Immunoinformatics design and in vitro evaluation of dendritic cell-targeting brucellosis multi-epitope vaccine based on Omp28 and GroEL

Brucellosis is a zoonotic infectious disease caused by bacteria of the genus Brucella, with an estimated global annual incidence of approximately 2.1 million cases. Currently, there is no safe and effective human vaccine available. Utilizing a reverse vaccinology strategy, this study screened 16 cytotoxic T lymphocyte epitopes, 10 helper T lymphocyte epitopes, and 3 B cell epitopes derived from Brucella Omp28 and GroEL proteins. These epitopes were fused with a dendritic cell-targeting peptide to design a stable, hydrophilic, non-toxic, and non-allergenic multi-epitope vaccine. Molecular docking and dynamics simulations demonstrated that the vaccine could stably bind to major histocompatibility complex molecules and Toll-like receptor 4 (TLR4). Immune simulations using C-ImmSim indicated that the vaccine could elicit a robust immune response. Subsequently, the recombinant plasmid pET-28a(+)-D-OG was constructed and introduced into Escherichia coli for induced expression and purification. Further in vitro experiments, including indirect enzyme-linked immunosorbent assay, cytometric bead array assay, and flow cytometry, were conducted to validate the immunogenicity of the vaccine ( P < 0.01). The D-OG constructed in this study exhibits excellent structural stability and strong immunogenicity, effectively activating dendritic cells and enhancing antigen presentation efficiency to elicit robust specific immune responses. Combined bioinformatic analyses and multiple in vitro experiments in this study collectively suggest that the candidate vaccine exhibits robust immunogenicity. It holds promising prophylactic potential against brucellosis and offers a valuable blueprint for developing multi-epitope vaccines targeting other intracellular bacterial pathogens.