The Research Of Different Immunization Schedules Of BCG Priming And AMM Subunit Vaccine Boosting

Tuberculosis, which is the second biggest killer after AIDS in infection diseases, has now become a serious public health problem. Bacillus Calmette-Guerin (BCG), at present, as the only approved anti-TB vaccine in many countries of the world, has been used for more than 80 years. However, in different regions, BCG's protective effect for adolescents and adults have a significant difference, BCG has little effect on adult tuberculosis. Therefore, exploring new vaccine strategy is an important means for TB controlling. At present, the subunit protein vaccine is one of the most promising new vaccines. It has made a good immune protective effect that using this kind of vaccine to boost BCG priming in animal model.Many research has confirmed that the subunit vaccine, constructed by fusion protein Ag85B-Mpt64190-198-Mtb8.4 (AMM) and the adjuvant dimethyl-36 alkyl bromide (DDA), BCG-polysaccharide nucleic acid (BCG PSN), is an ideal subunit vaccine, and it could induce a strong immune protective effect as a booster after the BCG priming.Objective:In this study, the subunit vaccine composed of AMM, DDA and BCG PSN was applied to boost BCG priming mice in order to compare the immune and the protective effects with different boosting schedules.Methods:1. Comparison of the immune effect induced by BCG priming, boosted with AMM subunit vaccine once in 10th week and twice in 8th,10th weeks.The AMM subunit vaccine constructed by fusion protein AMM, adjuvants DDA and BCG PSN. The experimental group 1 was immunized with BCG first, then boosted with the AMM subunit vaccine in 10th week; The experimental group 2 was boosted with the AMM subunit vaccine in 8th week and 10th week respectively after the primed with BCG. Two control groups were treated respectively with physiological saline alone and BCG alone. ELISPOT and ELISA were used for the detection of the cell-mediated and humoral immune response in 14th and 22nd week after the BCG priming. Furthermore, the immunized mice were challenged with live BCG to mimic tuberculosis infection in 22nd week. Subsequently the T cell typing and humoral response were detected by cytometry assay and ELISA respectively in 26th week.2. Comparison of the immune and the protective effect induced by BCG priming, boosted with AMM subunit vaccine once in 10th week, twice in 8th,10th week and thrice in 6th,8th,10th weeks.The immunization protocol of the experimental group 1 and 2 was the same as the first part. The experimental group 3 was added:mice were primed with BCG first, then boosted with the AMM vaccine in 6th,8th,10th week, respectively. Two control groups were treated respectively with PBS alone and BCG alone. The cell-mediated and humoral immune responses were detected with ELISPOT and ELISA in week 14. The immunized mice were challenged with H37Rv strain in 22nd week. Then the load of M. tuberculosis in mice and the pathological sections were detected after 6 weeks.Results:1. The results showed the boosting twice in the 8th,10th weeks induced stronger INF-γsecretion capacity than boosting once at the 10th week; boosting twice induced significantly higher IgGl antibody titers than boosting once; and the ratio of IgG2a to IgGl which reflected thl-type immune response was lower in the boosting twice group than that in the boosting once group. After challenging with live BCG strain, the CD4+CD25+T cells proportion was higher in the both boosting once and twice groups than that in the BCG alone group.2. The results showed the twice boosting in 8th,10th weeks with AMM subunit vaccine induced stronger IFN-y secretion capacity than the once or thrice boosting; the IgGl antibody titer revealed a positive correlation with the frequency of boosting;the IgG2a antibody levels were not affected by the frequency of boosting; the ratio of IgG2a to IgG1 which reflect the Thl-type immune response here showed a negative correlation. The twice boosting induced stronger protection against TB than the once or thrice boosting, which was demonstrated by the least loading of bacterium in the boosting twice group. In pathological section, it was found using AMM subunit vaccine did not increase the pathological injury in mice.Conclusion:the strategy that priming with BCG first, then boosting twice with AMM subunit vaccine in 8th,10th weeks not only induced stronger cell and humoral immune responses, but also induced stronger protective effect. Boosting thrice in 6th,8th,10th weeks did not show better effect than boosting twice. Increase of the boosting frequency could strengthen the Th2-style immune reaction. The strategy of boosting BCG with AMM subunit vaccine did not increase the pathological injury.