Identification of novel B-cell epitopes on Hirame novirhabdovirus (HIRRV) G protein and development of a multi-epitope vaccine.

Hirame novirhabdovirus (HIRRV) is a significant viral pathogen affecting marine fish, and its glycoprotein (G) serves as a major immunogenic protein capable of inducing protective immunity. Given the critical role of B lymphocytes-mediated humoral and cellular immune responses in combating viral infections, we designed a recombinant multi-epitope antigen vaccine targeting the HIRRV-G protein (rGBLE) and evaluated its efficacy in Japanese flounder (Paralichthys olivaceus). Bioinformatic analysis identified 14 high-antigenicity B-cell epitopes, which were linked via GPGPG spacers to construct rGBLE. The recombinant protein exhibited enhanced hydrophilicity (GRAVY: 0.578) and structural stability (instability index: 16.6) compared to the full-length recombinant G protein (rG). rGBLE was successfully expressed in E. coli and purified as a 28.6 kDa His-tagged protein. Immunization assays revealed that rGBLE elicited significantly higher specific serum IgM titers against HIRRV and rG at week 4 post-vaccination compared to the rG vaccine, and induced neutralizing antibody titers (97.57 ± 14.04) that were higher than those elicited by rG (68.65 ± 12.24). Flow cytometry demonstrated robust proliferation of CD4+ T and mIgM+ B lymphocytes in peripheral blood, spleen, and head kidney tissues, with rGBLE inducing delayed but sustained CD4-2+ T lymphocytes responses and elevated mIgM+ B lymphocytes levels in specific tissues. In challenge experiment, rGBLE exhibited an RPS of 85.2 % versus 70.4 % for rG, both conferring effective protection, but the survival curve difference was not statistically significant. These findings demontrated that rGBLE could elicit strong humoral and cellular immune reponses and generate high neutralizing antibodies, suggesting its potential for the development as a potent and promising vaccine against HIRRV in aquaculture.