Activation of murine peritoneal macrophages by Streptococcus pneumoniae type II capsular polysaccharide: involvement of CD14-dependent pathway

In this study we examined the ability of capsular polysaccharide type 2 (PS) from Streptococcus pnemoniae to induce secretory and cellular responses in peritoneal macrophages. Tumour cytotoxicity induced by preincubation with PS was demonstrated to be concentration-dependent. PS-induced tumouricidal activity was partially abrogated by anti-tumour necrosis factor (TNF)-alpha and inhibitor of nitric oxide, whereas anti-interferon (IFN)-alpha/beta antibody and the scavengers of reactive oxygen intermediates had no effect. In addition, supernatants from macrophages treated with PS contained TNF-alpha, and their iNOS-enzymatic activity was significantly increased. Thus, the tumouricidal activity induced by PS appeared to be mediated by the production of TNF-alpha and nitrite. Treatment of macrophages with PS increased the expression of CD14, the receptor for lipolysaccharide (LPS)/LPS-binding protein. Moreover, blocking antibody to CD14 abrogated partially TNF-alpha and nitrite induction by PS, suggesting that the PS-induced CD14 upregulation was correlated with secretion of TNF-alpha and nitrite. Taken together, these results demonstrate that PS may induce macrophage-secretory and cellular activities, in part, possibly via CD14-dependent pathway.     

Current trends in capsular polysaccharide biosynthesis of Streptococcus pneumoniae

The capsular gene cluster (cap/cps) of 13 out of the 90 known pneumococcal types has been sequenced. The cap/cps operon, located between dexB and aliA in the Streptococcus pneumoniae chromosome, contains some of the genes responsible for the synthesis of the type-specific polysaccharide flanked by four conserved open reading frames. The biochemical function of only a few capsular genes has been established, whereas the role of the flanking regions is controversial. Remarkably, only one gene (tts) located outside the cap locus is required for the synthesis of type 37 capsule. Moreover, other genes not linked to the cap gene cluster are also needed for capsule synthesis in pneumococcus.     

Immunogenic properties in mice of hexasaccharide from the capsular polysaccharide of Streptococcus pneumoniae type 3.

Hexasaccharide (HS) containing 3 U of cellobiuronic acid was isolated from Streptococcus pneumoniae type 3 capsular polysaccharide S3 and coupled to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or tetanus toxoid (TT). The immunogenicity of these HS-protein conjugates in BALB/c mice was studied by measuring the production of circulating antibodies and the induction of protective immunity to viable S. pneumoniae type 3. Immunization of BALB/c mice with 0.5 micrograms of S3 resulted in the induction of immunoglobulin M (IgM) antibodies and complete protection against 25 U of a mean lethal dose of S. pneumoniae type 3 for 19 weeks after immunization. BALB/c mice immunized with 100 micrograms of HS9-BSA (containing 12 micrograms of HS) were also protected due to circulating IgM antibodies. Repeated injections with either 100 micrograms of HS9-BSA (three immunizations) or 100 micrograms of HS6-KLH (two immunizations) resulted in high levels of circulating IgG antibodies. These HS-protein conjugates induced complete protection which lasted at least 14 (HS9-BSA), 23 (HS6-KLH), or 8 (HS16-TT) weeks after the last immunization. Protection against viable S. pneumoniae type 3 could be passively transferred to nonimmunized mice by antisera containing IgM or IgG antibodies or both. Sera containing both IgM and IgG antibodies gave better protection than sera containing only IgM antibodies. The specificity of the induced protection was confirmed by challenge with the non-cross-reacting S. pneumoniae type 11.     

Immunogenic properties of Klebsiella pneumoniae type 2 capsular polysaccharide.

The immunoprotective activity of Klebsiella pneumoniae K2 cell surface preparations and purified capsular polysaccharide was tested in mice. The 50% protective dose (PD50), expressed as capsular polysaccharide content, was 2 ng for cell surface preparations and 50 ng for purified capsular polysaccharide. Both preparations lost their immunoprotective activity after alkali treatment. Immune sera were raised in rabbits immunized with cell surface preparations. The precipitating and hemagglutinating capacity of these antisera was tested against either purified capsular polysaccharide or alkali-treated capsular polysaccharide. No difference was observed between the reactivity of the antisera against each antigen. The protective activity of these sera was tested on mice in passive transfer experiments, before and after absorption with either purified capsular polysaccharide or alkali-treated capsular polysaccharide. The sera lost their protective activity after absorption with purified capsular polysaccharide and after absorption with alkali-treated capsular polysaccharide. These experiments show that the difference in immunoprotective activity of cell surface preparations, purified capsular polysaccharide, and alkali-treated capsular polysaccharide is not due to a difference in their antigenic determinants. Cell surface preparations and purified capsular polysaccharide were fractionated by gel filtration on Sepharose 4B and by ultracentrifugation on cesium chloride density gradients. Three forms of capsular polysaccharide have been characterized. (i) A form of capsular polysaccharide with a very high protective activity (PD50 = 2 ng) that copurified with protein and lipopolysaccharide and was characterized by a low coefficient of distribution (Kd = 0.20) and a low density (1.5 to 1.6 g/cm3). (ii) A form of capsular polysaccharide with an intermediate protective activity (PD50 = 50 ng), contamined by less than 3% protein and 1% lipopolysaccharide, with a Kd of 0.35, and a density of 1.7 to 1.8 g/cm3. (iii) A nonimmunoprotective capsular polysaccharide obtained after alkali treatment of either cell surface preparations or purified capsular polysaccharide. The Kd of these fractions varied from 0.20 to 0.90 and their density from 1.7 to 1.8 g/cm3.     

Effect of culture conditions on the structure of Streptococcus pneumoniae type 19A(57) capsular polysaccharide.

The structural modifications and immunochemical activities of several Streptococcus pneumoniae type 19A polysaccharide (PS) preparations have been studied by sugar compositional analysis and immunodiffusion. The 19A PS preparations Lab-A-1 and Lab-A-3 and one PS isolated from 19A strain OB contained fucose (Fuc) and galactose (Gal) in addition to rhamnose (Rha) and glucose (Glc). In contrast, 19A PSs Lab-A-2 and Lab-B contained only Rha and Glc. Despite their different sugar compositions, these 19A preparations appeared to be identical in serologic activity as measured by immunodiffusion with rabbit 19A and 19F antisera. The 19A PS Lab-A-1 was separated into three fractions by DEAE-Sepharose CL-6B column chromatography with a NaCl gradient. Fraction II was the major peak with a yield of 72.9%. Fraction Ia contained Fuc and Gal, while fraction II contained Fuc, Gal, Rha, and Glc. Fractions Ia and Ib did not react with rabbit 19A antiserum. In contrast, 19A PS Lab-A-2 displayed only one peak, which was eluted by a NaCl gradient (0 to 0.6 M NaCl), and contained only Rha and Glc. The 19A PSs prepared from Lab-A and Centers for Disease Control (CDC) strains and grown in pneumococcal inoculum medium (PIM) and modified Holt medium were chromatographed on a DEAE-Sepharose CL-6B column, and the separated fractions were examined for their sugar composition. The fractions obtained from the 19A PSs Lab-A-PIM and CDC-PIM exhibited four sugar components, as observed for the PS Lab-A-1, while the separated fractions from the 19A PSs Lab-A-Holt and CDC-Holt displayed two sugar components, a pattern similar to that of PS Lab-A-2. Thus, the sugar compositions of 19A PS appeared to vary according to the type of culture medium used to grow the 19A organisms.     

Isolation and characterization of type IV group B Streptococcus capsular polysaccharide.

An antigenically distinct serotype, type IV, has recently been added to the recognized serotypes of group B streptococci (GBS). We isolated and purified the capsular polysaccharide antigen from a prototype type IV GBS strain. The type IV capsular polysaccharide formed a precipitin line with rabbit antiserum to type IV GBS organisms but not with antiserum to organisms of GBS serotype Ia, Ib, II, or III. Enzyme-linked immunosorbent assay inhibition experiments showed no cross-reaction between type IV antiserum and other GBS serotypes. Capsular polysaccharide released from the bacterial cells with mutanolysin and that isolated from the culture supernatant had similar elution profiles on Sepharose CL-6B, with a Kav of 0.30 and an estimated Mr of 200,000. The purified type IV polysaccharide was found to contain galactose, glucose, N-acetylglucosamine, and N-acetylneuraminic acid (sialic acid) as exclusive sugars. The polysaccharide contained 23% (by weight) sialic acid and galactose, glucose, and N-acetylglucosamine in a relative ratio of (1):1.10:0.55. These results are compatible with a repeating structure of six monosaccharide residues containing galactose, glucose, N-acetylglucosamine, and sialic acid in a molar ratio of 2:2:1:1. Unlike type Ia, II, and III GBS polysaccharides, desialylation of the type IV polysaccharide produced an antigen which formed a line of identity with the native type IV antigen in double diffusion in agar against homologous antiserum. This result suggests that sialic acid is not as critical to the immunodeterminant structure of the type IV antigen as it is for other GBS capsular types.     

Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae

Streptococcus pneumoniae protects itself from components of the human immune defense system by a thick polysaccharide capsule, which in most serotypes is covalently attached to the cell wall peptidoglycan. Members of the LytR-Cps2A-Psr (LCP) protein family have recently been implicated in the attachment of anionic polymers to peptidoglycan in Gram-positive bacteria, based on genetic evidence from Bacillus subtilis mutant strains and on the crystal structure of S. pneumoniae Cps2A containing a tightly bound polyprenol (pyro)phosphate lipid. Here, we provide evidence that Cps2A and its two pneumococcal homologs, LytR and Psr, contribute to the maintenance of normal capsule levels and to the retention of the capsular polysaccharide at the cell wall in the capsular type 2 S. pneumoniae strain D39. GFP fusions of all three LCP proteins showed enhanced localization at mid-cell, indicating a role in cell wall growth. Single cps2A or psr mutants produced a reduced amount of capsule. A cps2A lytR double mutant showed greatly impaired growth and cell morphology and lost approximately half of the total capsule material into the culture supernatant. We also present the crystal structure of the B. subtilis LCP protein YwtF and provide crystallographic evidence for the phosphotransferase activity of Cps2A, supporting an enzymatic function in the attachment of capsular polysaccharides to cell wall peptidoglycan.     

Streptococcus pneumoniae types 19A and 19F and Streptococcus suis capsular type 8 share common capsular epitopes.

Two monoclonal antibodies (MAbs) to Streptococcus pneumoniae types 19A and 19F were tested with the 35 reference strains and 334 field strains of Streptococcus suis by dot blotting. Both MAbs reacted with the capsular type 8 reference strain, and one reacted with 69% and one reacted with 100% of 81 S. suis capsular type 8 field strains tested. Epitopes recognized by both MAbs are capsular in origin.     

Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae.

To assess the role of capsular serotypes in the virulence of Streptococcus pneumoniae, we have constructed isogenic derivatives differing only in the type of capsule expressed. Strains of types 2, 5, and 6B were converted to type 3 by transformation and selection for an erythromycin resistance marker linked to the type 3 capsule locus. Characterization studies revealed that these type 3 derivatives were indistinguishable from the type 2, type 5, and type 6B parental strains in terms of restriction enzyme fragment pattern and expression of pneumococcal surface protein A (PspA). Expression of the type 3 capsule did not alter the mouse virulence of the similarly virulent type 2 strain. However, alteration of capsule type had a profound effect on the virulence of the type 5 and type 6B derivatives. The highly virulent type 5 strain was essentially avirulent when expressing the type 3 capsule. Conversely, the 50% lethal dose of the relatively avirulent type 6B strain was reduced > 100-fold when the type 3 capsule was expressed. Thus, the serotype of capsule expressed has a major effect on virulence, and this effect is dependent upon the genetic background of the recipient strain.     

Immunochemical cross-reactions between type III group B Streptococcus and type 14 Streptococcus pneumoniae.

Serological cross-reactions between certain streptococci and some serotypes of Streptococcus pneumoniae have been reported. These studies detail the serological cross-reactivity observed between hot HCl-extracted group b streptococcus type III (GBS III) antigens and S. pneumoniae type 14 (Pn 14) polysaccharide. Similar electrophoretic migration patterns of GBS III and Pn 14 were observed when either type-specific BGS III antisera or pneumococcal omniserum was utilized to precipitate these antigens. Both the GBS III antigen and the Pn 14 polysaccharide migrated toward the cathode, whereas all other pneumococcal polysaccharides migrated toward the anode. No cross-reactions were observed between GBS III antisera and the 11 other types of pneumococcal polysaccharides. Lines of identity were observed between type-specific GBS III antisera and monospecific Pn 14 antiserum with either GBS III antigens or purified Pn 14 polysaccharide. The cross-reacting antigens of GBS III and Pn 14 appear to be identical by immunodiffusion and immunoelectrophoresis.     

Dendritic cell-derived exosomes express a Streptococcus pneumoniae capsular polysaccharide type 14 cross-reactive antigen that induces protective immunoglobulin responses against pneumococcal infection in mice

Exosomes activate T cells in vivo, but whether exosomes are able to induce humoral immune responses is still unknown. We found that dendritic cells, but not other immune cells, constitutively release an exosome-associated glycoconjugate that is cross-reactive with the capsular polysaccharide of Streptococcus pneumoniae type 14 (Cps14-CRA). Cps14-CRA was localized to the cholesterol-enriched microdomains or rafts of the exosomes and was mapped to the beta1-->6 branched N-acetyl-lactosamine derivatives of the Cps14-CRA. Injection of CFA-primed naive mice with purified dendritic cell exosomes induced immunoglobulin (Ig) anti-Cps14 responses composed predominantly of IgM, IgG3, and IgG1. These responses were associated with protection against a lethal challenge with live S. pneumoniae type 14, but not with type 3 bacteria, and was correlated with the titer of elicited IgM and IgG3 anti-Cps14. These data show, for the first time, that exosomes can induce a humoral immune response to an associated unprocessed, autologous antigen. Although anti-Cps14 Ig responses are specifically demonstrated, these could reflect a broader mechanism that modulates both natural immunity and autoimmunity to other glycotopes.     

Oligoclonality of serum immunoglobulin G antibody responses to Streptococcus pneumoniae capsular polysaccharide serotypes 6B, 14, and 23F.

Serum antibodies (Abs) specific for the capsular polysaccharides of Streptococcus pneumoniae provide protection against invasive pneumococcal disease. Previous studies indicate that Abs to pneumococcal polysaccharide (PPS) serotypes 1 and 6B have limited clonal diversity. To determine if restricted diversity was a feature common to other PPS specificities, we examined the light (L)-chain expression and isoelectric heterogeneity of type 6B, 14, and 23F Abs elicited in 15 adults following PPS vaccination. At the population level, both PPS-6B and PPS-14 Abs expressed kappa and lambda chains, although 6B Abs more frequently expressed lambda chains lambda and 14 Abs more frequently expressed kappa chains. In individual sera, Abs were generally skewed towards either kappa or lambda expression. 23F-specific Abs had predominantly kappa chains. Isoelectric focusing analyses showed that sera contained one or at most a few immunoglobulin G Ab spectrotypes to all three respective capsular serotypes, a result indicative of oligoclonality. A sequence analysis of a purified PPS-14-specific Ab having a single spectrotype gave uniform amino-terminal sequences for both the heavy chain (V(H)III subgroup) and the L chain (kappaIII-A27 V region). From these results we conclude that within individual adults, serum Ab responses to PPS serotypes 6B, 14, and 23F derive from a small number of dominant B-cell clones, and consequently variable-region expression is probably individually limited as well. Oligoclonality appears to be a general characteristic of human PPS-specific Ab repertoires, and we suggest that this property could lead to individual differences in Ab fine specificity and/or functional activity against encapsulated pneumococci.     

Type III group B streptococcal polysaccharide induces antibodies that cross-react with Streptococcus pneumoniae type 14

Covalent linkage of a bacterial polysaccharide to a protein greatly enhances the carbohydrate's immunogenicity and its binding to solid surfaces in immunoassays. These findings have spurred the development of glycoconjugate vaccines to prevent serious bacterial infections as well as the use of glycoconjugates as coating antigens in bioassays. We evaluated sera from women immunized with unconjugated group B streptococcal (GBS) type III (GBS III) polysaccharide (IIIPS) or with IIIPS covalently linked to tetanus toxoid to assess specificity, sensitivity, and parallelism in dilution curves in two GBS III enzyme-linked immunosorbent assays (ELISAs). One assay used IIIPS mixed with methylated human serum albumin (IIIPS + mHSA) as the coating antigen, and the other used IIIPS covalently linked to HSA (III-HSA). Each coating antigen was associated with a highly specific GBS III bioassay. The sensitivity was higher in the III-HSA ELISA, in which conjugated IIIPS is bound to the plates. Parallelism in titration curves was observed in the III-HSA but not in the IIIPS + mHSA ELISA. The excellent correlation between the concentrations of GBS IIIPS-specific immunoglobulin G (IgG) and the opsonophagocytic activity of these antibodies indicated that the III-HSA assay can predict functionality of vaccine-induced IgG against GBS III disease. The structure of the repeating unit of the capsular polysaccharide of GBS III differs from that of Streptococcus pneumoniae type 14 (Pn14 PS) only by the presence on GBS III of a sialic acid residue at the end of the side chain. The majority of healthy adults responding to GBS III vaccines with a fourfold or greater increase in GBS III-specific IgG antibodies developed antibodies cross-reacting with Pn14 PS (i.e., desialylated GBS IIIPS). The proportion of GBS vaccine responders who developed IgG to the desialylated IIIPS did not depend on whether IIIPS was given in the unconjugated or conjugated form. When present, these vaccine-induced cross-reacting antibodies conferred in vitro antibody-mediated opsonophagocytosis and killing of both GBS III and Pn14, two pathogens that cause invasive disease in young infants.     

Cell growth rate regulates expression of group B Streptococcus type III capsular polysaccharide.

The capsular polysaccharide (CPS) of group B streptococci (GBS) is an important virulence factor that also serves to protect cells from nonspecific host defense mechanisms. Expression of CPS by GBS, as with other encapsulated bacterial pathogens, is not constitutive but varies during growth in vitro and in primary cultures isolated from different sites of infection. Despite this understanding, little is known about regulation of this surface-expressed carbohydrate antigen in GBS. Here we report that expression of type III CPS by GBS strain M781 grown in continuous culture with a modified chemically defined medium is regulated by growth rate. Cells in steady state at mass doubling times (tds) of 0.8, 1.4, and 1.6 h expressed an average of sixfold more cell-associated CPS than did cells held at tds of 2.3 and 11 h. Strain M781 grown at a td of 1.4 h repeatedly produced more type III CPS than those held at a td of 11.0 h, even when limited for glucose, pyridoxamine, or thiamine. In our studies, > or = 93% of the total CPS expressed by strain M781 was cell associated. Strain M781 grown at a td of 11.0 h (i.e., lowered CPS expression) was susceptible to in vitro complement-mediated opsonophagocytosis and killing by human peripheral blood leukocytes, whereas cells grown at a td of 1.4 h (i.e., higher CPS expression) were not killed unless type III CPS-specific antibody was present. Factors that allow GBS to asymptomatically colonize women yet cause invasive infection to both mother and infant are poorly understood. Our results shed new light on parameters that regulate the pathogenic potential of GBS and may also serve as a way to discern more fully the genetics and biochemistry of GBS capsule synthesis.     

Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters

Capsular polysaccharide (CPS) is a major virulence factor in Streptococcus pneumoniae. CPS gene clusters of S. pneumoniae types 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5' end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3' end of the gene cluster. Comparison of the four dTDP-L-rhamnose pathway genes (rml genes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rml genes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rml genes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5' ends and the relatively conserved rml genes at the 3' ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzx and wzy genes for all sequenced CPS gene clusters by use of motifs.     

Molecular and functional characteristics of a protective human monoclonal antibody to serotype 8 Streptococcus pneumoniae capsular polysaccharide.

The structural characteristics and biological activity of human antibodies that are reactive with the capsular polysaccharides of most serotypes of Streptococcus pneumoniae, including serotype 8, are unknown. This paper describes the generation, molecular structure, and protective efficacy of a human monoclonal antibody (MAb) reactive with the capsular polysaccharide of serotype 8 Streptococcus pneumoniae. We generated the immunoglobulin M(kappa) [IgM(kappa)] MAb D11 by Epstein-Barr virus transformation of peripheral lymphocytes from a Pneumovax recipient. Nucleic acid sequence analysis revealed that MAb D11 uses V3-15/V(H)3 and A20/V(kappa) gene segments with evidence of somatic mutation. In vitro studies revealed MAb D11-dependent complement deposition on the capsule of serotype 8 organisms via either the classical or the alternative complement pathway. In vivo, MAb D11 prolonged the survival of both normal and C4-deficient mice with lethal serotype 8 S. pneumoniae infection. Our findings demonstrate that a serotype-specific human IgM with certain structural and functional characteristics was protective in mice lacking a functional classical complement pathway and show that alternative complement pathway activation is an important determinant of pneumococcal protection.     

14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗研究

目的：探讨制备肺炎球菌荚膜多糖（PNCPS）－蛋白结合疫苗适宜条件。方法：采用碳二亚胺法将14型PNCPS与破伤风类毒素（TT）结合，将结合物免疫NIH小鼠，用ELISA法检测小鼠血清中抗14例PNCPS的IgG抗体滴度。结果：结合反应产率和结合物中多糖，蛋白含量的测定显示试验成功合成了14型PNCPS－TT结合物，该结合物具有14型PNCPS的血清学特异性，将之免疫小鼠诱生的抗14型PNCPS特异性IgG抗水平显著高于单纯14型PNCPS。结论：试验成功地合成了14型PNCPS－TT结合物，本试验采用的结合方法可行。     

Characterization of antibodies to capsular polysaccharide antigens of Haemophilus influenzae type b and Streptococcus pneumoniae in human immune globulin intravenous preparations

The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.     

Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus pneumoniae type 23F capsular polysaccharide

The human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F is predominated by antibodies which express either VkappaL6 or VkappaA23 light chain genes. In this report, we demonstrate that the antigenic epitopes recognized by these two families can be differentiated based on their specificity for the hapten l-rhamnose, a constituent sugar of the bacterial capsule polymer. The VkappaL6 family utilizes light chains with an unusually long third complementarity-determining region. These VkappaL6-encoded Fabs are 100-fold more sensitive to inhibition with l-rhamnose than VkappaA23 Fabs. VkappaL6 Fabs preferentially recognize the l-isomer of the hapten and can distinguish l-rhamnose from the structurally similar sugar l-mannose. We also demonstrate that the VkappaL6 family component of the antibody response recognizes the polysaccharide antigens of Group B streptococci (GBS), thereby accounting for the previously reported cross-reaction between PPS 23F-specific antisera and Group B streptococci.