Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

Bacterial strains and growth condition

A smooth virulent B. abortus biovar 1 strain of B. abortus 544 was kindly provided by Animal, Plant and Fisheries Quarantine and Inspection Agency in Korea and Escherichia (E.) coli DH5α cells was purchased from Invitrogen (USA). B. abortus was routinely cultured overnight in Brucella broth (BD Biosciences, USA) at 37℃ in a gyratory shaker (Waver Digital Platform; VWR International, USA) at 10 × g. When needed, solid medium was made by supplementing Brucella or Luria-Bertani (LB) broth (Becton Dickinson, USA) with 1.5% (w/v) agar (Takara, Japan). E. coli DH5α cells were used for producing the necessary plasmid constructs. E. coli cultures were routinely grown at 37℃ in LB broth or agar supplemented with 100 µg/mL of ampicillin (Sigma, USA).

rOMP expression

Total genomic DNA was prepared from B. abortus. Briefly, B. abortus cells were cultured at 37℃ overnight in Brucella broth with shaking. Next, 5 mL of the culture were collected and genomic DNA from the cells was recovered with a bacteria genomic DNA purification kit (iNtRON, Korea). The B. abortus Omp28 gene was amplified by PCR with the following primer pair: 5'-GATC GGA TCC AAC ACT CGT GCT AGC AAT TTT-3' (BamHI site underlined) and 5'-GATC AAG CTT TTA CTT GAT TTC AAA AAC GAC-3' (HindIII site underlined). The amplified DNA was digested with appropriate restriction enzymes (BamHI and HindIII; Takara, Japan) and ligated into a pMAL vector (New England Biolabs, USA). The recombinant plasmid was then used to transform the E. coli DH5α host cells. An exponential-phase culture of a rOmp28 clone confirmed in ampicillin-containing media was spread onto LB agar plates containing isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM; Amresco, USA) and ampicillin (100 g/mL).

Purification of rOMP protein

One liter of LB broth supplemented with 100 µg/mL of ampicillin was inoculated with 10 mL of an overnight culture of bacteria expressing the fusion plasmid. IPTG was then added at a final concentration of 0.3 mM and the culture was further incubated at 37℃ for 2 h. Bacterial cells were harvested by centrifugation at 2,560 × g for 20 min. The supernatant was then discarded, and the cells were resuspended in 50 mL column buffer (20 mM Tris HCl, 200 mM NaCl, and 1 mM EDTA, pH 7.4). The sample was frozen at -70℃ and thawed in cold water three times. The cells were then was sonicated (Bandelin electronic, Germany) at 10,000 Hz in an ice-water bath and centrifuged at 5,200 × g for 30 min to collect the supernatant. The supernatant was diluted with column buffer (1 : 5) and loaded into maltose resin (Bio-Rad Laboratories, USA) according to the manufacturer's instructions. The purified protein was stored at -20℃.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting

SDS-PAGE was performed according to previously described methods [11]. The purified rOmp28 was boiled for 10 min at 100℃ and diluted in 2× Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris HCl, pH 6.8). After electrophoresis, the separated proteins were transferred onto Immobilon-P membranes (Milipore, USA) in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) with a constant current of 2 mA/cm² for 30 min using a semi-dry electroblot assembly (Bio-Rad, USA). The membrane was blocked with 1% bovine serum albumin (Sigma, USA) for 1 h at 4℃, washed three times with 0.05% PBS-Tween 20, and incubated with Brucella-positive mouse sera (produced from mouse immunized by virulent B. abortus, 1 : 1,000 dilution) in blocking buffer at 4℃ for 2 h. The membrane was then washed with 0.05% PBS-Tween 20 and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1 : 10,000 dilution; Sigma, USA) in blocking buffer for 1 h at 4℃, and finally washed with 0.05% PBS-Tween 20. The proteins were detected with a luminol-coumaric acid-H₂O₂ detection solution (iNtRON, Korea) and exposure to X-ray film (Fuji, Japan).

Immunization and bacterial challenge in mice

Six-week-old female BALB/c mice (Japan SLC, Japan), two groups of five BALB/c mice each were immunized intraperitoneally with rOmp28 (100 µg/mL) or PBS in incomplete Freund's adjuvant (IFA) (Sigma, USA) on days 0, 14, and 35. Serum samples were obtained from all animals 14, 35, and 49 days after the first immunization via retro orbital bleeding. Serum titers of immunoglobulin G1 and IgG2a isotypes specific for rOmp28 were determined with an ELISA (BioTek, USA) according to the manufacturer's instructions. The cutoff value for the assay was calculated as the mean specific optical density for samples from nonimmunized mice diluted 1 : 100 as previously described [27].

To test the protective effects of rOmp28, mice were challenged with approximately 10⁴ colony forming unit (CFU) of B. abortus 14 days after the last immunization. Ten days post-infection, all mice were scarified and the spleens were removed, weighed, and homogenized (Wisd homogenizer; Witeg, Germany) in saline. The homogenates were serially diluted (1 : 100~1,000) with PBS and plated on Brucella agar and then incubated for 3 days at 37℃. The number of CFUs for each spleen sample was determined to assess the virulence of the bacteria in each group of animals.

Statistical analysis

The results for each of experiment are expressed as the mean ± SD. Student's t-test was used to compare the groups. p-values < 0.05 were considered significantly different.

PCR amplification and immunoreactivity of rOmp28

A specific band (753 bp) corresponding to B. abortus Omp28 was amplified by PCR. The PCR-amplified gene fragment encoding Omp28 was integrated into the pMAL expression vector and used to transform competent E. coli cells. The cloned product sequence was confirmed and found to completely match that of the reported Omp28 gene sequence for B. abortus (Genbank accession No. NC_007618.1). E. coli harboring the Omp28 plasmid were grown in LB medium, and 1 mM IPTG was used to induce Omp28 protein expression.

The protein product of the cloned Omp28 gene in the pMAL expression system was purified and was approximately 28 kDa (Fig. 1A). To evaluate rOmp28 protein immunoreactivity, Western blotting was performed (Fig. 1B). Our results showed that the purified rOmp28 strongly reacted with Brucella-positive mouse serum (1 : 1,000 dilution).

Fig. 1
Expression of recombinant Brucella (B.) abortus Omp28 (rOmp28) via a maltose fusion protein (pMAL) expression system. Total bacterial cell lysate proteins were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue (A). The proteins were then transferred to a membrane and incubated with Brucella-positive mouse serum (B). pMAL (arrowheads) and rOmp28 (arrows) are indicated. M: marker, Lane 1: pMAL, Lane 2: rOmp28.

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IgG1 and IgG2a responses to rOmp28 immunization

To test the protective effect of rOmp28, two groups of mice were immunized with purified rOmp28 protein or PBS with IFA on days 0, 14, and 35. Blood was collected from both groups 14, 35, and 49 days after the first immunization, and the IgG1 and IgG2a titers were determined. Throughout the entire experiment, the mean log titers of both IgG1 and IgG2a were significantly higher for the rOmp28-immunized mice compared to the animals injected with PBS (Figs. 2A and B). Our results clearly indicated that administration of rOmp28 increased both humoral and cell mediated immune response by inducing IgG1 and IgG2a production.

Fig. 2
Kinetics of the immune response elicited by immunization with rOmp28. The IgG1 (A) and IgG2a (B) log titers were determined by ELISA. Significant differences between the rOmp28-immunized and control (PBS-treated) mice are indicated by asterisks (^*p < 0.05 and ^***p < 0.001).

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Protection of rOmp28 against B. abortus challenge

To analyze the ability of rOmp28 to protect against infection with virulent B. abortus, an in vivo protection study was performed in BALB/c mice (two groups with five mice each). In this experiment, protection was defined as a significant reduction (p < 0.05) in the mice receiving the vaccine when compared to animals injected with PBS. Mice treated with PBS developed splenomegaly that was enlarged 2~3 times its normal size as a consequence of the host inflammatory response, but this was not observed in the rOmp28-immunized group (Fig. 3). Vaccine efficacy was calculated by comparing the log units of bacterial burden in the spleens of the PBS-treated or rOmp28-immunized mice after B. abortus challenge. The mean log titers were 5.49 ± 0.49 for the PBS-treated animals and 2.89 ± 0.17 for the rOmp28-immunized (Fig. 4). These finding showed that immunization with rOmp28 conferred protection against challenge with virulent B. abortus.

Fig. 3
Spleen weight of mice treated with PBS or immunized with rOmp28 and infected intraperitoneally with B. abortus. Spleen weights of the mice 10 days post-infection are shown. Data are presented as the mean ± SD (n = 5 per group). Spleen weight differed significantly between the rOmp28-immunized group and control (PBS-treated) animals as indicated by asterisks (^**p < 0.01).

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Fig. 4
Bacterial proliferation in the spleen of mice treated with PBS or immunized with rOmp28 and infected intraperitoneally with B. abortus. The numbers of viable bacteria recovered from spleens of the mice 10 days post-infections are shown. Data are presented as the mean ± SD (n = 5 per group). Bacteria proliferation differed significantly between the rOmp28-immunized group and control (PBS-treated) animals as indicated by asterisks (^**p < 0.01).

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