Chitosan microspheres enhance the immunogenicity of an Ag85B-based fusion protein containing multiple T-cell epitopes of Mycobacterium tuberculosis

To develop novel delivery system for tuberculosis (TB) subunit vaccine, biodegradable chitosan microspheres were prepared and used to deliver a fusion protein, Ag85B-MPT64(190-198)-Mtb8.4 (AMM for short), made from three Mycobacterium tuberculosis genes. AMM-loaded microspheres were first characterized for their morphology, size, zeta potential, loading efficiency, and in vitro release of AMM. C57BL/6 mice were immunized at weeks 1, 3 and 5 subcutaneously with AMM formulated in chitosan microspheres, in incomplete Freund's adjuvant (IFA), or in phosphate-buffered saline (PBS), respectively. Three weeks after the last immunization, humoral and cell-mediated immune responses were examined. It was shown that the microspheres bound AMM quite efficiently (loading efficiency: >99%). AMM-loaded chitosan microspheres were observed as aggregated shapes with the average particle size of 5.78+/-0.65 microm and zeta potential of 32.77+/-1.51 mV. In vitro release studies revealed that only small amount of antigen was released in 16 days. Following subcutaneous administration, splenocytes immunized with AMM in chitosan microspheres produced higher levels of IFN-gamma compared to administration of AMM in PBS upon stimulation with Ag85B and synthetic peptide MPT64(190-198). The levels of Ag85B-specific IgG (H+L), IgG1 and IgG2a in sera of mice immunized with AMM in chitosan microspheres were also higher than those with AMM in PBS. These results indicate that chitosan microspheres when used as a carrier for fusion protein AMM could elicit strong humoral and cell-mediated immune responses.