Substantial Protective Immunity Conferred by a Combination of Brucella abortus Recombinant Proteins against Brucella abortus 544 Infection in BALB/c Mice
Gyeongsang National University, Republic of KoreaCorrespondence to:
J. Microbiol. Biotechnol. 2019; 29(2): 330-338
Published February 28, 2019
Copyright © The Korean Society for Microbiology and Biotechnology.
Recent advances in the development of brucellosis vaccine are still underway. The availability of current live attenuated vaccines for animals such as RB51 and
Preliminary evaluation in the selection of potential
In this study, we evaluated the protective efficacy of a combination of five selected
Materials and Methods
Wild-type strains derived from
Recombinant Expression and Protein Purification
The sequences of the five
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis
Induced cells were lysed and purified products were boiled at 100°C for 5 min in 2x SDS buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue and 0.125 M Tris HCl, pH 6.8) subjected to SDS-PAGE [19, 20]. Following gel electrophoresis, separated proteins were subjected to western blot analysis. Proteins were transferred to Immobilon-P membranes (Milipore, USA) emerged in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, pH 8.3) for 30 min using ATTO Semi-dry transfer machine (WSE-7210, Japan). Membranes were blocked in 5% skim milk at room temperature (Difco, USA) for 30 min, washed with 0.05% Tween-20 (PBS-T) and incubated with
Immunization and Bacterial Challenge
The protective effect of a combination of recombinant proteins; SodC, RibH, Ndk, L7/L12 and MDH was evaluated. Twenty, ten- week-old female BALB/c mice (Japan SLC, Japan) were allocated to four groups. All groups of mice were intraperitoneally injected with 1:1 volume ratio of recombinant protein and incomplete Freund’s adjuvant (IFA) (Sigma) in a total volume of 200 μl. The groups of five mice each were immunized IP with PBS, maltose- binding protein (MBP) (100 μg), RB51 (1 × 106 CFU) or a combination of five
The method of handling and sacrifice conducted in this experiment was approved by the Animal Ethical Committee of Gyeongsang National University (Authorization Number GNU- 170331-M0017).
Cytokine and Humoral Immune Responses
The levels of IL-12p70, IL-10, IFN-γ, TNF, MCP-1, and IL-6 in sera were determined by cytometric bead array (BD CBA Mouse Inflammation Kit, USA) and analyzed using a FACS Calibur flow cytometer (BD Biosciences, USA). On the other hand, IgG1 and IgG2a isotypes were measured through indirect ELISA. Briefly, purified proteins were diluted in coating buffer (carbonate buffer; pH 9.6) comprising 1.5 μg/ml of each protein, coated into a 96- well plate (Maxibinding, SPL Life Sciences, Korea) in 100 μl per well followed by overnight incubation at 4°C. The wells were washed with 0.5% PBS-T and blocked with 5% skim milk in PBS-T at room temperature for 2 h. The wells were then incubated with sera in blocking buffer at dilutions reaching cut off values. The wells were washed and incubated with HRP-conjugated IgG1 or IgG2a (Abcam, USA) for 1 h. Finally, the wells were washed and O-phenyldiamine (OPD) was added into the wells and the absorbance was read after 15 min at 450 nm through an ELISA reader (Biotek, Korea). Cut-off value was computed as the mean specific OD plus standard deviation for non-immunized mice diluted at 1:100 . Titers were determined as the reciprocal value of the dilution that yields an absorbance higher than the cut off value.
Bacterial Clearance Efficiency
The protective effect of the combined vaccine was evaluated as previously reported . Mice were sacrificed via cervical dislocation at two weeks post-infection. Spleens were collected, weighed and homogenized in PBS. Homogenized spleens were serially diluted, plated on Brucella agar and incubated at 37°C for 3 days. Log protection was computed as the mean log10 CFU of PBS group minus Log10 CFU of the vaccinated group. Finally, the log10 (number of CFUs/g) was calculated.
The results for each experiment are expressed as the mean ± SD. One-way ANOVA program was utilized in all assays.
Protein Purification and Immunoreactivity of Recombinant Proteins
After induction and purification steps, purified proteins were analyzed via SDS to check for target molecular masses. The approximate molecular masses for recombinant proteins; SodC, RibH, Ndk, L7/L12, and MDH were approximately 68.1, 67.3, 65.27, 62.5, and 73 kDa, respectively (Fig. 1A). Immunoblot analysis showed immunoreactivity of rSodC, rRibH, rNdk, rL7/L12, and rMDH with both
Expression and immunoreactivity of purifiedSDS-PAGE analysis of purified proteins stained with Coomasie Brilliant Blue (A) Immunogenicity of each recombinant protein against B. abortusrecombinant proteins. Brucella-positive mouse sera (B) or -negative mouse sera (C). B. abortusdot blots are provided as reference for determining molecular weight.
Combined Recombinant Vaccine Engendered Significant Protection against
The rate of infection was assessed by measuring CFU in the spleen two weeks post-infection. The mean log10 CFU of the spleen of the combined protein-immunized group was significantly reduced than with the PBS (
Degree of protection conferred by the administration of combined recombinant proteins against a virulent strain ofProtection log unit in the combined protein-immunized group (A). Reduction in the weight of the spleen in the combined immunized group (B). rSodC+rRibH+rNdk+rL7/L12+rMDH: Combination of B. abortus recombinant proteins (Cu-Zn superoxide dismutase, riboflavin synthase subunit beta, nucleoside diphosphate kinase, 50S ribosomal protein and malate dehydrogenase). Statistical differences are analyzed from the values of the PBS (non-immunized group). Asterisks indicate significant difference: * B. abortus. p <0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.
Combined Antigens Induce Substantial Th1 Immune Response
The production of (Th1)-type, cell-mediated immunity cytokines that is responsible for anti-
IFN-γ concentration (pg/ml) in the sera of mice challenged withrSodC+rRibH+rNdk+rL7/L12+rMDH: Combination of B. abortus recombinant proteins (Cu-Zn superoxide dismutase, riboflavin synthase subunit beta, nucleoside diphosphate kinase, 50S ribosomal protein and malate dehydrogenase). Asterisks indicate significant difference: * B. abortusinfection at 3- and 4-week time point after first immunization. p<0.05, ** p< 0.01, *** p< 0.001.
IL-10 concentration (pg/ml) in the sera of mice challenged withrSodC+rRibH+rNdk+rL7/L12+rMDH: Combination of B. abortus recombinant proteins (Cu-Zn superoxide dismutase, riboflavin synthase subunit beta, nucleoside diphosphate kinase, 50S ribosomal protein and malate dehydrogenase). Asterisks indicate significant difference: * B. abortusinfection at 3- and 4-week time point after first immunization. p<0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.
Humoral Immune Response in Combination Protein Immunized Mice
The presence of rSodC, rRibH, rNdk, rL7/L12, and rMDH-specific antibodies in the sera of mice collected at three and four weeks post-immunization was analyzed through ELISA. As shown in Figs. 5A andombined antigens elicited significant levels of both IgG1 (
Humoral immune response in the combined protein-immunized group.The levels of IgG1 (A) and IgG2a (B) elicited by the combined protein-immunized group after 3- and 4-week time points after the first immunization were determined by ELISA. Mice infected with 1 × 106 RB51 were included as positive control. rSodC+rRibH+rNdk+rL7/ L12+rMDH: Combination of B. abortus recombinant proteins (Cu-Zn superoxide dismutase, riboflavin synthase subunit beta, nucleoside diphosphate kinase, 50S ribosomal protein and malate dehydrogenase). Data are represented as the mean ± SD ( n= 5 mice/group). Asterisks indicate significant difference: * p <0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.
The search for the ideal vaccine will continue to be the primary goal in the effort to eradicate brucellosis as vaccination still remains the most efficient method in the control of this infectious agent. RB51, a widely used live attenuated vaccine, offers major advantages such as non- interference in the diagnosis of brucellosis, however it also causes residual virulence in both animals and humans . Several antigenic proteins such as periplasmic, cytoplasmic, inner and outer membranes have been previously identified according to previous studies.
It is our goal in this study to evaluate the potential of these single immunogenic recombinant proteins hypothesized to confer superior protection against
In summary, the combination of these five antigens predominantly boosted cellular immune responses and humoral immunity in a mouse model through upregulation of IFN-
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI16C2130).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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