OMP16-specific IgM antibodies D3 and F5 protect mice from Brucella infection and enhance macrophage killing

Brucellosis is a debilitating zoonotic infectious disease with significant global economic and health impacts. Its persistent nature stems from the bacteria's ability to survive intracellularly, particularly within macrophages, evading host immune responses and complicating disease control. Current therapeutic strategies often fall short, necessitating novel immunotherapeutic approaches. Targeting conserved bacterial outer membrane proteins like OMP16 with specific antibodies represents a promising avenue for developing effective antibody-based therapies or multi-epitope vaccines to overcome this challenge.

Researchers generated two IgM monoclonal antibodies, D3 and F5, against the conserved Brucella outer membrane protein OMP16 using hybridoma technology. They identified the linear epitopes (D3: 77TLSKQAQW84; F5: 120RDFLASRG127) via peptide scanning and Western blot. Integrated approaches, including molecular docking, alanine-scanning mutagenesis, and dot-blot assays, pinpointed key residues crucial for antibody-epitope binding. In vitro, the antibodies' functional activity was assessed by their ability to activate the complement system and enhance macrophage-mediated opsonophagocytosis and intracellular killing of Brucella abortus A19. For in vivo evaluation, a mouse infection model was used, where passive immunization with either D3 or F5 was administered to assess protection against Brucella infection.

The generated IgM antibodies, D3 and F5, specifically targeted highly conserved linear epitopes on Brucella OMP16 (D3: 77TLSKQAQW84; F5: 120RDFLASRG127), which are present across major Brucella species. Molecular analysis confirmed that key residues at the epitope interface form stable bonds with the antibody complementarity-determining regions (CDRs). Functionally, both antibodies activated the complement system, with F5 demonstrating significant complement-dependent bacteriolytic activity in vitro. Furthermore, in the presence of complement, both D3 and F5 enhanced macrophage-mediated opsonophagocytosis and intracellular killing of Brucella abortus A19. In a mouse infection model, passive immunization with either antibody significantly alleviated infection-induced weight loss and splenomegaly.

Importantly, passive immunization with D3 or F5 also significantly reduced bacterial load in the spleen, demonstrating in vivo protective efficacy.