Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu–Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice

Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu–Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4+ T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus.

simple fever to major complications in which function in the nervous, digestive, genital-urinary, cardiovascular, and muscular systems is compromised, sometimes leading to death (3). Brucellosis can impose a significant economic burden on animal production (reduction in milk production, abortions, delayed in conception). In cattle, it has been estimated that more than 300,000 animals, out of the 1.4 billion in the world, are infected (4). Brucellosis is one of the most common zoonotic diseases in humans, with more than 500,000 cases reported annually. However, depending upon the system of controls and the socioeconomic conditions, official reports only account for a fraction of the true incidence of this disease, and different countries have reported from 0.09 to 1603 cases per million inhabitants (5). Infection by Brucella spp. is usually associated to an acute inflammatory reaction, the principal mechanism against local proliferation of Brucella organisms. Infection initially prompts an innate immune response that reduces the number of bacteria (6). The innate response activates immunity mediated by cells, in which CD4+ and CD8+ T lymphocytes, macrophages (MΦ), dendritic cells (DCs), and pro-inflammatory cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), participate to confer protection (7). Although the host raises strong immune response, Brucella abortus has the capacity to survive inside MΦ and DCs, expressing a number of virulence factors that allow it to reach its replicative niche and to avoid immune-mediated destruction (8). It has been shown that antigen O protects Brucella from intracellular death mechanisms, while lipid A is involved in evasion of the innate immune system during the first stages of infection (9). Some additional virulence factors have been reported to be involved in intracellular replication and immune evasion. These include VirB type IV secretion (10), BTP1, a seven-helix transmembrane protein that prevents maturation of DCs (11), a BvrR/BvrS regulatory system of two components (12), and the RNA chaperone protein Hfq (13). B. abortus contains a Cu-Zn superoxide dismutase (SOD1), an homodimeric metalloenzyme (14). SOD1 catalyzes the dismutation of the superoxide anion O 2 − to O2 and H2O2, detoxifying superoxide radicals generated during the host antimicrobial immune response (15). It has been observed that a DNA vaccine with the gene sequence for this protein (sodC) is highly immunogenic (16,17).
One characteristic of Brucella is a limited genetic diversity. This is manifested by the small number of differences responsible for host preferences and by virulence restrictions (18). The Brucella genome incorporates transfer-acquired mobile elements referred to as genomic islands (GIs). Nine GIs have been identified in Brucella (19). GI-3 is present in B. abortus, Brucella melitenses, and Brucella ovis. This GI contains 25 genes, many of which have unknown function, and several pseudogenes (20). GI-3 in B. abortus 2308 includes open reading frame (ORF) BAB1_0260, BAB1_0270, BAB1_0273, BAB1_0278, and BAB1_0278a. Our group has reported that BAB1_0270, described as a zincdependent metallopeptidase protein, and BAB1_0278, which has homology with the GcrA superfamily, are involved in Brucella virulence. Their deletion affects the capacity of Brucella to invade phagocytic cells and to survive within them (21,22). Furthermore, DNA vaccine encoding BAB1_0270 (23), BAB1_0278 (24), and BAB1_0278a, a hypothetical ABC-type transporter (25), were able to induce an immune response and protection against B. abortus 2308 infection. Immunization with the recombinant flagellar protein (BAB1_0260) also induced protection (26). Based on bioinformatics analysis BAB1_0273, a possible DNAbinding protein, is a putative antigen.
Despite the existence of effective commercial vaccines against brucellosis and a diagnostic test, it has not been possible to eradicate the disease. The main vaccines currently used for cattle are based on live bacteria attenuated to decrease their pathogenicity. Cattle are often vaccinated with B. abortus S19 or RB51, which, although providing good protection, may induce abortion if administered to gravid females (27), and are potentially infectious to humans (28). Recent advances in genomics, proteomics, recombinant DNA techniques, and vaccinology have made possible the development of safer vaccines, which overcome the drawbacks associated with live-attenuated vaccines. For example, DNA vaccines offer the possibility of inducing both humoral and cellular immune responses, and potentially can prolong the expression of an antigen (29). The use of epitopes in the design of this type of vaccine is a new alternative in the development of multi-epitope DNA vaccines (30)(31)(32). In this strategy, an informed selection of antigenic determinants that correlate with immunogenicity was used.
In this study, we have predicted antigenic determinants using bioinformatics tools from any ORF codified in GI-3 from B. abortus and designed a multi-epitope chimeric DNA vaccine. Humoral, cell-mediated, and protective immunity induced by this multi-epitope DNA vaccine was examined in BALB/c mice.

MaTerials anD MeThODs animals
Seven-to eight-week-old female isogenic BALB/c mice (obtained from the Instituto de Salud Pública, Santiago, Chile) were randomly allocated to three groups. Mice were kept in conventional animal facilities and received water and food ad libitum. All animals were handled in accordance with the regulations of the Bioethics Committee of the Faculty of Biological Sciences, Universidad de Concepción regulations. The Bioethics and Safety committee of the Faculty of Biological Sciences of the Universidad de Concepción approved this study. All efforts were made to minimize animal suffering.

Bacterial strains
Escherichia coli strain BL21 (DE3) pLys (Novagen, Madison, WI, USA) was used as the host strain for expression of recombinant multi-epitope protein and E. coli DH5α (Invitrogen, San Diego, CA, USA) was used for obtaining plasmids. Both strains were grown at 37°C in LB broth. The virulent B. abortus 2308 and the attenuated strain RB51 were obtained from our culture collection. Bacterial cells were grown under aerobic conditions in Trypticase soy broth (Difco Laboratories, Detriot, MI, USA) for 72 h at 37°C.   BamHI-PstI-digested pVAX1 or BamHI-PstI-digested pQE80L. We obtained pV-MEB (multi-epitope DNA vaccine for Brucella) and pQE80L-MEB plasmids and confirmed them by restriction digestion analysis (Figure 1). We observed 1140 base pairs (bp) corresponding to MEBm fragment (A) and 1098 bp corresponding to MEBe fragment (B), respectively.
immunization BALB/c mice were randomly divided into three groups consisting of 10 mice per group. Group 1 was injected with 100 µg of the pV-MEB vaccine in 100 µl of phosphate buffer saline (PBS), divided into two injections of 50 µl, in each posterior tibialis muscle. As negative controls, groups of mice received either 100 µg pVAX1 in 100 µl of PBS or 100 µl of PBS, injected as described above for the experimental group (23). All groups were immunized three times at 15-day intervals.

Purification of recombinant Multi-epitope Protein
To obtain the multi-epitope recombinant protein, E. coli BL-21 were chemically transformed with pQE80L-MEBe, and we standardized the protocols to carry out the purification of the rMEB protein.

Protection experiment
The protection experiments were performed as previously described (16). Briefly, 4 weeks after last vaccination, four mice from each group were challenged by intraperitoneal injection of 10 4 CFU B. abortus 2308 per animal. Two weeks later, infected mice were euthanized and their spleens were homogenized in PBS, with the homogenate serially diluted and cultured in Petri dishes containing agar Columbia supplemented with 5% sheep blood (bioMériex, Santiago, Chile) for 72 h at 37°C. Bacterial counts were recorded and the number of CFU per spleen calculated. This experiment was repeated twice. When the immunizations were initiated, one reference-vaccinated control group was immunized with 1 × 10 8 CFU B. abortus RB51 per mouse. Results are reported as units of protection represented by the difference between mean ± SD of log10 CFU/spleen of the PBS control groups with respect to mean ± SD of log10 CFU/spleen values of experimental groups.

statistical analysis
The immune response in mice was analyzed using a two-way analysis of variance (ANOVA) and the protective response was analyzed using a one-way ANOVA. Data were analyzed using Prism 5.0 (GraphPad software). Differences were considered significant if P < 0.05.  Table 2). Peptides were selected based on having a lower percentile rank score. Non-redundant peptides were selected to construct the DNA vaccine. An immunodominant peptide of the Cu, Zn superoxide dismutase protein from B. abortus, described previously in the literature (39), was also included in the vaccine sequence. Finally, 21 epitopes were used to construct the DNA vaccine. To connect the epitopes, we used a GDGDG linker sequence, a rationally designed sequences used to link multi-epitope vaccines (40). The final design of the multiepitope vaccine is shown in Figure 2A.

Production of recombinant Multi-epitope Protein of Brucella
To construct the recombinant protein, E. coli BL21 (DE3) cells were transformed with the pQE80L-MEB plasmid and expression of the 6xHis-Tagged protein was induced. The recombinant protein was mainly expressed in the soluble fraction of the transformed bacteria after their sonication (Figure 2B, lane 4). The recombinant protein of B. abortus (rMEB) was induced and detected by Western blot. Its weight was ~37 kDa, the expected mass ( Figure 2C).

humoral immune response of immunized Mice
Specific antibodies for rMEB and CBPs were measured in order to evaluate the humoral immune response. We performed ELISAs to detect specific IgG, IgG1, and IgG2a antibodies induced in mice against rMEB and CBP. Serum from mice immunized with multi-epitope DNA vaccine for Brucella (pV-MEB) contained  significant titers of IgG specific for rMEB at 30 days after the first immunization. IgG titers were higher at 45 days, compared to the negative-control groups pVAX and PBS ( Figure 3A). The same pattern was observed for IgG against CBPs ( Figure 3B). No rMEB-or CBP-specific IgG1 was detected in serum from mice immunized with pV-MEB (Figures 3C,D). Titers of IgG2a antibodies specific for rMEB proteins significantly differed between the pV-MEB groups immunized (P < 0.05) only after the second immunization between pV-MEB groups immunized, compared to PBS and pVAX controls ( Figure 3E). On the other hand, a significant titer of IgG2a specific for CBP was observed after the third immunization with pV-MEB, compared to PBS and pVAX negative controls ( Figure 3F).

cellular immune response
To evaluate the cellular immune response, splenocytes were obtained from mice immunized with pV-MEB, p-VAX, or PBS at 30 days after the last immunization. In vitro stimulation using splenocytes from pV-MEB mice immunized with 10 or 2 µg/ ml rMEB protein resulted in a significant increase in cell proliferation in relation to the control group (P < 0.001 and P < 0.05, respectively; Figure 4A). In vitro stimulation using splenocytes from pV-MEB mice immunized with 10 and 2 µg/ml of CBP also induced a significant increase in splenocytes proliferation (P < 0.001 and P < 0.01, respectively; Figure 4B). In this assay, 10 µg/ml of ConA was used as lymphoproliferation control. It induced a strong lymphoproliferative response in all experimental groups (data not shown). In vitro stimulation of splenocytes with 10 µg/ml crude E. coli protein and 10 µg/ml albumin did not induce proliferation across the different experimental groups (Figure 4C).
IFN-γ levels in supernatants from cultures of splenocytes obtained from the pV-MEB immunization group re-stimulated with rMEB or CBP were significantly higher than those in the negative-control groups (P < 0.001, respectively) ( Figure 5A). There were no significant difference in levels of IL-4 secretion between the experimental and control groups ( Figure 5B).

Protection against Virulent B. abortus challenged
The protective capacity provided by the pV-MEB DNA vaccine was evaluated 6 weeks after the last immunization. Immunized mice were challenged with 10 4 CFU B. abortus 2308, and after 2 weeks their spleens were removed, homogenized, and cultured. The results showed that pV-MEB DNA vaccine confers protection against B. abortus 2308. The DNA vaccine induced 1.14 log10 units of protection (P > 0.005; Figure 6) compared to the PBS control group. By comparison, vaccination with live B. abortus strain RB51 induced 2.85-log units of protection.

DiscUssiOn
Brucellosis is a worldwide zoonotic disease of increasing incidence. Vaccination of livestock is considered the best prevention method, but it is necessary to generate safer and more effective vaccine formulations (41). The availability of bioinformatics tools and databases allow the design of vaccines without the need for in vitro manipulation of a pathogenic microorganism. Using "reverse vaccinology" approach, in silico genomic databases are screened to identify antigenic sequences for new vaccines (42). This allows the identification of antigens that would be difficult     using traditional methods (42). It has been observed that recombinant protein vaccines induce a humoral/Th2 immune response and it is suggested that a boost with a protein improve the protective efficacy of the antibodies (43). The effect of DNA vaccines, however, is based toward a Th1 response (44)(45)(46). DNA vaccine have a number of advantages, including ease storage, flexibility of antigen codification, and the presence of CpG motifs, which improve the immune response (45). Monoantigenic DNA vaccines induce a good immune response, but tend to induce less protection against pathogens compared to poly-antigenic vaccine (47,48). In the case of B. abortus, the three antigens: BCSP31, Cu-Zn SOD, and L7/L12 ribosomal proteins, when giving together as part of a formulation, improve the immune response against pathogenic B. abortus (49).
Within the last few years, the use of epitopes in vaccines has become a valid alternative for improving the efficacy of traditional vaccine, based on a "natural" form of the pathogen (50). Multi-epitope peptide DNA vaccines are effective against some viruses (30,51,52) and they are potentially effective against some bacteria such as Helicobacter pylori (53) and in cancer prevention (54,55).
Multi-epitope DNA vaccines more faithfully mimic antigen processing and presentation during natural infection (30). In addition, multi-epitope DNA vaccines induce more potent immunoreaction than whole protein vaccine (30). Since the epitopes are derived from multiple antigens and packaged into a relatively small delivery vehicle, the vaccine can induce powerful cross-reactive responses toward multiple antigens and elicit a strong humoral and cellular immune response (56). In this study, we used bioinformatics methods to identify epitopes on antigenic proteins of B. abortus and to design a multi-epitope chimeric DNA vaccine. We performed the epitope prediction using bioinformatics resources available online, including NetChop, SYFPEITHI, or BIMAS. However, these database had a 50% prediction assertiveness about the prediction (57). The predictive power of the IEDB has been expanded with the provision of a large number of published epitopes and full-scale MHC-binding peptides (58), so we opted to use the IEDB server. In order to design a rational vaccine against Brucella, we focused on finding MHC class I or MHC class II binding peptides known to orchestrate primarily a T-cell immune response, since B. abortus is known as a facultative intracellular pathogen (59). We selected 21 dominant epitopes from ORFs present within GI-3 and proteins described as antigens of immunological interest ( Table 1). Peptides were selected based on their low percentile score using observed redundant sequences as a further selection criterion, choosing peptides with non-redundant sequences ( Table 2). For the theoretical binding of peptides we used the "GDGDG" sequence as spacer (Figure 2A) (40). The introduction of GDGDG spacers does not preclude the possibility that such linear arrangements of epitopes might contain other cryptic epitopes. The presence of this spacer at 15-20 residue intervals might help create some secondary and possibly tertiary structure, thereby facilitating antigen expression (40). Then, we constructed the B. abortus multi-epitope chimeric DNA vaccine using of chemical gene synthesis (pV-MEB).
At systemic level pV-MEB promotes the stimulation of MEBspecific IgG2, indicating an adequate induction of a Th1 response. In vitro stimulation of splenocytes from pV-MEB immunized mice induced the highest proliferation in response to antigen, confirming the in vivo translation of the MEB synthetic gene and subsequent induction of a cell-mediated immune response. We used albumin and E. coli proteins as control of proliferation and in both cases splenocytes did not proliferate. Therefore, the immune response inducing by pV-MEB DNA vaccine was specific to MEB protein and Brucella antigens. Antigen-stimulated splenocytes from vaccinated mice produced IFN-γ. The level of IFN-γ and the in vitro proliferation of splenocytes stimulated by MEB recombinant protein demonstrated that pV-MEB DNA vaccine induces a strong immunoreaction and a polarized Th1 response against Brucella infection, which is associated to effective clearance of intracellular pathogens, so essential feature for a Brucella vaccine (7,59). The immunogenicity induced by pV-MEB DNA recombinant plasmid was evaluated by challenging immunized mice with B. abortus 2308 strain. Our results confirmed that immunization with pV-MEB induced immunogenicity associated with significant protection, but protection induced by attenuated B. abortus RB51 was more robust.
In conclusion, we have shown that a B. abortus multi-epitope chimeric DNA vaccine (pV-MEB) elicits strong humoral and cellular protective immunity. Future studies must include an array of epitopes or combination of peptides and adjuvants as alternatives to conventional vaccine design. This study provides a starting point for the development of multi-epitope DNA vaccines against B. abortus. aUThOr cOnTriBUTiOns EE: construction of recombinant plasmid, analysis in silico of determination of epitopes, evaluation of immune response, evaluation of antibodies specificity by ELISA, and writing and discussion of the results. DS: immunization trail, lymphocyte proliferation assays, and protection experiment. AO: programming and monitoring the experiments, performing the analysis, and discussion and writing the manuscript. AO is principal investigator at the FONDECYT grant that funded this work.