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. 2024 Jun;300(6):107395.
doi: 10.1016/j.jbc.2024.107395. Epub 2024 May 19.

Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV)

Yusheng Jia  1 , Qingxin Wu  1 , Yilin Li  1 , Mulin Ma  1 , Wei Song  1 , Rongmao Chen  1 , Yongxiu Yao  2 , Venugopal Nair  3 , Nianzhi Zhang  4 , Ming Liao  5 , Manman Dai  6
Affiliations

Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV)

Yusheng Jia et al. J Biol Chem. 2024 Jun.

Abstract

B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) in vitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9 Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens in vivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

Keywords: H9N2 AIV; MHC B2 haplotype; T-cell epitope; crystal structure; peptide-binding motif.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
The peptide-binding motif of BF2∗02:01 was determined by RPLD-MS analysis. The synthetic random nonapeptide library was renatured with chβ2M and BF2∗02:01 using the serial dilution method. A and B, the complexes were purified using molecular sieve chromatography (A) and ion-exchange chromatography (B). The pBF2∗02:01 was concentrated, incubated with 0.2 N acetic acid, and concentrated using 3-kDa filter devices to collect the bound peptides. C and D, the eluted peptides were sampled for liquid chromatography-tandem mass spectrometry (LC-MS/MS) (C) and de novo analysis (D). Peptides with a score of >60 were collected in the statistics for the de novo analysis. E, the peptide-binding motif of BF2∗02:01 was displayed using WebLogo 3. The RPLD-MS analysis was repeated twice. chβ2M, chicken β2-microglobulin; RPLD-MS, random peptide library-based MHC-I LC-MS/MS analysis.
Figure 2
Figure 2
Crystal structure analysis of BF2∗02:01-WV9 and the comparison with BF2∗04:01, and BF2∗21:01.A, the overall structure of the crystal cell. B, the overall structure of BF2∗02:01-WV9. C, surface mode of the peptide-binding groove of BF2∗02:01-WV9, which binds the peptide WIIRNWETV. D, compositions of amino acids of the A–F pockets of BF2∗02:01-WV9. The pockets are shown as surface representations in light yellow. The residues comprising these pockets and the bound peptide WV9 are labeled. The hydrogen bonds between WV9 and the pockets are shown as dashed red lines. E and F, the structural comparison of the B and F pockets of BF2∗04:01, BF2∗02:01, and BF2∗21:01 were performed. The color indicates the surface charge and hydrophobicity (red, negatively charged; blue, positively charged; and white, hydrophobic). The residues which cause significant changes in the B and F pockets are marked. G, amino acid species and percentages of top 9 amino acid in H9N2 AIV proteins in H9N2 AIV proteins. H, peptide number of each protein from H9N2 AIVs based on the BF2∗02:01, BF2∗04:01, and BF2∗21:01 peptide-binding motifs. AIV, avian influenza virus.
Figure 3
Figure 3
Detection of immune response in B2 haplotype chickens after H9N2 AIV infected.A, gating strategy of T lymphocyte from PBMCs after H9N2 AIV infection. B–D, four chickens from the infected and control groups were randomly selected for sampling to detect the percentage of CD3+CD8α+ T cells (B), the percentage of CD3+CD4+ T cells (C), and the ratio of CD3+CD4+ CD8α+ T cells (D). 1.5 × 105 cells per sample were collected for flow cytometric analysis. Expressions of immune-related genes in PBMCs were detected by quantitative real-time polymerase chain reaction (qRT-PCR). In the H9N2 AIV-infected experiments, the total RNA of PBMC was extracted from chickens at 5 DPI. E–G, the transcriptional expressions of immune-related genes, including cytotoxicity-associated genes (E), innate immunity-related genes (F), and Th2 genes (G) were analyzed. Furthermore, data were collected from three biological samples in each group, and each sample performed in triplicate. The results were presented as means ± SEM, and the unpaired t test was used for statistical comparison of T lymphocyte percentage, and the paired t test was used for statistical comparison of expressions of immune-related genes. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. AIV, avian influenza virus; DPI, days postinfection; IFN-γ, interferon-γ; PBMCs, peripheral blood mononuclear cells.
Figure 4
Figure 4
Identification of T-cell epitopes with MHC B2 restriction on H9N2 AIV via IFN-γ ELISpot assays.A–E, the splenocytes isolated from H9N2 AIV-infected chickens were stimulated with 85 peptide pools (A and B)and 20 individual peptides (C, D, and E) for 24 h at 37 °C and 5% CO2. As a positive control, splenocytes were stimulated with PMA and ionomycin; as a negative control (NC), splenocytes were supplemented with DMSO. Spot counts are expressed as spots/1 × 106 cells. Furthermore, data were collected from three biological samples, each sample performed in triplicate. An unpaired t test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. AIV, avian influenza virus; DMSO, dimethyl sulfoxide; MHC, major histocompatibility complex; NP, nucleoprotein; PBMCs, peripheral blood mononuclear cells; PMA, phorbol 12-myristate 13-acetate.
Figure 5
Figure 5
Conservation of the sequences between circulating strains for the newly identified four peptides. The Global Initiative of Sharing All Influenza Data (gisaid.org) was used with the search criteria set as Asia, PB2/NP/NS1, and H5N1/H5N6/H5N8/H7N9/H9N2. A, protein sequences were aligned using the CLUSTALW tutorial and ESPript3.0 and peptide regions were mapped. B–E, 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.
Figure S1
Figure S1
Figure S2
Figure S2
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