PROJECT SUMMARY Ixodes scapularis saliva proteins that elicit tick immunity Acquired resistance to Ixodes scapularis, commonly known as “tick immunity”, prevents transmission of Borrelia burgdorferi, the agent of Lyme disease. We hypothesize that tick proteins expressed in saliva confer tick immunity, and that their identification and characterization can be utilized as the basis of a tick vaccine to prevent transmission of B. burgdorferi. The goal of this proposal is to identify the specific proteins in I. scapularis saliva that elicit tick immunity. Our published data demonstrate that tick immunity can be generated in guinea pigs repeatedly exposed to I. scapularis, and consequently prevent tick- borne B. burgdorferi transmission. Our preliminary data show that: (a) immunization of guinea pigs with I. scapularis saliva can induce robust tick immunity. (b) protease treatment of saliva eliminates tick immunity. (c) tick immune guinea pigs immunized with I. scapularis saliva elicit antibodies recognizing the salivary protein tick salivary lectin pathway inhibitor (TSLPI) and salivary protein 14 (Salp14). (d) guinea pigs immunized with recombinant TSLPI and Salp14 induce partial tick immunity. We will now delineate the relative importance of TSLPI, Salp14 and additional saliva proteins in the induction of tick immunity (Specific Aim 1). We will also use bulk and single-cell RNAseq to carefully determine the host response to saliva and saliva proteins at the tick bite site, and elucidate the responses associated with tick immunity (Specific Aim 2). These studies will lead to new knowledge to aid in the development of a novel vaccine which may prevent Lyme disease and other tick-borne infections.