Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis

Veterinary Microbiology 102 (2004) 203–213

Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis Silvia M. Estein a,∗ , Pablo C. Cheves b , Mar´ıa A. Fiorentino b , Juliana Cassataro c , Fernando A. Paolicchi b , Raúl A. Bowden a,1 a

Laboratorio de Inmunoqu´ımica y Biotecnolog´ıa, Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, 7000 Tandil, Argentina b Laboratorio de Bacteriolog´ıa, Departamento de Producción Animal, Instituto Nacional de Tecnolog´ıa Agropecuaria, 7620 Balcarce, Argentina c Laboratorio de Inmunogenética, Hospital de Cl´ınicas “José de San Mart´ın”, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Received 29 July 2003; received in revised form 27 February 2004; accepted 5 May 2004

Abstract Detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli was previously identified as a protective immunogen against B. ovis in mice. In this study, we evaluated the immunogenicity of rOmp31extract in rams. This immunogen was emulsified in an oil adjuvant and administered three times with 4 and 8 weeks intervals. Antibody response was measured in serum by whole B. ovis ELISA. Specific antibodies to purified rOmp31 (pET-Omp31) were detected by Western blotting and indirect ELISA. In addition, isotype specific antibodies were measured in tears. Serum bactericidal activity against B. ovis in the presence of complement was measured in vitro. Cellular immune response was explored by intradermal testing with purified rOmp31. Immunization with rOmp31 extract induced IgG specific antibodies in serum able to bind to whole B. ovis cells. Furthermore, strong inhibition in a competitive ELISA (with an Omp31-specific monoclonal antibody) suggested that a proportion of Omp31-specific antibodies were directed against a loop containing a protective epitope. Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum. Tears had both IgG and IgA antibodies to equivalent titers. Finally, immunized rams showed skin reactivity to Omp31. These data demonstrate that B. melitensis Omp31, a protective antigen identified in the mouse model, induces antibody and cellular immune mechanisms in sheep. © 2004 Elsevier B.V. All rights reserved. Keywords: Brucella ovis; Vaccine; Omp31; Immunogenicity; Sheep; Bacteria

1. Introduction

∗ Corresponding author. Tel.: +54 2293 422 357; fax: +54 2293 426 667. E-mail address: [email protected] (S.M. Estein). 1 Dead on October 6th, 2003.

B. ovis and B. melitensis are the etiologic agents of ovine brucellosis. B. ovis causes an infectious disease characterized by epididymitis and decreased ram fertility, abortions in ewes and increased lamb mortality, with severe economic losses (Blasco, 1990;

0378-1135/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.05.004

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Bulgin, 1990). Control measures in flocks include culling of animals positive to serological tests and/or bacteriological culture. Vaccination is recommended when prevalence is high. Among available vaccines, B. melitensis Rev. 1, an attenuated smooth strain used to control B. melitensis infection in small ruminants, gives heterologous protection against B. ovis and is currently considered the best vaccine for the prophylaxis of ovine brucellosis (Mar´ın et al., 1990). However, there are significant limitations associated with its use, namely (i) the development antibodies interfering with serodiagnosis, (ii) the occasional induction of abortion when given during pregnancy, (iii) its virulence for humans and (iv) that its use is not allowed in countries free of B. melitensis. Thus, better vaccines are needed. Attenuated smooth B. suis S2 or rough B. abortus RB51, are inefficacious against B. ovis in rams (Blasco et al., 1993a; Jiménez de Bagués et al., 1995). Recently, a B. ovis mutant containing a disrupted omp25 gene (BO25) has been found attenuated in mice and afforded greater protection against B. ovis than Rev. 1, but the safety and efficacy in sheep are being investigated (Edmonds et al., 2002b). In the past, several B. ovis bacterins with different adjuvants have been used with less success (Swift and Maki, 1968). In addition, they led to the development of local lesions and, as with live Rev. 1, intense antibody responses affected the specificity of serodiagnosis (Swift and Maki, 1968). Accordingly, research is under way to develop subcellular vaccines avoiding the problems of B. melitensis Rev. 1; Brucella surface molecules are good candidates for such a vaccine. A hot saline (HS) extract from B. ovis has been shown to protect mice (Jiménez de Bagüés et al., 1994) and rams (Blasco et al., 1993b) against a B. ovis challenge. In addition, transfer to naive mice of immune serum raised against HS extract demonstrated an important involvement of antibodies specific for HS extract molecules in protective immunity (Jiménez de Bagüés et al., 1994). The HS extract of B. ovis is a molecular complex mainly composed of R-LPS and outer membrane proteins (OMPs); among the latter group 3 OMPs (Gamazo et al., 1989; Suárez et al., 1988) which includes Omp25 and Omp31, the major OMPs of Brucella (Cloeckaert et al., 1996, 2002). In addition, other immunogenic proteins were found in this extract namely

Omp1, Omp2b and lipoproteins Omp10, Omp16 and Omp19 (Bowden et al., 2000). The demonstration that HS extract depleted of R-LPS retained protective activity in mice (Jiménez de Bagüés et al., 1994) underlined the contribution of OMPs to protective immunity; however, in rams, this was less protective than total HS extract (Blasco et al., 1993b). Moreover, passive protection experiments in mice have shown that mixtures of Mabs to well-exposed OMPs and R-LPS were highly protective in mice against B. ovis infection (Bowden et al., 1995b, 2000). In particular, a single anti-Omp31 Mab, conferred passive protection as strong as that obtained with an anti-HS extract serum (Bowden et al., 2000). The epitope recognized by this Mab is well exposed at the bacterial surface and conserved among strains (Vizca´ıno et al., 1996, 2001). In addition, Omp31 was also shown to be immunodominant in serological responses of B. ovis infected rams (Kittelberger et al., 1995, 1998). Molecular cloning has made easier the isolation of individual antigens for testing as vaccines. B. melitensis Omp31 has been cloned and expressed on the surface of E. coli (Vizca´ıno et al., 1996) and was shown to protect mice against a B. ovis challenge (Estein et al., 2003). These features made Omp31 a potential component for a subcellular vaccine against B. ovis. In this paper, we studied the immunogenicity of B. melitensis Omp31 in sheep, and the bactericidal activity of Omp31-specific antibodies in presence of complement against B. ovis.

2. Materials and methods 2.1. Bacteria and plasmids B. ovis PA-76250 (PA, for short) was obtained from the Brucella collection (INRA-Nouzilly, France) and was used for serological determinations and bactericidal assay. For Western blotting (WB), HS extract from B. ovis REO198 was prepared as described previously (Alton et al., 1988). Recombinant E. coli cells (obtained from INRANouzilly, France) were used for obtaining detergent extracts for immunizations. Plasmid pNV3123 containing the omp31 gene of B. melitensis 16M was constructed as described previously (Guilloteau et al., 1999; Vizca´ıno et al., 1996). E. coli (pUC19) was

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used as negative control. To check for Omp31 expression, the recombinants were subjected to SDS-PAGE and WB with the anti-Omp31 Mab A59/10F09/G10 (Cloeckaert et al., 1990). As expected, Omp31 was detected in E. coli (pNV3123) and not in E. coli (pUC19) extract (Vizca´ıno et al., 1996). For antibody determinations by ELISA, analysis by WB and intradermal injections, purified rOmp31 was used. For this, the Omp31 gene was cloned in pET22 + vector (Novagen, Madison, WI, USA) as reported elsewhere, with the sequence information previously described (Vizca´ıno et al., 1996). The resultant plasmid (pET-Omp31) contained Omp31 gene with the addition of a sequence for a poly-H tail. The rOmp31 was successfully expressed as inclusion bodies in competent cells E. coli strain BL21(DE3) (Stratagene, La Jolla, CA, USA). The inclusion bodies, containing Omp31, were resuspended in 50 mM Tris–5.0 mM EDTA–8.0 M urea (pH 8.0) during 24 h. After centrifuging (20,000 × g, 30 min at 4 ◦ C), soluble protein was purified by chromatography through Ni-agarose (Quiagen, Dorking, UK). The presence of rOmp31 in eluates was checked as indicated above. Purity was assessed by SDS-PAGE and Coomassie blue staining. 2.2. Experimental design 2.2.1. Animals and fluids samples Eleven Romney Marsh rams, 10–12 months of age were used. All animals were clinically normal and bacteriologically negative to B. ovis. In addition, sera were negative by R-LPS ELISA (Vigliocco et al., 1997). Rams were randomly distributed in three groups: group 1 (n = 4) vaccinated with rOmp31 extract, group 2 (n = 4) vaccinated with E. coli (pUC19) extract as a negative control and group 3 (n = 3) unvaccinated control, injected with phosphate buffered saline (PBS). Blood samples for serum obtention were collected by jugular venipuncture. Serum samples were obtained at different immunization intervals: during the first 13 weeks, samples were taken weekly and, every 2 weeks or once a month until 19 weeks after the immunization. Additional samples were obtained from the animals at weeks 29 and 40. In addition, samples of tears were obtained by placing sterile cotton swabs in the conjunctival sacculus; proteins were recovered from the swabs by overnight

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diffusion at 4 ◦ C in 1 mL of PBS added with sodium azide (0.01%). After centrifugation, supernatants were stored at 4 ◦ C. 2.2.2. Preparation and administration of vaccines Extraction/separation of recombinant E. coli with a non-ionic detergent was carried out as previously described (Estein et al., 2003). Briefly, 1.0 g of each E. coli (pNV3123) and E. coli (pUC19) were mixed with 10 mL of 4% Triton X-114 in TBS (Sigma, St. Louis, MO, USA) and stirred overnight at 4 ◦ C. The mixtures were centrifuged and the supernatants of each mixture were heated at 37 ◦ C and centrifuged at 10,000 × g for 20 min at 25 ◦ C. Detergent-rich phases were recovered and designed as rOmp31 extract and E. coli (pUC19) extract, respectively. Protein was precipitated by adding three volumes of cold acetone (−20 ◦ C) (Robyt and White, 1987) and recovered by centrifugation. Protein was quantified by the bicinchoninic acid method (BCA Protein Assay, Pierce, Rockford, IL, USA). Protein concentrations in the extracts were 0.12 and 0.01 mg/ml for rOmp31 extract and E. coli (pUC19) extract, respectively. Immunogens were solubilized by sonication in 1 mL of PBS and then mixed by double-hubbed needle method (Herbert, 1978) with an equal volume of Marcol 52 (kindly provided by Bayer, Argentina). The immunogens were injected subcutaneously three times. In the first and second vaccination, 4 weeks apart, animals received 50 ␮g of immunogen in each application. Seven weeks later, rams received the third vaccination containing 500 ␮g immunogen. Reactions at injection sites were evaluated visually and by palpation following each immunization. 2.3. Immunological procedures 2.3.1. ELISA tests All ELISAs were carried out in 96-well polystyrene plates (NUNC 2-69620, Roskilde, Denmark). 2.3.1.1. Indirect ELISA for B. ovis surface-binding antibodies in sera (iELISA-PA). B. ovis PA cells were grown for 48 h, harvested, washed, heatinactivated (65 ◦ C, 60 min) and were adjusted to an absorbance value (A6 0 0 ) of 1.0. B. ovis cells were adsorbed to plates by overnight incubation at 4 ◦ C in sodium bicarbonate coating buffer (pH 9.6). Ram sera

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were diluted 50-fold in PBS-EDTA-EGTA-Tween 20 (15.0 mM EDTA, 15.0 mM EGTA and 0.05% Tween 20 in PBS) were incubated for 2 h at 37 ◦ C. Bound antibodies were detected by a donkey anti-sheep IgG (whole molecule) conjugated to horseradish peroxidase (Sigma, St. Louis, MO, USA) (1/2000; 1 h at 20 ◦ C). Enzyme activity was revealed by incubation with 1 mM 2,2 -azino-di (3-ethylbenzothiazolinesulphonic acid) (ABTS) and 4 mM hydrogen peroxide (H2 O2 ) in citrate buffer pH 4.5. After 45 min with shaking at room temperature, colour was read at 405 nm in automatic ELISA reader (Titertek, Multiskan EX, Labsystems). Antibody levels (IgG) were expressed as the arithmetic mean ± S.D. of the OD obtained for rams included in each group. 2.3.1.2. ELISA with purified rOmp31. In sera. The following two enzyme immunoassays were also used with pET-Omp31 as antigen: an indirect ELISA (iELISA-Omp31) and a competitive ELISA (cELISA-Omp31) with Mab A59/10F09/G10 for competition. Briefly, in both ELISAs the plates were sensitized with 100 ␮l per well with pET-Omp31 (0.1 ␮g/well) in PBS pH 7.2, at 4 ◦ C overnight. Then, plates were blocked with PBS-containing 1% bovine serum albumin (BSA). In the iELISA-Omp31, ram sera were diluted 1/50 in PBS-BSA-EDTA-EGTA-T and were incubated for 2 h at 37 ◦ C. Bound antibodies were detected by donkey anti-sheep IgG (whole molecule) conjugated to peroxidase (1/2000, 1 h). Enzyme activity was revealed as indicated in Section 2.3.1.1. The plates were shaken for 30 min at room temperature before read-out. In the cELISA-Omp31, 50 ␮l of ram sera (diluted 1/50) were added to the wells along with 50 ␮l of the anti-Omp31 Mab (1/3000). The plates were shaken for 2 h at room temperature. A rabbit anti-mouse IgG (whole molecule) conjugated to peroxidase (1/4000) (ICN, Irvine, USA) was used for detection. The plates were shaken for 45 min at room temperature before reading. Results were expressed as the percentage of inhibition of the Mab binding, and were calculated from the mean absorbance values for each sample by the following formula: percent inhibition = [1 − (mean absorbance value of test sample/mean absorbance value of the Mab without serum)] × 100.

The procedure to analyze antibody isotypes was similar to the iELISA-Omp31 described above, but the second antibodies used were rabbit antisera to sheep IgG, IgA and IgM in appropriate dilutions (Heavy chain; Bethyl, Montgomery, TX, USA) (1 h at 37 ◦ C). Protein A peroxidase diluted 1/2000 (1 h at room temperature) was used for detection. Tears. The procedure for detecting specific antibody response in tears to Omp31 was similar to that described above in iELISA-Omp31 for isotype determination, except that pools of each fluids were diluted 1:2 in the same buffer. 2.3.2. SDS-PAGE and WB 2.3.2.1. In sera. The humoral immune response to Omp31 from all rams was assessed by WB using HS extract (rich in group 3 OMPs) (20 ␮g/lane) and rOmp31 (10 ␮g/lane). SDS-PAGE was carried out in 12.0% acrylamide–bisacrylamide gel. Antigens were boiled for 10 min in sample buffer, sonicated briefly and centrifuged. The supernatant was loaded in gel and electroblotted as described previously (Bowden et al., 2000). Blots were blocked for 2 h at room temperature with TBS (50 mM Tris–HCl, 0.15 M NaCl, pH 7.5) containing 3.0% skim milk and 0.05% Tween 20 (TBS-SM-T). Strips were incubated overnight with pooled sera from immunized rams (diluted 1:5 in TBS-SM-T). The detection was done by incubating strips with donkey anti-sheep immunoglobulin G, diluted 1:500. Mab to Omp31 (A59/10F09/G10), used as control for antigen position, were incubated at a 1:500 dilution and detected with protein A peroxidase conjugate diluted 1:2000 and developed as described elsewhere (Bowden et al., 2000). 2.3.2.2. In tears. The presence of antibodies to Omp31 in tears (diluted 1 in 2) was assessed by WB using pET-Omp31 as described in Section 2.3.2.1. 2.4. Complement-mediated bacteriolysis assay This assay was performed in 96-well, flat bottomed polystyrene micro-titre plates (Linbro, Italy). Bacterial suspensions were used in non-agglutinating mixtures with sera, to avoid clumping that could interfere with colony counting. Duplicate assay wells received 50 ␮l of ram pooled serum (heat-inactivated at 56 ◦ C, 30 min) and 30 ␮l of bacterial inoculum

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(1.0 × 104 UFC/mL) suspended in PBS-containing 0.5 mM MgCl2 y 0.15 mM CaCl2 . The mixtures were shaken at 100 rpm for 30 min at 37 ◦ C to allow antibody–Brucella interaction to occur. As the source of complement, 40 ␮l of guinea pig complement or 40 ␮l of fresh sheep serum were dispensed in appropriate wells. Sheep serum was collected and stored as recommended (Oswald et al., 1990). Mixtures were shaken at 100 rpm for 1 or 2 h at 37 ◦ C. Controls, in duplicate, included antibody–Brucella mixtures with (i) both heat-inactivated complement sources and (ii) without complement. Next, 25 ␮l of each well were plated (duplicate) onto triptein soy agar–yeast extract (TSAYE) with 5.0% equine serum. The mean percentage of bacterial killing (%K) was calculated as: %K = 100 × 1 − (mean number of CFU mL−1 after incubation/mean number CFU mL−1 before incubation). 2.5. Intradermal reaction to Omp31 Seventeen weeks after the third vaccination, all rams were injected intradermally into wool-free region of the inside thigh with pET-Omp31 (10 ␮g in 0.1 mL). The skin thickness was measured with a Vernier calliper just before injection (initial skin thickness) and at 48, 96 and 120 h later. 2.6. Statistical analysis Data from all the ELISAs and intradermal test were analyzed by ANOVA and Dunnett’s and Tukey’s post hoc tests using Prism 3.0 (Graph-Pad, CA, USA). Correlation analysis between levels of serum and tears antibodies and graphs were done with the same program.

3. Results 3.1. Antibody response specific to Omp31 induced in rams by rOmp31 extract The capacity of rams immunized with rOmp31 extract (group 1) to produce antibodies to Omp31 was investigated by WB in sera collected 1 week after the third immunization, using a B. melitensis recombinant Omp31 (pET-Omp31) or HS extract from B. ovis as antigens. Only pooled sera from group 1 recognized bands at the same molecular mass level than the anti-Omp31 Mab, when Omp31 was used as anti-

Fig. 1. Western blot of recombinant Omp31 (15 ␮g/lane) was developed with Mab A59/10F09/G10 (specific to Omp31) (lane 1); pooled sera from rams immunized with rOmp31 extract (1 week after the last injection) (lane 2); pooled sera from rams immunized with E. coli (pUC19) extract (lane 3); pooled sera from rams injected with PBS (lane 4). On the left, arrow indicates the position of Omp31.

gen (Fig. 1). When the HS extract was incubated with each individual serum of all rams, additional weak low molecular weight bands were observed in some sera from group 2 (data not shown). To measure the specific antibody response to Omp31, sera from all rams collected after each immunization were tested in ELISA with pET-Omp31. Rams immunized with rOmp31 extract presented significantly higher antibody levels (P < 0.001) than both control groups at 3 weeks after the first vaccination that remained stable until week 40 without significant differences within the group. Sera from rams belonging group 2 and group 3 exhibited low, background absorbances throughout the study (Fig. 2).

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Fig. 2. Antibody response to Omp31 were determined in pooled ram sera from each immunization group at 0, 4, 6, 13, 19, 29 and 40 weeks after the first immunization. Sera were diluted 1/50 and assayed against purified rOmp31. Data are expressed as mean ± S.D. absorbances units. Values significantly different from non-immunized group are indicated by ∗∗∗ P < 0.001.

To analyze the antibody isotypes generated by immunization, ELISA with antiserum to sheep IgG, IgA and IgM were used. IgG antibodies predominated throughout the study whereas IgA and IgM had low levels (data not shown). The same sera were also investigated in cELISAOmp31 by using Mab A59/10F09/G10 specific for an exposed epitope located in an hydrophilic loop of Omp31 (Vizca´ıno et al., 1996, 2001). Rams immunized with rOmp31 extract presented the highest inhi-

Fig. 3. Antibody response to Omp31 were evaluated in an competitive ELISA using purified rOmp31 as antigen and a mouse Mab 10/F09/G10 for competition. Samples were taken at 0, 2, 5 and 13 weeks after pi. Data are expressed as mean ± S.D. absorbances units. Values significantly different from non-immunized group are marked by ∗∗∗ P < 0.001.

bition percentage, significantly different respect both control groups (P < 0.001) (Fig. 3); this indicated that a proportion of the antibodies raised were against or close to the above epitope. 3.2. Evolution of antibody response to B. ovis PA-76250 in ELISA The ability of antibodies induced by immunization with rOmp31 extract to bind whole B. ovis PA cells, was tested in an iELISA (Fig. 4). At 3 weeks

Fig. 4. Evolution of antibody response to Brucella ovis surface-exposed antigens. Rams were first immunized s.c. at day 0 and boosted on weeks 4 and 12. Serum antibodies (IgG) were measured by indirect ELISA onto heat-killed whole cells of B. ovis PA-76250. Data are expressed as mean ± S.D. absorbance units. Values significantly different from non-immunized group are marked: ∗ P < 0.05; ∗∗ P < 0.01.

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with little differences among group 1 rams. Thereafter antibody levels remained stable throughout the study. Ram sera from controls exhibited low absorbance values throughout the experiment. 3.3. Specific antibodies in tears

Fig. 5. Bactericidal activity of pooled immunized ram sera against B. ovis in presence of guinea pig and ovine sera as a sources of complement after 2 h of incubation. Serum samples were taken after the each boost. %Killing (i.e. the percentage of bacteria killed) = 100 × 1 − (the mean number of CFU mL−1 after incubation/mean number CFU mL−1 before incubation). Data are representative of two separated experiments.

post-immunization the level of B. ovis-specific antibodies in rams immunized with rOmp31 extract (group 1) increased significantly in comparison to that obtained in group 2 injected with E. coli (pUC19) extract (P < 0.05) or group 3 (PBS) (P < 0.01). The second immunization performed at 4 weeks with the same dose resulted in a peak at week five with wide variations among individuals (2.495 ± 0.927), decreasing thereafter. Two weeks after the third immunization (at week 12 pi) antibodies peaked again (2.708 ± 0.050)

Tear IgA, IgG and IgM antibodies specific to Omp31 were investigated at weeks 2, 5, and 13 pi by iELISA-Omp31. Rams from group 1 developed specific antibodies of the isotypes IgA and IgG with similar kinetics (r2 = 0.99) while IgM was not detected (data not shown). None of the animals of both control groups had antibodies to Omp31 in tears (Fig. 6). 3.4. Bactericidal activity of immune sera Pooled sera collected at weeks 0, 4, 6, 13 after the first injection, were tested for the ability to promote in vitro complement-mediated killing of B. ovis. Sera from rams immunized with rOmp31 extract (group 1) showed bactericidal activity. The percentage of killing increased in each sample, with similar values whatever guinea pig or ovine complement were used (Fig. 5). Sera from groups 2 and 3 rams did not exhibit bactericidal activity indicating that Omp31specific antibodies were essential for bacterial killing. Heat inactivation of the complement sources completely abolished bactericidal activity, confirming that complement was essential.

Fig. 6. Determination of IgA (a) and IgG (b) antibodies in tears (diluted 1:2) to purified rOmp31 measured by indirect ELISA. Values represent mean ± S.D. absorbance units. Values significantly different from non-immunized group are indicated: ∗∗∗ P < 0.001.

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skin thickeness mean (mm.)

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7.5

***

***

***

5.0

2.5

0.0

0

48

96

120

time post-injection (hs) rOmp31extract

E.coli (pUC19) extract

PBS

Fig. 7. Increase in the skin thickness following an intradermal injection with purified rOmp31 at 20 weeks after the last boost. Measure were taken at 48, 96 and 120 h after the injection. Values represent the mean differences between skin thickness after and before injection in the same animal. Values significantly different from non-immunized group are indicated: ∗∗∗ P < 0.001.

3.5. Omp31 induced an in vivo cellular immune response To test in vivo correlates of cellular mediated immunity (CMI), rams were intradermally tested with rOmp31. As illustrated in Fig. 7, at 17 weeks after the third boost, only rams immunized with rOmp31 extract exhibited a marked local increase in skin thickness when compared with rams from group 2 or 3. The swelling was maximal at 48–90 h post-injection and then decreased.

4. Discussion We had already reported that recombinant B. melitensis Omp31 extract was a protective immunogen in the B. ovis mouse model of infection (Estein et al., 2003). In the present work, we examined the immunogenicity of this molecule in the natural host and characterized a protective mechanism against B. ovis. Rams immunized with adjuvanted rOmp31 extract developed serum IgG antibodies to high levels. Furthermore a Mab-based cELISA revealed the presence of serum antibodies to an epitope located to an hydrophilic loop of this protein (Vizca´ıno et al., 1996, 2001). Of note is that the Mab used in this assay was previously shown to afford passive protection against B. ovis infection in mice; this activity was as strong as that obtained with an anti-HS extract sera not inter-

ferred by non-protective Mabs (Bowden et al., 1995b, 2000). Moreover, this epitope is shared between B. melitensis and B. ovis and was recognized by antibodies of several B. ovis infected rams (Vizca´ıno et al., 2001). Therefore, potential protective antibodies were induced in rams by immunization with rOmp31. Serum antibodies showed good binding to whole B. ovis cells indicating that the antigenicity in this vaccine formulation was preserved. The binding to OMP-specific antibodies has been previously associated with the accessibility of the epitopes/antigens at the surface of B. ovis (Bowden et al., 1995a) and a relationship between accessibility and protective activity was found for Omp25 in B. melitensis (Bowden et al., 1998). Mechanisms mediating the protective effect of antibodies against B. ovis have not yet been elucidated. Although Brucella spp. are intracellular pathogens, they are transiently in the extracellular space, where antibodies could act by opsonization for an enhanced intracellular killing or by promoting antibody-dependent cellular toxicity by NK or other killer cells and/or complement-mediated lysis (Jiménez de Bagüés et al., 1994). Moreover, since Brucella is a parasite that transits through phagocytic vesicles (Pizarro-Cerdá et al., 1998), antibodies and other extracellular immune molecules could still act inside cells. According to Rittig study, the presence of heat-inactivated Brucella immune serum increased the number of CFU ingested although its persistence within the cells was

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not affected (Rittig et al., 2001). For several intracellular pathogens, mechanisms were shown by which antibody can influence the course of infection. For example, antibody binding to Ehrlichia risticii in the extracellular space can inhibit its intracellular replication (Messick and Rikihisa, 1994). This mechanism is different from that reported for Listeria monocytogenes, where antibody is active within macrophages neutralizing listeriolysin O (Edelson and Unanue, 2001). We wished to know whether antibodies to rOmp31 extract raised in rams were able to mediate complement bacteriolysis of B. ovis. Previously we found that Mabs against R-LPS or different OMPs including the anti-Omp31 Mab, promoted killing of B. ovis in presence of guinea pig complement (Estein, unpublished data). In the present study, both sources of complement (guinea pig and sheep) did not kill B. ovis in the absence of specific antibody indicating that B. ovis could not activate in vitro the alternative complement pathway. This result is in agreement with others studies (Corbeil et al., 1988; Eisenschenck et al., 1995) which showed that smooth and rough B. abortus were not killed in the absence of a functional classical complement pathway. Corbeil et al. (1988) demonstrated that, at later stages of infection, killing increased for the rough B. abortus strain but decreased for the smooth one, because blocking antibodies to S-LPS predominated. In contrast, in the same work, immunization with cell envelope from rough strain (rich in porin and group 3 proteins) induced a significant increase in killing, which correlated with the increase in antibody levels to major OMPs. In agreement, in our experiment, bactericidal activity showed a high correlation with the level of antibodies to Omp31, either with ovine or guinea pig complement (r2 = 0.75 and r2 = 0.73, respectively). Moreover, Omp31 antibodies were not efficacious unless in combination with complement indicating that bactericidal activity was due to lytic complement and not to other putative antibodymediated effect. Further studies are needed to elucidate the complement pathways involved in killing or opsonization of B. ovis. Although genital secretions were not studied, the presence of IgA and IgG anti-Omp31 antibodies in tears suggested the induction of a mucosal immune response. Foster et al. (1988) demonstrated the presence of different immunoglobulin classes and immunocom-

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petent cells in tissues and fluids of the male genital tract infected with B. ovis. These immunoglobulins could prevent bacterial colonization and subsequent inflammation of genital mucosae when this bacterium enters by the venereal route. Protective mechanisms involved could be the bacterial agglutination by IgA, which abounds in secretions, or the activation of alternative complement pathway with IgA–antigen complex (Butler, 1983). Likewise, bovine IgG1 antibodies could efficiently fix both guinea pig and ovine complement (Butler, 1983; Griebel et al., 1998) thus damaging bacteria. Experiments are needed to evaluate the role of local immunity against B. ovis in rams. It is well established that Brucella spp. are facultative intracellular pathogens capable to survive in macrophages. In the BALB/c model, protection against B. ovis is partially attributable to CMI (Jiménez de Bagüés et al., 1994). It is worth to mention that splenocytes from mice immunized with recombinant E. coli expressing the omp31 gene of B. melitensis proliferated when stimulated in vitro with a cell fraction rich in group 3 proteins (Guilloteau et al., 1999). In the present work, we observed that immunization with rOmp31 extract from B. melitensis elicited an in vivo hypersensitivity response when purified rOmp31 was injected. Therefore, Omp31 could have also induced cellular mechanisms which, if mouse model data applied, might be relevant for protection in sheep. Because, the important drawbacks of the current B. melitensis Rev.1 vaccine, safer and efficacious vaccines are sought against ovine brucellosis. Recently, a mutant that carry a defined disruption in the omp25 gene of B. melitensis (BM25), has shown to be attenuated and to afford protection against B. melitensis 16 M in pregnant goats. Although this mutant did not induce abortion and elicited antibodies against LPS (Edmonds et al., 2002a). Interestingly, an analogous ∆Omp25 mutant of B. ovis (BO25) was shown to provide sterilizing immunity against B. ovis challenge in mice, but its protective activity, and safety in sheep was not evaluated (Edmonds et al., 2002b). In conclusion, we showed that B. melitensis Omp31, was highly immunogenic in rams and induced lytic antibodies. The protective activity of this subcellular vaccine in challenged sheep remains to be investigated.

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Acknowledgements We thank Mar´ıa R. Ort´ız for media preparation (UNICEN), R. Malena for your efficient assistance and A. Llamas and S. Dinino for animal care (INTA). S.E. is a member of C.I.C. (Buenos Aires) and M.A.F. is doctoral fellow of CONICET. Special thanks go to A. Cloeckaert (INRA, Nouzilly) and N. Vizca´ıno (University of Salamanca) for helpful comments and for providing some of the recombinants used. Work supported by UNICEN, CIC and Project 522004 of INTA, Argentina. This article is dedicated to the memory of Dr. R.A. Bowden.

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