Tuberculosis (TB) is a chronic infectious disease that infects approximately one-third of the world's population. Eighty-five percent of the estimated 9.4 million new cases of TB in 2008 occurred in resource-limited countries located in Asia and Africa. In 2010, there were 1.1 million deaths from TB among HIV-negative patients and 350,000 additional TB deaths among HIV-positive patients. In some tuberculosis-endemic areas, fewer than 40% of TB cases are diagnosed due to the lack of accurate and easy-to-use diagnostic assays. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis, in clinical specimens by serial sputum smear microscopy and culture. These methods lack sensitivity, are time consuming, are expensive, and require trained personnel. An alternative approach is to develop an efficient immunoassay to detect M. tb. antigens in bodily fluid, such as serum or urine. Current commercial immunoassay kits that detect M. tb. glycan lipoarabinomannan (LAM) in urine exhibit poor sensitivity perhaps due to low abundance. Alternative biomarkers need to be identified to facilitate accurate diagnosis and treatment of tuberculosis. Our overall hypothesis is that M. tb. biomarkers are shed into the urine and/or serum during infection and that high-affinity monoclonal antibodies can be generated in rabbits to these biomarkers. Subsequently, we hypothesize that through the use of these mAbs, a sensitive and specific immunoassay can be constructed to detect these biomarkers in patients. The first specific aim is to identify novel M. tb. biomarkers shed into serum and urine during infection. This will be accomplished by immunizing na?ve rabbits with serum and urine from infected rabbits, a technique known as InMAD. Unlike rabbits immunized with crude antigens, such as M. tb. whole cell lysate or culture filtrate, rabbits immunized using this approach will create antibodies to only clinically relevant biomarkers that are shed into the serum or urine. The key to this aim is that rabbits will be infected with a highly virulent strain of M. tb. (W-Beiing strain 5097 sublineage RD207) that has high transmission rates and represents endemic disease. The polyclonal antibodies will be used in proteomic analysis of serum and urine samples of infected rabbits in the second aim, which is to validate the identified biomarkers. The third specific aim is to produce rabbit monoclonal antibodies to the validated M. tb. biomarkers. While several M. tb. immunoreactive proteins have been previously described, the approach that we will use in all three aims will facilitate the identification of new biomarkers. Using the rabbit for both the disease model and immunization will provide relevant disease state serum and urine as well as antibodies that will have higher affinity and greater epitope recognition than their mouse counter-parts. If the goals of this Phase I are achieved, Phase II will use mAbs from Phase I to construct and evaluate an immunoassay in POC format. Our preferred assay platform would be the lateral flow immunochromatographic (dipstick) assay, as it will meet all of the World Health Organization's A.S.S.U.R.E.D. criteria for developing world diagnostic assays. If successful, this translational research project could dramatically decrease mortality from tuberculosis through accurate diagnosis, allowing for appropriate treatment. Importantly, this can be done at the low cost needed in resource-limited countries.

Public Health Relevance

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, the bacillus that infects approximately one-third of the world's population. Current diagnostic methods either lack effective sensitivity or are too labor- and resource-intensive for regions where tuberculosis burden is greatest. This is a translational research study whose initial goal is to identify biomarkers that are shed into the urine or serum of M. tuberculosis-infected rabbits with the ultimate goal of developing an immunoassay that will facilitate accurate and timely diagnosis of tuberculosis. If successful, this project could dramatically decrease the morbidity, mortality and health care costs associated with tuberculosis through earlier diagnosis.

National Institute of Health (NIH)
Small Business Technology Transfer (STTR) Grants - Phase I (R41)
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Special Emphasis Panel (ZRG1)
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Jacobs, Gail G
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Immuno-Mycologics, Inc.
United States
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