High-throughput analysis of the antigenic effects of mutations to influenza hemagglutinin

Abstract

Influenza virus rapidly evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the effects of viral mutations on antibody neutralization vary across the human population, and how this heterogeneity might affect virus evolution. In my dissertation, I address this question by mapping the antigenic effects of influenza mutations against sera from defined age cohorts. First, I describe the development of an improved deep mutational scanning system to measure how mutations in hemagglutinin (HA) affect neutralization by human sera. I engineer a chimeric, barcoded HA construct, which both improves sample throughput while also allowing for absolute quantification of both escape and sensitizing mutations. I show that the resulting barcoded libraries can be used to map the HA epitopes targeted by monoclonal antibodies, antibody cocktails, and polyclonal sera, and that these measurements are consistent with results from traditional neutralization assays. In the remainder of my thesis, I use a barcoded library in the background of the A/Hong Kong/45/2019 H3 HA protein to analyze heterogeneity in serum antibody targeting across individuals and age cohorts. I find that the effects of HA mutations on serum neutralization differ across age groups, and that these differences can be partially rationalized in terms of exposure histories. For instance, mutations that revert to amino acids found in the HAs of older viral strains often increase neutralization sensitivity in older individuals, but not young children. I incorporate data from similar experiments using the earlier, non-barcoded A/Perth/16/2009 H3 HA library, which also found substantial differences between child and adult escape maps. Natural mutations that fixed in influenza variants after 2020 cause the greatest escape from sera from children and teenagers, suggesting that antigenic pressure from younger age groups play a more prominent role in driving viral evolution. Overall, my graduate research demonstrates that influenza faces distinct antigenic selection regimes from different age groups, and that this heterogeneity may have a substantial impact on viral evolution. More rigorous characterization of this immune heterogeneity has the potential to improve both evolutionary forecasting and vaccine effectiveness.