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D L Kasper

Publications and source records attributed to D L Kasper.

At least 19 recordsLinked to original sources
PubMed1992

Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci.

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C protein alpha antigen of GBS is thought to have a role in both virulence and immunity. We previously cloned the C protein alpha antigen structural gene (named bca for group B, C protein, alpha) into Escherichia coli. Western blots of both the native alpha antigen and the cloned gene product demonstrate a regularly laddered pattern of heterogeneous polypeptides. The nucleotide sequence of the bca locus reveals an open reading frame of 3060 nucleotides encoding a precursor protein of 108,705 Da. Cleavage of a putative signal sequence of 41 amino acids yields a mature protein of 104,106 Da. The 20,417-Da N-terminal region of the alpha antigen shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein. Each repeating unit is identical and consists of 82 amino acids with a molecular mass of 8665 Da, which is encoded by 246 nucleotides. The size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins expressed by GBS. The C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of Gram-positive surface proteins. The large region of identical repeating units in bca defines protective epitopes and may play a role in generating phenotypic and genotypic diversity of the alpha antigen.

Amino Acid Sequence↗
PubMed1992

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.

Bacteroides fragilis↗
PubMed1992

Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy.

The capsule of Bacteroides fragilis is unusual in that it consists of two distinct capsular polysaccharides. Using a combination of high-resolution NMR spectroscopy, theoretical calculations, and as few chemical procedures as required, the structure of both polysaccharide antigens (polysaccharides A and B) was elucidated. Using the above procedures, it was possible to obtain the complete structures using minimal quantities of polysaccharides A and B (8 and 5 mg, respectively). Only small amounts of each subjected to chemical analysis were not recoverable. Polysaccharide A is composed of the following repeating unit: [----3)alpha-D-AATp(1----4)[beta-D-Galf(1----3)]alpha-D- GalpNAc(1----3)beta-D-Galp(1----], where AAT is 2-acetamido-4-amino-2,4,6-trideoxygalactose. A pyruvate substituent having the R configuration spans O-4 and O-6 of the beta-D-galactopyranosyl residue. Polysaccharide B is composed of the following repeating unit: [----4)alpha-L-QuipNAc(1----3)beta-D-QuipNAc(1----4)[alpha-L - Fucp(1----2)beta-D-GalpA(1----3)beta-D-GlcpNAc(1----3)]alpha -D-Galp(1----]. A 2-aminoethylphosphonate substituent is situated on O-4 of the N-acetyl-beta-D-glucopyranosyl residue.

Bacteroides fragilis↗
PubMed1992

Neonatal mouse model of group B streptococcal infection.

Neonatal mice were infected with type III group B streptococcal (GBS) strain M781 by the intraperitoneal route. Age-related susceptibility to challenge was seen within the first 5 days of life. Quantitative blood cultures demonstrated a rapid increase in bacterial numbers during the first 30 h after challenge. Infected pups showed clinical signs of septicemia, and most succumbed within 48 h of challenge. Histopathologic evaluation of the neonates showed bacterial infection within 1 day after challenge. Pregnant adult mice were given a single inoculation of serum raised in rabbits against a tetanus toxoid-conjugated type III GBS polysaccharide vaccine. This serum passively protected 100% of the offspring. This neonatal mouse model of GBS infection and protection may be suitable for study of various forms of intervention.

Aging↗
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

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

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

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↗
PubMed1979

Rapid diagnosis of Bacteroides infections by indirect immunofluorescence assay of clinical specimens.

43 specimens from a variety of sites were directly examined by indirect immunofluorescence assay (I.F.A.) with specific antisera against the capsular polysaccharide of Bacteroides fragilis and pooled antisera against a number of serotypes of Bacteroides sp. (all of the former B. fragilis subspecies). The findings were compared with those of routine anaerobic bacteriology and gas liquid chromatography for short chain fatty acids. Examination by I.F.A. was a sensitive (100%) and specific (90.3%) means of identifying B. fragilis. Use of the pooled serum was sensitive (100%) but less specific (64.3%) than the capsular antiserum (90.3%) although it had the advantage of detecting Bacteroides species other than B. fragilis. The capsular serum I.F.A. gave 9.7% false positives and no false negatives. The predictive value of a positive identification of B. fragilis in a clinical specimen using this anticapsular serum I.F.A. test was 80%; with the pooled Bacteroides group-serum it was 60%. The predictive value of a negative test was 100% for both sera, indicating that a negative I.F.A. test is a reliable index of the absence of Bacteroides from the culture I.F.A. of clinical material provides a rapid (less than 2 h) specific and sensitive means for the diagnosis of B. fragilis infections and would be of use in a clinical laboratory.

Abdomen↗
PubMed1979

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.

Antibodies, Bacterial↗
PubMed1979

Group B streptococcal colonization and antibody status in lower socioeconomic parturient women.

This investigation was undertaken to determine the prevalence of group B streptococcal vaginal and throat colonization among lower socioeconomic pregnant women and the antibody concentration to the capsular polysaccharide antigen of type III group B streptococcus in their sera. Group B streptococci were recovered from 28.6 per cent of the 112 women studied; vaginal colonization was detected in 23.4 per cent, throat colonization in 4.7 per cent, and colonization at both sites in 0.9 per cent of the patients, respectively. Among these isolates of group B streptococci, serotypes III (39.5 per cent) and II (30.3 per cent) predominated. No differences were found between colonized and noncolonized women with respect to age, race, marital status, or parity. The majority of all women studied had low concentration of antibody in serum (less than 1.0 microgram per milliliter). However, women with isolation of type III group B streptococci from cultures at the time sera were collected had significantly higher concentrations than did women without group B streptococci from cultures at the time sera were collected had significantly higher concentrations than did women without group B streptococcal colonization.

Adolescent↗
PubMed1979

Rapid diagnosis of type III group B streptococcal meningitis by latex particle agglutination.

A latex particle agglutination assay was developed to detect type III group B streptococcal antigen in cerebrospinal fluid. Agglutination was observed in each of 12 initial and 54% of 26 subsequent CSF specimens obtained a mean of 53 hours after the institution of antimicrobial therapy. Latex agglutination was more sensitive than countercurrent immunoelectrophoresis for the detection of type III group B streptococcal antigen in CSF from neonates and young infants with meningitis.

Antigens, Bacterial↗