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M R Wessels

Publications and source records attributed to M R Wessels.

At least 55 records · Page 3Linked to original sources
PubMed1994

Functional activity of antibodies to the group B polysaccharide of group B streptococci elicited by a polysaccharide-protein conjugate vaccine.

Group B streptococci (GBS) are a major cause of sepsis and meningitis in infants. While antibodies directed to the type-specific GBS capsule have been shown to be protective, it is less clear whether antibodies to the group B polysaccharide, a noncapsular, cell wall-associated antigen, may play a role in immunity. To investigate the functional activity of group B polysaccharide-specific antibodies, we tested sera from rabbits vaccinated with group B polysaccharide coupled to tetanus toxoid (B-TT). Anti-B-TT was weakly opsonic in vitro for a highly encapsulated type III strain, while antiserum elicited by vaccination with type III capsular polysaccharide linked to tetanus toxoid (III-TT) was a very effective opsonin. In contrast to anti-III-TT, anti-B-TT given before or after bacterial challenge was only marginally effective in protecting newborn mice against lethal infection with type III GBS. The number of C3 molecules bound to type III GBS was augmented by anti-III-TT but not by high antibody concentrations of anti-B-TT. These results suggest that the difference in opsonic activity between anti-B-TT and anti-III-TT may be due to a difference in their ability to deposit C3. In addition, the maximum number of antibody molecules bound to the bacterial surface was greater for anti-III-TT than for anti-B-TT. That anti-B-TT binds to fewer sites than anti-III-TT may explain the differences in complement activation and in opsonic and protective efficacy of antibodies to group B polysaccharide compared with antibodies to the type-specific capsular polysaccharide.

Animals↗
PubMed1994

Neonatal mouse protection against infection with multiple group B streptococcal (GBS) serotypes by maternal immunization with a tetravalent GBS polysaccharide-tetanus toxoid conjugate vaccine.

Most cases of neonatal sepsis and meningitis caused by group B streptococci (GBS) are attributable to one of four major capsular serotypes: Ia, Ib, II, or III. Because resistance to infection with GBS has been correlated with the presence of serum antibodies to the type-specific capsular polysaccharides in both experimental animals and human neonates, efforts have been made to elicit protective immunity with GBS capsular polysaccharide vaccines. However, the GBS capsular polysaccharides alone are not highly immunogenic in either animals or human volunteers. Therefore, we and other investigators have attempted to enhance immunogenicity by coupling individual capsular polysaccharides to a carrier protein. Here we report the synthesis and immunogenicity in rabbits of a GBS type Ib polysaccharide-tetanus toxoid vaccine prepared by the direct, covalent attachment of tetanus toxoid to a selected number of sialic acid residues on the type-specific polysaccharide. In addition, the Ib polysaccharide-tetanus toxoid conjugate vaccine was combined with similar tetanus toxoid conjugates of GBS type Ia, II, and III polysaccharides to form a tetravalent GBS conjugate vaccine. Protective efficacy of the GBS tetravalent conjugate vaccine was demonstrated in a mouse maternal immunization-neonatal challenge model of GBS infection. The results support testing in human subjects of a multivalent GBS conjugate vaccine of this design, with the eventual goal of protecting newborns against GBS infection.

Animals↗
PubMed1994

Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.

Cytidine Triphosphate↗
PubMed1993

Identification of cpsD, a gene essential for type III capsule expression in group B streptococci.

We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild-type strain of type III GBS, COH1, by transposon mutagenesis with Tn916 delta E. The mutant, COH1-13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild-type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916 delta E insertion site in COH1-13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31-15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1-13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O-polysaccharide synthesis. A particulate fraction of a lysate of wild-type strain GBS COH1 mediated the transfer of galactose from UDP-galactose to an endogenous acceptor. The galactose-acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane-associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1-13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.

Amino Acid Sequence↗
PubMed1993

Stimulation of protective antibodies against type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine.

Antisera elicited by type Ia group B streptococci (GBS) contain antibodies that react with both type Ia and type Ib strains. Previous studies suggested that antibodies elicited by type Ia organisms recognized a carbohydrate antigen or epitope common to Ia and Ib strains. We now report the synthesis and immunogenicity testing of a type Ia polysaccharide-tetanus toxoid (Ia-TT) conjugate vaccine. Ia-TT elicited type Ia polysaccharide-specific immunoglobulin G antibodies in all three of the rabbits inoculated. In competitive enzyme-linked immunosorbent assay, these antibodies reacted with high affinity to type Ia polysaccharide and with lower affinity to the structurally related GBS type Ib polysaccharide. Despite the lower binding affinity of the Ia-TT-induced antibodies for the type Ib polysaccharide, Ia-TT antiserum opsonized not only type Ia GBS but also type Ib GBS for killing by human blood leukocytes. Ia-TT antiserum was also evaluated in a mouse model designed to test the efficacy of maternal antibodies in protecting neonates against GBS infection. Pups born to dams that had received Ia-TT antiserum were protected against lethal challenge with either type Ia or Ib GBS. These studies using a polysaccharide-protein conjugate as an immunogen support the view that the carbohydrate immunodeterminant recognized on Ib strains by Ia antisera is a common epitope contained within the structurally related Ia and Ib capsular polysaccharides. Although antibodies elicited by Ia-TT had protective activity against both Ia and Ib strains, these antibodies reacted with lower affinity to Ib than to Ia polysaccharide.

Animals↗
PubMed1993

Capsular polysaccharide regulates neutrophil complement receptor interactions with type III group B streptococci.

The capsular polysaccharide of type III group B streptococci contributes substantially to the virulence of this organism. We explored the extent to which capsular polysaccharide influences neutrophil complement receptor interactions by using a poorly encapsulated strain (COH 31r/s), two well-encapsulated strains (M732 and M912), and strains produced from COH 31r/s by transposon mutagenesis that lacked capsule (COH 31-15) or had capsular polysaccharide lacking terminal sialic acid residues (COH 31-21). When tested with normal human serum, each strain had initially high bactericidal indices (85 to 96%). Monoclonal antibody blockade of neutrophil complement receptor 3 (CD11b/CD18) inhibited opsonophagocytosis to a significantly greater extent for the well-encapsulated strain than for the poorly encapsulated, asialo, or unencapsulated mutant strain. The addition of antibody with specificity for capsular polysaccharide reduced the inhibitory effect significantly for the encapsulated but not for the mutant strains. Blockade of neutrophil complement receptor 1 (CD35) effected only low-level inhibition. However, simultaneous blockade of complement receptors 1 and 3 augmented the inhibitory effect. When hypogammaglobulinemic serum was used as an antibody-free complement source, the initial bactericidal index was low (30% +/- 15%) for an encapsulated strain and was not affected for the mutant strains. Blockade of either neutrophil complement receptor 1 or 3 or the combination fully inhibited killing of the encapsulated strain. These results demonstrate that the type III group B streptococcal capsular polysaccharide regulates interactions with neutrophil complement receptors. We conclude that efficient phagocytic killing of encapsulated group streptococci in nonimmune serum requires ligation of complement receptors 1 and 3.

Adult↗
PubMed1992

Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.

Animals↗
PubMed1992

Group B Streptococcus type II polysaccharide-tetanus toxoid conjugate vaccine.

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.

Animals↗
PubMed1992

Identification of a genetic locus essential for capsule sialylation in type III group B streptococci.

The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916 delta E. In contrast to the wild-type strain, the asialo mutant strain COH1-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COH1-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COH1-11. One of the two transposon insertions in the asialo mutant COH1-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.

Bacterial Toxins↗
PubMed1992

Group B streptococcal opacity variants.

Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown.

Bacterial Capsules↗
PubMed1992

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.

Animals↗
PubMed1991

Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci.

Mucoid strains of group A Streptococcus have been associated with recent outbreaks of acute rheumatic fever. The mucoid colony morphology of these strains is a result of abundant production of capsular polysaccharide, which is composed of hyaluronic acid. To study the role of the hyaluronic acid capsule in virulence, we derived an acapsular mutant from a mucoid strain of group A Streptococcus by transposon mutagenesis. M protein expression was not altered in the mutant strain. The mucoid wild-type strain grew in fresh human blood and was resistant to phagocytic killing in vitro. In contrast, the acapsular mutant failed to grow in fresh human blood and was sensitive to phagocytic killing in vitro. Loss of capsule was associated with a 100-fold reduction in virulence of the organisms in mice. We conclude that the hyaluronic acid capsule protects mucoid group A streptococci from phagocytosis and has an important role in virulence.

Animals↗
PubMed1991

Structural determination and immunochemical characterization of the type V group B Streptococcus capsular polysaccharide.

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.

Bacterial Capsules↗
PubMed1990

An oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus.

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.

Animals↗
PubMed1990

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.

Animals↗