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The L3 immunotype lipopolysaccharide (LPS) of Neisseria meningitidis was subjected to degradation procedures, which produced a number of different oligosaccharide fragments. The high resolution 1H and 13C NMR spectroscopic analyses of these oligosaccharides yielded structural information on a number of different regions of the LPS. For example, from one oligosaccharide, it was found that the endogenous sialylation of the meningococcal LPS occurs at O-3 of the terminal beta-D-galactopyranosyl residue of its lacto-N-neotetraose antenna in the alpha-D-configuration. From another, it was also established that the dominant structural feature responsible for L3 epitope specificity is the presence of a phosphorylethanolamine substituent at O-3 of the penultimate heptopyranosyl residue of its other antenna. In addition from information obtained with another oligosaccharide the structure of the 3-deoxy-D-manno-octulosonic acid disaccharide region of the L3 LPS was also elucidated. From all the above cumulative data plus some published data, it was then possible to reconstruct the complete structure of the entire native L3 LPS.

The immunological properties of alpha(2-->8) polysialic acid have been rationalized in terms of the presence of an epitope situated on a unique extended helical segment (n approximately 9) of the polymer. The critical importance of the carboxylate group to the stability of the extended helical epitope can be ascertained from NMR spectrocopic studies and potential energy calculations on the carboxyl reduced alpha(2-->8) polysialic acid. These studies indicate that the extended helix (n approximately 9) is not stabilized in the reduced polymer and that the majority of conformers can only have helical parameters with n = 2 and 3. This result is also consistent with the fact that the reduced alpha(2-->8) polysialic acid, contrary to its acidic counterpart, exhibits conventional immunological properties. Only five to six reduced oligomers are required to inhibit the binding of the reduced polysialic acid to its homologous antiserum. NMR spectroscopic analysis and potential energy calculations on the N-propionyl, N-butanoyl, N-isobutanoyl, N-pentanoyl, N-hexanoyl, and N-glycolyl derivatives of alpha(2-->8) polysialic acid indicate that, despite the bulk of some of these substituents, they did not disrupt the extended helical conformer. The presence of the extended helical epitope in some of these N-acyl derivatives has also been confirmed from immunological data.

Group B streptococci (GBS) are the most common cause of bacterial sepsis and meningitis in neonates in the United States. Although the capsular polysaccharide of GBS is an important virulence factor, it is variably immunogenic in humans. In this report, we have increased the immunogenicity of GBS type II polysaccharide by coupling it to tetanus toxoid (TT). Like other GBS capsular polysaccharides, the type II polysaccharide has side chains terminating in sialic acid. Controlled periodate oxidation of native II polysaccharide resulted in the conversion of 7% of sialic acid residues to an analog of sialic acid, 5-acetamido-3,5-dideoxy-D-galactosyloctulosonic acid. TT was conjugated to free aldehyde groups created on the oxidized sialic acid residues by reductive amination. Serum from rabbits vaccinated with type II-TT conjugate (II-TT) vaccine contained antibodies specific to type II polysaccharide as well as to TT, whereas rabbits vaccinated with uncoupled native type II polysaccharide failed to produce a type-specific antibody response. Antibodies elicited by II-TT vaccine were serotype specific and mediated phagocytosis and killing in vitro of type II GBS by human peripheral blood leukocytes. Serum from rabbits vaccinated with II-TT vaccine provided 100% protection in a mouse model of GBS type II infection. Antibodies induced by II-TT vaccine were specific for the native but not desialylated type II polysaccharide, suggesting that an important antigenic epitope of II-TT vaccine was dependent on the presence of sialic acid. Therefore, the coupling strategy which selectively modified a portion of the sialic acid residues of types II polysaccharide before coupling the polysaccharide to TT preserved the epitope essential to protective immunity and enhanced the immunogenicity of the polysaccharide.

A single-site ELISA for group B streptococcal polysaccharide based on a monoclonal antibody against an immunodominant trirhamnoside epitope was inhibited at high capture antibody coating densities. The inhibition was eliminated when less antibody was coated or when high antigen concentrations were tested. This antigen is polyvalent with respect to the terminal trirhamnoside epitope and therefore it appears that closely spaced capture antibodies bound the epitope completely, leaving no sites for attachment of the enzyme-labeled second antibody with the same specificity. To make use of the trirhamnoside epitope feasible, a two-site ELISA was evaluated with this monoclonal antibody and a polyclonal antibody isolated by affinity chromatography. ELISA inhibition studies using oligosaccharides derived from the group B polysaccharide were used to evaluate the specificity of the polyclonal antibody. This showed that the antibody recognized both alpha-L-rhamnose (1----3)-D-galactose and alpha-L-rhamnose(1----3)-D-glucitol side chains, which together represent 30 potential binding sites per antigen molecule. A two-site ELISA with the anti-trirhamnoside monoclonal antibody to capture the antigen and the polyclonal antibody as enzyme conjugated second antibody reacted with only group B streptococci when tested against a panel of bacteria representative of the vaginal flora and was able to detect 3 x 10(4) cfu/test of group B streptococci. This two-site ELISA, based on well defined oligosaccharide epitopes had the sensitivity and specificity necessary to identify women at risk of infecting their newborns with group B streptococcus.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.

The immunological properties of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide have been rationalized in terms of a model where the random coil nature of the polymer can be described by the presence of local helices. The conformational versatility of the alpha NeuAc(2-8)alpha NeuAc linkage has been explored by NMR studies at 600 MHz in conjunction with potential energy calculations for colominic acid, an alpha(2-8)NeuAc polymer, and the trisaccharide alpha NeuAc(2-8)alpha NeuAc(2-8)beta NeuAc. Potential energy calculations were used to estimate the energetically favorable conformers and to describe the wide range of helices which the polymer can adopt. No unique conformer was found to satisfy all NMR constraints, and only ensemble averaged nuclear Overhauser enhancements could correctly simulate the experimental data. Conformational differences between the polymer and the trisaccharide could be best explained in terms of slight changes in the relative distribution of conformers in solution. Similar helical parameters for the alpha(2-8)NeuAc polymer and poly(A) were proposed as the basis for their cross-reactivity to a monoclonal antibody IgMNOV. The unusual length dependency for binding of oligosaccharide to group B specific antibodies was postulated to arise from the recognition of a high-order local helix with an extended conformation which was not highly populated in solution.

The capsule of Bacteroides fragilis is unusual in that it consists of two distinct capsular polysaccharides. Using a combination of high-resolution NMR spectroscopy, theoretical calculations, and as few chemical procedures as required, the structure of both polysaccharide antigens (polysaccharides A and B) was elucidated. Using the above procedures, it was possible to obtain the complete structures using minimal quantities of polysaccharides A and B (8 and 5 mg, respectively). Only small amounts of each subjected to chemical analysis were not recoverable. Polysaccharide A is composed of the following repeating unit: [----3)alpha-D-AATp(1----4)[beta-D-Galf(1----3)]alpha-D- GalpNAc(1----3)beta-D-Galp(1----], where AAT is 2-acetamido-4-amino-2,4,6-trideoxygalactose. A pyruvate substituent having the R configuration spans O-4 and O-6 of the beta-D-galactopyranosyl residue. Polysaccharide B is composed of the following repeating unit: [----4)alpha-L-QuipNAc(1----3)beta-D-QuipNAc(1----4)[alpha-L - Fucp(1----2)beta-D-GalpA(1----3)beta-D-GlcpNAc(1----3)]alpha -D-Galp(1----]. A 2-aminoethylphosphonate substituent is situated on O-4 of the N-acetyl-beta-D-glucopyranosyl residue.

Three different oligosaccharides were identified following mild acid hydrolysis of the lipopolysaccharide obtained from Neisseria meningitidis serotype 2 and their structures elucidated by combined chemical and physical techniques. The use of 500 MHz 1H nmr in both one- and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The oligosaccharides were all triantennary nonasaccharides in which the longest antenna terminates in lacto-N-neotetraose. Two of the nonasaccharides (major components), not separable by column chromatography, were distinguishable only by their different patterns of phosphorylethanolamine substitution and the third minor component by the absence of this substituent.

One method to improve the immunogenicity of polysaccharide antigens is the covalent coupling of the native polysaccharide or a derivative oligosaccharide to a carrier protein. In general, T cell-dependent properties are enhanced in conjugates of smaller saccharides, but a conformational epitope of the native polysaccharide may be better expressed in conjugates of larger saccharides. We have reported previously the synthesis and immunogenicity in animals of an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. In this study, we sought to determine the optimal size of group B Streptococcus type III oligosaccharide for use in a conjugate vaccine by evaluating the relative immunogenicity of conjugate vaccines containing oligosaccharides that were twofold smaller (7,000 Mr) or larger (27,000 Mr) than that reported previously (14,500 Mr). All three type III oligosaccharide conjugate vaccines were immunogenic in rabbits, in contrast to native, uncoupled group B Streptococcus type III polysaccharide. However, with respect to eliciting specific antibodies that were protective in vivo, the vaccine containing the intermediate-size oligosaccharide was superior to the smaller or larger conjugate vaccine. Analysis of opsonic activity of vaccine-induced antibodies demonstrated a predominance of IgG antibodies, thought to reflect T cell dependence, in response to shorter chain length conjugates, while the conformational epitope of the native polysaccharide was maximally expressed on longer chain length conjugates. These opposing trends may account for the optimal immunogenicity of an intermediate-size group B Streptococcus type III oligosaccharide conjugate vaccine.

The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune response against L1 and L3,7,9 LPS and part of a cross-reactive epitope between these two immunotypes. For L2-specific immune responses, oligosaccharide structures terminating in beta-D-Glcp(1----4), alpha-D-GlcNAcp(1----2), or L-alpha-D-Hepp(1----5) are needed. The results suggest that it is possible to prepare an oligosaccharide structure with the ability to evoke an immune response against L1, L2, and L3,7,9 LPS. A feasible structure for such a "hybrid" oligosaccharide is discussed.

A number of epitope specificities associated with the group antigen (group B polysaccharide) of group B streptococci have been identified in a polyclonal antiserum induced in rabbits by a nonencapsulated variant strain of group B streptococci. This was achieved by using a series of oligosaccharide inhibitors, obtained by both synthetic and degradative procedures, to inhibit the binding of the group B polysaccharide to the polyclonal antiserum. While the dominant epitope expressed in the antiserum was alpha-L-Rhap(1----2)alpha-L-Rhap(1----2)alpha-L-Rhap, specificities associated with alpha-L-Rhap and alpha-L-Rhap(1----3)alpha-D-Galp(1----3)beta-D-Glcp-NAc(1----4)alp ha-L-Rhap were also identified. The dominant expression of the former epitope is consistent with its terminal location on the group antigen and also with highly branched multiantennary structure of this antigen. Antibodies specific for the alpha-L-trirhamnopyranoside epitope were purified by affinity chromatography, using the synthetic trisaccharide glucitol as the hapten. Oligosaccharide inhibition studies indicate that the specificity of these antibodies is identical to that of a murine monoclonal antibody induced by the same nonencapsulated strain of group B streptococci.

The type V capsular polysaccharide of group B Streptococcus has been isolated and purified, and its repeating unit structure determined. The native type V polysaccharide contains D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and sialic acid in a molar ratio of 3:2:1:1. Methylation analysis and 1H NMR and 13C NMR analysis of the native type V polysaccharide and of its specifically degraded products permitted the determination of the repeating unit structure of the type V polysaccharide: [formula: see text] The type V polysaccharide has certain structural features in common with other group B streptococcal capsular polysaccharides but is antigenically distinct: no immunologic cross-reactivity was observed between type V and types Ia, Ib, II, III, or IV polysaccharides. Studies of antibody binding to the partially degraded forms of the type V polysaccharide indicated that the native epitope is complex, involving most if not all of the sugar residues of the repeating unit.

The native capsular polysaccharide of type III group B Streptococcus elicits a specific antibody response in only 60% of nonimmune human subjects. To enhance the immunogenicity of this polysaccharide, we coupled the type III polysaccharide to tetanus toxoid. Prior to coupling, aldehyde groups were introduced on the polysaccharide by controlled periodate oxidation, resulting in the conversion of 25% of the sialic acid residues of the polysaccharide to residues of the 8-carbon analogue of sialic acid, 5-acetamido-3,5-dideoxy-D-galactosyloctulosonic acid. Tetanus toxoid was conjugated to the polysaccharide by reductive amination, via the free aldehyde groups present on the partially oxidized sialic acid residues. Rabbits vaccinated with the conjugate vaccine produced IgG antibodies that reacted with the native type III group B streptococcal polysaccharide (3/3 rabbits), while rabbits immunized with the unconjugated type III polysaccharide failed to respond (0/3 rabbits). Sera from animals receiving conjugate vaccine opsonized type III group B streptococci for phagocytic killing by human peripheral blood leukocytes, and protected mice against lethal challenge with live type III group B streptococci. The results suggest that this method of conjugation to a carrier protein may be a useful strategy to improve the immunogenicity of the type III group B Streptococcus polysaccharide in human subjects.

We have developed an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. Purified group B streptococcal type III capsular polysaccharide was depolymerized by enzymatic digestion using endo-beta-galactosidase produced by Citrobacter freundii. Following enzymatic digestion, oligosaccharides were fractionated by gel filtration chromatography on Sephadex G-75. An oligosaccharide pool of average Mr = 14,500 (corresponding to 13.6 repeating units of the type III polysaccharide) was used for conjugation to tetanus toxoid. Tetanus toxoid was covalently coupled via a synthetic spacer molecule to the reducing end of the oligosaccharide by reductive amination. The oligosaccharide-tetanus toxoid conjugate elicited type III-specific anticapsular antibodies (measured in enzyme-linked immunosorbent assay) in three out of three rabbits whereas the unconjugated native type III polysaccharide was nonimmunogenic. Antiserum from rabbits vaccinated with the oligosaccharide-protein conjugate protected mice against lethal challenge with live group B streptococci (16 out of 16 mice survived) and opsonized group B streptococci for phagocytosis in vitro. No protection was conferred by preimmune serum nor by serum from rabbits vaccinated with unconjugated native type III polysaccharide. An oligosaccharide-protein conjugate vaccine of this design may prove to be an effective immunogen for protection against group B streptococcal infection in humans. In addition, the approach to vaccine design utilized in these studies will facilitate further definition of the structural parameters that determine immune response to glycoconjugate vaccines.

Syntheses of the propyl glycosides (1-3) of beta-D-Galp-(1----4)-beta-D-GlcpNAc, beta-D-Glcp-(1----6)-[beta-D-Galp-(1----4)]-beta-D-GlcpNAc, and beta-D-Galp-(1----4)-beta-D-Glcp-(1----6)-[beta-D-Galp-(1----)]-beta-D- GlcpNAc, respectively, are reported. Reaction of allyl 2-acetamido-3-O-benzyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranos ide with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide under Hg(CN)2 catalysis, followed by oxidative removal of the 4-methoxybenzyl group, gave allyl 2-acetamido-3-O-benzyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-beta-D-glucopyranoside (10) O-deacetylation of which, followed by hydrogenolysis/hydrogenation, gave 1. Reaction of 10 with beta-D-glucopyranose penta-acetate and beta-lactose octa-acetate, under catalysis by trimethylsilyl trifluoromethanesulfonate, and treatment of the products as for 10 gave 2 and 3, respectively. Attempted glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl bromide under catalysis by Hg(CN)2 or silver trifluoromethanesulfonate gave an orthoester. Complete assignments of the 1H- and 13C-n.m.r. spectra of 1-3 are reported together with their carbon spin-lattice relaxation times which indicate that 3 assumes a compact instead of an extended shape.

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the larger.

A mass-spectrometric approach is presented for the analysis of the structures of lipopolysaccharide-derived oligosaccharides, which are frequently difficult to define by classical methods since they contain chemically labile components. The method involves f.a.b.-m.s. of the oligosaccharides, their peracetylated and permethylated derivatives, their deuterioacetylated and methylated analogues, and the fragments obtained during graded methanolysis of the methylated analogues. Data obtained from two representative meningococcal LPS oligosaccharides define the sequence, patterns of branching, and the extent and location of the phosphorylethanolamine and O-acetyl substituents.

Outer membrane material was extracted from a serogroup 1 strain of Legionella pneumophila using ethylenediaminetetraacetate. Ferritin-conjugated antiserum reacted only at the surface of the organism, as seen by electron microscopy. Outer membrane material was fractionated into five peaks by molecular sieve chromatography using a gel that had been equilibrated in a buffer containing sodium deoxycholate. One resolved peak (approximately 4x10(4) daltons) was highly active serologically. When rechromatographed in the absence of sodium deoxycholate, material from this peak reaggregated to approximately 10(6) daltons. Ther serologic activity of this antigen was restricted to L. pneumophila serogroup 2, although minor cross-reactions with strains of serogroups 2 and 4 were detected using an enzyme-linked immunosorbent assay. The antigen was less than 10% carbohydrate, 15% protein, 1.1% phosphate, and the remainder lipid of unknown composition. Neither 2-keto-3-deoxyoctonate nor heptose was detected, and the antigen did not induce a Shwartzman reaction. Only one band was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The circular dichroic spectra of the acid and sodium salt forms of several sialic acid-containing homo- and hetero-polysaccharides have been measured. The spectra are shown to be influenced by the state of ionization of the carboxyl groups contained in the sialic acid, the location within the individual sialic acid residues of the inter-saccharide linkages, and changes in the configuration of hydroxyl groups remote to the carboxyl group of the sialic acid.

The capsular polysaccharide antigen from Neisseria meningitidis serogroup 29-e contains equimolar quantities of 2-acetamido-2-deoxy-D-galactose and 3-deoxy-D-manno-octulosonic acid (KDO), the latter of which is rarely found in biopolymers other than lipopolysaccharides. Carbon-13 nuclear mangetic resonance in conjunction with other chemical data indicated that the polysaccharide is composed of an alternating sequence of these two residues, the linkages being at C-3 of galactosamine and C-7 of KDO in the alpha-D and beta-D configuration, respectively. The native 29-e polysaccharide is O-acetylated, the O-acetyl groups being located at C-4 and C-5 of the KDO residues. Assignments of signals in the 13C nuclear magnetic resonance spectrum of the 29-e polysaccharide were made by consideration of those in the spectra of the monomer models, which necessitated the first recorded syntheses of methyl-alpha- and beta-D-3-deoxy-manno-octulopyranosonic acid. Like the methyl alpha- and beta-D-ketosides of sialic acid (Na+ salts), the equivalent methyl alpha- and beta-D-ketosides of KDO exhibit large chemical shift differences in the exocyclic C-8 position dependent on anomeric configuration. This can again be attributed to hydrogen bonding between the axial carboxylate group of the methyl beta-D anomer of KDO (C1 conformation) and the primary hydroxyl group at C-8. This phenomenon is also exhibited by the beta-D-linked KDO units of the 29-e polysaccharide.

The protective properties of antibodies induced by immunization of mice with a conjugate of tetanus toxoid and the N-propionyl derivative of group B meningococcal polysaccharide (N-Pr-GBMP-TT) have been investigated. Mice immunized with the conjugate produced antibodies which were bactericidal for Neisseria meningitidis strains B:2b:P1.Ham and B:15:P1.16. Passive protection studies indicated that the conjugate serum completely eliminated or reduced considerably levels of bacteremia by the same strains in mice. There was no bactericidal activity or passive protection against a strain of N. meningitidis C:2b:P1.2. Following absorption of the conjugate serum with GBMP the non-absorbed antibody, directed to N-Pr-GBMP, was bactericidal and protected mice against bacteremia with group B meningococci. Thus N-Pr-GBMP antibodies which do not bind to the GBMP are protective in vitro and in vivo.

The structure of the Pneumococcus type 19A (57) capsular polysaccharide has been reinvestigated by using methylation analysis and n.m.r. spectroscopy. It is composed of residues of 2-acetamido-2-deoxy-D-mannose, D-glucose, L-rhamnose, and phosphate in the molar ratios of 1:1:1:1. The polysaccharide is linear, and is composed of these components in a repeating unit of the following structure. ---- 4)-beta-D-ManpNAc-(1 ---- 4)-alpha-D-Glcp-(1 ---- 3)-alpha-L- Rhap-(1-PO4-) ---- The type 19A polysaccharide (Na+ salt) was depolymerized by heating it in water at 100 degrees, conditions that also hydrolyzed the newly formed phosphoric monoesters.

Condensation of methyl 2,3-O-isopropylidene-alpha-L-rhamnopyranoside with methyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside activated by nitrosyl tetrafluoroborate gave an excellent yield of the protected disaccharide 9, which was transformed into glycosyl acceptor 11. Methyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-galactopyranoside, obtained from D-galactose penta-acetate and methyl trimethylsilyl sulfide, under catalysis by boron trifluoride etherate, was converted into glycosyl donor 25, which was condensed with 11 under halide-ion catalysis to give the trisaccharide derivative 26. Rhamnosylation with 28 of 27, obtained by selective deprotection of 26, gave the protected tetrasaccharide 29. Deprotection of 10, 26, and 29 gave di- (2), tri- (3) and tetra-saccharide (4) methyl glycosides which form part of the group-specific polysaccharide antigen of Group B Streptococci.

A meningococcal group B-specific horse antiserum contains at least two distinct populations of antibodies with specificities for determinants on the group B capsular polysaccharide antigen. These two populations were differentiated on the basis of the ability of only one of them to be absorbed from the antiserum by the structurally related colominic acid. The nature of the colominic acid-specific determinant was elucidated by a radioimmunoassay inhibition technique with the use of a series of linear alpha-(2----8)-linked oligomers of sialic acid as inhibitors. Colominic acid was labeled by prior removal of its N-acetyl groups, followed by their replacement with the use of [3H]acetic anhydride. The conformational nature of the determinant was proposed because of the unusually large size (10 sialic acid residues) of the oligomer required to function as an efficient inhibitor. The structure of the determinant responsible for the second population of group B-specific antibodies has not been determined, but it is obviously based on an as yet undefined conformational or structural feature peculiar to the group B meningococcal polysaccharide. In contrast to the colominic acid-specific group B determinant, the determinant responsible for the group C polysaccharide-specific rabbit antibodies proved to be more conventional. Inhibitory properties of the alpha-(2----9)-linked oligomers maximized with those containing four or five sialic acid residues, which is consistent with the approximate estimated maximal size of an antibody site.

The type III polysaccharides of group B Streptococcus in its native state chemically consists of glucose, galactose, glucosamine, and sialic acid. The core of this polysaccharide lacks sialic acid and precipitates with type III antiserum to give a partial identity with the precipitate between the native antigen and this serum. The core determinant is immunochemically similar to the capsular polysaccharide of type XIV Streptococcus pneumoniae, while the native type III group B streptococcal polysaccharide does not cross-react with type XIV pneumococcal antiserum. In human sera, it is antibody directed to the native antigen which correlates very highly with opsonic immunity (r = 0.94) while a poorer correlation exists between antibody to the core antigen and opsonins (r = 0.51 P less than 0.001). In natural infections, as association exists between low levels of maternal antibody to the native antigen and risk of disease in the infant. This association is not true for antibody to the core structure, where both infected infants and their mothers have much higher levels of antibody to the core than the native antigens. Infected infants are also more likely to respond to infection by developing antibody to the native antigen. Immunization of 12 adults with multivalent pneumococcal polysaccharide induced significantly better antibody response to the core antigen than to the native, and this vaccine induced opsonic activity in only one recipient. Immunization of adults with type III group B streptococcal antigens induced antibody to the native determinant which correlated with opsonic activity. Therefore, it would appear that native group B streptococcal polysaccharides will provide the best candidate antigens for immunization.

The successful coupling of the meningococcal groups A, B, and C polysaccharides to tetanus toxoid to yield water soluble conjugates is described. Reactive aldehyde groups were strategically introduced into the terminal residues of the polysaccharides by the controlled periodate oxidation of the native groups B and C polysaccharides and of the group A polysaccharide previously modified by the reduction of its terminal reducing N-acetyl-mannosamine residue. This produced essentially monovalent polysaccharide molecules, which were subsequently covalently linked to tetanus toxoid by means of reductive amination. Although the groups A and C polysaccharides proved to be poor immunogens in rabbits and mice, their tetanus toxoid conjugates produced high levels of polysaccharide-specific antibodies in both animals. By contrast, even in the form of its tetanus toxoid conjugate, the group B polysaccharide failed to elicit homologous polysaccharide-specific antibodies in either animal; a major proportion of the antibodies actually produced had a specificity for the linkage area of the conjugate. This evidence is compatible with the hypothesis of the poor immunogenicity of the group B polysaccharide being structure related. Hyperimmunization of mice with the groups A and C polysaccharide-tetanus toxoid conjugates produced antisera with good bactericidal activity against their respective homologous organisms, and indicated the potential of these conjugates as potential human vaccines.

The group-specific antigen was isolated from a type Ia group B streptococcal strain and is a complex polysaccharide composed of alpha-L-rhamnopyranosyl, alpha-D-galactopyranosyl, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, D-glucitol, and phosphate residues. The complexity of the group B polysaccharide antigen is evident from the fact that when depolymerized by basic hydrolysis it yielded three structurally related, but nevertheless significantly different, oligosaccharides. These oligosaccharides were obtained in different molar quantities as their monophosphate esters. This evidence strongly suggests that they are linked by phosphodiester bonds in the original group B antigen. If these oligosaccharides are in fact randomly situated throughout the linear polysaccharide, then this type of heterogeneous repeating unit is unusual for a polysaccharide of bacterial origin. However, this structural arrangement of the oligosaccharides has yet to be unambiguously established because the alternate explanation of there being three different polysaccharides in the group B antigen cannot be discounted in the evidence presented here. The oligosaccharides were enzymatically dephosphorylated, and the structures of two of the three oligosaccharides are (formula: see text) Despite their structural differences, the two oligosaccharides are related by the smaller being an integral part of the larger. In the structural analysis of the group B antigen, methylation analysis, periodate oxidation, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, fast atom bombardment mass spectrometry, and various specific chemical and enzymatic degradations were the principal methods used. Of particular interest was the use of an alpha-rhamnosidase to selectively degrade the larger oligosaccharide. This facilitated the assignment of signals in its 1H and 13C NMR spectra.

Effective meningococcal vaccines have to induce protection against groups A, B, C, W-135 and Y meningocci. Polysaccharides are the most effective vaccine candidates against groups A, C, W-135 and Y meningococci. The vaccine potential of polysaccharides is enhanced by conjugating the polysaccharides to carrier proteins. This paper describes the preparation and immunological characterization of two types of group C polysaccharide - tetanus toxoid conjugate. Both types of conjugate were produced by different procedures. Procedure i. depends on cross-linking of both components with N-ethyl-N'-(3-dimethylaminopropyl) carbodimide. Procedure ii. is a more monofunctional way of conjugating partially depolymerized polysaccharide with tetanus toxoid by reductive amination. The antigenic activity of the tetanus toxoid component of the conjugates containing this carrier protein was studied in ELISA and showed differences between the conjugate preparations. The immunogenic activity of all conjugate preparations was tested in mice. All conjugates produced IgG antibodies to the polysaccharide component, although the booster effects of procedure ii. conjugates were less pronounced than those of procedure i. conjugates. The immunogenic activity of the tetanus toxoid component of the various conjugate preparations correlated rather well with their antigenic activities as measured in ELISA.

The fine structure of the combining site of human mAb IgMNOV to poly-alpha(2----8)linked NeuNAc, the epitope of the group B meningococcal and E. coli K1 polysaccharides, has been probed using RIA and ELISA. Inhibition by oligomers ranging from 2 to 12 residues was used to assay binding to IgMNOV by group B meningococcal polysaccharide preparations (GBMP) or by poly(A). The inhibitory properties of the oligomers were almost identical in both assays of the binding of GBMP to horse IgM (H46). This evidence and the finding that both GBMP and poly(A) precipitated IgMNOV equally per unit weight indicated that the epitope of poly(A) must mimic an equivalent epitope on GBMP despite the absence of any apparent common structural features in the two molecules. Unlike most carbohydrate-anticarbohydrate systems in which the site is saturated by oligomers of up to six or seven sugars, all the anti-alpha(2----8)NeuNAc systems above required much larger oligomers. Because these oligomers are larger than the maximum size of an antibody site the epitope must be conformationally controlled, and this has been confirmed by nuclear magnetic resonance spectroscopy. However, despite the above similarities, GBMP and poly(A) were differentiated in that only GBMP bound to H46. Smaller linear molecules obtained by delipidating the GBMP, as well as periodate-oxidized GBMP with its nonreducing end oxidized or linked covalently to BSA, bound to and precipitated IgMNOV and H46. This showed that, despite their differences, terminal nonreducing ends were not involved and that both epitopes were located in the conformationally controlled inner residues of the GBMP. The difference thus must reside in the ability of IgMNOV and H46 to recognize different structural aspects of the same conformationally controlled inner residues. The ELISA data indicate that both IgMNOV and H46 have groove-type sites that bind exclusively to an epitope located on the acidic side of the inner residues. The differences determining the ability of IgMNOV and the failure of H46 to cross-react with poly(A), poly(I), and denatured DNA, may depend on differences in the degree of protonation required by each antibody, and this may be clarified by a study of the effects of pH on the precipitin behavior of IgMNOV and H46.

The group-specific antigen of group B Streptococcus is composed of four different oligosaccharide units of Mw 766 (III), 1277 (II), 1462 (IV), and 1788 (I). The major constituent sugars of the oligosaccharides are alpha-L-rhamnopyranose, alpha-D-galactopyranose, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, and D-glucitol except that III does not contain alpha-D-galactopyranosyl or 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues and IV contains no D-glucitol but has one additional beta-L-rhamnopyranosyl residue. The structures of II and III have been previously elucidated [Michon, F., Katzenellenbogen, E., Kasper, D. L., & Jennings, H. J. (1987) Biochemistry 26, 476-486]. In the group B antigen all the oligosaccharides are linked by one type of phosphodiester bond from O6 of the D-glucitol residue of one oligosaccharide to O6 of the alpha-D-galactopyranosyl residue of the next to form a complex and highly branched multiantennary structure. However, despite the heterogeneous nature of its component oligosaccharides, some order has been identified in the biosynthesis of the group B antigen from chemical and enzymatic sequence studies. Because III lacks an alpha-D-galactopyranosyl residue but has a D-glucitol residue, it is situated at the reducing terminus of all the branches of the group B antigen where it is always adjacent to a II moiety. Conversely, IV has an alpha-D-galactopyranosyl residue but has no D-glucitol and is therefore located at the reducing terminus of the group B antigen where it probably functions as a linker molecule between the group B polysaccharide and the cell wall peptidoglycan of the group B streptococcal organisms. Oligosaccharide I contains two alpha-D-galactopyranosyl residues and one D-glucitol residue and thus constitutes the branch point in the group B antigen, whereas II contains one of each of the above residues and therefore is situated in linear interchain positions. The group B antigen is highly branched and probably has a unique multiantennary structure.

Capsular polysaccharides have assumed an important role as vaccines against disease caused by bacteria in humans. The concept of using pure definable polysaccharides devoid of their accompanying complex bacterial mass is technically elegant and is obviously capable of extension into other areas of immunoprophylaxis. However, problems have been identified which will need to be solved in order that the concept may be more widely adopted. Focusing on the meningococcal polysaccharides, possible solutions to two of these important problems, namely, the poor immunogenicity of the A and C polysaccharides in infants, and the poor immunogenicity of the B polysaccharide in all humans, are proposed. These solutions involve the use of a new generation of artificial synthetic antigens for modulating the immune response. For instance, conjugation of the A and C polysaccharides to tetanus toxoid converted them to T-cell dependent antigens in mice, thus making these conjugates potential infant vaccine candidates. Although a similar conjugation of the B polysaccharide failed to substantially enhance its immunogenicity in mice, this could be achieved by further chemical manipulation of the basic structure of the B polysaccharide. N-propionylation of the B polysaccharide, followed by its conjugation to tetanus toxoid, yielded an antigen, which when injected in mice, induced in them high titers of cross-reactive B polysaccharide-specific IgG antibodies. The chemical modification of polysaccharides requires an understanding of the interrelation between their structures and immunospecificities, and the structural elucidation of polysaccharides and the resultant monitoring of their structural modifications, can be conveniently accomplished using a wide range of NMR spectroscopic techniques. The capsular polysaccharides of many of the bacteria which cause meningitis in humans contain sialic acid and have extensive structural homology with human tissue. As a result of this homology the immunospecificities of these polysaccharides are complex, being based on unconventional conformational determinants.

The alpha-(2----8)-linked sialic acid oligosaccharides (NeuAc)n exhibit an unusual degree of heterogeneity in the conformation of their linkages. This was diagnosed by observation in their 13C NMR spectra of an equivalent and unique heterogeneity in the chemical shifts of their anomeric carbons and subsequently confirmed by more comprehensive 1H and 13C NMR studies. In these studies both one-dimensional and two-dimensional experiments were carried out on the trisaccharide (NeuAc)3 and colominic acid. In addition to the unambiguous assignment of the signals in the spectra, these experiments demonstrated that both linkages of (NeuAc)3 differed in conformation from each other and from the inner linkages of colominic acid. The NMR data indicate that these conformational differences extend to both terminal disaccharides of oligosaccharides larger than (NeuAc)5, a result that has considerable physical and biological significance. In the context of the group B meningococcal polysaccharide, it provides an explanation for the conformational epitope of the group B meningococcal polysaccharide, which was proposed on the evidence that (NeuAc)10, larger than the optimum size of an antibody site, was the smallest oligosaccharide able to bind to group B polysaccharide specific antibodies. Because the two terminal disaccharides of (NeuAc)10 differ in conformation to its inner residues, the immunologically functional part of (NeuAc)10 resides in its inner six residues. This number of residues is now consistent with the maximum size of an antibody site.

A strain of neisseria meningitidis (LCDC 78189) isolated from the mother of a 3-year-old male with meningococcal meningitis was found to be antigenically distinct from the known serogroups A, B, C, D, H, I, K, X, Y, Z, 29E, and W135; it was designated serogroup L. Anti-78189 serum specifically agglutinated the homologous strain and three other strains which were isolated from the father and two other contacts of the child. Only those strains isolated from the contacts produced immunoprecipitates with the anti-78189 serum by the antiserum-agar method. A structurally unique capsular polysaccharide which was obtained from strain 78189 in a highly purified state was demonstrated to be the antigen responsible for the serological properties of the strain. The polysaccharide formed a precipitin band with the anti-78189 serum but not with the meningococcal grouping sera, and it was also able to absorb both the agglutinating and precipitating activity from the anti-78189 serum.

The complex cell-wall polysaccharide, C-substance, was isolated from Streptococcus pneumoniae type 1 and purified by DEAE-cellulose (HCO3(-) form) and Sephadex column chromatography. The complete structure of this antigen was obtained by the application of methylation and 1H NMR and 13C NMR spectroscopic techniques to a series of oligosaccharide fragments obtained by the selective degradation of the N-acetylated antigen. Native C-substance is composed of the following repeating unit: beta-D-Glup-1 leads to 3-alpha-AAT-Galp-1 leads to 4-alpha-D-GalNAcp-1 leads to 3-beta-D-GalNH2p-1 leads to 1'-ribitol-5-phosphate where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose. Phosphocholine substituents are situated at O(6) of the unacetylated galactosamine residues, and the repeating units are linked through a diphosphate ester from ribitol to O(6) of the beta-D-glucopyranose residue. This structure has also been shown to be common to C-substances prepared from a number of other pneumococcal types based on the criterion of their identical 13C NMR spectra.

Conjugation of the group B meningococcal polysaccharide to tetanus toxoid failed to substantially enhance its immunogenicity in mice. Therefore, additional chemical manipulation of the basic structure of the group B meningococcal polysaccharide was attempted, on the premise that a synthetically derived artificial antigen might be capable of modulating the immune response in mice to produce elevated levels of cross-reactive group B meningococcal polysaccharide-specific antibodies. To achieve this, the antigenicity of the modified polysaccharide to group B meningococcal polysaccharide-specific antibodies had to be preserved, and this criterion could only be satisfied in modifications in which the carboxylate and N-carbonyl groups of the sialic acid residues of polysaccharide remained intact. Therefore, the most successful modifications were accomplished by N-deacetylation of the group B meningococcal polysaccharide with strong base to yield a precursor that could then be N-acetylated or N-arylated with different substituents. For example, the introduction of N-propionyl groups, followed by conjugation of the resultant N-propionylated group B meningococcal polysaccharide to tetanus toxoid, yielded an antigen that when injected in mice induced in them high levels of cross-reactive group B meningococcal polysaccharide-specific IgG antibodies. The T cell dependency of this antigen was established when it was demonstrated that the levels of these B polysaccharide-specific antibodies could be significantly boosted by using both the N-propionylated- and native N-acetylated-group B meningococcal polysaccharide-tetanus toxoid conjugates.

The structures of the largest dephosphorylated oligosaccharides (OS) obtained by mild acid hydrolyses of the L2, L3 and L5 serotype lipopolysaccharides (LPS) of Neisseria meningitidis have been elucidated. The OS have extensive regions of structural similarity of which some are responsible for cross-reactivity among the meningococcal LPS. However, the fact that the LPS are predominantly serotype-specific antigens implies that the terminal lacto-N-neotetraose unit, common to all the above OS, is not immunodominant, and that the major LPS serotype specificity originates in the inner core region of the OS.

The alpha-D configuration of the 3-deoxy-D-manno-2-octulosylonic acid residues in the E. coli LP1092 polysaccharide was definitively assigned by a comparison of its c.d. spectrum with those of methyl 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosidonic acid. The c.d. spectrum of the polysaccharide showed a negative n----pi transition at 211 nm, associated with carboxyl groups, which correlated well with the negative band at 218 nm exhibited in the c.d. spectrum of the methyl alpha-D-glycoside. In contrast, the methyl beta-D-glycoside gave a positive band in the same region of its c.d. spectrum, at 221 nm. Treatment of the E. coli LP1092 polysaccharide with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride yielded a modified polysaccharide containing O-acylisourea groups and intramolecular lactones, in addition to some unesterified carboxyl groups. Both forms of ester could be reduced to hydroxymethyl groups by sodium borohydride. Although immuno-precipitation of an antiserum specific for the E. coli LP1092 polysaccharide is not sensitive to the introduction of O-acylisourea groups into the polysaccharide, precipitation was completely eliminated when approximately half of the carboxyl groups of the polysaccharide were converted either into lactone or hydroxymethyl groups. Failure to precipitate the antiserum can be attributed to significant conformational changes in the polysaccharide brought about by the introduction of the latter two groups.