znuA is known to be an important factor for survival and normal growth under low Zn 2؉ concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (⌬znuA) was constructed and found to be lethal in low-Zn 2؉ medium. When used to infect macrophages, ⌬znuA B. abortus showed minimal growth. Further study with ⌬znuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the ⌬znuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain. Zn2ϩ is an essential mineral required by bacteria as either a structural or catalytic cofactor (32). Bacterial survival and proliferation in the environment and within animal hosts are critically dependent on the uptake and sequestration of transition metals, such as Zn 2ϩ (4). This is problematic, because free Zn 2ϩ concentrations in mammalian hosts are very low, so as to prevent bacterial colonization. To acquire the necessary Zn 2ϩ for its metabolism, bacteria have evolved several types of proteins that are involved in binding and transporting zinc (9).The translation products of the znuABC operon found in Escherichia coli (5, 31), Haemophilus spp. (27), Neisseria gonorrhoeae (8), Pasteurella multocida (15), and Synechocystis sp. strain 6803 (4) constitute a high-affinity periplasmic binding protein-dependent and ATP-binding cassette (ABC) transport system for Zn 2ϩ . In gram-negative bacteria, ABC transporters are involved in the active transport of molecules from periplasm to the cytosol (19). In addition, znuA mutants in H. ducreyi (27) and P. multocida (15) were found to be significantly less virulent than wild-type strains when tested in animal models.B. abortus is a gram-negative facultative, intracellular pathogen capable of infecting both wildlife and livestock (7), and it is able to cause severe zoonotic infection in humans (3, 34). Currently, there are no human Brucella vaccines, and current livestock vaccines such as S19 and RB51 are virulent in humans. Attempts to develop live brucellae vaccines have met with varied success. For instance, inactivation of the amino acid biosynthesis pathway genes pheA, trpB, and dagA displayed little or no attenuation in cultured murine macrophages or in mice (1). The mutants of purine biosynthesis pathway genes purL, purD, and purE (1) displayed significant attenuation in BALB/c mice, but live brucellae remained viable after 12 weeks, suggesting that their virulence was not sufficiently attenuated for adoption as...

Zoonotic transmission of brucellosis often results from exposure to Brucella-infected livestock, feral animals, or wildlife or frequently via consumption of unpasteurized milk products or raw meat. Since natural infection of humans often occurs by the oral route, mucosal vaccination may offer a means to confer protection for both mucosal and systemic tissues. Significant efforts have focused on developing a live brucellosis vaccine, and deletion of the znuA gene involved in zinc transport has been found to attenuate Brucella abortus. A similar mutation has been adapted for Brucella melitensis and tested to determine whether oral administration of ⌬znuA B. melitensis can confer protection against nasal B. melitensis challenge. A single oral vaccination with ⌬znuA B. melitensis rapidly cleared from mice within 2 weeks and effectively protected mice upon nasal challenge with wild-type B. melitensis 16M. In 83% of the vaccinated mice, no detectable brucellae were found in their spleens, unlike with phosphate-buffered saline (PBS)-dosed mice, and vaccination also enhanced the clearance of brucellae from the lungs. Moreover, vaccinated gamma interferon-deficient (IFN-␥ ؊/؊ ) mice also showed protection in both spleens and lungs, albeit protection that was not as effective as in immunocompetent mice. Although IFN-␥, interleukin 17 (IL-17), and IL-22 were stimulated by these live vaccines, only RB51-mediated protection was codependent upon IL-17 in BALB/c mice. These data suggest that oral immunization with the live, attenuated ⌬znuA B. melitensis vaccine provides an attractive strategy to protect against inhalational infection with virulent B. melitensis.Brucellae are Gram-negative intracellular bacterial pathogens of both humans and animals. Brucellosis, caused primarily by Brucella melitensis, Brucella abortus, Brucella ovis, and Brucella suis (12,22

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against ∼1000 LD50 Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD50 Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein 1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein 1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cellmediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500-fold, with more than 50% of vaccinated mice showing no detectable brucellae. Furthermore, tenfold more brucellae-specific, IFN-γ-producing CD8+ T cells than CD4+ T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8+ T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44hiCD62LloCCR7lo) T cells producing elevated levels of IFN-γ, TNF-α, perforin, and granzyme B. To assess the relative importance of these increased numbers of CD8+ T cells, CD8−/− mice were challenged with virulent B. melitensis, and they showed markedly increased bacterial loads in organs in contrast to similarly challenged CD4−/− mice. Only ΔznuA B. melitensis- and Rev-1-vaccinated CD4−/− and wild-type mice, not CD8−/− mice, were completely protected against Brucella challenge. Determination of cytokines responsible for conferring protection showed the relative importance of IFN-γ, but not IL-17. Unlike wild-type mice, IL-17 was greatly induced in IFN-γ−/− mice, but IL-17 could not substitute for IFN-γ’s protection, although an increase in brucellae dissemination was observed upon in vivo IL-17 neutralization. These results show that nasal ΔznuA B. melitensis vaccination represents an attractive means to stimulate systemic and mucosal immune protection via CD8+ T cell engagement.

The common mucosal immune system may be compartmentalized because lymphocyte homing to the upper respiratory tract appears to be mediated by L-selectin interactions rather than α4β7 interactions, as is the case for gut-associated lymphoreticular tissue. To assess the role of L-selectin in effector B cell immunity, L-selectin-deficient mice were intranasally immunized with cholera toxin (CT), and mucosal immune responses were compared with C57BL/6 mice. The absence of L-selectin correlated with a reduction in CT-specific secretory-IgA responses in nasal passages and reproductive tract, but not intestinal lamina propria. Cell sorting experiments showed that an L-selectin-dependent subset was responsible for CT-specific responses in nasal passages and reproductive tract, whereas an αEβ7+ B cell subset was responsible for L-selectin-independent intestinal immunity. This study provides evidence for compartmentalization of the common mucosal immune system into “intestinal” vs “nonintestinal” effector sites.

The Brucella melitensis 16M genome was examined for proteins in excess of 100 amino acids and for immunogenicity-associated genes. One subset of 32 annotated genes or open reading frames was identified, and each of these were cloned into the eukaryotic vector pcDNA3.1. Purified recombinant plasmids were used to intramuscularly (i.m.) immunize BALB/c mice. After challenge with B. melitensis 16M strain, two protective antigens were found: the periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). Protective efficacy was confirmed with DNA vaccines for these two B. melitensis proteins and, when combined, protection against wild-type challenge was significantly enhanced. Both proteins were found to be immunogenic since elevated serum immunoglobulin G (IgG) antibodies without a specific IgG subclass bias were induced subsequent to i.m. DNA immunization. Antigen-restimulation assays revealed that bp26 and TF stimulated gamma interferon and only bp26 induced interleukin-4 (IL-4), IL-5, and IL-6 cytokines as measured by cytokine enzyme-linked immunospot assay. These collective results suggest that both bp26 and TF are excellent candidates for use in future vaccination studies against brucellosis.Brucella spp., facultative intracellular pathogens, are the etiological agents of brucellosis, a disease that affects livestock and humans (9). The attenuated strains such as Brucella melitensis Rev1 and B. abortus S19 and RB51 are used to control brucellosis in domesticated animals. However, these are less than ideal because of their limited efficacy and potential to cause disease in humans. Moreover, both B. abortus S19 and B. melitensis Rev1 strains induce antibodies to their lipopolysaccharide (LPS), making it difficult to differentiate vaccinated animals from those naturally infected (3,17,20). Recently, Brucella spp. have also been recognized as a bioterror threat by the Centers for Disease Control (16). Therefore, a subunit vaccine that is protective against B. melitensis is desirable.DNA vaccines offer a promising approach because they can stimulate both cellular and humoral immunity (13, 26). Furthermore, DNA vaccines have many advantages over traditional protein-based vaccines, including ease of development, induction of long-lived immunity, and minimal preparation costs. With regard to effectiveness, previous studies have already shown that DNA vaccination with sodC (22), lumazine synthase gene (27), and P39 (2) can elicit partial protection against Brucella challenge. Furthermore, in contrast to live attenuated vaccines, there are no concerns of induced disease, and the DNA vaccines are stable.With the completion of sequencing the Brucella genome, identification of novel protective antigens is feasible. In the present study, we applied a search strategy to screen the B. melitensis 16M genome for potential immunogenic antigens. By cloning these potential antigen candidates into the pcDNA3.1 vector and testing their efficacy in BALB/c mice, two protective antigens were identified.

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