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PubMed1995

Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps.

While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.

Antithrombin III↗
PubMed1996

Comparison of serum antibodies to pneumolysin with those to pneumococcal capsular polysaccharides in children with acute otitis media.

BACKGROUND: Streptococcus pneumoniae is a major bacterial pathogens in acute otitis media. Pneumolysin is a species-specific protein toxin produced intracellularly by all clinically relevant pneumococcal strains, and antibodies to pneumolysin should therefore represent pneumococcal involvement in the disease, regardless of the serotype. METHODS: Antibodies to pneumococcal pneumolysin and capsular polysaccharides were measured by enzyme immunoassay in acute and convalescent sera of 121 children with acute otitis media. A pneumococcal otitis episode was defined by a positive middle ear fluid culture and/or pneumolysin PCR. RESULTS: Median age of the 10 children who developed a seroconversion response to pneumolysin was 1 year 8 months, and of the 21 children responding to polysaccharides it was 2 years 9 months. Eight of the 10 seroconversion responses to pneumolysin were of IgA class alone, whereas 17 of the 21 polysaccharide responses were of IgG class alone or IgG together with IgM and/or IgA. Of the 41 children with a pneumococcal otitis episode, 13 (39%) showed a seroconversion response, 3 (7%) to pneumolysin and 11 (27%) to capsular polysaccharides. The children with a pneumococcal otitis episode had lower titers of acute phase IgG to the capsular polysaccharide pool of S. pneumoniae (containing types 6B, 14, 19F and 23F), as compared with the titers in children with otitis caused by other pathogens and pneumococci only in the nasopharynx or not found at all (P = 0.04). CONCLUSIONS: Serum antibodies to pneumolysin can be detected at an earlier age than those to the capsular polysaccharides. However, a seroconversion is rare and therefore of no diagnostic value. The presence of serum IgG to the pneumococcal capsular polysaccharides seems beneficial in the prevention of pneumococcal otitis.

Acute Disease↗
PubMed1996

Immunologic priming of young children by pneumococcal glycoprotein conjugate, but not polysaccharide, vaccines.

BACKGROUND: Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children. Licensed pneumococcal polysaccharide vaccines are not reliably immunogenic in children younger than 2 years of age; therefore pneumococcal glycoprotein conjugate vaccines are currently being evaluated for safety, immunogenicity and efficacy in various age groups. METHODS: During a 12-month period we determined the kinetics of pneumococcal IgG antibody in 60 children who received primary immunization with one dose of bivalent (serotypes 6A and 23F) pneumococcal polysaccharide-CRM197 vaccines at 18 to 30 months of age. To assess immunologic priming a subgroup of 20 subjects received secondary immunization with pneumococcal polysaccharide vaccine, including serotypes 6B and 23F, at 11 to 20 months after primary immunization. Pneumococcal-specific IgG subclass distributions were also evaluated in the subgroup. RESULTS: In the 12 months after primary immunization with glycoprotein conjugate vaccine, geometric mean pneumococcal IgG antibody concentrations to 6B and 23F serotypes remained stable. Pneumococcal polysaccharide vaccine induced a greater anamnestic response in children primed with glycoprotein conjugate vaccines (13- to 40-fold increases to geometric mean concentrations of 6 to 30 micrograms/ml for type 23F), than in those primed with polysaccharide (2- to 4-fold increases). A greater IgG response to pneumococcal serotype 23F than to 6B was observed with both primary and secondary immunization. The serotype-specific pneumococcal IgG antibody response was virtually restricted to the IgG1 subclass after primary immunization, but secondary immunization elicited antibodies of IgG1 and IgG2 subclasses. CONCLUSIONS: These glycoprotein conjugate vaccines appear to prime for anamnestic IgG antibody responses to subsequent immunization with polysaccharide vaccine, suggesting that the polysaccharide-CRM197 vaccine effectively induces a predominantly T cell-dependent immune response. The greater IgG response to 23F than to 6B indicates that pneumococcal serotype is a major determinant of immunogenicity of pneumococcal glycoprotein conjugate vaccines.

Bacterial Proteins↗
PubMed1997

A glycoprotein pneumococcal conjugate vaccine primes for antibody responses to a pneumococcal polysaccharide vaccine in Gambian children.

BACKGROUND: Streptococcus pneumoniae is a major cause of acute respiratory infections and acute bacterial meningitis in children. Pneumococcal polysaccharide vaccines are poorly immunogenic in this highly vulnerable group, but protein polysaccharide conjugate vaccines are likely to be more effective. OBJECTIVES: To determine whether immunization of infants with a pneumococcal conjugate vaccine induces immunologic memory. METHODS: Eighty-four Gambian children, who had been vaccinated previously with two or three doses of a pentavalent pneumococcal conjugate vaccine (CRM197) or with a Haemophilus influenzae type b (Hib) conjugate vaccine were immunized when approximately 2 years old with a 23-valent pneumococcal polysaccharide vaccine, and a blood sample was obtained 10 days later. Pneumococcal antibody titers in prevaccination and postvaccination sera were measured by enzyme-linked immunosorbent assay and by an opsonophagocytic assay. RESULTS: On revaccination with a pneumococcal polysaccharide vaccine, children who had previously received pneumococcal conjugate vaccine had higher antibody concentrations to each of the five polysaccharide components of the conjugate vaccine than did control children. For type 6B polysaccharide, which is poorly immunogenic in young children, postvaccination antibody concentrations were 0.37, 27.6 and 50.9 microg/ml in children who had received no previous pneumococcal immunization or two or three doses of conjugate vaccine, respectively. Type 14 antibodies produced after revaccination were of high avidity and had opsonic activity. CONCLUSION: Vaccination of young infants with two or three doses of a pneumococcal conjugate vaccine primes the immune system to respond strongly and rapidly on subsequent exposure to pneumococcal polysaccharide.

Antibodies, Bacterial↗
PubMed1995

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.

Amino Acids↗
PubMed1995

Polysaccharide composition of unlignified cell walls of pineapple [Ananas comosus (L.) Merr.] fruit.

The polysaccharides of cell walls isolated from the fleshy, edible part of the fruit of the monocotyledon pineapple [Ananas comosus (L.) Merr.] (family Bromeliaceae) were analyzed chemically. These cell walls were derived mostly from parenchyma cells and were shown histochemically to be unlignified, but they contained ester-linked ferulic acid. The analyses indicated that the noncellulosic polysaccharide composition of the cell walls was intermediate between that of unlignified cell walls of species of the monocotyledon family Poaceae (grasses and cereals) and that of unlignified cell walls of dicotyledons. Glucuronoarabinoxylans were the major non-cellulosic polysaccharides in the pineapple cell walls. Xyloglucans were also present, together with small amounts of pectic polysaccharides and glucomannans (or galactoglucomannans). The large amounts of glucuronoarabinoxylans and small amounts of pectic polysaccharides resemble the noncellulosic polysaccharide composition of the unlignified cell walls of the Poaceae. However, the absence of (1-->3,1-->4)-beta-glucans, the presence of relatively large amounts of xyloglucans, and the possible structure of the xyloglucans resemble the noncellulosic polysaccharide composition of the unlignified cell walls of dicotyledons.

Carbohydrate Sequence↗
PubMed2005

Carbohydrate engineering of the recognition motifs in streptococcal co-aggregation receptor polysaccharides.

The cell wall polysaccharides of certain oral streptococci function as receptors for the lectin-like surface adhesins on other members of the oral biofilm community. Recognition of these receptor polysaccharides (RPS) depends on the presence of a host-like motif, either GalNAcbeta1-3Gal (Gn) or Galbeta1-3GalNAc (G), within the oligosaccharide repeating units of different RPS structural types. Type 2Gn RPS of Streptococcus gordonii 38 and type 2G RPS of Streptococcus oralis J22 are composed of heptasaccharide repeats that are identical except for their host-like motifs. In the current investigation, the genes for the glycosyltransferases that synthesize these motifs were identified by high-resolution nuclear magnetic resonance (NMR) analysis of genetically altered polysaccharides. RPS production was switched from type 2Gn to 2G by replacing wefC and wefD in the type 2Gn gene cluster of S. gordonii 38 with wefF and wefG from the type 2G cluster of S. oralis J22. Disruption of either wefC or wefF abolished cell surface RPS production. In contrast, disruption of wefD in the type 2Gn cluster or wefG in the type 2G cluster eliminated beta-GalNAc from the Gn motif or beta-Gal from the G motif, resulting in mutant polysaccharides with hexa- rather than heptasaccharide subunits. The mutant polysaccharides reacted like wild-type RPS with rabbit antibodies against type 2Gn or 2G RPS but were inactive as co-aggregation receptors. Additional mutant polysaccharides with GalNAcbeta1-3GalNAc or Galbeta1-3Gal recognition motifs were engineered by replacing wefC in the type 2Gn cluster with wefF or wefF in the type 2G cluster with wefC respectively. The reactions of these genetically modified polysaccharides as antigens and receptors provide further insight into the structural basis of RPS function.

Amino Acid Motifs↗
PubMed2005

Complement dependency of splenic localization of pneumococcal polysaccharide and conjugate vaccines.

The immune response to polysaccharides is initiated when polysaccharides bind complement factor C3d, and these polysaccharide-C3d complexes subsequently localize on splenic marginal zone B cells strongly expressing CD21 (complement receptor 2). Infants and children under the age of 2 years have low or absent expression of CD21 on their marginal zone B cells, and consequently do not adequately respond to polysaccharides. In contrast, polysaccharide-protein conjugate vaccines are able to induce antibodies at this young age. Conjugate vaccines apparently overcome the necessity for CD21-C3d interaction for an antipolysaccharide immune response. We demonstrate in a rat model that localization of pneumococcal polysaccharides on splenic marginal zone B cells indeed is complement dependent. We also show that pneumococcal conjugates do not specifically localize on splenic marginal zone B cells and that splenic localization of polysaccharide conjugates is independent of the presence of complement. Thus, the induction of antipolysaccharide antibodies by conjugate vaccines apparently can occur independently of CD21-C3d interaction. These basic findings may explain the effectiveness of conjugated vaccines in young children and may open the way for their application in other patient groups.

Animals↗
PubMed1986

Biosynthesis of the wall neutral polysaccharide in Bacillus cereus AHU 1356.

The pathway for the biosynthesis of a cell wall polysaccharide, composed of glucosamine, mannosamine, galactosamine and glucose in a molar ratio of 4:1:1:1, was studied with a membrane system from Bacillus cereus AHU 1356. In this system a glycolipid characterized as GalNAc(alpha 1----4)ManNAc(beta 1----4)GlcNAc-PP-undecaprenol was formed from GlcNAc-PP-undecaprenol by sequential transfer of N-acetylmannosamine and N-acetylgalactosamine residues from UDP-ManNAc and UDP-GalNAc respectively. An additional N-acetylglucosamine residue and a glucose residue were individually transferred from their UDP derivatives to the trisaccharide-linked lipid with the formation of tetrasaccharide-linked lipids, which seem to serve as intermediates in the polysaccharide synthesis. Incubation of membranes with the trisaccharide-linked lipid even in the absence of sugar-linked nucleotides led to the formation of polysaccharide. These results, together with the data on Smith degradation of the synthesized polysaccharide, indicate that the repeating trisaccharide units of the main chain of the polysaccharide arise from the GalNAc-ManNAc-GlcNAc moiety of the glycolipid intermediates and that the sugar residues in the lateral branches of the polymer are at least partly introduced through oligosaccharide-linked lipid intermediates. In addition, the structure of native polysaccharide was re-examined, and the presence of the disaccharide sequence ManNAc(beta 1----4)GlcNAc in the polysaccharide chain was confirmed.

Affinity Labels↗
PubMed1988

Pyruvic-acid-containing polysaccharide in the cell wall of Bacillus polymyxa AHU 1385.

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacillus polymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HC1 buffer, pH 2.5, at 100 degrees C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I-III, which contained different amounts of pyruvic acid (0,0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-II, PS-III and polysaccharide-linked glycopeptides by treatment with 10 mM HC1 at 100 degrees C for 1 h. Results of analyses of these polysaccharide preparations by 1H-NMR and 13C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, ----3)[4,6-O-(1-carboxyethylidene)]ManNAc(beta 1----4)GlcNac(beta 1----, for the acidic polysaccharide of this strain.

Acetylglucosamine↗
PubMed1984

Polysaccharide production by cell free transferases in saliva in relation to salivary microflora.

The supernatant of centrifuged whole saliva was incubated with radiolabeled sucrose to measure polysaccharide production by cell free transferases and to examine whether water-insoluble polysaccharides were produced. Amounts of polysaccharides were considered to reflect the level of cell free transferases in saliva. Plating samples on blood agar, MS and MSB plates gave salivary counts of total CFU, total streptococci, S. salivarius and S. mutans. The capability of the cell free portion of saliva to produce polymers was confirmed and it appeared that the cell free transferases were able to produce water-insoluble polysaccharides. Significant correlations were found between the total and the insoluble polysaccharides from 14C-G'-sucrose and total CFU, total streptococci, S. salivarius and S. mutans, respectively. Heavily S. mutans infected subjects seemed to produce particularly large amounts of water-insoluble polysaccharides from 14C-G'-sucrose. The apparently water-insoluble 14C-F'-polysaccharides correlated significantly to the number of S. salivarius. It was thus concluded that the constitution of the oral microflora and particularly the levels of S. mutans and S. salivarius were of importance for the level of cell free transferase activity.

Cell-Free System↗
PubMed1987

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein.

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.

Acinetobacter↗
PubMed1975

Extracellular polysaccharide from the black yeast NRRL Y-6272: improved methods for preparing a high-viscosity, pigment-free product.

When the extracellular polysaccharide from the black yeast NRRL Y-6272, composed of two parts N-acetyl-D-glucosamine and one part N-acetyl-D-glucosaminuronic acid, is isolated at maximum culture viscosity, adhering black pigment gives the polysaccharide preparations a gray-to-black appearance. Precipitation of the polysaccharide from cell-free culture supernatants with either ethanol of hexadecyltrimethylammonium bromide failed to remove the pigment. Various other methods were therefore tried for obtaining a high-viscosity polysaccharide product free of pigment. By systematically varying ingredients of defined and semidefined media, an improved medium was found that not only gave polysaccharide preparations of increased viscosity, but also increased yield. A key ingredient in this medium is L-asparagine. Also, adding autoclaved bovine serum albumin or egg albumin to this medium at the time of inoculation allowed a pigment-free polysaccharide to be isolated by standard procedures. None of several other proteins of synthetic polyamides tested were as effective as bovine serum albumin or egg albumin. In an alternate approach, pink mutants obtained by irradiation of the parent black strain with ultraviolet light, apparently produce the same extracellular polysaccharide free of any pigment but in lower yields or inferior in quality.

Acetylglucosamine↗
PubMed1979

Opsonization of Cryptococcus neoformans by human immunoglobulin G: masking of immunoglobulin G by cryptococcal polysaccharide.

Previous studies have shown that attachment of non-encapsulated cryptococci to macrophages is highly dependent on opsonizing immunoglobulin G (IgG) and that cryptococcal polysaccharide inhibits the attachment phase of phagocytosis. We investigated various mechanisms by which cryptococcal polysaccharide might interfere with the opsonizing action of IgG. Cryptococcal polysaccharide did not appreciably prevent binding of opsonizing IgG to the yeast. Furthermore, cryptococcal polysaccharide acted as a noncompetitive inhibitor with respect to the opsonizing action of IgG. These experiments suggested that cell wall-bound IgG is masked in some manner such that it is unable to participate in Fc-mediated phagocytosis. This appeared to be the case, since cryptococcal polysaccharode inhibited agglutination of IgG-opsonized yeast cells by antiserum to IgG. There was good dose-response correlation between the amount of polysaccharide needed to inhibit phagocytosis of non-encapsulated Cryptococcus neoformans and the amount of polysaccharide needed to prevent agglutination of IgG-opsonized cryptococci by antiserum to IgG. The ability of cryptococcal polysaccharide to prevent agglutination of IgG-opsonized cryptococci by antiserum to IgG was lost if dextran, a substance known to enhance agglutination of several particles, was incorporated into the medium.

Agglutination↗
PubMed1981

Immunochemical characterization of cross-reactivity of pneumococcal group 9 capsular polysaccharide types 9N, 9A, 9L, and 9V.

The chemical composition and immunochemical characterization of the four cross-reactive pneumococcal capsular polysaccharides within group 9 (types 9N, 9A, 9L, and 9V) were investigated. Their serological reactions were studied by using unabsorbed antisera prepared by immunizing rabbits with pneumococci of each of the four group 9 capsular polysaccharide types. Type 9A antiserum showed the most extensive cross-reactions with the four group 9 polysaccharides. Absorption with type 9N, 9L, or 9V polysaccharide removed 63, 96, or 87%, respectively, of the heterologous antibodies from the type 9A antiserum. All four of the group 9 polysaccharides contained glucose, N-acetylmannosamine, and glucuronic acid. In addition, types 9N and 9L had N-acetylglucosamine, and types 9A, 9L, and 9V contained galactose. Reduction of the uronic acid residues of the type 9 polysaccharides removed most of their homologous and much of their heterologous reactivities, indicating an important role for the uronic acid component in their antigenicity. The four group 9 polysaccharide preparations had comparable molecular sizes and only traces of protein and nucleic acid. Further studies to evaluate the most protective type among the group 9 strains to be included in the current pneumococcal vaccine are discussed.

Amino Sugars↗
PubMed1982

Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded that the hydrolytic stability of the type 6B antigen would favor its use in the polyvalent pneumococcal vaccine rather than the cross-reactive, but comparatively unstable, type 6A polysaccharide, if all other factors are equal.

Chemical Phenomena↗
PubMed1982

Class-specific antibody response to group B Neisseria meningitidis capsular polysaccharide: use of polylysine precoating in an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed for measuring immunoglobulin class-specific antibodies to Neisseria meningitidis capsular polysaccharides. The polysaccharides were fixed to the solid phase by precoating the polystyrene surface with basic polyamino acids. Polylysine precoating was found to give optimal antibody values with the meningococcal polysaccharides studied. The enzyme-linked immunosorbent assay was used to measure antibodies against group B meningococcal polysaccharide in healthy adults and in patients with group B meningitis. Antibodies to group B polysaccharide in sera obtained from healthy adults were primarily immunoglobulin M (IgM). Although the antibody response to disease was mostly IgM, both IgG and IgM antibodies increased. Infection with group B organisms stimulated anti-group B polysaccharide antibodies even in young patients, aged 6 to 7 months, and the magnitude of the B polysaccharide response increased with the age of the patient.

Adolescent↗
PubMed1983

Immunological evaluation of meningococcal group C polysaccharide-tetanus toxoid conjugate in mice.

Neisseria meningitidis group C polysaccharide-tetanus toxoid conjugate was prepared to obtain the polysaccharide component in a thymus-dependent form and to preserve the immunogenic properties of the tetanus toxoid component. Biochemical and immunochemical analyses of this conjugate revealed that (i) it was composed of equal amounts of polysaccharide and protein; (ii) the antigenic activity of the polysaccharide component was greatly reduced; (iii) it contained about 10% free polysaccharide; (iv) the composition was not homogeneous; and (v) only 5% of the tetanus toxoid component was present at the surface of the conjugate molecules. In this study, the influence of these characteristics on the antibody response to both components in mice was investigated. The dose-response relationship, the persistence of antibodies, a possible antigenic competition, and the specificities of the antibodies induced were also studied. Our data suggest that the conjugate behaves as a pronounced thymus-dependent antigen, that the tetanus toxoid component is more immunogenic at lower dosages (0.8 and 20 ng) than equivalent doses of tetanus vaccine, that the presence of free polysaccharide does not influence the induction of antibodies to polysaccharide by the conjugate, and that no antibodies to new structures in the conjugate are induced. These characteristics favor the application of this conjugate as a vaccine for human use.

Animals↗