Type-specific Capsular Polysaccharide Research Articles

Role of the SaeRS Two-Component Regulatory System in Group B Streptococcus Biofilm Formation on Human Fibrinogen.

Streptococcus agalactiae, also known as Group B Streptococcus or GBS, is a commensal colonizer of human vaginal and gastrointestinal tracts that can also be a deadly pathogen for newborns, pregnant women, and the elderly. The SaeRS two-component regulatory system (TCS) positively regulates the expression of two GBS adhesins genes, but its role in the formation of biofilm, an important step in pathogenesis, has not been investigated. In the present study, we set up a novel model of GBS biofilm formation using surfaces coated with human fibrinogen (hFg). Biofilm mass and structure were analyzed by crystal violet staining and three-dimensional fluorescence microscopy, respectively. GBS growth on hFg resulted in the formation of a mature and abundant biofilm composed of bacterial cells and an extracellular matrix containing polysaccharides, proteins, and extracellular DNA (eDNA). Enzymatic and genetic analysis showed that GBS biofilm formation on hFg is dependent on proteins and eDNA in the extracellular matrix and on the presence of covalently linked cell wall proteins on the bacterial surface but not on the type-specific capsular polysaccharide. In the absence of the SaeR regulator of the SaeRS TCS, there was a significant reduction in biomass formation, with reduced numbers of bacterial cells, reduced eDNA content, and disruption of the biofilm architecture. Overall, our data suggest that GBS binding to hFg contributes to biofilm formation and that the SaeRS TCS plays an important role in this process.

Immune Response to Conjugates of Fragments of the Type K9 Capsular Polysaccharide of Acinetobacter baumannii with Carrier Proteins.

ABSTRACTThe clonal bacterial species Acinetobacter baumannii is an emerging multidrug-resistant pathogen which causes high-lethality infections. Cells of A. baumannii are surrounded by the type-specific capsular polysaccharide (CPS), which provides resistance to the protective mechanisms of the host and is considered a target for immunization. The conjugates of three inert carrier proteins and A. baumannii type K9 CPS fragments, which contained various numbers of oligosaccharide repeats (K-units), were synthesized by periodate oxidation and squaric acid chemistry. The conjugates were applied to immunize mice, and chemical synthesis by squaric acid was shown to significantly improve the immunogenic properties of glycoconjugate. In BALB/c mice, IgG antibodies were predominant among type K9 CPS reactive antibodies, and their total content was several times higher than that of IgM. Immune sera were characterized by their opsonization ability during practically the entire lives of the experimental mice. The sera were cross-reactive, but the highest specificity was observed against the antigen (type K9 CPS) used for immunization. The immunization of BALB/c and ICR-1 mice with a glycoconjugate without adjuvants led to varying degrees of stimulation of IL-10, IL-17A, and TNF-α production, but not IL-4 production in the ICR-1 mice. This is in contrast to the BALB/c mice, in which γ-IFN production was also activated. The protective effectiveness of the glycoconjugates obtained by squaric acid chemistry was demonstrated by experiments that involved challenging immunized and nonimmunized animals with a lethal dose of A. baumannii K9.IMPORTANCE Immunization by glycoconjugates with A. baumannii type K9 CPS fragments induced a high level of antibodies (predominantly IgG) in sera, which reacted specifically with the CPS of A. baumannii type K9, as well as a long immunological memory. The sera of immunized animals efficiently opsonized A. baumannii type K9. Immunization resulted in the balanced production of pro/anti-inflammatory lymphokines and protective antibodies to ensure the survival of the mice infected with A. baumannii. The level of specific antibodies was sufficient to provide protective immunity against the challenge by A. baumannii, making this approach applicable in the development of vaccine preparations.

Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies.

The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 10(7) CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.

Group B Streptococcal Infections

1. Laura Sass, MD* 1. *Division of Infectious Diseases, Children's Specialty Group, Children's Hospital of The King's Daughters, Norfolk, VA; Pediatrics, Eastern Virginia Medical School, Norfolk, VA. * Abbreviations: CDC: : Centers for Disease Control and Prevention CPS: : capsular polysaccharides CSF: : cerebrospinal fluid EOD: : early onset disease GBS: : group B Streptococcus IAP: : intrapartum antibiotic prophylaxis NAAT: : nucleic acid amplification test UTI: : urinary tract infection In 2010, revised guidelines were published with updated algorithms for group B Streptococcus screening, intrapartum prophylaxis, antibiotic doses, and revised management for newborns. After completing this article, readers should be able to: 1. Know the mode of transmission of group B Streptococcus (GBS) and the peripartum risk factors associated with a high risk of infection. 2. Know the major clinical manifestations of GBS and be able to differentiate the epidemiology and clinical presentation of early onset GBS disease from that of late-onset disease. 3. Know the laboratory testing for GBS: isolation, antigen detection, and susceptibility tests. 4. Know the treatment of GBS infections: drugs of choice, alternative drugs, and ineffective drugs. 5. Review the most recent guidelines for the prevention of perinatal GBS disease. 1. Understand the importance of maternal screening tests for GBS. 2. Know the criteria for the treatment of mothers known to be GBS carriers at the time of delivery. 3. Differentiate the prevention of early onset disease from that of late onset. Group B Streptococcus (GBS), or Streptococcus agalactiae , is an encapsulated gram-positive diplococcus that produces a narrow zone of β-hemolysis on blood agar and generally is resistant to bacitracin. GBS can be further differentiated by type-specific capsular polysaccharides (CPS) and protein antigens. Current circulating serotypes include Ia, Ib, Ia/c, II, III, IV, V, VI, VII, VIII, and IX. GBS initially was believed to be a bovine mastitis pathogen, but cases of puerperal sepsis in humans were described in the 1930s. The organism then became more prominent in the 1970s as a cause of maternal and neonatal disease. The most common maternal manifestations are asymptomatic bacteriuria, urinary tract infection (UTI), bacteremia, chorioamnionitis, and endometritis. In …

Serologically indicated pneumococcal pneumonia in children: a population-based study in primary care settings.

The aim of the study was to assess age-specific incidences of community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae and diagnosed serologically in a child population. The study was population-based, and prospective, and performed in primary health care settings. During a surveillance period of 12 months from 1981-1982, all pneumonia cases in a defined child population (57% urban residents) were registered prospectively. In total, 201 CAP cases were diagnosed (mean age 5.6 years; 57% boys; 58% urban residents). S. pneumoniae etiology was studied by antibody and immune complex (IC) assays to C-polysacchride (C-PS), type-specific capsular polysaccharides (CPS), and to pneumolysin (Ply), in acute and convalescent sera. Serologic evidence of S. pneumoniae etiology was indicated in 57(28%) cases, 35(61%) being mixed infections with other microbes. The distribution of pneumococcal cases was 44%, 30% and 26% in the three 5-year age groups, respectively. There were 33 (58%) males and 34 (60%) urban residents. In total, 26 cases were identified by antibody assays and 35 cases by IC assays, 26/35 being positive in acute sera. Responses to C-PS, CPS and Ply, when antibody and IC results are combined, were found equally often in 23-25 cases. The total annual incidence of pediatric S. pneumoniae CAP was 6.4/1000/year. S. pneumoniae etiology was found in 28% of the children and was similar at all ages. The incidence of pneumococcal CAP was assessed for the first time, being high (19/1000/year) in 0- to 4-year-old urban boys and rather stable (5-9/1000/year) in all other groups by age, sex and residence.

Intranasal Cry1Ac Protoxin is an Effective Mucosal and Systemic Carrier and Adjuvant of Streptococcus pneumoniae Polysaccharides in Mice

Streptococcus pneumoniae is a major respiratory pathogen in infants, children and the elderly. Available parenteral anti-pneumococcal vaccines based on type-specific capsular polysaccharides (CPSs) are useful in adults but do not elicit protective immunity in infants and young children. To enhance their immunogenicity, pneumococcal CPSs conjugated to proteins are being developed. Mucosal vaccines may induce mucosal and systemic immune responses, but their development has been hampered by the lack of effective, inexpensive innocuous mucosal adjuvants or immunogenic vaccine carriers. We have demonstrated that the recombinant Cry1Ac protoxin from Bacillus thuringiensis is highly immunogenic and has mucosal and systemic adjuvant effects on proteins coadministered in mice. In this work, we evaluated Cry1Ac as a carrier and adjuvant of S. pneumoniae CPS for the induction of mucosal and systemic antibody responses after intranasal and intraperitoneal immunization in mice. Our results demonstrate that intranasal application of pneumococcal polysaccharides either coadministered or conjugated with Cry1Ac induces higher systemic and mucosal specific antibody responses than those elicited by pneumococcal polysaccharides alone. Adjuvant effects of Cry1Ac on polysaccharides may be appropriate for vaccine design.

Group B Streptococcal Infections

1. Samuel P. Gotoff, MD* 1. *Professor and Chairman, Department of Pediatrics, Rush Children’s Hospital, Chicago, IL After completing this article, readers should be able to: 1. Delineate the epidemiologic factors in the transmission of group B Streptococcus (GBS) neonatal infections. 2. List the major factors that increase the risk of neonatal GBS infections. 3. Describe four clinical presentations of neonatal GBS infections. 4. List the laboratory methods for isolating and identifying GBS in mixed flora, normally sterile biologic fluids, and a patient treated with a penicillin. The group B Streptococcus (GBS) initially was isolated by Nocard in 1887 and described as Streptococcus agalactiae , a cause of bovine mastitis. The organism is an encapsulated gram-positive diplococcus that usually produces a narrow zone of beta-hemolysis on blood agar. Most strains are resistant to bacitracin. The streptococci were classified serologically in the 1930s by Lancefield, based on cell wall polysaccharides. The GBS organisms are differentiated further by type-specific capsular polysaccharides and protein antigens. Current serotypes include: Ia, Ib, Ia/c, II, III, IV, V, VI, VII, and VIII. Since the mid-1960s, GBS has become the major cause of bacterial infections in the perinatal period, including bacteremia, amnionitis, endometritis, and urinary tract infection in pregnant women as well as focal and systemic infections in newborns. It is a relatively rare cause of infection in older children and nonpregnant adults. GBS early-onset disease (EOD) occurs within the first week after birth, usually before 72 hours. GBS late-onset disease (LOD) accounts for about 20% of cases, usually occurring between 1 to 4 weeks after birth, but rarely seen up to 6 months of age. Late, late-onset infections, which occur from 1 to 6 months of age, and infections in nonpregnant adults often are associated with immunodeficiency. All of the serotypes have been associated with GBS EOD, with Ia, II, III, and V being most common in the United States. GBS LOD is caused by serotype III …

Identification of Sialyltransferases of Streptococcus agalactiae.

Group B streptococci, Streptococcus agalactiae , produce high-molecular-weight polysaccharides containing N -acetylneuraminic acid. Although the type-specific capsular polysaccharide (CP) synthesis ( cps ) genes of several S. agalactiae strains have been extensively analyzed, to date, no sialyltransferase activity has been detected from any gene product of the cps gene cluster. Among the cps genes, the cpsK gene products of S. agalactiae types la and Ib showed weak similarity to several bacterial sialyltransferases. In this study, the cpsIaK and cpsIbK gene products were found to show sialyltransferase activity specific for lacto- N -neotetraose and lacto- N -tetraose, respectively. This acceptor specificity seems to reflect the respective CP structure, since the repeating unit of type la CP is sialyllacto- N -neotetraose and that of type Ib CP is sialyllacto-Ntetraose. We also found that the C-terminal regions of CpsKs were almost completely conserved in various S. agalactiae strains.

Identification of sialyltransferases of Streptococcus agalactiae

Group B streptococci, Streptococcus agalactiae, produce high-molecular-weight polysaccharides containing N-acetylneuraminic acid. Although the type-specific capsular polysaccharide (CP) synthesis ( cps) genes of several S. agalactiae strains have been extensively analyzed, to date, no sialyltransferase activity has been detected from any gene product of the cps gene cluster. Among the cps genes, the cpsK gene products of S. agalactiae types la and Ib showed weak similarity to several bacterial sialyltransferases. In this study, the cpsIaK and cpsIbK gene products were found to show sialyltransferase activity specific for lacto- N-neotetraose and lacto- N-tetraose, respectively. This acceptor specificity seems to reflect the respective CP structure, since the repeating unit of type la CP is sialyllacto- N-neotetraose and that of type Ib CP is sialyllacto-Ntetraose. We also found that the C-terminal regions of CpsKs were almost completely conserved in various S. agalactiae strains.

The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon.

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.

Intranasal immunization with heat-inactivated Streptococcus pneumoniae protects mice against systemic pneumococcal infection.

In order to study the mucosal and serum antibody response to polysaccharide-encapsulated bacteria in mice, a preparation of heat-inactivated Streptococcus pneumoniae type 4 was administered, with and without cholera toxin, at various mucosal sites. It appeared that intranasal immunization of nonanesthesized animals was superior to either oral, gastric, or colonic-rectal antigen delivery with regard to the induction of serum immunoglobulin G (IgG) and IgA, as well as saliva IgA antibodies specific for pneumococci. The marked IgA antibody response in feces after intranasal, but not after oral or gastric, immunization is suggestive of a cellular link between the nasal induction site and the distant mucosal effector sites. Intranasal immunization also induced antibodies in serum and in mucosal secretions against type-specific capsular polysaccharide. IgA and IgG antibody levels in pulmonary lavage fluids correlated well with saliva IgA and serum IgG antibodies, respectively. Antibody determinations in pulmonary secretions may therefore be redundant in some cases, and the number of experimental animals may be reduced accordingly. After intraperitoneal challenge with type 4 pneumococci, mice immunized intranasally were protected against both systemic infection and death, even without the use of cholera toxin as a mucosal adjuvant. Thus, an efficient intranasal vaccine against invasive pneumococcal disease may be based on a very simple formulation with whole killed pneumococci.

Molecular characterization of type-specific capsular polysaccharide biosynthesis genes of Streptococcus agalactiae type Ia.

The type-specific capsular polysaccharide (CP) of a group B streptococcus, Streptococcus agalactiae type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1- ->3 )]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (cps) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated cpsIaA to cpsIaL, neu (neuraminic acid synthesis gene) A to D, orf1 and ung (uracil DNA glycosylase). The cps gene products of S. agalactiae type Ia were homologous to proteins involved in CP synthesis of S. agalactiae type III and S. pneumoniae serotype 14. Unlike the cps gene cluster of S. pneumoniae serotype 14, transcription of this operon may start from cpsIaA, cpsIaE, and orf1 because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3' end of this operon (downstream of orf1 and ung). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in Escherichia coli JM109 harboring plasmids containing various S. agalactiae type Ia cps gene fragments. Enzyme assays suggested that the gene products of cpsIaE, cpsIaG, cpsIaI, and cpsIaJ are putative glucosyltransferase, beta-1, 4-galactosyltransferase, beta-1,3-N-acetylglucosaminyltransferase, and beta-1,4-galactosyltransferase, respectively.

Characterization of pneumococcal specific antibodies in healthy unvaccinated adults

Unlike the elderly, healthy middle aged adults are at relatively low risk of acquiring serious pneumococcal disease. An explanation that has been proposed is that people in this age group have significant amounts of serum antibody (primarily IgG2) that react with many pneumococcal capsular polysaccharide serotypes. The level of antibody can be as high as several hundred micrograms per milliliter of blood for some serotypes. A significant component of this reactivity is directed toward the conserved C-polysaccharide depletion. Even after C-polysaccharide depletion, which is included as a routine part of the assay to determine antibody levels, resting antibody levels in a normal healthy adult population can vary widely. We have analyzed the reactivity of serum from 76 people to 16 pneumococcal capsular polysaccharide serotypes. The antibody reactivities to 13 of 16 serotypes are highly correlated with one another. Depletion of serum with C-polysaccharide and purified capsular polysaccharide inhibited antibody binding to type specific capsular polysaccharide. Cross-serotype inhibition of antibody binding was also observed. This indicates that there are materials contained within the pneumococcal polysaccharides that contribute to the cross-reactivity of serum antibodies in people that have not been vaccinated with the pneumococcal vaccine.

Pneumococcal immune complexes in the diagnosis of lower respiratory infections in children.

In recent years serologic methods have been applied to assess pneumococcal etiology of pneumonia and other respiratory tract infections. Antigen and antibody assays have shown to be insensitive, especially in young children. The aim of this study was to evaluate the usefulness of circulating immune complexes in the diagnosis of pneumococcal lower respiratory infection in children. Pneumococcal immune complexes (IC) containing antibodies to species-specific C-polysaccharide, to mixtures of type-specific capsular polysaccharides or to a protein antigen, pneumolysin, were studied in the sera of 449 children with lower respiratory tract infection. Circulating ICs were found in 68 (15%) children; 46 (68%) of them were demonstrated in acute and 43 in convalescent serum. In 5 (7%) of the 68 IC-positive patients pneumococcal antigen was present in acute serum; those patients formed 18% of the 28 cases with antigenemia. An antibody response between paired sera to any of the 3 pneumococcal antigens studied was observed in 14 (21%) IC-positive children; they formed 23% of the 60 cases with an antibody response. In total ICs were positive in 51% of all the 134 pneumococcal cases diagnosed by any method. We conclude that the measurement of circulating ICs is more sensitive than other serologic methods for the diagnosis of pneumococcal lower respiratory infection. In infants, however, it was as insensitive as antigen and antibody assays.

Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 10 determined by NMR spectroscopy

The complete primary structure of the type-specific capsular polysaccharide of Clostridium perfringens Hobbs 10 was determined. The polysaccharide was isolated from C. perfringens Hobbs 10 by cold-water extraction of whole, heavily encapsulated cells. The polysaccharide was purified, by ethanol precipitation, deproteination, selective precipitation with hexadecyltrimethylammonium bromide, ion-exchange chromatography and gel-filtration chromatography. The polysaccharide was comprised of d-glucose, d-galactose, N-acetylgalactosamine, and iduronic acid, in molar ratios of 2:2:1:1. Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear DQF-COSY, TQF-COSY and TOCSY, heteronuclear { 1H, 13C} single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments. The capsular polysaccharide of C. perfringens Hobbs 10 is a polymer composed of a hexasaccharide repeating unit with the following structure: ▪ This structure is novel among bacterial cell-surface polysaccharides, and it is only the second of many serotypically distinct capsular polysaccharides of C. perfringens to be described.

Prevalence of antibodies against group B streptococcal capsular polysaccharides in healthy Chinese neonates.

Prevalence of antibodies against group B streptococcal capsular polysaccharides in healthy Chinese neonates.

Role of complement and complement receptor C1qR in the antibody-independent killing of group B streptococcus.

Normal human serum (NHS) containing low levels of antibodies to the type-specific capsular polysaccharide still mediates killing of type III or Ia group B Streptococcus (GBS) in opsonophagocytic assays.1, 3, 4 The killing activity of the antibody-deficient serum can be abolished or strongly diminished by various conditions or treatments: deficiency in C2 complement protein, heat inactivation, preadsorption with living bacteria, and chelation with MgEGTA. Incubation of type Ia GBS with the antibody-deficient serum led to consumption of C3 and C4 complement proteins and the bacteria directly bound purified C1q, the first component of the classical complement pathway (CCP) in the absence of antibody.2 Preincubation of the antibody-deficient serum with soluble type Ia or III polysaccharides inhibited the killing activity of the serum and binding of C1 to GBS, and also decreased the levels of C4.6 These observations suggest that type III GBS can directly activate CCP without the need for antigen-antibody complexes. We investigated the involvement of C1q and CCP in the molecular interactions responsible for antibody-independent opsonophagocytic killing of GBS.KeywordsNormal Human SerumHuman Polymorphonuclear LeukocyteClassical Complement PathwayPhagocytic KillingCalcium Chelator EGTAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A protective surface protein from type V group B streptococci shares N-terminal sequence homology with the alpha C protein.

Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Historically, serotypes Ia, Ib, II, and III have been most prevalent among disease cases; recently, type V strains have emerged as important strains in the United States and elsewhere. In addition to type-specific capsular polysaccharides, many GBS strains possess surface proteins which demonstrate a laddering pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resistance to trypsin digestion. These include the alpha C protein, the R proteins, and protein Rib. Some of these proteins elicit protective antibodies in animals. We demonstrate a trypsin-resistant laddering protein purified from a type V GBS strain by mutanolysin extraction and column chromatography. This protein contains a major 90-kDa band and a series of smaller bands spaced approximately 10 kDa apart on SDS-PAGE. Cross-reactivity of the type V protein with the alpha C protein and with R1 was demonstrated on Western blot (immunoblot). N-terminal sequence analysis of the protein revealed residue identity with 17 of 18 residues at corresponding positions on the alpha protein. Western blot of SDS extracts of 41 clinical type V isolates with rabbit antiserum to the protein demonstrated a homologous protein in 25 isolates (61%); two additional strains exhibited a heterologous pattern which was also demonstrated with 4G8, a monoclonal antibody directed to the alpha C protein repeat region. Rabbit antiserum raised to the type V protein conferred protection in neonatal mice against a type V strain bearing a homologous protein. These data support the hypothesis that there exists a family of trypsin-resistant, laddering GBS surface proteins which may play a role in immunity to GBS infection.

Immunoblot analysis of bacterial polysaccharides: application to the type-specific polysaccharides of Streptococcus suis and Streptococcus agalactiae

A method for the immunoblot analysis of the type-specific capsular polysaccharides of streptococci was developed. The capsular polysaccharides were extracted by sonication and subjected to polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulphate (SDS). After electrophoresis the polysaccharides were transferred to charged PVDF-N membranes and probed with the type specific antibodies. A characteristic ladder-like pattern of polysaccharide bands specific for the serotype (1, 2, 4, 7) was observed for capsular extracts of Streptococcus suis. Human immune sera against type-specific group B streptococcal polysaccharides reacted most strongly with the immunizing polysaccharide type (Ia, II, III). The previously observed crossreactions between the group B streptococcal type-specific capsular preparations were shown to be due to binding to the isomeric polysaccharide molecules. Thus, gel electrophoresis combined with immunoblot analysis seems to provide a novel method for the molecular and immunochemical characterization of bacterial polysaccharides and for the study of the specificity and properties of antibodies to capsular polysaccharides.

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.