Human CD8+ T cell cross-reactivity across influenza A, B and C viruses

Marios Koutsakos, Patricia T. Illing, …Katherine Kedzierska

Abstract

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8⁺ T cells confer cross-protection against IAV strains, however the responses of CD8⁺ T cells to IBV and ICV are understudied. We investigated the breadth of CD8⁺ T cell cross-recognition and provide evidence of CD8⁺ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8⁺ T cell epitopes from IBVs that were protective in mice and found memory CD8⁺ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8⁺ T cells displayed tissue-resident memory phenotypes. Notably, CD38⁺Ki67⁺CD8⁺ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8⁺ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

Main

Although 2018 marked the 100th anniversary of the catastrophic Spanish influenza pandemic, influenza viruses remain a constant, global health threat. Three types of influenza viruses infect humans: type A (influenza A virus; IAV), type B (IBV) and type C (ICV). Two IAV subtypes (A/H3N2 and A/H1N1pdm09) and two IBV lineages (B/Yamagata/16/88-like and B/Victoria/2/87-like) co-circulate annually, causing seasonal epidemics of mild, severe or fatal respiratory disease, whereas ICV causes severe disease in children^1,2,3,4,5. Antigenically novel IAVs generated by reassortment of the segmented genome and derived from animal reservoirs can also infect humans with high rates of morbidity and mortality¹.

The search for a long-lasting, universal, broadly protective vaccine against influenza viruses is ongoing. Immune protection against influenza is mainly mediated by adaptive humoral and cellular responses^1,2. Antibodies induced by seasonal inactivated influenza vaccine typically provide strain-specific immunity by targeting the variable head domain of the surface glycoprotein hemagglutinin. Although these antibodies can provide neutralizing immunity, the constant antigenic drift of hemagglutinin makes them poor targets for cross-protection. Broadly cross-reactive antibodies predominantly targeted to the conserved stem of hemagglutinin or at neuraminidase⁶ can provide heterosubtypic cross-reactivity across multiple IAV subtypes⁷ or across IBVs, but not heterotypic cross-reactivity across IAVs and IBVs, with the exception of one rare antibody clone (CR9114)⁸. Conversely, cytotoxic CD8⁺ T cells provide cross-protection across seasonal IAVs^9,10 or IBVs¹¹ and pandemic^12,13,14,15 and avian^16,17,18 IAVs by recognizing conserved virus-derived peptides presented by major histocompatibility complex class-1 (MHC-I) glycoproteins (human leukocyte antigens; HLAs) on the surface of infected cells. So far, 195 CD8⁺ T cell epitopes restricted by 24 different HLA allotypes have been identified for IAVs, 7 epitopes (restricted by HLA-A*02:01 or HLA-B*08:01) for IBV and no T cell epitopes for ICV (Immune Epitope Database; accessed 2 January 2018). After recognition of the peptide–MHC-I complex, CD8⁺ T cells kill virally infected cells and release antiviral cytokines (interferon-γ (IFN-γ) and tumor necrosis factor (TNF)). The breadth of CD8⁺ T cell cross-reactivity across antigenically novel viruses renders these cells promising targets for a universal vaccine. However, the current inactivated influenza vaccine formulation does not boost CD8⁺ T cells¹⁹. Thus, new vaccines are needed to harness the potential of cross-protective CD8⁺ T cells.

The establishment of universal immune memory against influenza viruses requires prior knowledge of conserved antigenic regions to facilitate immunogen design and assessment of immune responses. Although antibodies can be first isolated from serum and used to map the epitopes, identification of antigen-specific CD8⁺ T cells requires prior knowledge of the antigenic epitopes, including both peptides and restricting HLAs. This can inform the antigenic composition of T cell–based vaccines to focus the immune response towards conserved and protective epitopes. Here, we defined the CD8⁺ T cell cross-reactome against influenza A, B and C viruses, and identified novel IBV CD8⁺ T cells using immunopeptidomics^20,21. We demonstrated that CD8⁺ T cells confer previously unrecognized, broad heterotypic cross-reactivity and characterized these responses in depth. Our data provide new insights into universal CD8⁺ T cells across IAVs, IBVs and ICVs, and suggest that combining universal CD8⁺ T cells with B cell–based vaccines might lead to a broadly protective influenza vaccine that does not require annual reformulation.

Results

Universal CD8⁺ T cell epitopes across IAV, IBV and ICV

To investigate the breadth of CD8⁺ T cell cross-reactivity across IAVs, IBVs and ICVs, we first assessed the conservation of previously identified IAV-specific CD8⁺ T cell epitopes across IAVs, IBVs and ICVs (Fig. 1a and Supplementary Fig. 1), as IAV-specific CD8⁺ T cells have been our the research focus so far. Our conservation analysis of >67,000 influenza segment sequences identified 31 conserved epitopes (with >70% amino acid identity) across IAV and IBV, and 8 epitopes across IAV, IBV and ICV types (Supplementary Table 1). Based on the prevalence of HLA-restricting molecules in the population and the nature of alterations within the peptide variants, we selected nine epitopes across both HLA-A (HLA-A*01:01, HLA-A*02:01 and HLA-A*03:01/A*11:01/*31:01/A*68:02) and HLA-B (HLA-B*07:02, HLA-B*44:02 and HLA-B*37:01) alleles (Table 1) for further investigation.

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