PROJECT SUMMARY New World Hemorrhagic Fever (NWHF) mammarenaviruses are category A pathogens that pose a grave threat to humans due their high infectivity, mortality between 15-30% when untreated, and our lack of therapeutic options. A subgroup of NWHF mammarenaviruses include the clade B pathogens Junin, Machupo, Guanarito, Chapare and Sabia, which collectively put thousands at risk per year and are considered potential bioweapons. Infection of human cells by New World hemorrhagic fever viruses is mediated by the binding of their surface glycoprotein (GP1) to the human Transferrin Receptor 1 (hTfR1/CD71). However, while all viral glycoproteins bind the same receptor, structural variability among GP1 proteins curtails efforts to develop broadly neutralizing therapeutics against the entire group of viruses; this is one of the most complex challenges faced by targeted therapies. We recently reported the use of a monoclonal antibody (ch128.1), which targets the ectodomain of hTfR1, to efficiently inhibit the infection of mammalian cells by pseudovirus expressing pathogenic NWHF GP1. We aim to develop an effective and broadly neutralizing therapeutic against NWHF mammarenaviruses and establish the structural foundation for its inhibition. To achieve this goal, we will generate and characterize single chain antibody fragments (scFv and scFab) with the variable region of the monoclonal antibody 128.1 that prevent binding of all pathogenic NWHF GP1 proteins to hTfR1. We will recombinantly produce and purify the single chain fragments and biochemically confirm their expected high affinity and specificity for hTfR1. We will also evaluate fragments for their ability to inhibit entry of pseudoviruses decorated with NWHF virus GP1 into cells expressing hTfR1. We will then crystallize our fragments alone and bound to hTfR1, and determine their atomic structure. Our elucidation of these structures will benefit from frontier crystallographic tools at our disposal, including microfocal x-ray diffraction and electron micro- diffraction (MicroED). The resulting structures will provide us with a template from which we can generate new generations of improved and broadly acting therapeutics against all clade B NWHF mammarenaviruses.