Lyme disease (LD), caused by the Ixodes tick-borne spirochete Borrelia burgdorferi (Bb), is the most common vector-borne disease in the United States. Despite public health preventive measures, the annual confirmed case incidence has risen to nearly 30,000, the vast majority of which occur in the Northeast. Disseminated infection causes disease in the skin, heart, joints and nervous system and can be more difficult to treat. Although antibiotics achieve clinical cure when administered in early stages of infection, delay in diagnosis and treatment can lead to substantial morbidity and health care expenditures for unexplained symptoms that may persist. Timely and accurate diagnosis of LD, therefore, is essential for optimizing treatment and preventing long-term sequelae of the disease. Currently, serologic tests that detect Bb-reactive antibodies are the mainstay of LD diagnostics, but have low sensitivity early in infection, do not distinguish previous exposure to Bb from active infectio, and cannot be used to assess response to therapy. This proposal seeks to improve upon current serologic tests by developing a new diagnostic cytokine assay for LD based on a novel panel of Bb antigens expressed in the skin and/ or required for Bb infection and persistence in the mammalian host. The R21 phase of the proposal will first use a multiplex cytokine assay to determine whether recombinant Bb proteins/synthetic peptides elicit cytokine production from whole blood obtained from patients with newly diagnosed, active LD. We will then optimize the assay conditions, including cytokine selection and Bb protein combination, to produce a refined whole blood cytokine assay that will be further validated in the R33 phase. In a prospective study of a large number of LD subjects and controls conducted during the R33 phase, we will define the sensitivity and specificity of the whole blood cytokine assay and determine whether positive responses in the assay are associated with persistent symptoms after antibiotic treatment. As a strategy that complements current serologic tests, the whole blood cytokine assay will improve the combined sensitivity and specificity of LD diagnostics, especially in situations in which the serologic tests alone under-perform.