Influenza A virus matrix protein 1-specific human CD8+ T-cell response induced in trivalent inactivated vaccine recipients

Abstract

Among 17 HLA-A2-positive healthy adults, CD8+ T-cell responses against an HLA-A2-restricted matrix protein 1 (M1) epitope increased after immunization with trivalent inactivated influenza vaccine (TIV) in two individuals. The presence of M1 in TIV was confirmed by Western blotting. T-cell cytotoxicity assays showed that TIV is processed and the epitope is presented by antigen-presenting cells to an M1 epitope-specific CD8+ T-cell line for specific lysis. These data show that TIV, which is formulated to contain surface glycoproteins to induce serotype-specific antibody responses, also contains M1, capable of inducing subtype cross-reactive CD8+ T-cell responses in some vaccinees.

FIG. 1.

Influenza virus M1_58-66 epitope-specific CD8⁺ T-cell response before and after vaccination quantitated by ELISPOT assays (A) and detection of M1 protein in autologous BLCL pulsed with TIV (2006/2007 formulation) by cytotoxic CD8⁺ and CD4⁺ T-cell lines (B and C). (A) Cryopreserved PBMC were tested in IFN-γ ELISPOT assays as described previously (3). Cells were incubated at 37°C for 15 h with peptides at a final concentration of 2 μg/ml or with live influenza A viruses (data not shown). Medium was used as a negative control. Assays were performed in triplicate. The frequency of peptide-specific IFN-γ-producing cells was calculated (average number of spots in the virus wells minus average number of spots in medium wells/number of cells/well) and converted to the number of IFN-γ-producing cells per 10⁶ PBMCs. (B) Detection of M1 protein by the CD8⁺ T-cell line, 1-7-K, specific to the M1_58-66 epitope. The effecter/target ratio was 10. The peptide concentration was 25 μg/ml for both the M1_58-66 and M1_17-31 peptides. Autologous BLCLs as targets were pulsed with peptides, the TIV alone, or the TIV and Iscomatrix. (−), negative control (medium alone). Results shown are representative of the two experiments. (C) Detection of M1 protein by the cytotoxic CD4⁺ T-cell line 1-3, specific to the peptide M1_17-31 (containing epitope M1_18-29). Results shown are representative of the two experiments.

FIG. 2.

Detection of M1 protein by Western blotting. A 12.5-μl amount of the TIV (2007/2008 formulation by Sanofi Pasteur) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a goat polyclonal antibody recognizing the N terminus of M1 was used (left panel). For NP, a rabbit polyclonal antibody specific to influenza A NP was used (right panel). NC (H1N1) and W (H3N2) are A/New Caledonia/20/99 IVR-166 (H1N1) and A/Wisconsin/67/2005X-161B (H3N2), used as positive controls, and allantoic fluid was used as a negative control.