INTRODUCTION

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The first report of the protective efficacy of a nucleic acid-based vaccine in an animal model was published by J. B. Ulmer et al. (57). Since then, several reports have shown that DNA vaccination engenders long-lived humoral and cellular immune responses in vivo against virus, bacterium, or parasite but could also be of potential interest for the treatment of autoimmunity, cancer, and allergy in a variety of animal models (10, 20, 22, 33, 54).

DNA vaccine provides prolonged antigen expression, leading to amplification of immune response and induces memory responses against infectious agents (23, 30). Moreover, endogenous expression of antigen from DNA introduced into host cells leads to processed peptides presented with the major histocompatility complex class I, able to induce cytotoxic T-lymphocyte (9, 58). Finally, specific nucleotidic sequences present in the plasmid play a critical role in the immunogenicity of these vaccines by acting as adjuvant (39, 47). This type of vaccine is capable of eliciting the strong cell-mediated immunity that is required for control of infection by many intracellular agents (24, 35, 44). This kind of immune response is of paramount importance against Brucella spp. (19, 41, 50).

Brucella are gram-negative facultative intracellular bacteria that cause brucellosis in animals and humans. Their ability to survive and replicate within host phagocytic and nonphagocytic cells seems to be responsible for the duration of the disease, which may remain active for years (8, 45). The protection against this infection requires a long-lived cellular immune response, depending on the processing of the bacteria by macrophages (3, 4). Survival of the vaccine-strain should be crucial for the development of a protective cellular immune response against B. abortus in cattle. Understanding the mechanisms by which long-lived cellular immune responses are generated following vaccination will be important for the rational design of vaccines against brucellosis.

The vaccination against bovine brucellosis with live attenuated B. abortus strain S19 is used in most of the world because it has been useful for the control and eradication of this disease (18). However, S19 presents several drawbacks: it is difficult to differentiate between vaccinated and naturally infected animals, because S19 elicit antibodies against smooth lipopolysaccharide, and S19 can also cause abortion in pregnant cattle and is still fully virulent for humans (7, 50). In order to avoid these drawbacks alternative vaccine approaches are needed.

Our laboratory has previously described bacterioferritin (BFR) (13) and P39 (a putative periplasmic binding protein) (11) as T-cell immunodominant Brucella antigens (12) that elicit both a strong delayed-type hypersensitivity reaction in guinea pigs sensitized with brucellin and in vitro proliferation or gamma interferon (IFN-) production by peripheral blood mononuclear cells from infected cattle. The potential of these antigens to induce a Th1-oriented immune response makes them attractive candidates for DNA vaccination. We have recently demonstrated that these recombinant antigens (BFR and P39) adjuvanted with CpG oligodeoxynucleotide (ODN) induced a Th1-type immune response, but only the recombinant P39 plus CpG ODN induced a significant level of protection against B. abortus 544 challenge (2).

The present study shows that intramuscular (i.m.) immunization of mice with pCIBFR or pCIP39 (expression vectors for BFR and P39, respectively) generates a strong and long-lived specific T-cell response, whereas only pCIP39 induces a strong humoral response. However, the Th1 immune response induced by pCIBFR does not protect mice against B. abortus 544 following intraperitoneal (i.p.) challenge, whereas pCIP39 induced a moderate level of protection compared to the S19 vaccine.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. B. abortus 544 (virulent strain) was obtained from J.-M. Verger (Institut National de la Recherche Agronomique, Pathologie Infectieuse et Immunologie, Nouzilly, France), and B. abortus S19 was obtained from J. Godfroid. Brucella cells were grown on 2YT agar. For vaccination or challenge, the colonies were suspended in a sterile phosphate-buffered saline (PBS), bacterial cells were washed twice, and the number of bacteria was measured by determining the CFU on 2YT (10 g of yeast extract, 10 g of tryptone, and 5 g of NaCl per liter) agar plates.

Plasmid preparation. The coding sequences of the Brucella p39 gene (flanked by XbaI/EcoRI sites) and the Brucella bfr gene (flanked by XbaI/XhaI sites) were ligated into the multiple-cloning site of the mammalian expression vector pCI (Promega, Madison, Wis.), giving pCIP39 and pCIBFR, respectively. Escherichia coli DH5 cells (Stratagene, La Jolla, Calif.) were transformed with the plasmids and grown in Luria-Bertani broth containing ampicillin (100 µg/ml). Plasmid DNA for in vitro transfection or mouse immunization, was extracted from a 16-h culture and purified using the Endo-Free Plasmid Giga kit (Qiagen, Chatsworth, Calif.). The concentration and purity of the plasmid was determined by measuring the optical density ratio A₂₆₀/A₂₈₀. Plasmid DNA was adjusted to a final concentration of 1 mg/ml in PBS and stored at 80°C.

Polyclonal antibodies against BFR. Purified BFR (0.1 mg) was mixed with 1 ml of Freund's adjuvant (complete for the initial injection; incomplete for subsequent intramuscular injections) and injected into a rabbit that was previously bled to collect preimmune serum. Three inoculations were performed at 3-week intervals. Antisera were collected 10 days after the last injection; antisera used in this work were used at working dilutions of 1:500.

Antigen expression in COS-7 cell line. Monkey kidney COS-7 epithelial cells were grown at 37°C in 5% CO₂ in six-well plates (Falcon) containing Dulbecco's modified Eagle's medium (DMEM) (Gibco, BRL) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 U/ml), and gentamicin (50 µg/ml), and subconfluence monolayers were washed once with serum-free DMEM; afterwards, 500 µl of DMEM (supplemented as above, but without FBS) was added. Then, 100 µl of transfection mixture (100 µl of serum-free DMEM containing 6 µg of Fugene6 [Boehringer, Mannheim, Germany]) and 1 µg of plasmid DNA were held at room temperature (RT) for 5 min and added to the cells, which were then incubated at 37°C in 5% CO₂ overnight. Expression of BFR and P39 proteins was detected by immunoblotting.

SDS-PAGE and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as previously described (56, 61). After 24 h of transfection COS-7 cells were lysed by freeze-thawing cycles. Cell lysates were centrifuged at 12,000 × g for 5 min and then analyzed by immunoblotting, by using anti-BFR or anti-P39 antibodies (37).

Mice DNA vaccination and challenge. Specific-pathogen-free 4-week-old BALB/c female mice were purchased, from Iffa-Credo, Brussels, Belgium. Six-week-old mice randomly allocated in three groups of 16 mice, received i.m. injections in the tibialis anterior muscles with 100 µg of pCIBFR, pCIP39, or pCI as a negative control in 50 µl sterile saline (PBS), by using a 1-ml insulin syring with a 28-gauge needle. Three vaccinations at 3-week intervals were performed. The immune response (four mice per group) was analyzed 3 and 12 weeks after the last DNA vaccination. Positive control mice (n = 8) received an intraperitoneal (i.p.) injection with 5 × 10⁴ CFU of B. abortus vaccine strain S19 in 100 µl of sterile PBS 4 weeks before the challenge.

Quantitation of bacteria in the spleen. At 4 and 8 weeks postchallenge, mice (n = 4) were killed by cervical dislocation, and their spleens were removed, homogenized in 2 ml of sterile PBS, serially 10-fold diluted, and plated in triplicate. B. abortus 544 colonies were counted after 3 days of incubation at 37°C with 10% CO₂.

Isotype-specific Ig ELISAs. Expression and purification of the recombinant P39 and recombinant BFR were described previously (2, 37). Briefly the respective genes were cloned into a pET-15b expression vector (Novagen, Madison, Wis.), and the resulting plasmid was introduced in E. coli BL21 (DE3). After induction with IPTG (isopropyl--D-thiogalactopyronoside) (Promega) the His-tagged proteins were purified from the pellet lysate on a Ni-affinity chromatography column (Pharmacia Biotech).

Lymphocyte proliferation assay. Proliferation is determined by measuring the level of incorporation of [³H]thymidine into the DNA of actively dividing cells. The spleens were removed and homogenized with RPMI 1640 medium (Gibco, BRL) containing 10% FBS, the cells were centrifuged for 10 min at 1,200 × g, and the cell-containing pellets were washed two times with RPMI 1640 supplemented with 5% FBS. Erythrocytes in spleen cell preparations were lysed with Gey's solution, by incubating the mixture for 10 min at 4°C. The lymphocytes were washed two times in RPMI 1640 containing 5% FBS by centrifugation. Splenocytes resuspended in complete medium (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, penicillin-streptomycin [100 U/ml], gentamicin [50 µg/ml], and 5 × 10⁵ M 2-mercaptoethanol) were cultured at 2 × 10⁵ cells per well in 96-well flat-bottom microwell plates. Cultures in quadruplicate were stimulated with 1 µg of BFR or P39 or 3 µg of B. abortus 544 or B. melitensis 16M. Concanavalin A (ConA) (Sigma, St. Louis, Mo.) at a concentration of 0.5 µg per well and RPMI were used as positive and unstimulated controls, respectively. After 72 h at 37°C in a humidified 5% CO₂ incubator, cells were pulsed with 0.5 µCi of [³H]thymidine/well (ICN) for 18 h. The cells were harvested onto glass filter strips (Skatron Inc., Sterling, Va.). Tritiated-thymidine incorporation was counted by liquid scintillation spectroscopy with a Betaplate counter (WALLAC Oy, Turku, Finland). The mean number of counts per minute and standard error of the mean for each quadruplicate set of cells were determined.

Cytokine quantitation. Levels of IFN- and interleukin-5 (IL-5) in murine splenocyte culture supernatants were measured after 96 h of incubation with antigen or mitogen as described for the lymphocyte proliferation assay. IFN- and IL-5 were assayed according to the manufacturer's instructions using specific enzyme-linked immunosorbent assay (ELISA) kits (Pharmingen, San Diego, Calif.).

Statistical analysis. The P value was calculated by using the Student t test, and a P value of <0.05 was considered to be significant.

	RESULTS

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Construction of pCIBFR and pCIP39 expression vectors. To evaluate the relative role of the BFR and P39 proteins in inducing an immune response and protective immunity against brucellosis, the bfr and p39 coding sequences were inserted into eukaryotic expression vector, pCI, to produce plasmids pCIBFR and pCIP39, respectively.

View larger version (46K): [in this window] [in a new window]   FIG. 1.   In vitro expression of BFR and P39 was tested by transient transfection of COS-7 cells. The cells were transfected with either pCI (lanes 2 and 4), pCIBFR (lane 1), or pCIP39 (lane 3). After 24 h, the COS-7 cells were washed and lysed. The proteins were separated by SDS-PAGE, and the antigens were then detected by immunoblotting, using rabbit anti-BFR polyserum (lanes 1 and 2), or anti-P39 monoclonal antibody (lanes 3 and 4). Molecular mass standards (lane M.M.) are indicated in kilodaltons.

The humoral response to BFR or P39 induced by DNA vaccination. Sera collected 3 and 12 weeks after the last vaccination were assayed for the presence of BFR- or P39-specific antibodies by indirect ELISA using the relevant purified recombinant proteins, and Brucella whole-cell extracts as antigens. As shown in Table 1, 3 weeks after the last immunization, mice injected with pCIBFR have weak antibody IgG2a titers to BFR protein and Brucella extracts while no IgG1 could be detected. The pCIP39 immunizations induced antibody IgG2a responses to P39 and Brucella extracts that were 1.5 to 2 logs higher than these induced by pCIBFR injections. This antigen also induced IgG1 titers which were slightly lower than the IgG2a titers. Antibodies against P39 could already be detected 1 week after the first pCIP39 DNA injection (data not shown). Immunization with pCI or pCIBFR did not induce any production of anti-P39 antibodies. Humoral response measured 12 weeks postvaccination indicates that pCIP39 DNA vaccine leads to the generation of long-lived IgG1 and IgG2a responses (Table 2). By contrast, at 12 weeks postvaccination, no more antibody against BFR could be demonstrated.

T-cell-proliferative response by splenocytes from DNA-vaccinated mice. To further investigate the cellular immune response induced by the plasmid vectors we analyzed the proliferative T-cell response.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Lymphocyte proliferation assay. BALB/c mice were immunized with pCI, pCIBFR, or pCIP39. T-cell-proliferative responses were measured at week 3 (A) and 12 (B) after the last immunization. Splenocytes from each mouse were prepared and stimulated in vitro with purified recombinant BFR or P39 (1 µg/ml) and Brucella extracts (3 µg/ml) as the antigen. After 72 h of culture, [3H]thymidine was added; 18 h later, cells were harvested and the counts per minute were determined. Each sample was assayed in quadruplicate. Data represent the mean ± standard deviation (error bars) from the four mice.

IFN- and IL-5. Cytokine production profiles from splenocytes of four vaccinated mice per group, 3 and 12 weeks after the last vaccination, were examined after restimulation with different antigens. As shown in Fig. 3, splenic lymphocytes from pCIP39-vaccinated mice produced up to 2.6 ng of IFN- per ml 3 weeks postvaccination or up to 1.7 ng per ml 12 weeks postvaccination upon restimulation with P39. Lower, while significant, IFN- levels were obtained upon stimulation with Brucella extract. By contrast, little if any IL-5 was measured (data not shown). Spleen cells from animals immunized with pCIBFR also secreted large amounts of IFN- upon restimulation with BFR, 2.4 ng per ml at 3 weeks and 1 ng per ml at 12 weeks (Fig. 3A). We could not detect IL-5 secretion from cells obtained from this group (data not shown). Mice vaccinated with pCI produced neither IFN- or IL-5 upon restimulation with the antigens. The RPMI did not induce IFN- or IL-5 production, whereas ConA induced a large quantity of IFN- (Fig. 3) and a minimal amount of IL-5 (data no shown). Taken together, these results indicate that there was a significant Th1 biased response after the immunization with pCIBFR or pCIP39.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Quantitative ELISA analysis of IFN- secretion by murine lymphocytes isolated after DNA immunization (pCI, pCIBFR, or pCIP39) and subsequent stimulation in culture using different antigens. Splenocytes from the immunized mice (four mice/group) were harvested at week 3 (A) and 12 (B) after the last immunization. Assays were performed on supernatants of 2 × 106 lymphocytes per ml. Lymphocytes were restimulated with the indicated antigens. At 96 h after restimulation, supernatants were analyzed for IFN- levels. Samples were tested in duplicate.

Protection against B. abortus 544 challenge in DNA-immunized mice. To determine whether the immunization with the candidate DNA vaccines induced protection against B. abortus 544 infection, mice immunized three times with either pCIBFR or pCIP39 or pCI were infected 4 weeks later with B. abortus 544. Four and eight weeks after challenge, mice were killed and CFU in the spleens were quantitated. The mice which were immunized by pCIP39 showed only a slight but significant level of protection at 8 weeks after challenge (Table 3). No significant difference in the number of the bacteria isolated from the spleens of the nonvaccinated and pCIBFR- or pCI-vaccinated animals was observed (Table 3). In contrast, at a vaccination-to-challenge interval of 4 weeks, mice vaccinated with strain S19 demonstrated a significant protection, which increased between 4 (2 log₁₀) and 8 weeks postinfection (3.08 log₁₀).

	DISCUSSION

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Vaccination continues to be the most successful procedure for preventing losses in animals due to infectious diseases. The development of new-generation vaccine systems to prevent brucellosis is important to avoid the disadvantages of the currently used live vaccines. These new vaccines will be designed to generate immune responses mimicking those found in animals during natural infection. It is well established that protection against infection by an intracellular pathogen, including Brucella, requires the generation of potent cell-mediated immunity linked to the induction of a Th1-type immune response over time (19, 41). IFN-, which is a key Th1 cytokine, plays a prominent function in up-regulation of macrophage anti-Brucella activity and, with tumor necrosis factor alpha, is considered crucial for protection against Brucella spp. (5, 6, 28, 29, 52, 62).

The achievement of this goal for a new brucellosis vaccine implies both the identification of adequate delivery or adjuvant system and the analyses of major immunodominant antigens. Many antigens of Brucella have been tested with or without a variety of adjuvants. Among these antigens, Cu-Zn superoxide dismutase, Yajc, L7-L12, and GroELs could induce a humoral or/and cellular immune response in mice, but only L7-L12 and certain epitopes of the Cu-Zn superoxide dismutase could induce some level of protection (38, 42, 53, 60). We recently described that the addition of CpG ODN adjuvants to the recombinant P39 protein can induce a strong anti-Brucella Th1 response that is able to significantly reduce the splenic bacterial load after a challenge with B. abortus 544 (2). With the P39-CpG ODN vaccine the level of protection was high (2.48 log₁₀) 4 weeks after the challenge and similar to the protection conferred by the S19 vaccine control. Nevertheless, at 8 weeks postinfection while the S19 induced protection remained high, the protective activity of the P39 subunitary vaccine vanished (1.21 log₁₀). In order to achieve a long-term protection with this promising P39 antigen, we investigated the naked DNA vaccine approach.

DNA vaccines, because of prolonged in situ antigen production, can elicit long-lasting humoral and cellular immune responses (21, 25, 36, 57). DNA vaccines also produce antigen in a highly immunogenic form because of the processing via major histocompatibility complex class I and II (14, 16, 48, 51). Furthermore, the plasmidic vector contains a built-in adjuvant effect (51). The CpG motifs in the bla gene stimulate the innate immune system to create a cytokine milieu (e.g., IL-12, IFN-, IFN-, IL-2, and tumor necrosis factor beta [1, 15, 59]) that favors the generation of Th1 response against the antigen encoded by the plasmid (26, 32).

In this study, we constructed eukaryotic expression vectors for BFR and P39 Brucella proteins, and we showed that the BFR and P39 proteins were expressed intracellularly in COS-7 cells transfected with pCIBFR and pCIP39 plasmids, respectively. We evaluated the capacity of these constructs to elicit immune responses and protective immunity in BALB/c mice.

Three weeks after the last vaccination we found a weak titer of specific IgG2a in mice immunized i.m. with plasmid pCIBFR (Table 1). In contrast, pCIP39 induced high titers of anti-P39 antibodies (IgG1 and IgG2a), also reacting against Brucella extracts that were still present up to 12 weeks after the last vaccination (Table 2). The IgG1/IgG2a ratio was, approximately, equal to 1. Since the isotype profile of antibody response is a reflection of the T-helper-cell types (27, 40), these results suggest that i.m. pCIP39 or pCIBFR DNA vaccination induced Th1 responses. Mice vaccinated i.m. with pCI did not produce detectable antibodies against BFR or P39 antigens (Tables 1 and 2). Those data paralleled the results obtained with the adjuvanted recombinant proteins (2), the BFR antigen being less able to induce a strong antibody response than P39. This is also in agreement with the demonstrated potency of recombinant P39 protein to serve as serological diagnostic antigen in ELISA (38).

The induction of T-cell immune responses after DNA immunization was then evaluated by measuring T-cell-proliferative and cytokine responses after in vitro stimulation of splenic cells with purified recombinant BFR or P39 proteins or Brucella total extracts. Both BFR and P39 induced a high T-cell-proliferative response (Fig. 2) and high levels of IFN- (Fig. 3) but no IL-5. While we do not look for IL-4 synthesis, the existence of IgG1 antibodies specific for P39 indicates that IL-4 is also somehow involved in this response. Furthermore, this DNA immunization induced memory T cells since we still found a good immune response 12 weeks postvaccination. Even a single dose of DNA immunization (pCIBFR and pCIP39) was able to elicit a detectable immune response (data not shown). Once against these results are in agreement with those obtained with the CpG adjuvanted recombinant proteins, which were both able to induce a strong Th1 type immune response.

On the basis of these data we started to analyze the protective efficacy of the pCIBFR or pCIP39 DNA vaccines against a B. abortus challenge.

Whatever the time postchallenge considered, neither the pCIBFR vaccine nor pCI vector alone was able to confer a significant protection level (Table 3). Those results were reminiscent of the results gained with recombinant BFR, which was unable to protect, while inducing a strong specific Th1 immune response (2).

The level of protection conferred by the pCIP39 vaccine was low becoming only significant (0.73 logs) at 8 weeks postchallenge, while still remaining less efficient than the S19 vaccine. We also found that coinjection of both expression vectors did not enhance the immune response or the level of protection (data non shown).

It was previously reported that a DNA vaccine encoding the L7-L12 ribosomal protein induced an appropriate immune response, and conferred, in a B. abortus 2308 challenge experiment, a significant level of protection of 0.53 and 1.26 log units at 20 and 30 days after the last immunization, respectively (34). On the other hand the recombinant L7-L12 adjuvanted with monophosphoryl lipid A induced 1.13 and 1.21 log units of protection at 14 and 28 days after the last immunization, respectively (42). These results demonstrated that these two vaccine preparations induced approximately the same level of protection 4 weeks postinfection. Surprisingly, in our case, the pCIP39 vaccine was less efficacious than the recombinant P39 protein adjuvanted with CpG ODN. Considering the overall similarity (as far as we can see) of the immune response induced by the two different vaccinal preparations, the discrepancy between the level of protection achieved is striking.

Reasonable explanations for the low protection conferred by pCIP39 may be searched for at the level of the antigen (quantity, localization, etc.), at the level of the adjuvantation or may be caused by subtle differences between immune response elicited by the two vaccinal preparations.

The production of P39 detected in transiently transfected COS-7 cells and the immune response induced in vaccinated mice argue for an in vivo expression. Nevertheless, the amount of antigen produced in vivo after DNA immunization is usually in the picogram or the nanogram range (23, 43). So the total level of P39 protein production in vivo, while believed to be sustained, could still be less than the amount of recombinant protein administered (three administrations of 20 µg) in the previous experiment, although the systemic immune parameters we measured were almost undistinguishable from the parameters measured when the mice were immunized with recombinant proteins. So, notwithstanding the quantity of antigen, the naked DNA immunization, compared to the adjuvanted recombinant protein, could also evoke an immune response subtly different in quality or localization, leading to the observed difference in the protective efficacy.

Alternatively, we demonstrated previously that neither the recombinant protein nor the CpG ODN given alone was able to induce protection and that the combination of both was required. So, another explanation could be that the adjuvant effect of the CpG in the naked DNA vaccine is less potent than the CpG ODN used with the recombinant protein, probably because it is less abundant.

In spite of the relative failure of the pCIP39 vaccine as used in this study, we remain confident of the potential of this antigen, and the testing of new formulations of the pCIP39 plasmid (e.g., adding CpG ODN as adjuvant [32], combining the DNA plasmid with a final protein boost [55], or using a live delivery vector [18, 50]) offers the opportunity to increase the potency of this candidate vaccine. This is currently under investigation in our laboratory. In addition, it would also be interesting to use several antigens as DNA-encoded vaccines (e.g., encoding L7-L12 and P39) in order to increase the level of protection against the Brucella challenge.