Understanding influenza CD8+ T cell immune escape from pre-existing immunity and T cell receptor cross-strain recognition

© 2015 Dr. Sergio Manuel Quiñones ParraProtective immunity against influenza virus infections relies heavily in neutralising antibodies (nAbs) and CD8+ T lymphocytes (CTLs). Current nAbs-based influenza vaccines are greatly undermined by the antigenic adaptation of influenza A viruses (IAVs). In contrast, protective CTLs recognize more conserved antigenic determinants and thus, provide potential for broadly protective vaccines. However, how influenza-specific CTL immunity is established, maintained and recalled, is far from clear. Therefore, this PhD thesis investigated key factors shaping CTL responses against seasonal, pandemic and newly emerged IAVs. CTL escape was investigated using a mouse model and a human case of persistent IAV infection. Analysis of viral isolates from the clinical case revealed a sequential emergence and accumulation of influenza virus variants with mutations in relevant CTL epitopes. These findings were validated in mice experiments where it was demonstrated that CTL escape variants can be generated promptly after influenza infection. The frequency of viral variants coincided with the magnitude of the ongoing CTL responses. The mechanism of viral escape in influenza is thus similar to that of chronic human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV), indicating that immunocompromised patients may play a significant role in the establishment of influenza quasispecies. We then investigated the emergence of influenza CTL escape variants at the population level and its potential role in determining influenza susceptibility. To this end, we performed conservation analysis of CTL antigenic peptides across the 40 years preceding the emergence of the pandemic H2N2 virus in 1957; when children were ten times more susceptible than adults to undergo symptomatic influenza virus infection. Our work showed extensive conservation of influenza CTL peptides and no evidence of major immune escape. In contrast, experiments in mice lacking B cells showed that (irrespective of antibodies) influenza-associated morbidity was significantly reduced following a tertiary influenza virus infection, coinciding with an increase in the number of lung CD8+ tissue-resident memory cells (CD8+ TRM). These findings suggest that repeated influenza infections drive the accumulation of protective CD8+ TRM in the lung, in turn better predicting pandemic influenza demographic outcome. The lack of nAbs against the H7N9 virus that emerged in March of 2013 raised pandemic concerns. We investigated the extent of pre-existing CTL immunity against this virus and identified universally conserved antigenic peptides with the capacity to mount robust CTL responses against any IAV, including H7N9. We also revealed novel mutations that abrogated CTL recognition and epitopes with diminish capacity to reactive memory CD8+ T cells. The Human Leukocyte Antigen Class-I (HLA-I) molecules that present universally conserved influenza peptides varied in prevalence depending on ethnicity. Thus, our findings suggest that compared to other ethnicities, pre-existing CTL immunity against H7N9 could be compromised in Alaskan and Australian Indigenous people. To understand a mechanism that can counteract influenza CTL escape, we investigated human αβ T cell receptors (TCRs) capable of recognizing multiple CTL peptide variants, derived from serologically distinct IAVs. This work unravelled how the TCR clonotype determines fine specificity and cross-reactive capacity while compromising peptide + HLA-I (pHLA-I) avidity. This work also provided insights into the molecular basis underpinning TCR cross-reactivity of IAVs and has led to ongoing structural and affinity studies. In summary, the findings described in this PhD thesis demonstrated the emergence of acute CTL escape variants at the individual and population level. Further, we find extensive conservation of influenza CTL peptides and no evidence of major immune escape that could explain increased influenza susceptibility. Our identification of universally conserved influenza peptides provides a measure of minimal pre-existing immunity against a novel influenza subtype. Finally, we document the nature of TCR influenza cross-strain recognition. Our findings have implications for the feasibility, design and delivery of a potential broadly-protective CTL-based vaccine against IAVs