Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities

Abstract

The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus-specific CD8(+) T lymphocytes (CTLs) can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16-57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701, B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development.

Keywords: CD8 T cells; HLA types.

Fig. 1.

High level of conservation for H7N9 CTL peptides. CTL antigenic peptides within (A) NP and (B) M1 were obtained from the Immune Epitope Database (IEDB, www.iedb.org; April 2013) and analyzed using the IEDB’s Epitope Conservancy Analysis tool (http://tools.immuneepitope.org/tools/conservancy/iedb_input). (C) Summary of numbers and percentages of conserved, unique and variable epitopes within NP and M1. Conservation at 100% match was determined by comparing the corresponding CTL peptides in H7N9 to those of representative strains that have circulated in the human population (Table S1). Black, CTL peptides conserved over the last century; red, variable epitopes; *, unique CTL peptides for the H7N9 IAV; white, conserved H7N9-NP₃₈₃ peptide that binds to HLA-B*2705 (escape mutants were identified in H3N2 strains for NP₃₈₃).

Fig. 2.

CTL peptide map for NP and M1 across all human IAV lineages. The analyses spanned the full protein-coding region of the NP and M1 proteins to deduce changes in the conserved, unique and variable epitopes (Tables S2–S7). Green, blue, and red bars on the left of the peptides refer to conserved, unique, and variable CTL peptides, respectively. The horizontal gray bars throughout the alignments highlight the nonsynonymous substitutions established in H1N1, H2N2, and H3N2 viruses through their evolutionary history in human population. CTL peptides within (A) NP and (B) M1, which do not fall on the gray bars, show regions that have not changed in human influenza A viruses (the exception being NP₂₅₉), indicating lesser selection pressure on those sites.

Fig. 3.

CTL responses to conserved immunodominant H7N9 peptides. PBMCs from healthy donors were peptide-stimulated and cultured for 10 d. CTL responses were determined by an IFNγ/TNFα intracellular cytokine staining (ICS). Representative FACS plots for (A) A*0301⁺NP₂₆₅, (B) A*0201⁺M1₅₈, (C) B*2705⁺NP₃₈₃, (D) B*5701⁺NP₁₉₉, (E) B*1801⁺NP₂₁₉, (F) B*0801⁺NP₂₂₅, (G) B*0702⁺NP₁₇₂, and (H) A*2402⁺NP₃₉ are shown. Values for no peptide are in brackets. Graphs show pooled data from multiple donors. Background (no peptide controls) was subtracted.

Fig. 4.

H7N9 escape mutants for A*0101-NP₄₄ and A*6801-NP₈₉. (A–F) The Y9N mutation in the immunodominant H7N9 NP₄₄ peptide abrogates CTL recognition by reducing thermal stability. (A) Representative FACS plots for CTL responses to different A*0101-NP₄₄ variants. (B) CTL responses (IFNγ ICS, n = 7) against four NP₄₄ variants. (C) Thermal stability for the A*0101-NP₄₄ variants. (D and E) Crystal structures of HLA-A*0101 (cartoon) bound to the NP_44-WT peptide (pink) and to the NP₄₄-S7N peptide (orange), respectively. Only the α1-helix of the HLA is shown for clarity. (F) Superposition of the HLA-A*0101 binding cleft to NP₄₄-WT (pink) and NP₄₄-S7N (orange), with the Arg156 and Trp147 of the HLA represented in stick; the H bond shown as dashed lines. (G) An ICS response to a unique H7N9 peptide NP₈₉ restricted by HLA-A*6801, following stimulation with the NP₈₉-H7N9 variant or a pool of NP₈₉ seasonal and pandemic variants (Table S4). Confirmation of prior IAV exposure was determined by assessing the reactivity to A*0201-M1₅₈.