Anthracis Live Attenuated Vaccine A16r Mucosal Immunity And Cytokine Role

Objective To observe the effect of the intranasal and subcutaneous routes to the immunogenicity of live attenuated anthrax vaccine A16R and provide proof for the new immune route. In the meanwhile,try to illustrate the effect of cytokines at different times after immunization through RT-PCR method.Methods Divide the Balb/c mice aged 6-8 weeks into 3 groups randomly, 5 mice per group.Mice were immuned respectively with 5×107CFU live attenuated vaccine 4 times with an interval of one week by intranasal or subcutaneous route. 7, 30, 60 and 90 days after the last immune, serum,nasopharynx wash,lung wash and vaginal wash were collected .The amounts of IgA ,IgG and its subclass were measured by ELISA. Lymphocytes were isolated from nasal associated lymphoid tissue(NALT), nasal passage(NP), spleen(SP) and Peyer's patches(PP), the changes of lymphocytes phenotype were analyzed by FACS. Immune mice were challenged 10 LD50 by celiac injection. Mice were immuned respectively with the same amount live attenuated vaccine by subcutaneous route. At 4, 16 and 24 hours after the first immunization and the second and third times with an interval of one week and 12 days after challenged by Vollum strain, total RNA of mice's livers were extracted and inverse transcripted into cDNA, using PCR method, cytokines such as IL-6 ,MCP-1 ,CD86 ,CD64 ,IL-1β,TNF-α,IL-4 and IL-2 were amplificated and observed by agarose gel electrophoresis.Results The levels of specific IgG antibody were detected in both immune groups. IgA in serum and sIgA in mucosal wash were induced by intranasal route, but not induced by subcutaneous route. The ratio of IgG1/IgG2a in both groups were significantly higher than the control group. Afer challenged by celiac injection, mice of the subcutaneous group were all living, the ratio of intranasal group is 80%. The ratios of CD4+/CD8+ T lymphocytes were various in different lymphoid nodes. In NA, the intranasal group was higher than the subcutaneous group. In PP and SP, the former was lower than the latter one. The ratios of CD3+/CD45+T lymphocytes were various in different lymphoid nodes too. In NA, PP and SP, the subcutaneous group was lower than the intranasal group and in NP, the former was higher than the latter one. In contrast, in 7 days after the last immunization, the levels of specific antibody were dramatically decreased in 7 days to 30 days after the last immunization and the decreasing levels were mild in the following days. There was regularity of decreasing levels of specific anbody in the mucosal sites, that is the antibody in nasopharynx wash was higher than the one in lung wash, and the latter one was higher than the one in vaginal wash. As far as cytokines were concerned, IL-1β, CD64 and CD86 were detected in all times after immunization. MCP-1 were not detected in the earlier times after immunization, but continusly founded in livers of mice in 1 days after the first, second and third immunization. All the molecules were detected in the second time and the...