| Influenza virus mainly infects the epidemic cells of respiratory tract, which is the entrance and first site of influenza viral replication. Therefore, mucosal immunity against influenza virus infection in the field appears to be particularly important.In this report, the influenza whole virus antigen and the influenza split virus were respectively formulated with the cholera toxin B subunit (CTB), alumina (Al (OH) 3) and proteosome. The studies reported here were conducted to determine which vaccine was efficient and which adjuvant could be used as an inflenza mucosal adjuvant.1. Influenza virus type A virus were cultured in 33°C incubator for 56 h, and B virus were cultured in 33°C incubator for 72 h, harvest virus, then inacitivated with the formalin(final concentration 0.02%) in 4°C for 12 h, concentrated by ultrafiltration (membrane molecular weight: 10 ku) and purified through sucrose density gradient centrifugation(from the bottom upwards centrifuge tube followed 60%, 45%, 30% sucrose solution) in 4°C, 30000r/min for 2 h, dialysis and PEG concentrated. The virus split by 5% Triton X-100 in 20°C for 12 h. After inactivated certification test, individual immune diffusion test, electron microscopy analysis and so on, the antigen produced by above methods can be used for follow tests.2. Influenza whole virus antigen influenza split virus antigen were respectivly formulated with the adjuvants above mentioned, and used to immunized mice intranasally. The results show that I.N. lonely with influenza whole virus antigen induced high serum IgG, serum HAI and sIgA, I.N. with influenza whole virus antigen not only stimulate humoral immune response, but also induce high level mucosal immune response. Howerver, the proteosome influenza split virus induced high serum IgG, serum HAI and sIgA, note that I.N. with influenza split virus antigen not only stimulate humoral immune response, but also induce high level mucosal immune response.3. I.N. with different does of proteosome influenza split virus and influenza split virus, the results show that, in adjuvant group, 4μg vaccine can significantly increase serum IgG, IgM, HAI and sIgA, the CD4+/CD8+ of splenic T lymphocyte was significantly higher than the rest of the group, note that this group can stimulate high level humoral and mucosal immune responses, but not cause immunosuppression. However, in non-adjuvant group, serum IgG, HAI and sIgA of all groups maintain at lower level, demonstrated that influenza split virus only stimulate weak immune response. In the conditions of same dose, adjuvant group can induce significantly higher immune responses than those without adjuvant group. Suggested that proteosome was a efficient mucosal adjuvant, which can significantly improve system immune response and mucosal immune response.4. I.N. with different doses of trivalent influenza virus antigen of mice, at a certain antigen dose range, the serum IgG, HAI and sIgA antigen showed a dose-dependent, means that with the increasing dose of antigen, the indicators levels increse, when reach to 8μg, serum IgG, sIgA and HAI reach the peak, antigen dose increase again, the indices declined, note that 8μg vaccine can significantly increase the system immune response and respiratory mucosal immune response. While the CD4+/CD8+ of splenic T lymphocyte decrease when antigen dose values increase, when the dose reach to 8μg, CD4+ value decrease to the lowest, again raise antigen dose, the value rebounded. 8μg vaccine group can significantly enhance cell-mediated response. I.N. with different doses of trivalent influenza virus antigen of guinea pigs, indicators of 6μg vaccine group significantly lower than 10μg and 20μg vaccine group, and all indices of 10μg and 20μg dose group get equivalent level, so lower dose was the best dose, 10μg for the best.
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