Molecular Organization and Antibody Recognition of the New World Arenavirus Glycoprotein Complex

Abstract

Of the multiple arenaviruses that cause hemorrhagic fevers in the Americas, all lack reliable therapeutic options, and only one has a vaccine. The arenavirus glycoprotein complex (GPC) mediates cellular entry through pH-dependent fusion and is the sole target of neutralizing antibodies. GPC is a unique class I viral fusion protein comprising a tripartite assembly of GP1, GP2 and stable signal peptide (SSP) subunits. Currently all characterized New World arenavirus neutralizing antibodies are derived from animal studies or human vaccination and target GP1 to prevent receptor binding. A lack of trimeric GPC reagents has hindered a broader understanding of GPC immunogenicity, and their development has been complicated by a lack of New World GPC structures. Understanding the prefusion GPC architecture is therefore critical for the rational design of stabilized GPC trimers useful for examining humoral responses to arenavirus infection. In this dissertation, we use JUNV GP1 to isolate 203 monoclonal antibodies from the peripheral blood mononuclear cells (PBMCs) of four survivors of Argentine hemorrhagic fever (AHF). Functional and structural analyses show that a majority of these antibodies are neutralizing and that a subset closely mimic the host receptor (TfR1) by inserting a CDR H3 tyrosine into the GP1 receptor-binding pocket. Importantly, select survivor-derived antibodies provide significant therapeutic protection in lethal murine and guinea pig models of JUNV infection. We also report high-resolution cryo-EM structures of Junin virus (JUNV) and Machupo virus (MACV) GPC stabilized in the prefusion conformation using a single amino acid substitution in SSP. These structures provide the comprehensive architecture of the arenavirus GPC, including previously unresolved transmembrane and cytosolic segments. Functional assays reveal how contacts in the membrane-proximal and transmembrane regions regulate GPC metastability and fusogenicity. Importantly, these data reveal the molecular source of attenuation for the Candid#1 vaccine and demonstrate the need for the development of next-generation vaccine platforms with improved safety and immunogenicity profiles. Altogether, Chapter 1 highlights current understanding and knowledge gaps related to New World arenavirus biology, GPC structure, and antibody interactions. Chapter 2 describes our efforts to characterize protective anti-GP1 humoral responses during JUNV infection in humans and provides a framework for further inquiry into antibody responses against the full-length New World arenavirus GPC. Chapter 3 presents our structural and functional interrogation of JUNV and MACV GPC, providing comprehensive high-resolution models that may aid in the design of stabilized trimeric antigens, therapeutic antibody cocktails and small-molecule inhibitors.