDC-VP1 group(A group): mice were immunized with 1.2×10~7pfu/100μl of Ad/MDC-VP1 for two times at an interval of two weeks; pcDNA3/MDC-VP1 group(B group): mice were immunized with 100μg plasmid for three times at the interval of three week; pcDNA3/MDC-VP1+Ad/MDC-VP1 group (C group): mice were immunized with the plasmid at the first time, and then followed by two immunizations with Ad/MDC-VP1 at a two week interval three weeks later;Ad/MDC-VP1+pcDNA3/MDC-VP1 group (D group): mice were immunized with Ad/MDC-VP1 two times at a two week interval, then followed by immunization with plasmid three weeks later; Ad group(E group):mice were immunized with Ad two times at a two week interval; PBS group(F group):mice were immunized with 100μl PBS three times at a three week interval. Fourteen days after each injection, sera of each group were collected, titers of CVB3-specific neutralizing antibodies and specific VP1 antibodies were measured. Three weeks after the last immunization, splenocytes of the mice in each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, other twelve mice were challenged with 4LD50 CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice in each group were challenged with 3LD50 CVB3 and sacrificed seven days later to evaluate the viruses titers in the blood.Results: 1 Recombinant adenoviral transfer plasmids pAdTrack-CMV/MDC-VP1 was constructed successfully.2 Recombinant adenoviral plasmid pAd/MDC-VP1 was generated successfully. Restriction digest with Pac I yielded a large fragment of approximately 30 kb, and a smaller fragment of 4.5 kb. The lengths of the fragmentswere the same as expected. 3 Proved by the expression of GFP, which could be screened 48h post infection, recombinant adenovirus Ad/MDC-VP1 was generated successfully. The infected cells were lysed by freezing/thawing cycles, and supernatant was used to reinfect noive HEK 293 cells.4 The titers of recombinant adenovirus Ad/MDC-VP1 from passage 1 to passage 4 were 2.2×10~3 pfu/ml, 1.3×10~4 pfu/ml , 4×10~6 pfu/ml and 5.8×10~8 pfu/ml respectively.5 Secreted protein MDC-VP1 was verified by Western blotting analysis, the target protein was expressed in the 293 cell culture medium after infected by Ad/MDC-VP1.6 The mean titers of specific CVB3 VP1 IgG antibody after every immunization were as follows respectively: A group: 1:75.78,1:527.72;B group: 1:66.07,1:199.99,1:459.41;C group: 1:65.97,1:229.72,1:696.31;Dgroup:1:65.98,1:459.41,1:1392.52;while the titers of the mice were always lower than 1:50 in E and F group. The statistics analysis showed that except for the E and F group, differences of specific VP1 IgG antibody titers between each group were significant after every immunization. More specifically, after the last immunization, the antibody titers of each group can be arranged in a descending order: D group, C group, A group, B group. 7 The mean titers of neutralizing antibody after every immunization were as follows respectively: A group: 1:8.41,1:53.38;B group:1:7.07,1:25.2,1:50.39;C group:1:7.49,1:28.28,1:67.26;D group:1:8.41,1:50.39,1:71.26;while the titers of the mice were always lower than 1:5 in E and F group. The statistics analysis showed that excepting E and F group, the differences of neutralizing antibody titers of every group were significant after every immunization. More specifically, after the second immunization, the antibody titers of each group can be arranged in a descending order: A group, D group, C group, B group. And no difference in antibody titers between groups was found after the last immunization.8 Splenocytes from the vaccinated mice showed different proliferative activity to different stimulants of CVB3 or ConA. When stimulated by CVB3, there was no difference in the proliferative activity between groups. However, after stimulated by ConA, the proliferative activities of mice in group C and D were higher than those of others.9 The specific lymphocytic CTL cytotoxic activities of each group could be arranged in a descending order: C group, D group, A group, B group, E group, F group. However statistic analysis showed that both the difference between group C and D and the difference between D and A were not significant(P>0.05).10 After 3LD50 CVB3 challenge, the virus titers in the blood of group A, B, C and D were significantly lower than those of group E and F (P<0.05). Besides, the blood virus titers of group C and D were much lower than those of group B.11 Up to the 21th day after CVB3 challenge, the survival rates of group A, B, C, D, E, F were as follows respectively: 25%, 25%, 33.33%, 41.7%, 0, 0. Kaplan-Meier survival curve showed that the differences of survival time between groups were significant(P<0.01).Conclusions: 1 The recombinant adenovirus Ad/MDC -VP1 was constructed and packaged successfully. The target protein was verified by Western blotting analysis.2 Humoral immune response, nonspecific lymphocyte proliferation activity and specific CTL cytotoxic activity of the mice could be significantly enhanced after two times of immunization with Ad/MDC-VP1, and the sera virus titers decreased .3 Higher humoral immune response, nonspecific lymphocyte proliferation activity and specific CTL cytotoxic activity could be induced by combined immunization.4 The survival time of mice after lethal dose CVB3 challenge can be significantly prolonged by DNA vaccine immunization combined with adenovirus vaccine. |
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