Enrichment and validation of urine and serum-specific antigens from acute Lyme disease patient samples

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ABSTRACT Lyme borreliosis is the leading vector-borne disease in the United States. Early diagnosis and treatment of Lyme disease is critical to prevent the development of severe complications such as extensive arthritis, chronic neurological impairments, and carditis. A critical barrier in the clinic is the inability to diagnose Lyme disease early during infection in patients that do not develop a distinct erythema migrans. The only FDA cleared tests for Lyme disease are two-tier serologic assays that are laborious to perform. More importantly, such assays are unable to diagnose Lyme disease in its early stages due to the time that is required for a patient to develop an antibody response to the infecting Borrelia burgdorferi (Bb), the causative agent of Lyme disease. In this proposal, our objective is to identify and validate urine and serum-specific antigens in samples collected from patients with Lyme disease. Validated antigens will be used, as part of a future proposal, to develop Bb antigen detection assays for the early diagnosis of Lyme disease. This is a challenging research endeavor that we believe will require an unwavering focus on the following two hypotheses: First, development of a diagnostic test detecting only one Bb antigen will not result in a clinically-useful test for early Lyme disease. We strongly believe that targeting multiple antigens, as opposed to a single antigen, will be required for the development of a highly-sensitive assay. During our preliminary studies we optimized methods to discover antigens that are present within samples collected from Lyme patients as well as a tick-borne macaque model of Lyme disease. Six candidate Bb antigens were identified within serum; these antigens will be considered for validation in patient samples through this R21 proposal. To demonstrate that the use of the DIA-MS approach, described in this proposal, will result in the consistent detection of potential antigens results from a preliminary study in which 5 acute patient serum samples were analyzed using the proposed method. The analysis found that 243 proteins were detected in all 5 samples, confirming the reliability of the approach. The reliable detection of Bb-specific antigens from biological samples is a significant diagnostic breakthrough, however, detection of multiple Lyme antigens will be essential to achieve high clinical sensitivity. Second, a diagnostic test for early Lyme disease will require patient sample enrichment prior to downstream assays. This has become clear from our preliminary studies with Lyme samples, as well as our studies to detect other infectious agents from patient samples. The development of sample enrichment protocols that require minimal processing will ensure that enrichment is compatible with the workflow of a clinical laboratory. A collaboration with John Hopkins Lyme Disease Research Center will allow for the inclusion of paired human urine and serum samples in the discovery and validation stages of the project. Ultimately, simplifying the diagnosis of Lyme disease will allow for an earlier diagnosis, improved patient outcome, and an easement of Lyme-related healthcare burdens.