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 sub-

Previous studies have shown that human serum containing anti-group A streptococcus carbohydrate (GAS CHO) antibodies were opsonic for different M protein-carrying serotypes. To investigate the role that anti-GAS CHO antibodies play in passive and active protection, mice were immunized subcutaneously or intranasally with GAS CHO conjugated to tetanus toxoid, and mortality and oral colonization were monitored after challenge with live GAS. Compared with control mice, immunized mice were significantly protected against systemic or nasal challenge with GAS. Furthermore, studies of serum samples and throat cultures from Mexican children revealed an inverse relationship between high serum titers of anti-GAS CHO antibodies and the presence of GAS in the throat. Anti-GAS CHO antibodies were also tested for cross-reactivity with human tissues and cytoskeletal proteins. No cross-reactivity was observed in either assay. The present study demonstrates that GAS CHO is both immunogenic and protective against GAS infections.Previous work from our laboratory has shown that serum samples from healthy children contain antibodies to group A streptococcus carbohydrate (GAS CHO) and that the titers of these antibodies increase with increasing age. These antibodies were also shown to be opsonic for several M protein-carrying (M+) serotypes of GAS in an in vitro phagocytic assay. In addition, the specificity of these antibodies for GAS CHO was clearly demonstrated, because removal of them by absorption with N-acetyl glucosamine coupled to Sepharose beads

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.

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.

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. Can. J. Chem. 68, 1029 (1990). The core oligosaccharides obtained from the lipopolysaccharides of Neisseria menirlgitidis serotypes 1 and 6 were isolated by mild acid hydrolysis, and their structures elucidated by combined chemical, biochemical, and physical techniques. Use was made of 500 MHz 'H nuclear magnetic resonance measurements in both one-dimensional and two-dimensional modes as well as of nuclear Overhauser enhancement experiments. To assist in the structural assignments, the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The L1 oligosaccharide is a biantennary partially 0-acetylated heptasaccharide in which the larger antenna terminates in a a-D-Galp 1+4P-~-Galp l+4P-~-Glcp unit, whereas the smallest disaccharide antenna carries phosphorylethanolamine groups at 0 -3 of its heptosyl moiety. The L6 oligosaccharide is also a biantennary partially 0-acetylated heptasaccharide but, unlike the L1 core, is terminated by a P -D -G~c~N A c~+~P -DGalp I+4P-~-Glcp unit in its larger antenna. The heptosyl moiety in the small disaccharide antenna bears a phosphorylethanolamine group at 0-7, unlike 0 -3 for the L1 determinant.Key words: lipopolysaccharides, Neisseria meningitidis, oligosaccharide epitopes, structure, NMR spectroscopy. On a procCdC a une hydrolyse douce en milieu acide pour isoler les oligosaccharides de base des lipopolysaccharides du Neisseria meningitidis des strotypes 1 et 6; on a aussi ClucidC leurs structures en combinant des techniques chimiques, biochimiques et physiques. On a utilisC la rksonance magnCtique nucltaire du 'H dans les modes mono-et bi-dimensionnels ainsi que des expkriences d'effet Overhauser nuclCaire. Afin de faciliter les attributions de structure, on a aussi utilist des mCthodes chimiques ainsi qu'enzymatiques pour dkgrader les oligosaccharides en fragments plus petits. L'oligosaccharide L1, un heptasaccharide partiellement 0-acCtylC, portant deux antennes, dans lequel l'antenne la plus importante est une unit6 a -~-G a l p l + 4 P -~-G a l p l + 4 P -~-G l c p alors que la plus petite antenne est un disaccharide portant des groupements phosphorylCthanolamine en position 0 -3 de sa portion heptosyle. L'oligosaccharide L6 est aussi un heptasaccharide partiellement 0-acttylC portant deux antennes; toutefois, par opposition au produit L1, il se termine avec antenne portant une unit6 P-D-GlcpNAcl+3P-D-Galp 1+4P-~-Glcp. La portion heptosyle du disaccharide de la petite antenne porte un groupement phosphorylCthanolamine qui se trouve en position 0-7 plutdt qu'en 0 -3 comrne dans le dCterminant L1.

A T cell-dependent immune response to group C meningococcal capsular polysaccharide (CPS) can be elicited when CPS is conjugated to the class 3 neisserial porin (CPS-porin). Treatment of CPS-porin-immunized mice with B7-2 blocking monoclonal antibody (MAb) caused a dramatic reduction in the CPS-specific IgG response, treatment with anti-B7-1 MAb had no effect, and concurrent blockade of B7-1 and B7-2 resulted in a synergistic abrogation of the CPS-specific IgG response while the CPS IgM response was unaffected. Anti-CD40L MAb treatment caused a significant reduction of both CPS-specific IgG and IgM levels. In contrast, blockade of CTLA4 interactions resulted in increases in both CPS IgG and IgM responses in CPS-porin-immunized mice. These data support the hypothesis that the ability of neisserial porins to improve the immune response to poorly immunogenic antigens (e.g., polysaccharides) is related to porin-induced increases in B7-2 expression on antigen-presenting cells and enhanced B/T cell interactions.

Abstract