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

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.

Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.

Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial insemination. Endometrial biopsies were taken during consecutive cycles: (i) at estrus, when follicles reached 35 mm and at diestrus (7 +/- 1 days after ovulation); (ii) at 24 h post-AI, with dead semen (estrus) and in diestrus; (iii) at 24 h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) preparation and AI (with dead semen), and at diestrus. mRNA expression was quantitated by real time PCR. Under basal conditions, SM had significantly higher mRNA expression of all cytokines in estrus and of IL-1beta and TNF-alpha in diestrus, compared to RM. After AI, there were no differences between RM and SM in estrus; however, mRNA expression for all three pro-inflammatory cytokines was higher than under basal conditions. In diestrus, RM showed significantly lower IL-1beta and TNF-alpha mRNA expression than SM. When MCWE was administered at time of AI, no differences between cytokine induction from RM and SM were found. Globally, mRNA expression for all three cytokines correlated well among themselves when expression was high. The present study showed that (i) in basal conditions RM had lower mRNA expression of pro-inflammatory cytokines than SM with no effect of estrous cycle; (ii) AI upregulated mRNA expression for all three cytokines in both RM and SM, with persistance in diestrus in the latter; (iii) treatment with MCWE at time of AI down-regulated mRNA expression of IL-1 with significant effects in SM which behaved like RM. Immunomodulation with MCWE could be of help in restoring homeostatic local inflammatory mechanisms, thus assisting in the prophylaxis of post-breeding endometritis in mares.

Brucella lumazine synthase elicits a mixed Th1-Th2 immune response and reduces infection in mice challenged with Brucella abortus 544 independently of the adjuvant formulation used.

The immunogenicity and protective efficacy of recombinant lumazine synthase from Brucella spp. (rBLS) administered with different adjuvants was evaluated in mice. Mice were immunized with rBLS in the absence or the presence of aluminum hydroxide gel (BLS-Al), monophosphoryl lipid A (BLS-MPA), or incomplete Freund's adjuvant (BLS-IFA). rBLS per se induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. All the adjuvants increased this response; the BLS-IFA formulation was the most effective at inducing BLS-specific IgG antibodies. In addition, after in vitro stimulation with rBLS, spleen cells from BLS-IFA-, BLS-Al-, or BLS-MPA-immunized mice proliferated and produced interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-10, and IL-4, suggesting the induction of a mixed Th1-Th2 response. Immunization with rBLS protected mice against challenge with B. abortus 544. The levels of protection in the spleen were similar for all adjuvants, but only BLS-Al and BLS-IFA were effective in the liver. Our results indicate that BLS might be a useful candidate for the development of subunit vaccines against brucellosis, since it elicits antigen-specific cellular responses, with production of IFN-gamma and protection, independently of the adjuvant formulation used.

The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB/c mice.

Immunogenicity and protective activity against Brucella ovis of detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli, purified rough lipopolysaccharide from B. ovis (R-LPS) and a mixture of rOmp31 extract and R-LPS (rOmp31 extract + R-LPS) were assessed in BALB/c mice. The experimental vaccines were compared with a hot saline extract (HS extract) from B. ovis mainly composed of outer membrane proteins (OMPs) and R-LPS, and known to be protective in mice against a B. ovis infection. Serum antibodies to Omp31 and R-LPS were detected in the corresponding mice using Western blotting with B. ovis whole-cell lysates and ELISA with purified antigens. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. ovis. A significantly lower number of B. ovis colony-forming units in spleens relative to unimmunized (saline injected) controls were considered as protection. Mice immunized with rOmp31 extract or rOmp31 extract mixed with R-LPS developed antibodies that bound to the B. ovis surface with similar titers. Vaccination with rOmp31 extract plus R-LPS provided the best protection level, which was comparable with that given by HS extract. Similar protection was also obtained with rOmp31 extract alone and, to a lesser degree, with R-LPS. Comparisons between groups showed that an extract from E. coli-pUC19 (devoid of Omp31) provided no protection relative to either HS extract, rOmp31 extract or rOmp31 extract mixed with R-LPS. In conclusion, the recombinant Omp31 associated or not with B. ovis R-LPS, could be an interesting candidate for a subcellular vaccine against B. ovis infection.

Major outer membrane proteins of Brucella spp.: past, present and future.

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 and 25-27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.

Comparison of serological tests based on outer membrane or internal antigens for detecting antibodies to Brucella ovis in infected flocks.

The aim of this work was to compare the performance of 6 serological tests using outer or internal antigens from Brucella for the diagnosis of Brucella ovis infection in sheep in an endemic area. Outer membrane antigens included a hot saline extract (HS) and the rough lipopolysaccharide (R-LPS) from B. ovis. Internal antigens were LPS-free total cytosolic proteins (CP) and an 18-kDa cytosolic protein (p18) from Brucella spp. Sera from 200 sheep from naturally infected flocks were assayed by agar gel immunodiffusion test (AGID) and by complement fixation test (CFT), both using HS, and by 4 ELISA using HS, R-LPS, CP, and p18, respectively. The percentage of positive results was 45.5% for ELISA with HS, 42.0% for ELISA with p18, 39.5% for CFT, 33.5% for ELISA with R-LPS, 29.0% for ELISA with CP, and 18.0% for AGID. Taking CFT as the reference test for calculating relative test parameters, the ELISA with HS had the best sensitivity (96.2%), while AGID and the ELISA with R-LPS had the best specificity (96.6%). The ELISA with CP was not more sensitive than the ELISA with p18 (67.1% vs. 79.7%) in spite of the higher number of antigens in CP. The lower relative sensitivity of tests using internal antigens might reflect a lack of antibodies to cytosolic proteins in some infected animals or a shorter persistence of these antibodies relative to antibodies to outer membrane components after recovery from infection.

Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucella ovis infection in rams.

To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA). The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E. coli cells in a soluble form. This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography. After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein. The degree of purity appeared satisfactory so that it could be directly used in I-ELISA. Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B. ovis hot saline (HS) extract as antigen, the high number of sera from B. ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B. ovis infection in rams.

A DNA vaccine encoding lumazine synthase from Brucella abortus induces protective immunity in BALB/c mice.

This study was conducted to evaluate the immunogenicity of the Brucella abortus lumazine synthase (BLS) gene cloned into the pcDNA3 plasmid, which is driven by the cytomegalovirus promoter. Injection of plasmid DNA carrying the BLS gene (pcDNA-BLS) into BALB/c mice elicited both humoral and cellular immune responses. Antibodies to the encoded BLS included immunoglobulin G1 (IgG1) IgG2a, IgG2b, IgG3, and IgM isotypes. Animals injected with pcDNA-BLS exhibited a dominance of IgG2a over IgG1. In addition, spleen cells from vaccinated animals produced interleukin-2 and gamma interferon but not IL-10 or IL-4 after in vitro stimulation with recombinant BLS (rBLS), suggesting the induction of a Th1 response. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. abortus 544. Immunization with pcDNA-BLS- reduced the bacterial burden relative to those in the control groups. Mice immunized with rBLS produced a significant humoral response but did not show a specific cellular response or any protection from challenge. Altogether, these data suggest that pcDNA-BLS is a good immunogen for the production of humoral and cell-mediated responses in mice and is a candidate for use in future studies of vaccination against brucellosis.

Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA.

A study was conducted to evaluate the immunogenicity of the Brucella melitensis ribosome recycling factor (RRF)-homologous protein (CP24). The CP24 gene was cloned, expressed in Escherichia coli and purified. The resulting purified recombinant protein (rCP24) produced delayed-type hypersensitivity (DTH) reactions in B. melitensis-infected mice but not in naive controls. Thus, we decided to characterise the immune responses generated with DNA vaccination (pcDNACP24) or immunisation with the rCP24 in adjuvant. Animals injected with pcDNACP24 exhibited a dominance of IgG2a to IgG1 while mice injected with rCP24 developed a higher response of IgG1 than IgG2a. Both immunisation protocols were capable of eliciting CP24-specific gamma interferon (IFN-gamma) producing cells. Spleen cells from pcDNACP24-immunised mice did not produce interleukin (IL)-4, IL-10 or up-regulation of IL-2 mRNA. Cells from rCP24-immunised mice produced IL-10, up-regulated IL-2 mRNA but did not produce IL-4. Neither immunisation with purified CP24 nor injection of pcDNACP24 protected mice against challenge with live smooth B. melitensis. However, the potential of CP24 for a Brucella diagnostic test based on an in vitro antigen (Ag)-specific IFN-gamma production or DTH test would be worth testing.