One of the most difficult tasks in developing a subunit vaccine is the identification of the antigens that will stimulate the most effective immune response against the pathogen, particularly when the genome of the infectious organism is large. A technology developed by Gene Therapy Systems Inc. under a previously funded SBIR grant (R43 AI47641-01) will be applied to the general problem of identifying potent vaccine antigens from tuberculosis. The technology called Transciptionally Active PCR (TAP) is a method for generating functional PCR fragments that can be used directly in in vitro transfection assays, and in vivo. TAP fragments can be used as templates in cell free in vitro trascription/translation reactions generating >50 micrograms of protein/50 microliter reaction volume, and the TAP system has been placed onto a robotics workstation enabling 384 different purified proteins to be generated in 1 day. This system will be used to amplify and purify all 3,924 proteins encoded by the genome of Mycobacterium tuberculosis. This operation will take, nominally, 10 days. The identity of each protein will be confirmed by electrospray mass spectrometry, and the reactivity of the proteins to the cellular immune response will be assessed using the murine model of tuberculosis and high throughput in vitro T cell assays.
