PROJECT SUMMARY Borrelia miyamotoi is a newly emerging relapsing fever (RF) spirochete transmitted by Ixodes tick spp that also transmit the Lyme disease agent Borrelia burgdorferi. B. miyamotoi has recently been found to cause human disease in areas where Lyme disease is endemic. Our group has isolated B. miyamotoi from a person with disseminated Lyme disease, demonstrating that coinfection of B. miyamotoi and B. burgdorferi is occurring. The full spectrum of B. miyamotoi-associated disease is not known, however other RF Borrelia cause blood disorders, multiorgan system dysfunction, and fetal demise in humans. This project will use a mouse model to define B. miyamotoi pathogenesis, host immune responses and risk factors for disease, and to examine the impact of B. miyamotoi/B. burgdorferi coinfection on disease associated with each of these pathogens. Studies will be conducted with our clinical isolate of I. scapularis-borne B. miyamotoi from Connecticut and a newly generated tick isolate from Wisconsin. The duration of tick attachment for B. miyamotoi transmission will be determined as well as kinetics of bacteremia, spectrum of disease and sites of persistence. As the greatest B. miyamotoi diversity is seen among isolates from different Ixodes spp, we will evaluate disease expression after infection with an I. pacificus-borne B. miyamotoi isolate. Studies in immunodeficient mice and mice with altered immunity due to splenectomy, immunosuppressive agents, and pregnancy will be conducted to delineate risk factors for more severe disease and adverse maternal-fetal outcomes. Based on preliminary data, we hypothesize that the early innate defense against B. miyamotoi may differ from other RF Borrelia. We will employ the advanced technology of cytokine time-of-flight mass spectrometry (CyTOF) to broadly immunophenotype responding cells and the cytokines they produce during infection. We will determine whether antibodies elicited by infection can prevent challenge infection in a naïve host or in the same host challenged with tick-transmitted spirochetes. We will assess the impact of B. burgdorferi/B. miyamotoi coinfection on disease expression associated with each of these pathogens. CyTOF will be conducted to phenotypically and functionally characterize the host immune response to coinfection with B. miyamotoi and B. burgdorferi, pathogens that thrive differentially in blood and connective tissue. The results of these studies will provide clinically useful information regarding tick transmission dynamics, B. miyamotoi tissue tropism and disease, and immune correlates of protection to predict risk factors for human disease and adverse outcomes. !