Selection of protective epitopes for Brucella melitensis by DNA vaccination

Abstract

The Brucella melitensis 16M genome was examined for proteins in excess of 100 amino acids and for immunogenicity-associated genes. One subset of 32 annotated genes or open reading frames was identified, and each of these were cloned into the eukaryotic vector pcDNA3.1. Purified recombinant plasmids were used to intramuscularly (i.m.) immunize BALB/c mice. After challenge with B. melitensis 16M strain, two protective antigens were found: the periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). Protective efficacy was confirmed with DNA vaccines for these two B. melitensis proteins and, when combined, protection against wild-type challenge was significantly enhanced. Both proteins were found to be immunogenic since elevated serum immunoglobulin G (IgG) antibodies without a specific IgG subclass bias were induced subsequent to i.m. DNA immunization. Antigen-restimulation assays revealed that bp26 and TF stimulated gamma interferon and only bp26 induced interleukin-4 (IL-4), IL-5, and IL-6 cytokines as measured by cytokine enzyme-linked immunospot assay. These collective results suggest that both bp26 and TF are excellent candidates for use in future vaccination studies against brucellosis.

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.

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]).

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.

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.