PROJECT SUMMARY Lyme disease, due to infection with the tick-transmitted spirochete Borrelia burgdorferi, is a multisystem disorder that can present with either localized skin infection or with disseminated infection involving multiple skin sites and other organs systems, including the heart, nervous system and joints. Humoral immunity is a critical host defense against B. burgdorferi infection, and a strong early B cell response in the blood is associated with symptom resolution. B. burgdorferi infection can also trigger autoantibody production and immune dysregulation associated with autoimmune pathologies. Despite the importance of antibody production to host defense, little is known about their role in the skin where tick-transmitted spirochetes first establish infection. We have applied advanced transcriptome technologies to study the sparse B cell populations found in the primary erythema migrans lesion and nonlesional skin of subjects who presented with localized or disseminated Lyme disease. Using 10X single cell immune repertoire and gene expression profiling, we identified antibody sequences from B cells that have the characteristics of a response to an immune challenge such as infection (i.e. clonal expansion, somatic hypermutation and class switching). These sequences were not present in nonlesional skin, but in some cases were also found in the blood of the same patient. In preliminary data, we have produced 11 recombinant human monoclonal antibodies (mAb) based on these antibody sequences and to date, have found that 2 mAb react with a surface exposed Bb antigen, 15-20kD in size. In this proposal, we will expand the recombinant mAb production and determine if the mAb react with B. burgdorferi or self-antigens. We will use the newly developed and innovative Rapid Exoproteome Antibody Profiling (REAP) discovery tool for high throughput screening of recombinant mAb and immune sera for reactivity against proteins in the exoproteome. Archived sera collected prospectively from Lyme subjects at diagnosis and several time points in convalescence will be screened for reactivity against identified Bb antigens as well as self antigens over time. The results of these studies will be the first tissue-specific analysis of the antigen specificity of B cells in early Lyme disease, and provide insight into the durability of immune responses and capacity of the antibodies to influence infection outcomes. It may also provide identities of new target antigens for use in diagnostic tests for early Lyme disease or distinguish new infection from previously treated infections. In addition, these studies may provide insight into when self-reactivity first arises after B. burgdorferi infection and the evolution of the response to targeted antigens. Successful completion of these studies will create a model for investigation of the skin B cell response to other vector-borne pathogens of public health significance.