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. 2014 Nov;21(11):1474–1480. doi: 10.1128/CVI.00554-14

Use of S-[2,3-Bispalmitoyiloxy-(2R)-Propyl]-R-Cysteinyl-Amido-Monomethoxy Polyethylene Glycol as an Adjuvant Improved Protective Immunity Associated with a DNA Vaccine Encoding Cu,Zn Superoxide Dismutase of Brucella abortus in Mice

Angello Retamal-Díaz a, Roberto Riquelme-Neira a, Darwin Sáez a, Alejandra Rivera a, Pablo Fernández a, Alex Cabrera b, Carlos A Guzmán b, Ángel Oñate a,✉
Editor: D L Burns
PMCID: PMC4248768  PMID: 25165025

Abstract

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.

INTRODUCTION

Brucella is the causative agent of brucellosis, one of the most important and widespread zoonoses in the world (1). Brucella abortus is a Gram-negative facultative intracellular bacterial pathogen. In cattle, it leads to abortion and infertility, resulting in serious economic losses, especially in developing countries where no rigorous vaccination programs exist (2). In humans, the disease is characterized by nonspecific symptoms, including undulant fever, weight loss, hepatomegaly, splenomegaly, and other severe complications such as neurobrucellosis (3).

In the infected host, B. abortus multiplies within the phagosomes of reticuloendothelial cells, avoiding the killing effect of the macrophages by inhibiting phagosome-lysosome fusion (4). Like other facultative intracellular bacterial pathogens, resistance to B. abortus depends on acquired cell-mediated immunity (CMI) (5). The Th1 subset of CD4+ lymphocytes that secrete gamma interferon (IFN-γ), which upregulates macrophage anti-Brucella activity, seems to present a critical bacterial clearance mechanism (6, 7). Concerning the role played by CD8+ cells, there are contrasting reports in the literature. Some studies concluded that CD8+ T cells are critical for the resolution of infection (7, 8), whereas others suggested that they are dispensable (9). However, the protective response against Brucella does not rely only on CMI; antibodies also have been shown to contribute toward bacterial clearance (10, 11). Although the contributing role of secretory IgA (sIgA) against B. abortus is still unclear, it may be relevant for protection against mucosal transmission. Therefore, while the induction of systemic immune responses following immunization represents a major goal for the ideal vaccine against brucellosis, stimulation of the mucosal immune response following vaccination may represent a true asset. To achieve this aim, one might need to use adjuvants, substances that exhibit immunopotentiating and immunomodulatory properties, which are coadministered with the antigen in the vaccine formulations. One of these adjuvants is S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG), a pegylated synthetic derivative of macrophage-activating lipopeptide-2 (MALP-2). This compound exhibits improved solubility with respect to MALP-2 and retains its Toll-like receptor 2/6 agonistic capacity on antigen-presenting cells (12, 13). In addition, it induces proinflammatory chemokines and cytokines (14, 15) and promotes cross-priming and T-helper cell-dependent cytotoxic responses (16). BPPcysMPEG exerts its activity when administered by either the parenteral or the mucosal route (16–18).

One of the antigens of B. abortus recognized by the immune system is an 18.5-kDa periplasmic protein called Cu,Zn superoxide dismutase (SOD). Intramuscular administration of a plasmid vector coding for SOD conferred protection in mice and strong immune responses in calves (19, 20). However, parenteral administration of a vaccine does not mimic the natural infection route. Thus, in this study, we evaluated if intranasal (i.n.) administration of a plasmid coding for Cu,Zn SOD combined with BPPcysMPEG is able to promote immune responses conferring protection to the animals challenged with the pathogenic B. abortus 2308 strain.

MATERIALS AND METHODS

Mice.

Experimental groups were composed of 2-month-old female BALB/c mice (acquired from the Instituto de Salud Pública, Santiago, Chile), which were randomly distributed, acclimated, and kept under controlled conditions. All mice received food and water ad libitum, and their management and disposal were performed according to the guidelines of the Institutional Ethical Committee of the Universidad de Concepción.

Bacterial strains.

The Escherichia coli BL21 (Novagen, Madison, WI) and E. coli DH5α (Invitrogen, San Diego, CA) strains were grown in Terrific broth at 37°C, supplemented with 50 μg/ml of ampicillin when required. The attenuated B. abortus strain RB51, virulent B. abortus strain 2308, and B. abortus strain RB51 overexpressing SOD (RB51-SOD) were grown under aerobic conditions in Brucella broth (Difco) for 72 h at 37°C. Every experiment with Brucella was performed under biosafety level three conditions.

Construction of the Cu,Zn SOD DNA vaccine.

The vector pcDNA3.1 (Invitrogen) was used as the backbone for the DNA vaccine construct. The region encompassing the Cu,Zn SOD-encoding gene (sodC) was amplified from the B. abortus strain 2308 by PCR using a custom-designed primer pair based on the corresponding sequence data available in GenBank (NCBI gene identification no. 3827840). The primer sequences were 5′-CCA AGC TTG CCA CCA TGA AGT CCT TAT TTA T-3′ (forward) and 5′-CCG GAT CCT TAT TCG ATC ACG-3′ (reverse). The fragment amplified was double digested with HindIII/BamHI and ligated into the expression vector pcDNA3.1, thereby generating pcDNA-SOD. Large-scale plasmid DNA isolation was performed using the EndoFree plasmid giga kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Immunization.

Mice (n = 12) were immunized by the i.n. route with 50 μg of plasmid coadministered with 0.5 μg of BPPcysMPEG as a mucosal adjuvant when required. The animals were vaccinated at weeks 0, 2, and 4 with pcDNA-SOD, pcDNA-SOD/BPPcysMPEG, and pcDNA3.1/BPPcysMPEG. As a negative control, a group of mice was immunized with pcDNA3.1 empty vector or phosphate-buffered saline (PBS). In protection experiments, the positive-control group was vaccinated intraperitoneally with 2 × 108 CFU of the B. abortus strain RB51 in 0.1 ml of PBS on day 0.

Enzyme-linked immunosorbent assay.

Sera were obtained from five mice per group 2 days before each immunization and 15 days after the last boost. The presence of SOD-specific serum IgG, IgG1, and IgG2a was determined by an indirect enzyme-linked immunosorbent assay (ELISA) (19). Recombinant SOD (rSOD) protein (2 μg/ml) (21) or crude extracts of Brucella proteins (CBPs) (10 μg/ml) (22) were diluted in carbonate buffer (pH 9.6) and used to coat the wells of a polystyrene plate (Nunc-Immuno plate with a MaxiSorp surface). After overnight incubation at 4°C, the plates were blocked with 5% gelatin in Tris-buffered saline for 1.5 h at 37°C. The plates were then incubated for 3 h at room temperature with serial 2-fold dilutions of sera. Isotype-specific goat anti-mouse horseradish peroxidase (HRP) conjugates (ICN Biomedicals, Inc., Aurora, OH) were added at a 1:1,000 dilution. Endpoint titers were expressed as retrograde values of the last dilution that gave an optical density at 450 nm (OD450) of two times above the values of the negative controls. The cutoff value for the assay was calculated as the mean specific OD450 plus standard error of the mean (SEM) for 10 serum samples from nonimmunized mice assayed at a dilution of 1:100. The titer of each serum sample was calculated as the last serum dilution yielding a specific optical density higher than the cutoff value.

The amount of total antigen-specific IgA present in bronchoalveolar lavage (BAL) and nasal lavage (NAL) fluids was also determined by ELISA. Antibody titers were estimated as the reciprocals of the last sample dilution giving an absorbance (A450) value above the cutoff. To compensate for potential variations in the efficiency of recovering secretory antibodies among the animals, we normalized the results according to the total IgA content of the sample. Thus, results were expressed as ELISA units (EUs), namely, the endpoint titer of antigen-specific IgA divided by the total concentration (in μg) of the IgA present in the sample (23, 24). To establish the IgA standard curve, we coated plates with anti-mouse IgA (Sigma) and further incubated them with serial dilutions of purified mouse IgA (Sigma). As a secondary antibody, HRP-conjugated goat anti-mouse IgA (ICN Biomedicals, Inc., Aurora, OH) was used; plates were developed as described above. For calculation purposes, samples negative for SOD-specific IgA were assigned an arbitrary titer of the lowest dilution measured.

Cell-mediated immune responses.

Mice were euthanized 14 days after the last immunization, and spleens were aseptically removed. The spleens were used to prepare single-cell suspensions by mechanical disaggregation, and the erythrocytes were lysed using ammonium-chloride-potassium (ACK) buffer. Cells were later washed with RPMI medium (19). Then, the splenocytes were resuspended at a concentration of 4 × 105 viable cells/well in 96-well flat-bottom plates for culturing at 37°C with 5% CO2 in the presence of one of the following stimulants: 10 μg/ml CBPs, 4 μg/ml purified rSOD, 2.5 μg/ml concanavalin A, or no additive (unstimulated control). The cells were cultured in complete RPMI medium for 72 h and pulsed for 8 h with 0.4 μCi of [3H]thymidine (50 μCi/mmol; Amersham, United Kingdom) per well. The radioactivity incorporated into the DNA was measured using a scintillation counter. Results are expressed as the mean of counts per minute of triplicate cultures from a cell pool obtained from each group (five mice per group).

Cytokine assays.

Cell suspensions of spleens from five control or immunized mice in RPMI medium were plated at 2 × 105 cells/well in flat-bottom microwell plates (Nunc, Roskilde, Denmark). The cells were stimulated in vitro with either 0.4 μg/ml rSOD, 5 μg/ml CBPs, or medium alone and incubated at 37°C in 5% CO2. After 48 h of culture, supernatants were taken and stored until use at −80°C. To measure the production of IFN-γ, interleukin 4 (IL-4), and IL-10, we used an antigen-capture ELISAReady-SET-Go! kit (eBioscience, San Diego, CA). Every experiment was performed in triplicate, and the cytokine concentration from the supernatants was calculated using a linear regression equation from the absorbance values of the standards.

Cytotoxicity assay.

The cytotoxicity assay was carried out as described previously (25). Briefly, stimulator cells were prepared by infecting J774.A1 macrophages with live B. abortus strain RB51-SOD at a ratio of 1:100 (cells to RB51-SOD) and incubated for 5 h in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Then, extracellular bacteria were rinsed away with RPMI medium containing 50 μg/ml of gentamicin. Macrophages were scraped off and centrifuged at 200 × g for 5 min, and then cells were suspended and incubated for 45 min in 5 ml of RPMI medium supplemented with 35 μg/ml of mitomycin C. After incubation, the cells were washed by centrifugation with RPMI medium supplemented with 5% FBS. The previously pulsed antigen-presenting cells and the lymphocytes of immunized mice were distributed to the wells of 24-well cell culture plates (Corning) at a 10:1 ratio and incubated at 37°C with 5% CO2 for 5 days in order to generate specific cytotoxic T lymphocytes (CTLs) (effector lymphocytes). To test the cytotoxic activities against target cells expressing SOD antigen, we incubated effectors and target cells (B. abortus RB51-SOD-infected J774.A1 macrophages which were not treated with mitomycin C) for 16 h at 37°C at 50:1, 10:1, 5:1, and 2.5:1 effector-to-target ratios. Specific cell lysis was determined by a fluorimetric assay using CytoTox-ONE (Promega Corporation, WI) according to the manufacturer's instructions. The percentage of specific lysis was established by applying the following formula: % lysis = [(fluorescence of experimental group − fluorescence background)/(fluorescence control − fluorescence background)] × 100.

Protection studies.

As previously described (22), 5 weeks after the last vaccination (day 60), each mouse was challenged by an intraperitoneal injection of 1 × 104 CFU of B. abortus strain 2308 (5 mice per group). Fourteen days later, the mice were euthanized, and their spleens were homogenized. Then, dilutions were plated to establish the number of Brucella CFU per spleen. The average results of two independent experiments are presented in Table 1. The log10 units of protection were calculated by subtracting the mean log10 CFU for the experimental group from the mean log10 CFU for the corresponding control group.

TABLE 1.

Protection of mice against challenge with B. abortus strain 2308 after immunization with DNA vaccine formulations

Vaccine (dose) Mice (n = 5) challenged intraperitoneally with 104 CFU of B. abortus strain 2308
Log10 CFU of B. abortus 2308 in spleen (mean ± SD) Log10 units of protection
PBS 6.02 ± 0.52 0
pcDNA3.1 (50 μg) 6.16 ± 0.48 0
pcDNA-SOD (50 μg) 3.86 ± 0.35 2.16a,b
pcDNA3.1 (50 μg) and BPPcysMPEG (5 μg) 6.12 ± 0.39 0
pcDNA-SOD (50 μg) and BPPcysMPEG (5 μg) 2.73 ± 0.22 3.29a,b
B. abortus strain RB51 2.47 ± 0.30 3.55a
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a

α = 0.05 compared to the negative-control group receiving PBS.

b

The difference between pcDNA-SOD and pcDNA-SOD/BPPcysMPEG is statistically significant, with values compared using Sidak's multiple-comparisons test (α = 0.05).

Statistical analysis.

The data derived from lymphocyte proliferation, detection of cytokines and cytotoxicity, and evaluation of antibody levels were analyzed using the analysis of variance (ANOVA) test and Bonferroni adjustment (a P value of ≤0.05 was considered statistically significant). The data derived from the protection experiment were analyzed using the two-way ANOVA test and Sidak's multiple-comparison test with a confidence interval of 95% (α = 0.05).

RESULTS

Humoral immune responses.

To evaluate humoral immune responses, we determined rSOD-specific antibodies by ELISA in sera from mice immunized by the i.n. route with pcDNA-SOD or pcDNA-SOD/BPPcysMPEG and from controls that received pcDNA3.1, pcDNA3.1/BPPcysMPEG, or PBS. As shown in Fig. 1A, i.n. immunization of mice with pcDNA-SOD or pcDNA-SOD coadministered with BPPcysMPEG resulted in detectable titers of SOD-specific IgG by day 15, which sustainably increased until the last sampling on day 45. The responses in these two groups were significantly (P < 0.001) different than the those in the animals that received PBS, pcDNA3.1, or pcDNA/BPPcysMPEG (i.e., negative-control groups). When mice were immunized with pcDNA-SOD alone, we observed that on days 15, 30, and 45, the SOD-specific IgG antibody titer was significantly lower than that in animals from the pcDNA-SOD/BPPcysMPEG group (P < 0.01 on day 15 and P < 0.001 on days 30 and 45).

FIG 1.

FIG 1

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SOD-specific serum antibodies. Five mice in each group were immunized by the i.n. route with pcDNA-SOD, pcDNA-SOD/BPPcysMPEG, pcDNA/BPPcysMPEG, control pcDNA3.1, or PBS. Sera were obtained on days 0, 15, 30, and 45 postimmunization, and SOD-specific IgG (A), IgG1 (B), and IgG2a (C) titers were determined. Endpoint titers are expressed as retrograde values of the last dilution that gave an OD450 of two times above the value of the negative controls ± SEM. These results are representative of data from two independent experiments. Statistical significances are represented by asterisks (*, P < 0.05, **, P < 0.01, and ***, P < 0.001, as compared between the pcDNA-SOD and pcDNA-SOD/BPPcysMPEG groups).

Then, SOD-specific IgG isotypes (IgG1 and IgG2a) were analyzed. In samples taken on days 15, 30, and 45 after priming, the SOD-specific IgG1 titers (Fig. 1B) in mice immunized with pcDNA-SOD/BPPcysMPEG were observed to be significantly increased over those in all the other groups (P < 0.001). Sera obtained on days 30 and 45 from mice immunized with pcDNA-SOD showed lower but still significantly increased titers of anti-SOD IgG1 compared to those in the negative-control groups (P < 0.001). The values for SOD-specific IgG2a (Fig. 1C) titers in sera from the pcDNA-SOD/BPPcysMPEG group were significantly increased in samples taken on days 15, 30, and 45 postpriming compared to those from the negative-control groups (P < 0.001). The sera taken on days 30 and 45 from mice immunized with pcDNA-SOD/BPPcysMPEG also showed significantly higher titers of anti-SOD IgG2a compared to those in the mice immunized with pcDNA-SOD (P < 0.05 and P < 0.001, respectively).

To evaluate the mucosal immune responses stimulated in vaccinated animals, we determined the SOD-specific sIgA response in BAL and NAL fluids. As demonstrated in Fig. 2, the mice immunized with pcDNA-SOD or pcDNA-SOD/BPPcysMPEG showed significantly higher levels of SOD-specific mucosal IgA in BAL (Fig. 2A) and NAL (Fig. 2B) fluids than did the negative-control mice that received PBS, pcDNA3.1, or pcDNA3.1/BPPcysMPEG (P < 0.01). The mice immunized with pcDNA-SOD/BPPcysMPEG also showed significantly higher sIgA levels in BAL and NAL fluids than did the animals immunized with pcDNA-SOD (P < 0.01).

FIG 2.

FIG 2

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SOD-specific mucosal sIgA antibodies in bronchoalveolar lavage (A) and nasal lavage (B) fluids. SOD-specific antibody titers were estimated as the reciprocals of the last sample dilution giving an A450 value above the cutoff value; results were normalized according to the total IgA content of the sample. Results are expressed as ELISA units (EUs), namely, the endpoint titer of SOD-specific IgA divided by the total concentration of IgA (in μg) present in the sample. Data are shown as the mean ± SEM values from two experiments. Statistical significances are represented by asterisks (P < 0.01). Bar-connecting lines indicate significant differences between the corresponding groups.

Cellular immune responses.

In order to evaluate the induction of cell-mediated immunity, we first analyzed the proliferative responses on splenocytes. As shown in Fig. 3, following i.n. immunization with either pcDNA-SOD or pcDNA-SOD/BPPcysMPEG, significant inductions of proliferative responses to rSOD and CBPs were observed (P < 0.001 compared to the induction levels after the immunization give to the mice in the negative-control groups). Responses from the splenocytes of mice immunized with pcDNA-SOD/BPPcysMPEG were also significantly higher than those of the animals vaccinated with pcDNA-SOD alone after restimulation with either rSOD (P < 0.001) or CBPs (P < 0.01). Only low levels of spontaneous proliferation were observed when testing cells obtained from the mice in the control groups.

FIG 3.

FIG 3

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Lymphocyte proliferation assay. BALB/c mice were immunized with pcDNA-SOD, pcDNA-SOD/BPPcysMPEG, pcDNA/BPPcysMPEG, pcDNA3.1, or PBS. SOD-specific T-cell proliferative responses were measured 2 weeks after the last immunization by [3H]thymidine incorporation. Splenocytes derived from animals in each group were pooled, and 4 × 105 cells were restimulated in vitro with purified rSOD (0.4 μg/ml) or CBPs (5 μg/ml). Each bar indicates the average number of counts per minute for triplicate cultures of cells ± standard deviation (error bars) obtained from five mice per group. Statistical significances are represented by asterisks (**, P < 0.01, and ***, P < 0.001). Bar-connecting lines indicate significant differences between the corresponding groups.

The cytokine profiles of supernatants from cell cultures of spleen cells from the pcDNA-SOD and pcDNA-SOD/BPPcysMPEG immunization groups restimulated with rSOD showed significantly higher levels of IFN-γ (P < 0.001 and P < 0.01, respectively) than those from the negative-control groups (Fig. 4A). Cells from animals immunized with pcDNA-SOD also exhibited a significantly higher production of IFN-γ than did those from pcDNA-SOD/BPPcysMPEG-immunized mice (P < 0.01). In contrast, significant production of IL-4 (P < 0.001) was observed only in the supernatant fluids from cultures of splenocytes derived from pcDNA-SOD/BPPcysMPEG-immunized mice compared to those from pcDNA-SOD-immunized mice or from the negative-control groups (Fig. 4B).

FIG 4.

FIG 4

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IFN-γ (A) and IL-4 (B) secreted by lymphocytes upon stimulation with rSOD. Spleen cell suspensions from five mice immunized with pcDNA-SOD, pcDNA-SOD/BPPcysMPEG, pcDNA/BPPcysMPEG, pcDNA3.1, or PBS were stimulated in vitro with purified rSOD (0.4 μg/ml) as antigen. Data are shown as means ± SEM and are representative of data from two independent experiments. Statistical significances are represented by asterisks (*, P < 0.01, and **, P < 0.001).

SOD-specific cytotoxic response.

DNA immunization has been shown to be effective in inducing CTLs (26). Therefore, the inductions of SOD-specific CTLs in pcDNA-SOD- or pcDNA-SOD/BPPcysMPEG-immunized mice were evaluated and compared to those in mice receiving PBS, pcDNA3.1, or pcDNA3.1/BPPcysMPEG (Fig. 5). Specific lysis of RB51-SOD-infected J774.A1 macrophages was observed only at effector-to-target ratios of ≥5:1 using cells from mice immunized with pcDNA-SOD or pcDNA-SOD/BPPcysMPEG (P < 0.001). Cytotoxic responses were significantly higher at ratios of 5:1 to 50:1 using cells from mice immunized with pcDNA-SOD/BPPcysMPEG than when using cells obtained from the mice immunized with pcDNA-SOD (P < 0.001).

FIG 5.

FIG 5

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SOD-specific cytotoxic activity. Four weeks after the immunization, splenocytes from mice immunized with pcDNA-SOD, pcDNA-SOD/BPPcysMPEG, pcDNA/BPPcysMPEG, pcDNA3.1, or PBS were stimulated for 5 days with J774.A1 macrophages infected with B. abortus RB51-SOD at a ratio of 1:100 (cells to RB51-SOD). These effector cells were then incubated with either J774.A1 cells or J774.A1 cells infected with RB51-SOD. A CytoTox-ONE assay was used to measure target lysis. The data are means from experiments performed in triplicate, and standard deviations did not exceed 20% of the means. E, effector; T, target. *, statistically significant (P < 0.001) compared to negative-control groups or between pcDNA-SOD and pcDNA-SOD/BPPcysMPEG groups.

Protection experiments.

Four weeks after the last vaccination, the mice were challenged by an intraperitoneal injection of the virulent B. abortus strain 2308 and euthanized 15 days later to determine the number of CFU in their spleens (Table 1). Data from two independent protection experiments indicated that immunization with pcDNA-SOD or pcDNA-SOD/BPPcysMPEG induced significant degrees of protection (2.73- and 3.29-log-unit increases in protection, respectively) compared to the PBS control group (α = 0.05). In comparison, vaccination with live B. abortus strain RB51 induced a 3.55-log-unit increase in protection. Furthermore, immunization with pcDNA-SOD/BPPcysMPEG induced a higher degree of protection against challenge (1.13-log-unit increase in protection) than did immunization with pcDNA-SOD (α = 0.05).

DISCUSSION

Mucosal surfaces are the portal of entry for Brucella species in humans and animals. Therefore, large epithelial surfaces, such as the respiratory, intestinal, and urogenital tracts, need to be protected against penetration to prevent systemic dissemination to target organs. This has led to the concept that a successful vaccine should be able to trigger not only agent-specific immunity at a systemic level (27) but also an efficient local mucosal immunity (28). Unfortunately, soluble antigens administered via the mucosal route are usually very poorly immunogenic (29). Thus, different strategies have been developed to increase immunogenicity, including the use of mucosal adjuvants (30, 31). BPPcysMPEG was proven to be an efficient compound for promoting immune responses at the systemic and mucosal levels when coadministered with a vaccine antigen (23, 32–34).

Sufficient experimental evidence supports the ability of pcDNA-SOD, a plasmid containing the Brucella Cu,Zn SOD-encoding gene, to promote immune protection in mice (19, 35) and SOD-specific immune responses in cattle (20) following vaccination. In the present study, we evaluated the immunogenicity and protective efficacy conferred in mice upon i.n. inoculation with a vaccine formulation containing pcDNA-SOD coadministered with BPPcysMPEG. The results show that immunization with pcDNA-SOD or pcDNA-SOD/BPPcysMPEG triggers SOD-specific humoral and cellular immune responses in BALB/c mice. At a systemic level, pcDNA-SOD/BPPcysMPEG promoted the stimulation of SOD-specific IgG2a and IgG1 responses, suggesting elicitation of a balanced Th1/Th2-type cellular immune response (36). In agreement with previous studies, immunization with pcDNA-SOD appears to promote a Th1-biased immune response (19). At the mucosal level, i.n. immunization with pcDNA-SOD/BPPcysMPEG resulted in the induction of antigen-specific sIgA in BAL and NAL fluids.

Protection against infection with Brucella, an intracellular bacterium, requires cell-mediated immunity, which includes IFN-γ-producing CD4+ T cells and CD8+ T lymphocytes (37). Splenocytes from pcDNA-SOD- and pcDNA-SOD/BPPcysMPEG-immunized mice showed increased rSOD-specific proliferative responses, as well as the production of IFN-γ, which was more abundant in pcDNA-SOD-vaccinated mice. Significantly increased secretion of IL-4 was observed only in splenocytes derived from mice immunized with pcDNA-SOD/BPPcysMPEG. Conversely, restimulated splenocytes from pcDNA-SOD- and pcDNA-SOD/BPPcysMPEG-immunized mice showed weak CBP-specific proliferative responses, which can be explained by low-level expressions of SOD in the CBPs compared to that in the total protein content. Thus, pcDNA-SOD preferentially stimulates Th1 responses (19), whereas the inclusion of BPPcysMPEG in the formulation results in stimulation of a more balanced Th1/Th2 response.

As mentioned above, CD8+ T cells with a cytotoxic capacity seem to play a key role against persistent infection with Brucella spp. (38–40). Thus, we performed a comparative evaluation of the capacity to induce a cytotoxic T-lymphocyte response from pcDNA-SOD when administered alone or together with BPPcysMPEG. These two vaccine formulations promoted cytotoxic responses, but the strongest response was observed with the BPPcysMPEG-adjuvanted formulation. This is in agreement with results from previous studies demonstrating that BPPcysMPEG induces maturation of CD8α+ and CD8α− dendritic cells, resulting in enhanced cross-presentation and a robust helper-cell-dependent cytotoxic T-lymphocyte response (16). To confirm that the observed immunological response induced upon vaccination with pcDNA-SOD or pcDNA-SOD/BPPcysMPEG translates into effective protection against B. abortus infection, we performed challenge studies. The obtained data demonstrate that only the vaccinated mice were protected against infection and that coadministration of BPPcysMPEG resulted in superior protection. In conclusion, our results show that i.n. immunization with pcDNA-SOD coadministered with BPPcysMPEG leads to SOD-specific systemic and mucosal immunity, which in turn confers protection against B. abortus infection, with an improved response compared to that of immunization with pcDNA-SOD alone.

ACKNOWLEDGMENT

This work was supported by grant 1100032 from the “Fondo Nacional de Investigación Científica y Tecnológica” (FONDECYT), Chile.

Footnotes

Published ahead of print 27 August 2014

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