Estimation of group B streptococcus type III polysaccharide-specific antibody concentrations in human sera is antigen dependent.

The presence of immunoglobulin G (IgG) antibodies against group B streptococcus (GBS) type III polysaccharide (PS) has been correlated with protection against GBS disease. The GBS type III PS is structurally similar to the pneumococcal type 14 PS, differing only in the presence of sialic acid residues. Four different preparations of GBS type III PS were evaluated for their specificity in enzyme-linked immunosorbent assay (ELISA): free PS, free PS mixed with methylated human serum albumin (mHSA), PS conjugated to biotin and PS conjugated to human serum albumin. Three groups of human sera were used to evaluate these PS preparations: sera from recipients of a GBS PS vaccine, sera from women receiving a GBS type III PS-tetanus toxoid conjugate vaccine, and sera from nonimmunized healthy women of childbearing age. Estimated antibody concentrations were different depending on the PS preparation used. Using any of the four preparations, we were able to measure

Relation between maternal age and serum concentration of IgG antibody to type III group B streptococci.

Coded serum samples collected from healthy obstetric patients at delivery were examined by ELISA for IgG antibody to the purified type III polysaccharide of group B streptococci. When 217 patients were divided into 4 groups according to age (group I =16-20 years, n = 56; group II = 21-25, n = 53; group III = 26-30, n = 54; group IV = 31-35, n = 54), antibody concentrations were significantly lower in group I than in older patients. Fewer subjects in group I had measurable antibody levels (> or = 0.05 microgram/mL) than in groups II-IV (41% vs. 76%, P < .001). The geometric mean in group I (0.09 microgram/mL) was significantly lower (P < .001) than in the older groups (0.23, 0.19, and 0.20 microgram/mL, respectively) with little or no overlap of the 95% confidence limits (1.96 SE) about the means. These findings may be relevant to the observation of a significantly greater risk of both early- and late-onset group B streptococcal disease in infants of teenage mothers.

Solid-phase antigen density and avidity of antibodies detected in anti-group B streptococcal type III IgG enzyme immunoassays.

Two enzyme immunoassays which measure anti-group B streptococcal type III capsular carbohydrate IgG antibodies were compared. One utilised poly-L-lysine conjugated coating antigen while the other used tyraminated coating antigen. Both carbohydrate antigens appeared to be antigenically identical but the poly-L-lysine based assay gave significantly lower values for some sera. Sera were identified which had low and high avidity anti-group B streptococcal type III IgG antibodies by the thiocyanate elution method. These antibodies gave results on a dilution range of coating concentrations consistent with their relative avidity. Comparison of dilution ranges of the two conjugates used for coating suggests that the poly-L-lysine conjugate coats with a ten-fold lower efficiency than the tyramine conjugate and therefore detects only higher avidity antibodies. Four fractions containing different relative avidities of affinity-purified IgG were produced from a single serum. These fractions behaved in the same manner as sera containing antibodies of different avidities. The results of this study suggest that the method of polysaccharide conjugation in enzyme immunoassays may affect the antigen concentration on the solid phase and thence the detection of antibodies of various avidities.

Comparison of group B streptococcal hyperimmune globulin and standard intravenously administered immune globulin in neonates.

Standard intravenously administered immune globulin (IVIG) contains varying amounts of group B streptococcus (GBS) antibody. A GBS hyperimmune IVIG was produced by immunizing plasma donors. The GBS type-specific opsonic activity was > or = 90% in the hyperimmune IVIG at a 1280 dilution-1 versus at a 10 dilution-1 in standard IVIG. Suckling rat survival after GBS type-specific infection was 100% when the rats were treated with hyperimmune IVIG versus < or = 20% with standard IVIG. To evaluate the effect of this product on GBS antibody levels and clinical toxic effects, we randomly administered either GBS hyperimmune IVIG, 500, 250, or 100 mg/kg, or standard IVIG, 500 mg/kg, to 20 neonates with suspected sepsis. No adverse effects were observed. Total and subclass serum IgG levels reflected only the dose; serum GBS type-specific IgG and opsonic activity reflected both the product and dose of IVIG administered. Standard IVIG did not significantly increase serum GBS type-specific IgG, whereas hyperimmune IVIG, 500 mg/kg, produced a fourfold rise for > 6 weeks; more variable increases were observed after 250 and 100 mg/kg doses were given. Serum GBS type-specific opsonic activity correlated with serum GBS type-specific IgG levels (R2 = 0.74; p < 0.0001). Further studies of this or similar products will be necessary to determine whether GBS type-specific antibody improves the outcome of GBS-infected neonates.

Intravenous immune globulin therapy for early-onset sepsis in premature neonates.

Newborn infants may have IgG deficiencies that increase their susceptibility to bacterial infection. To determine whether intravenous immune globulin (IVIG) therapy improves survival rates in early-onset sepsis, we prospectively entered 753 neonates (birth weight 500 to 2000 gm, gestation less than or equal to 34 weeks, age less than or equal to 12 hours) into a multicenter, double-blind, controlled trial. Blood culture specimens were obtained and infants randomly assigned to receive 10 ml (per kilogram) intravenously of a selected IVIG (500 mg/kg) or albumin (5 mg/kg) preparation. Maternal and neonatal risk factors were not different between groups. Thirty-one babies (4.2%) had early-onset sepsis; the causative organisms were group B streptococcus (12 babies), Escherichia coli (6), and others (13). Of these 31 neonates, 7 (23%) died. Total serum IgG was higher for 7 days after IVIG therapy than after albumin treatment (p less than 0.05). During these 7 days, 5 (30%) of 17 albumin-treated and none of 14 IVIG-treated patients died (p less than 0.05). The survival rate at 56 days of age, however, was not significantly improved. Group B streptococcus type-specific IgG antibody was significantly increased after IVIG treatment and appeared to be related to the amount of IVIG specific antibody. Infusion-related adverse reactions were less frequent in patients receiving IVIG therapy (0.5%) than in those receiving albumin. The IVIG therapy in neonates with early-onset sepsis, while reducing the early mortality rate, did not significantly affect the overall survival rate. Further studies are necessary to confirm these findings and to determine more effective therapeutic regimens.

Opsonic effect of jacalin and human immunoglobulin A on type II group B streptococci.

This study examined the effect of immunoglobulin A (IgA) and the IgA-binding lectin jacalin on the phagocytosis of type II group B streptococci (GBS). Strains possessing the trypsin-sensitive and trypsin-resistant components of the c protein (II/c) and type II GBS lacking the c protein (II) were examined by radiolabeled bacterial uptake, bactericidal assays, and electron microscopy. Type II/c GBS resisted phagocytosis by monocytes (4.9% +/- 0.8% uptake, mean +/- SE, n = 25) compared with type II GBS (8.5% +/- 1.4% uptake, n = 14, P = 0.03). Phagocytic killing by polymorphonuclear leukocytes was also less for the type II/c strain 78-471 than for the type II strain 79-176 (68% +/- 5% versus 86% +/- 4% reduction in CFU at 45 min, P = 0.03). IgA binding did not explain the resistance of type II/c GBS to phagocytosis. The uptake of type II/c GBS was not significantly different after opsonization in cord sera lacking endogenous IgA (5.93% +/- 1.4%) than in the same cord sera after addition of exogenous IgA (5.48% +/- 1.4%, P = 0.69, n = 9). Attempts to remove serum IgA with the IgA-binding lectin jacalin resulted in the binding of IgA-jacalin complexes to II/c GBS. This combination of nonspecific IgA and jacalin increased uptake of II/c GBS from 4.9% +/- 0.8% to 11.8% +/- 1.9% (P = 0.002). Jacalin also combined with specific, immune, monoclonal IgA bound to the surface of Haemophilus influenzae and promoted the uptake of these bacteria. Jacalin and IgA mediated phagocytosis of II/c GBS via receptors that were not dependent on divalent cations and that were not modulated by plating monocytes on antigen-antibody complexes.

Nonimmune binding of human immunoglobulin A to type II group B streptococci.

The binding of 125I-labeled human myeloma immunoglobulin A (IgA) to four type II strains and one nontypable strain of group B streptococci was measured after streptococcal chains were broken by brief sonication. Some IgA binding was observed with all strains, but specific binding (binding that was inhibited by excess unlabeled IgA, was dose dependent, and was saturable) occurred only with those strains possessing the trypsin-sensitive beta component of the c protein. Similar amounts of binding were observed with myeloma IgA and IgA1 purified from normal serum. Specific binding was more rapid at 25 degrees C than at 0 or 37 degrees C and reached a plateau in 6 to 8 h. Binding was drastically reduced (85 to 90%) when streptococci had been heated (90 degrees C for 1 h). Most radioactivity bound to group B streptococci could be displaced (greater than 60% in 3 days) by the addition of excess unlabeled IgA. The binding capacity of one strain (10(8) streptococci in 1 ml of buffer) was saturated by approximately 24 micrograms of IgA. When transformed for Scatchard analysis, these data indicated that there was a specific binding capacity of 16,000 molecules of monomeric serum IgA per single streptococcal cell. The dissociation constant for IgA binding was 19.3 nM. Since enzyme-linked immunosorbent assay studies showed that the myeloma IgA used for the studies described above was IgA1, our quantitative data apply only to the binding of this subclass to group B streptococci. However, an enzyme-linked immunosorbent-filtration assay showed that both IgA1 and IgA2 bound to a type II group B streptococcus bearing the c protein.

Opsonic activity of human IgG and IgM antibody for type III group B streptococci.

We separated IgG and IgM from 10 adult sera containing measurable (greater than or equal to 0.05 micrograms/mL) antibody of both isotypes to the type III polysaccharide of group B streptococci. Ig preparations were used to preopsonize 3H-labeled type III group B streptococci which were added to adherent monolayers of human, monocyte-derived macrophages. Reproducibly significant phagocytosis, measurable by accumulation of radioactivity and confirmed by electron microscopy, required both antibody (from adult serum or Ig preparation) and complement (from cord serum deficient in specific antibody). All adult IgG and IgM preparations were opsonic in the presence of complement. When a cord serum containing IgG but no IgM antibody was separated by the same procedure, the IgG fraction was opsonic but the fraction that would contain IgM was not. The opsonic activity of IgM in adult sera persisted after the removal of contaminating IgG4. These observations indicate that both IgG and IgM antibody in adult sera are opsonic for type III GBS and support the hypothesis that IgM deficiency in newborns may be a contributing factor to certain bacterial infections.

Demonstration of opsonic and protective activity of human cord sera against type III group B streptococcus that are independent of type-specific antibody.

In an effort to further understand the host defense against group B streptococcus (GBS), we examined 71 human cord sera for their content of type III GBS IgG antibody by enzyme-linked immunosorbent assay and correlated the results with opsonic and protective activity against type III GBS. Most cord sera (67%) containing greater than 0.1 microgram/ml of type III GBS IgG antibody promoted phagocytosis and killing in vitro and protection against type III GBS in neonatal rats. However, 26% of cord sera containing less than 0.1 microgram/ml of type III IgG antibody exhibited similar activity in vitro and in vivo against type III GBS. This opsonic and protective activity was retained in IgG fraction of whole serum, and was not directly associated with complement activity or with fibronectin. Further studies are needed to understand the mechanisms responsible for the opsonic and protective activity of some cord sera against type III GBS that may be independent of antibody to the type-specific polysaccharide antigen.

Gram-positive bacteria: an overview and summary of session.

The more pathogenic gram-positive bacteria present a complex array of surface structures to the human or animal host. The cell wall of Staphylococcus aureus has a pattern of surface proteins; the predominant one is protein A. Virulent S. aureus strains may also produce polysaccharide capsules in vivo that impede opsonization and phagocytosis in the absence of anticapsular antibody. Coagulase-negative staphylococci commonly elaborate an exopolysaccharide slime that may promote adherence to plastic surfaces and interfere with host responses. Structure-function relationships for some antiphagocytic M proteins of group A streptococci are now well understood, and recombinant techniques offer the prospect of multivalent vaccines. The best known surface protein of group B streptococci is the c (Ibc) protein, which stimulates protective antibody in animals and may be an important virulence factor. Monoclonal antibodies to types Ib, II, and III group B streptococci have also confirmed the presence of multiple immunodeterminants on these antiphagocytic polysaccharides. A protein on the surface of pneumococci has been shown to induce protective antibody and to enhance pneumococcal virulence in mice, suggesting a potential alternative or adjunct to pneumococcal polysaccharide vaccines. Listeria also possess a variety of cell surface structures important in pathogenesis. Surface components are, therefore, critical determinants of the interaction of gram-positive bacteria with the host.

Relative functional activity of purified human immunoglobulin G against a type III group B streptococcal strain.

Human immunoglobulin G (IgG) separated from whole serum by a quaternary aminoethyl-Sephadex A-50 ion exchanger was evaluated for its activity against a type III group B streptococcal strain in the newborn rat model. Separated IgG yielded approximately 70 to 80% of whole serum IgG and did not contain detectable IgM or IgA. This IgG preparation also contained similar ratios of specific type III group B streptococcal antibody to total IgG in comparison with whole serum. In vivo, 50% protection from death was achieved by 3.9 ng of type III-specific antibody per 10 g of rat body weight. This value was considerably lower than 50% protective doses obtained in our previous studies with different human IgG preparations. Further studies are needed to understand the mechanisms responsible for these differences in the functional activity of IgG antibody.

Functional activities of various preparations of human intravenous immunoglobulin against type III group B Streptococcus.

Two preparations of human immunoglobulin modified for intravenous use (iv immunoglobulin) by different methods (reduction-and-alkylation, or pH 4 treatment) were evaluated for in vitro and in vivo activity against a strain of type III group B Streptococcus (GBS). Both preparations contained similar amounts of total IgG and specific IgG antibody against the type-specific polysaccharide. In vitro, opsonophagocytic studies revealed that pH 4-treated iv immunoglobulin was significantly more effective than reduced-and-alkylated iv immunoglobulin in supporting neutrophil-mediated killing of the type III GBS strain. In vivo, both preparations resulted in similar levels of serum antibody in newborn rats, but pH 4-treated iv immunoglobulin was significantly more protective against the type III GBS strain. This was demonstrated by the lower magnitude of bacteremia, improved survival, and lower protective dose (PD50) in recipients of pH 4-treated iv immunoglobulin. Thus, reduced-and-alkylated iv immunoglobulin was less effective in vitro and in vivo against the strain of type III GBS than was pH 4-treated iv immunoglobulin. Further studies are needed to elucidate the mechanisms for this apparent discrepancy in functional activities of iv immunoglobulin.

The role of specific antibody in neonatal bacterial infections: an overview.

Neonatal infections caused by GBS are commonly associated with a deficiency of IgG antibody to the type-specific polysaccharide of the infecting organism. The prevalence and importance of human antibody to specific surface proteins, which are very common in clinical GBS strains, are unknown. Type-specific antibody is sufficiently prevalent that immunoglobulin preparations for both intramuscular and intravenous use contain animal-protective, opsonic antibody for many GBS strains. The availability of polysaccharide vaccines suggest that even more potent, "hyperimmune" preparations can be prepared from immunized volunteers. The importance of selective antibody deficiency in K1 E. coli infections of newborns is less clear. Protective, opsonic antibody is more difficult to demonstrate in human serum, but activity has been shown in some IGIV preparations. The specificity of the active antibody and the feasibility of producing hyperimmune IGIV against K1 E. coli are unknown at this time.

Human antibody to the group-specific polysaccharide of group B Streptococcus.

IgG antibody to purified group polysaccharide of group B Streptococcus was detected with a specific, reproducible enzyme-linked immunosorbent assay in sera of 178 human subjects: 106 parturient patients, 67 of their healthy infants, and 5 adults with invasive infection. Antibody concentrations in 44 parturient carriers of group B streptococci were significantly greater than in 44 noncarriers (geometric mean level, 3.5 and 1.2 micrograms/ml, respectively). Cord serum levels agreed closely with maternal serum levels. The sera of 18 mothers of infants with early-onset streptococcal infection contained levels (geometric mean level, 5.5 micrograms/ml) similar to those of carriers and significantly higher than noncarriers. Of five adults with invasive group B streptococcal infection, three demonstrated significant increases in antibody titer in consecutive sera, and four had antibody concentrations greater than most (93%) noncarriers. These findings suggest that group B polysaccharide is immunogenic in humans and that levels of specific IgG antibody increase with colonization or infection of adults with group B streptococci.