Identification of a dual-specific T cell epitope of the hemagglutinin antigen of an h5 avian influenza virus in chickens

Abstract

Avian influenza viruses (AIV) of the H5N1 subtype have caused morbidity and mortality in humans. Although some migratory birds constitute the natural reservoir for this virus, chickens may play a role in transmission of the virus to humans. Despite the importance of avian species in transmission of AIV H5N1 to humans, very little is known about host immune system interactions with this virus in these species. The objective of the present study was to identify putative T cell epitopes of the hemagglutinin (HA) antigen of an H5 AIV in chickens. Using an overlapping peptide library covering the HA protein, we identified a 15-mer peptide, H5(246-260,) within the HA1 domain which induced activation of T cells in chickens immunized against the HA antigen of an H5 virus. Furthermore, H5(246-260) epitope was found to be presented by both major histocompatibility complex (MHC) class I and II molecules, leading to activation of CD4+ and CD8+ T cell subsets, marked by proliferation and expression of interferon (IFN)-gamma by both of these cell subsets as well as the expression of granzyme A by CD8+ T cells. This is the first report of a T cell epitope of AIV recognized by chicken T cells. Furthermore, this study extends the previous finding of the existence of dual-specific epitopes in other species to chickens. Taken together, these results elucidate some of the mechanisms of immune response to AIV in chickens and provide a platform for creation of rational vaccines against AIV in this species.

Figure 1. Identification of stimulatory peptide pools and individual peptides.

P2a chickens were vaccinated with TROVAC^™-AIV H5 or vaccine diluent. (1a) In two replicate experiments, at days 10 and 21 post-secondary vaccination, spleen mononuclear cells from control and vaccinated chickens were pooled and used for peptide screening. A peptide library covering the entire HA antigen of an H5 virus was used in 12 pools. Cells were stimulated with peptide pools at concentrations of 1.0 and 10 µM for 24, 48 or 72 hours. (1b) Peptides from pool 5 of the peptide library were used individually at day 21 post-secondary vaccination to stimulate spleen mononuclear cells of vaccinated P2a chickens. In each of three replicate experiments, cells from vaccinated birds were stimulated using peptide for 48 or 72 hours. (1c) Spleen mononuclear cells from vaccinated birds in 4 independent replicates were used to determine dose-dependent proliferative response to H5_246–260. Cells were incubated with peptide or peptide solvent for 72 hours. For all figures, cell proliferation, as measured by average stimulation index, (mean cpm of treatment/mean cpm of peptide solvent cultures for all independent experimental replicates), is shown for spleen mononuclear cells harvested at 21 days post-secondary vaccination and cultured for 72 hours. SI values >3 for both concentrations of peptides were considered significant.

Figure 2. Cytokine mRNA expression in splenocytes in response to H5_246–260.

In three replicate experiments, spleen mononuclear cells from vaccinated birds were incubated with peptide or peptide solvent for 24 hours before RNA extraction and cytokine analysis using real-time PCR. Relative expression of IFN-γ (A), IL-10 (B) mRNA normalized to β-actin expression in stimulated and unstimulated spleen mononuclear cells are presented. Comparisons were made using the REST program and considered significant at P≤0.05 (*).

Figure 3. Determination of MHC restriction of response to H5_246–260.

Antibodies against MHC I and MHC II (A) and anti-CD4 and anti-CD8 (B) were used to block proliferative response to peptide, H5_246–260. In at least 5 replicate experiments, spleen mononuclear cells from vaccinated birds were incubated with 5 µg/ml antibodies or isotype control before addition of peptide or solvent control. There were two additional groups of control cells, one being treated with the peptide without addition of any antibodies (peptide alone) and the second being treated with peptide solvent without addition of any antibodies (solvent alone). Proliferation was measured at 72 hours. Data are presented as counts per minute and error bars represent standard errors of the mean.

Figure 4. Proliferative response to H5_246–260 in sorted CD4+ and CD8+ spleen cells.

Spleen mononuclear cells from vaccinated birds, in 6 replicate experiments, were sorted into CD4+ and CD8+ T cell subsets followed by co-culture with APC and peptide, H5_246–260 (10 µM) at an APC to responder ratio of either 2∶1 or 10∶1. T cell subsets were also cultured with or without the peptide in the absence APC (no APC). In another control group (APC alone), APCs were cultured in the absence or presence of the peptide. Proliferative response was measured at 72 hours. Results for CD4+ (A) and CD8+ (B) are presented as an average stimulation index, (mean cpm of treatment/mean of control for all independent experimental replicates).

Figure 5. Cytokine mRNA expression in CD4+ and CD8+ T cell subsets.

Spleen mononuclear cells from vaccinated birds in three replicate experiments, were co-cultured with peptide solvent or peptide, H5_246–260 (10 µM) for 24 hours followed by magnetic cell sorting into CD4+ and CD8+ T cell subsets. Sorted cells were used for RNA extraction and cytokine analysis using real-time PCR of cDNA. Relative expression of IFN-γ (A), IL-10 (B) mRNA normalized to β-actin in CD4+ and CD8+ T cells treated with the peptide or peptide solvent are presented. Comparisons between peptide-treated and control cells were made using the REST program and considered significant at P≤0.05 (*).

Figure 6. Granzyme A and perforin mRNA expression in CD8+ T cell subsets.

Spleen cells from vaccinated birds in three replicate experiments, were co-cultured with peptide solvent or peptide, H5_246–260 (10 µM) for 24 hours followed by magnetic cell sorting into CD4+ and CD8+ T cell subsets. Sorted cells were used for RNA extraction and cytokine analysis using real-time PCR. Relative expression of granzyme A (A), perforin (B) mRNA expression normalized to β-actin in CD4+ and CD8+ T cells treated with the peptide or peptide solvent are presented. Comparisons between peptide-treated and control cells were made using the REST program and considered significant at P≤0.05 (*).