PROJECT SUMMARY/ABSTRACT The overall goal of this project addresses the molecular and structural basis of neutralization utilized by human monoclonal antibodies (mAbs) against Eastern equine encephalitis virus (EEEV). The North American lineage of EEEV (NA-EEEV) is the most virulent of the encephalitic alphaviruses with up to 70% human and 90% equine mortality rate. In addition, of those that survive infection, up to 80% show signs of neurological deterioration. On average, eight human cases a year in the US are reported. However, the recent rise in detection of NA-EEEV in human-feeding mosquito species raises concern for a large outbreak in the eastern US. Moreover, NA-EEEV is considered a NIH Category B priority pathogen and USDA/CDC Select Agent due to its potential threat as a bioterrorism agent. There are no approved antiviral drugs or licensed human vaccines available for NA-EEEV. The antibody response to alphaviruses has been shown to be an important part of the immune response in conferring protective immunity and aiding in the clearance and recovery from infection. However, the fundamental molecular and structural mechanisms of action of antibodies in humans to NA-EEEV remain poorly defined. To fill this gap in knowledge, the objective of this study is to determine the mechanisms by which human mAbs neutralize NA-EEEV. The overarching hypothesis of this study is that the principle mode of action of human neutralizing mAbs is to stabilize the virus particle and inhibit viral fusion to host cells. To test this hypothesis, I will isolate human mAbs from naturally infected survivors of NA-EEEV to the BSL-2 chimeric virus, Sindbis/EEEV (SINV/NA-EEEV) and recombinant NA-EEEV structural proteins. I will characterize the panel of human mAbs isolated for specific reactivity and neutralization potency against SINV/NA-EEEV. Of the human mAbs that neutralize NA-EEEV, I then will determine the step(s) in the replication cycle the mAbs neutralize the virus through in vitro mechanistic assays. To identify neutralizing antigenic determinants, I will use epitope mapping, alanine scanning mutagenesis, and structural biology techniques. Determination of the neutralizing antigenic determinants recognized by human NA-EEEV mAbs will inform how mAbs interact with NA-EEEV. The results obtained from this project will fill major gaps in knowledge about the human antibody response to NA-EEEV, will identify potential correlates of protection, and may facilitate the design of efficient therapeutics and vaccines against this clinically relevant alphavirus. The proposal outlines a comprehensive fellowship training plan that will prepare me for a high-impact career in the field of viral immunology. To accomplish this program and research, we have assembled a collaborative team consisting of principal investigators and staff scientists with exceptional expertise in the immunology, virology, and structural biology fields to provide support and guidance for this study.