Abstract
Abstract BCG, the vaccine against tuberculosis (TB), is one of the mandatory vaccines administered to infants in several parts of the world. However, BCG conferred immunity wanes when children reach adulthood. To develop booster vaccines, we engineered recombinant Bacillus subtilis spores to deliver Mycobacterium tuberculosis (Mtb) antigens. MTAG1 recombinant strain was designed to express a fusion protein of CotC-Ag85B-CFP10 on the spore coat, while MTAG2 and MTAG3 strains were designed to express Ag85B-CFP10 fusion protein in vegetative cells. The difference between MTAG2 and MTAG3 strains is that the latter expresses secreted listeriolysin (LLO) in addition to Ag85B-CFP10. Immunoblot analysis revealed that MTAG1 strain showed positive signals for Ag85B and CFP10 in the spore-coat and other strains in the vegetative cell extracts, indicating that antigens are expressed in these strains as expected. Splenocytes of mice immunized with recombinant spores, through intranasal route, displayed significantly higher antigen specific proliferation of IFN-γ producing cells compared to the splenocytes of control mice. Also, the culture fluids of splenocytes from recombinant spores immunized mice exhibited higher antigen specific release of Th1 cytokines than the culture fluids of splenocytes from control mice. These results indicate that antigens delivered via recombinant spores can be processed and presented to the immune cells. Additional in vivo studies may reveal whether these recombinant spores can be used to boost immunity in BCG vaccinated individuals.
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