This Small Business Innovation Research (SBIR) Phase I project aims to develop recombinant monoclonal antibodies using "mosaic" antigens derived from Leptospira spp proteins identified in the urine of clinical patients. The mosaic antigens are designed to artificially combine important antigenic epitopes in order to maximize the coverage of potential epitopes in one structure and to provide broader coverage than those afforded by multiple synthetic peptides. Our approach involves the use of computational biology and bioinformatics to create, score, and select "mosaic" antigens from Leptospira spp. Antigenic properties of the mosaic antigens are evaluated by indirect ELISA using a panel of well-characterized human sera from clinical patients and apparently healthy individuals. We will then use recombinant DNA and protein engineering techniques to derive cognate chimeric proteins, followed by the generation of recombinant epitope-specific monoclonal antibodies that target potential epitopes (rather than a few dominant epitopes) generated from the "mosaic" protein. The diagnostic potential of the recombinant antibody is evaluated by immunoblotting and ELISA using leptospirosis specimens from early and convalescent phases of the illness.
The broader impact/commercial potential of this project, if successful, will be the development of a rapid, non-invasive, one-step, multiplex test that will have general applicability in the timely diagnosis of a complement of febrile illnesses, including leptospirosis. This is aimed at tropical and subtropical countries where the disease incidences are higher and where current tools are mostly inadequate. Early leptospira diagnosis is essential because antibiotic treatment is most effective when initiated early in the course of the disease. Current diagnostic tools are unsuitable for use in resource-limited settings especially in the tropical regions where other similar acute febrile illnesses are common. It expands the multiplex diagnostic test panel we are presently developing by iterative expansion.
Pathogenic species of Leptospira cause the global zoonotic disease leptospirosis – a potentially serious but treatable disease whose symptoms may mimic those of a number of other unrelated infections. The disease is considered as one of the most widespread zoonotic diseases in the world, and ranked as the 7th highest global risk-severity index (GRSI) disease by the Armed Forces Medical Intelligence Center (AFMIC) among 53 infectious diseases. Due to its global distribution, the health risk posed by the disease directly affects the United States’ national interests as US military and civilian personnel deployed overseas are at high risk of being infected. Symptoms of leptospirosis are easily confused with a variety of other pathogens such as dengue, malaria and Q fever that require different treatment regimens, therefore, early specific diagnosis is essential. Current diagnostic methods rely on clinical presentation, serology or isolation of the pathogen from the specimen, which are inaccurate and/or labor-intensive, time-consuming, and requiring substantial training/expertise, especially in resource-limited settings. Progress in proteomic analyses of differentially expressed proteins in the urine of clinical Leptospira patients have allowed the identification of biomarkers that may be useful for the development of more efficient and accurate diagnostic tests for leptospirosis. In this project, we developed diagnostic recombinant antibodies from chimeric leptospiral protein antigens. Our approach involved the use of computational biology and bioinformatics to create, score and select the chimeric antigen from Leptospira spp. Using this approach, we generated a leptospira antigen cocktail that combined reactive antigenic epitopes to enhance the coverage of potential epitopes in one structure and provide broader coverage than those afforded by multiple synthetic peptides. The antigenic properties of the antigen and diagnostic potential of the recombinant antibody were evaluated by indirect ELISA and immunoblotting. In Phase II of the project, we aim to evaluate the diagnostic utility of the reagents on clinical samples from leptospira patients.
