Gérard Dubray

A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels

Abstract A highly sensitive silver stain for visualizing proteins in polyacrylamide gels was adapted for staining 1,2-diol groups of carbohydrates. This periodic acid-silver stain is 64 times more sensitive than the periodic acid-Schiff stain, and the lower limit of detection is about 0.4 ng of bound carbohydrates. Its lower limit of detection is comparable to autoradiography of radioactively labeled lipopolysaccharides.

Outer-membrane protein- and rough lipopolysaccharide-specific monoclonal antibodies protect mice against Brucella ovis

Brucella ovis, a naturally virulent rough Brucella species, is the aetiological agent of ram epididymitis. The identification of protective antigens is necessary to obtain a safe, specific subcellular vaccine. Monoclonal antibodies (MAbs) directed at both brucella outer-membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS) in a mouse protection test were used to identify potential targets for humoral immunity. Mixtures of MAbs directed at the 16.5-, 25-27-, 31-34- and 36-38-kDa OMPs conferred significant protection 7 days after challenge with reference strain B. ovis 63/290 compared with controls receiving either saline or an anti-brucella O-polysaccharide MAb. Furthermore, an anti-R-LPS MAb tested alone conferred protection at a level comparable with that obtained with the mixture of anti-OMP MAbs. The combination of protective OMP MAbs with the anti-R-LPS MAb was also strongly protective. One combination of OMP MAbs, which bound intensely to B. ovis in vitro, was ineffective. These results indicate that B. ovis OMPs and R-LPS are targets for protective antibodies and that they can be regarded as candidates for ram epididymitis subcellular vaccines.

Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies.

A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.

Protection conferred on mice by combinations of monoclonal antibodies directed against outer-membrane proteins or smooth lipopolysaccharide of Brucella

The effect of monoclonal antibodies (MAbs) injected alone or in combination on brucella splenic infection in CD-1 mice was tested 7 and 21 days after a challenge with virulent Brucella abortus 544. Passive immunisation of mice with anti-25-27-kDa MAb alone, or mixed with protective anti-16.5 and anti-36-38-kDa MAbs, or with MAbs of the same specificity which were previously demonstrated to have no activity on CD-1 mice, produced a significant reduction of spleen counts of B. abortus (p less than 0.01). Other combinations of MAbs did not reduce splenic infection in comparison with the untreated control group. BALB/c mice were used to test the possible interference of the immune response of CD-1 mice against MAbs that were produced in BALB/c mice. No reduction of splenic infection was shown with anti-25-27- or -36-38-kDa MAbs, whereas anti-lipopolysaccharide (LPS) MAb which was produced in CBA mice was effective. Combination of anti-protein MAbs with the anti-LPS MAb produced only the effect of the anti-LPS MAb at 7 and 21 days after challenge.

Induction of antibody and protective responses in mice by Brucella O-polysaccharide-BSA conjugate

A Brucella conjugate vaccine was synthesized by covalently coupling the O-polysaccharide (PS) obtained from Brucella melitensis 16M, to bovine serum albumin (BSA). The conjugate (PS-BSA) was non-toxic for mice. When injected at two doses (10 or 1 micrograms) in mice, it induced the production of antibodies and the boost was shown to be efficient if administered after 10 weeks. Immunization of mice with 10 micrograms of conjugate conferred significant protection against virulent challenge with B. melitensis H38, similar to that obtained with the reference vaccine H38.

Characterization of O-polysaccharide specific monoclonal antibodies derived from mice infected with the rough Brucella melitensis strain B115.

Twenty-two monoclonal antibodies (mAbs) specific for smooth lipopolysaccharide (S-LPS) were generated by fusion of spleen cells from mice infected with the rough Brucella melitensis strain B115 with the NSO myeloma. According to reactivity in enzyme-linked immunosorbent assay (ELISA) with O-polysaccharide (O-PS) and absence of reactivity with rough lipopolysaccharide (R-LPS), it was postulated that these mAbs recognized epitopes present on the O-PS. Most of the mAbs reacted equally well in ELISA and immunoblotting with S-LPS types of Brucella A and M dominant strains and were designated as specific for common (C) epitopes. Three mAbs were highly specific for M dominant S-LPS. All these mAbs, in contrast to a mAb specific for the A epitope, showed little or no cross-reactivity with Yersinia enterocolitica O:9 S-LPS. S-LPS of B. melitensis B115 was extracted and analysed by immunoblotting and ELISA with mAbs specific for A, M and C epitopes. Reactivity of the mAbs with this S-LPS was compared to reactivity with S-LPS of A and M dominant smooth Brucella strains. The results suggest that S-LPS of B. melitensis B115 bears mainly C epitopes and a few M epitopes. The very weak reactivity of this S-LPS with the mAb specific for the A epitope and the fact that the mAbs specific for C and M epitopes showed little or no cross-reactivity with Y. enterocolitica O:9 S-LPS suggest that O-PS from this rough strain could be used to distinguish Y. enterocolitica O:9 infection from Brucella infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive enzyme-linked immunosorbent assay using monoclonal antibodies to the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev.1 vaccinated sheep

Competitive enzyme-linked immunosorbent assay (C-ELISA) was performed using 15 monoclonal antibodies (MAbs), specific for Brucella BP26 (previously also called CP28), a periplasmic protein antigen, to investigate antibody responses in naturally and B. melitensis H38 experimentally infected and B. melitensis Rev.1 vaccinated sheep. The antigen preparation consisted of cytosoluble protein extract (CPE) of B. melitensis B115. By combining the C-ELISA results of several MAbs, a high percentage of naturally infected animals were detected which showed different status in the current conventional diagnostic tests. Indeed, 90% of sheep which were positive in the conventional bacteriological and serological tests were positive in C-ELISA. 72% of the bacteriologically negative but serologically and delayed type hypersensitivity positive sheep were also positive in the C-ELISA. Moreover, 79% of the bacteriologically and serologically negative sheep but delayed type hypersensitivity positive were also detected by C-ELISA. Thus, these results confirmed the importance of BP26 as a frequently recognized target of the humoral immune response of infected sheep. The 8 B. melitensis H38 experimentally infected sheep showed various degrees of antibody responses at the 90th day after infection, which was delayed in comparison to that against O-polysaccharide (O-PS). Of the 15 MAbs tested, only one MAb was weakly inhibited (20 to 35% inhibition) by 56% of negative control sera. Furthermore, no antibody response against BP26 was detected in B. melitensis Rev.1 vaccinated sheep. Results of the C-ELISA with the 15 MAbs showed individual variability of the antibody responses against BP26. Thus, it is suggested that several epitopes of BP26 are of interest for diagnosis of B. melitensis infection in sheep.

Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/c mice

BALB/c mice were immunized with recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis, a gene coding for a major outer membrane protein. Immunization resulted in the production of specific antibodies to B. melitensis in the serum, the production of which was considerably increased after boosting with a dose ten times lower than the first. A significant specific proliferative response of immune spleen cells to B. melitensis was observed 5 weeks after the first immunization but this response did not persist. Despite the induction of systemic humoral and cellular immune responses by recombinant E. coli expressing the B. melitensis omp31 gene, no significant protection against a challenge with smooth B. melitensis H38S was observed in immunized mice. These results demonstrate that despite the strong antibody response induced in mice, immunization with the recombinant Omp31 of B. melitensis does not confer any protective effect against a virulent smooth B. melitensis. However, its potential protective effect for protection against rough Brucella would be worth testing.

O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy.

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)