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Infect Immun. 2009 November; 77(11): 5181–5189.
Published online 2009 August 24. doi:  10.1128/IAI.00231-09
PMCID: PMC2772552

Nondividing but Metabolically Active Gamma-Irradiated Brucella melitensis Is Protective against Virulent B. melitensis Challenge in Mice[down-pointing small open triangle]


Brucella spp. are gram-negative bacteria that cause the most frequent zoonotic disease worldwide, with more than 500,000 human infections yearly; however, no human vaccine is currently available. As with other intracellular organisms, cytotoxic mechanisms against infected cells are thought to have an important role in controlling infection and mediating long-term immunity. Live attenuated strains developed for use in animals elicit protection but retain unacceptable levels of virulence. Thus, the optimal design for a brucellosis vaccine requires a nonliving vaccine that confers effective immunity. Historically, inactivation methods such as chemical or heat treatment successfully impair Brucella reproductive capacity; nevertheless, metabolically inactive vaccines (subunit or killed) present very limited efficacy. Hence, we hypothesized that bacterial metabolism plays a major role in creating the proper antigenic and adjuvant properties required for efficient triggering of protective responses. Here, we demonstrate that inactivation of Brucella melitensis by gamma-irradiation inhibited its replication capability and yet retained live-Brucella protective features. Irradiated Brucella possessed metabolic and transcriptional activity, persisted in macrophages, generated antigen-specific cytotoxic T cells, and protected mice against virulent bacterial challenge, without signs of residual virulence. In conclusion, pathogen metabolic activity has a positive role in shaping protective responses, and the generation of inactivated and yet metabolically active microbes is a promising strategy for safely vaccinating against intracellular organisms such as B. melitensis.

Brucellosis is the most frequent zoonotic disease worldwide, with over 500,000 new human infections every year (30). Despite that astonishing fact, brucellosis is often neglected, being underdiagnosed and under-reported (17). Caused mainly by the intracellular bacteria species Brucella abortus, Brucella suis, and Brucella melitensis, manifestations of human brucellosis are inconsistent, making clinical diagnosis difficult. During initial stages of the disease, fever is a common symptom, while later outcomes are often manifested by focal infections, and almost every organ can be affected (29). Disease transmission occurs through the consumption of infected meat and dairy products, through inhalation of aerosols, or through skin wounds. There are currently no human vaccines. Treatment exists but can be costly and often prolonged, lasting at least 6 weeks in moderate cases and extending for years depending on complications. Nevertheless, even with extended treatment, disease relapse occurs in 16% of the patients treated with the antibiotic regimen recommended by the World Health Organization (40). PCR data indicate that bacteria are frequently detectable after therapy since 70% of individuals are tested positive more than 2 years after concluding treatment (43). Because of high cost, prolonged duration, and significant disease relapse and reinfection rates, antibiotic treatment is not an efficient solution for disease control worldwide. A vaccine may be more effective for individuals in high-risk groups. B. melitensis is the most virulent Brucella species to humans (29). Therefore, efforts to control human brucellosis should target this organism.

Live attenuated strains currently used as animal vaccines against B. melitensis are protective but still retain virulent traits. B. melitensis Rev-1 vaccine persists in animals and can cause fever, abortion, and granuloma formation (6, 20, 26, 28, 31). Furthermore, no gene deletion mutant has yet yielded a safe human vaccine (18). As a consequence, human brucellosis vaccines may benefit from inactivated vaccines that trigger comparable responses to immunity engendered by living organisms.

Living, but not inactivated Brucella spp., induces long-term protective immunity (25). Although it is clear that metabolically active brucellae support an appropriate environment for engendering host immunity, it remains unknown how the metabolic activity of the pathogen shapes downstream host-adaptive responses. In addition, the bacterial antigen correlation with immune protection and memory T-cell activation is unknown. Since the molecular characterization of protective immunity remains elusive, vaccines eliciting a broad repertoire of immune responses, comparable to responses engendered by living organisms, should provide an optimal Brucella vaccine.

Based on this concept of living organism mimicry, we generated a nondividing, but still metabolically and transcriptionally active vaccine by gamma irradiating B. melitensis. Irradiation has long been used as a method to prevent mammalian cell proliferation without inhibition of cellular activity (39). Irradiated microorganisms also retain metabolic activity, as suggested by researchers utilizing the irradiated bacteria B. abortus strain RB51 (38). Furthermore, other researchers demonstrated that the gamma-irradiated protozoan Toxoplasma gondii retains “morphology, metabolism, and cell invasion properties,” suggesting that the cellular functions were not abrogated by irradiation (19). The principal gamma-irradiation effect mediating loss of bacterial replication is accumulation of double-strand breaks by free radicals causing fragmentation of DNA (42). Nevertheless, a large portion of the genome remains intact after irradiation and, accordingly, the bacteria has the potential to express genes in these segments and synthesize and secrete proteins. Consequently, gamma-irradiated B. melitensis should likely contain the proper antigenic and adjuvant determinants necessary for an efficient immunization.

We demonstrate here that inactivation of B. melitensis by gamma irradiation leads to replication incompetence and yet retained all of the live protective features of Brucella. Irradiated brucellae possessed metabolic and transcriptional activity, persisted in macrophages, generated antigen-specific cytotoxic T lymphocytes (CTLs), and protected mice against virulent bacterial challenge without signs of residual virulence. Thus, gamma irradiation is a promising strategy for safely vaccinating against intracellular organisms such as B. melitensis.



Four- to six-week-old female BALB/c or four- to eight-week-old interferon regulatory factor 1 (IRF-1)-deficient mice on the C57BL/6 background (IRF-1−/−) (24) were used. All animal experiments were conducted with approval from the Institutional Animal Care and Use Committee and housed in AAALAC-accredited facilities.

Bacterial strains, growth, and gamma-irradiation conditions.

B. melitensis 16M (ATCC 23456) and the engineered bioluminescent strain GR023 (33) were grown in brucella broth (BB) alone and BB supplemented with kanamycin (50 μg ml−1), respectively. Log-phase cultures were collected and measured by spectrophotometry at an absorbance of 600 nm, and 1-ml aliquots in 1.5-ml microcentrifuge tubes were pelleted, resuspended in fresh BB or phosphate-buffered saline (PBS), and irradiated at room temperature at the indicated amounts using a Cs137 Mark I irradiator (J. L. Shepherd, San Fernando, CA). Irradiated bacteria samples were kept at 4°C until assayed.

Replicative viability and metabolic assays.

The replicative viability of irradiated bacteria was determined as growth on brucella agar. After irradiation, dose/kill curves were made by pelleting 1-ml aliquots and plating serial dilutions for up to 7 days before counting. Metabolic activity was assayed by using Alamar Blue (BioSource International, Camarillo, CA), incorporating a colorimetric growth indicator based on the detection of metabolic activity. Specifically, the system incorporates an oxidation-reduction indicator that changes color (blue to red) in response to the chemical reduction of growth medium resulting from bacterial metabolic activity. Briefly, irradiated samples were incubated in fresh medium (BB) at 37°C in 96-well optical plates with 10% Alamar Blue dye added. The absorbance was monitored at 570 nm (reduced) and 600 nm (oxidized) over time from 0 to 120 min. The percent reduction (equivalent to the metabolic activity) was determined by subtracting the 600-nm absorbance from the 570-nm absorbance and multiplying that value by 100. The replicative ability of heat-treated brucellae was performed with 1-ml aliquots of B. melitensis in 1.5-ml microcentrifuge tubes as with gamma irradiation. Samples were incubated in a 65°C water bath for 0, 15, 30, 45, 60, 75, and 90 min and then quenched on ice. Heat-treated samples were kept at 4°C until assayed. The replicative viability was assessed by dilution plating on agar (11), and the metabolic activity was measured by using an Alamar Blue assay as with the irradiated samples above.

Luminescent promoter assays.

Bioluminescent Brucella sp. strain GR023 (33) or B. melitensis carrying the plasmid pARL07 (PvirB BMEI0025-lux) (34) was used as a reporter system to evaluate de novo transcription and protein synthesis. Irradiated GR023 (350 and 1,000 kilorads) were replated in fresh medium in the presence or absence of a protein synthesis inhibitor (chloramphenicol, 50 μg/ml). The luminescence intensity was measured every 5 min for 1 h by using a Turner Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA) or biophotonic imaging (Caliper Biosciences, Hopkinton, MA). For VirB promoter assays, 5 × 104 RAW 264.7 cells were seeded in 96-well plates and left to adhere overnight. Cells were then washed (RPMI plus 10% fetal bovine serum) and incubated with live and irradiated B. melitensis (PvirB BMEI0025-lux) at a multiplicity of infection of 500. Bacteria samples preincubated for 24 h with gentamicin (50 μg/ml) to permanently inhibit protein synthesis were used as controls.

Cell infection and electron microscopy.

At 1 day prior to infection, splenic dendritic cells were separated by using CD11c+ magnetic beads (Miltenyi Biotec, Germany) according to the manufacturer's protocol, and 3 × 105 cells/well were seeded on sterile glass coverslips in a six-well plate in 2 ml of RPMI/well with 10% fetal calf serum (Invitrogen, Carlsbad, CA). The following day, the bacteria were counted by 600-nm absorbance spectrometry and added to washed eukaryotic cells in fresh medium without antibiotic at the specified multiplicity of infection. Bacteria were then centrifuged onto the monolayer at 2 krpm for 5 min at 4°C. The cells were incubated for 24 h with bacteria and washed with fresh medium supplemented with 30 μg of gentamicin/ml to kill the extracellular bacteria. After 48 h of infection, the cells were washed in PBS and fixed in Karnovsky fixative (Electron Microscopy Sciences, Hatfield, PA) according to the manufacturer's protocol. Transmission electron microscopy was performed at the University of Wisconsin Medical School Electron Microscope Facility ( Figures were imported using Adobe Photoshop CS3 10.0.1.

IRF-1−/− mouse survival.

To assess the potential virulence of the irradiated vaccine, IRF-1−/− mice were infected intraperitoneally (i.p.) with 108 CFU equivalents of gamma-irradiated B. melitensis 16M (n = 4) resuspended in 100 μl of PBS. In addition, IRF-1−/− mice infected with 106 CFU equivalents of live B. melitensis 16M (n = 4) were used as a control. Mouse survival was evaluated for 28 days postinfection.

Immunizations, mouse infection, and follow-up.

To evaluate protection generated by immunization with inactivated Brucella, BALB/c mice were vaccinated i.p. with 108 CFU equivalents of gamma-irradiated (350 kilorads) B. melitensis 16M (n = 4) or 108 CFU equivalents of heat-killed (65°C; 60 min) B. melitensis 16M (n = 4). PBS-injected mice were used as an unvaccinated control. At day 15, a booster vaccination was given using the dosage described above. Vaccinated BALB/c mice were challenged 30 days after vaccination with 107 CFU equivalents of B. melitensis GR023 in 100 μl of PBS i.p. The bacterial load was evaluated by biophotonic imaging (Caliper Biosciences) and was performed at the indicated times until 2 weeks postinfection. For long-term protection studies, BALB/c mice (n = 7/group) were vaccinated with 106 live or 108 irradiated or heat-killed CFU equivalents of B. melitensis Rev-1 and challenged with 2 × 104 CFU equivalents of B. melitensis 1 week later. Bacterial CFU counts were performed on individual spleens 2 weeks after challenge.

Cell-mediated cytotoxicity.

Splenocytes from 4-week-immunized mice were isolated, gradient purified, and T cell enriched for use as effector cells. Pooled splenocytes from four mice per immunization group were isolated and density gradient purified (Fico/Lite-LM [mouse]; Atlanta Biologicals, Lawrenceville, GA). Leukocytes were subjected to non-T-cell depletion by using a Pan T Cell isolation kit and MACS separation (Miltenyi Biotec) according to the manufacturer's protocol. Target cells were RAW 264.7 cells infected with live B. melitensis or uninfected controls. Target cells were infected for 24 h and then incubated with gentamicin for 90 min and thoroughly washed as with cell infection and electron microscopy described above. Cells were counted and assayed by using a CytoTox 96 nonradioactive cytotoxicity kit (Promega, Madison, WI) according to the manufacturer's protocol with 4 h of incubation.

Statistical analysis.

Data graphs were generated by using the statistical software Prism (GraphPad Software, Inc., La Jolla, CA), and statistical tests are indicated when appropriate. P values of ≤0.05 were considered significant.


Irradiated B. melitensis does not replicate but still has metabolic activity.

Inactivation by gamma irradiation was evaluated as an alternative method to generate metabolically active, replication-incompetent B. melitensis. First, the effect of heat and irradiation on survival of B. melitensis was analyzed, and an expected decrease in viability was observed with increasing heat or irradiation doses (Fig. (Fig.1A).1A). Likewise, the metabolic activity is reduced with heat or irradiation treatment (Fig. (Fig.1B).1B). At treatment times where 65°C incubation inactivated most of the bacteria (>40 min), B. melitensis showed a complete loss of metabolic activity (Fig. 1A and B). In contrast, B. melitensis inactivated by irradiation (>~3.5 × 105 rads) (Fig. (Fig.1A)1A) retained considerable metabolic activity (Fig. (Fig.1B,1B, asterisk).

FIG. 1.
Irradiation inhibits B. melitensis reproductive capacity but does not impair metabolic activity. (A and B) Effect of increasing exposure time to heat (65°C) (left) or radiation (right) on Brucella viability (A) and metabolic activity (B). Bacterial ...

Irradiated B. melitensis retains de novo transcription and protein synthesis activity.

Immune responses elicited by killed vaccines are confined to a limited expression profile, since the bacteria are incapable of producing newly synthesized antigens. Having established that gamma-irradiated B. melitensis retained active metabolism, we investigated its transcriptional and translational activity. Bacterial bioluminescence is an excellent marker of active metabolism and a model for studying protein synthesis. Luciferase reactions are highly dependent on available energy, and the decay of luminescence also reflects protein turnover (24a). We used a bacterial lux operon (32) as a gene reporter to evaluate the effects of irradiation on brucella transcriptional and translational activity. Bioluminescent B. melitensis (GR023) expresses lux genes under a constitutive promoter (BMEI0101) (32), and change in the luminescence intensity relates directly to the transcription or translation ability under similar medium environments. Consistent with the previous finding, irradiated but not heat-killed bacteria preserved light emission evaluated by biophotonic imaging (Fig. (Fig.2A).2A). Furthermore, light emission was detectable even at high irradiation doses (1,000 kilorads). In addition, the light intensity was reduced in the presence of the translation inhibitor chloramphenicol (Fig. (Fig.2B),2B), affirming that the observed light emission was a result of de novo synthesis. These results suggest the presence of integral transcriptional and translational mechanisms occurring in B. melitensis after replication is inactivated by irradiation.

FIG. 2.
Irradiated B. melitensis preserves de novo protein synthesis activity. Bioluminescent (BMEI0100::lux) B. melitensis GR023 was used as a reporter system for transcription and translation activity. (A) Bioluminescence imaging of live, 350 kilorad-treated, ...

Irradiated B. melitensis induces virulence factor expression.

We investigated the ability of nonreplicating metabolically active B. melitensis to coordinate the expression of type IV secretion system (T4SS) virulence genes during host cell infection. Recent evidence demonstrated that T4SS is critical for Brucella intracellular persistence and trafficking (9, 12, 15). Furthermore, secreted bacterial antigens are reported to be more protective than cytosolic antigens (47). We took advantage of a virB (T4SS) promoter construct expressing lux as a reporter system to evaluate changes in bioluminescence intensity upon in vitro contact with macrophage cells. To assess the induction of virB promoter, we incubated live and irradiated B. melitensis (PvirB BMEI0025-lux) with RAW 264.7 cells and evaluated the emitted luminescence during the initial hours of infection (Fig. (Fig.3).3). As controls, we used bacterial samples preincubated with gentamicin to permanently inhibit protein synthesis, since antibiotics could have reduced activity against intracellular bacteria resulting from the lack of membrane permeability. To compare the magnitude of virB promoter induction in the different groups, the results were normalized to the relative luminescence intensity at time zero (measured intensity/initial observation). The virB promoter was induced early in macrophage infection with live B. melitensis (Fig. (Fig.3).3). Likewise, irradiated B. melitensis sustained an increased expression of virB promoter during the initial hours of infection. As expected, the capacity of inducing the gene reporter expression was also dependent on de novo protein synthesis, since live and irradiated bacteria previously treated with the protein synthesis inhibitor gentamicin did not show signs of an increase in bioluminescence. Although irradiated Brucella sustained an increased expression of the reporter gene, this expression was shorter in duration (Fig. (Fig.3)3) compared to live bacteria. This raised the possibility that irradiated and live bacteria have differential uptake kinetics; however, a similar macrophage uptake was determined by flow cytometry at early infection times (data not shown).

FIG. 3.
Irradiated B. melitensis induces T4SS promoter activity upon macrophage cell infection. Live or irradiated bacteria (B. melitensis PvirB BMEI0025; multiplicity of infection of 500) were incubated in the presence or absence of a protein synthesis inhibitor ...

Irradiated B. melitensis retains wild-type morphology after dendritic cell infection.

Expression of virulence genes is essential for proper host infection, since VirB mutants fail to persist intracellularly (9). Since irradiated B. melitensis possess the potential to express necessary virulence factors encoded in the genome as suggested by the data in Fig. Fig.3,3, we examined its intracellular fate in primary dendritic cells by transmission electron microscopy. Dendritic cells are an important reservoir of Brucella in the host and are critical in triggering adaptive immunity (5). Previous reports indicate that metabolically inactive Brucella (3) and T4SS knockouts (9) do not modify the Brucella-containing vacuole and are killed by fusion with lysosomes. As shown in Fig. Fig.4A,4A, nonreplicating but metabolically active irradiated B. melitensis had an intact phenotype within the phagosome, even after 48 h postinfection. In contrast, heat-killed, metabolically inactive B. melitensis organisms were digested within the phagosome since membrane fragments were observed (Fig. (Fig.4B).4B). These results suggest that replication incompetent but metabolically active B. melitensis persists intracellularly longer than killed bacteria.

FIG. 4.
Irradiated B. melitensis preserves membrane morphology after cell infection. Splenic dendritic cells were infected (200 bacteria/cell) with irradiated (A) and heat-killed (B) B. melitensis. These electron micrograph images of infected cells were taken ...

Nondividing but metabolically active B. melitensis is not virulent for susceptible mice.

To assess the attenuation of nonreplicating but metabolically active B. melitensis, Brucella-sensitive IRF-1−/− mice (22) were infected i.p. with high doses (108 bacteria) of irradiated B. melitensis (Fig. (Fig.5).5). Using biophotonic imaging, the persistence of the nondividing vaccine was tracked in vivo after infection. Mice infected with live luminescent B. melitensis (106 bacteria/mouse) died at between 7 and 8 days of infection, whereas none of the mice infected with irradiated B. melitensis (108 bacteria/mouse) showed signs of bacterial proliferation or disease for 4 weeks (Fig. (Fig.5).5). Irradiated B. melitensis organisms were also efficiently cleared by immunocompetent BALB/c mice (not shown), and the bacteria were not detected in the spleens of IRF-1−/− and BALB/c mice infected with irradiated B. melitensis (not shown). Together, these results provide compelling evidence that irradiated B. melitensis is attenuated despite possessing metabolic activity.

FIG. 5.
Irradiated B. melitensis is not virulent in brucellosis-susceptible IRF-1−/− mice. Bioluminescent (BMEI0100::lux) B. melitensis-infected mice (106 live bacteria/mouse and 108 irradiated bacteria/mouse) were evaluated for bacterial dissemination ...

Irradiated B. melitensis protects BALB/c mice against virulent challenge.

Traditionally, metabolically inactive vaccines (subunit or killed) demonstrate very limited efficacy (25). Since irradiated B. melitensis retained wild-type features, such as metabolic activity, protein synthesis, and intracellular persistence, the ability to elicit protective immunity was investigated. Bioluminescence imaging is a powerful approach for measuring bacterial burden in vivo (16) and allows tracking pathogen dissemination in individual mice. Therefore, bioluminescence imaging was used to evaluate the protection offered by irradiated brucellae. We determined previously that infected mice emit light proportionally to CFU counts from tissue (32, 33). To test the immunogenic efficacy of nonreplicating but metabolically active irradiated B. melitensis, BALB/c mice were vaccinated i.p. with 108 irradiated or heat-killed wild-type B. melitensis and challenged 8 weeks later with live bioluminescent B. melitensis. The bacterial burden and dissemination were monitored in real time by using biophotonic imaging (Fig. 6A and B). Mice vaccinated with irradiated Brucella presented reduced pathogen colonization, with notable reduction on the bacterial spread throughout the mice (Fig. 6A and B). Long-term protection was assessed on BALB/c mice vaccinated with live, irradiated, and heat-killed B. melitensis Rev-1 at 2 weeks postchallenge. Importantly, mice vaccinated with replication-incompetent metabolically active (irradiated) brucellae conferred enhanced clearance (4.75 ± 0.20 log10 CFU/spleen ± the standard error) compared to metabolically inactive (heat-killed) bacteria (5.328 ± 0.06 log10 CFU/spleen) (Fig. (Fig.6C6C).

FIG. 6.
Irradiated brucella vaccine protects mice against virulent challenge. BALB/c mice were immunized i.p. with 108 live, irradiated, or heat-killed brucellae and challenged with virulent bioluminescent B. melitensis 4 weeks after vaccination. (A) The course ...

Irradiated B. melitensis generates CTL responses.

Inactivated vaccines induce humoral responses but fail to activate sufficient cellular immune components that are necessary to clear infected cells (41). CD4+ and CD8+ T cells control intracellular bacterial infections in vivo (41). Cytolytic activity against Brucella-infected cells (27) is thought to be a major mechanism controlling the disease. We assayed the ability of gamma-irradiated B. melitensis to trigger specific CTL responses. T cells isolated from the splenocytes of immunized mice were used as effector cells in cytotoxicity assays against B. melitensis-infected RAW 264.7 macrophage targets. As shown in Fig. Fig.7,7, irradiated B. melitensis elicited marked CTL response against target cells. As expected, cells from mice immunized with metabolically inactive heat-killed brucellae or from mock-immunized mice (PBS) had minimal levels of CTL induction. To optimize the immunization protocol, we repeated this experiment with mice vaccinated with different doses of irradiated vaccine ranging from 104 to 108 cells. The results (not shown) demonstrated that the highest vaccine dose (108) elicited the highest percent target cell killing.

FIG. 7.
Irradiated B. melitensis generates CTL responses. T-cell-enriched splenocytes from 4-week-immunized BALB/c (H-2d) mice were isolated as effector cells against RAW 264.7 target cells (H-2d) infected with live B. melitensis or uninfected controls. Live ...


In this report, we sought to retain the antigenic and adjuvant properties of live Brucella necessary for an efficient immunization in an avirulent, nonreplicating vector achieved by gamma irradiation. Although live attenuated Brucella vaccines are too virulent for human use (18), metabolically inactive bacteria elicit very limited immunity (25). We hypothesized that bacterial metabolism plays a major role in creating proper stimuli required for efficient triggering of protective responses. A nonreplicating metabolically active vaccine vector would ensure that pathogen-specific responses are generated in the physiological context of the infection, including presentation of bacterial antigens induced in response to host factors and native triggering of host immune receptors. Traditional inactivation methods such as heat or chemical agents impair microbial replicating capability by denaturing proteins and nucleic acid, resulting in the loss of metabolic activity. Alternatively, gamma irradiation provides an effective way to inactivate bacteria, since it impairs microbial replication by DNA fragmentation (42). Importantly, considerable portions of the genome remain amplifiable after irradiation, and the population of irradiated bacteria retains the potential to express virtually all genes with subsequent neo-protein synthesis and secretion (42). Therefore, gamma-irradiated B. melitensis should contain the proper determinants for an efficient immunization, without the residual virulence of living vaccines. Our results demonstrate that irradiation rendered B. melitensis metabolically active and replication incompetent. We showed that gamma-irradiated B. melitensis retained de novo protein synthesis and transcriptional activity. Since the interactions of Brucella with host cells involve gene transcription, protein synthesis and effector protein secretion (7, 9, 14), the ability to transcribe new proteins is critical to generate and maintain relevant targets for immune recognition.

The paradigm for immunity against bacterial pathogens is cell-mediated immunity, specifically T-cell-mediated responses considered essential for protection (41, 46). Crucial events for CD8+ T-cell priming occur upon contact of naive T cells with antigen-presenting cells (4). Thus, an appropriate cellular context during pathogen recognition and antigen presentation (i.e., precise triggering of innate receptors and presentation of relevant antigens) should be required for the generation of protective immunity. Infection of the host cell is characterized by the expression of virulence factors empowering the bacteria to establish a successful intracellular replicative niche. As a consequence, these novel synthesized antigens are likely to be expressed after infection and be presented by infected cells, serving as targets for the host response. We showed here that replication-incompetent metabolically active B. melitensis induced the T4SS upon exposure to macrophages. The roles of secreted Brucella proteins in host protection are still not characterized, but secreted antigens are reported to elicit stronger protective responses in intracellular bacterial infections (41). Thus, possessing T4SS activity and persisting inside host cells are remarkable features of irradiated B. melitensis, enabling the display of naturally expressed antigens during an infection. In addition, Brucella T4SS activity has been implicated in activation of the innate immune system (35, 36). virB mutants fail to elicit a vast array of inflammatory pathways, and vaccination with virB mutants does not elicit long-term protection (31, 35, 36), demonstrating that expression of this virulence factor is critical to eliciting protective responses. Since metabolic active Brucella had the potential to express virtually all virulence factors, in addition to T4SS, we were interested in knowing how this expression would affect the interaction of irradiated B. melitensis and the phagocyte cell. Others have monitored the interaction of phagosomes containing dead and live B. abortus and observed that live bacteria divert its intracellular fate and preserve the morphological structure after infection (3). More recent findings established the critical role of the T4SS machinery in this process (9, 12, 15). Like previous reports, heat-killed bacteria were completely digested shortly after exposure to dendritic cells (3). However, we were able to detect irradiated B. melitensis with preserved morphology even at 48 h after infection. Together, our findings suggest that replication-incompetent metabolically active B. melitensis expressed all of the effector proteins required for intracellular persistence. Importantly, these metabolism-dependent processes in the pathogen shape the immune recognition by not only exposing distinctive antigens but also triggering cellular responses through innate receptors and influencing downstream potency of adaptive immunity (1).

To test the concept that metabolic activity could influence the generation of host protective responses, we immunized and challenged mice with irradiated and killed vaccines. Importantly, susceptible mice challenged with nonreplicating metabolically active Brucella did not develop any signs of disease, underscoring that metabolic activity per se is not sufficient for pathogenesis. Consistent with the literature, heat-killed preparations provided some levels of protection in the first week of infection but not at later time points (25). It is noteworthy that irradiated Brucella demonstrated enhanced protection compared to metabolically inactive heat-treated bacteria. Our results are consistent with previous reports using gamma-irradiated B. abortus strain RB51 and gamma-irradiated Listeria monocytogenes (13, 38) Importantly, different approaches generating nonreplicating metabolically active organisms result in effective protection against intracellular pathogens (8, 13, 23, 38). Together, these findings suggest that despite the inactivation method, metabolic activity is the key component mediating effective immunity. Future modifications to further improve metabolic activity when using inactivated vaccines might enhance long-term efficacy of killed vaccines.

Mice vaccinated with live bacteria provided the best observed protection among all groups in long-term protection experiments, as expected. The nature of the immune response changes as disease develops; at 1 week postinfection antibodies play a large role in controlling the bacteria (25), and T-cell activity gains importance with the progression of brucellosis (2). Therefore, the long-term immunity observed in animals vaccinated with the irradiated vaccine is of seminal importance. The fact that irradiated Brucella protected mice at a later time point indicates that this vaccine is capable of triggering the differential immune responses responsible for long-term immunity.

Developing effector T cells is critical for generating and maintaining effective long-term immunity against intracellular bacteria (21), and cytotoxic mechanisms mediated by T cells are considered an essential component of immunity against Brucella (27, 46). However, it is not clear how Brucella activity impacts the development and function of T cells. We decided to evaluate the influence of pathogen metabolism on the function of specific cytotoxic T cells. We observed a marked difference in the cytotoxic ability of T cells elicited by metabolically active Brucella compared to metabolically inactive heat-killed Brucella. In addition, other researchers have shown that cells infected with gamma-irradiated Brucella are appropriate targets for cytotoxic activity generated by B. abortus (44, 45). Potentially, irradiated B. melitensis triggers danger signals that enhance the potency of CTL responses but do not impose an energy burden on the antigen-presenting cells, allowing efficient processing and presentation of antigens. In addition, metabolism and protein secretion are known to be important components in triggering host T cells, since secreted bacterial proteins are more efficient in eliciting protective T cells than nonsecreted antigens (10, 37, 47). Moreover, vaccination against intracellular L. monocytogenes with a metabolically active but replication-incompetent vector induces protective T-cell immunity (8, 13).

In conclusion, gamma irradiation preserved B. melitensis adjuvant and antigenic properties that are destroyed by other inactivation methods. We observed metabolic and transcriptional activity by irradiated Brucella. Importantly, metabolically active B. melitensis had enhanced protection and T-cell responses compared to metabolically inactive controls. Since the antigenic and immunological correlates of protection are unknown, a replication-incompetent but metabolically active vector has the potential to elicit a broad repertoire of immune responses in a physiological context. This approach provides a promising strategy of safely vaccinating against intracellular organisms, such as B. melitensis. Future work needs to identify the most relevant B. melitensis antigens for protection in order to further enhance the protective responses.


This study was supported by the NIH/NIAID Regional Center of Excellence for Bio-defense and Emerging Infectious Diseases Research (RCE) Program. We acknowledge membership within and support from the Region V “Great Lakes” RCE (NIH award 1-U54-AI-057153 and NIH 1R01AI073558). Further support was provided by the U.S.-Israel Binational Agriculture Research and Development Fund (BARD grant US-3829-06 R).


Editor: R. P. Morrison


[down-pointing small open triangle]Published ahead of print on 24 August 2009.


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