Understanding how mutations shape the evolution of seasonal influenza virus hemagglutinin

Abstract

Influenza virus hemagglutinin (HA) is under strong selective pressure. These selective forces constrain and drive the evolution of HA in nature. As it evolves, HA rapidly accumulates mutations that can affect its function and antigenicity; the effects of these mutations are a key determinant of the evolutionary success of viral lineages. However, we have a very limited understanding of how mutations to HA affect viral fitness and thereby shape viral evolution in nature. Here I describe my work in which I completely examined the effects of mutations to HA by leveraging the high-throughput techniques of deep mutational scanning and mutational antigenic profiling. Briefly, these techniques enabled me to introduce all of the approximately 10,000 single amino-acid mutations to HA and experimentally test the variants under functional or immune selective pressure. First, I measure the effects of mutations to a recent seasonal H3N2 HA on viral growth in cell culture and relate these measurements to the evolutionary success of mutations in nature. I find that mutations that reach high frequencies in nature tend to be measurably favorable for viral growth, whereas mutations measured as highly deleterious in our experiments rarely reach high frequencies in nature. Previously generated measurements from a diverged H1 HA do not show a strong relationship with success of viral lineages. These findings suggest that experimental measurements of the functional effects of mutations to an H3 HA can help distinguish successful H3N2 viral lineages from those that quickly die out. Next, I apply mutational antigenic profiling to evaluate the ease of viral escape from several strain-specific and broadly-neutralizing antibodies against an H1 HA. Although the virus can readily escape the narrow antibodies and the broad receptor-binding site targeting antibody via mutations with large effect sizes, it is quantifiably harder to escape the stalk-targeting antibodies that we tested. Antibody breadth therefore does not reflect the ease of viral escape by single mutations. Finally, I extend antibody mapping approaches to profile immune selection on H3 HA by polyclonal serum. I observe direct evidence of individuals with highly immunodominant serum responses against HA such that there is selection for single amino-acid mutations with relatively strong effects on neutralization resistance. Mutations in nature have recently emerged at several sites of escape in the experiments, indicating that individuals with immunodominant targeting drive antigenic drift of HA. Overall, this work demonstrates the utility of experimentally mapping mutational effects for improving our understanding of the dynamics of influenza virus evolution in nature.