Candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: developmental chemistry and investigation of immunological responses following immunization of mice and rabbits.

Glycoconjugates were prepared by covalently linking the immunogenic protein carrier CRM(197) to O-deacylated lipopolysaccharide (LPS) derived from Neisseria meningitidis (strain H44/76), immunotype L3 galE LPS. This mutant strain elaborates a truncated LPS structure that displays immunological epitopes characteristic of 76% of Group B meningococcal (NmB) strains. CRM(197) was covalently linked either to the reducing glucosamine residue of the lipid A region of the O-deacylated LPS or to a 2-keto-3-deoxy-octulosonic acid (Kdo) residue in the inner core region of the O-deacylated LPS. In both rabbits and mice a much stronger IgG response to the immunising antigen was generated in those animals that received conjugates linked via the lipid A region. Sera from mice that were immunized with these conjugates were assayed for their reactivity with LPS, both mutant and wild-type, of several homologous and heterologous NmB strains. Sera obtained from mice immunized with conjugates in which the carrier protein was linked via the Kdo moiety were only able to react with O-deacylated, but not fully acylated (native), LPS from the homologous strain. However, sera obtained from mice that were immunized with conjugates, in which the carrier protein was coupled to the lipid A region, reacted predominately with inner core epitopes that contained phosphoethanolamine at the same 3-position of the distal heptose residue (HepII) of the inner core LPS as was present on the immunising antigen. Additionally it was observed that sera from rabbits immunised with lipid A linked conjugates, unlike the mice responses, were generally not as specific for LPS antigens that contained phosphoethanolamine at the same 3-position as was present on the immunising antigen, but showed a broader inner core recognition, whereas those rabbits that received the Kdo-linked conjugates gave only a very weak non-specific response to all immunotypes. Finally, the sera from two out of six mice that had received lipid A linked conjugates had bactericidal activity against L3 wild-type NmB strain 8047 and one of these was able to passively protect against meningococcal infection in an infant rat model. This study demonstrates evidence towards the proof-in-principle that by using Nm inner core LPS conjugates coupled via the lipid A region with an intact phosphoethanolamine at the O-3 position of the HepII of the inner core LPS, it is possible to elicit functional and protective antibodies against meningococcal infection.

Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in approximately 70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5-6% and inter-assay CV was 19-22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occurring serum antibodies (specific to inner core LPS) between healthy adults (18-65 years, N=54) and healthy infants (3-4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5. 0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5. 7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26-113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3. 1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5-181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis.

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B. 15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. meningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

Molecular basis for the lack of mimicry of Brucella polysaccharide antigens by Ab2gamma antibodies.

The crystal structure of the complex of an anti-Id Fab with an Fab specific for a Brucella polysaccharide antigen has previously been reported (Evans et al., 1994, J. Mol. Biol. 241, 691-705). To complement this study, the binding characteristics and immunological properties of this Ab2 and two others raised with a second anti-Brucella antibody were investigated, including quantitative kinetic measurements by surface plasmon resonance. The affinities of the Fabs from the Ab2s for the Ab1s were three orders of magnitude greater than those estimated for the antigen, but the Ab2s failed to induce antigen-binding Ab3s, that is, they were of the Ab2gamma type. The avidities of the Ab1s for antigen were however within one order of magnitude of their avidities for Ab2. Tests of 16 other anti-Brucella polysaccharide antibodies showed that the two idiotopes were not present in them, and in confirmation of the lack of a dominant idiotope, N-terminal sequencing of their H and L chains showed a wide variety of V genes were employed in the immune response to the Brucella polysaccharides. The failure of the Ab2 to induce antigen-reactive Ab3 thus appears to be due to neither intrinsic affinity nor idiotope frequency, but arises instead from structural reasons, for example, the incomplete penetration of the Ab2 into the binding-site cleft of the Ab1. The surface topography of polysaccharide antigens and their binding-sites thus appears to be especially difficult for Ab2s to mimic and will restrict their routine use as surrogates for T-cell independent polysaccharide antigens.

Conformational epitope of the type III group B Streptococcus capsular polysaccharide.

The protective epitope of the type III group B streptococcal polysaccharide (GBSPIII) is length dependent and conformational. To obtain a more accurate characterization of the conformational epitope, ELISA inhibition and surface plasmon resonance studies were conducted on two GBSPIII-specific mAbs using a large panel of oligosaccharide probes. The results of the studies confirmed that 2 repeating units (RU) is the minimum binding unit and that, while increases in chain length from 2 RU to 7 RU caused further optimization of the epitope, it remained monovalent. A 3-fold increase in affinity was observed between 7 RU and 20 RU, which, by surface plasmon resonance studies on a Fab, was shown to be due to both further optimization of the individual epitope and the occurrence of multivalency of epitope. The data support our hypothesis that the conformational epitope is an extended helical segment of the GBSPIII. GBSPIII exists mainly in the random coil form, which structurally mimics short oligosaccharide self Ags, but it can infrequently and spontaneously form extended helices. Although not prevalent in GBSPIII, the immune system preferentially selects these helical epitopes because they are unique to the polysaccharide. Contrary to a previously proposed model of GBSPIII binding in which the binding of the first Ab propagates a continuum of helical epitopes, our binding kinetics are consistent only with the helical epitope's being discontinuous and infrequent.

Production and characterization of monoclonal antibodies to Actinobacillus pleuropneumoniae serotype 5b.

Monoclonal antibodies specific for capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of A. pleuropneumoniae serotype 5b were generated by hybridoma cells and selected by indirect ELISA of culture supernatants with purified and structurally defined LPS and CPS preparations and their synthetic conjugates. It was shown in this study that at least one monoclonal antibody, 3B4, presented 100% specificity and recognized all A. pleuropneumoniae serotype 5 field strains tested in a dot-ELISA assay.

Molecular recognition of a Salmonella trisaccharide epitope by monoclonal antibody Se155-4.

The binding site of monoclonal antibody Se155-4, which has been the object of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1, rather than to the tetrasaccharide repeating unit alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1-->4) alpha-L-Rhap(1- of the bacterial antigen. Specificity for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi B O-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic oligosaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives showed that complementary surfaces and three antibody-saccharide hydrogen bonds are essential for full binding activity. Both hydroxyl groups of the 3,6-dideoxy-D-galactose residue were obligatory for binding and consistent with the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-dideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mannose, tyvelose were not bound by the antibody. Titration microcalorimetry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy that accompanies binding of the native trisaccharide determinant. The protein sequences determined for the antibody VL and VH domains reveal somatic mutation of the VL germ line gene, implying that this antibody-binding site results from a mature antibody response.

Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.

Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.

Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.

A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.

Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides.

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and O polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica O:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A- and M-antigen structures, an interpretation supported by competitive binding studies with O polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous alpha 1,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by alpha 1,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element alpha-D-Rha4NFo(1----3)alpha-D-Rha4NFo bracketed by alpha 1,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated alpha 1,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.

Identification of Escherichia coli serotype O157 strains by using a monoclonal antibody.

The O157 antigenic determinant of Escherichia coli serotype O157:H7, an important bacterial pathogen, resides in the polysaccharide portion of its cellular lipopolysaccharide component which, from structural studies, was identified as a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, L-fucose, 2-acetamido-2-deoxy-D-galactose, and 4-acetamido-4,6-dideoxy-D-mannose residues (1:1:1:1). Hybrid cells producing monoclonal antibodies against the E. coli O157 antigen were obtained by fusion of myeloma cells with lymphocytes from BALB/c mice immunized with killed E. coli O157:117 cells. Clones were selected for binding specificity with purified O polysaccharide. One monoclonal antibody used in direct slide agglutinations or in coagglutination reactions with Staphylococcus aureus Cowan 1 cells sensitized with the affinity column-purified antibody accurately detected all strains of E. coli O157 tested. This selected monoclonal antibody did not agglutinate E. coli strains such as E. coli O7 and E. coli O116 or other bacteria which are known to give positive agglutinations with conventional polyclonal E. coli antisera. These results indicate that the monoclonal antibody is a superior specific-typing reagent.

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods.

An O-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli O157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli O157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli O157:H7, the organism was isolated at levels of up to 10(3)/g. The lower limit of sensitivity was 10 E. coli O157 per g of meat. Specific typing for E. coli O157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.

Characterization of Shigella flexneri-specific murine monoclonal antibodies by chemically defined glycoconjugates.

Chemically defined glycoconjugates are demonstrated to have considerable potential for selecting hybridoma antibodies directed toward O-antigenic determinants, especially when used in combination with a panel of well-characterized LPS molecules. Monoclonal antibodies specific for the Shigella flexneri O-antigens of serogroup 5b, variants X and Y, were generated after immunization of BALB/c mice with killed bacterial cells, and active hybrids were selected on the basis of ELISA performed with the purified serotype-specific LPS antigen. Subsequent screening with a variety of glycoconjugates, derived from synthetic oligosaccharides and larger structures obtained by phage Sf6/endo-rhamnosidase hydrolysis of purified LPS established a detailed profile of binding characteristics for Shigella flexneri variant Y-specific antibodies. Together with the results of precipitin analysis and heavy chain isotyping experiments, a limited number of antibodies were selected as candidates for detailed studies of the antibody combining site.

Development and characterization of monoclonal antibodies to pregnancy-specific beta 1-glycoprotein.

Six monoclonal antibodies were developed to pregnancy-specific beta 1-glycoprotein (PS beta 1G). Studies of ascitic fluid antibodies by a double-antibody radioimmunoassay (RIA) included an evaluation of titers, dose-response parameters, and mass action properties. Four of the antibodies demonstrated moderate to high titers ranging from 1/40 000 to greater than 1/120 000, as determined by the specific binding of 125I-labeled PS beta 1G. In inhibition studies utilizing a standard containing known quantities of placental PS beta 1G, two of the antibodies (AR#11 and B#2) were highly sensitive and only slightly lower in this regard than a high affinity polyclonal antiserum. The binding affinities of AR#11 and B#2 monoclonals were greater than 10(9) mol-1 which underline their importance as potential clinical reagents for the RIA of PS beta 1G. The Scatchard plots, for several of the antibodies, were linear and in full agreement with a single order of binding sites predicted for specific monoclonal reagents. Immunodiffusion results provide preliminary evidence that at least three distinct determinants on PS beta 1G are recognized by a number of the monoclonal antibodies. Further studies on the fine specificities of the antibodies by solid-phase RIA, as well as a detailed evaluation of their clinical applications, are in progress.

Serological confirmation of Brucella abortus and Yersinia enterocolitica O:9 O-antigens by monoclonal antibodies.

Murine monoclonal antibodies that bind the O-antigens of Yersinia enterocolitica serotype O:9 and Brucella abortus 1119-3 were generated after immunization of BALB/c mice with killed, whole cells. Highly purified lipopolysaccharide preparations from each organism were used to screen for antigen-specific antibodies. Immunization with B. abortus cells induced 56 antigen-specific hybrids, and 10 of the highest antibody-producing clones were selected for further study. Seven of these clones secreted immunoglobulin G, and three secreted immunoglobulin M antibodies. Immunization with Y. enterocolitica cells resulted after fusion in 76 antigen-specific hybrid cell lines; from these, seven immunoglobulin G-secreting clones were selected for study. The serological cross-reactivity of the B. abortus and Y. enterocolitica O-antigens was established by enzyme-linked immunosorbent assay, immunoprecipitation, and agglutination tests with the monoclonal antibodies induced by each bacterium. This serological cross-reactivity is consistent with the structural identity of the two O-antigens established by chemical analysis.

Hybridomas specific for carbohydrates; synthetic human blood group antigens for the production, selection, and characterization of monoclonal typing reagents.

A general method for the production of carbohydrate-specific hybridoma antibodies is illustrated by generation of monoclonal antibody to the antigenic determinant of human blood group B. This trisaccharide determinant was chemically synthesized and covalently coupled to bovine serum albumin and human blood group O red cells. Soluble protein antigen and the 'artificial' B red cells were used to immunize BALB/c mice before fusion of spleen cells with the Sp2/0 plasmacytoma cell line. ELISA screening of putative hybrids for B-specific binding activity was facilitated by the availability of a second synthetic conjugate, B-horse hemoglobin. IgM-producing clones were identified by class-specific ELISA reagents and by hemagglutination assay. In this way, clones suitable for blood typing were rapidly identified. The precise antigenic specificity and Ig class of such monoclonal antibodies were defined by inhibition of precipitation and by gel filtration. Hybridoma antibodies were obtained from two separate fusion experiments. One of these, clone 3E-4, was of the IgM class and possessed a binding site that was completely satisfied (100% inhibition) by the trisaccharide determinant of the B blood group. This antibody is shown to be suitable for use in blood typing.