Reverse Vaccinology and Immune Simulation of a Novel Multiepitope Vaccine Targeting <em>Brucella </em>Virulence

Background/Objectives: Brucella is a major global One Health threat, causing an estimated 2.1 million human infections and substantial livestock losses annually, with no vaccine currently available for humans, underscoring the urgent need for a safe and effective vaccine. Methods: Employing a reverse vaccinology approach, a novel 175-mer multiepitope vaccine (Mvax) targeting Brucella FrpB was computationally designed in this study, incorporating two B-cell, two MHC class I (MHC-I), and three MHC class II (MHC-II) epitopes selected for their high predicted antigenicity, safety, and IFN-γ-inducing potential. To further enhance immune activation, human beta-defensin-3 was fused to the N-terminus as an adjuvant, followed by comprehensive in silico evaluation of the construct. Results: Population coverage analysis showed the selected epitopes provide 99.59% global coverage for MHC class combined, suggesting broad immunogenic potential. Mvax is predicted to be substantially more soluble (Protein-SOL score 0.808 vs. 0.275) with greater antigenicity (VaxiJen score 1.06 vs. 0.61) than native FrpB. Dissociation constant (Kd) analysis at 37ºC predicts stronger binding of Mvax to human TLR4/MD2 and TLR2/TLR6 receptors. Immune simulations (over 100 days and three years) indicate that a single dose of Mvax may elicit a strong Th1 response, generate durable T-cell memory lasting up to three years, and produce elevated IL-12, IFN-γ, and IL-2 levels, along with approximately ten-fold higher IgM responses compared with FrpB. Conclusions: In silico data from this study suggest that Mvax could serve as a safe and effective vaccine candidate for human brucellosis, with the potential to induce long lasting immune memory; however, experimental validation is still required.