Influenza A viruses exhibit extreme diversity via multiple serotypes of the hemagglutinin (HA 1-16) and neuraminidase (NA 1-9) surface antigens. To date, only three of the possible 144 combinations found in bird and animal reservoirs have been associated with human pandemics (H1N1, H2N2, H3N2). Recently, a distinct lineage of influenza A viruses has been identified in bats, further increasing the spectrum of possible zoonotic viruses that could infect humans. This proposal seeks to elucidate at the structural level, key sites of vulnerability on influenza virus for development ofa sustainable cross-serotype immune response, and understand activity relationships of the surface glycoproteins (HA, NA) and ribonucleoprotein (RNP) complex, including the polymerase (PA, PB1, PB2), that underlie the pathogenicity and transmissibility of pandemic and seasonal influenza viruses. Antibody-mediated neutralization of influenza virus is a complex combinatorial problem for the human immune system as it is presented with diverse, highly variable and constantly evolving viruses. While neutralizing antibodies against human flu are traditionally regarded as being strain specific, recent studies have shown that a much broader response can be mounted over decades of evolution of a particular subtype (e.g. H3N2), across group 1 or group 2, and even across two major phylogenetic groups (I and 2). While these examples provide compelling evidence that the immune system is capable of mounting a sustained, cross-serotype response against influenza, how to elicit broadly neutralizing antibodies by vaccination is poorly understood. Therefore, we propose to determine the structural basis of broad neutralization and delineate the sites of vulnerability on the HA to enable development of novel vaccine scaffolds and even small molecule inhibitors that ameliorate or prevent disease progression. Furthermore, because we do not understand why certain viruses, such as the recent H1N1 2009 swine flu, are able to enter the human population and cause pandemics, we will also study the molecular basis of pathogenicity. Using a novel strategy to investigate both HA and NA substrate specificity and activity using newly designed glycan microarrays, we will assess the functional relationships between NA and HA activity. In this way, we will test the hypothesis that efficient infection of humans by influenza viruses requires a functional balance between the binding and specificity of HA and enzymatic activity of the NA with host glycan receptors. Another key factor in host-specific pathogenicity is the replication machinery composed of the RNP with associated polymerase, where mutations can alter polymerase activity and interaction with host cell factors. Structural and functional understanding of the RNP will enable other approaches to combat influenza infection. A combined biophysical and biochemical approach from three laboratories employing state of the art x-ray crystallography, electron microscopy and glycan array technologies will be used to provide key insights into influenza virus neutralization, tropism and pathogenesis, to reveal novel strategies to control and combat future pandemics.
We will investigate structural correlates of human pandemic influenza viruses (H1N1, H2N2, H3N2), as well as other emerging viruses (such as H5N1, H7N7, H9N2, and recently discovered novel flu A viruses from bats) that have the potential to cause severe or lethal human infection, to elucidate the key factors that contribute to host specificity, antigenicity, replication and virulence. This proposal seeks to provide valuable insights into influenza virus neutralization, tropism and pathogenesis that can be used to design novel strategies to control and combat highly lethal influenza virus infections in the event of a new pandemic, as well as aid in preventing epidemics that arise from seasonal flu.
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