A high throughput protein expression platform has been developed which can be used to produce the complete proteome from F. tularensis, and the resulting proteome will be applied to the general problem of identifying potent vaccine antigens affective against F. tu/arensis infection. The proteome expression platform uses our GeneActivation by PCR (GAP) approach and a novel high throughput in vivo recombination cloning method for generating transcriptionally active genes that can be used directly in in vitro transfection assays, and in vivo. The system allows for the addition of poly-Histidine tags and other tags into the protein products so that they can be conveniently purified and detected, and the system has been adapted to a robotics workstation enabling 384 different purified proteins to be produced and purified in 1 day. The F. tularensis proteome will be prepared in 2 forms. The first form will be on protein """"""""chips"""""""" and the chips will be used to quantify serum antibody titers from infected or vaccinated, humans and animals, against each of the -2000 individual F. tularensis antigens. For the second proteome format, each individual protein will be purified and stored in 96-well microtiter plates in a form that will enable them to be delivered to, and processed by antigen presenting cells. These antigen presenting cells will then be used as targets in an ELISPOT assay to determine the level of antigen specific T-cells against each individual antigen from infected or vaccinated, humans and animals. This quantitative humoral and cellular immune response scan will produce a complete profile of the immune response against F. tularensis in humans and experimental animals. Mice will be immunized with the Live Vaccine Strain (LVS) of F. tularensis, and the humoral and cellular immune responses of the surviving animals will be scanned. The animals will be challenged with a highly virulent strain of F. tularensis (strain SchuS4) and the immune responses of the surviving animals will be re-scanned. Comparing the immune responses from animals that survive lethal challenge with those that don't survive will identify key antigens that are important for conferring protection. These data will be used together with other immunological and bioinformatics criteria to identify likely candidate antigens for a DNA vaccine or a protein subunit vaccine. The assays developed in mice will be adapted to human tissue samples, and the humoral and cellular vaccine antigen potency indexes will be determined in a group of humans previously immunized with the LVS vaccine. Since the immune response induced by the LVS vaccine is known to have protective activity against F. tularensis infection, the responsive antigens identified by the Vaccine Antigen Scan will be candidates for use in a DNA or subunit vaccine.
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