Cross-reactive CD8+ T-cell immunity between the pandemic H1N1-2009 and H1N1-1918 influenza A viruses

Abstract

Preexisting T-cell immunity directed at conserved viral regions promotes enhanced recovery from influenza virus infections, with there being some evidence of cross-protection directed at variable peptides. Strikingly, many of the immunogenic peptides derived from the current pandemic A(H1N1)-2009 influenza virus are representative of the catastrophic 1918 "Spanish flu" rather than more recent "seasonal" strains. We present immunological and structural analyses of cross-reactive CD8(+) T-cell-mediated immunity directed at a variable (although highly cross-reactive) immunodominant NP(418-426) peptide that binds to a large B7 family (HLA-B*3501/03/0702) found throughout human populations. Memory CD8(+) T-cell specificity was probed for 12 different NP(418) mutants that emerged over the 9 decades between the 1918 and 2009 pandemics. Although there is evidence of substantial cross-reactivity among seasonal NP(418) mutants, current memory T-cell profiles show no preexisting immunity to the 2009-NP(418) variant or the 1918-NP(418) variant. Natural infection with the A(H1N1)-2009 virus, however, elicits CD8(+) T cells specific for the 2009-NP(418) and 1918-NP(418) epitopes. This analysis points to the potential importance of cross-reactive T-cell populations that cover the possible spectrum of T-cell variants and suggests that the identification of key residues/motifs that elicit cross-reactive T-cell sets could facilitate the evolution of immunization protocols that provide a measure of protection against unpredicted pandemic influenza viruses. Thus, it is worth exploring the potential of vaccines that incorporate peptide variants with a proven potential for broader immunogenicity, especially to those that are not recognized by the current memory T-cell pool generated by exposure to influenza variants that cause successive seasonal epidemics.

Fig. 1.

Loss of interepitope cross-reactive T-cell immunity for the NP_418–426 variant with the emergence of the A(H1N1)-2009 strain. Lymphocytes were taken from healthy donors who had no serological or clinical evidence of exposure to the pandemic A(H1N1)-2009 strain [D1, HLA-B*3501⁺ (A); D2, HLA-B*3501⁺ (B); D3, HLA-B*3503/01⁺ (C); D4, HLA-B*3503⁺ (D); D5, HLA-B*3503⁺(8) (E); and D6, HLA-B*0702⁺ (F)] and from patients hospitalized following infection with this virus [D7, HLA-B*0702 (G) and D8, HLA-B*0702 (H)]. Individual PBMC populations were cultured in vitro for 10 d with a pool of 12 variant NP₄₁₈ peptides (Table 1) and then analyzed for CD8⁺ T-cell specificity in a standard IFN-γ intracellular cytokine staining assay. The final 7-h stimulation was with HLA-B*3501⁺CIR-B35 cells (A–E) or with autologous HLA-B*0702⁺ PBMCs (F–H) pulsed with either a pool of 12 peptides (black bars) or single NP₄₁₈ variants (white bars). The percentage of IFN-γ–producing cells in the CD8⁺ T-cell fraction is shown with the no-peptide control background subtracted. Background IFN-γ production for restimulated CD8⁺ T cells was typically 0.046–0.62%. Experiments were repeated with similar results unless limited by cell numbers. CD8⁺ T-cell reactivity of HLA-B*0702⁺ D6, D7, and D8 against the pandemic A(H1N1)-2009 NP₄₁₈ variant in single-peptide PBMC cultures is shown in Fig. 2. Plasma antibody titers are listed in Table S2.

Fig. 2.

Natural infection with A(H1N1)-2009 elicits CD8⁺ T cells specific for 2009-NP₄₁₈ that are cross-reactive with 1918-NP₄₁₈. CD8⁺ T-cell reactivity against the A(H1N1)-2009 NP₄₁₈ variant is shown for single-peptide PBMC cultures obtained from a healthy donor (A) and A(H1N1)-2009–infected HLA-B*0702⁺ donors (B and C). PBMCs were obtained from D6 (A), D7 (B), and D8 (C) and stimulated for 10 d with the 2009-NP₄₁₈ peptide. On day 10, PBMCs were restimulated with autologous PBMCs pulsed with either a pool of 12 or single NP₄₁₈ peptides. CD8⁺ T-cell reactivity was determined following a 7-h restimulation (5 h with Brefeldin A) by IFN-γ production. The percentage of IFN-γ production by CD8⁺ T cells is shown, with background subtracted of no-peptide controls. Background IFN-γ production was 0.2, 0.26, and 0.029% for D6, D7, and D8, respectively. Black bars show the corresponding original restimulation 2009-NP₄₁₈ peptide, and white bars show cross-reactive CD8⁺ T-cell responses.

Fig. 3.

Patterns of NP₄₁₈-specific cross-reactive T-cell immunity in the healthy HLA-B*3501⁺ donor in single-peptide cultures. Individual PBMC populations from D1 HLA-B*3501 were stimulated for 10 d with either the 12 pooled (Table 1) or individual NP₄₁₈ peptides (shown above each panel). On day 10, PBMCs were restimulated with peptide-pulsed CIR-B35 cells and assayed for IFN-γ production, as described in the legend to Fig. 1. The black bars give the results for the homologous restimulation, whereas the white bars show the cross-reactive CD8⁺ T-cell responses. Background IFN-γ production was 0.34–0.82% (subtracted from the samples).

Fig. 4.

Chronological mutations in the NP_418–426 variants occur at the most solvent-exposed residues. Crystal structures of the binary HLA-B*3501 complexes with the NP_418–426 variants emerging chronologically between 1918 and 2009 were resolved: 1918-NP₄₁₈ LPFERATIM (A), 1934-NP₄₁₈ LPFDRTTIM (B), 1947-NP₄₁₈ LPFDKTTIM (C), 1972-NP₄₁₈ LPFDKSTIM (D), 1980-NP₄₁₈ LPFEKSTVM (E), and 2009-NP₄₁₈ LPFERATVM (F). Further characteristics of pMHC-I interactions are presented in Fig. S1.