TONSILS: AN UNRECOGNIZED ROLE IN INFLUENZA VIRUS EVOLUTION

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Identifying the host factors that contribute to influenza virus (IV) pathogenesis are critical for disease control and preventing pandemics. The tonsils comprise 2 lymphoid organs located in the nasopharynx of mammals and in the cecum of birds, in which IVs are enteric. The tonsils are an initial site of various viral infections and transmission, but their role in IV pathogenesis is unclear. My proposed studies will provide new insights on how the tonsils contribute to IV pathogenesis, immune responses, and adaptation by identifying the role of the tonsils in IV pathogenesis, performing tonsillectomy in ferrets to recapitulate the heterogeneous responses to IV infection severity and vaccination in subpopulations of humans with/without tonsils, and exploring the role of the tonsils in IV adaptation. My recent study demonstrated that human tonsillar epithelial cells (HTECs) are susceptible to IV infections, with effective replication of different IV subtypes in vitro. I will expand upon these findings by performing time-intensive monitoring of IV infection dynamics and distribution in the tonsils of ferrets. I collected and analyzed human tonsillectomy data over the last 60 years in the US and found that the percentage of human subpopulations with tonsillectomies is high in different age groups. The prevalence, disease severity, and level of immunity of IV infections in these subpopulations are unknown, and the role of the tonsils in epidemic or pandemic spread of IVs is undetermined. I previously found that HTECs induce chemokine and cytokine release during IV infection. Previous clinical studies reported no differences in salivary IgA immune responses to IV live attenuated vaccine in individuals before and after tonsillectomy, but these studies were limited by low patient numbers and confounded by original antigenic sin. I will use ferrets to ascertain how tonsillectomy affects immunity resulting from the IV infection and vaccine. I will measure the quantity and quality of antibody responses after infection and/or immunization and compare T- cell and B-cell activity in ferrets with and without tonsils. The soft palate is an important site of IV adaptation, extending downward in the oral cavity and passing anterior and posterior to the tonsils. The replicative fitness of IVs in tonsillar tissues may induce rapid selection, ostensibly preventing infection and reducing their pandemic potential. Therefore, I propose to verify the role of the tonsils in IV adaptation a tissue not typically sampled in animal models of IV and investigate the extent of different human IV replication in the tonsils. Acquiring mutations and switching receptor-binding specificity between avian and human sialic acid (SA) preferences are key for IV transmission and adaptation. I previously found that both human and avian tonsil epithelial cells are rich in both human α2,6 and avian α2,3–linked SA receptors and support IV replication. I will use genetically engineered IVs with altered SA preferences to conduct transmission studies and analyze the viral fitness within the tonsils that may select for transmissible IVs more rapidly than the soft palate by using a loss-of-function approach. The mentored phase of this proposal will occur at St. Jude Children Research Hospital under the auspices of Richard Webby and will elucidate the role of the tonsils in IV pathogenesis and immune responses to infection. The independent phase will focus on immune responses to IV vaccines and IV adaptation. The institutional resources, academic environment, and educational opportunities outlined in my proposal will ensure my successful transition to independence.