Effect Of Humoral Immunity Immune Efficacy On Nucleic Acid Vaccine Immunized Beagle Dog 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 spreads 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. Besides, there are other vaccines such as the bigeminy vaccine of ICH and Canine parvoviral enteritis, the composition vaccine of canine distemper, Minute virus, parainfluenza, Coronavirus and ICHV.Nucleic acid vaccine is a new immunization technique developed in recent years and has many advantages compared with conventional vaccines . It was called the third generation vaccine. DNA vaccine can stimulate the bodies of animals to produce humoral immune response which is protective to them. B cells that are stimulated by antigens can excrete antibodies after differentiation and proliferation which constitute the main component of humoral immunity. Antibodies can bind to specific antigens and moreover have a wide range of immune function, therefore, testing the level of antibodies in bodies after immunization becomes an important method that can evaluate the level of humoral immunity in bodies and vaccine effect. neutralizing antibody are produced when pathogenic microbes attack the body, which can eliminate pathogenic microbes and their toxic products (toxins) and make them loss of infectious ability or virulence. So neutralizing antibody are reckoned as important tools for research of virus infection mechanism. The existence of neutralizing antibody is an important indicator of humoral immunity that can reflect vaccine immunogenicity.Methods1 Plasmid isolation and purificationThe plasmid pVAX1-CpG-Loop was isolated by the method of alkaline lysis, and purified by polyethylene glycol precipitation. The fusion protein was purified by Ni-NTA affinity chromatography column.2 ImmunizationSix Beagle dogs were randomly numbered. No.1,No.2,No.3 were immunized from a single litter were injected with doses of 400μg,600μg or 800μg pVAX1-CpG-Loop in a total volume of 1 ml phosphate buffered saline (PBS, 0.067 M, pH 7.2) respectively on week 0,8 and 16.and then followed by the same doses of antigen protein on last immunized. No.4,No.5,No.6 were immunized from a single litter were injected with doses of 400μg,600μg or 800μg pVAX1-CpG-Loop respectively on week 0,8 and 16. No.7 was used to inoculated Conventional vaccines( Table1). All injections were pretreated 15min before immunization by intramuscular injection of 500μl of 25% degerming cane sugar into quadriceps muscle, and Loop protein were administered multidrop in cervical part hypodermic .3 ChallengeSix weeks after the last vaccination, all dogs were given Virus infection through the oral mucosa under anesthesia , with a dose of 1.0 ml (TCID50 of 10-4.23/0.1ml) .4 Assessment of immune efficacy4.1.ELISA of IgG antibodySera from all animals were collected. Ninety-six-well plates were coated with 1:5 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 1 hour, 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.4.2. Indirect immunouorescence assayMadin-Darby canine kidney (MDCK) cells grown on glass coverslips in 6-well plates were infected with CAV-1 (100 TCID50) and incubated at 37℃, 12h cell monolayers were rinsed with PBS andfixed with pre-cooled methanol for 10min. After incubation with 0.5% Triton-X 100 in PBS for 10min, the cell monolayers were incubated with antisera (1:10,1:50,1:100,1:200,1:400,1:800dilution in PBS)After incubation for 30 h at 37℃, the supernatants were removed and the cells were rinsed three times (5min each wash) with PBS;FITC-conjugated goat anti-dog IgG (H+L) antibody (1:200 dilution in PBS) was then added, and the cells were incubated for 30 h at 37℃and washed with PBS. and counterstained with (1:2000 dilution in PBS) Evan's blue. After washing and drying, the slides were read with a fluorescence microscope .4.3. Neutralization assaySerum-virus neutralization (SN) antibody assays were performed byheat-inactivating all serum samplesat 56℃for 30 min. Assays were performed in 96-well microtiter plates by combining 0.05 ml of sera in serial twofold dilutions in cell culture medium (DMEM) with an equal volume of CAV-1 (100TCID50)also diluted in cell culture medium. The serum-virus mixture was then preincubated at 37℃for 1 h. Madin– Darby canine kidney (MDCK) cells were plated in a 96-well plate (DMEM containing 10% newborn calf serum, 37℃, CO2 incubator). When the density of MDCK cells reached more than 90%, the cells were washed with PBS. One hundred microliters of the mixture (serum and virus) was added to each well, and kept at 37℃for 1 h. The mixture was then replaced with culture medium (DMEM containing 10% newborn calf serum). Cytopathic effect (CPE) was observed 72 h later and the neutralization titer was calculated according to the Reed and Muench method. Titers, expressed as the reciprocal of the serum dilution, were considered positive if no CPE was observed.Results1 The recombinant plasmid pVAX1-CpG-Loop extracted abundantlyThe purity and concentration of the extracted recombinant plasmid detected by grating spectrophotometer demenstrated that the ratio of OD260/OD280 was 1.82,1.9,1.88.2 Induced and purification the Loop proteinThe recombinant plasmid pET28aLoop was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE analysis showed an induced product band about 36kDa; The purity of Loop protein was between 95%~98% after purification with Ni-NTA affinity chromatography column.3 Determination of the serum IgG level of immunized dogs by indirect ELISAPrior to challenge, all test dogs produced no significant changes in specific antibodies. The antibody titer generated by NO.1 dog and NO.7 dog was higher than that of other dogs(data not shown).After challenge, antibody titers of all dogs produced were significantly increasing,the antibody of NO.7 dog reached the highest antibody titer at 1 week,and other dogs reached their highest antibody titer at 3 week,and then gradually declining to 27 weeks.4 Determination of the total serum IgG level of immunized dogs by IFAAfter received three immunizations described above . the test dogs NO.1,4,5,7 developed a rather low IFA IgG titers 1:10.and other dogs remained antibodies were not detectable. after challenge,all dogs developed strong anti-CAV responses. The antibody levels were increasing on the fifth day,The dogs NO.1,5,7 had increased IFA IgG titers1:50,and other dogs had an IFA IgG titers 1:10. From the first week after Challenge ,All of these vaccinated dogs showed an anti-CAV antibody ranging from 1:50 to 1:200 . Apparently ,The dogs NO 3,4 showing the highest titer of 1:400 at 3 week respectively. and other dogs reached their highest antibody titer reached 1:200 at 1week . The titer of anti-CAV IFA IgG showed gradually declining to 7 weeks,and were not detectable from week 14 to week 27.5 Determination of the serum neutralizing antibody level of immune dogs by neutralization testPrior to challenge, all immunized dogs except the dog NO. 7 (sustained neutralizing antibody titers 1:8) did not show neutralizing antibody response. Strong anamnestic neutralizing antibody responses were present 5 days after challenge of virus in vaccinated dogs. The neutralizing antibody titer of NO.1 dog was 1:128 higher than that of other dogs.The titer of neutralizing antibody increased transiently from week 1 to week 3 ,and all dogs reached thire the highest antibody titer during that time, significantly declined at the 5 week and then dropped gradually from week 5 to week 27 .Conclusions1 The recombinant vaccine of Infectious Canine Hepatitis our research lab constructed pVAX1-CpG-Loop can stimulate the body to produce a good humoral immune response.2 The recombinant vaccine of Infectious Canine Hepatitis our research lab constructed pVAX1-CpG-Loop can stimulate the body to produce neutralizing antibodies.