PROJECT SUMMARY - RP2 Picornaviruses are an ever-present threat to human health, causing myriad diseases ranging from mild (common colds, hand, foot, and mouth disease) to more severe (meningitis, acute flaccid myelitis, myocarditis, and neonatal sepsis). Fortunately, one of the most successful vaccine programs in history targeted picornaviruses; the poliovirus vaccines completely eradicated two of three types and nearly eliminated the third. Learning from this experience, neutralizing antibodies in the sera of vaccinees are a well-established correlate of protection against paralytic poliomyelitis. Therefore, to prepare for a possible picornavirus pandemic in the future, we must have tools in place to comprehensively analyze the antibody response to an emerging pathogen to inform adaptable and streamlined picornavirus vaccine platforms. To accomplish those goals, we have assembled a team of experts in picornaviruses and human antibodies with an established record of collaboration. We will focus our studies on four prototype picornaviruses with antigenic diversity and different sites of infection: respiratory (enterovirus D68 [EV-D68], rhinovirus C [RV-C]) and gastrointestinal (enterovirus A71 [EV-A71], echovirus 11 [E11]). Studying these diverse viruses will best prepare us to respond to an emergent picornavirus with unknown properties. Our first specific aim focuses on understanding the polyclonal humoral immune response to picornavirus infections. We will leverage biobanked samples from a previous study, new samples from a recently funded cohort, and our Virologic and Immunologic Sample Acquisition Core (VISAC) samples to identify children with high neutralizing serum titers against the prototype viruses. We will use cryo-electron microscopy to map the binding of polyclonal antibodies from these sera to purified viruses to identify epitopes. We will supplement these data with tiled peptide arrays to comprehensively analyze linear B cell epitopes of RV-C proteins. We will use these advanced techniques to analyze sera before and after defined infections to define antibody binding patterns before and after children develop high neutralization titers in their serum. Our second specific aim focuses on monoclonal antibody (mAb) discovery. Starting with our established EV-D68 human mAb isolation methods, we will speed the time of discovery from months to about two weeks, then use these methods to isolate EV-A71 and E11 mAbs. Our prior screening methods (identifying mAbs that bind to native virions in ELISA) are suboptimal for RV-C, so we will develop new screening methods for this virus that will better prepare us to respond to emergent pathogens. As we identify new antiviral mAbs, we will select strongly neutralizing mAbs for study in our Structural Biology Core and test them in models of infection developed in Project 1 of this program. We will use protective mAbs as quality control reagents to ensure that new candidate vaccines maintain antigenici...