This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.

In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens of Brucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-␥), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4؉ -and CD8 ؉ -T-cell populations. However, only the CD4 ؉ population was able to produce IFN-␥ and only the CD8 ؉ population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulent B. abortus strain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-␥ production and cytotoxic activity against infected cells by SOD-specific CD4 ؉ and CD8 ؉ T cells, respectively.Brucellosis is a zoonotic disease that is endemic in some regions of the world. In human populations, the major cause of the disease is Brucella melitensis, but several cases have also been attributed to Brucella abortus, which otherwise primarily affects bovines. Because of the economic losses to the cattle industry caused by B. abortus, as well as because of the zoonotic infections by these bacterial species (8), great efforts are being made to eradicate bovine brucellosis all over the world. In order to achieve this objective, vaccine strains of B. abortus 32) have been used with relatively good results. However, even these vaccine strains are far from ideal, since they present some disadvantages, e.g., causing reactions in humans, inducing abortion in pregnant cattle, and showing a likelihood of changing to a virulent form (33).Brucella is an intracellular pathogen; therefore, cellular immune response is critical in generating protection against infection (42). It is well documented that gamma interferon (IFN-␥) production by CD4 ϩ T cells is essential to the protective response; IFN-␥ activates macrophages by enhancing their ability to kill bacteria (18,20,34,43). It is still unknown if there is a correlation between the degree of in vitro cytotoxic-Tlymphocyte (CTL) activity and in vivo levels of pro...

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We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.Brucella abortus is gram-negative, facultative intracellular bacterial pathogen that causes brucellosis in humans and cattle. In the infected host B. abortus multiplies within the phagosomes of reticuloendothelial cells, avoiding the killing effect of the macrophage cells by inhibiting phagosome-lysosome fusion (22). Like resistance to other facultative intracellular bacterial pathogens, resistance to B. abortus depends on acquired cellmediated immunity (CMI) (51). In this respect, the development of the Th1 subset of CD4 ϩ lymphocytes that secrete gamma interferon (IFN-␥), a crucial cytokine that up-regulates the macrophage anti-Brucella activity, and the development of CD8 ϩ T lymphocytes that are able to lyse Brucella-infected cells (33,34) are the two main components of the protective response. Live, attenuated vaccines, such as B. abortus strains S19 and RB51, that can stimulate a strong CMI response are usually very effective against brucellosis and have been used to control brucellosis in domestic animals. However, these vaccine preparations are far from ideal, since they have disadvantages; e.g., they are considered virulent or unsafe for human use, and they induce abortion in pregnant cattle (43). In the search for methods that can provide immunity against brucellosis, DNA-based vaccines have been shown to be effective for the delivery of antigenic proteins (3, 22) and to generate a strong cellular response (28). In previous reports, we showed that a DNA vaccine encoding the Cu,Zn superoxide dismutase (SOD) protein of B. abortus is able to induce cytotoxic Tlymphocyte (CTL) activity, high levels of IFN-␥ production, and a significant level of protection against challenge with B.abortus virulent strain 2308 in BALB/c mice (30, 37). However, repeated doses and high c...