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.
