Immune Responses to the R4 Protein Antigen of Group B Streptococci and Its Relationship to Other Streptococcal R4 Proteins

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1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1996, p Vol. 3, No X/96/$ Copyright 1996, American Society for Microbiology Immune Responses to the R4 Protein Antigen of Group B Streptococci and Its Relationship to Other Streptococcal R4 Proteins ELIZABETH L. FASOLA, AUREA E. FLORES, AND PATRICIA FERRIERI* Department of Laboratory Medicine and Pathology and Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota Received 19 September 1995/Returned for modification 18 October 1995/Accepted 9 February 1996 The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 protein is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II or III and from 26 noncolonized mothers; 92.5% of the colonized mothers had anti-r4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-dnase B titers. Of those individuals with an anti-dnase B titer of >640, 64% had a positive WB for anti-r4 antibody, compared with 30% of individuals with low anti-dnase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-gbscolonized individuals may be due to immunologic responses to past exposure to the R antigen present in GBS or other streptococcal groups. Group B streptococci (GBS) are the most common cause of neonatal sepsis and meningitis in the United States (5, 20). They are also an important causative pathogen of severe infections in peripartum women and immunocompromised adults (20). At this time, intrapartum chemoprophylaxis is the most effective strategy available for the prevention of neonatal disease (1), but the morbidity and mortality from GBS disease remain high despite adequate treatment. Because of this, the development of immunologic prevention and study of antibody responses to the various antigens of GBS are of major interest. Protection against GBS has been attributed to specific antibody responses to the GBS type polysaccharide (2, 10). More recently, some studies have suggested that antibodies against GBS surface proteins also may play an important role in protection against GBS disease (5, 14, 17). The R antigen, a trypsin-resistant, surface-localized protein observed in group A, C, F, G, and L streptococci, has also been found in GBS (6, 18, 19). Although four distinct immunologic species of R antigen have been described for GBS, R4 is the most prevalent in isolates recovered from humans. There appears to be no serological cross-reactivity among these antigenically distinct proteins (6, 7, 19). We previously reported that the R4 protein was expressed almost exclusively in GBS serotypes II and III (6). In contrast to the protein of GBS, R4 does not bind immunoglobulin A (IgA), nor does it bind IgG (unpublished observations). Although the specific role of these protein antigens is not completely understood, it is thought that they may play an important role in the pathogenicity of * Corresponding author. Mailing address: University of Minnesota Hospital and Clinics, Mayo Memorial Building, 420 Delaware St., S.E., Minneapolis, MN Phone: (612) Fax: (612) GBS. Some investigators (12) have reported that anti-r protein antibodies protected mice against experimental infection with type II but not type III GBS strains. However, the prevalence of R4 antibodies in human sera and their role in immunity have not been studied comprehensively (13). The initial purpose of this study was to examine the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in mothers colonized with GBS serotype II or III with and without expression of surface-localized R4 antigen, as well as the concordance of the presence of antibodies to R4 in GBS-colonized mothers and their infants. The results from this evaluation, showing a high prevalence of antibodies to the GBS R4 antigen, prompted us to analyze further the antigenic relationship of the GBS R4 protein antigen to other streptococcal R4 proteins. Therefore, this report represents a comprehensive study of the prevalence of GBS R4 antibodies in human sera by WB and the interaction of these antibodies with R4 proteins expressed by group A streptococci (GAS). This study also evaluated the WB profiles and the immunologic reactions of the GBS R4 antigen compared with those of the GAS R4 protein antigen as an initial exploratory tool for understanding the possible role of the streptococcal R4 protein antigen in the immune response to streptococcal infections. (This work was presented in part at the XII Lancefield International Symposium on Streptococci and Streptococcal Diseases, 6 10 September 1993, St. Petersburg, Russia.) MATERIALS AND METHODS Bacterial strains. The GBS isolates (from this laboratory) and their serotypes were as follows: (serotype III/R4), (III/R1), (II/R4), (III/R4), and (II/R4). The GAS isolates used were B960, a laboratory prototype strain, and 56 clinical pharyngeal isolates recovered from 56 patients. 321

2 322 FASOLA ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 1. Frequency of antibody to R4 in GBS-colonized and noncolonized mothers as determined by WB. The difference between the results for GBScolonized and noncolonized mothers was significant (P by Fisher s exact test). These GAS isolates were of diverse M types, including M types 1 to 6, which are commonly isolated from the pharynx. The GAS isolates were studied for R1 and R4 protein expression. Since information on the expression of R4 among many different M types is limited, with reports of expression confined to M-2, M-4, and T-28 strains in published studies (19), we chose many different M types for this study, including two isolates of M type 2. Human sera and patient population. The serum samples were obtained from our collection of sera from pregnant women and their infants at delivery. The patients were participants in mother-infant GBS prevalence studies for which written informed consent was obtained. With rare exceptions, the infants had 37 weeks of gestation. The sera were maintained in the laboratory at 80 C and diluted 1:500 before being tested. Sera from 40 GBS-colonized mothers, 26 noncolonized mothers, and 14 pairs of mothers and infants (the maternal sera were from the group of colonized mothers) were tested. Maternal GBS colonization status was assessed in the third trimester of pregnancy and at the onset of labor. Cultures were taken from all infants at multiple sites at birth (anterior nares, external ear canal, and umbilicus) and at discharge from the hospital (nares and umbilicus). All cultures were done with broth enrichment techniques and antibiotic-containing blood agar plates inhibitory to normal bacterial flora. The mothers and their infants colonized with GBS did not show signs of infection. Sera from 11 healthy adult individuals were also tested for antibody to the R4 antigen. Serum samples previously tested for GAS anti-dnase B titers (for clinical diagnostic purposes) were also tested for antibody to the R4 antigen (see below). In each experiment, positive and negative human sera were included as controls. The anti-r4 antibody-negative human serum control was the only negative serum from the 11 healthy adults tested; however, it was reactive with the antigen of the GBS c protein, an unrelated protein. GBS antigen. The R4 protein was extracted from GBS , a type III/R4 strain, by a modification of the trypsin digestion method previously described by us (6). Briefly, cultures were grown overnight at 35 C in Carey s chemically defined medium. Cells were harvested by centrifugation, washed once with saline, and extracted for 30 min at 37 C with 0.1% trypsin (Sigma Chemical Co., St. Louis, Mo.) in Sorensen s buffer (ph 8.2). Detection of antibodies to R4 antigen. Antibodies against the R4 protein of GBS were detected by WB as follows. The R4 antigen from the control GBS strain was first electrophoresed in a 7.5% nondenaturing polyacrylamide minigel and then transferred to Immobilon-P membranes (Millipore, Bedford, Mass.). After the membrane strips were blocked overnight in phosphate-buffered saline (PBS) plus Tween 20, they were reacted with the diluted sera. Biotinconjugated anti-human IgG (heavy plus light chains) (Vector Laboratories, Burlingame, Calif.) was used as the secondary antibody and was followed by streptavidin-alkaline phosphatase (Zymed Laboratories Inc., San Francisco, Calif.). The strips were developed with 5-bromo-4-chloro-3-indolylphosphate (BCIP) plus nitroblue tetrazolium (Sigma), and the intensity of blotting was graded visually on a scale of 1 to 4. A WB was considered positive when the intensity of blotting was 2. This was chosen as the cutoff to eliminate any weak reactions that might be due to background. Relationship of GBS R4 to other streptococcal R4 proteins. To study the relationship between R4 proteins from GBS and GAS, 23 serum samples from individuals with low ( 50) and 25 serum samples from individuals with high ( 640) GAS anti-dnase B titers were examined by WB. The WB was performed as described above with the R4 antigen extracted from GBS strain The R4 protein from the prototype GAS strain B960 (NT/T28/R1,R2,R4) was compared by immunodiffusion in agarose and WB with the R4 proteins from several GBS strains by using a rabbit anti-r4 antibody prepared in this laboratory against the R4 protein from GBS. GAS R4 antigen characterization. To evaluate the prevalence of the R4 antigen in GAS, 56 strains of several M serotypes of GAS were examined under code for the presence of R4 protein by the following three methods of extraction and detected by immunodiffusion and WB: (i) trypsin extraction as used for GBS and described above; (ii) HCl extraction, previously used for typing of GAS (16); and (iii) an adaptation of mutanolysin extraction (15). For the last method, 25 ml of inoculated Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) was incubated overnight at 35 C. The cells were spun down, washed twice with 0.01 M PBS (ph 7.4), and resuspended in 500 l of PBS containing mutanolysin (Sigma) at a final concentration of 350 U/ml. The digestion was allowed to occur for 4 h at 37 C in a water bath. The mixture was then centrifuged, and the supernatant was stored at 20 C until it was tested. Detection of R4 protein by double immunodiffusion (DD). Slides were prepared with 0.8% agarose in 0.01 M phosphate buffer with 1% polyethylene glycol (9). Antigen and antibody were placed in the appropriate wells and were allowed to react overnight at room temperature (9). The slides were stained with 0.1% Coomassie blue R-250 (Sigma). GBS strain (serotype III/R4) was the positive control, and GBS strain (serotype III/R1) was the negative control. Detection of R4 protein by WB. The R4 antigen extracts obtained from each of the GAS strains, as well as from the GBS control strains, by the three methods were electrophoresed and transferred to a membrane according to the method described above for the detection of antibodies against the R4 antigen. A rabbit monospecific anti-r4 antibody prepared in this laboratory was used as the primary antibody. Alkaline phosphatase-conjugated anti-rabbit IgG [F(ab ) 2 ] (Jackson Immunoresearch Laboratories, West Grove, Pa.) was used as the secondary antibody. The R4 antigen was detected by developing the membrane strips with a solution of BCIP plus nitroblue tetrazolium. GBS strain was used as the positive control, and GBS strain was used as the negative control as described above for R4 protein detection by DD. The streptococcal strains in the study were considered to express R4 antigen when the extracted proteins probed with specific serum revealed reactive bands of any molecular weight. Trypsin extracts displaying one reactive band were concentrated, and when the WB was repeated with the concentrated extracts, well-defined and reproducible patterns were observed. Statistical analysis. Results from WB were statistically analyzed by chi-square analysis and Fisher s exact test. Linear regression analysis was done to evaluate the concordance of the mother-infant paired serum samples. RESULTS The prevalence of antibodies against the R4 antigen in sera from 40 mothers colonized with GBS serotype II (n 19) or serotype III (n 21) and from 26 noncolonized mothers was examined by WB. Of the colonized mothers, 37 (92.5%) had anti-r4 antibodies, compared with 14 (54%) of the noncolonized mothers (P 0.001) (Fig. 1). In fact, 27 colonized mothers (68%) had a strong positive WB ( 3 ), compared with 5 noncolonized mothers (19%) (P 0.001). Of mothers known to be colonized with GBS serotype II/R4 or III/R4, 100% (n 12) had antibodies against R4 by WB. Ten of the 11 serum samples from healthy adults showed a positive WB, suggesting past exposure to R4-positive streptococcal strains (data not shown). These sera had the same banding pattern as those from GBS-colonized individuals. Fourteen pairs of mother and infant sera were also examined. The mothers and their infants were colonized with GBS serotype II (n 7 pairs) or serotype III (n 7 pairs). Concordant results were obtained in 71% of mother-infant pairs colonized with GBS serotype II (five infant sera positive) and in 57% of those colonized with serotype III (four infant sera positive). The intensity of the WB reaction of the infant serum was in agreement with the intensity seen in the maternal serum (Fig. 2). Therefore, a relatively high degree of correlation (r 0.68) between maternal and infant sera in the intensity of the antibody response was observed. Since it is known that the R4 antigen is present in other streptococcal groups (4, 6, 19) and since positive reactions were found in noncolonized mothers and healthy adult controls by WB, this study evaluated the prevalence of antibodies to the GBS R4 antigen in 48 sera from individuals with high and low GAS anti-dnase B titers, as an indication of past exposure to GAS (3). Of those individuals with an anti-dnase B titer of 640, 64% had a positive WB for anti-r4 antibody,

3 VOL. 3, 1996 STREPTOCOCCAL R4 ANTIBODY IN HUMAN SERA 323 FIG. 2. WB of R4 protein reacted with maternal (M) and infant (I) paired sera. C, positive control; C, negative control; M.W. Std., molecular mass standards. compared with 30% of individuals with low anti-dnase B titers (P 0.05) (Fig. 3). On the basis of this finding, the presence of the R4 antigen in GAS was investigated preliminarily by immunodiffusion studies. The extracted antigen from strain B960, a prototype GAS strain, showed a reaction of identity with that from GBS strain , indicating shared immunodeterminants in the GBS and GAS R4 proteins (Fig. 4). To further evaluate the role of the R4 protein in GAS and its relationship to the GBS R4 protein, 56 GAS strains of several M serotypes were tested for the R4 antigen. The results demonstrated that six (10.7%) of the GAS strains had the R4 antigen as demonstrated by one or more extraction methods (Table 1). When the code was broken, it was found that the GAS-positive strains were two of M type 2, two of M type 58, and two of M type 77. Trypsin extraction gave cleaner and more distinct immunoblots, but concentrated samples were needed in order to detect the antigen. The amount of antigen extracted by trypsin was probably too low to be detected by DD. Therefore, DD testing with trypsin extracts was not suitable for screening of the R4 protein in GAS. This is in contrast with GBS, for which the results for trypsin extracts tested by DD were almost always in concordance with WB results (unpublished observations). Unconcentrated mutanolysin and HCl extracts of GAS gave positive reactions with the R4 antibody by WB, although three of the six samples positive by WB had negative reactions by DD (Table 1). Heterogeneity of the R4 protein among the GAS strains was seen by WB (Fig. 5), as seen previously by us with GBS strains (7). The trypsin-extracted R4 protein in GAS exhibited a main FIG. 4. Immunologic relationship of R4 antigens from GBS and GAS as determined by DD in agarose. Wells: Ab, antiserum to R4; 1, GBS (III/R4); 2, GBS (II/R4); 3, GBS (III/R4); 4, GBS (III/R4); 5, GBS (II/R4); 6, GAS B960 (NT/T28/R1,R2,R4). dense band and three or more less intense bands with lower molecular masses. The patterns resembled the size ladder seen in GBS, but the higher protein band of the ladder in GAS in general had a lower molecular mass (110 versus 190 kda, respectively) than those seen in R4 proteins from the GBS control strain (results not shown). Mutanolysin extraction of R4 from GAS strains also gave a ladder profile by WB, but the protein bands displayed a wider range of molecular mass (150 to 41 kda) than those obtained after trypsin extraction (110 to 60 kda). It is of interest that the densest band seen in the WB after extraction with trypsin or mutanolysin had the same molecular mass but that this band was associated with lowermolecular-mass fragments in the trypsin extracts and with higher-molecular-mass bands in mutanolysin extracts. HCl-extracted proteins also revealed a ladder profile for the R4 antigen, but the larger number of bands seen with these samples made comparison among the strains more difficult than with extracts obtained by the other two methods (results not shown). Each extraction method evaluated for R4 protein detection gave reproducible results when the same samples were retested. DISCUSSION The present findings, along with our previous studies (8), enhance our understanding of the prevalence of antibodies against various surface-localized protein antigens of GBS in colonized and noncolonized mothers and their babies. A high prevalence of antibody to the c protein in maternal sera was previously reported (8), but its presence in serum seemed to be independent of the colonization status of the individual tested, except for the very high titers (8). We chose to evaluate the TABLE 1. Detection of R4 protein in GAS by WB and DD with three extraction methods Detection of R4 protein extracted with a : Strain no. Trypsin HCl Mutanolysin DD WB DD WB DD WB FIG. 3. GAS anti-dnase B titers of sera from patients with positive and negative WBs for GBS R4 antigen. The difference between individuals with low and high anti-dnase B titers was significant (P 0.05 by Fisher s exact test) a HCl and mutanolysin extracts were unconcentrated; trypsin extracts were 4 concentrated.

4 324 FASOLA ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 5. WB of mutanolysin extracts of GAS probed with rabbit anti-r4 antibody. Lanes: MW, molecular mass standards; 1, GAS B960 (positive control for GAS); 2 to 4, wild-type GAS strains; 5, GBS (positive control for R4); 6, GBS (negative control). presence of anti-r4 antibodies in sera by WB instead of by the enzyme-linked immunosorbent assay technique, because WB was a more sensitive method for detecting antibodies to surface proteins of GBS (8). In addition, since WB revealed electrophoretic profiles, this allowed the examiner to evaluate the binding characteristics of the antibodies immediately and to compare the banding profiles among the R4 antigens extracted from different streptococcal isolates. The present data indicated that although the prevalence of antibodies against the R4 protein was high, the intensity of the reaction by WB was significantly higher in the colonized maternal group than in the noncolonized maternal group. This finding suggested that the R4 protein antigen was able to elicit antibodies in individuals colonized with GBS. However, antibodies against the R4 antigen were also observed in the control group, since 10 of the 11 serum samples from healthy individuals tested also exhibited a positive WB. The GBS colonization status of these individuals was unknown, and therefore, antibodies against R4 present in the sera could be the result of previous colonization or of cross-reactive antibodies evoked by other streptococcal groups expressing R4 protein. Similar explanations could account for the presence of antibodies against the GBS R4 protein found in half of the noncolonized mothers. The high correlation in the intensity of the WB reaction seen in mother-infant paired sera (as an indication of the level of antibody present in the serum) demonstrated that the R4 antibodies indeed crossed the placenta. It was found that in some of the cases when results for the maternal and infant sera were different, the intensity of reaction of the maternal serum was just above the breakpoint for being considered positive, while that of infant serum was just below the breakpoint. In most cases, however, the infant s serum revealed an intensity similar to or one rank lower than that of the mother s serum. This finding also supported the transfer of maternal antibodies to the fetus during pregnancy. The present study did not investigate whether the colonized infants who had antibodies against the R4 antigen became infected later or whether they were protected against GBS infection. However, some investigators (11, 12, 14, 17) have suggested that maternal antibodies to cell surface proteins may be important for protective immunity, as demonstrated in animal studies in which a monospecific rabbit antiserum against the R protein protected animals from infection by type II GBS strains carrying the R antigen but not from that by type III/R-positive GBS strains (12). The latter finding could not be fully explained, but the sialic acid of type III polysaccharide has been suggested as a possible factor preventing anti-r antibody from facilitating bacterial opsonization (12). The present study did not find any significant differences in the intensities of the WB in serum samples from mothers colonized with type III/R4 versus mothers colonized with type II/R4. The correlation of presence of antibody in maternal and infant sera was slightly higher for the mothers colonized with GBS type II strains than for the mothers colonized with GBS type III strains, but the number of samples studied was too small for this finding to be significant. A protective role of antibodies against other GBS protein antigens has been reported (14). In that study, the investigators identified and characterized two groups of genetic clones that encoded the and components of the c protein. They found that antisera raised against each of these clones provided protective immunity to GBS infection in a mouse model. This protection was specific for strains carrying the c proteins, and there was no crossreactivity between the clones (14). As discussed above, the protective role of antibody to the c protein against GBS infection seems to be very important. However, the role of anti-r4 antibodies still remains unclear, and further studies should address this issue. Our findings that the R4 protein antigen induces an immune response in the host and that there is transplacental passage of these antibodies suggest that the R4 protein may be a potentially important component of a GBS polysaccharide-protein conjugate vaccine to induce type-specific immunity in mothers. Another interesting finding was the high prevalence of antibody against R4 seen in individuals previously infected with GAS, as indicated by the high titers of anti-dnase B in their sera. These studies also revealed that the R4 protein of GBS had a reaction of identity in immunodiffusion with the R4 antigen from GAS, suggesting shared immunodeterminants. Our previous research (6) revealed that the R antigen was a stable antigen and that it was expressed consistently on the strains that carry it. The present results suggested that perhaps the antibodies against the GBS R4 antigen could cross-protect against other streptococcal groups expressing the R4 antigen. The prevalence of the R4 antigen in GAS strains was lower than expected, given the prevalence of antibodies against this antigen found in sera from individuals with putative GAS infection (elevated GAS anti-dnase B titers). However, the GAS isolates tested were isolated during a different time period than were the serum samples studied for GAS anti-dnase B antibodies, and the expression of the R4 protein in GAS could have varied during these periods. Another possible explanation could be that antibody presence may be easier to detect than antigen presence. The GAS strains studied carried M protein on their surface, and this could be a factor preventing efficient and complete extraction of the R4 protein from these strains. The specific rabbit antiserum used for the detection of R4 antigens in GAS strains was prepared against the R4 protein from GBS, and although R4 antigens from GAS and GBS cross-reacted extensively in DD, there may be some immunochemical differences between the R4 proteins produced by these streptococcal groups. The different molecular patterns observed in the WB for the GAS and GBS strains seemed to support this. In conclusion, these findings demonstrated that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers. The high correlation seen with mother-infant paired sera suggested that the R4 antibodies crossed the placenta. The presence of R4 antibodies in non-gbscolonized individuals may be due to immunologic responses to

5 VOL. 3, 1996 STREPTOCOCCAL R4 ANTIBODY IN HUMAN SERA 325 past exposure to the R antigen present in GBS or other streptococcal groups, particularly GAS as demonstrated in the present report. A role for the R4 protein in the pathogenicity of GBS has been suggested (13), but the role of antibodies to R4 in protecting against GBS infection is undefined. ACKNOWLEDGMENTS We thank Edward L. Kaplan and Dwight R. Johnson from the WHO Streptococcal Reference Laboratory for providing the GAS strains, and we thank Sharyn Erickson for preparation of the manuscript. This work was supported by Public Health Service grant RO1 AI13926 from the National Institute of Allergy and Infectious Diseases and research grant NO1 AI REFERENCES 1. American Academy of Pediatrics Committee on Infectious Diseases and Committee on Fetus and Newborn guidelines for prevention of group B streptococcal (GBS) infection by chemoprophylaxis. Pediatrics 90: Baker, C. J., and D. L. Kasper Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294: Ferrieri, P Immune responses to streptococcal infections, p In N. R. Rose, H. Friedman, and J. L. Fahey (ed.), Manual of clinical laboratory immunology, 3rd ed. American Society for Microbiology, Washington, D.C. 4. Ferrieri, P Surface-localized protein antigens of group B streptococci. Rev. Infect. Dis. 10:S363 S Ferrieri, P Neonatal susceptibility and immunity to major pathogens. Rev. Infect. Dis. 12:S394 S Flores, A. E., and P. Ferrieri Molecular species of R-protein antigens produced by clinical isolates of group B streptococci. J. Clin. Microbiol. 27: Flores, A. E., and P. Ferrieri Comparison of trypsin resistant surface localized proteins of group B streptococci (GBS), abstr. I-67, p In Abstracts of the 93rd General Meeting of the American Society for Microbiology American Society for Microbiology, Washington, D.C. 8. Flores, A. E., J. A. Nelson, X. Y. Wu, and P. Ferrieri Antibody profiles to the group B streptococcal beta antigen in maternal and infant paired sera. APMIS 101: Johnson, D. R., and P. Ferrieri Group B streptococcal Ibc protein antigen: distribution of two determinants in wild-type strains of common serotypes. J. Clin. Microbiol. 19: Lancefield, R. C., and E. H. Freimer Type-specific polysaccharide antigens of group B streptococci. J. Hyg. 64: Lancefield, R. C., M. McCarty, and W. N. Everly Multiple mouseprotective antibodies directed against group B streptococci. Special reference to antibodies effective against protein antigens. J. Exp. Med. 142: Lindén, V Mouse-protective effect of rabbit anti-r-protein antibodies against group B streptococci type II carrying R-protein. Lack of effect on type III carrying R-protein. Acta Pathol. Microbiol. Immunol. Scand. Sect B 91: Lindén, V., K. K. Christensen, and P. Christensen Correlation between low levels of maternal IgG antibodies to R protein and neonatal septicemia with group B streptococci carrying R protein. Int. Arch. Allergy Appl. Immunol. 71: Michel, J. L., L. C. Madoff, D. E. Kling, D. L. Kasper, and F. M. Ausubel Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity. Infect. Immun. 59: Stålhammar-Carlemalm, M., L. Stenberg, and G. Lindahl Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections. J. Exp. Med. 177: Swift, H. F., A. T. Wilson, and R. C. Lancefield Typing group A hemolytic streptococci by M precipitin reactions in capillary pipettes. J. Exp. Med. 78: Valtonen, M. V., D. L. Kasper, and N. J. Levy Isolation of a C (Ibc) protein from group B Streptococcus which elicits mouse protective antibody. Microb. Pathog. 1: Wibawan, I. W. T., and C. Lämmler Distribution of B streptococcal type antigens among streptococci of serological groups B, G, and L. Zentralbl. Bakteriol. 273: Wilkinson, H. W Comparison of streptococcal R antigens. Appl. Microbiol. 24: Zangwill, K. M., A. Schuchat, J. D. Wenger, and Group B Streptococcal Disease Study Group Group B streptococcal disease in the United States, 1990: report from a multistate active surveillance system. Morbid. Mortal. Weekly Rep. 41: Downloaded from on March 18, 2019 by guest