Identification of novel avian influenza virus derived CD8+ T-cell epitopes

Abstract

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.

Figure 1. CD8+ T-cell frequencies in tissues of LPAIV infected chickens.

Percentages of CD8αα+CD3+ T cells (A–C) and CD8βα+CD3+ T cells (D–F) were analysed by flowcytometry in lung, spleen and PBMC at several days post infection. Absolute numbers were calculated by multiplying the percentage of CD8αα+CD3+ T or CD8βα+CD3+ T cells with the total number of cells isolated from lung (G, I) and spleen (H, J). Mean plus SEM is shown. In white: uninfected controls (UNINF, n = 12), in grey infected birds (n = 3 per time point).

Figure 2. Identification of MHC B12-restricted CD8+ T-cell epitopes using peptide pools.

Lung cells were stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis. Results for three individuals birds are shown at 5 dpi (A–C), 7 dpi (D–F), 10 dpi (G–I) and 14 dpi (J–L). Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.

Figure 3. Identification of MHC B4-, B15-, B19- and B21-restricted CD8+ T-cell epitopes using peptide pools.

Lung cells isolated at 10 dpi were stimulated with B4 restricted peptide pools (A) or, B15 (B), B21 (C) and B19-restricted peptide pools (D–F) and IFNγ producing cells were determined by IFNγ Elispot analysis. Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.

Figure 4. Screening of MHC B12-restricted CD8+ T-cell epitopes using individual peptides.

Lung cells were isolated, in vitro re-stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis at 7 dpi (A) and 10 dpi (B). Mean plus SEM is shown, n = 4 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.

Figure 5. Identification of MHC B4-restricted CD8+ T-cell epitopes using individual peptides.

Lung cells isolated at 10 dpi were thawed and re stimulated with B4 restricted peptides and IFNγ producing cells were determined by IFNγ Elispot analysis. Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.

Figure 6. Novel AIV-specific CD8+ T cell epitopes within the LPAI H7N1 virus.

Epitope mapping resulted in 11 novel CD8+ T-cell epitopes in the nucleoprotein (A) and 5 epitopes in the matrix 1 protein (B) of the LPAI H7N1 virus. In black, B12-restricted epitopes, in grey, B4-restricted epitopes (A) or B19-restricted epitopes (B).