*4.3. Immunoblotting*

For immunoblotting, the recombinant proteins were resolved by 12% SDS-PAGE followed by electroblotting onto nitrocellulose membrane. The membrane was further blocked using 3% BSA followed by incubation with anti-Omp25+L7/L12 antibody (1:5000 dilution, raised in mice) for 1 h. After subsequent washing, binding specificity was checked using AP-conjugated goa<sup>t</sup> anti-mouse IgG antibody (Catalog no. sc-2047, Santa Cruz Biotechnology, USA) [52–54].

### *4.4. Immunization of Respective Proteins in Mice*

Four to six week old female BALB/c mice (inbred) were obtained from the National Centre for Laboratory Animal Sciences, Hyderabad, India. Recommendations from the Institutional Animal Ethics and Biosafety Committee were regularly followed during mouse experiments. In brief, mice were caged under sterile conditions in micro-isolators, fed with pathogen-free food and water *ad libitum* during consecutive immunizations. Once infected with *B. abortus* 544, mice were maintained at the BSL-3 animal facility of JNU for evaluation of protective efficacy.

For immunization of rL7/L12 and rOmp25, the optimized dose of each antigen was considered as mentioned in earlier reports [1,20]. Briefly, mice were grouped and immunized through the intraperitoneal route, either with Omp25 (30 μg) or L7/L12 (40 μg) alone or in combination as a divalent vaccine candidate with alum as an adjuvant. Two boosters were administered at regular intervals of 2 weeks, and 1X PBS with alum and *B. abortus* S19 immunized mice groups were taken as controls. For prime immunization and subsequent booster immunization, 100 μl emulsion of the required antigen and alum in 1X PBS was injected in each mouse. The blood was collected from each mouse on day 0, 14, 28, and 42 from tail veins and sera was extracted through centrifugation at 15,600 *g* for 20 min, followed by storage at −80 ◦C for further analysis.

### *4.5. Elucidation of End-Point Antibody Titer*

An enzyme-linked immunosorbent assay (ELISA) was used to analyze serum antibody titer. In brief, 96-well microtiter plates (NuncMaxiSorp) were coated overnight with 500 ng/well of capture antigen (rOmp25 or rL7/L12) in PBS at 4 ◦C. The plates were washed three times using PBST (PBS with 0.1% tween 20) followed by blocking using 2% BSA in PBS for 2 h at 37 ◦C. The antibody titer in the sera of respective antigen immunized mice along with the divalent vaccine immunized mouse group was assessed by priming dilutions of the same, in triplicates, at 37 ◦C for 1 h. Washing of the plates was done using PBST followed by addition of horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (Catalog no. sc-2005, Santa Cruz Biotechnology, USA) at 1:10,000 dilution for 1 h at 37 ◦C [53,54]. The plates were further incubated with OptEIA TMB substrate (BD Biosciences, USA) for calorimetric assay and the reaction was stopped using 1N HCl. Absorbance of the plate was measured at 450 nm through Tecan's Sunrise absorbance microplate reader. End point titer was evaluated as the reciprocal of highest dilution giving absorbance greater than the threshold value. Threshold value was calculated as the mean of absorbance plus three times standard deviation of 1:1000 dilution of the control group (PBS + alum).

### *4.6. Analysis of IgG Isotypes in Immunized Mice*

The IgG isotypes (IgG1, IgG2a and IgG2b) were detected in immunized mice using ELISA as described above. For secondary antibodies, anti-mouse IgG1-HRP (Catalog no. sc-2060), anti-mouse IgG2a-HRP (Catalog no. sc-2061) and anti-mouse IgG2b-HRP conjugated antibodies (Catalog no. sc-2062) (raised in goat; Santa Cruz Biotechnology, USA) were used and absorbance at 450 nm was measured [53].

### *4.7. Evaluation of Protective <sup>E</sup>*ffi*cacy of Vaccine Candidate*

Two weeks after the final booster immunization (day 42), mice groups immunized with PBS, rOmp25, rL7/L12, and divalent vaccine candidate (rOmp25+rL7/L12) were challenged with 2 × 10<sup>5</sup> cells of *B. abortus* 544 through the intraperitoneal route. *B.abortus* S19 was injected on day 0 in respective group, and challenge was done after 21 days with 2 × 10<sup>5</sup> cells of virulent *B. abortus* 544. After 4 weeks of infection, mice from each group were euthanized through cervical dislocation. Their spleen was extracted under sterile conditions and finally homogenized in PBS using probe homogenizer. For CFU count, various dilutions of the spleen homogenate were prepared and plated on tryptic soya agar followed by incubation at 37 ◦C for 48 h in the presence of 5% CO2. Total splenic load was calculated and represented as Log10 CFU mean ± standard deviation (SD). Log10 units of protection were determined by calculating the difference between the log10 CFU of PBS injected group (control) and vaccinated group.

### *4.8. Statistical Analysis*

The results are represented as mean ± SD and are reported as data of three di fferent sets of experiments. The statistical significance in antibody titer was calculated using two-tailed Student's t-test. (\* represents *P* < 0.05; \*\* represents *P* < 0.01; \*\*\* represents *P* < 0.001, \*\*\*\* represents *P* < 0.0001).

**Ethical statement:** All mice experiments were performed while abiding by the rules of Institutional Animal Ethics Committee (IAEC), Jawaharlal Nehru University, New Delhi, India guidelines. All experiments involving virulent *Brucella abortus* 544 and *Brucella abortus* S19 strain have been performed in Biosafety level-3 (BSL-3) facility.

**Author Contributions:** Each author has made valuable contributions to this work. Conceptualization, S.G. and R.B.; methodology, S.G., RB; validation, S.G., S.M., V.K.S., S.A. and R.B.; formal analysis, S.G., V.K.S., S.A.; investigation, R.B.; resources, R.B.; data curation, S.G.; writing—original draft preparation, S.G.; writing—review and editing, S.G., S.M., V.K.S., S.A., R.B.; visualization, S.G., S.M., V.K.S. and S.A.; supervision, R.B.; project administration, R.B.; funding acquisition, R.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** We would like to thank the Department of Biotechnology, Government of India for providing us with financial support and specifically, we acknowledge members of "Network Project on Brucellosis" for their valuable guidance and support. S. Gupta is a Senior Research Fellowship recipient from the Council of Scientific & Industrial Research (*CSIR*), Government of India (Grant no. 09/263(1085)2015-EMR-1).

**Conflicts of Interest:** The authors declared no conflict of interest.
