**3. Discussion**

Brucellosis is still a major public health concern and endemic in many countries, mainly in the Mediterranean region, eastern and western Africa, and parts of South and Central America. There is a substantial requirement to control and eradicate this disease caused by the *Brucella* genus [4,5,7,35,36]. The current vaccines in use, Strain 19 and RB51 despite being popular are still far from ideal [12,37]. They prevent infection in animal but offer potential risks such as attenuation reversal and virulence in susceptible hosts. In this context, subunit vaccines are better options as compared to live attenuated vaccines, since they are safe and do not revert back to pathogenic strain upon administration [13–15]. Further, recombinant subunit vaccines protect against a given pathogen by activating humoral and cellular arms of immunity based upon a specific antigen along with used adjuvant, which makes them competent and useful in the vaccine field. There are certain proteins in *Brucella* species which can provide significant protection against the disease and are conserved throughout, such as L7/L12 [17], Omp19, Omp31 [18], BLS [19], BP26 [15], and Omp25 [20,21]. Among these, *omp25* and *l7*/*l12* genes in *Brucella* species encode for Omp25 and L7/L12 immunodominant proteins respectively, and have the potential to stimulate a strong humoral immune response along with providing protection against *Brucella* infections in mice models [15,17,20,28]. Earlier studies have suggested that outer membrane proteins from *Brucella* such as Omp25 [38], Omp10 [39], Omp19 [39], BP26 [40], Omp28 [41,42], and Omp31 [30] can distinguish between *Brucella*-infected animals and non-infected ones in an efficient and accurate way, withdrawing the false positive results in the field due to cross-reacting antibodies. Further multivalent subunit vaccine formulations possess the capability to generate a wide range of immunogens that may result in better protection than their univalent counterpart [24–26]. In this study, we evaluated humoral immune response and protective efficacy of a divalent vaccine candidate consisting of rOmp25 and rL7/L12 against *Brucella* infection in mice. The foremost point to be explored in this study was whether two components combined together in a divalent vaccine have the capability to show a synergistic response and promote a heightened immune response, or if some kind of competitive inhibition occurs among them? Aluminum hydroxide (Alum) is beneficial since it is inexpensive and has been certified as the safest adjuvant for use by the United States Food and Drug Administration [31,32,43]. Alum creates a depot effect at the site of injection, resulting in a slow release of adsorbed antigens and an elevation in the immune response. The intraperitoneal route of administration was chosen because it helps to quickly absorb antigens into the vasculature, which leads to a rise of antigen drainage into the spleen and activation of immune cells circulating in the lymph nodes [44].

The results in this study exhibited that humoral immune response was elevated in mice immunized with the divalent vaccine Omp25+L7/L12 as compared to the control group (PBS + alum). The divalent vaccine (Omp25+L7/L12) was found immunogenic with high IgG levels against both of its components, rOmp25 and rL7/L12 (Figure 2), depicting that divalent vaccine has the potential to exhibit synergy among its individual components and elevate the immune response against virulent *Brucella abortus* 544 challenge. During bacterial infection, Th2-mediated immune response is characterized by synthesis and increase in the level of IgG1 antibodies [45,46] whereas Th1 immune response is represented by levels of IgG2a antibody along with IFN-γ cytokine levels. The IgG subclass (IgG1, IgG2a and IgG2b) detection in Omp25+L7/L12 immunized mice exhibited that IgG1 levels were significantly higher as compared to IgG2a levels, predicting a more prominent Th2 immune response in case of anti-Omp25 and anti-L7/L12 antibodies (Figure 3). Although individual vaccinated group generated antibodies specifically to single antigen immunized whereas divalent vaccine candidate resulted in generation of antibodies against both antigens, rOmp25 and rL7/L12. Further, immunization with alum as an adjuvant has also been suggested to enhance antibody response in mice [43,47]. It helps in enhancement of antigen uptake and presentation to antigen-presenting cells, which results in promotion of Th2 immune responses [47,48]. Therefore, it is possible that alum has helped to increase antibody titer and elevate the humoral immune response in divalent vaccine immunized mice [49]. The analysis of protective efficacy in different mice groups after infection with virulent *Brucella abortus* 544 showed that Omp25+L7/L12 immunized mice exhibited a significant increase in log10 protection (1.98) as compared to the control (i.e. alum immunized mice) at 28 days after challenge (*p* value < 0.001; Table 1). This specifies that Omp25+L7/L12 immunized mice were capable of eliminating pathogenic *B. abortus* 544 compared to the control group. On comparing log10 units of protection at 28 days after challenge in individual protein immunized mice, rOmp25 (1.46) and rL7/12 (1.75) with alum as the adjuvant, it was observed that Omp25+L7/L12 immunized mice showed a superior level of protection against *B. abortus* 544 infection, however S19 exhibited the maximum.

It is noteworthy that although *B. abortus* recombinant subunit vaccines show very promising results in mice models, the immune responses recognized in mice models may not reflect the protection achieved in natural hosts such as cattle after immunization [11]. Therefore, further studies determining protective efficacy in other animal models such as rats, guinea pigs, and monkeys are also encouraged before proceeding towards cattle administration [50]. Recombinant vaccines also need multiple booster administrations along with adjuvants and a combination of several antigens, which makes them economically unsuitable for cattle immunization [51]. Hence, there is a need to decrease the production cost, search for effective and affordable adjuvants, and reduce the expense of recombinant protein purification in order to make these vaccines economical for mass administration.

In a nutshell, this preliminary study shows that the combination of rOmp25 and rL7/L12 elicited steady immune responses against both antigens in mice. Further, when mice were immunized with the Omp25+L7/L12 vaccine candidate, a significant reduction in *B. abortus* 544 load in mice spleens was observed, implying the use of divalent vaccine (Omp25+L7/L12) as an improved vaccine candidate against brucellosis. Nevertheless, this study illustrates the potential of a divalent vaccine in providing host immunity and protection against *B. abortus* challenge, suggesting the use of a divalent recombinant vaccine candidate as an advanced approach in the future against brucellosis.

### **4. Materials and Methods**

### *4.1. Plasmids and Bacterial Strains*

*E. coli* DH5α was used for propagation of recombinant plasmids. *E. coli* BL21 (DE3) and C43 strains were used for expression of rL7/L12 and rOmp25 proteins, respectively. *E. coli* strains were cultured using Luria–Bertani (LB) medium. Kanamycin was added to the medium at a final concentration of 50 μg/mL. *B. abortus* 544 and S19 strains were obtained from the Indian Veterinary Research Institute, Bareilly, India. *Brucella abortus* 544 was cultured in tryptic soy medium. Experiments involving *B. abortus* 544 and S19 strains were performed in a biosafety Level 3 laboratory at Jawaharlal Nehru University (JNU), Delhi, India.

### *4.2. Expression and Purification of Recombinant Proteins*

For formulation of the divalent vaccine, Omp25 and L7/L12 antigens of *Brucella abortus* were PCR amplified using gene specific primers and cloned in pET28(a) vector (Table 2). The expression of proteins was done in *E. coli*. To purify rOmp25, recombinants were grown in terrific broth until OD600 ~ 0.5–0.6, and then induction was done using 1 mM IPTG for 5 h at 37 ◦C. Further purification of rOmp25 was done from the insoluble inclusion bodies fraction, using the urea-denaturing method and on-column refolding [20]. To purify rL7/L12, recombinants were grown in LB medium containing kanamycin to OD600 ~ 0.7–0.8 followed by induction using 1 mM IPTG for 5 h at 37 ◦C. Both the proteins were affinity purified using immobilized nickel-nitrilotriacetic acid (Ni-NTA) agarose columns equilibrated in PB buffer (100 mM potassium phosphate buffer, pH 8.0) and eluted using 100–500 mM imidazole in PB. Purified proteins were analyzed by SDS-PAGE for content and purity. The dialysis of purified proteins was done against phosphate buffer saline (pH 7.4).


