Network pharmacology-based identification and validation of novel vaccine candidates against brucellosis.

Brucellosis remains one of the most prevalent zoonotic diseases worldwide, causing substantial economic losses and significant human morbidity. Despite decades of research, no licensed human vaccine against Brucella infection exists, and current veterinary vaccines exhibit considerable safety limitations including residual virulence, pregnancy complications, and diagnostic interference. Here we report the development of a multi-epitope messenger RNA (mRNA) vaccine candidate targeting Brucella melitensis, the most pathogenic species responsible for human brucellosis. Using an integrated immunoinformatics pipeline, we screened ten outer membrane proteins (OMPs) and identified Omp25, Omp31, and BP26 as the most immunodominant antigens. A fusion construct incorporating 24 HLA class I and 31 HLA class II predicted epitopes achieved 87.3% global HLA population coverage. Codon optimization improved the codon adaptation index from 0.55 to 0.93. Molecular dynamics simulations over 200 ns confirmed structural stability of the vaccine-TLR4 complex, with a binding free energy of -65.7 kcal/mol. In vitro assays demonstrated robust Th1-biased immune activation, with IFN-gamma reaching 125.6 pg/mL. In a BALB/c mouse challenge model, the mRNA vaccine conferred 80.0% protection, comparable to the live-attenuated S19 vaccine (92.3%) but without associated safety risks. These findings establish a promising platform for Brucella vaccine development warranting further evaluation in large animal models and human clinical trials.