Human immunity to group B streptococci measured by indirect immunofluorescence: correlation with protection in chick embryos.

An indirect immunofluorescence (IF) assay has been developed as a useful semiquantitative method for determination of type-specific IgG antibody in human sera to the five serotypes of group B Streptococcus. Antibody titers measured by IF correlated with passive protection in chick embryos, and antibody titers associated with chick embryo protection were delineated. Except for types Ia and Ic, IF antibody to each of the streptococcal types was completely absorbed by homologous strains, and antibody titers were unchanged by incubation with heterologous bacteria. For types Ia and Ic, IF antibody was absorbed by either the Ia or the Ic strain and by native Ia carbohydrate antigen. Antibody titers measured by IF and chick embryo protection against types Ia and Ic were similar, but were divergent for Ib and Ic, a finding suggesting that antibody is predominantly directed to the major carbohydrate determinants. In addition, 29 of 31 sera that had been tested in chick embryos yielded comparable results in mice against challenge with type Ia group B Streptococcus, a finding further validating the chick embryo assay. Sera from all of 43 mothers of infants infected with group B streptococci had antibody titers by IF that were less than titers associated with protection in chick embryos.     

Antibodies to capsular polysaccharides of group B Streptococcus in pregnant Canadian women: relationship to colonization status and infection in the neonate.

In a cohort study of 1207 pregnant women in Alberta, Canada, the serotype distributions of vaginal-rectal group B Streptococcus (GBS) isolates were compared with all isolates from neonates with invasive GBS disease identified by population-based surveillance. Serum concentrations of Ia, Ib, II, III, and V capsular polysaccharide (CPS)-specific IgG also were determined, according to serotype of the vaginal-rectal colonizing GBS strain. GBS colonization was detected in 19.5% (235 of 1207) of women. Serotype III accounted for 20.6% (48 of 233) of colonizing strains available for typing but for 37% (27 of 73) of invasive isolates from neonates (P<.01). Maternal colonization with type III was least likely to be associated with moderate concentrations of III CPS-specific IgG. Serotype III GBS is more invasive than other serotypes in this population; this may be due, at least in part, to poor maternal type III CPS-specific antibody response.     

Level of maternal antibody required to protect neonates against early-onset disease caused by group B Streptococcus type Ia: a multicenter, seroepidemiology study

Because of the difficulty of conducting efficacy trials of vaccines against group B streptococcus (GBS), the licensure of these vaccines may have to rely on studies that measure vaccine-induced antibody levels that correlate with protection. This study estimates the level of maternal antibody required to protect neonates against early-onset disease (EOD) caused by GBS type Ia. Levels of maternal serum IgG GBS Ia antibodies, measured by ELISAs in 45 case patients (neonates with EOD caused by GBS Ia) and in 319 control subjects (neonates colonized by GBS Ia but without EOD) born at > or =34 weeks gestation were compared. The probability of developing EOD declined with increasing maternal levels of IgG GBS Ia antibody (P = .03). Neonates whose mothers had levels of IgG GBS Ia antibody > or =5 microg/mL had an 88% lower risk (95% confidence interval, 7%-98%) of developing type-specific EOD, compared with those whose mothers had levels < 0.5 microg/mL. A vaccine that induces IgG GBS Ia antibody levels > or =5 microg/mL in mothers can be predicted to confer a high degree of type-specific immunity to EOD to their infants.     

Group B streptococcal vaccines

In recent years group B Streptococcus (GBS) has been recognized as a major perinatal pathogen. As with other encapsulated bacteria, protective immunity appears to correlate with serum antibody specific for the homologous capsular polysaccharide antigen of each serotype. Since susceptibility of the young infant to disseminated GBS infection relates to type-specific antibody deficiency in maternal serum, immunization of women with purified GBS type-specific polysaccharides has been proposed as a method for the prevention of infant disease through placental transport of protective antibodies. Candidate native polysaccharides from GBS have been purified, immunochemically and structurally characterized, and employed as immunogen in healthy adult volunteers. Native type Ia, II, and III polysaccharides have been shown to be nontoxic, safe, and immunogenic in approximately 65%, 95%, and 70%, respectively, of nonimmune adults. Antibody response to immunization approaches 100% in previously immune volunteers. Vaccine-induced type-specific antibodies to these candidate polysaccharide vaccines promote in vitro opsonophagocytosis, protect animals given a lethal challenge of homologous organisms, and are predominantly of the IgG isotype. Once similar results can be documented in women immunized during the last half of pregnancy, efficacy of these candidate GBS polysaccharide vaccines in the prevention of neonatal and young infant GBS disease should be evaluated.     

ELISA for type-specific group B streptococcal antibody and its clinical significance

In order to investigate the neonatal infection of group B streptococci (GBS), vaginal and anal cultures, and measurement of type-specific antibodies to GBS were carried out on 461 pregnant women. Levels of antibody to GBS were measured with ELISA plates coated with type-specific antigen of GBS. These plates were furnished by Toyo Jozo Co., Ltd. The results were as follows: 1) Antibodies to type Ia, Ib, II and III were detected in 41.9, 34.7, 31.7 and 40.1% of subjects, respectively. 2) GBS was isolated in 78 (16.9%) of subjects. 3) Antibody levels against GBS in the sera of colonized mothers and cord blood of their infants were well correlated. 4) Among the colonized mothers, 4 out of 19 (Ia), 9 out of 18 (Ib) 5 out of 8 (II) and 5 out of 17 (III) showed low levels of antibody. 5) Those who had low levels of antibody were administered antibiotic delivery, and there was no case of crisis in both treatment (antibody levels were negative) and non-treatment (antibody levels were positive) groups.     

Type-specific antibody to type Ia, Ib, II and III group B Streptococcus in maternal and neonatal sera measured by ELISA

In order to determine the type-specific antibody to group B streptococcus (GBA) type Ia, Ib, II and III, the ELISA system was established in Research laboratories, Toyo Jozo Co., Ltd. We assayed type-specific antibody by this ELISA system in both maternal and neonatal (or cord) sera. The cut off levels were determined by the antibody levels of maternal and neonatal sera of 26 infected cases and 90 GBS carriers, that type Ia, Ib, II were 0.20 and type III was 0.15. Prevalence of type-specific antibody levels were studied in 356 maternal sera (14 affected cases, 100 GBS carriers and 242 non carriers). Antibody levels were positive in 47.2% of maternal sera to type Ia, 34.0% to type Ib, 46.9% to type II and 45.5% to type III. Antibody levels to type Ia, Ib, II and III were positive, respectively, in 100%, 88.2%, 25.0% and 42.9% of the sera of carrier mothers whose infants were not affected. Antibody levels in 50 pair sera of maternal and cord blood were well correlated.     

Group B streptococcal serotype distribution of isolates from colonized pregnant women at the time of delivery in United Arab Emirates

OBJECTIVE: To determine the maternal colonization rate with group B streptococcus (GBS) and to identify the most frequent GBS serotypes occurring in UAE women during labour. STUDY DESIGN: From February 1998 to January 1999, five hundred and sixty three pregnant women from a similar socio-economic and ethnic population were enrolled for the study. High vaginal swab cultures for GBS were obtained at the time of admission for delivery. Isolates were classified according to their capsular polysaccharide types (Ia, Ib, Ic, II-V) and c protein antigen compound. RESULTS: Fifty-seven (10.1%) of 563 mothers were found to be carriers of GBS. Among the isolates, serotype IV (26.3%) predominated followed by type Ia (21.0%), type III (17.6%), type V (12.3%) and nontypeable, which accounted for 15.8%. CONCLUSIONS: In view of the unknown status for GBS carrier rates in our community, this study suggests that about 10% of UAE women are colonized with group B streptococcus at delivery. The serotype distribution of the isolates in this population is different than those reported elsewhere with type IV predominating followed by type Ia and III.     

Safety and immunogenicity of a bivalent group B streptococcal conjugate vaccine for serotypes II and III

To determine whether 2 monovalent group B streptococcus (GBS) serotype II or III capsular polysaccharide (CPS)-tetanus toxoid (TT) conjugate vaccines combined in a single intramuscular dose would elicit immune responses comparable to those of monovalent vaccines, 75 healthy adults were randomized to receive GBS II-TT (3.6 micro g of CPS), GBS III-TT (12.5 micro g of CPS), or a bivalent mixture of GBS II-TT/III-TT vaccine (double-masked design). Vaccines were well tolerated. Four-fold or greater increases in GBS II or III CPS-specific IgG, respectively, were noted in postimmunization serum samples from 80%-90% of bivalent conjugate vaccine recipients, and these responses were similar to those of recipients of GBS II-TT or GBS III-TT monovalent vaccines. Immune serum samples promoted the opsonophagocytic killing of types II and III GBS in vitro. Unexpectedly, some recipients of these vaccines developed cross-reactive antibodies to the structurally similar heterologous polysaccharide. These results support the feasibility of a multivalent vaccine for the 5 prevalent invasive disease-causing GBS CPS serotypes.     

Human IgG antibody to group b Streptococcus type III: comparison of protective levels in a murine model with levels in infected human neonates

We determined the serum concentration of human IgG antibody to the native capsular polysaccharide of group B Streptococcus (GBS) type III needed to passively protect mice against lethal homologous challenge. Antibody was measured by an ELISA, standardized by two methods, and corrected for nonprecipitating antibody. A concentration of 1.3 micrograms of IgG antibody to GBS type III/ml protected 126 (97%) of 130 mice from an 80%-96% lethal dose bacterial challenge. Concentrations of IgG antibody to GBS type III in sera from 42 infected infants were less than or equal to 0.3 micrograms/ml. Concentrations of antibody ranged from less than 0.02 to 21.7 micrograms/ml in sera from 102 unselected pregnant women (median, 0.05 microgram/ml); 13% had concentrations greater than or equal to 1.3 microgram/ml. Levels in 25 women colonized with GBS type III who gave birth to normal infants were significantly higher and ranged from 0.1 to 10.7 microgram/ml (median, 0.78 micrograms/ml). In a study of transplacental passage of antibody, protective levels were found in a number of infants with gestational ages between 28 and 36 weeks.     

Role of complement receptors in opsonophagocytosis of group B streptococci by adult and neonatal neutrophils

The role of complement receptors in bactericidal activity for types III and Ia group B streptococci (GBS) by adult or neonatal polymorphonuclear leukocytes (PMNL) was explored using polyclonal and monoclonal antibodies to complement receptors one (CR1) and three (CR3). In an opsonophagocytic assay, selective blockade of the CR3 sugar or lectin-like binding site on adult or neonatal PMNL effected a significant reduction in killing of both GBS serotypes that was more pronounced for type III. In contrast, blockade of the iC3b binding site effected greater inhibition of bactericidal activity for type Ia than for type III GBS. When both the CR3 sugar or lectin-like binding site and CR1 were blocked, inhibition was enhanced for type Ia GBS by adult PMNL and for both serotypes by neonatal PMNL. These results demonstrate a role for both CR1 and CR3 in the opsonophagocytosis of GBS. Possibly, differences in CR3 epitope utilization contribute to the differential virulence among GBS serotypes in neonates.     

Isolation and characterization of an age-related antigen present on senescent human red blood cells

Autologous membrane-bound IgG was isolated from a subpopulation of human red blood cells (RBC) with specific density greater than 1.110, by affinity chromatography of purified RBC membrane glycoprotein preparations using immobilized wheat germ agglutinin and immobilized anti-human immunoglobulin (Ig) as immunoabsorbents. The Ig-containing population thus obtained, when further separated by chromatography on Sephadex G-200 in the presence of chaotropic agents, yielded four peaks (Ia, Ib, II, and III). Double immunodiffusion revealed the presence of Ig in the first three peaks (IgM in peak Ia, IgA in Ib, and IgG in II) but not in peak III. Peak III was precipitated by the Ig-containing peaks (Ia, Ib, and II) in immunodiffusion assays, suggesting that the antigenic membrane determinants responsible for the binding of autologous Ig to senescent human RBC were contained in this peak (III). Peaks Ia, Ib and II precipitate purified asialoglycophorin; peak III was reactive with purified autoantibodies directed against asialoglycophorin. These results suggest that an age-related antigenic determinant(s) present on senescent human RBC is exposed by desialylation of the major sialoglycoprotein component of the RBC membrane.     

The platelet glycoprotein Ib-von Willebrand factor interaction activates the collagen receptor alpha2beta1 to bind collagen: activation-dependent conformational change of the alpha2-I domain

Integrin alpha2beta1 (glycoprotein [GP] Ia/IIa) is a major platelet receptor for collagen, containing its collagen-binding site within the alpha2 I domain. alpha2beta1 changes conformation upon platelet activation, increasing its affinity for collagen. We observed that 2 antibodies known to bind within the alpha2I domain, 12F1 and 6F1, bound preferentially to adenosine diphosphate (ADP)-activated platelets. Interestingly, when whole blood was perfused over a surface coated with either 12F1 or 6F1, only 6F1 supported the adhesion of unstimulated platelets. To test whether the interaction of GP Ib with von Willebrand factor (VWF) directly activates alpha2beta1, we used 12F1 as a probe of integrin activation. We perfused blood over a surface coated with a mixture of VWF-A1 domain (a GP Ib ligand) and 12F1 or VWF-A1 and mouse immunoglobulin G (IgG). Platelets rolled and did not attach stably on the A1/IgG surface, but they firmly bound and covered the A1/12F1 surface. We corroborated that 12F1 binds an active conformation of the I domain by showing that it binds with higher affinity to a gain-of-function mutant than to either wild-type I domain or a loss-of-function mutant. These results strongly suggest that the interaction of platelet GP Ib with VWF mediates the activation of alpha2beta1, increasing its affinity for collagen.     

Quantitative determination of immunoglobulin G specific for group B streptococcal beta C protein in human maternal serum.

The beta C protein of group B streptococci (GBS) elicits antibody that is protective against GBS challenge in animals and is considered to be a potential component of a GBS conjugate vaccine. We developed a quantitative enzyme-linked immunosorbent assay to measure beta-specific serum immunoglobulin G (IgG) levels and used it to compare beta-specific IgG in a group of mothers of neonates with invasive type Ib/beta GBS disease and a group of mothers colonized with Ib/beta strains whose neonates remained well. beta-Specific IgG concentrations from these 2 groups were similar. To investigate differences in beta-specific antibody in animals and humans, protein fragments were generated that corresponded to major regions within the beta C protein. A single major region was predominantly recognized in human and rabbit serum samples. Thus, in contrast to immunized animals, no relationship was seen between levels of naturally acquired human beta-specific IgG and protection from neonatal disease. This difference was not explained by a major difference in epitope specificity.     

Functional activities of monoclonal antibodies to the O side chain of Escherichia coli lipopolysaccharides in vitro and in vivo

Mouse hybridoma antibodies (IgG and IgM) to O side chain determinants of Escherichia coli strain Bort (O18ac:K1:H7) were evaluated for their in vitro and in vivo activities against E. coli strains. Both IgG and IgM were opsonic in vitro and protected newborn rats challenged with a K1 E. coli strain, but their activities were strain specific. The antibodies protected against a K1 strain possessing a homologous O serotype but not against one possessing a heterologous O serotype. These antibodies were not effective against the K5-encapsulated O18 E. coli strain (possessing a homologous O type) but protected against its unencapsulated derivative. The opsonic and protective activities of these antibodies were significantly greater with IgG than IgM. Both IgG and IgM, however, required complement for their activities. When IgM to lipopolysaccharide was given to newborn rats in conjunction with IgM monoclonal antibody to the group B meningococcal polysaccharide, the protective effect was significantly greater than that of either antibody alone. Combinations of two (or more) antibodies to different cell wall components may be more beneficial in preventing and treating E. coli infection.     

Value of genetic studies and immunologic markers in diabetes. Prediction of insulin dependence

A new classification based on physiopathological criteria distinguishes Type I diabetes, observed in patients with stigmata of anti-islet of Langerhans auto-immunity, from Type II diabetes without these autoimmune changes. Type I diabetes is sub-divided into Classes Ia and Ib, Class Ib comprising those cases associated with other auto-immune diseases. Serological analysis of 76 patients with clinical type Ia diabetes and 215 healthy, first degree relatives showed that the distinction between Classes Ia and Ib was not clear-cut and that patients classified clinically Ia were in fact infraclinical Ib subjects. Forty-one per cent of patients with Class Ia diabetes had anti-gastric, anti-adrenal or anti-thyroid antibodies, and 28 p. 100 of their healthy relatives also had the same types of antibodies. Several prospective studies of the families of patients with Type I diabetes have shown that anti-islet of Langerhans antibodies were associated with a high risk of diabetes. Similarly, the presence of these antibodies in patients apparently with Type II diabetes (non-insulin dependent) was associated with an increased risk of developing insulin dependence. These results illustrate the value of immunological investigations in diabetic patients. They may influence the choice of treatment in the future.     

Cross-reactivity of rabbit antibodies to lipopolysaccharides of Escherichia coli J5 and other gram-negative bacteria

Antiserum to rough gram-negative mutants such as Escherichia coli J5 and Salmonella minnesota Re595 is thought to neutralize the toxic effects of lipopolysaccharides (LPSs). To verify that such antisera are capable of binding heterologous endotoxins, we examined IgG and IgM class antibodies induced in rabbits to a variety of LPSs. Immunization with rough mutants or lipid A induced high IgG antibody responses to the homologous purified LPS and relatively low but significant responses to heterologous LPSs. Increases in IgM antibodies were also primarily to homologous LPS. Immunization with smooth organisms induced little or no antibody to heterologous LPSs. Soluble LPS, outer membrane vesicles, and whole bacteria produced strong homologous inhibition but little or no heterologous inhibition in enzyme-linked immunosorbent assays. Cross-adsorption of antisera to rough mutants suggested that the IgG and IgM antibodies induced to heterologous LPS were adsorbed by the heterologous LPS and not by the core LPS used to immunize the animals. Rabbit antibody directed to J5 or Re595 LPS fails to bind to any substantial degree to heterologous LPS. Immunization with whole bacterial vaccines, particularly the rough mutants and lipid A, does increase antibody to a wide variety of antigens. The possibilities that the protective effects of antisera to rough mutants are due to a polyclonal antibody response or to the induction of as yet unidentified factor(s) deserve further investigation.     

Serotypes VI and VIII predominate among group B streptococci isolated from pregnant Japanese women

Infection by group B streptococcus (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Whereas serotypes Ia, Ib, II, III, and V are most commonly associated with colonization and disease in the United States, strains of other serotypes have been isolated from patients in Japan. By use of an inhibition ELISA, the serotypes of 73 vaginal colonizing GBS strains isolated from healthy pregnant Japanese women were investigated. Twenty-six (35.6%) were type VIII, 18 (24.7%) were type VI, and the remaining 29 were distributed among more traditional serotypes. Strains were also tested by immunoblot for the presence of GBS surface proteins. Fifty-three (72.6%) of the 73 strains expressed one or more laddering GBS proteins. These data show that type VI and VIII GBS strains are common vaginal isolates in pregnant Japanese women and that one or more laddering proteins are present in most GBS strains.     

Quantitation of IgG antibodies to type Ia group B streptococcal type-specific polysaccharides measured by enzyme-linked immunosorbent assay

The type-specific polysaccharide antigen of the group B streptococcus (GBS) type Ia as extracted and purified according to the procedures of Kane and Karakawa. Using this purified polysaccharide antigen, we made a sensitive and specific assay system of enzyme-linked immunosorbent assay (ELISA) and measured the titres of of the type-specific antibodies in maternal sera and cord blood sera. The titres of antibodies in 78 pregnant women (26 Ia carriers, 18 other types of GBS carriers and 34 non carriers) were compared. A mother of an infant affected by early onset infection of GBS Ia had a titre of antibody 1:10 at delivery, while 2 years later she became a non carrier and had a titre of antibody over 1:160. The titres of antibodies in 27 pair sera of mothers and cords were well correlated.     

A novel method for simultaneous analysis of specific platelet antibodies: SASPA

Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.     

Bacterial evasion of the antibody response: human IgG antibodies neutralize soluble but not bacteria-associated group B streptococcal C5a-ase.

Most strains of group B streptococci (GBS) possess an enzyme that inactivates the human anaphylatoxin C5a by cleaving a heptapeptide from the carboxyl terminus of C5a. This enzyme, called GBS C5a-ase, has been purified to homogeneity and cleaves and inactivates C5a in physiologic buffer. The enzymatic activity of soluble C5a-ase is completely inhibited, however, in the presence of plasma or serum from normal human adults. The neutralization of soluble C5a-ase by plasma and serum results largely from naturally occurring IgG antibodies directed against C5a-ase. IgG does not neutralize C5a-ase present on intact encapsulated type III GBS but does neutralize the C5a-ase activity associated with a transposon-induced mutant strain of type III GBS that lacks capsule. The location of GBS C5a-ase on the surface of encapsulated type III GBS permits the C5a-ase to inactivate C5a while evading neutralization by IgG antibodies.