OMP16-targeting monoclonal antibodies H4 and E6 reveal essential linear epitopes for Brucella immunoprotection.

Brucellosis, a global zoonosis, faces significant challenges from lacking safe vaccines and therapies. OMP16, a key Brucella virulence factor essential for bacterial survival, also functions as a protective antigen capable of activating host immunity. However, not all OMP16-targeting antibodies confer immunoprotection; antibodies against non-protective epitopes may consume immune resources and even exacerbate infection. To validate the protective potential of two OMP16-targeting monoclonal antibodies (H4/E6) and identify their corresponding epitopes, we established in vitro models showing both mediate macrophage opsonophagocytosis, bactericidal activity, and NK cell-mediated ADCC. Notably, E6 additionally triggered complement-dependent bacteriolysis. Murine challenge studies confirmed that both mAbs reduce bacterial loads and mitigate splenic/hepatic pathology. Based on this protection, we then employed integrated epitope mapping, interaction modeling, and alanine-scanning mutagenesis, revealing two linear B-cell epitopes essential for immunoprotection, alongside critical binding residues. This work not only provides foundational evidence for OMP16-based antibody therapy but also reports the first two validated protective OMP16 epitopes. By deciphering molecular mechanisms of these mAbs, we deliver precision epitopes for next-generation multi-epitope vaccines, offering targeted solutions against brucellosis.