Experimental Immune Study On H5N1 Influenza Virus Inactivated Vaccine And DNA Vaccine Of HA Gene

It has been proved that tigers could be infected by H5N1 subtype influenza virus, and TIV is the H5N1 influenza virus isolated from the lung of dead tigers. Four strains of TIV, HAB/01, HAB/03, SX/02 and SH/04 had been isolated, identified and purified to this day. In this paper, the KM mouse was used as a model to study the pathogenesis to the virus, 50% mouse lethal dose of (MLD50) the virus was determined and comparison of immunity to these four strains was conducted. On the basis we studied the preparation of inactivated vaccine whose protection effects was evaluated through immunization tests. TIV DNA vaccine was constructed and its immunogenicity was evaluated in the mice. Initial investigation on the immune effects of the combined immunization with inactivated vaccine and DNA vaccine was made. The mice of different groups were infected respectively, intranasally with serial 10-fold dilutions of HAB/01 virus grown in embryonated eggs. The average weight of mice of every inoculated group was on a down trend seen as a whole, and the total number of white cells declined evidently. The MLD₅₀ is about 10^-7.25/0.1mL, which was similar to EID₅₀ (fifty percent embryonated eggs infectious dose), and it indicated that TIV was highly pathogenic to mice. There was spotted bleeding on the lungs. Bleeding on the meninges and denaturalization of brains could be found with the mice behaving neural symptom. Viruses were isolated from the brains, the livers, et al, but the positive rate of virus isolation from the lungs was the highest. Besides, the HI antibody titer against TIV in the mice surviving from the infection rose evidently in contrast with that of mice before the inoculation. These results indicated the mouse could be used as a good model for study on vaccines of TIV. Based on the mouse model of TIV, TIV-AF (infectious allantoic fluid of TIV) of HAB/01, HAB/03, SX/02 and SH/04 was inactivated with suitable amount of methanal, and then it was used to vaccine the mice. A control group immunized with Al(OH)₃ adjuvant was set up. The results showed the TIV-AF was inactivated absolutely with 0.05% methanal under the temperature of 37℃for 24 hours. After three immunizations the average HI antibody titer in the mouse serum immunized with HAB/01 strain was notably higher than that of the other three groups, which indicated that the immunogenicity of HAB/01 strain was the best one and should be the candidate for TIV vaccines. Suitable amount of Al(OH)3 substantially augmented the antibody response so it is a useful adjuvant to H5N1 TIV inactivated vaccine. Then the HAB/01 was used as vaccine strain. It was propagated in embryonated eggs, the TIV-AF was inactivated with the methanal and then condensed with differential centrifugation. The results showed that the TIV-AF was condensed 8-16 times by centrifugating at the rate of 5000r/min for 15 minutes and 30000r/min for 4 hours. The recovery rate of virus is above 95% and the TIV-AF was purified to some degree in the same time. Bacterias in the condensate were killed completely with U.V. irradiation. The prepared inactivated vaccine was injected into animals with the Al(OH)3 adjuvant. The results demonstrated that high level immune response was stimulated after a second injection and absolute immunoprotection from challenge with HAB/01 strain for the mice and the cats was to be required. High titer HI antibody in the sera of chicks maintained for a long time. HI and SN antibody of high titer against TIV were generated in tigers or lions. No side effect was founded in the animals immunized with the inactivated vaccine. Specific primers were designed referring to the HA gene nucleotide sequence in GenBank and the complete open reading frame of the HA gene of HAB/01 strain was amplified by RT-PCR. Then the HA gene was cloned into pMD18-T. The recombinant, named pT-HA, was sequenced and analyzed. As a result, the ORF was made up of 1707 bp. coding 568 amino acids. Then the HA gene was sub-cloned into the vector pVAX1 at the downstream of CMV directedly. The recombinant, named pV-HA, was used as TIV DNA vaccine. F81 cells transfected with pV-HA did encode TIV HA protein specifically reacting to anti-TIV sera. In the following study the mice in different groups were immunized with pV-HA, the inactivated vaccine and pV-HA plus the inactivated vaccine respectively. The mice in the latter combined immunization group were divided into two groups. The mice in the one group were immunized with gene vaccine 2 times and then were immunized thirdly with inactivated vaccine (2G+Ina.), while the mice in the other group were...