Integrated immunoreactivity analysis of MHC-I epitopes derived from the Marburg virus glycoprotein

Marburg virus (MARV) is a hemorrhagic fever virus, and its glycoprotein (GP) represents an important target for vaccine research. MHC class I (MHC-I)-restricted T cell epitopes are critical for inducing cytotoxic CD8⁺ T cell responses. In this study, we aimed to identify MHC-I-restricted T cell epitopes derived from the MARV Angola strain GP through an integrated bioinformatics and experimental approach.

We retrieved the MARV Angola strain GP sequence and divided it into 673 overlapping 9-mer peptides. By integrating five computational prediction algorithms, immunogenicity scoring tools and BLASTp conservation analysis, we screened 9-mer peptides from the MARV Angola GP. Molecular docking was performed with MHC-I structures using HPEPDOCK and molecular dynamics (MD) analysis. Bidirectional hierarchical clustering was used to analyze binding similarity across MHC-I molecules. Mutations identified in epitope regions were tested for their effect on affinity. Toxicity and allergenicity of the epitopes were predicted using dedicated tools. Finally, BALB/c and C57BL/6 mice were immunized with a DNA vaccine encoding MARV GP. IFN-γ secretion in mouse splenocytes was then measured by ELISpot, serum IgG by ELISA, and CD8⁺ T cell cytokine production by flow cytometry.

Among 673 GP peptides, eight epitopes (GAFFLYDRI, REGDMFPFL, DMFPFLDGL, NENDCDAEL, AGLSWIPFF, NQNNLVCRL, TGWGLGGKW, SDWGVLTNL) were predicted to exhibit high-affinity MHC-I binding, strong immunogenicity, and intraspecies conservation. Bidirectional hierarchical clustering analysis, molecular docking, sequence alignment, and toxicity/allergenicity predictions suggested binding across selected alleles, structural stability, limited impact from identified mutations, and low predicted toxicity. In subsequent ELISpot validation, eight epitopes elicited strong IFN-γ responses. In addition, serum from GP-immunized mice contained IgG that bound the corresponding linear peptide sequences.

An integrated computational-experimental pipeline identified eight conserved, immunogenic MHC-I epitopes from MARV GP. These selected epitopes may serve as promising candidates for future vaccine design.