P-1804. Deep Mutational Scanning of the RSV Fusion Protein Reveals Mutational Constraint and Antibody Escape Mutations

Abstract Background Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections of infants and older adults. Recent advances in RSV prevention include new vaccines for adults including one to protect infants via passive transfer of maternal antibodies and a new monoclonal antibody (mAb) for infants that target the fusion protein (F). However, efforts to combat RSV must deal with antigenic variability in F which can lead to escape from prophylactic measures. Viral escape as a vulnerability of mAb prophylaxis has been demonstrated, and RSV surveillance efforts have identified circulating strains that contain mutations in key antigenic sites of F. Here we perform deep mutational scanning (DMS) of RSV F to comprehensively map the functional and antigenic effects of mutations.Graphical overview of RSV F deep mutational scanning for measuring cell entry and antibody escape(A) Strategy to make genotype-phenotype-linked pseudoviruses. A library of barcoded and mutagenized F variants in a lentiviral vector are transfected into 293T cells with additional lentiviral helper plasmids and a plasmid encoding VSV-G to create pseudoviruses that lack a genotype-phenotype link. To establish a link between the F on the virion surface and the genotype, we then infect LentiX cells at a low multiplicity of infection (MOI99% of all possible mutations. Most F variants carried a single amino-acid mutation. Our DMS reveals regions of mutational constraint in RSV F likely due to protein folding and conformational change requirements for function (Figure 2A-B). Our study also identifies mutations at known antigenic regions that do not impact cellular entry, suggesting the possibility of viral escape from antibodies targeting these regions (Figure 2B-C). We map escape mutations for clinically relevant mAbs including nirsevimab (Figure 3). Nirsevimab escape mutations include mutations not previously identified and mutations that are functionally tolerated. These mutations exist among natural RSV sequences at low frequency. Conclusion The DMS maps of mutational effects further our understanding of RSV F function and facilitate interpretation of the functional and antigenic consequences of F mutations observed by RSV surveillance efforts in the setting of more widespread use of vaccines and mAbs. Disclosures Jesse Bloom, PhD, Apriori Bio: Advisor/Consultant|Apriori Bio: I am inventor on patents licensed to Apriori Bio by the Fred Hutch|GSK: Advisor/Consultant|Invivyd: Advisor/Consultant|Pfizer: Advisor/Consultant