Cellular Immune Response And Combined Immunization Of H5Subtype Avian Inlfuenza DNA Vaccine

Avian Influenza is a serious disease, which causes huge economic losses for the domestic poultryin rescent years. H5subtype Highly pathogenic avian influenza virus(HPAIV) not only infect poultryand wild birds, but also threat our human health.Therefore, it is essential to research new vaccineagainst Avian Influenza. DNA vaccine has lots of advantages. It can induce both humoral and cellimmune responses. It is also easier to store, transport and manufacture. Otherwise, it will inducelong-lasting protective immune response.DNA vaccine pCAGGoptiHA5which express HA gene has good immune effect and has completedits clinical tests. DNA vaccine is applying for a new veterinary drug registration certificate. In view ofmany influent facters of immune condition and H5subtypes of AI different antigen coexist, evalution ofcell immune response and discussion of different DNA vaccine joint immunity will provide useful datasupports for the popularization and application of DNA vaccine.For eukaryotic expression of avian influenza virus (AIV) hemagglutinin (HA), the HA gene ofA/Goose/Guangdong/1/96(H5N1)(GS/GD/1/96)was cloned into the baculovirus transfer vectorpFastBacHTa and the resultant recombinant plasmid was transformed into DH10Bac competent cells togenerate recombinant bacmid (rBacmid-HA), which was identified by PCR and sequencing. Therecombinant baculovirus stock was prepared by transfecting rBacmid-HA into the Sf9insect cells. Theexpression of the recombinant HA in sf9cells was identified by indirect immunofluorescence assay andwestern blot, which shown that the recombinant HA were about64ku. Further identificationsdemonstrated that the HA was able to induce high titer of HI antibody and effectively stimulateproliferation of lymph cells in peripheral blood of SPF chicken detected by MTT assay. The preparationof the recombinant protein in sf9cells was facilitated the further study of HA functions, preparation ofthe subunit vaccine against AIV infection and evaluation of DNA vaccines cellular immune responses.To verify cellular immune responses in SPF chickens after immunization, SPF chickens wereimmunized with15μg AIV HA gene DNA vaccine pCAGGoptiHA5.The percent of CD3+,CD4+andCD8+T lymphocytes and proliferation of lymph cells in periphery blood and spleen were detected byflow cytometer and MTT assay, respectively. The dynamic changes of IFN-γ、IL-2in sera werechecked with ELISA assay.The results demonstrated that the percent of CD4+and CD8+T lymphocytesin periphery blood and spleen had the same tendency. The percent of CD4+and CD8+T lymphocytes ofimmunized groups were higher than that of the control groups and had significant difference betweentwo groups(p<0.05).The amount of CD4+T lymphocytes reached the peak one week after boostingimmunization, then decreased slightly until the experiment finished. In the first immunization, thepercent of CD8+T lymphocytes consistently increased and reached the peak one week after boostingand maintained in a steady level two weeks after challenge. The ratios of CD4+/CD8+increasedgradually in the first immunization and reached the peak one week after boosting. The amount of CD4+and CD8+T lymphocytes as well as ratios of CD4+/CD8+from unchickens had increasing trend butlower than that of vaccinated chickens and then all chickens died one week after challenge. The lymphocytes in vaccinated group and control group stimulated by ConA were proliferation, but nosignificant difference between two groups(p>0.05). While the HA stimulation made significantdifference between two groups(p<0.05). The amount of IFN-γ and IL-2in sera of vaccinated groupswere higher than that of the control groups and reached the peak one week after boosting and thenincreased slightly after challenge.While the amount of IFN-γ and IL-2in control chickens hardly had nochanges and chickens died one week after challenge.The two groups had significant difference(p<0.05).These results also showed that DNA vaccine could induce strong cellular immune responses inSPF chickens.For evaluating the protective efficacy of H5subtype avian influenza vaccine pCAGGoptiHA5、pCAGGoAHHA and pCAGGSXHA, groups of3-week-old SPF chickens were intramuscular inoculatedwith different doses of three DNA vaccines in15μg、30μg、45μg、60μg and90μg. Groups of chickenswere injected with200μl PBS as controls. Two weeks after the boost, all chickens were challenged with105EID50of highly pathogenic A/Goose/GuangDong/1/96strain, A/Duck/FuJian/31/07strain andA/Chicken/ShanXi/2/06strain, respectively. Oropharyrigeal and cloacalswab specimens were collectedfrom all chickens3,5and7days after inoculation for titration of virus in eggs and chickens wereobserved daily for disease signs and deaths for two weeks. Sera were collected weekly after vaccinationand challenged for detecting HI antibodies. Results demonstrated that60μg and90μg vaccines boostedchickens were completely protected from virus challenge (no disease sign, no virus shedding and nodeath).The protective rates in15μg groups which challenged by A/Goose/GuangDong/1/96(H5N1) orA/Duck/FuJian/31/07(H5N1) were90.0%(9/10)and80%(8/10), respectively. The protective rates in15and30μg groups which challenged by A/Chicken/ShanXi/2/06(H5N1) were80.0%(8/10)and90.0%(9/10), respectively.45μg doses of the vaccine can provide100%of the lethal protection.Resultsshow that the combination of vaccine immune can protecte chickens from different lethal H5N1viruschallenge to achieve purpose of cross protection. The joint DNA vaccine is a highly efficient geneengineering vaccine and can be used for preventing HPAIV effectively in near future.In summary, the study imply that DNA vaccine pCAGGoptiHA5can stimulate strong cellularimmune response in SPF chickens.The effect of three plasmid joint immune is good and this willprovide necessary scientific basis and support for industrialization and application of AI DNA vaccine.