Project Summary Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative diseases affecting both humans and animals. The clinical manifestations of the disease include rapid progressive dementia, motor and psychiatric abnormalities, and short disease durations. Decades of research have firmly established that the pathogenic form, PrPSc, of the host-encoded prion protein (PrPC), is the causative agent of prion disease. Thus far, there is no prophylactic or therapeutic treatment available, and all forms of prion disease are invariantly fatal. The long-term goal of our study is to elucidate the pathogenic mechanism and to develop effective prophylactic and therapeutic strategies against this group of fatal neurodegenerative diseases. In the past few years, we have established a platform to generate the PrPSc conformer with bacterially expressed recombinant PrP in the presence of cofactors. Our rigorous studies have revealed that the in vitro-generated recPrPSc recapitulate all the properties of naturally occurring prions, which provided the strongest evidence supporting the prion hypothesis. Besides the infectious recPrPSc, we have also generated and characterized a series of recPrP conformers, which resembles PrPSc in conformation but do not cause any disease or abnormality in animals. Built upon our expertise in generating and characterizing these recombinant PrP aggregates with distinct biological activities in vivo, we propose to use these well-characterized recPrP conformers to develop novel intervention strategies against prion disease. First, we propose to determine the prophylactic potential of in vitro-generated, non-infectious, PrPSc-like recPrP conformers using animal bioassays. Second, we will determine, characterize, and optimize the capability of a novel PrP-binding nanobody against prion disease. Third, we have outlined a strategy to develop novel nanobodies with great diagnostic and therapeutic potential. Findings from these studies will greatly impact our efforts to develop an effective therapy against these devastating and incurable neurodegenerative disorders. In addition to their practical usefulness, the tools developed in this study will also greatly help us in exposing the molecular mechanism of prion infectivity and neutotoxicity.