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The only available tuberculosis vaccine, bacille Calmette Guérin (BCG) has highly variable efficacy. Antigen 85B (Ag85B) is a protein secreted by Mycobacterium tuberculosis (MTB) in the early culture and a promising subunit antigen. Pulmonary vaccination is an attractive immunization route. It was proposed that Ag85B/adjuvants in microparticle formulations intended for pulmonary delivery would target macrophages, elicit related cell-mediated immune response sufficient to protect guinea pigs from tuberculosis. Three specific aims were adopted to test the hypotheses. (1) To express recombinant Ag85 (rAg85B) from E. coli suitable for inclusion in polymeric microparticles for (2) in vitro screening of particle formulations with respect to related cell-mediated immunity and for (3) assessment of protection from infection in a guinea pig model of tuberculosis. rAg85B was expressed from two E. coli strains and encapsulated by spray-drying in poly(lactide-co-glycolide) (PLGA) microparticles with adjuvants muramyl dipeptide (MDP) or trehalose dibehenate (TDB) to be delivered as dry powder aerosols. The antigenicity of the recombinant forms was evaluated by monitoring IL-2 secretion from T hybridoma cells DB-1 after recognition of Ag85B97-112 epitope presented by THP-1, antigen presenting cells. The powders were administered as single or multiple doses to guinea pigs to evaluate protection from a low dose aerosol infection with MTB strain H37Rv. Spray-dried microparticles exhibited mass median aerodynamic diameters (MMAD) of 2.5-3.0[mu]m, suitable for inhalation aerosols. The amount of IL-2, an indicator of antigenicity, released from T hybridoma cells following PLGA-Ag85B microparticle administration to THP-1 phagocytic cells, was 92-360 fold that of rAg85B solutions. Surface-associated rAg85B played a significant role in the observed IL-2 release. This formulation extended epitope presentation as demonstrated by duration and extent of IL-2 production. BCG prime: PLGA-Ag85B aerosol boosts (x2) appeared to enhance protection from infection with virulent MTB strain H37Rv compared to BCG alone. However, further work is required to demonstrate this effect conclusively. This is a clinically meaningful observation since many susceptible individuals in the developing world have been immunized with BCG. The results indicate that microparticles encapsulating rAg85B were immunologically effective, with preliminary evidence for protection and, potential for pulmonary vaccination to prevent MTB infection.