**1. Introduction**

Brucellosis, one of the major bacterial zoonoses across the globe, is caused by members of the *Brucella* genus, (*B. abortus*, *B. melitensis*, *B. suis* and *B. canis*). It is still considered one of the seven "neglected zoonoses" worldwide, in spite of a huge public health burden in many countries with low income [1]. The transmission of brucellosis in humans occurs through coincidental exposure to pathogenic bacterium from infected animals or animal products [2–6]. Domestic animals a ffected by brucellosis are more prone to abortions whereas human brucellosis leads to debilitating symptoms such as recurrent fevers, spondylitis, joint pains, and osteomyelitis [7–9]. The successful vaccines available commercially against brucellosis are Strain 19 and RB51 [10]. Strain 19 is an attenuated *B. abortus* strain with smooth morphology that has the capability to induce antibody responses and protect cattle against brucellosis. *B. abortus* RB51 is a rifampicin-resistant strain with rough morphology which provides protection against infection and abortion [11]. But there are several disadvantages associated with these vaccines such as occurrence of abortions in pregnan<sup>t</sup> cows, restriction on age of vaccination, and reversion of vaccine strain back to pathogenic strain upon administration [12]. Further, antibodies are directed majorly against the lipopolysaccharide O-side chain during natural infection or S19 immunization which causes obstruction during a brucellosis diagnostic test. Therefore, development of an e ffective and safe vaccine is required.

In this context, recombinant protein-based subunit vaccines have an advantage over traditional live-attenuated vaccines as being protective and safe for human administration [13–15]. Recombinant subunit vaccines rely on specific parts of the pathogenic microorganism such as proteins or capsular polysaccharides containing protective epitopes to result in a protective immune response. Since these vaccines cannot replicate in the host, they are not pathogenic on administration [16]. Numerous intracellular components and surface proteins from bacteria have been researched as potential protective antigens against *Brucella* infection, such as L7/L12 [17], Omp19, Omp31 [18], BLS [19], BP26 [15] and Omp25 [20,21], and some have shown to be e ffective in providing significant protection against *Brucella* infection [15,17,20,21]. Although recombinant subunit vaccines o ffer no residual virulence but these require administration of multiple boosters along with providing lower levels of protection [22]. Further, when these *Brucella* protective antigens were administered in combination, the induced immune responses were superior in clearing intracellular *Brucella* as compared to their univalent counterpart [14,15,18,23–27]. Earlier studies have suggested that Omp25 and L7/L12 can serve as e fficient protective antigens by inducing strong humoral and cellular immune response [17,20,28]. Outer membrane protein 25 (Omp25) of *Brucella* species has been identified as a potential antigen [20,28] capable of inhibiting TNFα production [21]. Importantly, *omp25* gene is highly conserved in multiple *Brucella* species, strains, and biovars [8]. In addition, L7/L12 is a conserved protein which is found to be immunogenic and a stimulant of Th1 type CD4+ cellular response in mice [17], making both these antigens relevant as components for divalent vaccine formulation against brucellosis. Further, many protective antigens from *Brucella* have been classified as specific and novel diagnostic target as compared to LPS based conventional tests [29,30].

Aluminum hydroxide (Alum) has been validated as an economical and safe adjuvant by U.S. Food and Drug administration for veterinary and human use. Aluminum salts form a short-term depot at the site of injection and slowly release antigen to the body's immune system [31,32]. In this study, we co-immunized Omp25 and L7/L12 of *Brucella abortus* using alum as an adjuvant. Elicited antibody titers and antibody subtype profile were analyzed when administered as a divalent vaccine candidate in BALB/c mice. Further, the protective e fficacy of individual proteins and the divalent vaccine candidate against virulent *B. abortus* 544 challenge were determined.
