Abstract
Brucellosis remains a persistent global health threat. While the live attenuated Rev-1 vaccine is effective, it is associated with safety concerns, including residual virulence and interference with diagnostic tests. This study aimed to evaluate the immunogenic potential and safety profile of a sonicated Brucella melitensis Whole Antigen (WA) compared with its derived subunit fractions (Lipopolysaccharide [LPS] and Protein) and the standard live vaccine. A virulent B. melitensis isolate was confirmed via Vitek-2 Compact System (99% probability). Forty-eight male Wistar rats (200-250g) were randomised into six groups (n=8 each): Negative Control, Protein Antigen, LPS Antigen, Whole Antigen (WA), Pathogenic Group (infected with virulent B. melitensis), and Vaccine Group (Rev-1). All antigens were standardised to 3 mg/mL protein concentration using the Bradford assay before immunisation. Blood samples were collected at days 1, 2, 7, and 14 post-primary immunisation, and at the same intervals after booster injection. Humoral (IgG, IgM) and cellular (IFN-γ, TLR2, TLR4) immune responses were quantified by ELISA. The Whole Antigen group elicited a superior and balanced immune response. At 14 days post-booster, the WA group maintained the highest IgG levels (11.16 ± 1.0 μg/mL), significantly outperforming the Vaccine group (7.41 ± 0.6, P<0.001). Critically, with respect to safety, although the Vaccine and Pathogenic groups exhibited high inflammatory IFN-γ levels (197.78 and 212.36 pg/mL, respectively), the WA group showed a regulated decline to near-baseline (103.33 ± 6.2 pg/mL), thereby preventing chronic inflammation. In conclusion: The sonicated Whole Antigen, using standardised protein content, demonstrates superior immunogenicity compared to live vaccine under the conditions of this study. It effectively decouples immunogenicity from immunopathology, presenting a promising candidate for a safer acellular vaccine.

Key words: Brucella melitensis, IFN-γ, Immunopathogenesis, Vitek-2, Vaccine development, Whole Antigen.