**2. Results**

### *2.1. Expression and Purification of Recombinant Proteins rOmp25 and rL7*/*L12*

The recombinant proteins rOmp25 and rL7/L12 were expressed in *E. coli* C43 cells and *E. coli* BL21 (DE3) cells respectively and purified using Ni-NTA chromatography. Further, the size of the expressed recombinant proteins was verified using 12% SDS-PAGE electrophoresis (Figure 1a). The immunoreactivity of purified proteins was confirmed by immunoblotting using (anti-Omp25 + L7/L12) mice serum signifying that serum from mice immunized with Omp25 +L7/L12 consisted of antibodies specifically against its component proteins rOmp25 and rL7/L12 (Figure 1b).

Further, the virulence potential of rOmp25 and rL7/L12 was analyzed using bioinformatics analysis using VirulentPred and VaxiJen. VirulentPred is a tool used for prediction of virulent protein sequences in bacteria based on bi-layer cascade support vector machine (SVM) [33]. The SVM classifiers in this tool were trained and optimized using individual protein sequence features such as their amino acid and dipeptide composition along with position-specific iterated blast (PSI-BLAST). This tool distinguishes virulent proteins from non-virulent bacterial proteins with an accuracy of 81.8% [33]. On the basis of VirulentPred, rOmp25 and rL7/L12 were concluded to be virulent with predicted scores of 1.0411 and 0.2440, respectively (Figure 1c). In addition, we used the VaxiJen tool [34], which uses an alignment-independent approach for prediction of protective antigens. The antigen classification is

purely based on the physiochemical properties of proteins without applying sequence alignment [34], and depends on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of amino acid properties. VaxiJen results categorized Omp25 and L7/L12 as vaccine antigens with predicted scores of 0.7506 and 0.6442, respectively, at the threshold value of 0.4 (Figure 1d).

**Figure 1.** (**a**) Purification of rL7/L12 and rOmp25: SDS-PAGE gel stained with coomassie blue stain showing purification of rL7/L12 and rOmp25 recombinant proteins corresponding to 17 KDa and 25 KDa, respectively. (**b**) Immunoblotting with polyclonal sera of mice immunized with divalent vaccine (Omp25+L7/L12): The reactivity of purified proteins was confirmed by immunoblotting using anti-Omp25+L7/L12 mice serum. Negative control (lane 1; *E. coli* BL21 (DE3) cells with pET28a only), marker (lane 2) Precision Plus Protein™ Dual Color Standards, BIORAD #1610374, rOmp25 (lane 3), and rL7/L12 (lane 4). (**c**) Prediction of virulent proteins in a bacterium using VirulentPred: The sequences for *Brucella abortus* protein, Omp25 and L7/L12, have been submitted as input and their predicted scores have been calculated using VirulentPred software. (**d**) Prediction of vaccine antigens using VaxiJen: The sequences for *Brucella abortus* protein, Omp25 and L7/L12, have been submitted as input and the probability of these proteins being vaccine antigens has been predicted using VaxiJen.

### *2.2. Determination of IgG Antibody Titre upon Divalent Vaccine Immunization*

To assess the levels of IgG antibody titer generated in each of the immunized mice groups, sera was collected at day 28 and 42 post-priming and levels were estimated using Enzyme linked immunosorbent assay (ELISA). Our results revealed that immunization with Omp25+L7/L12 supported a robust anti-L7/L12 IgG response that was detectable at day 28 and remained stable until day 42. At day 28, anti-L7/L12 antibodies were observed to be higher in Omp25+L7/L12 mice compared to L7/L12 only immunized mice (*p* < 0.05; Figure 2a). At day 42, anti-L7/L12 levels were found to be similar in both L7/L12 and Omp25+L7/L12 immunized mice (approximately 6 × 10<sup>5</sup> in both). Immunization with divalent vaccine elicited a vigorous anti-Omp25 IgG response as well. Antibody levels were observed to be similar in Omp25+L7/L12 and Omp25 only immunized mice at day 28 and day 42 (Figure 2b). Therefore, the antigen alone vaccinated group generated antibodies only against a single antigen, whereas mice immunized with divalent vaccine (Omp25+L7/L12) produced antibodies against both components (rOmp25 and rL7/L12) in a cumulative manner, indicating that co-immunization of two proteins didn't hinder the immune response and supported generation of antibodies against its individual components.

**Figure 2.** IgG antibody response elicited after immunization with L7/L12, Omp25, and divalent vaccine (Omp25+L7/L12): The mice were immunized with proteins rOmp25, rL7/L12 and rOmp25+rL7/L12 followed by isolation of serum samples from tail veins on day 14, 28, and 42. Estimation of IgG antibody end point titer was done through Enzyme linked immunosorbent assay (ELISA) and data is plotted as (mean ± SD).

### *2.3. Evaluation of IgG Isotype Levels upon Divalent Vaccine Immunization*

In order to predict the Th1/Th2 bias of immune response, the relative IgG isotypes levels (IgG1, IgG2a and IgG2b) were analyzed in Omp25+L7/L12 immunized mice along with mice immunized solely with L7/L12 and Omp25. In the case of anti-L7/L12 antibodies, IgG1 levels were found to be significantly higher than IgG2a levels in the divalent vaccine as well as L7/L12 only immunized mice group, indicating a Th2 biased immune response in both (Figure 3a). Similarly, in the case of anti-Omp25 antibody levels, IgG1 levels were found to be higher than IgG2a (IgG1/IgG2a = 1.86), suggesting a Th2 biased immune response in divalent vaccine immunized mice. Interestingly, levels of IgG2a and IgG2b antibodies in Omp25+L7/L12 immunized mice were noteworthy (Figure 3b), predicting an elicitation of Th1 immune response in divalent vaccine immunized mice as well.

**Figure 3.** IgG antibody isotypes (IgG1, IgG2a, and IgG2b) elicited after immunization with rL7/L12, rOmp25, and divalent vaccine (Omp25+L7/L12): The recombinant *B. abortus* proteins rL7/L12, rOmp25, and Omp25+ L7/L12 were immunized in mice and isolation of serum samples was done from tail veins on day 42. Estimation of IgG isotype levels in serum of immunized mice was done through ELISA. The antibodies used for ELISA were horseradish peroxidase (HRP) conjugated anti-mouse IgG1, IgG2a, and IgG2b antibodies and data is plotted as (mean (OD450 ± SD)).

### *2.4. Evaluation of Protective Efficacy Conferred by Divalent Vaccine Candidate against B. abortus 544 Challenge*

The protective efficacy of the Omp25+L7/L12 vaccine candidate along with groups immunized solely with Omp25 and L7/L12 was analyzed against *B. abortus* 544 infection. Two weeks after last immunization, the immunized mice were challenged with virulent *B. abortus* 544 through intraperitoneal route. The mice were sacrificed four weeks post-infection, and bacterial colony forming units (CFU) were determined. As shown in Table 1, the level of log10 CFU per spleen at 28 days post-challenge with *B. abortus* 544 was (4.820 ± 0.18) in Omp25+L7/L12 immunized mice. Consecutively, log10 protection conferred by the Omp25+L7/L12 group was 1.98 at 28 day post-challenge as compared to (PBS + alum) immunized mice indicating that the Omp25+L7/L12 vaccine candidate was effective at eliminating pathogenic *B. abortus* 544 from a mice model. Mice immunized with Omp25 and L7/L12 alone exhibited log10 units of protection as 1.46 and 1.75, respectively at 28 days post-challenge with *B. abortus* 544 as compared to (PBS + alum) immunized mice. Overall, upon analyzing the levels of protection of the divalent vaccine candidate against *B. abortus* 544 challenge, it was found that Omp25+L7/L12 immunized mice exhibited efficacious log10 units of protection against *B. abortus* 544 challenge along with its individual components, however S19 exhibited the maximum.

**Table 1.** Bacterial proliferation in the spleen of mice immunized with rOmp25, rL7/L12, divalent vaccine candidate (Omp25+L7/L12) and control, using alum as adjuvant. The mice were infected with *B. abortus* 544 through intraperitoneal route and the splenic bacterial load was determined by plating dilutions of the splenocytes suspension on the tryptic soya agar plates followed by incubation at 37 ◦C in the presence of 5% CO2 for 48 h. Data is represented as mean ± S.D.

