Tuberculosis remains one of the most significant public health challenges the modern world faces. Improved vaccines are needed for prevention of infection and improved immunotherapies are needed to combat existing or recurring disease. An better vaccine would induce a concerted protective response by CD4+ and CD8+ T cells together. Our hypothesis is that CD4+ and CD8+ T cells sample different bacterial compartments differently. Information about which compartment is optimal for effective presentation of ClassII and Class I epitopes and generation of protective responses will help create better vaccines. To do this we will use a novel approach while building on the strengths of previous experimental systems. TCR transgenic mice will be infected with recombinant Mycobacterium bovis strain bacille Calmette Guerin (BCG), the current vaccine strain. These rBCG will express T cell epitopes in the context of the same fusion proteins located in different subcellular compartments of the bacteria. A parallel series of rBCG strains will be constructed for both class I or class II presentation using either Lymphochoriomeningitis Virus (LCMV) gp33 peptide or pigeon cytochrome C peptide (PCC) respectively. We will study how access of each epitope to its respective presentation pathway is influenced by its location in different bacterial compartments. Subsequently, we will study the activation and recruitment of antigen specific cells both systemically and in the BCG induced liver granulomas in response to various rBCG using adoptively transferred antigen specific T cells. Our final analysis will be to study how bacteremia is effected when antigen is presented in different bacterial compartments with or without prior peptide specific immunization. In this manner we hope to define how the different epitopes in different bacterial compartments effect T cell responses and protection. We chose PCC (CD4+ specific) and gp33 (CD8+ specific) for this work because they are both widely studied model antigens and a multitude of reagents are available, including T cell clones, hybridomas, TCR transgenic mice, and MHC tetramer reagents. The mouse model of BCG infection was chosen because we wish to improve the vaccine capacity of this attenuated strain and also because infection of mice with BCG has been widely employed and many of the characteristics of this model are well understood. The experimental results from this proposal should have direct relevance to improving vaccine design for protection against tuberculosis, and will also provide knowledge about how bacterial antigen access different antigen presenting pathways.
