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Clinical and Vaccine Immunology, October 2007, p. 1296-1301, Vol. 14, No. 10
1071-412X/07/$08.00+0     doi:10.1128/CVI.00459-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Partial Protection against Brucella Infection in Mice by Immunization with Nonpathogenic Alphaproteobacteria

M. Victoria Delpino,¹ Silvia M. Estein,² Carlos A. Fossati,¹ and Pablo C. Baldi^1*

Instituto de Estudios de la Inmunidad Humoral (IDEHU, CONICET-UBA), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina,¹ Laboratorio de Inmunología, Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, 7000 Tandil, Argentina²

Received 7 December 2006/ Returned for modification 2 April 2007/ Accepted 10 August 2007

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Previous findings indicate that Brucella antigens and those from nonpathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system. We hypothesized that immunization with NPAP would protect mice from Brucella infection. Mice were immunized subcutaneously with heat-killed Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38 (standard positive control) before intravenous challenge with Brucella abortus 2308. Cross-reacting serum antibodies against Brucella antigens were detected at the moment of challenge in all NPAP-immunized mice. Thirty days after B. abortus challenge, splenic CFU counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 than in the phosphate-buffered saline controls (protection levels were 0.80, 0.66, and 1.99 log units, respectively). In mice immunized intraperitoneally with cytosoluble extracts from NPAP or Brucella abortus, protection levels were 1.58 for the latter, 0.63 for O. anthropi, and 0.40 for M. loti. To test whether the use of live NPAP would increase protection further, mice were both immunized and challenged by the oral route. Immunization with NPAP induced a significant increase in serum immunoglobulin G (IgG), but not serum or fecal IgA, against Brucella antigens. After challenge, anti-Brucella IgA increased significantly in the sera and feces of mice orally immunized with O. anthropi. For all NPAP, protection levels were higher than those obtained with systemic immunizations but were lower than those obtained by oral immunization with heat-killed B. abortus. These results show that immunization with NPAP, especially O. anthropi, confers partial protection against Brucella challenge. However, such protection is lower than that conferred by immunization with whole Brucella or its cytosoluble fraction.

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Brucellosis, an infectious disease affecting livestock and humans, is caused by different species of the genus Brucella, which belongs to the alpha-2 subgroup of the Proteobacteria. This subgroup also includes genera normally nonpathogenic for humans, such as Agrobacterium, Rhizobium, and Ochrobactrum (24). In contrast to Brucella species, which infect animals and humans, other alphaproteobacteria usually live in the soil (Ochrobactrum), establish symbiotic relationships with plants (Rhizobium, Mesorhizobium, and Sinorhizobium species) (19), or are phytopathogens (Agrobacterium) (29). These related alphaproteobacteria are not pathogenic for immunocompetent individuals or show only a limited pathogenicity. Human infections with Agrobacterium tumefaciens (Agrobacterium radiobacter) and Ochrobactrum anthropi have been reported almost exclusively to occur in immunocompromised patients or in those with debilitating underlying diseases or undergoing chemotherapy (2, 3, 21). There are no reports of human disease by Rhizobium and other nodule-forming bacteria.

The close genetic and antigenic relationship between Brucella and nonpathogenic alphaproteobacteria (NPAP) has been revealed by several studies. A total of 1,902 Brucella suis open reading frames are conserved in Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens, and 2,408 B. suis open reading frames are conserved in at least one of these three genomes (25). Similar relationships have been observed between NPAP and Brucella melitensis and Brucella abortus, since the genomes of the last two species are highly similar to that of B. suis (13, 17).

Few studies have investigated the immunological cross-reactivity between proteins from Brucella and those from related alphaproteobacteria. Velasco et al. (30) found an extensive cross-reactivity between cytosolic proteins from Brucella melitensis and those from Ochrobactrum anthropi and also some cross-reactions at the level of outer membrane proteins and lipopolysaccharide. More recently, we have shown the existence of cross-reactivities between Brucella and other NPAP, including Sinorhizobium meliloti and Agrobacterium tumefaciens (12). Moreover, we showed that some of these cross-reactivities could potentially be exploited for the development of serological tests for brucellosis based on antigens from NPAP.

Live attenuated Brucella strains are still widely used for the vaccination of animals against brucellosis. While these vaccines have reduced virulence for animals, they still can produce disease in humans (4, 5, 6, 27). Therefore, large-scale production of such vaccines requires biosafety level 3 facilities (11), which are seldom available in developing countries, where brucellosis is more prevalent. This is one of the reasons that has led many researchers to investigate alternative vaccination strategies for brucellosis, including the use of subunit vaccines based on recombinant proteins (1, 15) or the use of DNA vaccination (9, 20). Taking into account our previous findings that Brucella antigens and those from NPAP are cross-recognized by the immune system, we hypothesized that the immune response elicited by immunization with the latter could protect mice from Brucella infection. Here we report the results of the studies conducted to address this hypothesis. We show that systemic immunization with heat-killed NPAP or their cytosoluble fractions partially protects mice from Brucella challenge. However, protection levels are higher in mice orally immunized with live NPAP and challenged with Brucella by the oral route than in mice immunized systemically with NPAP.

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Bacteria and culture conditions. Brucella abortus 2308, Brucella melitensis H38, Ochrobactrum anthropi, and Agrobacterium tumefaciens were initially grown in tryptic soy agar (TSA) and expanded in tryptic soy broth to obtain inocula for infections or to prepare heat-killed bacteria. Sinorhizobium meliloti and Mesorhizobium loti were initially grown on solid TY medium (Bacto tryptone, 5 g; yeast extract, 3 g; CaCl₂, 1.3 g; agar, 7 g in 1 liter of distilled water) and expanded in TY broth (same medium but without agar). All liquid cultures were performed overnight at 28°C with constant agitation.

Antigens. (i) Heat-killed bacteria. After overnight growth, liquid cultures were placed in a water bath at 80°C for 4 h. Samples were plated on TSA or solid TY medium to check for sterility.

(ii) CYTs. Cytosoluble fractions were obtained as described previously (12). Liquid bacterial cultures were centrifuged at 15,000 x g, and the pellets were washed twice with sterile phosphate-buffered saline (PBS). Bacteria were killed with heat, and the suspensions were centrifuged at 15,000 x g for 10 min and washed three times with 10 mM Tris-HCl (pH 8) (Tris buffer). The cells were suspended in Tris buffer (0.1 g of cells [wet weight] per ml) and disrupted with a French press (SIM-AMINCO-Espectronic Instruments). Bacterial cells were broken by two passages and then digested for 1 h at 37°C with DNase and RNase (10 µg/ml). Unbroken cells were separated by centrifugation. Particulate matter was pelleted by centrifugation at 105,000 x g for 4 h, and the resulting supernatant (cytosoluble extract [CYT]) was stored at –20°C until use.

Systemic immunization and challenge. Mice (five animals per group) were immunized with three doses (at 2-week intervals) of heat-killed bacteria (1 x 10⁹ CFU per dose) mixed with incomplete Freund adjuvant administered subcutaneously in the back or with three doses of CYTs (40 µg per dose) administered intraperitoneally. Control groups received PBS by the same routes and at the same times. For the experiment with heat-killed bacteria, the Brucella group was immunized with B. melitensis H38 since this strain is considered a good laboratory reference for evaluating experimental vaccines in mice challenged with B. melitensis or B. abortus (8, 10, 16, 31). One month after the last immunization, mice were challenged by the intravenous route with 1.3 x 10⁴ CFU of live B. abortus 2308. One month after challenge, mice were killed, their spleens were aseptically removed and homogenized, and homogenates were plated on TSA for CFU counting.

Oral immunization and challenge. Two separate experiments of oral immunization were performed. Mice (five animals per group) were inoculated intragastrically at weekly intervals with three doses of live NPAP (1 x 10⁸ CFU) or heat-killed B. abortus (HKBA) strain 2308 (1 x 10⁸ CFU) or PBS by using an intragastric feeding tube. Twenty days after the last infecting dose, mice were challenged by the same route with live B. abortus 2308 (1.25 x 10⁴). Mice were killed at 30 days postinfection, and CFU counts in spleens were determined as described above.

Assessment of the antibody response. Blood samples were obtained before each immunization, before challenge, and at sacrifice. At the same time points, mice immunized by the oral route were housed in separate cages, and five fecal pellets were collected from each animal. Fecal samples were mixed with 0.5 ml of extraction buffer (30 mM disodium EDTA, pH 7.6, 100 µg/ml soybean trypsin inhibitor, and 10 mg/ml bovine serum albumin in PBS). Pellets were homogenized and centrifuged at 4°C, and supernatants were stored at –20°C until immunoglobulin A (IgA) measurements were performed.

Specific antibodies to CYT antigens were measured in serum samples and fecal extracts by an indirect enzyme-linked immunosorbent assay. Polystyrene plates (NUNC MaxiSorp) were sensitized at 0.5 µg/well with each of the CYTs (obtained as described above). Plates were blocked with 200 µl per well of PBS containing 3% skim milk. After a wash with PBS-T (PBS containing 0.05% Tween 20), sera diluted 1:200 were dispensed in PBS-T containing 1% skim milk, while fecal extracts were used undiluted. Specific antibodies were detected with horseradish peroxidase-conjugated antibodies against mouse IgG or mouse IgA (Santa Cruz Biotechnology, Santa Cruz, CA). The reaction was developed by adding ortho-phenylenediamine (2 µg in 0.1 M citrate-phosphate buffer containing 0.03% H₂O₂) and was stopped with 4 N H₂SO₄. The optical densities were read at 492 nm in a model 960 microplate reader (Metertech Inc., Taiwan).

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Protection conferred by immunization with heat-killed bacteria. Subcutaneous immunization with heat-killed bacteria elicited in each case a strong antibody response against CYT antigens of the homologous bacteria (not shown). In most cases, antibody levels were maximal after the second immunization. In mice immunized with nonpathogenic species, the presence of antibodies capable of reacting with Brucella antigens was also tested. As shown in Fig. 1, cross-reacting antibodies were detected in serum samples obtained at the moment of challenge in all groups of mice immunized with heterologous bacteria. Anti-Brucella antibody levels were significantly augmented in all groups compared to preimmunization levels but tended to be higher in animals immunized with S. meliloti or O. anthropi. These results suggested that an immune response capable of recognizing Brucella antigens was present in all groups of mice at the time of challenge. Mice were challenged intravenously with B. abortus and were killed 30 days later to count CFU in spleens. As shown in Table 1, spleen counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 (positive control) than in mice injected with PBS (negative control). However, protection levels obtained with the nonpathogenic bacteria were lower than that obtained with Brucella immunization. Counts of CFU in mice immunized with B. melitensis were significantly lower than those in mice immunized with any other bacteria (P < 0.01, Dunnett's multiple-comparison test). Anti-Brucella antibodies were detected in all groups at 30 days postchallenge at levels similar to those found at the moment of challenge.

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FIG. 1. Serum reactivity (IgG) against B. abortus cytosolic antigens in mice immunized subcutaneously with heat-killed S. meliloti (SM), O. anthropi (OA), M. loti (ML), A. tumefaciens (AT), or B. melitensis (H38). Antibodies were measured at challenge with B. abortus 2308 and at sacrifice for protection evaluation (30 days after challenge). The reactivities of preimmunization sera from all groups were pooled (Pre). *, significantly different from the value for Pre; #, significantly different from the value for H38; NS, not significantly different from reactivities measured at challenge; OD, optical density.

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TABLE 1. Protection against B. abortus infection conferred by immunization with heat-killed NPAP

Protection conferred by immunization with CYTs. As with whole bacteria, intraperitoneal immunization with CYTs from the different species elicited in each case a strong antibody response against the homologous antigens, which in most cases reached maximum levels after the second immunization (not shown). Cross-reacting antibodies against Brucella CYT antigens were detected in serum samples obtained at the moment of challenge in all groups of mice immunized with heterologous bacteria (Fig. 2). Anti-Brucella antibody levels were significantly augmented in all groups compared to preimmunization levels but were lower in animals immunized with M. loti or A. tumefaciens than in groups receiving S. meliloti, O. anthropi, or B. abortus. Thirty days after challenge with B. abortus, numbers of CFU were significantly lower in the spleens of mice immunized with the different CYT fractions than in mice injected with PBS (Table 2). However, the reduction in CFU counts was more significant in mice immunized with CYT from B. abortus or O. anthropi than in mice immunized with fractions from other bacteria. In addition, counts of CFU in mice immunized with B. abortus were significantly lower than those in mice immunized with any other bacteria (P < 0.01, Dunnett's multiple-comparison test). Anti-Brucella antibodies were detected in all groups at 30 days postchallenge at levels similar to those found at the moment of challenge.

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FIG. 2. Serum reactivity (IgG) against B. abortus cytosolic antigens in mice immunized intraperitoneally with cytosolic fractions from S. meliloti (SM), O. anthropi (OA), M. loti (ML), A. tumefaciens (AT), or B. abortus (BA). Antibodies were measured at challenge with B. abortus 2308 and at sacrifice for protection evaluation (30 days after challenge). The reactivities of preimmunization sera from all groups were pooled (Pre). *, significantly different from the value for Pre; #, significantly different from the value for BA; NS, not significantly different from reactivities measured at challenge; OD, optical density.

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TABLE 2. Protection against B. abortus infection conferred by immunization with cytosoluble fractions from alphaproteobacteria

Protection conferred by oral immunization with live bacteria. The experiments described above suggested that immunization with NPAP conferred some protection against Brucella challenge. In addition, for the heterologous species conferring the best protection (O. anthropi), immunization with whole bacteria seemed to protect better than immunization with the CYT fraction. Therefore, we decided to test whether infection with live NPAP would increase the level of protection against Brucella challenge. Taking into account the facts that mucosae are the most common portals of entry for natural Brucella infections and that a systemic infection with a large inoculum of live NPAP could potentially produce adverse effects, the oral route was chosen. Mice infected by intragastric delivery of live bacteria did not exhibit evident physical or behavioral alterations. A group of mice orally immunized with HKBA was also included. At the time of Brucella challenge, mice had developed both IgG and IgA antibodies against the homologous bacteria (not shown). Anti-Brucella IgG levels were significantly higher than preinfection levels in all groups of mice infected with NPAP but were lower in animals infected with A. tumefaciens than in the other NPAP groups (Fig. 3, upper panel). Before challenge, animals immunized with HKBA by the oral route exhibited low levels of anti-Brucella IgG in serum, not significantly different from preimmunization levels. After challenge, mice preinfected with A. tumefaciens and mice immunized with HKBA exhibited a significant rise in anti-Brucella IgG in serum. Regarding serum IgA against Brucella, no significant increase was found at the time of challenge in mice infected with NPAP or immunized with HKBA. After challenge, however, serum levels of anti-Brucella IgA increased significantly in mice previously infected with O. anthropi and in mice orally immunized with HKBA (Fig. 3, middle panel). Anti-Brucella IgA was also measured in fecal samples from the same groups of mice. As in the case of serum IgA, fecal levels of anti-Brucella IgA were not significantly increased after oral infection with NPAP or oral immunization with HKBA but increased significantly after oral challenge with live B. abortus in animals preinfected with O. anthropi or immunized with HKBA (Fig. 3, lower panel). Anti-Brucella IgA levels in serum and feces also tended to increase after challenge in mice preinfected with S. meliloti and A. tumefaciens, but the differences did not reach statistical significance. As shown in Table 3, Brucella CFU counts in spleens were significantly lower in mice previously infected with NPAP or immunized with HKBA than in the PBS controls, with no significant difference among the NPAP groups. In addition, counts of CFU in mice immunized with HKBA were significantly lower than those in mice immunized with A. tumefaciens (P < 0.05, Dunnett's multiple-comparison test). Protection levels tended to be higher than those obtained with the same bacteria in the experiments of systemic immunization and challenge, and the difference was especially evident for A. tumefaciens and S. meliloti.

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FIG. 3. Serum reactivity against B. abortus cytosolic antigens in mice orally infected with S. meliloti (SM), O. anthropi (OA), or A. tumefaciens (AT) or orally immunized with HKBA. Antibodies were measured at challenge with B. abortus 2308 and at sacrifice for protection evaluation (30 days after challenge). The reactivities of preimmunization sera from all groups were pooled (Pre). *, significantly different from the value for Pre; #, significantly different from reactivities measured at challenge; NS, not significantly different from reactivities measured at challenge; OD, optical density. The experiment was performed in duplicate; data from a representative experiment are shown.

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TABLE 3. Protection against oral B. abortus infection conferred by oral immunization with live NPAP or HKBA

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Vaccination of animals to prevent brucellosis is currently performed with live attenuated strains of Brucella, including B. melitensis Rev1, B. abortus S19, and B. abortus RB51. However, these vaccines are unsafe for humans, and several cases of human illness by accidental inoculation, inhalation, or splashing have been reported. Different strategies have been explored to develop safer vaccines against brucellosis (1, 9, 15, 20). The goal of the present study was to assess whether administration of whole NPAP or their subcellular fractions through different routes could protect mice from a subsequent challenge with live B. abortus. The rationale behind this strategy was that the immune response elicited against antigens or antigenic determinants shared by NPAP and Brucella would confer some protection against challenge with B. abortus.

Previous studies have assessed the reciprocal cross-protection conferred by immunization with bacterial species from different genera. The rationale behind most of these studies has been the similarity between the lipid A region of the lipopolysaccharides of different gammaproteobacteria (7, 23). Thus, cross-protection was assessed, for example, for Proteus spp. versus Providencia spp. (26), the Escherichia coli J5 mutant versus Haemophilus influenzae type b (22), and the Salmonella enterica serovar Minnesota Re mutant versus Pseudomonas aeruginosa (28). To the best of our knowledge, however, no studies of cross-protection between heterologous genera have been performed with the specific aim of reducing the biosafety conditions required for the production of live vaccines against highly hazardous agents, such as Brucella spp., and the risks inherent to the use of such vaccines.

To the best of our knowledge, there is only one previous study that explored the use of an NPAP to prevent brucellosis. He et al. (18) inoculated mice by the intraperitoneal route with wild-type O. anthropi or recombinant O. anthropi expressing the Brucella Cu,Zn superoxide dismutase and challenged the animals with B. abortus 2308. In that study, no protection was obtained unless unmethylated CpG motifs were administered as adjuvants. No studies have been performed with other NPAP, and none has explored whether oral immunization with these bacteria can confer protection against challenge by the same route.

The results of the current study show that, in general, the NPAP assayed confer some degree of protection against Brucella challenge. However, O. anthropi cells or antigens conferred the highest levels of protection for all the immunization strategies evaluated. A notable finding was that, for all the nonpathogenic species, the protection level obtained with orally administered viable bacteria against challenge by the same route was higher than that attained with systemic immunization with whole bacteria or CYT antigens. This difference was especially evident for immunizations with A. tumefaciens and S. meliloti. This result is especially important since the oral route is one of the most frequent portals of entry for Brucella species both in humans and animals. Therefore, a potential strategy for protecting animals from Brucella infection could be to add these NPAP in appropriate quantities to the usual diet or the drinking water of such animals. Further studies will be needed to explore the efficacy of these strategies. Vaccination with a live attenuated Brucella suis strain (S2) administered through drinking water has been used in China since the beginning of the 1970s for sheep, goats, and other animals and has been proven to confer up to 3 years of protection (14). As with other Brucella vaccine strains, however, this strain is not innocuous for humans and must be produced under biosafety level 3 conditions.

An important implication of the results obtained with oral immunization is that no adjuvants were required. In the study by He et al., no protection was obtained with systemic immunization with live O. anthropi in the absence of adjuvants. In the current study, oral vaccination with live O. anthropi without adjuvants conferred a protection level of 0.88 to 0.96 log₁₀ units.

While this study did not explore the immune mechanisms involved in protection against Brucella challenge, the finding of anti-Brucella antibodies in mice immunized or infected with NPAP indicates that these animals had developed an immune response capable of recognizing Brucella antigens. Particularly noticeable were the increases in serum and fecal IgA levels upon Brucella challenge of mice previously infected with O. anthropi by the oral route. In contrast, no increase in anti-Brucella IgG levels was detected after challenge of animals immunized with heat-killed bacteria or CYT fractions. Nevertheless, a boost of IgG levels may have occurred early after challenge, and by the time the animals were examined again (30 days postchallenge), antibody levels may have returned to prechallenge levels.

Experiments of oral immunization with live bacteria included a control group of mice immunized with HKBA. Before challenge, this group exhibited low levels of anti-Brucella antibodies in both their sera and their feces. This result is not surprising if we take into account the fact that these animals received killed bacteria by the oral route in the absence of adjuvants. Upon oral challenge with live B. abortus, in contrast, this group exhibited a marked increase in anti-Brucella antibody levels.

This is the first study to assess cross-protection as a strategy for reducing the risks associated with the production and handling of live vaccines against hazardous agents. Our results suggest that immunization with NPAP, especially O. anthropi, confers some degree of protection against Brucella infection. Inoculation of live NPAP seems to confer higher levels of protection than immunization with heat-killed bacteria or CYT fractions. However, even in this case, the protection obtained is lower than that conferred by the homologous species. New studies with other immunization protocols will be needed to assess whether protection conferred by NPAP immunization can be improved further.

 
We are very grateful to Antonio Lagares from the Universidad Nacional de La Plata for providing the strains of NPAP. We thank Susana Insua and Alejandro Dussio for technical assistance and Sergio Islas and Fabián Amaya (UNCPBA, Argentina) for animal care.

This work was supported by grant UBACYT B038 from the Universidad de Buenos Aires and by grant PICT 05-14305 from the Agencia Nacional de Promoción Científica y Tecnológica, Argentina. M.V.D. was supported by a fellowship of the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) from Argentina. S.M.E. was supported by the Comisión de Investigaciones Científicas (CIC), Provincia de Buenos Aires, Argentina. C.A.F., P.C.B., and S.M.E. are members of the Research Career of CONICET. C.A.F. is also a member of the Facultad de Ciencias Exactas, Universidad Nacional de La Plata.

 
* Corresponding author. Mailing address: IDEHU, Facultad de Farmacia y Bioquímica, UBA, Junín 956 4to Piso, 1113 Buenos Aires, Argentina. Phone: 54-11-4964-8259. Fax: 54-11-4964-0024. E-mail: pablobal{at}ffyb.uba.ar

Published ahead of print on 22 August 2007.

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Clinical and Vaccine Immunology, October 2007, p. 1296-1301, Vol. 14, No. 10
1071-412X/07/$08.00+0     doi:10.1128/CVI.00459-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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