Generation of protective immunity against severe influenza virus infection in Indigenous Australians

Abstract

Morbidity and mortality rates from seasonal and pandemic influenza virus infections occur disproportionately in high-risk groups, including Indigenous people globally. Although adaptive immunity is essential for combating viral infections, it is largely understudied in Indigenous populations, including Aboriginal Australians. To rationally design a protective vaccine against both seasonal and pandemic influenza viruses for both Indigenous and non-Indigenous people, in-depth knowledge about B cell and T cell responses is needed. Therefore, the aim of this PhD thesis was to identify and characterise CD8+ T cell responses restricted by HLA alleles dominant in Indigenous Australians as well as understand CD4+ T cell, B cell and humoral responses to the inactivated influenza vaccine in Indigenous and non-Indigenous Australians. The data generated in this PhD thesis provide key insights into a rational design of a cross-protective influenza vaccine.

The HLA-A*24:02 allele was associated with a higher mortality during the 2009 influenza pandemic and is highly expressed in Indigenous populations globally. In Chapter 3, we identified novel influenza A and B virus (IAV and IBV, respectively) CD8+ T cell targets presented by HLA-A*24:02 and characterised their immunogenicity in HLA-A*24:02 transgenic mice, and HLA-A*24:02+ Indigenous and non-Indigenous individuals. We discovered the first IBV-derived CD8+ T cell epitopes presented by HLA-A*24:02. A total of 6 novel immunogenic IBV epitopes were identified, with one epitope, A24/PB2550-558 being cross-reactive between IBV and the IAV variant, making it especially important for a cross-protective universal vaccine. We defined epitope-specific CD8+ T cells ex vivo in the blood of healthy Indigenous, non-Indigenous and acutely-infected donors. These newly identified epitope-specific CD8+ T cells were readily detected during acute infection, mainly of an effector memory phenotype and expressed activation markers such as PD-1, CD38, HLA-DR and CD71, thus highlighting their involvement during influenza virus infection. Sub-cutaneous vaccination of transgenic mice with three immunogenic IBV peptides resulted in reduced viral titres, reduced pro-inflammatory cytokines MIP-1a, MIP-1b and RANTES in the lung as well as reduced weight loss in comparison to unvaccinated mice. These data highlight the importance of identifying novel CD8+ T cell epitopes for their potential use as a universal influenza vaccine in the context of a highly prevalent HLA allele in Indigenous people globally.

Indigenous Australians express a unique HLA class I profile which includes high frequencies of HLA-A*24:02, A*11:01, A*34:01, B*13:01 and B*15:21. Except for HLA-A*24:02 and HLA-A*11:01, no influenza virus-derived epitopes have been identified for the Indigenous-associated HLAs. In Chapter 4, we generated single HLA-expressing cell lines to use as antigen-presenting cells for identifying and characterising CD8+ T cell responses towards the Indigenous-associated HLAs. We optimised an influenza virus infection assay, instead of using peptide pool cultures, to stimulate and expand rare influenza-specific CD8+ T cells, and thus allow sufficient numbers for the sequential dissection of single epitope candidates. As such, we identified the first IAV and IBV epitopes presented by HLA-B*13:01. While responses to IAV were dominated by the highly variable B13/NP404-412 epitope, IBV-responses were directed towards a variety of epitopes, including the dominant and highly conserved B13/HA371-379 and B13/HA427-435 peptides. We sequenced IAV- and IBV-specific CD8+ T cell receptors and identified unique TCR signatures between the different epitopes. While B13/NP404-412-specific CD8+ T cells showed a limited bias for the expression of TRBV19 with a public TCR, B13/HA371-379-specific CD8+ T cells almost uniquely expressed TRAV3 paired with a variety of different TCRb-chains. The developed tools proved to be an efficient method to identify and characterise IAV and IBV epitopes presented by Indigenous-associated HLAs to further develop a universal influenza vaccine that can protect Indigenous Australians.

Current influenza inactivated vaccine strategies delivered intramuscularly induce mainly B cell and antibody responses. Responses to the influenza vaccine are studied extensively but not fully understood in high-risk populations. Despite the strong recommendations for annual influenza vaccination, Indigenous Australians are completely underrepresented in studies that analyse vaccine responses. In Chapter 5, we defined cellular and humoral responses to the inactivated influenza vaccine in Indigenous Australians vaccinated between 2016 and 2018, and compared these responses to non-Indigenous Australians. We identified robust antibody responses to the vaccine that were comparable to non-Indigenous donors and were cross-reactive with viruses circulating more than 10 years ago. Antibody responses on day 23+ post vaccination correlated with acute activation of circulating T follicular helper cells type 1 (cTFH1) in line with previous studies. System serology of selected Indigenous and non-Indigenous donors revealed significant reduction of the contribution of IgG3 to influenza-specific antibodies in Indigenous Australians, which correlated with a higher frequency of the G3m21 allotype. The generated data are important to support vaccine recommendations for Indigenous Australians, but also highlight the need to improve influenza vaccinations by harnessing the protective capacity of CD8+ T cells in future vaccine designs.

Overall, this PhD thesis provides highly important knowledge of T and B cell immunity to IAV and IBV in Indigenous Australians. The findings of this PhD thesis provide key insights into the development of a universal influenza vaccine that also protects Indigenous Australians, one of the high-risk groups of developing severe influenza disease.