Effect Of Immune Efficacy On Nucleic Acid Vaccine Of Canine Infectious Hepatitis

Objective Canine infectious hepatitis is an acute and highly contagious viral disease of dogs and puppies, and is caused by canine adenovirus type 1 (CAV-1). This disease is transmitted by direct contact with the infected dogs and spread- -s all over the world in each season especially in spring and winter. CAV-1 has caused acute-infection and high dying in all breeds of young dogs, which severely harmed the canine cultivation.The best way to prevent canine infectious hepatitis is to advance the management and health measure and immunization by vaccine. Currently, vaccinations with attenuated vaccine provide an effective approach in preventing the disease. But the attenuated vaccine, however, have several drawbacks:(ⅰ)potential induction of atavism;(ⅱ)higher cost and complicated preparation in the vaccine production;(ⅲ) immunization in lifetime. In addition, humoral immune response induced by attenuated vaccine has the leading role in resisting virus infection.Nucleic acid vaccine is a new vaccine technique following inactivated vaccines and attenuated vaccines in 1990s . Nucleic acid vaccine is capable of eliciting both cellular and humoral immune responses more complete than conventional vaccination and have the advantage of mass-production easily. It has been actively developed in the past few years against a variety of infectious agents including virus, bacteria and parasites. In this article, we immunized BALB/c mice using constructed DNA vaccine of canine infectious hepatitis, and determined its primary immune efficacy. We hope our study can explore the possibility of DNA vaccine acting as canine infectious hepatitis vaccine, lay some foundations for deep understanding the immunology of our constructed vaccine.Methods1 Extracted eukaryotic recombinant plasmid abundantly E.coli that contained the eukaryotic recombinant plasmid pVAX1-CpG-Loop were cultivated for 36h by shake culture method. Then the sediment of E.coli was collected by centrifugalization and recombinant plasmids were extracted by SDS-alkaline lysis. The purity and concentration of the extracted recombinant plasmid were determined by grating spectrophotometer.2 Immunization of miceThirty-five BALB/c mice (female, 6-8 weeks old) were randomly divided into seven groups. Groups 1 and 2 were inoculated intramuscularly (i.m) with recombinant plasmid pVAX1-CpG-Loop of 100μg and 200μg respectively. Groups 3 were also inoculated intramuscularly with recombinant plasmid pVAX1-CpG-Loop (prime) and recombinant antigen protein (boost). Groups 4 were immunized by three ways of intramuscular injection, hypodermic injection and nasal instillation. Groups 5 and 6 were inoculated by electronic transfection with recombinant plasmid pVAX1-CpG-Loop of 100μg and the strategy of prime-boost. Groups 7 were negative control and were inoculated with pVAX1-CpG vector. All of the mice were immunized three times at 2-week intervals, on 0d, 14d and 28d.3 Assessment of immune efficacyELISA of IgG antibody Sera from all animals were collected from tail weekly. Ninety-six-well plates were coated with 100μg/ml canine infectious hepatitis per well diluted in carbonate buffer overnight at 4℃. After washed three times and blocked for 1h, serum samples diluted were added to the plate and incubated for 60 min, followed by the addition of anti-mouse IgG coupled to HRP, which was developed with OPD. Optical density readings at 492 nm were carried out in an ELISA processor.Constituent ratio of T lymphocyte subgroup Mice were killed by eyeball extirpation after three immunization and spleens were removed aseptically. Lymphocytes were harvested from spleens and were adjusted to a concentration of 1×106 cells/ml in DMEM medium with 10% new-born calf serum. Fluorescent-labeled antibody was added and constituent ratio of CD4+ T cells and CD8+T cells counted by flow cytometry. Lymphocyte proliferation Two methods MTT and CCK-8 cell counting kit were used to evaluate the activity of lymphocytes proliferation.Induction of cytokines splenocyte suspensions from immunized mice were diluted in 10% bovine calf serum-supplemented DMEM to a final concentration of 5×106 cells/ml. The contents of IFN-γin supernatants were detected by ELISA kit.4 Neutralizing antibody testSera collected from all the mice were inactivated and detected the neutralizing antibody titer by fixed virus-diluted sera.Results1 The recombinant plasmid pVAX1-CpG-Loop extracted abundantly by alkaline lysis was identified by double enzyme digestion. The electrophoretic band about 860 bp could be observe under ultraviolet light about 1.2﹪agarose gel electrophoresis. The purity and concentration of the extracted recombinant plasmid detected by grating spectrophotometer demenstrated that the ratio of OD260/OD280 was between 1.8 and 2.0.2 Specific IgG antibody against CAV-1 could be measured by indirect ELISA after immunized mice with recombinant plasmids pVAX1-CpG-Loop by different routes and strategy. Antibody titer induced by recombinant plasmids was higher considerably than that immunized with pVAX1-CpG vector(P<0.05).The IgG antibody titer induced by three routes combined immunization was obviously higher than other groups. According to the flow cytometry analyzing, either group's constituent ratio of CD4+T cells and CD8+T cells of mice spleen lymphocytes immunized with recombinant plasmids pVAX1-CpG-Loop was higher than that of control group (P<0.05). MTT and CCK-8 cell counting kit were used to assess the activity of lymphocytes proliferation. It came to the approximately same conclusion that: either group's the activity of lymphocytes proliferation was higher than that of control group (P<0.05);②the activity of lymphocytes proliferation immunized with the same immunized route and dose, as well as the different immunized strategy showed that: the group of immunized by electroporation B (prime-boost) was higher than the group of electroporation A (P<0.05); the group of immunized by intramuscular injection C (prime-boost) was higher than that of the group of intramuscular injection A;③immunized with the same immunized route and strategy, as well as the different immunized dose showed that: the group of immunized by intramuscular injection B (200μg/mouse) was higher than that of the group of intramuscular injection A (100μg/mouse) (P<0.05);④it showed no statistics significance immunized with the same immunized strategy and dose, as well as the different immunized route. The amount of IFN-γsecreted by spleen lymphocytes of either recombinant plasmid pVAX1-CpG-Loop immunized groups was also higher than that of control group by ELISA kit(P<0.05). 3 The micro-serum neutralization assay showed that the neutralizing antibodies could be detected in the inactive sera of all the groups immunized with recombinant plasmids pVAX1-CpG-Loop. The highest titer of neutralizing antibodies is 1:8.The sera of control group immunized with pVAX1-CpG vector didn't show any neutralizing activity.Conclusions1 Specific IgG antibody against CAV-1 could be detected by indirect ELISA after immunized BALB/c mice with recombinant plasmids pVAX1-CpG-Loop by different routes. It indicated that inoculation with recombinant plasmid pVAX1-CpG-Loop induced not only the specific humoral immunity response, but formed the immunologic memory and generated the sustained IgG antibody. And immunized through several routes could highly prove the humoral response. The ratio of T cell subgroups from either group's spleen lymphocytes immunized with recombinant plasmids pVAX1-CpG-Loop were higher than that of control group by flow cytometry analysis. Results of spleen lymphocytes proliferation using two methods of MTT and CCK-8 indicated that experimental groups were higher than that of control group. And contents of the IFN-γsecreted by spleen lymphocytes came to the same conclusion with that of the spleen lymphocytes proliferation. The results above all indicated that humoral and cellular-mediated immunity could be induced successfully by immunized BALB/c mice with recombinant plasmids pVAX1-CpG-Loop .2 Neutralizing antibody test demonstrated that neutralizing antibody capable of inhibiting virus infecting cells in vitro could elicit by immunization of recombinant plasmid pVAX1-CpG-Loop.The above results proved that the recombinant plasmid pVAX1-CpG-Loop constructed contained the neutralizing epitope and might play a significant role of immunoprotection in preventing virus infection.