Fusion Antigen Expression Of Mycobacterium Tuberculosis Recombinant Saccharomyces Cerevisiae Immune Effects Research

Tuberculosis (TB) is a devastating infectious disease wordwide. Vaccine is the most effective means to treat TB. Currently, BCG is the only licensed and widely used anti-TB vaccine, but its protection effecacy is limited and wanes over time. Therefore, a more effective vaccine is urgently needed. Ongoing anti-TB vaccine development is mainly focused on subunit and live-attenuated vaccines, some of which have already entered clinical stage. A novel recombinant Saccharomyces cerevisiae vaccine has emerged recently, which demonstraed therapeutical potential in treating hepatitis C virus infection and in curing colon carcinoma.We had constructed recombinant Saccharomyces cerevisiae expressing Interferon-y-ESAT-6-Ag85B and ESAT-6-Ag85B (here named Yeast-IEA, Yeast-EA respectively) using our patented pHR system and confrimed the protein expression in western blotting experiments. Then C57/BL6 mice were immunized subcutaneously by heat-killed recombinant Saccharomyces cerevisiae and humoral and cellular responses were investigated. We also studied the protection efficiency of the recombinant Saccharomyces cerevisiae vaccine by BCG challenging test. The main results were as follows:(1) Recombinant Saccharomyces cerevisiae expressing tuberculosis antigens could elicit Ag85B-specific IgG expression and Ag85B-specific IgG titre was increased as the inoculation time increased. After 4 weeks'vaccination, IgG titre of Yeast-IEA group was significantly higher than those of Yeast-EA group and BCG group while PBS group and Yeast group showed no detectable IgG titre. Additionally, long time immunization period could also enhance IgG2a,IgGl expression and increase IgG2a/IgGl ratio substantially. After 4 weeks'vaccination, IgG2a/IgGl ratio of Yeast-IEA group was significantly higher than those of Yeast-EA group and BCG group. These results indicated recombinant Saccharomyces cerevisiae vaccine could activate mice humoral response and Yeast-IEA had a more potent effect on inducing Thl type cellular response than Yeast-EA and BCG. (2) ELISA results showed recombinant Saccharomyces cerevisiae expressing tuberculosis antigens could stimulate Ag85B-specific IFN-γand IL-2 secretion instead of IL-4 secretion and both the IFN-y and the IL-2 secretion levels of Yeast-IEA group were significantly higher than those of Yeast-IEA group and BCG group. Our intracellular cytokine staining results also showed, in total spleen cells, the percentage of CD4+ T cells expressing IFN-y or IL-2 of Yeast-IEA group was signigicantly higher than those of Yeast-EA group and BCG group, which was consistent with the ELISA results. These findings suggested recombinant Saccharomyces cerevisiae vaccine could activate Thl type cellular response and compared with Yeast-EA, Yeast-IEA was a more effective activator.(3) Our surface marker staining experiments showed, recombinant Saccharomyces cerevisiae immunization contributed to the elevated percentages of CD80,CD86,MHCⅠand MHCⅡpositive dendritic cells(DC) in mice spleen cells. Further. we co-incubated MACS(Magnetics Assisted Cell Sorting)-purified DCs with respective inducer in vitro and observed elevated percentages of CD80,CD86 positive dendritic cells(DC) in mice spleen cells as well. These experiments proved direct contact with yeast could stimulate the maturation of DCs.(4) In order to ascertain the protective function of recombinant Saccharomyces cerevisiae vaccine on mice infected by BCG, mice were challenged by tail intravenous injection of BCG. CFU (Colony Forming Units) experiments results demonstrated, compared with the control group, pulmonary bacteria loads were drastically reduced in recombinant Saccharomyces cerevisiae vaccination groups, suggesting recombinant Saccharomyces cerevisiae vaccine could mitigate tuberculosis infection in mice.In conclusion, subcutaneous immunization by recombinant Saccharomyces cerevisiae expressing TB antigens can activate both humoral and cellular responses and help to protect mice from tuberculosis infection. This study provides foundations for new anti-TB vaccine development.