Comprehensively mapping the functional and antigenic effects of mutations to the Lassa virus glycoprotein complex

Project Summary/Abstract Lassa virus is estimated to cause thousands of human deaths each year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, I propose to use pseudovirus deep mutational scanning to comprehensively map how amino-acid mutations to GPC affect cell entry and neutralization by monoclonal and polyclonal antibodies. Pseudovirus deep mutational scanning involves creating large libraries of biosafety-level-2 single-round replicative pseudotyped lentiviruses carrying all mutations to a protein of interest (i.e., Lassa GPC), applying a selection pressure, and then deep sequencing the initial and selected libraries to quantify the effects of each mutation. Aim 1 of this proposal will characterize functional constraints throughout GPC as well as across Lassa lineages by mapping the effects of mutations on cell entry. The insight provided by this aim will aid the design of immunogens for candidate vaccines and therapeutics. Aim 2 of this proposal will quantify how mutations affect antibody-based neutralization from both monoclonal antibodies and polyclonal sera. Monoclonal antibodies are being developed as therapeutic cocktails; therefore, prospectively identifying escape mutations can guide development of antibody-based therapeutics against current Lassa diversity and possible future evolution. Furthermore, little is known about the polyclonal humoral response to natural Lassa infections and candidate vaccines, so mapping GPC mutations that escape neutralization from polyclonal sera will provide insights into the specificity and breadth of antibody based immunity as well as the person-to-person variation of immune responses. Aim 3 will leverage the comprehensive antibody escape measurements to assess how the natural diversity affects the neutralizing capability of potential antibody-based therapeutics. Analyzing the antigenicity of the current natural Lassa virus strains will provide valuable insight for the development of region-specific and pan-Lassa virus therapeutics. To facilitate the use of this data by other researchers, I will integrate these maps into an interactive Lassa virus phylogenetic tree publicly available on Nextstrain. Overall, this work will provide information vital for understanding the forces that drive GPC variation, enabling the design of therapeutics robust to viral escape, and understanding the potential for future GPC evolution.