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Abstract
Influenza A virus (IAV) remains a major threat to a wide range of animal hosts. The hemagglutinin (HA) of IAV initiates virus attachment and entry by binding to terminal sialic acid (SA) residues on host cells. HA gradually accumulates amino acid substitutions that allow IAV to escape population immunity through a mechanism known as antigenic drift. Recent work has identified seven amino acid positions (145, 155, 156, 158, 159, 189 and 193, H3 numbering) near the receptor-binding site (RBS) of H3N2 IAVs as the major determinants of antigenic drift. In the first part of the dissertation, we took an in-depth look at the amino acid plasticity at residue 145 and its impact on receptor binding and antibody recognition. We generated a panel of HA mutant viruses carrying substitution at residue 145 (H3 numbering) representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated and stably maintained in vitro. All substitutions retained receptor binding specificity, but frequently led to decreased receptor binding avidity. Glycan microarray analysis showed substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. In the second part of the dissertation, we combined deep mutational scanning with reverse genetics to examine the in vitro amino acid plasticity of these key antigenically relevant residues in the H3 HA. We envisioned a H3 HA antigenic virus library as an alternative IAV vaccine strategy that potentially induces broader protection. Our approach was efficient in producing H3 HA antigenic virus libraries in different donor virus strains. Despite limited diversity in nature, high-throughput targeted next-generation sequencing revealed that these residues exhibited extraordinary amino acid plasticity in vitro. Nonetheless, virus library diversity is severely reduced following virus rescue. To validate our experimental approach, a novel H3 HA variant virus was isolated and shown to possess distinct receptor binding and antigenic properties relative to wt H3 HA virus. The findings obtained in this dissertation have important implications for understanding virus evolution and aiding the development of novel vaccine design approaches.