Screening and identification of HLA-A*02: 01-restricted CTL epitopes in the glycoproteins Gn and Gc of Dabie bandavirus

Dabie bandavirus (DBV) infection causes severe fever with thrombocytopenia syndrome (SFTS), which has a high fatality rate and for which no specific vaccine is currently available. The DBV glycoprotein precursor (GP) is an important immunological target, however, its cytotoxic T lymphocyte (CTL) epitopes remain unclear. This study aims to screen and identify CTL epitopes with strong immunogenicity from DBV GP using bioinformatics tools for prediction, combined with in vitro animal experiments for validation, providing a theoretical basis for research on DBV-specific cellular immune mechanisms and epitope-based vaccine design.

Using the amino acid sequences of glycoprotein n (Gn) and glycoprotein c (Gc) of the DBV HB29 strain as templates, 3 bioinformatics tools, namely IEDB, NetMHCpan 4.1, and STMHCpan, were employed to predict HLA-A*02:01-restricted T-cell epitopes with a length of 8 to 11 amino acids. Toxicity and allergenicity were evaluated using ToxinPred2 and AlgPred 2.0, and conservation was analyzed through sequence alignment of 20 domestic DBV isolates. The peptides ranked in the top 2% for binding affinity by at least 2 of the 3 tools were selected as epitope peptides. Female HLA-A*02:01 transgenic mice (6 to 8 weeks old) were randomly divided into a Gn group and a Gc group (n=2). The transgenic mice were immunized on days 0, 14, and 28 for a total of 3 immunizations with Gn (100 μg/mouse) or Gc (100 μg/mouse) combined with Freund’s adjuvant. In 7 d after the last immunization, the mice were sacrificed, and the spleens were harvested to prepare single-cell suspensions. Splenocytes (5×10⁵ cells/well) were stimulated in vitro with each epitope peptide at a final concentration of 25 μg/mL, and IFN-γ secretion was detected using ELISPOT assay. A spot-forming cell (SFC) value >100 per 10⁵ splenocytes was set as the positive criterion. To eliminate the potential interference of IFN-γ secreted by CD4⁺T cells, CD4⁺T cell-depleted splenocytes (CD4-SP) were obtained via magnetic bead-based positive selection, and ELISPOT assay was performed again under the same conditions, with an SFC value >100 per 10⁴ CD4-SP set as the positive criterion.

A total of 12 epitope peptides (Gn1 to Gn6 and Gc1 to Gc6) were screened out through cross-prediction using the 3 tools. All epitope peptides showed promising potential in immunogenicity, passed toxicity and allergenicity evaluation, and exhibited good conservation among isolates from multiple regions. Molecular docking showed that all 12 epitope peptides could significantly induce IFN-γ secretion by splenocytes from immunized mice (all SFC values >100, all P<0.0001). In CD4-SP, Gn5, Gn6, Gc3 and Gc4 still induced strong IFN-γ responses (all SFC values >100, all P<0.0001).

Four CTL epitopes (Gn5, Gn6, Gc3 and Gc4) that can specifically induce IFN-γ responses in CD8⁺T cells were successfully identified from DBV. These epitopes are highly conserved among domestic DBV isolates, providing experimental evidence for the development of multi-epitope vaccines against DBV.