Brucella abortus strain RB51 is an attenuated rough strain that is currently being used as the official live vaccine for bovine brucellosis in the United States and several other countries. We reasoned that overexpression of a protective antigen(s) of B. abortus in strain RB51 should enhance its vaccine efficacy. To test this hypothesis, we overexpressed Cu/Zn superoxide dismutase (SOD) protein of B. abortus in strain RB51. This was accomplished by transforming strain RB51 with a broad-host-range plasmid, pBBR1MCS, containing the sodC gene along with its promoter. Strain RB51 overexpressing SOD (RB51SOD) was tested in BALB/c mice for its ability to protect against challenge infection with virulent strain 2308. Mice vaccinated with RB51SOD, but not RB51, developed antibodies and cell-mediated immune responses to Cu/Zn SOD. Strain RB51SOD vaccinated mice developed significantly (P < 0.05) more resistance to challenge than those vaccinated with strain RB51 alone. The presence of the plasmid alone in strain RB51 did not alter its vaccine efficacy. Also, overexpression of SOD did not alter the attenuation characteristic of strain RB51.Intracellular bacteria are responsible for several important infectious diseases of animals and humans. Cell-mediated immune (CMI) responses play a critical role in resistance against intracellular bacterial infections (7). Live bacterial vaccines are considered essential for effectively inducing the appropriate protective CMI responses. Usually, attenuated strains of bacteria are used as live vaccines for intracellular bacterial infections. However, in many cases, even these live vaccines cannot provide high levels of protection. We hypothesized that overexpression of a bacterial protective antigen(s) in its vaccine strain would result in enhancement of the vaccine's efficacy. Our studies with Brucella abortus vaccine strain RB51 validate this hypothesis.Members of the genus Brucella are small gram-negative, facultatively intracellular bacteria of zoonotic importance (1). These bacteria are causative agents of brucellosis, a chronic disease of animals and humans. In animals, this disease often results in infertility and abortions leading to severe economic losses to livestock producers (5). Humans acquire the infection by coming in contact with the infected materials or by consuming contaminated meat or dairy products. B. abortus is primarily responsible for brucellosis in cattle. B. abortus strain RB51, an attenuated rough mutant developed in our laboratory (20), is presently being used in several countries as a live vaccine for the control and eradication of brucellosis in cattle. Similar to most of the intracellular bacterial infections, CMI appears to play a major role in acquired resistance to brucellosis, although antibodies to surface antigens, especially to the O antigen, can confer certain level of protection against a challenge infection in some host species, such as the mouse (2, 5). Studies of mice indicate that protection afforded by strain RB51 vaccination is primarily th...

A safe, more sensitive, nonradioactive, neutral red uptake assay was adopted to replace the traditional 51 Cr release assay for detection of Brucella-specific cytotoxic T lymphocyte (CTL) activity. Our studies indicated that Brucella abortus strain RB51 vaccination of mice induced specific CTLs against both strain RB51-and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigenspecific cytotoxic activity was exerted by T lymphocytes but not by NK cells. CD3 ؉ CD4 ؉ T cells secreted the highest level of gamma interferon (IFN-␥) and were able to exert a low but significant level of specific lysis of Brucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3؉ CD8 ؉ T cells secreted low levels of IFN-␥ but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis. These findings indicate that B. abortus strain RB51 vaccination of mice induces specific CTLs and suggest that CD3 ؉ CD4 ؉ and CD3 ؉ CD8 ؉ T cells play a synergistic role in the anti-Brucella activity.Brucella abortus is a gram-negative, facultative intracellular bacterial pathogen that causes brucellosis in humans and cattle (4). In the infected host, B. abortus multiplies within the phagosomes of phagocytic cells (e.g., macrophages) by inhibiting the phagosome-lysosome fusion (4). Similar to most of the intracellular bacterial infections, cell-mediated immunity seems to play a critical role in protection against virulent Brucella infection, although antibodies specific for the O polysaccharide of the lipopolysaccharide can confer a certain level of protection in some host species (1, 2, 17, 31). Passive transfer assays with mice indicated that both CD4 ϩ and CD8 ϩ T-cell subsets are involved in the protective immunity to brucellosis (1, 2). One mechanism by which immune T cells confer protection from B. abortus infection is by secreting molecules such as gamma interferon (IFN-␥), which stimulates the antimicrobial activity of macrophages, allowing intracellular bacterial killing (16,35). The crucial role of IFN-␥ in resistance to Brucella infection has been demonstrated for mice by in vivo antibody neutralization experiments (35). Another mechanism of T-cellmediated immunity is the lysis of infected cells by the specific cytotoxic T lymphocytes (CTLs) (23). Present knowledge about the role of CTLs in the acquired resistance to brucellosis is limited. The development of Brucella-specific CTLs in vaccinated animals and the phenotypic and functional characterization of such CTL's have not been studied in detail. The classic CTL assay is based on determining the level of 51 Cr released from lysed target cells. Unfortunately, this assay is not very sensitive (23,24), and the use of radioactive 51 Cr in a biosafety level 3 environment restricts the usefulness of this assay in Brucella research even further. Therefore, we have developed a highly sensitive, nonradioactive assay for Brucella...

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