Lipopolysaccharide heterogeneity in the atypical group of novel emerging Brucella species

Recently, novel Brucella strains with phenotypic characteristics that were atypical for strains belonging to the genus Brucella have been reported. Phenotypically many of these strains were initially misidentified as Ochrobactrum spp. Two novel species have been described so far for these strains, i.e., B. microti and B. inopinata, and other strains genetically related to B. inopinata may constitute other novel species as well. In this study, we analyzed the lipopolysaccharides (LPS) (smooth LPS [S-LPS] and rough LPS [R-LPS]) of these atypical strains using different methods and a panel of monoclonal antibodies (MAbs) directed against several epitopes of the Brucella O-polysaccharide (O-PS) and R-LPS. Among the most striking results, Brucella sp. strain BO2, isolated from a patient with chronic destructive pneumonia, showed a completely distinct S-LPS profile in silver stain gels that looked more similar to that of enterobacterial S-LPS. This strain also failed to react with MAbs against Brucella O-PS epitopes and showed weak reactivity with anti-R-LPS MAbs. B. inopinata reference strain BO1 displayed an M-dominant S-LPS type with some heterogeneity relative to the classical M-dominant Brucella S-LPS type. Australian wild rodent strains belonging also to the B. inopinata group showed a classical A-dominant S-LPS but lacked the O-PS common (C) epitopes, as previously reported for B. suis biovar 2 strains. Interestingly, some strains also failed to react with anti-R-LPS MAbs, such as the B. microti reference strain and B. inopinata BO1, suggesting modifications in the core-lipid A moieties of these strains. These results have several implications for serological typing and serological diagnosis and underline the need for novel tools for detection and correct identification of such novel emerging Brucella spp.     

Immunochemical identification of Brucella abortus lipopolysaccharide epitopes.

Sera from Brucella abortus-infected and -vaccinated bovines recognized four lipopolysaccharide (LPS) determinants: two in the O-polysaccharide (A and C), one in the core oligosaccharide from rough Brucella LPS (R), and one in lipid A (LA). From 46 different hybridomas secreting monoclonal antibodies (MAbs) against various LPS moieties, 9 different specificities were identified. Two epitopes, A and C/Y, were present in the O-polysaccharide. Two epitopes were found in the core oligosaccharide (R1 and R2) of rough Brucella LPS. MAbs against R1 and R2 epitopes reacted against LPS from different rough Brucella species; however, MAbs directed to the R2 epitope also reacted against enterobacterial LPS from deep rough mutants. Three epitopes (LA1, LA2, and LA3) were located in the lipid A backbone. Different sets of MAbs recognized two epitopes in the lipid A-associated outer membrane protein (LAOmp3-1 and LAOmp3-2). LPS preparations from smooth brucellae had small amounts of rough-type LPS. Although LPS from rough brucellae did not show smooth-type LPS in western blots (immunoblots), two hybridomas generated from mice immunized with rough B. abortus produced antibodies against smooth B. abortus LPS. Results are discussed in relation to the structure and function of B. abortus LPS and to previous findings on the epitopic density of the molecule.     

Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.

Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24.     

Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry.

Seven surface-exposed outer membrane proteins (OMPs) in Brucella supp. have been previously described (A. Cloeckaert, P. de Wergifosse, G. Dubray, and J. N. Limet, Infect. Immun. 58:3980-3987, 1990). OMPs were shown to be more accessible to monoclonal antibodies (MAbs) on rough (R) Brucella melitensis and B. abortus strains than to MAbs on their smooth (S) counterparts. In this work, we have extended this study to representatives of the main Brucella species, using MAbs specific for OMPs and S and R lipopolysaccharides (S-LPS and R-LPS). Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoelectron microscopy showed important differences between strains in the binding of OMP- and R-LPS-specific MAbs which were in part related to the particular expression of S-LPS, irrespective of the species. Results indicated that both the amount and the length of O polysaccharide on S-LPS greatly influenced the accessibility of OMP and R-LPS epitopes to MAbs. S-R B. melitensis EP and S B. suis 40, for instance, which express O-polysaccharide chains in small amounts and with short mean length, respectively, bound a greater number of OMP- and R-LPS-specific MAbs than the other S Brucella strains. The major 31- to 34-kDa OMP was the most exposed OMP on S strains of B. melitensis and B. suis. In most cases, flow cytometry results agreed with those of ELISA and supplied additional data, such as the homogeneity or heterogeneity of OMP expression at the strain level. However, there were some discordances between flow cytometry and ELISA results concerning the surface exposure of the 25- to 27-kDa and 31- to 34-kDa OMPs on S strains and that of minor OMPs in vaccine strain B. melitensis Rev.1. Immunoelectron microscopy confirmed the poor accessibility of OMPs to MAbs on the surface of S Brucella strains. The naturally R pathogenic species B. ovis and B. canis bound the majority of OMP-specific MAbs as well as the R-LPS-specific MAbs. Therefore, the conserved OMP and R-LPS epitopes could play a role as targets of protective antibody-mediated immunity in infections caused by naturally R B. ovis and B. canis.     

Protection conferred on mice by monoclonal antibodies directed against outer-membrane-protein antigens of Brucella

Twenty-six monoclonal antibodies (MAbs) directed against seven brucella outer-membrane proteins (OMPs) of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 Kda were screened for passive protection of mice; three MAbs (directed against 16.5, 25-27 and 36-38 Kda) reduced significantly the initial colonisation of the spleen measured 7 days after challenge. Although significant, the reduction in numbers of Brucella organisms in the spleen was low compared with that conferred by MAbs against lipopolysaccharide of smooth specificity (S-LPS). The three most protective MAbs belonged to two isotypes (IgG1 and IgG2a) and were specific for three different OMPs. No relationship between protection and binding of MAbs to the challenge S strain or its rough mutant was observed in the mouse model. The humoral protection depended mainly on antibodies directed against S-LPS, although some MAbs against OMPs had weak protective activity.     

Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene.

The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.     

Use of monoclonal antibodies to identify the distribution of A and M epitopes on smooth Brucella species.

The smooth types of Brucella species express two lipopolysaccharides (LPSs) (A and M) which are antigenically distinct. Their existence as cross-reactive antigenic complexes makes definitive serology difficult. Murine hybridomas were produced and selected for their ability to produce monoclonal antibodies to the specific A- or M-LPS epitopes. The specificity of the monoclonal antibodies was determined by microplate enzyme-linked immunoassay, binding inhibition assay, microplate agglutination, and dot blot assay. Monoclonal antibody 12AE6 was specific for an epitope on the A LPS of Brucella spp., which was also expressed on Yersinia enterocolitica O:9. A unique epitope of M LPS was detected by monoclonal antibody 33.1.5. The two monoclonal antibodies did not exhibit cross-reactions when assayed with whole Brucella cells or purified M- and A-LPS preparations. Cross-reactive serology with polyvalent sera can be attributed to the presence of common antigenic determinants on both molecules. The use of A- and M-LPS-specific monoclonal antibodies has the potential to replace the more laborious methods involved in the production of monospecific sera.     

Lipopolysaccharide heterogeneity in Brucella strains isolated from marine mammals

Smooth lipopolysaccharides (S-LPSs) from Brucella strains isolated from seals, dolphins, porpoises, an otter and a minke whale were characterized by ELISA using monoclonal antibodies (mAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes and by Western blot after SDS-PAGE. All strains studied were A-dominant as shown by specific polyclonal sera and this was also confirmed by the mAbs. However, binding patterns in ELISA of mAbs to the specific common (C) epitopes were rather heterogeneous, and for some strains, such as those isolated from striped dolphins, binding of these mAbs was much reduced or negative as had previously been shown for Brucella suis biovar 2 strains. Western blot after SDS-PAGE showed the typical A-dominant strain banding pattern for all marine mammal Brucella isolates, but the average S-LPS size was shorter in many of these compared to reference Brucella abortus strain 544. Thus, S-LPSs of the marine mammal isolates show heterogeneity with regard to their O-PS C epitope content and their average size.     

Partial protection against Brucella infection in mice by immunization with nonpathogenic alphaproteobacteria

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.     

布鲁氏菌OMP31 T-B联合表位抗原的免疫应答分析

目的：研究OMP31表位的抗原性和免疫应答特点,为布鲁氏菌表位疫苗的研制奠定基础。方法：利用生物信息软件筛选OMP31 T-B联合优势抗原表位,进一步合成肽段。用OMP31肽段免疫BALB/c小鼠,采用ELISA检测小鼠血清中特异性IgG1和IgG2a抗体水平,ELISPOT检测脾淋巴细胞中IFN-γ阳性的T淋巴细胞活化情况。结果：用OMP31合成肽段免疫BALB/c小鼠4次后,小鼠血清中IgG1和IgG2a抗体水平升高,小鼠脾淋巴细胞集落形成单元（SFU）增高。结论：OMP31 T-B联合表位抗原能增强小鼠Th1免疫应答和体液免疫应答,可以作为布鲁氏菌病的候选疫苗。     

Evaluation of purified UspA from Moraxella catarrhalis as a vaccine in a murine model after active immunization.

Moraxella catarrhalis causes otitis media, laryngitis, and respiratory infections in humans. A high-molecular-weight outer membrane protein from this bacterium named ubiquitous surface protein A (UspA) is present on all isolates. A monoclonal antibody (MAb) to UspA that recognizes a conserved epitope of this protein has been shown to promote pulmonary clearance of bacteria in passively immunized mice. In the present study, M. catarrhalis heterologous isolates were screened by dot blot with a panel of four additional MAbs specific for surface-exposed epitopes of UspA from M. catarrhalis isolate 035E. Three of the MAbs were specific for 035E, and the fourth reacted with 17 (74%) of the 23 isolates tested. Thus, UspA contains highly conserved, semiconserved, and variable surface-exposed epitopes. The UspA was purified from the 035E isolate by ion-exchange and size-exclusion chromatography, formulated with the adjuvant QS-21, and used to immunize BALB/c mice. Upon pulmonary challenge with either 035E or the heterologous isolate TTA24, significantly fewer bacteria were recovered from the lungs of immunized mice 6 h postchallenge than from control mice. The immune sera from mice or guinea pigs contained high titers of antibodies to the homologous isolate and heterologous isolates in a whole-bacterial-cell enzyme-linked immunosorbent assay. Sera against UspA, whether prepared in mice or guinea pigs, had complement-dependent bactericidal activity toward homologous and 11 heterologous M. catarrhalis isolates. These results indicate that the conserved epitopes of the UspA are highly immunogenic and elicit broadly reactive and biologically functional antibodies. UspA may offer protection against M. catarrhalis infections and is being further evaluated as a vaccine candidate.     

Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae.

Brucellae have been reported to be phylogenetically related to bacteria of the family Rhizobiaceae. In the present study, we used a panel of monoclonal antibodies (MAbs) to Brucella outer membrane proteins (OMPs) to determine the presence of common OMP epitopes in some representative bacteria of this family, i.e., Ochrobactrum anthropi, Phyllobacterium rubiacearum, Rhizobium leguminosarum, and Agrobacterium tumefaciens, and also in bacteria reported to serologically cross-react with brucella, i.e., Yersinia enterocolitica O:9, Escherichia coli O:157, and Salmonella urbana. In particular, most MAbs to the Brucella outer membrane lipoproteins Omp10, Omp16, and Omp19 cross-reacted with O. anthropi and P. rubiacearum, which are actually the closest relatives of brucellae. Some of them also cross-reacted, but to a lower extent, with R. leguminosarum and A. tumefaciens. The putative Omp16 and Omp19 homologs in these bacteria showed the same apparent molecular masses as their Brucella counterparts. None of the antilipoprotein MAbs cross-reacted with Y. enterocolitica O:9, E. coli O:157, or S. urbana.     

Characterization of a monoclonal antibody specific for Brucella smooth lipopolysaccharide and development of a competitive enzyme-linked immunosorbent assay to improve the serological diagnosis of brucellosis.

The reactivity of monoclonal antibody (MAb) 12G12 was analyzed in regard to the main biovars of Brucella species and some members of the families Enterobacteriaceae and Vibrionaceae which present serological cross-reactions with the smooth lipopolysaccharide (S-LPS) of Brucella species. This MAb was strictly directed against the common specific epitope of the Brucella S-LPS. It recognized all of the smooth Brucella strains and biovars except B. suis biovar 2. In order to improve the specificity of the serological diagnosis of brucellosis, a competitive enzyme-linked immunosorbent assay (cELISA) was developed with the horseradish peroxidase-conjugated MAbs 12G12 and S-LPS of B. melitensis Rev1. The specificity of the cELISA was analyzed with 936 serum samples from healthy cattle. The assay was evaluated with sera from heifers (n = 18) experimentally infected with B. abortus 544. After infection, the performance of the cELISA was in agreement with those of the complement fixation test and the rose Bengal plate test. Finally, the specificity of the assay was also evaluated in regard to false-positive serological reactions by using sera from heifers experimentally infected with Yersinia enterocolitica 0:9 (n = 4) and with field sera presenting false-positive reactions (n = 74). The specificity of the cELISA was greater than the specificities of the complement fixation test and the rose Bengal plate test. Indeed, the new assay detected only 31 of the 101 false-positive serum samples detected by at least one serological test.     

Monoclonal antibodies that define neutralizing epitopes of pertussis toxin: conformational dependence and epitope mapping.

The epitope specificities of 13 hybridomas secreting monoclonal antibodies (MAbs) specific for pertussis toxin (PT) is described. Hybridoma lines were derived by the fusion of spleen cells from mice immunized with native PT, Formalin-detoxified PT, or isolated PT subunits (S1 to S5) with the myeloma line X63-Ag8.653. Five MAbs showed a toxin-neutralizing ability, which was demonstrated by use of a Chinese hamster ovary cell assay system and by a NAD glycohydrolase assay. All five toxin-neutralizing MAbs demonstrated high specificities for and reactivities with native PT but were unable to bind to denatured PT. One MAb was able to neutralize the enzymatic activity of PT. The other four neutralizing MAbs inhibited the binding of PT or PT subunits to the surface of Chinese hamster ovary cells, as shown by an immunofluorescence assay. All neutralizing MAbs reacted with purified S2-S4 or S3-S4 dimers but not with S4 alone. Three MAbs which recognized a common epitope shared by S2 and S3 (which are about 70% homologous at the DNA level) and one MAb which recognized S4 were not neutralizing. Isolated S2-S4 and S3-S4 dimers bound to Chinese hamster ovary cells. These results indicate that the majority of critical epitopes which elicit neutralizing antibody are conformation dependent.     

Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide.

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.     

Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay.

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.     

Minor nucleotide substitutions in the omp31 gene of Brucella ovis result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella species

The gene coding for the major outer membrane protein Omp31 was sequenced in five Brucella species and their biovars. Although the omp31 genes appeared to be highly conserved in the genus Brucella, nine nucleotide substitutions were detected in the gene of Brucella ovis compared to that of Brucella melitensis. As shown by differential binding properties of monoclonal antibodies (MAbs) to the two Brucella species, these nucleotide substitutions result in different antigenic properties of Omp31. The antigenic differences were also evidenced when sera from B. ovis-infected rams were tested by Western blotting with the recombinant B. melitensis or B. ovis Omp31 proteins. Twelve available sera reacted with recombinant B. ovis Omp31, but only four of them reacted with recombinant B. melitensis Omp31. These results validate previous evidence for the potential of Omp31 as a diagnostic antigen for B. ovis infection in rams and demonstrate that B. ovis Omp31, instead of B. melitensis Omp31, should be used to evaluate this point. The antigenic differences between the B. melitensis and B. ovis Omp31 proteins should also be taken into account when Omp31 is evaluated as a candidate for the development of subcellular vaccines against B. ovis infection. No reactivity against recombinant B. melitensis Omp31 was detected, by Western blotting, with sera from B. melitensis-infected sheep. Accordingly, Omp31 does not seem to be a good diagnostic antigen for B. melitensis infections in sheep. Two immunodominant regions were identified on the B. ovis Omp31 protein by using recombinant DNA techniques and specific MAbs. Sera from B. ovis-infected rams that reacted with the recombinant protein were tested by Western blotting against one of these immunodominant regions shown to be exposed at the bacterial surface. Only 4 of the 12 sera reacted, but with strong intensity.     

Identification of Anti-Alpha Toxin Monoclonal Antibodies That Reduce the Severity of Staphylococcus aureus Dermonecrosis and Exhibit a Correlation between Affinity and Potency

Staphylococcus aureus alpha toxin (AT) is an important virulence determinant and may be a valid target for immunoprophylaxis against staphylococcal disease. Here we report the identification of potent inhibitory anti-AT monoclonal antibodies (MAbs) derived using B-cell hybridoma technology from VelocImmune mice engineered to produce IgG with a human variable domain. A small panel of inhibitory MAbs blocked AT-mediated lysis of rabbit red blood cells, A549 human lung epithelial cells, and THP-1 human monocytic cells, in a dose-dependent manner. Binding studies indicated that these MAbs recognize a similar epitope on AT and exhibit dissociation constants (K_D) ranging from 0.50 to 15 nM. In an S. aureus dermonecrosis model, mice passively immunized with anti-AT inhibitory MAbs exhibited significant reductions of lesion size relative to mice treated with an irrelevant IgG control. Interestingly, there was a correlation between MAb affinity for a single epitope, the 50% inhibitory concentration (IC₅₀) in the AT hemolytic assay, and lesion size reduction in the dermonecrosis model. A representative high-affinity MAb, 2A3.1, was demonstrated to significantly reduce lesion size following infection with three different clinical isolates (USA300, CC30, and CC5). Taken together, these results indicate that in vitro potency of anti-AT MAbs predicts in vivo potency in this model, supporting their continued preclinical evaluation as molecules for immunoprophylaxis against staphylococcal skin and soft tissue infections caused by diverse clinical isolates.     

DNA immunization followed by a single boost with cells: a protein-free immunization protocol for production of monoclonal antibodies against the native form of membrane proteins

Recent advancements in antibody-based therapies require the development of an efficient method for generation of monoclonal antibodies (MAbs) against the native form of membrane proteins. We examined DNA immunization followed by a single boost with cells as a protein-free immunization protocol for production of MAbs. Mice immunized with plasmid cDNAs encoding human CD30 or Ret tyrosine kinase were given a single boost with cells expressing the corresponding antigen prior to cell fusion. A total of nine cell fusion experiments revealed that the cell boost is necessary for efficient generation of hybridomas and the DNA-cell boost method gave good yields of specific MAbs (5–59 MAbs from one mouse). All IgG isotypes except IgG3 were generated, although IgG2a was the dominant isotype. All the MAbs reacted with native antigens expressed on cells in a fluorescence-activated cell sorter (FACS) analysis as well as with recombinant CD30 or Ret protein genetically fused with human Fc in an enzyme-linked immunosorbent assay (ELISA). The affinities of the anti-CD30 MAbs to CD30-Fc protein ranged from 0.9 to 12.4 nM K_ds, which were comparable to existing MAbs to these proteins, which range from 3.0 to 13.0 nM. Western blot analysis and topographical epitope mapping experiments based on the mutual competition of pairs of the anti-CD30 MAbs revealed that about 40% of the epitopes were linear epitopes and that each epitope was topographically classified into one of six groups. The large number of MAbs that react with high affinities to a variety of epitopes on the native form of antigens indicates that the method presented in this paper could be generally useful for generating MAbs to other membrane proteins.     

A bile salt hydrolase of Brucella abortus contributes to the establishment of a successful infection through the oral route in mice

Choloylglycine hydrolase (CGH), a bile salt hydrolase, has been annotated in all the available genomes of Brucella species. We obtained the Brucella CGH in recombinant form and demonstrated in vitro its capacity to cleave glycocholate into glycine and cholate. Brucella abortus 2308 (wild type) and its isogenic Deltacgh deletion mutant exhibited similar growth rates in tryptic soy broth in the absence of bile. In contrast, the growth of the Deltacgh mutant was notably impaired by both 5% and 10% bile. The bile resistance of the complemented mutant was similar to that of the wild-type strain. In mice infected through the intragastric or the intraperitoneal route, splenic infection was significantly lower at 10 and 20 days postinfection in animals infected with the Deltacgh mutant than in those infected with the wild-type strain. For both routes, no differences in spleen CFU were found between animals infected with the wild-type strain and those infected with the complemented mutant. Mice immunized intragastrically with recombinant CGH mixed with cholera toxin (CGH+CT) developed a specific mucosal humoral (immunoglobulin G [IgG] and IgA) and cellular (interleukin-2) immune responses. Fifteen days after challenge by the same route with live B. abortus 2308 cells, splenic CFU counts were 10-fold lower in mice immunized with CGH+CT than in mice immunized with CT or phosphate-buffered saline. This study shows that CGH confers on Brucella the ability to resist the antimicrobial action of bile salts. The results also suggest that CGH may contribute to the ability of Brucella to infect the host through the oral route.