Immunological Markers of the R4 Protein of Streptococcus agalactiae

This study focuses on immunological markers of R4, an important Streptococcus group B (GBS) protein. The results obtained by using rabbit antisera and purified proteins for antigens in enzyme-linked immunosorbent assay-based experiments provided evidence that R4 possesses two antigenic determinants. One of the determinants is shared with the alpha-like protein 3 (Alp3) of GBS, was named R4/Alp3 common, and was expressed by GBS, which possessed the Alp3-encoding gene alp3 or the R4-encoding gene rib. The other antigenic determinant was detected only in rib-positive GBS organisms and was named R4 specific. This determinant probably is an immunological marker unique to the R4 protein. Neither of the antigenic R4 determinants showed serological cross-reactivity with the GBS proteins Cα, Cβ, and R3 or with alpha-like protein 2. Of 60 clinical serotype III GBS strains, 56 (93%) isolates possessed the rib gene and 50 (89%) of the rib-positive isolates expressed levels of R4 detectable by antibody-based tests, consistent with R4 expression failure or low-level expression in ~10% of rib-positive GBS. alp3 was not detected in type III GBS but was possessed by six of eight type V strains and six of six type VIII strains. All alp3-positive strains were recognized by the R4/Alp3 common antibodies, but none of them were recognized by the R4-specific antibodies. NCTC 9828, a reference strain for R3 and R4, expressed the determinant R4/Alp3 common but not R4 specific. A monoclonal R4 antibody, previously considered to be R4 specific and used in GBS serotyping, targeted R4/Alp3 common and is thus not R4 specific. The results show that failure to discriminate between R4 specific and R4/Alp3 common by antisera designed for GBS serotyping can result in the false identification of Alp3 as R4 or vice versa, whereas anti-R4 antibodies targeting only the determinant R4 specific will detect only R4. Both R4 and Alp3 need further evaluation with respect to the immunobiological function of each distinct antigenic determinant, for instance, with regard to their potential as GBS vaccine components.     

Antigenic determinants of alpha-like proteins of Streptococcus agalactiae

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Calpha) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Calpha. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments.     

Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments.     

Streptococcus agalactiae alpha-like protein 1 possesses both cross-reacting and Alp1-specific epitopes

Most isolates of group B streptococci (GBS) express an alpha-like protein (Alp), Cα (encoded by bca), Alp1 (also called epsilon; alp1), Alp2 (alp2), Alp3 (alp3), Alp4 (alp4), or R4/Rib (rib). These proteins are chimeras with a mosaic structure and with antigenic determinants with variable immunological cross-reactivities between the Alps, including Alp1 and Cα cross-reactivity. This study focused on antigenic domains of Alp1, studied by using rabbit antisera in immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay (ELISA)-based tests and whole cells of GBS or trypsin-extracted and partially purified antigens from the strains A909 (serotype Ia/Cα, Cβ) and 335 (Ia/Alp1). Alp1 and Cα shared an antigenic determinant, Alp1/Cα common, not harbored by other Alps, probably located in the Alp1 and Cα repeat units, as these units are nearly identical in genomic sequence. An antigenic Alp1 determinant was Alp1 specific and was most likely located in the N-terminal unit of Alp1 in which an Alp1-specific primer site for PCR is also located. In addition, Alp1 possessed a domain with low immunogenicity which cross-reacted immunologically with Alp2 and Alp3, with unknown location in Alp1. Alp1 was partially degraded by trypsin during antigen extraction but with the antigenic domains preserved. The results indicate that Cα and Alp1 are immunologically related in the same manner that R4 (Rib) and Alp3 are related. The domain called Alp1 specific should be important in GBS serotyping as a surface-anchored serosubtype marker. The Alp1/Cα common determinant may be of prime interest as an immunogenic domain in a GBS vaccine.     

Survey of Immunological Features of the Alpha-Like Proteins of Streptococcus agalactiae

Nearly all Streptococcus agalactiae (group B streptococcus [GBS]) strains express a protein which belongs to the so-called alpha-like proteins (Alps), of which Cα, Alp1, Alp2, Alp3, Rib, and Alp4 are known to occur in GBS. The Alps are chimeras which form mosaic structures on the GBS surface. Both N- and C-terminal stretches of the Alps possess immunogenic sites of dissimilar immunological specificity. In this review, we have compiled data dealing with the specificity of the N- and C-terminal immunogenic sites of the Alps. The majority of N-terminal sites show protein specificity while the C-terminal sites show broader cross-reactivity. Molecular serotyping has revealed that antibody-based serotyping has often resulted in erroneous Alp identification, due to persistence of cross-reacting antibodies in antisera for serotyping. Retrospectively, this could be expected on the basis of sequence analysis results. Some of the historical R proteins are in fact Alps. The data included in the review may provide a basis for decisions regarding techniques for the preparation of specific antisera for serotyping of GBS, for use in other approaches in GBS research, and for decision making in the context of GBS vaccine developments.     

Phenotypes, genotypes, serotypes and molecular epidemiology of erythromycin-resistant Streptococcus agalactiae in Italy

The purpose of this investigation was to analyse Streptococcus agalactiae (group B Streptococcus, GBS) isolates collected in Italy from vaginal and urine samples in respect to their clonality, distribution of virulence factors and antimicrobial resistance determinants. Three hundred and eighty-eight GBS were recovered from clinical samples. They were analysed for antibiotic resistance profiling. Erythromycin-resistant strains were further characterised by multilocus sequence typing (MLST), serotyping and the detection of alp genes of the alpha-like protein (Alp) family. GBS isolates represented 40 different sequence types (STs), grouped in five clonal complexes (CCs) and belonged to seven serotypes. Most serotype V strains (81%) possessed alp2-3; serotype Ia carried mainly epsilon, while the serotype III mainly rib. All isolates were susceptible to penicillin, whereas resistance to erythromycin was detected in 15% of isolates. Most erythromycin-resistant GBS strains were of serotype V (56.8%) and belonged to the CC-1 group (50%). Macrolide resistance phenotypes were the cMLS(B) (46.5%) and the M phenotypes (46.5%) due to the presence of ermB and mefA/E genes, respectively. These results provide data which establish a baseline for monitoring erythromycin resistance in this region and also provide an insight into the correlation among clonal types, serotypes, surface protein and resistance genes. The increased prevalence of strains that displayed the M phenotype strengthens the importance of the epidemiological surveillance of macrolide resistance in GBS, which may also represent an important reservoir of resistance genes for other species.     

Distinctive features of surface-anchored proteins of Streptococcus agalactiae strains from Zimbabwe revealed by PCR and dot blotting

The distribution of capsular polysaccharide (CPS) types and subtypes (serovariants) among 121 group B streptococcus (GBS) strains from Zimbabwe was examined. PCR was used for the detection of both CPS types and the surface-anchored and strain-variable proteins Calpha, Cbeta, Alp1, Alp2, Alp3, R4/Rib, and Alp4. The R3 protein was detected by an antibody-based method using monoclonal anti-R3 antibody in dot blotting. The CPS types detected, Ia (15.7% of strains), Ib (11.6%), II (8.3%), III (38.8%), V (24.0%), and nontypeable (1.7%), were essentially as expected on the basis of data from Western countries. The type V strains showed distinctive features with respect to protein markers in that Alp3 was detected in only 6.9% of the isolates while R3 occurred in 75.9% and R4/Rib occurred in 37.9% of the isolates. R3 occurred nearly always in combination with one of the alpha-like (Alp) proteins, and it was the third most common of the proteins studied. These results show that type V GBS strains from Zimbabwe differed from type V strains from other geographical areas and also emphasize the importance of the R3 protein in GBS serotyping and its potential importance in the immunobiology of GBS, including a potential role in a future GBS vaccine.     

Characterisation of invasive group B streptococci based on investigation of surface proteins and genes encoding surface proteins

The joint distributions of the six genes bca , bac , ε/alp1 , alp2 , alp3 and rib (encoding α-C-protein, β-C-protein, ε /Alp1, Alp2, Alp3, and Rib, respectively) and the proteins α-C-protein, β-C-protein and Rib were investigated in invasive isolates of group B streptococcus (GBS). In total, 297 invasive isolates (123 from neonates, 174 from adults) from south-west Sweden were collected during a 13-year period. Genes were detected using multiplex and specific PCRs, and expression of the surface proteins was demonstrated using monoclonal antibodies. The genes studied were found alone or in combinations in 294 (99%) of the invasive isolates. The most common genes were rib ( n =  127 isolates, 43%), alp3 ( n  =   78, 26%) and ε/alp1 ( n  =   42, 14%). The bac gene was never found alone, but was found in combination with one other gene in 36 isolates. The surface proteins studied were detected alone or in combinations in 152 (51%) isolates, with the most common being Rib ( n  =   80, 27%), α-C-protein ( n  =   68, 23%) and β-C-protein ( n  =   24, 8%). Several genes were associated significantly with particular serotypes (e.g., ε/alp1 with serotype Ia; bca and bac with serotypes Ib and II; rib with serotype III; alp3 with serotype V). Overall, it was concluded that demonstration of different genes and surface proteins of GBS strains can be useful in epidemiological studies and in formulation of vaccines, but disappointingly, no single gene or surface protein included in the study was sufficiently common for it to be considered as the basis for a successful GBS vaccine.     

Molecular profiles of group B streptococcal surface protein antigen genes: relationship to molecular serotypes

The study of surface protein antigens of group B streptococci (GBS) is important for understanding of the pathogenesis and epidemiology of infection, and several of these antigens have been proposed as components of GBS conjugate vaccines. In a previous study, we developed a novel PCR-and-sequencing system for identification of GBS serotypes and serosubtypes based on the capsular polysaccharide synthesis (cps) gene cluster. In this study, we used published sequences to develop PCR assays for identification of genes encoding GBS surface proteins including C alpha (bca), C alpha-like proteins 2 and 3 (alp2 and alp3), Rib (rib), and C beta (bac). We showed that the prototype R reference strain, Prague 25/60, contained a novel alpha-like protein antigen gene (the proposed alp4), which presumably encodes an atypical, but antigenically similar, R-like protein. Initial evaluation of these gene-specific assays showed excellent specificity. By combining cps serotypes, serosubtypes, and surface protein gene profiles, we were able to divide 224 GBS isolates into 31 serovariants. GBS bac-positive strains could be further subtyped into 11 groups and 20 subgroups. Our results confirmed and extended reported associations between some cps serotypes and serosubtypes, on the one hand, and surface protein genes, on the other: serosubtypes III-1 and III-2 were associated with rib, serosubtype III-3 with alp2, serotype Ib with bca and bac, and serotype V with alp3. The associations between serotype Ia and bca, bca repetitive unit, and bca repetitive unit-like sequence-containing genes need to be studied further. These PCR-based methods will provide an alternative and objective tool for subtyping of GBS based on surface protein antigen genes.     

Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes

We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Calpha), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cbeta). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed that mPCR/RLB avoids the common problems of cross-reaction and nontypability associated with protein typing using antisera. It is as sensitive as, but more practical than, separate gene-specific PCRs and would be suitable for large molecular epidemiological studies of GBS.     

Molecular epidemiology and distribution of serotypes, surface proteins, and antibiotic resistance among group B streptococci in Italy

Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.     

Typing of human isolates of Streptococcus agalactiae (group B streptococcus,GBS) strains from Zimbabwe

Serotyping and genotyping are important tools in epidemiological studies of group B streptococcal (GBS) infections, which are important diseases in man, particularly in newborns. In the present study, 241 GBS isolates from Zimbabwe, comprising 124 carrier isolates from pregnant women and 117 isolates from patients hospitalised for various diseases, were serotyped. Antibodies specific for the capsular polysaccharide antigens (CPAs) Ia, Ib and II-V and antibodies specific for the surface-localised proteins, c(alpha), c(beta), R1, R3 and R4 were used for serotyping. Strains of the CPA types Ia (17%), III (47.7%) and V (23.2%) predominated. Of the various protein antigens, c(alpha) and R4 were expressed with highest frequency, c(alpha) by 100% of the CPA type Ia strains and R4 by 92% of the CPA type III strains. The R3 protein occurred frequently (24%), especially in type V strains (84%). A total of 25 serovariants was detected in the strain collection with the variants Ia/c(alpha) (16%), III/R4 (43.5%) and V/c(alpha), R3 (14.1%) occurring with the highest frequency. Serotype and subtype distribution of the carrier isolates were essentially similar to those of the disease-associated isolates. Genomic heterogeneity was demonstrated by pulsed-field gel electrophoresis of type III/R4 and type V/c(alpha), R3 isolates, but to a much lesser extent than recorded with Norwegian strains. These results demonstrate that many variants of GBS occur in the Zimbabwean population. The data obtained may assist in the formulation of a possible future GBS vaccine for Zimbabwe and perhaps for other African countries.     

Properties and distribution of the putative R3 protein of Streptococcus agalactiae.

Strain-variable Streptococcus agalactiae (group B streptococci; GBS) proteins exposed at the bacterial cell surface are important markers in GBS serotyping. These proteins include the c proteins c(alpha) and c(beta) and the R proteins R1 through R4, of which R1 and R4 have been studied most extensively. This study presents the characteristics of a protein which was expressed by a capsular antigen type V GBS strain shown by means of polyclonal and monoclonal antibody testing. Examination of a number of reference and prototype strains by fluorescent antibody testing and Western blotting provided evidence that the serotype V-derived protein was the R3 protein of GBS, previously defined on the basis of immunoprecipitation assays. The putative R3 protein formed ladder-like banding patterns on Western blotting with polypeptides in the 30 kDa to > or = 140 kDa range, was destroyed by pepsin digestion, and partially degraded by trypsin digestion. The protein was expressed by 10 (6.5%) of 153 clinical GBS strains tested, the expression being restricted to isolates of the capsular antigen types II, III, and V. Some isolates expressed both the c(beta) and the R3 protein. Expression in combination with c(alpha) or R4 protein synthesis was not detected. Inclusion of the anti-R3 monoclonal antibody among antibody reagents for GBS serotyping will enhance the discriminatory power of this typing method.     

A set of two monoclonal antibodies specific for the cell surface-exposed 39K major outer membrane protein of Haemophilus influenzae type b defines all strains of this pathogen.

Six murine plasma cell hybridomas producing monoclonal antibodies (mabs) directed against the 39,000-molecular-weight (39K) major outer membrane protein of Haemophilus influenzae type b were employed in the antigenic analysis of the 39K protein. The initial characterization of the mabs by radioimmunoprecipitation analysis showed that four of these mabs reacted with antigenic determinants of the 39K protein that are exposed on the bacterial cell surface and accessible to antibody. The other two mabs reacted with antigenic determinants of the 39K protein that are either not exposed on the H. influenzae type b cell surface or not accessible to antibody (internal determinants). A total of 126 clinical isolates of H. influenzae type b obtained from pediatric research centers throughout the United States were examined for reactivity with the six mabs by using a solid-phase radioimmunoassay in which bacterial colony growth from agar plates was placed on filter paper and used as antigen. The reactivities of these strains with two of the mabs recognizing cell surface-exposed antigenic determinants of the 39K protein were used to divide the 126 strains into four different groups. Group 1 strains reacted with mab 12D9, group 2 strains reacted with mab 4C4, group 3 strains reacted with both mabs 12D9 and 4C4, and group 4 strains (only one was found) did not react with either mab. The reactivities of two other mabs recognizing cell surface-exposed antigenic determinants of the 39K protein were used to further divide the four groups into eight subgroups. A single mab recognizing an internal antigenic determinant of the 39K protein reacted with every H. influenzae type b strain examined in this study. These data indicate that only limited antigenic heterogeneity exists among the cell surface-exposed antigenic determinants of the 39K outer membrane proteins among H. influenzae type b strains and that at least one internal antigenic determinant of the 39K protein is universally present in all H. influenzae type b strains. Radioimmunoprecipitation analysis also demonstrated that H. influenzae type b strains which lacked a 39K major outer membrane protein possessed a 38K major outer membrane protein which reacted with the anti-39K mabs, indicating that the 38K and 39K outer membrane proteins of different H. influenzae type b strains are antigenically related.     

Recombinant group B Streptococcus alpha-like protein 3 is an effective immunogen and carrier protein

Conjugate vaccines against pathogens of multiple serotypes are optimized when all components induce functional antibody, resulting in broadened coverage. While most clinical studies of vaccines against group B Streptococcus (GBS) have evaluated conjugates composed of capsular polysaccharide (CPS) coupled to tetanus toxoid, conjugates prepared with GBS proteins as carriers have also been efficacious in animals. Here, we report that recombinant GBS alpha-like protein 3 (rAlp3) is both a strong immunogen and a viable carrier protein for type III CPS. The type III CPS-specific immunoglobulin G (IgG) titer rose from <100 to 64,000 among mice that received type III CPS coupled to rAlp3 (III-rAlp3) compared with an absence of a specific response among mice that received an uncoupled mixture. Most (94%) newborn pups born to III-rAlp-vaccinated dams survived challenge with viable type III GBS, compared with 43% survival among those born to dams that received the uncoupled mixture (P < 0.0001). A tricomponent conjugate of type III CPS, rAlp3, and a GBS recombinant beta C protein lacking its IgA binding site (III-rAlp3-rBCP(DeltaIgA)) provided protection against a serotype III strain and a serotype Ia strain bearing beta C protein. High-titered anti-rAlp3 rabbit serum opsonized Alp3-containing strains of two GBS serotypes (types V and VIII) and invasive type III strains bearing the cross-reactive Rib protein for in vitro killing by human peripheral blood leukocytes. Thus, the potential exists for the inclusion of rAlp3 in a GBS vaccine formulated to provide multiserotype coverage.     

Isolation and characterization of a common antigen in Campylobacter jejuni and Campylobacter coli.

Flagellin protein was isolated and purified from two serotype reference strains of Campylobacter jejuni, Pen 1 and Pen 3. Each preparation was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of a diffuse band with a molecular mass of approximately 62 kilodaltons. Antisera were prepared against flagellin from Pen 1, and specific antibody was isolated by affinity chromatography with flagellin protein covalently bound to cyanogen bromide-activated Sepharose. The high-affinity antibody was used to immune blot purified flagellin from Pen 1 and Pen 3, as well as whole-cell preparations and acid-glycine extracts from the 60 reference strains of the thermostable antigen serotyping system. From each of the 60 strains, a protein with a molecular mass of approximately 62 kilodaltons was identified which shared a common antigenic determinant. When the affinity-purified antibody was used in a coagglutination assay, washed whole cells were not agglutinable unless they had been pretreated with an acid buffer (glycine-hydrochloride [pH 2.0]). This indicated that the antigenic determinant common to strains of both C. jejuni and Campylobacter coli may not be exposed in the native state.     

Purification and analysis with monoclonal antibodies of P2, the major outer membrane protein of nontypable Haemophilus influenzae.

The protein P2 comprises a large proportion of the outer membrane of nontypable Haemophilus influenzae and functions as a porin. In view of the importance of the protein as a surface antigen, the present study was designed to purify and analyze P2 with particular emphasis on detection of antigenic determinants expressed on the bacterial surface and identification of bactericidal targets on P2. The P2 protein was purified by using detergent solubility, anion-exchange chromatography, and gel-filtration chromatography sequentially. Two monoclonal antibodies to P2 were developed. One antibody (2E6) recognized a determinant expressed on the bacterial surface, whereas the other antibody (3F3) recognized an internal epitope. The surface-exposed 2E6 determinant was present on 12% of strains from a nationwide collection. P2 is a bactericidal target for antibody 2E6. Cyanogen bromide cleavage of P2 resulted in two fragments, as in type b strains. Both monoclonal antibodies recognized epitopes on the larger fragment. These observations have potentially important implications regarding the development of vaccines to prevent H. influenzae infections and the development of a serotyping system for epidemiologic studies.     

Molecular characterization of nontypeable group B streptococcus

Traditionally, the capsular polysaccharide (CPS) antigen has been used to distinguish between the nine known serotypes of group B streptococcus (GBS) by classical antibody-antigen reactions. In this study, we used PCR for all CPSs and selected protein antigens, multilocus sequencing typing (MLST), and pulsed-field gel electrophoresis (PFGE) to molecularly characterize 92 clinical isolates identified as nontypeable (NT) by CPS-specific antibody-antigen reactivity. The PCR and MLST were performed on blinded, randomly numbered isolates. All isolates contained the cfb gene coding for CAMP factor. While most (56.5%) contained a single CPS-specific gene, 40 isolates contained either two or three CPS-specific genes. Type V CPS-specific gene was present in 66% of the isolates, and all serotypes except types IV, VII, and VIII were represented. Most (44.5%) of the isolates contained a single protein antigen gene (bca, bac, rib, alp1, or alp3), and the remaining isolates had multiple protein antigen genes. Of the 61 isolates that had the V CPS-specific gene, 48 (78.6%) had the alp3 gene. PFGE analysis classified the isolates into 21 profile groups, while MLST analysis divided the isolates into 16 sequence types. Forty-two (69%) of 61 isolates with the V CPS-specific gene were in PFGE profile group 4; 41 of these 42 were sequence type 1 by MLST. These data shed new light on the antigenic complexity of NT GBS isolates, information that can be valuable in the formulation of an effective GBS vaccine.     

The putative R1 protein of Streptococcus agalactiae as serotype marker and target of protective antibodies

The streptococcal R1 protein was studied by means of anti-R1 antibodies prepared by appropriate cross-absorption of rabbit antiserum raised against the group B streptococcal (GBS) strain ATCC 12403 (D136C), serotype III/R1. The protein was a ladder-forming antigen according to banding patterns in immunoblotting, similar to several other GBS proteins, and was susceptible to digestion by both pepsin and trypsin. Antibody-based testing revealed that 10% of Norwegian GBS isolates expressed the R1 protein, most frequently capsular antigen type V strains (72%) and less frequently type III strains (3%). None of 132 GBS strains from Zimbabwe, including 39 type V strains, expressed the R1 protein. R1-specific rabbit antibodies showed protective activity in mice challenged with a GBS type V/R1 strain. The results show that the R1 protein is an important GBS serotype marker in strains from certain geographical areas, notably for the subtyping of capsular type V strains, and that this protein is a target of protective antibodies.     

Serological purification of polysaccharide antigens from Streptococcus mutans serotypes a and d: characterization of multiple antigenic determinants.

The polysaccharide antigen preparations from serotype a and serotype d strains of Streptococcus mutans contained both a serotype-specific antigenic determinant and a common a-d antigenic determinant, as demonstrated by agar gel diffusion studies and a quantitative cross-precipitin assay. The chromatographically purified antigens were isolated by a method which depended on their serological specificity to determine if these two antigenic determinants were located on the same molecule. The a and d polysaccharides were recovered from specific antigen-antibody complexes and characterized with respect to their immunological specificity and chemical composition. Agar gel diffusion tests demonstrated that, in both the a and d preparations, the serotype-specific antigenic determinant and the common a-d antigenic determinant were present in one molecule.