Revealing novel CD8+ T-cell epitopes from the H5N1 avian influenza virus in HBW/B1 haplotype ducks

Abstract

The duck CD8⁺ T-cell response effectively defends against H5N1 highly pathogenic avian influenza virus (HPAIV) infection, but the recognized peptide is rarely identified. Here, we found that the ratio of CD8⁺ T cells and the expression of IFN-γ and cytotoxicity-associated genes, including granzyme A/K, perforin and IL2, at 7 days post-infection in peripheral blood mononuclear cells (PBMCs) from B1 haplotype ducks significantly increased in the context of defending against H5N1 AIV infection in vivo. Moreover, similar results were observed in cultured and sorted H5N1 AIV-stimulated duck CD8⁺ T cells in vitro. Next, we selected 109 epitopes as candidate epitopes on the basis of the MHC-I restriction binding peptide prediction website database and further identified twelve CD8⁺ T-cell epitopes that significantly increased IFN-γ gene expression after stimulating B1 haplotype duck memory PBMCs. In particular, NP_338-346, NP_473-481, M_2-10, PB1_540-548 and PA_80-88 were highly conserved in H5N1, H5N6, H5N8, H7N9, and H9N2 AIVs. These findings provide directions for the development of universal T-cell epitope vaccines for AIV in ducks.

Keywords: CD8+ T-cell response; H5N1 AIV; MHC B1 haplotype ducks; MHC B1-restricted T-cell epitopes.

Figure 1

Monitoring of H5N1 AIV shedding, HI Ab levels, and T lymphocyte percentages post-infection. Virus (H5N1 AIV) shedding was monitored via detection of the viral load in oropharyngeal (A) and cloacal (B) swabs. Statistical analyses of the virus titre in swabs at various time points were performed using one-way ANOVA. C HI Ab levels were monitored in 1% chicken RBCs. A value > 4 (dotted line) was considered HI Ab positive. One-way ANOVA was used for statistical comparisons. The percentages of CD8⁺ T cells (D) and CD4⁺ T cells (E) in the control group and infection group at various time points were detected. The cells (2 × 10⁵) from each sample were collected for flow cytometric analysis. The results are presented as the means ± SEMs, and the unpaired t test was used for statistical comparison. H5N1 virus shedding and H5N1 HI Ab expression in the control group were negative at various time points (data not shown). Three ducks in the infected and control groups were randomly selected for sampling and detection. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Figure 2

qRT-PCR analysis of immune-related gene expression in B1 haplotype duck PBMCs after 3 and 7 days of infection. Total RNA was extracted from the PBMCs of three ducks in the infected and control groups. The data were collected from three biological samples in each group; each sample was analysed in triplicate. A Innate immune genes in PBMCs. B IFN-γ and cytotoxicity-associated genes in PBMCs. C Th2 cytokines in PBMCs. D Cell apoptosis- and proliferation-related genes in PBMCs. The results are presented as the means ± SEMs, and paired t tests were used for statistical comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

In vitro culture and response of H5N1 AIV-stimulated CD8⁺T cells from B1-line ducks. A Diagram of in vitro culture and response detection of H5N1 avian influenza virus-specific duck CD8⁺ T cells. B Flow cytometry detection of the proliferation of CFSE-labelled PBMCs stimulated with H5N1 AIV. The red sample indicates CFSE-labelled memory PBMCs without stimulation. The orange and blue samples represent CFSE-labelled PBMCs cultured for 6 and 9 days after H5N1 AIV stimulation, respectively. The percentage of CD8⁺ T cells (C) and the number of CD8⁺ T cells (D) in H5N1-stimulated and unstimulated cells after 7 days of culture. CD8⁺ T-cell percentage or number data were collected from three replicates in two independent experiments. The results are presented as the means ± SEM, and the unpaired t test was used for statistical comparison. E Changes in the transcription of H5N1 AIV-specific CD8⁺ T cells were detected via qRT-PCR. CD8⁺ T cells were sorted from H5N1-stimulated and unstimulated PBMCs after 7 days of culture. The data were collected from three biological samples in triplicate from each H5N1-stimulated and unstimulated group. The results are presented as the means ± SEM, and paired t tests were used for statistical comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Screening of immunodominant epitopes of H5N1 AIV-specific CD8⁺ T cells. A RT-PCR analysis of IFN-γ gene expression in B1 haplotype memory PBMCs stimulated with peptide pools. The data were collected from three replicates of three biological samples. B RT-PCR analysis of IFN-γ gene expression in B1 haplotype memory PBMCs with individual 9-mer peptide stimulation from positive peptide pools. The results are presented as the means ± SEM, and paired t tests were used for statistical comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. C Conservation of the sequences between circulating strains for the positive peptides. The Global Initiative of Sharing All Influenza Data (GISAID) (gisaid.org) was used with the search criteria set as Asia, 2019 to 2024, NA/NP/M/PB1/PA/NS1, and H5N1/H5N6/H5N8/H7N9/H9N2. Protein sequences were aligned with the MUSCLE algorithm. The frequency of mutation was determined. The protein sequences of influenza A (H5N1-H5N6-H5N8-H7N9-H9N2) viruses are represented by various colored bars, and the number above the bars indicates the number of virus strains.