The Role of Genomics in Tracking the Evolution of Influenza A Virus

Influenza A virus causes annual epidemics and occasional pandemics of short-term respiratory infections associated with considerable morbidity and mortality. The pandemics occur when new human-transmissible viruses that have the major surface protein of influenza A viruses from other host species are introduced into the human population. Between such rare events, the evolution of influenza is shaped by antigenic drift: the accumulation of mutations that result in changes in exposed regions of the viral surface proteins. Antigenic drift makes the virus less susceptible to immediate neutralization by the immune system in individuals who have had a previous influenza infection or vaccination. A biannual reevaluation of the vaccine composition is essential to maintain its effectiveness due to this immune escape. The study of influenza genomes is key to this endeavor, increasing our understanding of antigenic drift and enhancing the accuracy of vaccine strain selection. Recent large-scale genome sequencing and antigenic typing has considerably improved our understanding of influenza evolution: epidemics around the globe are seeded from a reservoir in East-Southeast Asia with year-round prevalence of influenza viruses; antigenically similar strains predominate in epidemics worldwide for several years before being replaced by a new antigenic cluster of strains. Future in-depth studies of the influenza reservoir, along with large-scale data mining of genomic resources and the integration of epidemiological, genomic, and antigenic data, should enhance our understanding of antigenic drift and improve the detection and control of antigenically novel emerging strains

Viral factors in influenza pandemic risk assessment

The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk

The Impact of Receptor Binding Avidity and Immune History on the Antigenic Determination of Influenza A Viruses

Most humans are repeatedly infected with new strains of influenza throughout their lifetime even though protective neutralizing antibodies against the viral hemagglutinin (HA) are generated after both natural infection and vaccination. This observed lack of protection against variant strains is largely attributed to a process termed `antigenic drift\u27, where accumulating mutations in HA quickly abrogate recognition by antibodies elicited by earlier strains. Consequently, current influenza vaccines must be updated frequently in an attempt to match the antigenic profiles of vaccine strains to those of circulating strains. However, the existing process of antigenic determination is imperfect: it fails to consider the effects of receptor binding avidity in the interpretation of hemagglutination inhibition (HAI) assays or the effects of pre-exposure history on how a novel virus is viewed antigenically by an altered immune system. Here, we designed a series of experiments to address these issues. First, we computationally modeled how variation in receptor binding avidity could affect the antigenic characterization of historic H3N2 strains and experimentally demonstrated that single point mutations in HA can skew HAI titers without actually affecting antibody binding. Additionally, using the same H3N2 system, we showed that a single amino acid mutation can significantly alter the immunodominance of the anti-HA antibody response. We then completed a series of studies to determine how immune history influences the specificity of antibody repertoires. In examining patient serology, we found that the specificity of the human antibody response against the 2009 pandemic H1N1 virus (pH1N1) was highly correlated with pre-exposure history to different seasonal H1N1 (sH1N1) strains during childhood. Using a ferret model, we demonstrate that the anti-pH1N1 antibody response can be shifted to highly conserved epitopes on HA when the animals were primed with sH1N1s that are otherwise antigenically distinct. Collectively, our studies demonstrate that accounting for receptor binding avidity and factors that alter antibody repertoires will improve influenza vaccination strategies in the future

Vaccine Strain Selection For Influenza A Viruses Is Complicated By Unique Pre-Exposure Histories And Rapid Mutation Of Glycoproteins

Influenza viruses cause millions of infections worldwide each year. Influenza viruses constantly acquire mutations in their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), through a process called antigenic drift. HA is the main target of anti-influenza neutralizing antibodies (Abs). Due to antigenic drift, the seasonal influenza vaccines must be updated annually to include the most antigenically relevant strains. Each year, the World Health Organization collects thousands of clinical influenza isolates, propagates them in cell culture, and performs both sequencing and serological analyses to assess the antigenic characteristics of circulating viral strains. In this dissertation, we investigate multiple factors associated with surveillance and vaccine strain selection that could be improved to produce more reliable and effective seasonal influenza vaccines. We first demonstrate that recent H3N2 subtype viral isolates rapidly acquire mutations in both HA and NA when propagated in cell culture, resulting in increased receptor binding avidity or NA-dependent receptor binding, respectively. These mutations impact antigenic analyses that are routinely used for viral surveillance. We then explore how a single mutation in HA antigenic site B contributed to the antigenic drift and subsequent vaccine mismatch of newly emerged H3N2 viruses during the 2014-2015 influenza season. Finally, we found that antisera collected from previously na�ve ferrets infected for the first time with influenza (that are commonly used for antigenic analyses during vaccine selection) do not accurately represent the Ab repertoires found in humans that have been infected or vaccinated multiple times with different influenza virus strains. We identified some individuals who have an Ab response targeted to a region of the HA of H1N1 viruses that recently acquired a mutation. Overall, our studies identify ways to improve the process of choosing seasonal influenza virus vaccine strains. We propose that the implementation of “sequence-first” surveillance, new cell culture systems, and the use of clinical human antisera for antigenic characterization of viruses will improve the process of selecting seasonal influenza vaccine strains

The antigenic evolution of human influenza A haemagglutinin

A detailed understanding of the B-cell response to influenza A haemagglutinin is key to the accurate matching of vaccines to seasonal strains, and may inform the development of broader spectrum vaccines. In this study, I develop techniques for predicting the location of the epitopes of protective antibodies by observing the physical locations of amino acid substitutions in human wild-type strains. By linking the understanding gained from this analysis with a large body of assay data, I present a model which can predict antigenic distance from HA1 amino acid sequences and which meets or exceeds the predictive power of previously developed models while retaining generality. An interesting conclusion from the epitope analysis discussed above is that antibodies to the HA head bind in two regions. The antigenic evolution of influenza H3N2 is more punctuated than its genetic evolution. I propose that the dual regions might contribute to the punctuated nature of antigenic evolution, and explore this through the use of a simple simulation. Stalk-binding antibodies to HA have attracted much interest in recent years: a number of broad-binding examples have been isolated, and the slower evolution of the stalk gives hope that these may provide broad protection against future strains. Stalk-binding neutralising antibodies to H3 are known to bind in two regions, and I use data from crystal studies to identify the constituent residues of these regions, which I term antigenic sites F and G, in a manner that is consistent with previous analyses of the constituent residues of HA1 antigenic sites A-E. I analyse the degree of conservation of residues in sites F and G, and conclude that there have been episodes of change in the H3 stalk which are consistent with antigenic evolution

Phylodynamic Patterns in Pathogen Ecology and Evolution.

The rapid evolution of viral pathogens requires us to consider epidemiological, ecological and evolutionary processes as coupled together and occurring at the same timescale. Rotavirus and influenza account for high levels of morbidity and mortality worldwide and are two important examples of such dynamics. In this work, I investigate the different evolutionary and ecological processes that shape the antigenic structure and phylogenetic characteristics of these two viruses. In the first part of my work, I use a theoretical model of influenza A/H3N2 to identify the relative importance of antigenic novelty, competition between lineages, and changes in the susceptibility of the host population to circulating strains in determining the evolutionary and epidemiological trajectory of the virus. I develop this model further to correspond with patterns of immunity and infection observed in rotavirus, and investigate how reassortment, the swapping of gene segments between viruses, influences the formation and replacement of rotavirus genotypes through immune mediated processes. In the second part of my work, I use a tool (SeasMig), which I developed, to infer alternative stochastically generated migration and mutation events along phylogenetic trees in a Bayesian manner. Using SeasMig, I first show how the seasonality of A/H3N2 influenza incidence corresponds to rates of immigration and emigration of the virus. Subsequently, I tease out the different evolutionary and ecological processes, which drive changes in the US rotavirus population following onset of routine vaccination. My work has implications for identifying likely evolutionary mechanisms, which may lead to reduced vaccine efficacy, and for vaccine strain selection.PhDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113494/1/dzinder_1.pd

Dissecting Human Antibody Responses Against Influenza A Viruses And Antigenic Changes That Facilitate Immune Escape

Influenza A viruses pose a serious threat to public health, and seasonal circulation of influenza viruses causes substantial morbidity and mortality. Influenza viruses continuously acquire substitutions in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These substitutions prevent the binding of pre-existing antibodies, allowing the virus to escape population immunity in a process known as antigenic drift. Due to antigenic drift, individuals can be repeatedly infected by antigenically distinct influenza strains over the course of their life. Antigenic drift undermines the effectiveness of our seasonal influenza vaccines and our vaccine strains must be updated on an annual basis due to antigenic changes. In order to understand antigenic drift it is essential to know the sites of antibody binding as well as the substitutions that facilitate viral escape from immunity. In this dissertation, we explore both the epitopes targeted in human antibody responses and how influenza viruses evade these responses. We first demonstrate that prior exposure shapes the sites targeted in human antibody responses, and show that many middle-age adults mounted an antibody response against H1N1 viruses that is focused against sites on HA conserved between contemporary strains and strains that circulated in early childhood. In addition, we demonstrate that a viral substitution in this epitope allows influenza viruses to evade neutralizing antibody responses. We next demonstrate that an H3N2 HA substitution introducing a glycosylation site prevents the binding of neutralizing antibodies present in a large number of individuals. Importantly, our egg-based vaccines lack this glycosylation due to culture-adaptive substitutions, but a vaccine containing this glycosylation motif more potently induced antibody responses against circulating strains. Finally, we identify and characterize antibodies that target conserved residues in the receptor-binding site (RBS) of HA. We demonstrate that in some individuals RBS antibodies in sera contribute to neutralization of antigenically distinct strains, even in the case of an antigenically mismatched vaccine. Overall, the work presented here helps address the complex interaction of influenza viruses and human immunity. Importantly, our work identifies shortcomings with our current process of vaccine strain selection and production and investigates epitopes of interest for universal influenza vaccine efforts

Allele dynamics plots for the study of evolutionary dynamics in viral populations

Phylodynamic techniques combine epidemiological and genetic information to analyze the evolutionary and spatiotemporal dynamics of rapidly evolving pathogens, such as influenza A or human immunodeficiency viruses. We introduce ‘allele dynamics plots’ (AD plots) as a method for visualizing the evolutionary dynamics of a gene in a population. Using AD plots, we propose how to identify the alleles that are likely to be subject to directional selection. We analyze the method’s merits with a detailed study of the evolutionary dynamics of seasonal influenza A viruses. AD plots for the major surface protein of seasonal influenza A (H3N2) and the 2009 swine-origin influenza A (H1N1) viruses show the succession of substitutions that became fixed in the evolution of the two viral populations. They also allow the early identification of those viral strains that later rise to predominance, which is important for the problem of vaccine strain selection. In summary, we describe a technique that reveals the evolutionary dynamics of a rapidly evolving population and allows us to identify alleles and associated genetic changes that might be under directional selection. The method can be applied for the study of influenza A viruses and other rapidly evolving species or viruses

Evolutionary Trends of A(H1N1) Influenza Virus Hemagglutinin Since 1918

The Pandemic (H1N1) 2009 is spreading to numerous countries and causing many human deaths. Although the symptoms in humans are mild at present, fears are that further mutations in the virus could lead to a potentially more dangerous outbreak in subsequent months. As the primary immunity-eliciting antigen, hemagglutinin (HA) is the major agent for host-driven antigenic drift in A(H3N2) virus. However, whether and how the evolution of HA is influenced by existing immunity is poorly understood for A(H1N1). Here, by analyzing hundreds of A(H1N1) HA sequences since 1918, we show the first evidence that host selections are indeed present in A(H1N1) HAs. Among a subgroup of human A(H1N1) HAs between 1918∼2008, we found strong diversifying (positive) selection at HA1 156 and 190. We also analyzed the evolutionary trends at HA1 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1) HA. Different A(H1N1) viruses appeared to favor one of these two sites in host-driven antigenic drift: epidemic A(H1N1) HAs favor HA1 190 while the 1918 pandemic and swine HAs favor HA1 225. Thus, our results highlight the urgency to understand the interplay between antigenic drift and receptor binding in HA evolution, and provide molecular signatures for monitoring future antigenically drifted 2009 pandemic and seasonal A(H1N1) influenza viruses