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ItemImmunity and protection against Influenza B virusesKoutsakos, Marios ( 2018)Broad immunity to influenza viruses can be conferred by both humoral and cellular components of the immune system. Thus, universal vaccines, need to elicit both antibody and T cell responses with broad cross-reactivity across both influenza A and influenza B viruses (IAV and IBV, respectively). To achieve this, a thorough understanding of the immune response to IAV and IBV is required at the cellular and molecular level. In contrast to IAV, immune responses to IBV are understudied, despite the clinical relevance of IBV. Therefore, the overall aim of this PhD thesis was to characterize B cell and T cell immunity to IBV, in comparison to IAV, following influenza vaccination and virus infection in order to inform the rational design of future universal influenza vaccines. Using longitudinal blood samples from healthy adults vaccinated with the inactivated influenza vaccine, the cellular events that precede the generation of protective immunity were dissected in Chapter 3. This was achieved using recombinant haemagglutinin probes to quantitively and qualitatively assess influenza- specific B cells directly ex vivo. While vaccination induced humoral immunity, comprising of a three-pronged B cell response (CXCR5-CXCR3+ antibody-secreting B cells, CD21hiCD27+ B cells and CD21loCD27+ B cells) and a specialized subset of ICOS+PD-1+CXCR3+ circulating T follicular helper cells, components of cellular immunity like CD8+ T cells were not elicited. Furthermore, vaccine-induced B cells were not maintained in peripheral blood at one year after vaccination. Furthermore, analysis of influenza-specific memory B cells across human tissue compartments demonstrated the enrichment of memory B cells in human secondary lymphoid organs (SLOs) and the lung. In Chapter 4, conservation analysis of known CD8+ T cell epitopes derived from IAV identified a set of conserved peptides across IAV, IBV and is some cases influenza C (the A2/PB1413-421 epitope), providing the first evidence of universal cross-reactivity across IAV, IBV and ICV. Such CD8+ T cell cross-reactivity across influenza A, B and C viruses is broader than any known antibody or T cell specificity known for influenza viruses. Cutting-edge immunopeptidomics approach was used to further identify immunodominant CD8+ T cell epitopes from IBV in the context of the highly prevalent HLA-A*02:01 allele, with high conservation in IBV and the ability to accelerate viral clearance in HLA-A2-transgenic mice. Prominent memory CD8+ T cells towards both universal and influenza type-specific epitopes were detected in blood, lungs and SLOs of healthy humans, with lung-derived CD8+ T cells displaying a tissue-resident memory phenotype. Importantly, in IAV- and IBV-infected pediatric and adult patients, CD8+ T cells against these novel epitopes were readily detected with an effector CD38+Ki67+ phenotype. In Chapter 5, the quality of broadly cross-reactive CD8+ T cell responses was assessed by analyzing their TCR/pMHC avidity, functional avidity, polyfunctionality and TCRab repertoire. The TCRab repertoires of CD8+ T cells specific for broadly cross-reactive influenza-specific CD8+ T cells varied in terms of the intra-individual clonality and inter-individual sharing, in a manner dependent on underlying TCRab genetics and/or cognate pMHC-I structure, without however, significantly compromising functionality. Having characterized CD8+ T cell responses to IAV and IBV, the ability of influenza viruses to downregulate MHC-I was assessed in Chapter 6, as this is a common immune evasion mechanism. While IAV causes a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the loss of MHC-I molecules only from the cell surface, whereby MHC-I trafficking to the cell surface is delayed by retaining MHC-I intracellularly during IBV infection. Overall, this PhD thesis investigated immunity to IAV and IBV viruses by dissecting humoral and cellular immunity in healthy individuals, vaccinated adults, influenza-infected patients as well as human lymphoid and mucosal tissues. This work provided novel insights for the rational design of universal influenza vaccines which do not require annual reformulation.