A Comparative Study On The Immune Efficacy Of Two Adjuvants Against Inactivated Pseudorabies Virus GE Gene Deletion Vaccine

The pseudorabies virus is a double-stranded DNA virus with a capsule and that is a member of the herpesvirus.It can infect a variety of animals and swines as its storage host,Vaccination is the main means of preventing and controlling the disease,and achieved good control effect.However,since 2011,the domestic pseudorabies epidemic has once again exploded in large areas;the new epidemic strain has undergone a certain degree of variation,resulting in the existing vaccine not providing complete protection against the infection of the new strain,therefore,it is necessary to develop a vaccine against the new strain.In this study tend use the gene deletion strains rPRV-AH-gI^-/gE^-and rPRV-AH-gI^-/gE^-/g C⁺as vaccine strains,and MONTANIDE Gel 01 and MONTANIDE ISA 201 VG were used as adjuvants to prepare two kinds of delet strain,The water adjuvant inactivated vaccine and the oil adjuvant inactivated vaccine of the strain,and compared with a commercial pseudorabies inactivated vaccine to evaluate the immune effect on mice and swines.The main research contents are as follows:1.Analysis of the virus biological characteristics of PRV-AH,rPRV-AH-gI^-/gE^-,rPRV-AH-gI^-/gE^-/g C⁺Inoculate the identified three viruses into BHK-21 cells,and measured TCID₅₀ at different time after infection,and draw a one-step growth curve for three viruses,the results showed that the recombinant viruses rPRV-AH-gI^-/gE^-and rPRV-AH-gI^-/gE^-/g C⁺has similar growth characteristics to the parent strain PRV-AH,the highest titers of the three viruses were:10^-8.44/m L,10^-8.76/m L,10^-8.69/m L.The recombinant viruses rPRV-AH-gI^-/gE^-,rPRV-AH-gI^-/gE^-/g C⁺were continuously transmitted on BHK-21 cells for 20 generations,and extract the genomic DNA of the 4th,8th,12th,16th and 20th generations as a template for PCR identification,the results showed that there was no gene loss in the two recombinant viruses,indicating that the two recombinant viruses have good genetic stability.2.Evaluation of immune efficacy of rPRV-AH-gI^-/gE^-and rPRV-AH-gI^-/gE^-/g C⁺inactivated vaccine in miceUsing rPRV-AH-gI^-/gE^-and rPRV-AH-gI^-/gE^-/g C⁺as vaccine strains and MONTANIDE Gel 01,MONTANIDE ISA 201 VG as adjuvants to prepare a water adjuvant inactivated vaccine and oil adjuvant inactivated vaccine with virus content of 10⁵TCID₅₀/100?L and 10⁶ TCID₅₀/100?L.The mice were immunized with 100?L of each vaccine,and Intensive immunization 4 weeks after the first exemption,at the same time,a commercial pseudorabies inactivated vaccine immunization group with a dose of 10⁶TCID₅₀/100?L was seted as a positive control,and 100?L DMEM was seted as a negative control.After the immunization,the blood was collected at intervals,and Determination of neutralizing antibody.Four weeks after the second immunization,mice were challenged with 100 LD₅₀ PRV-AH strains,and dissect the mice killed and sacrificed after the challenge to made Pathological sections from brain,liver,spleen and lung,and observe the histopathological changes.The results showed that the neutralizing antibody level and immune protection rate of 10⁶ TCID₅₀ immunized group were better than 10⁵ TCID₅₀immunization group,and the neutralizing antibody level and immune protection rate of water adjuvant vaccine group were better than oil adjuvant vaccine group.Among them,10⁶ TCID₅₀ rPRV-AH-gI^-/gE^-/g C⁺,rPRV-AH-gI^-/gE^-water adjuvant inactivated vaccine immune protection rate are 100%,the immune protection rate of a commercial inactivated pseudorabies virus vaccine group was 87.5%;compared with the commercial inactivated vaccine group,10⁶ TCID₅₀ rPRV-AH-gI^-/gE^-/g C⁺immunization group had mild changes in brain,liver and lung tissue diseases,The water adjuvant inactivated vaccine group was better than the oil adjuvant inactivated vaccine group.3.Evaluation of immune efficacy of rPRV-AH-gI^-/gE^-and rPRV-AH-gI^-/gE^-/g C⁺inactivated vaccines in swinesThe experimental swines were immunized twice with 10⁷ TCID₅₀rPRV-AH-gI^-/gE^-/g C⁺,rPRV-AH-gI^-/gE^-water adjuvant inactivated vaccine and oil adjuvant inactivated vaccine respectively,at intervals of 4 weeks.10⁷ TCID₅₀ commercial pseudorabies inactivated vaccine was used as a positive control group,1 m L DMEM was used as a negative control.Bloods was collected at intervals after immunization,and determine serum neutralizing antibody levels in swines.The nasal cavity of the immunized pigs was challenged with 10⁶ TCID₅₀ PRV-AH 6 weeks after the second immunization and Collect nasal swabs at different time to determine the detoxification of the swines.The results showed that rPRV-AH-gI^-/gE^-/g C⁺water adjuvant inactivated vaccine induced higher levels of neutralizing antibodies in swines than commercial vaccines and rPRV-AH-gI^-/gE^-inactivated vaccines,and Compared with commercialization and non-immunized challenge group,the rPRV-AH-gI^-/gE^-/g C⁺and rPRV-AH-gI^-/gE^-water adjuvant inactivated vaccines can greatly alleviate the clinical symptoms of infected swines,but all vaccines are not effective in preventing the detoxification of wild-type infections in pigs.In summary,in this study we have been prepared rPRV-AH-gI^-/gE^-/g C⁺,rPRV-AH-gI^-/gE^-water adjuvant inactivated vaccine and oil adjuvant inactivated vaccine,and have evaluated the immune effects of vaccines on mice and pigs.The results showed that the immune efficacy of the water adjuvant inactivated vaccines of the two strains were better than the oil adjuvant inactivated vaccine,and the rPRV-AH-gI^-/gE^-/g C⁺water adjuvant inactivated vaccine were better than rPRV-AH-gI-/gE-water adjuvant inactivated vaccine and commercial inactivated vaccine,and it is expected to be used as a good vaccine candidate for the prevention and control of new PRV strains.