Assessment of seasonal influenza A virus-specific CD4 T-cell responses to 2009 pandemic H1N1 swine-origin influenza A virus

Abstract

Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.

FIG. 1.

Seasonal influenza virus-specific CD4 T cells respond to naturally processed epitopes. [³H]thymidine incorporation results for 17 individual seasonal influenza virus-specific T-cell lines cocultured with dendritic cells derived from autologous monocytes in the presence of medium (white bars) or 10 μg/ml trivalent seasonal influenza virus vaccine (black bars) are shown. Results for T-cell lines incubated with vaccine in the absence of antigen-presenting cells are also shown (gray bars). Error bars represent 1 standard deviation.

FIG. 2.

Seasonal influenza virus-specific CD4 T cells respond to S-OIV peptides. [³H]thymidine incorporation for seasonal flu virus-specific T-cell lines restimulated with 10 μg/ml of peptides derived from the seasonal influenza virus strain (black bars), S-OIV (hatched bars), or medium control (white bars), in the presence of irradiated (50 Gy) autologous PBMC, are shown. Error bars represent 1 standard deviation.

FIG. 3.

Ex vivo detection of seasonal flu virus-specific and S-OIV-specific CD4 T cells. (A) Number of influenza virus-specific CD4 T cells per 1 × 10⁶ CD4 T cells estimated by ex vivo tetramer staining. Each colored bar segment represents the sum of the indicated Ag-specific T cells. Cells were gated on CD19⁻ CD14⁻ CD4⁺ tetramer⁺ populations. The number above each bar represents the total number of Ag-specific T cells investigated. (B and C) Relative frequencies of CD45RA⁺ and CD45RA⁻ subsets within seasonal influenza virus (B)- and S-OIV (C)-specific T cells. The cells were gated on CD19⁻ CD14⁻ CD4⁺ tetramer⁺ populations.

FIG. 4.

S-OIV peptides elicit full-scale cross-reaction. IFN-γ ELISPOT results from three HLA-DR0401⁺ donors in response to (s)NA370 (A to C) and (s)MP101 (D to F) are shown. Each plot represents the results from one donor for a single seasonal flu virus epitope (closed circles, solid lines) and the S-OIV homologue (open circles, dashed lines). Two-week in vitro (seasonal H1N1 virus peptide)-primed T cells were used as responders. The number of spots per 10,000 cells is plotted against the concentration of peptide. Error bars represent 1 standard deviation. The embedded fluorescence-activated cell sorter (FACS) density plot shows the dual-tetramer staining (of the same T cells used for ELISPOT), where the y axis represents seasonal H1N1 virus peptide-loaded tetramer and the x axis represents S-OIV homologue peptide-loaded tetramer. Gated populations represent the CD4⁺ and seasonal flu tetramer⁺ cells.

FIG. 5.

S-OIV peptides require an excessive dosage to induce full-scale cross-reaction. IFN-γ ELISPOT results from three HLA-DR0401⁺ donors for (s)MP9 (A to C) and two donors for (s)NP441 (D and E) are shown. Each plot represents the results from one donor for the seasonal flu virus epitope (closed circles, solid lines) and the S-OIV homologue (open circles, dashed lines). Two-week in vitro (seasonal H1N1 peptide)-primed T cells were used as responders. The number of spots per 10,000 cells is plotted against the concentration of peptide. Error bars represent 1 standard deviation. The embedded FACS density plot shows the dual-tetramer staining (of the same T cells used for ELISPOT), where the y axis represents seasonal H1N1 virus peptide-loaded tetramer and the x axis represents S-OIV homologue peptide-loaded tetramer. Gated populations represent the CD4⁺ and seasonal flu tetramer⁺ cells.

FIG. 6.

S-OIV peptides induce partial cross-reaction. IFN-γ ELISPOT results from three HLA-DR0401⁺ donors for (s)HA440 (A to C), (s)NA96 (D to F), (s)NA249 (G to I), and (s)NP321 (J to L) are shown. Each plot represents the results from one donor for a single seasonal flu virus epitope (closed circles, solid lines) and the S-OIV homologue (open circles, dashed lines). Two-week in vitro (seasonal H1N1 virus peptide)-primed T cells were used as responders. The number of spots per 10,000 cells is plotted against the concentration of peptide. Error bars represent 1 standard deviation. The embedded FACS density plot shows the dual-tetramer staining (of the same T cells used for ELISPOT), where the y axis represents seasonal H1N1 virus peptide-loaded tetramer and the x axis represents S-OIV homologue peptide-loaded tetramer. Gated populations represent the CD4⁺ and seasonal flu tetramer⁺ cells.