The Subtype Specific and Cross-Reactive T Cell Responses to Influenza Viruses in Humans: A Dissertation

Abstract

Human influenza is a contagious respiratory disease resulting in substantial morbidity and mortality worldwide. With the recent cases of avian influenza infections in humans and the heightened concern for an influenza pandemic arising from these infections, it is essential to understand host responses that would confer protective immunity to influenza. The cell-mediated immune responses to influenza virus play an important role during influenza infection. To analyze the specificity and diversity of memory T-cell responses, we performed a genome-wide screening of T cell epitopes to influenza A virus in healthy adult donors. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-(IFN-γ) enzyme-linked immunosorbent spot assays (ELISPOT) using peripheral blood mononuclear cells (PBMCs) from four healthy adult donors. We found that among 11 influenza viral proteins, hemagglutinin (HA) and matrix protein 1 (M1) had more T-cell epitopes than other viral proteins. The donors were not previously exposed to H5N1 subtype, but we detected H5 HA T cell responses in two of the four donors. To confirm that HA is a major target of T cell responses we also analyzed H1 and H3 HA-specific T-cell responses using PBMC of additional 30 adult donors. Fifteen out of thirty donors gave a positive response to H3 HA peptides, whereas five of thirty donors gave a positive response to H1 HA peptides. Because we detected T cell responses to the H5 HA peptides in donors without prior exposure to H5N1 subtype, we asked if cross-reactive T cells to H5 HA peptides can be attributed to a prior exposure to H2N2 subtype, the closest HA to the H5 based on their phylogeny. We compared younger donors who have no prior exposure to H2N2 subtype and older donors who were likely to be exposed to H2N2 subtype, and both groups responded H2N2 peptides at similar level, suggesting that memory T cells cross-reactive to H5 HA peptides can be generated by prior exposure to the H1N1 and H3N2 subtypes, and the exposure to H2N2 subtype is not necessary. We subsequently identified a CD4+ T cell epitope that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of HA of influenza A and the HA of the influenza B virus. A CD4+ T cell line specific to this epitope recognizes target cells infected with various influenza A viruses including seasonal H1N1 and H3N2, a reassortant H2N1, the 2009 pandemic H1N1, H5N1 and influenza B virus in cytotoxicity assays and intracellular cytokine staining assays. Individuals who have the HLA-DRB1*09 allele have ex vivo IFN-γ responses to this epitope peptide in ELISPOT. Although natural infection or standard vaccination may not induce strong T and B cell responses to this very conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4+ T cells which are cross-reactive to both influenza A and B viruses.