Route-dependent immunogenicity and protective efficacy of a chitosan nanoparticle-based recombinant Omp31 vaccine against Brucella abortus

Abstract Background Brucellosis remains an important zoonotic disease, highlighting the need for safe and effective vaccine strategies. This study evaluated recombinant Omp31 (rOmp31) formulated in chitosan nanoparticles and investigated the effects of different administration routes on immune responses and protection against Brucella abortus ( B. abortus ). Methods and Materials: The omp31 gene was characterized using immunoinformatics approaches, and rOmp31 was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA chromatography. The purified protein was encapsulated in chitosan nanoparticles, and their physicochemical properties, encapsulation efficiency, antigen-release profile, and cytocompatibility were evaluated. BALB/c mice were immunized through subcutaneous, oral, or combined subcutaneous-oral routes. Antigen-specific IgG and IgA responses, cytokine production, and protective efficacy against virulent B. abortus strain 544 were subsequently assessed. Results The nanoparticles showed a mean size of 440.3 nm, PDI of 0.732, zeta potential of + 16.2 mV, and approximately 90% encapsulation efficiency. Approximately 75% of the encapsulated antigen was released over 120 h, while cell viability remained approximately 90%. All rOmp31-immunized groups exhibited significantly increased antigen-specific antibody responses compared with controls. Cytokine profiles varied according to the vaccination route. Following challenge, the combined subcutaneous-oral regimen produced the greatest reduction in bacterial burden (1.55-log10 CFU), followed by oral (1.12-log10 CFU) and subcutaneous (0.92-log10 CFU) immunization. Conclusion Chitosan nanoparticle-formulated rOmp31 induced systemic and mucosal immune responses and provided measurable protection against B. abortus . The combined administration route showed the highest protective efficacy, supporting further investigation of this formulation as a candidate brucellosis vaccine.