Tuberculosis is the world's leading cause of death from an infectious agent. The World Health Organization (WHO) estimates that there are 8 million new cases annually and two to three million deaths. Current methods for diagnosis and treatment, particularly of multiple drug resistant strains, are inadequate. In 1993, WHO took the unprecedented step of declaring tuberculosis a global emergency. Mycobacterium tuberculosis, the cause of tuberculosis, is a difficult microorganism to study using conventional methods. Consequently, there is still much to be learned about the ways it causes disease. In this proposal we describe the application of a novel proprietary technology that we call in vivo induced antigen technology (IVlAT) to the study of M. tuberculosis. It is our intention in this Phase I SBIR to identify a select group of patentable M. tuberculosis targets for vaccine, diagnostic and antibiotic strategies. The platform IVlAT technology identifies genes of a pathogen that are specifically expressed during a human infection. IVlAT has proven itself superior to other related technologies in a number of ways, including the fact that it does not rely on animal models, it is readily applicable to essentially any bacterial or fungal pathogen, and it can identify transiently expressed genes. For these reasons, IVlAT will be employed to identify in vivo induced genes of M. tuberculosis strain CSU93. This will be accomplished in two specific aims. In the first specific aim, pooled sera from 10 tuberculosis patients representing a broad spectrum of disease states will be exhaustively adsorbed with in vitro grown whole M. tuberculosis cells and cell extracts. The resulting serum will be used in a colony lift method to probe a genomic expression library of M. tuberculosis in Escherichia coli.
In specific aim 2, the cloned DNA inserts in reactive clones will be sequenced and analyzed to determine the cloned open reading frames (ORFs) responsible for expression of the in vivo induced antigens. The pertinent ORFs will be subcloned into an appropriate expression vector and final confirmation of the clones will be obtained using polyacrylamide gel and Western blot methods. The in vivo induced antigens identified in this Phase I SBIR will be further characterized and analyzed during Phase II to identify ones best suited to serve as targets for diagnostics and vaccines and antibiotic therapies.
