SBIR-STTR Award

Early diagnosis and treatment of Lyme disease is associated with an improved clinical outcome, and is recommended to Drevent subsequent manifestations such as arthritis and cranial paralysis. Unfortunately, early diagnosis of Lyme disease has been hampered because B. burgdorferi antigens and the human humoral response to them have been only partially characterized, and because there are no reliable serological assays for diagnosis of the disease. We will develop immunodots and ELISA assays for diagnosis of Lyme disease based on cloned B. burgdorferi gene products that are human immunogens. We shall investigate sensitivity and specificity of these diagnostic assays. The B. burgdorferi gene-products to be used are distinct from all characterized antizens as OspA/OspB, OspC, flagellin and heat-shock proteins. In preliminary studies, we have identified a single clone from a lambdagtl l B.burgdorferi library expressed in E. coli that reacted only with sera from patients with Lyme disease and failed to react with anti-Osp>4/OspB, anti-OspC or anti-flagellin antibodies or with sera from normal controls. We will finish sequencing the DNA from this clone, characterize its gene-products biochemically, and partially purify them. Recombinant B. burgdorferi antigens will be analyzed in ELISA and immunodot binding assays for their ability to discriminate between sera from patients with Lyme disease, sera from patients with other infectious diseases, and sera from normal individuals.Awardee's statement of the potential commercial applications of the research:Lyme disease is the most common arthropod borne disease in the United States. There is a great need for reliable serological assays to diagnose this disease, as B. burgdoferi is difficult to culture from patients, and the current serological assays lack specificity and sensitivity. The total market for serological assays for the diagnosis of Lyme disease is estimated to be 2,000,000 units in this country and a similar number in Western and Eastern Europe.National Institute of Allergy and Infectious Diseases (NIAID)

Lyme disease is the most common arthropod-borne disease in the United States. It is a chronic, multisystem, tick-borne infection caused by the spirochete Borrelia burgdorferi. It is characterized by recurrent systemic and local manifestations involving the skin, heart, nervous system and joints. Serological assays appear to be a feasible and practical approach to diagnose B. burgdorferi infections. However, development of such assays has been hampered by the complexity of B. burgdorferi antigens, the antigenic variability of different genospecies and geographic isolates of this organism, the heterogeneity of patient immune response to it, the lack of reliable and standardized laboratory diagnostic tests for Lyme disease, and uncertainty about exactly which B. burgdorferi antigens should be present in a diagnostic test. As B. burgdorferi appear to be clonal, chromosomally-encoded gene products of B. burgdorferi are likely to be more genetically stable than plasmid gene products, and are more likely to be present in all genotypes and isolates of these bacteria. We have used molecular genetic technology to identify a B. burgdorferi chromosomally-encoded putative outer surface lipoprotein, BmpC. We will test the hypothesis that this protein can be used to develop a diagnostic assay for Lyme disease. To provide evidence for this hypothesis, we will express recombinant B. burgdorferi BmpC in Escherichia coli as a native, non-fused protein, characterize it biochemically and immunochemically, and confirm its presence and expression in a variety of B. burgdorferi genera and isolates using molecular genetic, biochemical and immunochemical techniques. Immunochemical analyses will use polyclonal and monoclonal antibodies raised to BmpC and other well characterized B. burgdorferi proteins. Finally, we will characterize human immune response to BmpC and its fragments using ELISA, immunodot and T-cell stimulation assays and compare responses of patients with early and late Lyme disease, with and without arthritis and with and without neurological complications, to those of patients with other febrile infectious diseases including spirochetal diseases, and to those of healthy control subjects.Proposed Commercial Applications:Lyme disease is the most common arthropod borne disease in the United States. There is a great need for reliable serological assays to diagnose this disease, as B. burgdorferi is difficult to culture from patients, and the current serological assays lack specificity and sensitivity. The total market for serological assays for the diagnosis of Lyme disease is estimated to be 2,000,000 units per year in this country and a similar number in Western and Eastern Europe.