N-propionylated group B meningococcal polysaccharide mimics a unique epitope on group B Neisseria meningitidis.

Antibodies induced in mice by the N-propionyl (N-Pr)-group B meningococcal polysaccharide (GBMP)-tetanus toxoid (TT) conjugate were bactericidal for GBM organisms independent of protein serotype. The antisera contained two populations of N-Pr-GBMP-specific antibodies, only one of which crossreacted with the GBMP. Particularly significant was the fact that the bactericidal activity was mainly associated with the population of antibodies that did not crossreact with the GBMP. Therefore it can be inferred from the above evidence that the N-Pr-GBMP mimics a unique epitope on the surface of GBM organisms that is not present on the exogenous GBMP.

N-Propionylated group B meningococcal polysaccharide mimics a unique bactericidal capsular epitope in group B Neisseria meningitidis.

The N-propionylated group B meningococcal polysaccharide (NPrGBMP) mimics a unique protective epitope on the surface of group B meningococci (GBM) and Escherichia coli K1. Using a series of monoclonal antibodies (mAbs) induced by the NPrGBMP-monomeric tetanus toxoid (TT) conjugate vaccine it was demonstrated that mAbs having specificities for both extended and conventional short segments of the NPrGBMP were formed, but only the former were bactericidal, and/or gave passive protection against live challenge by GBM. The failure of mAbs specific for short epitopes to protect was further established when (NeuPr)4-TT was used as the vaccine. Of all the mAbs produced that were specific for short internal segments of the NPrGBMP, none were protective, despite the fact that most of them cross-react with the GBM capsular polysaccharide. In contrast, most of the protective mAbs produced by NPrGBMP- TT did not recognize the group B meningococcal polysaccharide (GBMP) unless it was present in its aggregated high molecular weight form. The bactericidal epitope mimicked by the NPrGBMP was shown to be ubiquitous in the capsule of both GBM and E. coli K1 using immunogold labeling techniques and, because of its unique properties, its identification could be significant in the development of a comprehensive conjugate vaccine against group B meningococcal meningitis. This is because most known human alpha(2-8)-polysialic acid self-antigens can be accommodated in 30-50 alpha(2-8)-linked sialic acid residues, which is roughly equivalent to an 11-kD length of the GBMP. It has been hypothesized that the formation of the protective epitope on the surface of GBM is due to the interaction of helical segments of the GBMP with another molecule and that the protective epitope is mimicked by the NPrGBMP. Support for the above hypothesis is provided by the fact that the protective NPrGBMP epitope has a similar unusual length dependency to that of the GBMP epitope.

The epitope associated with the binding of the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1 to a human monoclonal macroglobulin, IgMNOV.

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.

Immunodeterminant specificity of human immunity to type III group B streptococcus.

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.

Immunochemical analysis and immunogenicity of the type II group B streptococcal capsular polysaccharide.

The relationship between group B streptococcal (GBS) type-specific antisera and the type II-specific polysaccharide is evaluated from a structural and immunologic viewpoint. Although all GBS type-specific polysaccharides are composed of the same monosaccharides, the type II antigen is more complex structurally and contains these sugars in a molar ratio different from the other antigens. Type II polysaccharide has two side chains. One contains only sialic acid and is less susceptible to acid cleavage than sialic acid residues found on types III, Ia, and Ib polysaccharides. The other side chain is composed of galactose as the only sugar. Immunochemical studies demonstrate that the type II polysaccharide has several immunodeterminants. One of these determinants is likely to be the side-chain galactose, while sialic acid appears to comprise part of another immunodeterminant, more complex than sialic acid alone. A series of cross-reactions is demonstrated between the type II native antigen and antisera to serotypes Ia, III, and Ib by a sensitive radioactive antigen-binding assay, which account for additional, complex immunodeterminants. The strongest of these cross-reactions is with type Ia antiserum and the weakest with Ib antiserum. Since Ia and Ib polysaccharides differ in only one linkage, these findings suggest that the trisaccharide beta D-N-acetyl-glucosamine-p(1 leads to 3) beta D-galactose-p(1 leads to 4) beta D-glucose-p [[beta D-GlcNAcp(1 leads to 3) beta D-Galp(1 leads to 4)beta D-Glcap]] is the likely common site responsible for the interaction of the type II native polysaccharide and type Ia antiserum. Another cross-reaction is observed between type III antiserum and type II native antigen. Inhibition studies indicate that the most likely cross-reactive determinant in this case is [beta D-Galp(1 leads to 4)beta D-GlcNAcp]. Type II polysaccharide has been utilized in a human vaccine trial to test safety and immunogenicity. The polysaccharide is highly immunogenic, inducing an antibody response in 95% of recipients, and nontoxic, with side-effects confined to minimal local reactions. Despite the cross-reactions observed between type-specific antigens and antibody prepared by immunization of rabbits with whole bacteria, which suggest shared immunodeterminants, similar cross-reactions were not detected in human sera after immunization with purified type II polysaccharide.

Immunogenicity in animals of a polysaccharide-protein conjugate vaccine against type III group B Streptococcus.

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.

Effects of chain length on the immunogenicity in rabbits of group B Streptococcus type III oligosaccharide-tetanus toxoid conjugates.

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.

Immune response to type III group B streptococcal polysaccharide-tetanus toxoid conjugate vaccine.

Group B Streptococcus (GBS) is an important perinatal pathogen. Because transplacentally acquired maternal antibodies to the GBS capsular polysaccharides (CPS) confer protection, prevention of infant disease may be possible after immunization of women. Unfortunately, the purified CPS of GBS are only variably immunogenic in adults; therefore to enhance immunogenicity we have designed and developed a CPS-protein conjugate vaccine. The lability of a conformationally dependent epitope on the III CPS containing a critical sialic acid residue was important to consider in vaccine design. 100 women were randomized to receive GBS type III CPS-tetanus toxoid conjugate (III-TT) vaccine at one of three doses; unconjugated GBS type III CPS; or saline. Serum samples were obtained before immunization and 2, 4, 8, and 26 wk thereafter, and specific antibody to type III CPS was measured. Vaccines were well tolerated. In sera from recipients of the highest dose of III-TT, CPS-specific IgG levels rose from a geometric mean of 0.09 microg/ml before immunization to 4.53 microg/ml 8 wk later, whereas levels in recipients of unconjugated type III CPS rose from 0.21 microg/ml to 1.41 microg/ml. Lower doses resulted in lower antibody levels. A > or = 4-fold rise in antibody concentration was achieved in 90% of recipients of III-TT compared with 50% of those that received III CPS (P = 0.0015). Antibodies evoked by the conjugate vaccine recognized a conformationally dependent epitope of the III-CPS, promoted opsonophagocytosis and killing of GBS, and, after maternal immunization, protected neonatal mice from lethal challenge with type III GBS. We conclude that directed coupling of type III GBS polysaccharide to a carrier protein yielded a conjugate vaccine with preserved expression of a highly labile conformational epitope involving sialic acid and enhanced immunogenicity compared with uncoupled CPS.

Use of defined oligosaccharide epitopes in an ELISA for group B streptococcus.

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.

Immunostimulant oxidized beta-glucan conjugates.

beta-Glucans are polysaccharides that act as nonspecific immune system stimulants. However, many beta-Glucans are sparingly soluble in water. This work describes an oxidative procedure, which solubilizes the beta-Glucan from Saccharomyces cerevisiae and maintains its immunostimulatory properties. Furthermore, the carboxylates at the site of oxidation allow for the conjugation of small molecule immunostimulants. Both the parent oxidized beta-glucan and its conjugates with O-beta-alanyl-5-[6-(N,N'-dimethylamino)purin-9-yl]pentanol stimulate cytotoxic T-lymphocytes (CTLs), B cells and macrophages. In addition, they both stimulate natural killer (NK) cells, a property which the small molecule purine does not possess.

Determination of the linkages in some methylated, sialic acid-containing, meningococcal polysaccharides by mass spectrometry.

The application of gas-liquid chromatography-mass spectrometric (g.l.c.-m.s.) analysis to a number of sialic acid-containing polysaccharides of meningococcal origin has been studied. Methylation of these polysacchardies by the Hakomori conditions resulted in both O- and N-methylation. Methanolysis of the methylated polysaccharides from serogroup C [(2 leads to 9)-linked], colominic acid [(2 leads to 8)-linked], and serogroups Y and W-135 [both (1 leads to 4)-linked], yielded the respective 4,7,8-, 4,7,9-, and 7,8,9-tri-O-methyl derivatives of methyl N-acetyl-N-methyl-beta-D-neuraminate methyl glycoside. As model compounds, methyl N-acetyl-4,7,8,9-tetra-O-methyl-alpha-D-neuraminate methyl glycoside and its N-methyl derivative were also synthesized. All of the methylated derivatives could be identified on the basis of their typical fragmentation-patterns, indicating that this method is applicable to the determination of the position of linkages to sialic acid residues in biopolymers.

Synthesis of a di-, tri-, and tetra-saccharide unit of the group B streptococcal common antigen.

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.

The circular dichroic spectra of several sialic acid-containing polysaccharides isolated from Neisseria meningitidis.

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.

Structural determination of the capsular polysaccharide of Streptococcus pneumoniae type 19A (57).

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.

Structural determination of the capsular polysaccharide of Streptococcus pneumoniae type 18C (56).

The specific capsular polysaccharide of Streptococcus pneumoniae type 18C (56) contains D-glucose, D-galactose, L-rhamnose, and glycerol residues, and phosphate and O-acetyl groups in the molar ratios of 3:1:1:1:1:1. Accumulated data from methylation analyses of the native and the specifically degraded, native polysaccharide indicated that it is composed of the repeating unit shown; it also contains O-acetyl groups, of undetermined location, in the molar ratio to L-rhamnose of 1:1. (formula; see text).

Synthesis of a tri- and a tetra-saccharide fragment of the capsular polysaccharide of type III group B Streptococcus.

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.

Oligosaccharide fragments of the type III group B streptococcal polysaccharide derived from S. pneumoniae type 14 capsular polysaccharide by a chemoenzymatic method.

Partial N-deacetylation fo the GlcNAc residues in S. pneumoniae type 14 capsular polysaccharide (Pn14-PS) backbone was achieved by treatment with base, and the product was subsequently enzymatically sialylated at the 3-O-positions of the terminal galactose residues. The resultant, partially N-deacetylated type III Group B streptococcus capsular polysaccharide (GBSIII-PS) was subjected to nitrous acid deamination, which resulted in the degradation of GBSIII-PS polysaccharide into oligosaccharides containing increasing numbers of the identical repeating units. The oligosaccharides were then separated by passage through a Superdex 30 column and characterized by ESIMS and NMR spectroscopic analysis.

The structure of an R-type oligosaccharide core obtained from some lipopolysaccharides of Neisseria meningitidis.

The main structure of a lipopolysaccharide R-type core oligosaccharide common to a number of different strains of Neisseria meningitidis has been elucidated. Methylation analysis, specific degradations, and nuclear magnetic resonance spectroscopic analysis of the dephosphorylated cores indicated that they all have the following structure. (formula see text) The determinants responsible for the L3, L7, and L9 meningococcal lipopolysaccharide serotypes are situated in this oligosaccharide.

Chemical modification and serological properties of the 3-deoxy-alpha-D-manno-2-octulosonic acid-containing polysaccharide from Escherichia coli LP1092.

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.

Construction of multivalent sialyl Le(x) from the type Ia group B Streptococcus capsular polysaccharide.

The type Ia group B Streptococcus (GBSIa) capsular polysaccharide was specifically degraded by partial Smith oxidation of 2,3-diol of the Glc in the backbone to fragments representing asialo core repeating units. Sialylation of these oligomers furnished GBSIa multiple repeating units. One, two and three repeating units of GBSIa were obtained pure, and the higher oligomers were obtained as mixtures. After enzymatic fucosylation oligosaccharides carrying bivalent, trivalent and other multivalent sialyl Le(x) epitopes presented as appendages on an oligolactoside scaffold were obtained.