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D L Kasper

Publications and source records attributed to D L Kasper.

At least 91 records · Page 5Linked to original sources
PubMed1992

Protection of neonatal mice from group B streptococcal infection by maternal immunization with beta C protein.

Group B streptococci (GBS) cause the majority of cases of neonatal sepsis and meningitis in the United States. Immunization of women of childbearing age is one strategy under consideration for the prevention of neonatal disease. The beta C protein, a 130-kDa antigen present in many clinical isolates of GBS, was purified from GBS by extraction into sodium dodecyl sulfate (SDS)-containing buffer, preparative SDS-polyacrylamide gel electrophoresis, and electroelution. Purified beta C protein antigen (25 micrograms) with Freund's adjuvant was used to immunize rabbits. Rabbits developed enzyme-linked immunosorbent assay titers of > 1:1.6 x 10(6), and sera from immunized rabbits were administered to pregnant mice. Their neonatal pups were then challenged with a strain of GBS expressing beta C protein; 68% of these pups were protected by immune antiserum, whereas no controls were protected (P < 0.001). The immune serum (diluted 1:100) facilitated opsonophagocytic killing of GBS strains expressing the beta C protein but not those that do not express the antigen (mean log kill +/- standard deviation = 0.71 +/- 0.8 log10 CFU for beta+ strains and 0.09 +/- 0.2 for beta- strains; P = 0.02). In subsequent experiments, adult female mice were actively immunized with two doses of 2, 5, or 10 micrograms of beta C protein 2 months prior to mating. One- to two-day-old offspring of these dams were challenged with GBS and were protected in a dose-dependent manner, with 96% survival in the high-dose (10-micrograms) group and 20% survival in a sham-immunized control group (P < 0.001). Thus, active immunization of mice with the GBS beta C protein confers protection against lethal infection with beta+ GBS to their offspring.

Animals↗
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

Group B streptococcus infection in mother and child.

Despite significant advances in obstetric and pediatric health care, group B beta-hemolytic Streptococcus (GBS) remains one of the most prevalent and devastating pathogens in peripartum women and their newborn infants. It may cause urinary tract infection, chorioamnionitis and endometritis, bacteremia, and cesarean wound infection in the peripartum period. Moreover, GBS accounts for nearly 50% of serious neonatal bacterial infections. Approximately three in every 1,000 children born in the United States acquire pneumonia, sepsis, or meningitis from GBS, with combined mortality and morbidity exceeding 50% despite appropriate antibiotic and supportive therapy. Estimates indicate that more than 10,000 infants are affected annually, at a cost of more than $300 million. Neonatal disease is divided into early- and late-onset syndromes: The illness emerging after six days of age differs in terms of GBS serotype, clinical manifestations, and outcome from the disseminated process seen in earlier onset. We describe two infants infected with GBS and discuss risk factors, pathogenesis, diagnosis, therapy, and options for disease prevention in the peripartum woman and her infant.

Adult↗
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↗
PubMed1991

A monoclonal antibody identifies a protective C-protein alpha-antigen epitope in group B streptococci.

Group B streptococci (GBS) are the leading causes of neonatal sepsis and meningitis in the United States, with a high rate of fatality and serious morbidity despite appropriate therapy. The C-protein antigens of GBS appear to be important in immunity to experimental infection, yet these antigens remain incompletely characterized with respect to their number, structure, and function. None of these proteins has yet been purified to homogeneity. We have developed a novel method for extraction of surface proteins from the A909 (Ia/c) strain of GBS by using mutanolysin. Antibodies raised in rabbits against these partially purified proteins conferred passive protection to lethal GBS infection in mice challenged with a GBS strain expressing C proteins with a heterologous capsule type. In addition, mouse monoclonal antibodies were produced and identified by reactivity with the mutanolysin-extracted proteins. One of these monoclonal antibodies (4G8) identifies an epitope on the alpha-antigen of the GBS C proteins (identified by protease susceptibility and mouse protection). On sodium dodecyl sulfate-polyacrylamide gels, this epitope appears as a series of regularly spaced bands ranging in apparent molecular mass from 160,000 to 30,000 Da. The monoclonal antibody 4G8 induces opsonic killing of GBS and protects mice from lethal challenge with GBS. Thus, the 4G8 monoclonal antibody identifies a fully protective epitope on the C-protein alpha-antigen of GBS.

Animals↗
PubMed1991

The alpha-L-(1----2)-trirhamnopyranoside epitope on the group-specific polysaccharide of group B streptococci.

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.

Animals↗
PubMed1991

Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity.

Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C proteins of GBS play a role in immunity, but their number, size, structure, function, and virulence properties have not been well characterized. A recombinant library of DNA fragments from GBS strain A909 (type Ia/C) was prepared in the plasmid pUX12, a specially constructed Escherichia coli expression vector. The library was screened with a rabbit antiserum shown to be protective for passive immunity to GBS infection in a mouse lethality model. Clones were divided into two distinct groups on the basis of DNA-DNA cross-hybridization, restriction enzyme analysis, and the expression of antigenic proteins in E. coli. A characteristic clone from each group was chosen for further study. Clone pJMS23 expresses gene products that biochemically and immunologically correspond to the trypsin-resistant, C-protein alpha antigen. Clone pJMS1 expresses a gene product that binds to immunoglobulin A and is similar to the trypsin-sensitive, C-protein beta antigen. Antisera raised in rabbits against E. coli containing each of the plasmid clones were able to elicit protective immunity in mice challenged by GBS strains carrying the C proteins but not by non-C-protein-bearing strains. Southern blot analysis shows no DNA homology between the clones, and there is no immunological cross-reactivity between the antigens they express. Therefore, pJMS23 and pJMS1 encode two different C proteins that define unique protective epitopes.

Animals↗
PubMed1991

Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis.

Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physiochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans.

Animals↗
PubMed1991

Phenotypic diversity in the alpha C protein of group B streptococci.

Group B streptococci (GBS) is the leading cause of neonatal sepsis and meningitis. C proteins are an immunologically important group of surface-associated antigens in GBS that remain incompletely characterized. Two C proteins have been designated alpha and beta on the basis of protease susceptibility. We recently used a monoclonal antibody to describe a protective epitope of the GBS alpha (or trypsin-resistant) C protein in the prototype Ia/c GBS strain. In the present study, we examined 51 GBS isolates for expression of C-protein alpha and beta antigens. The alpha antigen, as detected with monoclonal antibody in sodium dodecyl sulfate (SDS) extracts, appears as a heterogeneous series of proteins spaced 8 kDa apart on SDS-polyacrylamide gel electrophoresis, but has a maximum molecular mass that varies among strains from 62.5 to 167 kDa. By immunoblotting with human immunoglobulin A, polyclonal antiserum, or monoclonal antibody, the beta antigen, in contrast, appears as a single protein of molecular mass between 124 and 134 kDa. The amount of alpha antigen expressed by each strain was quantified by enzyme immunoassay inhibition and was found to vary markedly from strain to strain. The susceptibility of strains of GBS to opsonization and killing by human polymorphonuclear leukocytes in the presence of either complement alone or complement with alpha-specific monoclonal antibody was examined. Strains expressing the alpha antigen were less readily killed in the absence of specific antibody than were alpha-negative strains. Killing in the presence of alpha-specific monoclonal antibody was found to correlate directly with the maximum molecular mass of the alpha antigen and with the quantity of antigen on the bacterial cell surface. Isolates of GBS that express the alpha C protein vary widely in the quantity and molecular mass of the alpha antigen produced, and this heterogeneity appears to have biologic importance.

Antibodies, Bacterial↗
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↗
PubMed1990

Molecular analysis of two group B streptococcal virulence factors.

Molecular biology has provided new technology for evaluating the traits of bacterial pathogens that are important in the pathogenesis of infections. The ability to derive isogenic strains that differ by a single trait provides a powerful tool for investigating the interaction of a putative virulence factor with the host at any of the various steps in pathogenesis. Recombinant DNA techniques afford the opportunity to clone the genes involved in the biosynthesis of a particular virulence factor. Once the gene(s) are cloned, a vast amount of information can be learned about their composition, structure, and regulation, and similarity with genes in other organisms. Understanding the molecular biology of a virulence factor also provides information about potential targets for future therapies and preventive modalities. The molecular analysis of two virulence factors from the type III group B streptococcus has been reviewed to provide specific examples of how these techniques can be used. The data has shown that the capsular polysaccharide is an essential factor in GBS virulence. The structural influence of sialic acid on the capsule plays a major role in its virulence properties. The importance of the capsule has been tested in several assays to identify its role in pathogenesis. Its primary role appears to be evading host phagocytic mechanisms, but it does not appear to be essential in the vascular response observed during GBS sepsis. Using the isogenic strains, we have also learned that the capsule does not mask a fibronectin receptor on GBS. In contrast to the capsule, the beta-hemolysin of GBS does not appear to be essential for systemic disease once the organism has invaded. Its role in the initial invasive steps in GBS pathogenesis has not been tested, but the availability of isogenic mutants in beta-hemolysin production will allow this question to be answered once the model systems are available.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Antibody recognition of the type 14 pneumococcal capsule. Evidence for a conformational epitope in a neutral polysaccharide.

Oligosaccharides consisting of one or more tetrasaccharide repeating units were derived from the capsular polysaccharide of type 14 pneumococcus (Pn14) by endo-beta-galactosidase digestion. The relative affinity of anticapsular antibody binding to derivative oligosaccharides of different chain lengths was measured in a Pn 14 ELISA inhibition assay. The concentration of inhibiting antigen required to achieve 50% inhibition of IgG binding increased progressively from 5.6 x 10(-4) M to 7.0 x 10(-11) M as the inhibiting saccharide chain length increased from 1 tetrasaccharide repeating unit to 2,500 repeating units. These data indicate that antibodies directed against the Pn14 polysaccharide recognize a conformational epitope fully expressed only in high molecular weight forms of the antigen. Similar results were found for inhibition of Fab fragment binding, suggesting that recognition of the conformational epitope is largely dependent on the intrinsic affinity of the Fab combining region. Unlike previously reported polysaccharides for which conformational epitopes have been described, the Pn14 polysaccharide does not contain negatively charged residues, indicating that expression of conformational determinants is not limited to acidic polysaccharides. Antibody recognition of conformational epitopes may be a common mechanism by which the host immune response discriminates between bacterial polysaccharides and host oligosaccharides of similar structure.

Animals↗
PubMed1989

Definition of a bacterial virulence factor: sialylation of the group B streptococcal capsule.

Sialylation of bacterial capsules has been proposed as an important virulence factor for several species of encapsulated pathogens, including group B Streptococcus. We have constructed a transposon mutant strain of type III group B Streptococcus that expresses a capsular polysaccharide differing from the wild type only in that the mutant strain's capsule lacks sialic acid. The mutant polysaccharide is antigenically identical to the capsular polysaccharide of type 14 Streptococcus pneumoniae, as predicted by the structures of the type III group B Streptococcus and S. pneumoniae polysaccharides. Loss of capsular sialic acid was associated with loss of virulence in the mutant strain in a neonatal rat model of lethal group B Streptococcus infection. These studies demonstrate directly that capsular sialic acid is a critical virulence determinant for type III group B Streptococcus and support the general hypothesis that surface sialylation aids pathogenic microorganisms in evading host defenses.

Bacterial Toxins↗