Dissecting the viral and host constraints that govern influenza virus antigenic evolution

Abstract Seasonal influenza A viruses (IAV) cause hundreds of thousands of deaths every year, despite widespread pre- exposure and vaccination. IAV persists in the human population by continually evolving resistance to herd immunity through a process known as antigenic drift. The evolutionary potential of RNA viruses like IAV is often considered enormous due to their rapid replication and high mutation rates. In reality, the evolutionary potential of IAV is highly constrained by the need to maintain a wide array of molecular functionality in a tiny genome. The specific constraints limiting IAV evolution are very poorly characterized and defining them is critical for understanding and potentially predicting the specific evolutionary pathways most likely to be taken by these viruses. We discovered that phenotypic variation in the viral neuraminidase (NA) gene results in the viral hemagglutinin (HA) gene taking divergent mutational pathways to escape neutralizing antibody pressure. These data suggest that the need to maintain a functional balance between the opposing activities of the viral glycoprotein genes (HA and NA) significantly constrains how the virus evolves to escape from host immune pressure. We hypothesize that these viral constraints, along with additional constraints imposed by the host environment, play significant roles in shaping the specific pathways of IAV antigenic evolution that occur in humans. We will use a combination of in vitro and in vivo experimental evolution and mechanistic approaches to define the specific host and viral factors that constrain the antigenic evolution of the HA gene. In Aim 1, we will quantify phenotypic variation amongst recently circulating NA genes and quantify how this phenotypic variation alters the evolutionary landscape of the HA gene. In Aim 2, we will explore how natural phenotypic variation in NA influences antigenic escape in vitro and in vivo. Finally, in Aim 3, we will define how changes in the host environment and sialic acid profile influence the potential for recent human seasonal H1N1 viruses to escape from humoral immune pressure. Collectively, these studies will deepen our mechanistic understanding of the antigenic evolution of seasonal influenza viruses in humans.