Selection of Protective Epitopes for Brucella melitensis by DNA Vaccination

Animals.Specific-pathogen-free female BALB/c mice (National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD) or gamma interferon-deficient (IFN-γ^−/−) on a BALB/c background (29) at 6 to 9 weeks of age were used throughout the present study. All mice were maintained at Montana State University Animal Resource Center under pathogen-free conditions in individually ventilated cages under HEPA-filtered barrier conditions and were fed sterile food and water ad libitum. In conducting research with animals, the investigator(s) adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH publication no. 86-23, revised 1985). For challenge studies with B. melitensis strain 16M, mice were maintained under similar isolation conditions, but in the ABSL-3 facilities. All animal care and procedures were in accordance with institutional policies for animal health and well-being.

Bacterial strains and growth conditions. B. melitensis virulent strain 16M was obtained from the National Veterinary Services Laboratory, U.S. Department of Agriculture, Ames, IA. Bacteria were grown under aerobic conditions in potato infusion agar for 72 h or in brucella broth (Difco Laboratories, Detroit, MI) overnight at 37°C and 5% CO₂. For inoculation, the bacterial suspensions were adjusted spectrophotometrically to an optical density at 600 nm corresponding to 10⁴ CFU/200 μl. All experiments with live brucellae were performed in biosafety level 3 facilities. Escherichia coli strain DH5α (Life Technology, Gaithersburg, MD) was used for producing the necessary plasmid constructs. The E. coli cultures were routinely grown at 37°C in Luria-Bertani (LB) broth or agar supplemented, when required, with 50 μg of ampicillin and 10 μg of kanamycin per ml.

Construction of DNA vaccine candidates.DNA fragments of the 32 target genes from B. melitensis 16M were amplified by a PCR in which the EcoRI and XbaI or BamHI and XhoI sites were integrated into upstream and downstream primers. The Kozak sequence was introduced within the upstream DNA primer (18). These fragments were then cloned under the cytomegalovirus (CMV) promoter in the eukaryotic vector pcDNA3.1(+) (Invitrogen Corp., San Diego, CA).

The resulting plasmids (using the prefix pCMV_) were cultured in LB broth containing ampicillin (50 μg/ml) and kanamycin (10 μg/ml). Large-scale plasmid DNA isolation was performed using a Plasmid Giga Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Plasmids were finally resuspended in phosphate-buffered saline at a concentration of 1,000 μg/ml. DNA concentration and purity were determined by measuring the optical density, and the A₂₆₀/A₂₈₀ ratio was typically greater than 1.8. The recombinant plasmid construct was verified both by restriction enzyme digest and by sequencing the complete insert. In conducting work involving the use of recombinant DNA, the investigator(s) adhered to the Guidelines for Research Involving Recombinant DNA Molecules (notice, Federal Register, 5 July 1994, vol. 59, no. 127).

Production of recombinant bp26 and TF proteins. To enable the assessment of immunity to bp26 and TF, bp26 and TF gene fragments from pcDNA3.1 vector were amplified by PCR. EcoRI and ApaI sites were integrated to the 5′ and 3′ ends to ensure the fragments were in frame with the C-terminal myc and His tag epitopes of Pichia pastoris vector pPICZA (Invitrogen Corp.). These two plasmids were designated pIC-bp26 and pIC-TF, respectively. The plasmids were digested by PmeI. After precipitation and washing in 70% ethanol, the pellets were dried, resuspended in double-distilled water, and transformed into Pichia pastoris X-33 cells (Invitrogen Corp.). Cells were resuspended in 1 ml of 1 M sorbitol and incubated for 1 h at 30°C. They were then resuspended in 0.5 ml of medium (yeast extract, 10 g/liter; peptone, 20 g/liter; glucose, 20 g/liter; sorbitol, 1 M [final concentration]; agar [optional], 15 g/liter) and incubated for 2 h at 30°C. Transformants were applied to plates containing 100, 500, and 1,000 U of zeocin/ml, respectively. After 3 days, 9 colonies were selected from the 1,000-U/ml zeocin plate. Cells were lysed using glass beads (Sigma-Aldrich Chemical Co., St. Louis, MO), and their expression was examined by Western blot analysis. Proteins were transferred from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (wt/vol) polyacrylamide gel to 0.2-μm-pore-size nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were probed, first with the rabbit polyclonal His tag antiserum and then with a goat anti-rabbit IgG conjugated to horseradish peroxidase (Southern Biotechnology Associates, Inc. [SBA], Birmingham, AL). Detection of His tag fusion protein bands was achieved upon development with the substrate 4-chloro-1-naphthol chromogen and H₂O₂ (Sigma-Aldrich). The clones providing the best expression were selected and named pICbp26 and pICTF, respectively.

Large-scale expression and purification were performed as follows: X-33/pICbp26 and X-33/pICTF were grown in four flasks, each containing 500 ml of buffered minimal glycerol medium (100 mM potassium phosphate buffer [pH 6.0], 1.34% yeast nitrogen base, 4 × 10⁻⁵% biotin, 1% glycerol), in 2-liter shaker flasks for 48 h at 30°C and 200 rpm, with glycerol replenished after 24 h. Biomass was collected through centrifugation. Pellets were resuspended at a 1:2 ratio in 2-liter baffled flasks containing buffered minimal methanol media (100 mM potassium phosphate buffer [pH 6.0], 1.34% yeast nitrogen base, 4 × 10⁻⁵ % biotin, 0.5% methanol) and shaken for 24 h at 30°C 200 rpm. Pellets were collected by centrifugation, and cells were lysed using glass beads (425 to 600 nm) and acid washed (Sigma-Aldrich) in lysis buffer (300 mM NaCl, 50 mM Na₂H₂PO₄, and 10 mM imidazole [pH 7.2] containing 0.1% Triton X-100). Lysate was cleared through centrifugation and filtered through a 0.45-μm-pore-size bottle top filter (Nalge-Nunc International, Rochester, NY). Purification was accomplished by batch/gravity flow over Talon resin (BD BioSciences Clontech, Franklin Lakes, NJ). The 20-ml final bed volume was equilibrated with Talon wash-equilibration buffer (pH 7.2; 300 mM NaCl, 50 mM Na₂H₂PO₄, 10 mM imidazole) and then incubated with cell lysate for 1 h at 4°C with rotation. A 35-ml glass column was packed with lysate-resin suspension using gravity flow and washed with 10× bed volumes of Talon wash-equilibration buffer (pH 7.2). Protein was diluted with Talon elution buffer (pH 7.2; 300 mM NaCl, 50 mM Na₂H₂PO₄, 150 mM imidazole), and the pooled fractions containing the protein of interest were stored at −80°C. Protein purity was checked by SDS-PAGE/Coomassie staining and Western blot analysis with the anti-myc antibody (Invitrogen Corp.).

SDS-PAGE and Western blot analysis.SDS-PAGE and immunoblot analysis of purified bp26 and TF proteins were performed using standard procedures. For Western blot analysis, proteins were transferred from the SDS-PAGE (12% [wt/vol] polyacrylamide) gel to 0.2-μm-pore-size nitrocellulose membranes (Bio-Rad). Membranes were probed, first with a monoclonal mouse anti-myc antibody (Invitrogen Corp.) and then with a goat anti-mouse IgG conjugated to horseradish peroxidase (SBA). Detection of bp26 and TF antigens was achieved upon development with the substrate 4-chloro-1-naphthol chromogen and H₂O₂ (Sigma-Aldrich).

Immunization and colony counts.To screen the 32 constructs made in pcDNA3.1, BALB/c mice were anesthetized with inhaled isoflurane, and 300 μg of a single plasmid DNA construct plus 50 μg of CpG was injected at the anterior tibial tuberosity to ensure the vaccines were delivered to the muscle, as previously described (10). The sequence of CpG used was as follows: TCCATGACGTTCCTGACGTT (30). Vaccination was performed at weeks 0, 2, 4, and 6. For negative controls, mice were immunized with an equal amount of empty pcDNA3.1 vector plus 50 μg of CpG. At 4 weeks after the final immunization, mice were challenged intraperitoneally with 2.4 × 10⁴ CFU B. melitensis 16M in 200 μl of PBS buffer. At 4 weeks postchallenge, mice were euthanized, and the spleens were removed for CFU determinations. Spleens were Dounce homogenized in 1 ml of sterile water to completely lyse cells. Serial 10-fold dilutions in duplicate of homogenates in sterile water were grown on potato infusion agar. After incubation for 3 to 5 days at 37°C with 5% CO₂, Brucella colonies were enumerated, and the number of CFU per spleen was calculated from the dilutions. An arbitrary criterion of a reduction in splenic colonization by >0.6 log may identify potential candidates. For the repeat experiments, mice (five animals/group) were intramuscularly (i.m.) immunized with 50 μg of CpG plus 300 μg of vector, pCMVbp26, pCMVTF, or 150 μg of pCMVbp26 and 150 μg of pCMVTF; for the third experiment, mice (five animals/group) were i.m. immunized with 50 μg of CpG plus 80 μg of vector or 40 μg of pCMVbp26 and pCMVTF (total 80 μg of DNA).

ELISA.To determine induced antibodies to bp26 and TF, an enzyme-linked immunosorbent assay (ELISA) was used to measure immune serum IgG, IgG1, IgG2a, and IgG2b levels. Purified bp26 and TF proteins (5 μg/ml) were used to coat Maxisorp microtiter plates (Nunc, Roskilde, Denmark) at 100 μl/well overnight at 4°C. Plates were washed four times in a wash buffer (Tris-buffered saline [pH 7.4] with 0.05% Tween 20) and blocked with 2% milk in Tris-buffered saline for 2 h at 37°C and then incubated with serial dilutions of the sera from mice for 3 h at room temperature, and washed three times. Horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b antibodies (SBA) were used for detection. After 90 min of incubation at 37°C and a washing step, the specific reactivity was determined by the addition of an enzyme substrate, ABTS [2,2′azinobis(3-ethylbenzthiazolinesulfonic acid)] diammonium (Moss, Inc., Pasadena, CA) at 100 μl/well. The absorbance was measured at 415 nm on a Kinetics Reader model EL312 (Bio-Tek Instruments, Winooski, VT). Endpoint titers were defined as the highest reciprocal of dilution of sample giving an optical density at 415 nm of 0.100 U above negative controls after 1 h of incubation at 25°C.

Cytokine ELISPOT.Groups of BALB/c or IFN-γ^−/− mice (five to ten/group) were euthanized 4 weeks after the last immunization to collect spleens. Splenic lymphocytes were isolated by conventional methods using Dounce homogenization (28), yielding >95% viability using trypan blue exclusion. Total splenic mononuclear cells (5 × 10⁶/ml) were resuspended in complete medium (CM; RPMI 1640 [Gibco-BRL/Life Technologies, Grand Island, NY], 10% fetal bovine serum [Atlanta Biologicals, Atlanta, GA], 10 mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate, 100 U of penicillin/ml, 100 μg of streptomycin/ml) and restimulated with 20 μg of recombinant bp26 or TF/ml in the presence of 10 U of human IL-2 (PeproTech, Inc., Rocky Hill, NJ)/ml for 2 days at 37°C. Cells were washed and resuspended in CM. Stimulated lymphocytes were then evaluated by IFN-γ-, IL-4-, IL-5-, IL-6-, and IL-10-specific enzyme-linked immunospot (ELISPOT) assays as previously described (28). To control for nonspecific reactivity against bp26 and TF, naive BALB/c lymphocytes were cultured as described above with 20 μg of bp26 or TF/ml in the presence of 10 U of human IL-2/ml for 2 days.

Statistical analysis.An analysis of variance followed by Tukey's method was used to evaluate differences between variations in splenic colonization, antibody titers, and cytokine production levels. The P value for statistical differences between plasmid vector and vaccines and the performance of these vaccines stimulating B and T lymphocyte responses was discerned at the 95% confidence interval.

Evaluation of B. melitensis 16M genome to obtain vaccine targets for cloning into eukaryotic expression plasmid.Since the B. melitensis 16M genome sequence was published in 2001 (12), the entire 16M genome was blasted against the National Center for Biotechnology Information database for proteins with a sequence of >100 amino acids and designated immunogenic. If the annotation results matched these selection criteria, the gene coding for this protein was subsequently cloned into the pcDNA3.1(+) plasmid as a possible DNA vaccine candidate.

A total of 32 genes or open reading frames (ORFs) were termed as being antigenic or immunogenic as indicated by the BLAST search. These genes were amplified by PCR and cloned into pcDNA3.1 plasmid. The ribosomal gene L7/L12, previously known to be a protective antigen for Brucella abortus (19), was also inserted into pcDNA3.1(+) to use as a positive control (Table 1). The empty pcDNA3.1 vector was used as a negative control.

Evaluation of protective efficacy by 32 DNA vaccines.Individual BALB/c mice were vaccinated i.m. four times with a single DNA construct, and two mice were immunized with vector only as negative controls. After 4 weeks, mice were challenged with 2.4 × 10⁴ CFU of B. melitensis 16M, and 4 weeks later, splenic CFU levels were enumerated (Table 2). Compared to negative controls, TF provided the greatest protection, with no detectable (<100 CFU) bacteria isolated from the spleen. Bp26 also showed some efficacy as evidenced by a >1-log reduction in colonization. Other genes of potential interest were the genes for omp17, nlpD, integrase, dihydrolipoamide succinyltransferase, preC, and 19-kDa periplasmic protein since these showed greater reduction in colonization than the remaining 24 vaccine candidates.

Confirmation of protective efficacy of bp26 and TF.To validate the protection data from Table 2, subsequent studies were performed. BALB/c mice were immunized i.m. with either bp26, TF, or both DNA vaccines as previously described. It was evident that the TF and bp26 DNA vaccines were each significantly protective (P = 0.011 and P = 0.036) against B. melitensis 16M challenge (Fig. 1A), whereas coimmunization with both vaccines afforded greater protection (P = 0.016) by 20-fold reduction in splenic colonization. To test whether a lesser dose of the combined DNA vaccines would be efficacious, a 40-μg dose of each vaccine was used according to the dosing regimen described above; the vaccines by themselves were not tested. Again, a reduction in splenic colonization by B. melitensis 16M was observed (P = 0.029) showing that the combined vaccines afforded the best protection (Fig. 1B). To ascertain potential efficacy of the remaining vaccine candidates, one group of mice was dosed with the remaining six vaccines plus bp26; this combination failed to show any efficacy so these vaccines were not pursued any further (data not shown).

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FIG. 1.

BALB/c mice vaccinated with bp26 or TF DNA vaccines showed reduced splenic colonization following challenge with 2.4 × 10⁴ CFU of B. melitensis 16M. (A) Groups of mice (five/group) were immunized on days 0, 14, 28, and 42 with 300 μg of pcDNA3.1 vector, pCMVTF, or pCMVbp26 or with 150 μg of pCMVTF and pCMVbp26, and each group received a 50-μg CpG/immunization. At 4 weeks after the last immunization, mice were challenged intraperitoneally with B. melitensis 16M. Compared to the group immunized with pcDNA3.1 vector, the pCMVTF (P = 0.011) and pCMVbp26 (P = 0.036) immunized groups had significantly less splenic colonization, and the combination of pCMVTF and pCMVbp26 (P = 0.016) had the least colonization. (B) To ascertain whether a lesser dose of pCMVTF and pCMVbp26 would still be efficacious (five mice/group), 40 μg of each vaccine was given i.m. on days 0, 14, 28, and 42 and compared to mice (five/group) dosed with 80 μg of pcDNA3.1 vector. As above, each group received 50 μg of CpG/immunization. Compared to the vector-immunized group, the combination of pCMVTF and pCMVbp26 (P = 0.029) showed reduced colonization. These results show that a subunit vaccine for B. melitensis is possible.

Immunization with bp26 and TF DNA vaccines induces elevated IgG to these B. melitensis proteins.In order to investigate host responses to bp26 and TF subsequent to DNA vaccination, bp26 and TF cDNAs were cloned into a yeast (Pichia pastoris) expression system, and recombinant proteins were analyzed by SDS-PAGE (Fig. 2A and B, lane 1) and by Western immunoblotting (Fig. 2A and B, lane 2). Both the SDS-PAGE and the Western blot results demonstrated that the purified recombinant proteins had the expected molecular sizes: bp26 at 29.0 kDa (26.6 kDa plus C-terminal myc tag 2.5 kDa) and TF at 57.0 kDa (54.5 kDa plus C-terminal myc tag 2.5 kDa).

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FIG. 2.

bp26 and TF expression analysis. pICbp26 and pICTF transformed Pichia pastoris in buffered minimal glycerol media were induced by methanol (0.5%) for 24 h to express the recombinant proteins. (A and B) Expression of bp26 (A) and TF (B) was verified by SDS-PAGE (lane 1) and Western blotting with an anti-myc monoclonal antibody (lane 2) showing that bp26 had the expected molecular mass of 29 kDa (26.6 kDa plus myc tag [2.5 kDa]) and TF had the expected molecular mass of 57 kDa (54.5 kDa plus myc tag [2.5 kDa]).

To determine the type of antibody responses elicited by these two protective antigens, BALB/c mice were i.m. immunized with pCMVbp26 and pCMVTF with CpG, and the control group was immunized with pcDNA3.1. plus CpG. Vaccination was performed on days 0, 7, and 14. At 6, 8, and 10 weeks after the final immunization, serum samples were obtained to assess antigen-specific endpoint titers (Fig. 3). The results showed the pCMVbp26-vaccinated mice elicited a peak IgG titer of 2¹⁶; mice vaccinated with pCMVTF showed a lower IgG titer of 2^13.8, which peaked early at week 6. No detectable titers were observed for the vector immunized mice. Evaluation of anti-TF and anti-bp26 IgG subclass endpoint titers showed no significant differences between IgG1, IgG2a, and IgG2b subclass responses (Fig. 3C and D).

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FIG. 3.

Immunization with DNA vaccines for bp26 and TF elicits modest serum IgG antibody titers. BALB/c mice (five/group) were i.m. immunized with both pCMVbp26 and pCMVTF plus CpG on days 0, 7, and 14, and serum IgG anti-bp26 and TF titers were measured by standard ELISA methods on weeks 6, 8, and 10. (A) The IgG anti-bp26 titers peaked between weeks 6 and 8 after primary immunization, and (B) IgG anti-TF titers peaked at week 6 after primary immunization, but with less intensity. Control mice immunized with the control vector, pcDNA3.1 plus CpG, failed to elicit antibodies to bp26 or TF. IgG1, IgG2a, and IgG2b anti-bp26 (C) and anti-TF IgG (D) subclass responses were not significantly different. IgG subclass responses were determined using serum samples from week 6.

Immunization with bp26 and TF DNA vaccines induces a mixed T helper (Th) cell response.Since the IgG subclass profile is a reflection of the types of Th cells simulated, our results suggested that i.m. DNA vaccination induced a mixed Th1 and Th2 cell immunity. To confirm what the supportive Th cells induced, a cytokine-specific ELISPOT assay was conducted. BALB/c or IFN-γ-deficient (IFN-γ^−/−) mice (on a BALB/c background) were i.m. immunized with either pCMVbp26 or pCMVTF as described above and, 4 weeks after the last immunization, spleens from individual mice were harvested. Whole splenic cells were cultured with either bp26 or TF for 2 days and then evaluated for IFN-γ, IL-4, IL-5, IL-6, and IL-10 secretion by the ELISPOT method (Fig. 4). In BALB/c mice, both bp26 and TF were supported by IFN-γ (Fig. 4) compared to unstimulated cultures (P < 0.001 and P = 0.009, respectively). Although TF did not stimulate any other Th2-type cytokines, bp26 produced significant increases in IL-4 (P = 0.020), IL-5 (P = 0.034), and IL-6 (P < 0.001), but not IL-10 (Fig. 4). Since IFN-γ^−/− mice were unable to produce IFN-γ, only increases in the measured Th2-type cytokines were observed. Significant increases were observed in IL-4 (P < 0.001 and P = 0.002), IL-5 (P < 0.001), and IL-6 (P < 0.001) after restimulation with bp26 or TF, respectively, compared to unstimulated cultures (Fig. 4). A slight, but significant increase (P = 0.021) in IL-10 was obtained upon restimulation with only TF. The recombinant bp26 and TF proteins failed to stimulate naive lymphocytes (Fig. 4). Differences in the magnitude of the cytokine responses were also observed between IFN-γ^+/+ and IFN-γ^−/− mice (Fig. 4). Aside from the differences in IFN-γ in response to three proteins (P < 0.001), IL-4 was enhanced in IFN-γ^−/− mice against bp26 (P = 0.003) and TF (P < 0.001); IL-5, bp26 (P = 0.002) and TF (P = 0.021); and IL-6, TF (P = 0.015). Thus, these studies confirm that a mixed Th-type response was obtained to bp26 and TF.

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FIG. 4.

Serum antibody responses were supported by mixed Th1 and Th2 cells for bp26 and Th1 cells for TF. Whole splenic cells isolated from BALB/c or IFN-γ^−/− mice immunized with both bp26 and TF were cultured with or without 20 μg of recombinant bp26 or TF for 2 days, and then cells were analyzed for production of IFN-γ, IL-4, IL-5, IL-6, and IL-10 by the ELISPOT method. In IFN-γ^+/+ mice, IFN-γ, IL-4, IL-5, and IL-6 were induced against bp26, whereas only IFN-γ was induced against TF. In the absence of IFN-γ, the Th2 cytokines compensated, as evidenced by increased production of IL-4, IL-5, and IL-6 upon restimulation with bp26 or TF, and a slight induction of IL-10 was observed against TF only. As a control, naive BALB/c lymphocytes remained unstimulated when cocultured with recombinant bp26 or TF (depicted as dashed line). Thus, IFN-γ supports antibody responses to both bp26 and TF. The data depict results from five individual mice ± the standard error of the mean, and significant differences were determined between bp26- or TF-restimulated cultures and unstimulated cells: *, P < 0.001; **, P ≤ 0.009; ***, P < 0.05.