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.     

Extracellular antigens of serotype III group B streptococci.

Two sialic acid-containing type III group B streptococcal antigens were obtained from a supernatant growth medium, purified by anion exchange or gel filtration, and found to be free of group B reactivity. Quantitation of the high-molecular-weight extracellular type III antigen indicated that approximately 20-fold more antigen was recoverable from the growth medium than could be obtained by neutral buffer extraction of whole cells.     

Bacterial genetics and human immunity to group B streptococci

Serotype III group B Streptococcus agalactiae (GBS) are the most common cause of neonatal sepsis and meningitis. We have classified type III GBS by restriction digest patterns of chromosomal DNA and demonstrated that a subgroup of genetically related strains (RDP type III-3) causes the majority of type III GBS neonatal infection. Genetic differences between type III GBS strains contribute significantly to differences in virulence and host immune responses. While 100% of less virulent RDP type III-1 and III-2 organisms express C5a-ase, an inhibitor of neutrophil chemotaxis, only 63% of virulent RDP type III-3 isolates have functional C5a-ase. Functional differences in type III GBS C5a-ase are attributable to a shared genetic polymorphism, supporting our genetic classification. The mean capsular sialic acid content of virulent RDP type III-3 strains is significantly higher than that of less virulent strains, suggesting that capsular sialylation is also genetically regulated. C5a-ase is not critical for all RDP type III-3 strains to be invasive because the higher capsular sialic acid content of III-3 strains limits complement activation. The identification of these and additional genetic differences between GBS strains has important implications for our understanding of the pathogenesis of these important human infections.     

Characterization of a novel leucine-rich repeat protein antigen from group B streptococci that elicits protective immunity

Group B streptococci (GBS) usually behave as commensal organisms that asymptomatically colonize the gastrointestinal and urogenital tracts of adults. However, GBS are also pathogens and the leading bacterial cause of life-threatening invasive disease in neonates. While the events leading to transmission and disease in neonates remain unclear, GBS carriage and level of colonization in the mother have been shown to be significant risk factors associated with invasive infection. Surface antigens represent ideal vaccine targets for eliciting antibodies that can act as opsonins and/or inhibit colonization and invasion. Using a genetic screen for exported proteins in GBS, we identified a gene, designated lrrG, that encodes a novel LPXTG anchored surface antigen containing leucine-rich repeat (LRR) motifs found in bacterial invasins and other members of the LRR protein family. Southern blotting showed that lrrG was present in all GBS strains tested, representing the nine serotypes, and revealed the presence of an lrrG homologue in Streptococcus pyogenes. Recombinant LrrG protein was shown in vitro to adhere to epithelial cells in a dose-dependent manner, suggesting that it may function as an adhesion factor in GBS. More importantly, immunization with recombinant LrrG elicited a strong immunoglobulin G response in CBA/ca mice and protected against lethal challenge with virulent GBS. The data presented in this report suggest that this conserved protein is a highly promising candidate antigen for use in a GBS vaccine.     

Variation in repeat number within the alpha C protein of group B streptococci alters antigenicity and protective epitopes.

Variable expression of repeating units of the protective alpha C proteins among clinical isolates of group B streptococci (GBS) may have implications for vaccine development. In this study, alpha C protein genes containing various numbers of repeats (1,2,9, and 16) were cloned in a T7 overexpression vector in Escherichia coli. Expression was induced by isopropyl-beta-D-thiogalactopyranoside, and proteins were purified by anion-exchange, gel filtration, or affinity chromatography or by isoelectric focusing. Rabbits were immunized with purified 1-,2-,9-, or 16-repeat proteins. All proteins appeared to be highly immunogenic. Enzyme-linked immunosorbent assay inhibition with 9-repeat protein as the coating antigen and 9-repeat-antigen-elicited antiserum showed that a 200-fold-higher concentration of 1-repeat antigen than of 9- or 16-repeat antigen was required for 50% inhibition of antibody-antigen binding. The concentration of 2-repeat antigen required for 50% inhibition was intermediate relative to the concentrations of 1- and 9-repeat antigens. These results suggested that antibodies to 9-repeat antigen recognized predominantly a conformational epitope(s) contained in proteins with higher numbers of repeats (9 or 16) but lost considerable binding affinities for an epitope(s) contained in alpha C proteins with fewer repeats (1 or 2). Similar results were obtained with antiserum to 16-repeat antigen. However, antibodies to 1- and 2-repeat antigens recognized 1-,2-,9-,and 16-repeat antigens with equal binding affinities. This finding suggested that 1- and 2-repeat-elicited antibodies recognized an epitope(s) on individual repeats. Loss of repeating units from the alpha C proteins may result in decreased protection because the loss of epitopes (including conformational epitopes) gives the microorganisms the opportunity to escape host antibodies. If 1- and 2-repeat-elicited antibodies bind all alpha C proteins with equal affinity, regardless of their repeat number, they may prevent GBS strains with fewer repeats from escaping host immunity. Protection data obtained with antisera to the proteins with different repeat numbers support this hypothesis: mouse pups challenged with GBS strain A909 were better protected when immunized with 1- or 2-repeat-elicited antiserum (76 and 75%, respectively) than when immunized with 9- or 16-repeat-elicited antiserum (41 and 48%, respectively).     

Factors influencing release of type III antigens by group B streptococci.

The release of serotype III group B streptococcal polysaccharides into the supernatant fluid was examined under a variety of physiological conditions. Release of both high- and low-molecular-weight type III antigens was fairly constant throughout exponential growth, but increased markedly upon entering the stationary phase of growth. Increased glucose and decreased phosphate concentrations both caused a large increase in release of antigens. Inhibition of protein synthesis in exponentially growing cells by chloramphenicol (10 micrograms/ml) caused a condition of unbalanced growth in which antigen release was increased greatly over control values. Strain variability in antigen release was also observed. Strains which are known to be high neuraminidase producers released elevated levels of both low- and high-molecular-weight type III antigens. Non-neuraminidase-producing strains released considerably less high-molecular-weight antigen, but similar levels of the low-molecular-weight antigen compared with the high neuraminidase producers. Strain D136C, a type III non-neuraminidase producer, released negligible quantities of the high-molecular-weight antigen in the supernatant fluid. These results indicate that both the physiological environment and the type III strain are important in determining the quantity of type-specific antigen released into the culture fluid.     

Properties of group B streptococci with protein surface antigens X and R.

A total of 128 bovine and 134 human group B streptococci were serotyped by conventional methods. Among the bovine cultures, 60 (47%) had type antigen X, and among the human cultures, 53 (39%) had type antigen R. The occurrence of type antigens X and R was significantly related to the growth pattern of the bacteria in fluid media. Type X- and R-positive cultures and most of the nontypeable cultures predominantly formed long chains and grew as granular sediment with clear supernatant. In addition, group B streptococci with surface antigen X or R showed compact colony formation in soft agar and reacted positively in the salt aggregation test. These properties, possibly caused by the surface charges of the X- and R-positive cultures, might be related to bacterial pathogenicity.     

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.     

Colloidal gold immunolabeling of immunoglobulin-binding sites and beta antigen in group B streptococci.

We have characterized the immunoglobulin A (IgA)-Fc-binding properties and beta-antigen expression of several strains of group B streptococci by using ultrastructural immunocytochemistry. Colloidal gold-labeled tracers were used with intact and sectioned bacteria in order to gain information regarding the location and distribution of cell surface and cytoplasmic IgA-Fc-binding molecules and beta antigen. Colloidal gold (5- or 15-nm particles) was conjugated to IgA to characterize IgA-binding properties and to IgG to test for IgG binding. Rabbit anti-beta antiserum was reacted with the bacteria and then with protein G labeled with 15-nm colloidal gold particles. A double-labeling technique was used for simultaneous localization of IgA-Fc- and anti-beta-antibody-binding properties on sectioned bacteria. The data corroborated previous results which indicated that (i) IgA-Fc-binding and IgA-Fc-nonbinding forms of beta antigen can be secreted by strains which do not express beta antigen on the cell surfaces (HG806, VC75); (ii) differences in levels of expression of beta antigen and/or IgA-Fc-binding proteins can be detected among various group B isolates; (iii) group B streptococci do not express human IgG-Fc-binding proteins; and (iv) not all forms of beta antigen are capable of binding human IgA.     

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.     

Immunogenicity and protective efficacy of the alpha C protein of group B streptococci are inversely related to the number of repeats.

Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates. Alpha C protein is a protective cell surface-associated protein of GBS. This protein contains a repeat region flanked by N and C termini. Variable expression of tandem repeating units of alpha C proteins had been found among clinical isolates of GBS. We examined the effect of the number of repeats on the immunogenicity of the alpha C protein and its ability to elicit protection from GBS infection in a neonatal mouse model. Mice were immunized with purified alpha C proteins of constructs containing various numbers of repeats (n = 1, 2, 9, and 16) and the N- and C-terminal regions. Both the N-terminal and the repeat regions contain protective and opsonic epitopes. Antibody responses to the alpha C protein constructs with various numbers of repeats were tested with enzyme-linked immunosorbent assay plates coated with either native, nine-repeat alpha C protein or "repeatless" N-terminal antigen. An inverse relationship was found between the number of repeats and the immunogenicity of the alpha C protein; this effect was most pronounced on titers of antibody to the N-terminal region. An inverse relationship was also observed between the number of repeats and protective efficacy, i.e., mouse dams immunized with 5 microg of one- or nine-repeat alpha C protein transferred protective immunity to 65 or 11% of their pups, respectively (P < 0.0001). Thus, the presence of multiple repeats appears to lessen the antibody response to the complete alpha C protein, and especially the antibody response to its N-terminal region, and suggests a mechanism whereby repeat elements contribute to the evasion of host immunity.     

Molecular species of R-protein antigens produced by clinical isolates of group B streptococci.

Clinical isolates of group B streptococci from body fluids and mucosal surfaces were examined for production of a trypsin-resistant antigen known as R protein. R protein was extracted with 1% trypsin from cells grown in a semidefined medium. The extracts were tested by immunodiffusion in agarose with a panel of antisera for detection and precise identification of the four species of R protein described by Wilkinson. R antigen was present in 49 of 131 (37%) of the strains tested. Analysis by serotype revealed that 0 of 2 type Ia, 0 of 11 Ib, 1 of 16 (6%) Ia/c, 12 of 15 (80%) II, 0 of 20 II/c, 35 of 49 (71%) III, 0 of 6 IV, and 1 of 12 (8%) nontypeable strains produced R antigen. Production of the R protein and the trypsin-resistant or alpha component of the c protein appeared to be mutually exclusive. R antigen was more prevalent in isolates from blood (50%) than in those from mucosal sites (27%) for type II strains; no difference was seen for type III strains from these sites. Concordant results were obtained with five paired body fluid-mucosal surface isolates from individual patients and with isolates from 17 mother-baby pairs. The most frequent species of R antigen was R4 (45 of 49), followed by R1 (4 of 49). These two species of R protein were biochemically (trypsin resistant and pepsin sensitive) and immunologically identical to the R-protein antigens produced by prototype strains of groups A, B, and C streptococci.     

Soluble antigens from group B streptococci induce cytokine production in human blood cultures.

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.     

Faecal carriage of group B streptococci.

Consecutive stool samples from 116 female and 98 male patients (both adults and children), and rectal and vaginal swabs from 28 and 53 cases respectively, were quantitatively cultured for group B streptococci using Islam's medium. Group B streptococcus was recovered from 5% and 2% of faeces in female and male patients respectively, and the colony counts ranged from 10(2) to 10(3)/g. In women, the faecal carriage rate was 6%, which was significantly lower than the rectal carriage rate (p 0.02), suggesting that the higher recovery rate (27%) from rectal specimens may be due to contamination of swabs by perianal skin flora. Type II group B streptococcus was the only faecal isolate in adults (numbers involved are small for statistical significance), and we suspect that this type strain may be the only resident gut flora in adults, and the gastrointestinal tract is unlikely to serve as the main reservoir of all group B streptococci.     

Receptors for fibrinogen and aggregated beta 2-microglobulin detected in strains of group B streptococci.

Binding of radiolabeled human fibrinogen and aggregated beta-microglobulin was measured in 60 strains of beta-hemolytic group B streptococci. Positive fibrinogen binding was detected in seven of the strains. Six of the group B strains showed an uptake of aggregated beta 2-microglobulin. Four individual strains carried both receptors, indicating a positive correlation between their occurrence. Inhibition studies showed that fibrinogen competed sterically with beta 2-microglobulin binding. Receptors for both proteins were trypsin sensitive. The presence of receptors did not correlate with the serological type of the 49 group B strains tested. However, all seven type II strains were negative. No uptake of fibrinogen was noted in any of 40 group D strains tested. Binding structures for fibrinogen and aggregated beta 2-microglobulin detected in group B streptococci were similar to receptors for the same proteins in group A, C, and G streptococci in terms of mutual correlation and steric interference of binding. The occasional occurrence of these receptors also in group B strains might reflect a common origin of some types of surface proteins in gram-positive cocci.     

Common (non-type-specific) antigens of group A streptococci.

Two common (non-type-specific) antigens in HCl extracts prepared from group A streptococcal cultures of different types were analyzed by immunodiffusion methods. These antigens are sensitive to trypsin and are apparently related to the cell wall proteins. The common antigens tested belong to a category of nonprotective non-type-specific antigens. The presence of common antigens in the HCl extracts should be taken into consideration when M-proteins are determined. The detection of one of the common antigens may be used as a virulence index, since this antigen is characteristic of the group A streptococcal cultures with enhanced virulence.     

Demonstration of the capsular antigens of bovine group B streptococci by the serum-soft agar method.

The elaboration of type-specific capsular antigens by group B streptococci can be demonstrated by the serum-soft agar technique. Group B streptococci isolated from bovine mastitis, namely, strains 9F, 14Mi, 8Mo, 44B, and 4S, were shown to form diffuse and compact types of colony morphology in serum-soft agar. Immunochemical and chemical analyses of antigens isolated from diffuse and compact colonies of strain 9F indicated that the diffuse-type growth of this strain was due to the elaboration of a galactose-rich surface antigen, whereas the compact 9F strain was devoid of this antigen. Specific 9F antiserum was effective in converting the diffuse 9F colonies of the compact type, indicating the presence of capsular material. Preliminary evidence suggests that the serum-soft agar technique could also be used to determine the antigenic diversity of the surface antigens of group B streptococci, thus providing an effective means of typing those organisms.     

Cell-associated collagenolytic activity by group B streptococci.

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.