© 1977 by the University of Chicago. All rights reserved.

Perinatal Immunity to Group B ,B-Hemolytic Streptococcus Type Ia P. B. Stewardson-Krieger, K. Albrandt, T. Nevin, R. R. Kretschmer, and S. P. Gotoff

From the Divisions of Infectious Diseases and Immunology, Department of Pediatrics, Michael Reese Hospital and Medical Center, Ptitzker School of Medicine, University of Chicago, Chicago, Illinois

Group B ,a-hemolytic (GBBH) streptococci are a major cause of serious bacterial infections in the human newborn infant [1, 2]. These organisms have been subdivided into five serotypes, la, Ib, Ic, II, and III, based on their polysaccharide capsular antigens and minor protein determinants [3]. Types Ia and III are the two types most frequently isolated from septic infants in the first week of life [4]. Since transplacentally acquired opsonic antibodies are important in neonatal host defense against certain bacterial infections, including group A streptococci [5, 6], it seems likely that similar mechanisms may account for immunity to GBBH streptococci. Preliminary studies indicate that the offspring of mothers who lack anti-

bodies to GBBH streptococci type III are at greater risk for acquiring type-specific disease [7]. The classical studies of humoral immunity to group A streptococci employed the passive mouse protection and in vitro bactericidal tests, the results of which correlate well with human resistance to streptococcal infections [8-11]. We used these two methods to evaluate sera from 25 normal adult women and 31 normal mother-cord serum pairs for immune status against GBBH streptococci serotype Ia and to assess the prophylactic value of pooled human y-globulin against challenge with GBBH streptococci serotype Ia in the mouse protection test. Studies were restricted to type Ia, since no suitable animal model exists as yet for GBBH streptococcal type III infections.

Received for publication February 3, 1977, and in revised form May 5, 1977. This paper was presented in part at the plenary session of the meeting of the Central Society of Clinical Research, Chicago, Illinois, November 1976. This investigation was supported in part by research grant no. RR 5476 from the General Research Support Branch, National Institutes of Health, and in part by the Medical Research Institute Council of the Michael Reese Medical Center. We thank Dr. Antonio Scommegna, Chairman of the Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center, for his cooperation, and Diane April and Darla Park for secretarial help. Please address requests for reprints to Dr. S. P. Gotoff, Department of Pediatrics, Michael Reese Hospital and Medical Center, Chicago, Illinois 60616.

Materials and Methods

Preparation of bacteria. GBBH streptococci strains IaSS615 (type Ia) and IIISS620 (type III) were obtained from the Center for Disease Control (Atlanta, Ga.) through Dr. Hazel Wilkinson. Bacteria from overnight cultures of GBBH streptococci type Ia in Todd-Hewitt (TH) broth were passaged in adult white mice weighing 26-28 g (Institute of Cancer Research strain; Scientific Small Animal Laboratory and Farm, Arlington Heights, Ill.) by the method of Lancefield et al. [12]. After 28 passages in mice, the injection of 1-2 X 103 cfu of GBBH streptococci type Ia re-


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Sera from 14 of 56 adult women protected mice from intraperitoneal challenge with mouse-virulent group B Streptococcus serotype Ia, and sera from seven of 25 nonparturient women in this group were bactericidal for >99% of the organisms in the presence of normal polymorphonuclear leukocytes. There were no discrepancies between the in vivo and in vitro assays. Protective activity was found in the IgG class in seven sera, and in the IgM class in one. Opsonic activity was partially dependent on heat-labile serum factors. Of 31 mother-cord serum pairs studied, seven maternal sera were protective, but four of the corresponding cord sera were not. Pooled human y-globulin injected by either the intraperitoneal or the intramuscular route protected mice from bacterial challenge.

Stewardson-Krieger et al.


Controls consisted of 10 animals injected with 0.1 ml of bacterial suspension and 0.1 ml of PBS and 10 animals injected with 0.2 ml of PBS alone. Mice were followed for five days, and the number of deaths was recorded. Serum was designated as protective if at least 75% of the mice survived the bacterial challenge, and as nonprotective if fewer than 50% of the mice survived. Bactericidal assay. The assay was performed by a modification of the method of Quie et al. [14]. Polymorphonuclear leukocytes (PMNL) were obtained from healthy human adults and prepared at a concentration of 25 X 106 PMNL/ml in Krebs-Ringer phosphate buffer, pH 7.4 (KRP-AD). Mouse-virulent bacteria were thawed in a water bath (56 C) and adjusted to a concentration of 5.0 X 106 cfujml with KRP-AD. Duplicate screwcapped glass tubes were filled with 0.2 ml of the PMNL suspension, 0.2 ml of test serum, 0.1 ml of bacterial suspension, and 0.5 ml of KRP-AD. Tubes lacking serum, PMNL, or both were also prepared, and the volumes were adjusted to 1.0 ml with KRP-AD. The tubes were incubated at 37 C on an end-over-end rotator (Scientific Instruments, St. Lake Worth, Fla.) at 10 revolutions/min. After incubation for 90 min, O.l-ml aliquots were removed, placed in 4.9 ml of sterile distilled water, and agitated for 30 sec to lyse PMNL and disperse intracellular bacteria. The samples were then serially diluted, and the number of cfu was determined by plating in triplicate on trypticase soy agar containing 5% sheep blood. The bactericidal index (BI) was calculated as [1 - (number of cfu at 90 min/number of cfu at zero-time)] X 100. Statistical analysis was performed by the Mann-Whitney V-test for analysis by ranks [15]. Results

Mouse protection tests. Sera from 14 of the total of 56 adult women protected at least 75% of the mice challenged with 1 LD 90 of GBBH streptococci type Ia (table 1). Of the 31 mother-cord serum pairs studied, three pairs demonstrated mouse protection. In four instances, only the maternal serum was protective, and in one instance, only the cord serum was protective. Absorption of sera with the homologous Ia strain completely removed mouse-protective activity against GBBH

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suited in the death of 160 (92%) of 174 21-day-old mice (woo) within five days. For mouse protection studies, aliquots of GBBH streptococci type Ia after the 28th passage were cultured for 18 hr in TH broth containing 5% defibrinated sheep blood and stored at -70 C until used. For bactericidal assays, aliquots of GBBH streptococci type Ia in the midlogarithmic phase cultured in TH broth containing 0.5% dextrose were frozen in an acetone-dry ice bath and stored at -70 C until used. More than 95% of the organisms were viable after storage. Collection and preparation of test sera. Blood samples were obtained from 25 normal nonparturient women aged 20-40 years, and the sera were frozen immediately at -70 C. Blood samples were also obtained from the umbilical cords of 31 normal, full-term infants at delivery and from their mothers. Sera were frozen at -70 C as soon as possible within 72 hr of collection. For some studies serum was heat-inactivated for 30 min at 56 C. Pooled 16% human y-globulin (Cutter Laboratories, Berkeley, Calif.) was used for the y-globulin protection studies. Sera were absorbed by the method of Lancefield et al. [12] using overnight cultures of organisms in TH broth, heat-killed at 56 C for 30 min and packed by centrifugation. Sera were mixed in a 10:1 ratio with bacteria, incubated at 37 C for 30 min, centrifuged, and sterilized by passage through Millipore filters with 0.45-p,m pores (Millipore Corp., Bedford, Mass.). Sera were chromatographed on a 2.5- X 160-cm Sephadex G-200 column (Pharmacia Fine Chemicals, Piscataway, N.J.). Sera were treated with 0.2 M 2-mercaptoethanol (2-ME) (Eastman Kodak Co., Rochester, N.Y.) following the method of Deutsch and Morton [13]. Samples were concentrated to the original volumes and sterilized by passage through Millipore filters with 0.45-p,m pores. Mouse protection test. Four or more 21-dayold white mice were injected ip [12] with 0.1 mlof 1....2 X 103 C£u of mouse-virulent GBBH streptococci type Ia in phosphate-buffered saline (PBS) and simultaneously with 0.1 ml of test serum or y-globulin in doses of 0.005-1.2 ml/kg. For some studies, y-globulin in doses of 0.04-0.6 ml/kg' was injected im 24 hr prior to ip bacterial challenge.


Immunity to Group B Streptococci

Table 1. Protection of mice against group B ~­ hemolytic Streptococcus type Ia with serum from adult women.

Serum Nonprotective

o o

No. of animals surviving/ no. tested

13 25 25 36 75 83 88

27 2 2 6 4 1 2 1 1

3/4 5/6 7/8




94 100 100

1 4 3

32/34 4/4

0/4 0/8 1/8

1/4 2/8 5/14


streptococci type Ia, whereas absorption of sera with heterologous type III bacteria failed to remove the mouse-protective effect. Two adult protective sera were filtered through Sephadex G-200. In both instances, mouse-protective activity was found only in the second or 7S fraction. There was no loss of mouse-protective activity after 2-ME treatment of seven of eight adult protective sera. The single serum that lost its mouse-protective effect after 2-ME treatment had been obtained from a mother whose infant's cord serum lacked protective activity against GBBH streptococci type la. Of the remaining discordant mother-cord pairs, samples were insufficient for gel filtration or 2-ME treatment. Pooled human y-globulin protected 100% of the mice from an LD 90 challenge of GBBH streptococci type la at a dose of as low as 0.075 ml/kg by the ip route. When injected im 24 hr prior to bacterial challenge, pooled human y-globulin protected all of the mice at a dose of as low as 0.3 ml/kg, Bactericidal assays. Sera from 25 nonparturient women (seven protective sera and 18 nonprotective sera) were studied in the bactericidal assay. The combination of PMNL and fresh frozen protective serum was always bactericidal for >99% of GBBH streptococci type la organisms (figure 1). Heat inactivation of protective serum significantly reduced its opsonic effect (P < 0.001), but the


The mouse protection test and the in vitro bactericidal assay have been utilized in the past as models for the study of human immunity to Streptococcus pneumoniae and group A Streptococcus pyogenes [8-11]. Earlier studies have demonstrated that type-specific antibodies in rabbit antisera protect mice against GBBH streptococci serotypes Ia, Ib, Ic, and II [12, 16], but this model has not been adapted for the analysis of human sera. Rabbit antibody to the group B carbohydrate is not mouse-protective, and cross..reactions between the subgroups of type I have been demonstrated. Both rabbit immune sera and human sera have been studied with in vitro phagocytic assays against all five GBBH streptococcal serotypes, and these experiments have reiterated the role of both type-specific and cross-reactive antibodies in the opsonization of GBBH streptococci [17-21]. We have found the mouse to be a useful, reproducible model for the study of human immunity to GBBH streptococci type la. In addition, we

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Percentage of mice surviving No. of sera

protective serum remained partially opsonic (P < 0.01). The addition of 10% fresh frozen nonprotective serum restored the opsonic activity of heatinactivated protective sera, although this 10% nonprotective serum was not opsonic when used alone. There were significantly fewer cfu after incubation for 90 min in tubes containing protective sera than in tubes containing nonprotective sera (P < 0.001) or bacteria in buffer alone (P < 0.05). This finding was not affected by heat inactivation. The combination of PMNL and freshly frozen nonprotective serum was partially bactericidal, with a mean BI (±SE) of 43% ± 27%. Heat inactivation of nonprotective serum destroyed this opsonic effect. There were significantly more bacterial cfu after incubation for 90 min in tubes containing nonprotective sera than in tubes containing bacteria in buffer alone (P < 0.001), and this finding was not altered by heat inactivation (figure 1). A single non protective serum sample had a Bl of >90%. This serum protected five of 14 mice from bacterial challenge (table 1). The remaining 17 nonprotective sera analyzed in the bactericidal assay protected ~25% of the mice.


Stewardson-Krieger et al.











-i .....




i- Hl •

tJj -----

0 I()

• ••




- ---





~ ffi•










~ • ~----

• •

'MN ~









Figure 1. Bactericidal assay of group B p-hemolytic Streptococcus type la. The bactericidal index was calculated as [1- (number of cfu at 90 min/number of cfu at zero-time)] X 100. PMN = polymorphonuclear leukocytes; P = mouse-protective serum; and NP = mouse-nonprotective serum. The bars in each column represent mean bactericidal index ± SE.

have shown an excellent correlation between the in vivo mouse protection test and the in vitro bactericidal assay. Sera from 14 (25%) of 56 normal women protected mice from lethal challenge with type Ia, and this effect could be specifically absorbed with the homologous strain. The in vitro bactericidal assay consistently discriminated protective from nonprotective sera. Opsonic activity against GBBH streptococci type Ia was partially dependent on heat-labile serum factors, presumably complement. Unexpectedly, significantly fewer bacterial c£u were produced when type Ia organisms were grown in protective sera. This effect was due to the formation of longer streptococcal chains when organisms were grown in immune sera in contrast to nonimmune sera [22], a reaction previously reported with group A streptococci [23]. Although the role of antibody in the opsonization of most strains of GBBH streptococci has been convincingly demonstrated [17-21], previous

reports using rabbit hyperimmune sera and human sera have not resolved to what extent complement participates in opsonization of GBBH streptococci. Although complement was not required for opsonization of GBBH streptococci by rabbit hyperimmune serum in investigations by Anthony [20] and Hemming et al. [19], the latter study demonstrated that complement significantly enhances opsonization of serotypes Ia and II, but not III. Baltimore et al. have recently reported that rabbit immune sera, leukocytes, and complement are necessary for optimal killing of all serotypes of GBBH streptococci with the exception of one of 10 type III strains, although some reduction in number of c£u with strains of Ia, Ib, Ic, and II was noted in the absence of complement [21]. Lancefield and Freimer [16] and Klesius et al. [17] found that opsonization of serotype Ia by human serum requires complement, whereas opsonization of the other four serotypes of GBBH streptococci is unaffected by complement. Our re-

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Immunity to Group B Streptococci

cocci, then passive immunization of newborn infants might be utilized to prevent neonatal disease. We have shown that pooled human 'Yglobulin protected mice from ip challenge with I LDgO of type Ia when injected either ip at the time of challenge or im 24 hr before challenge in clinically acceptable doses [24]. Additional ariimal studies with other serotypes of GBBH streptococci should be carried out before trials of human pooled or immune 'Y-globulin are performed in newborn infants. References

I. Hood, M., Janney, A., Dameron, G. Beta hemolytic streptococcus group B associated with problems of the perinatal period. Am. J. Obstet. Gynecol. 82: 809-818, 1961. 2. Eickhoff, T. C., Klein, J. 0., Daly, A. K., Ingall, D., Finland, M. Neonatal sepsis and other infections due to group B beta-hemolytic streptococci. N. Engl. J. Med. 271:1221-1228,1964. 3. Lancefield, R. C. Cellular antigens of group B streptococci. In L. W. Wannamaker and J. M. Matsen [ed.]. Streptococci and streptococcal diseases: recognition, understanding, and management. Academic Press, New York, 1972, p. 57-65. 4. Patterson, M. J., Hafeez, A. E. B. Group B streptococci in human disease. Bacteriol. Rev. 40:774-792, 1976. 5. Quinn, R. W., Lowry, P. N. Streptococcal M protein antibodies acquired at birth. Pediatrics 39:778-780, 1967. 6. Zimmerman, R. A., Hill, H. R. Placental transfer of group A type-specific streptococcal antibody. Pediatrics 43:809-814, 1969. 7. Baker, C. J., Kasper, D. L. Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294: 753-756, 1976. 8. MacLeod, C. M., Hodges, R. G., Heidelberger, M., Bernhard, W. G. Prevention of pneumococcal pneumonia by immunization with specific capsular polysaccharides. J. Exp. Med. 82:445-465, 1945. 9. Diefendorf, H. W. A method for detecting in human serum protective bodies against hemolytic streptococci. Proc. Soc. Exp. BioI. Med. 48:56-60, 1941. 10. Maxted, W. R. The indirect bactericidal test as a means of identifying antibody to the M antigen of Streptococcus pyogenes. Br. J. Exp. Pathol. 37:415422, 1956. II. Denny, F. W., j-, Perry, W. D., Wannamaker, L. W. Type-specific streptococcal antibody. J. Clin. Invest. 36:1092-1100, 1957. 12. Lancefield, R. C., McCarty, M., Everly, W. N. Multiple mouse-protective antibodies directed against group B streptococci: special reference to antibodies effec-

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sults with human sera indicate that both antibody and heat-labile serum factors contribute to opsonization and killing of type la. Since only a small fraction of colonized human neonates develop invasive GBBH streptococcal disease, it has been suggested that infants who lack transplacentally acquired antibody are at greater risk for developing GBBH streptococcal infection [7]. Baker and Kasper [7] and Hemming et al. [19] have shown that infants infected with GBBH streptococci and their mothers lack type-specific antibodies. In two earlier studies utilizing a slide phagocytic assay, it was reported that whereas only 5%-10% of human sera possess type-specific opsonic activity for serotype la, serotypes Ib, Ie, II, and III are nonspecifically opsonized by 90~o-95% of human sera [17, 18]. In contrast, Hemming et al. [19] demonstrated that opsonization of serotypes Ia, II, and III with human sera is type-specific. In their study, 74~o of adult human sera opsonized serotype III, 40% opsonized type II, and 12% opsonized type la. Regarding the ability of human opsonins for GBBH streptococci to cross the placenta, two studies have found comparable levels of antibody to GBBH streptococci in all mother-cord serum pairs [7, 19]. Hemming et al. [19] demonstrated one discordant pair for serotype II and one for serotype III and also showed that maternal opsonin levels are frequently higher than corresponding cord levels. In our studies, maternal protective activity was absent in cord sera in four of seven mothercord serum pairs. In one pair, the cord serum, but not the maternal serum, showed protection, an observation for which we presently have no explanation. All of the GBBH streptococcal opsonins studied by Hemming et al. [19] belonged to the IgG class. In the present series, protective activity was usually found in the IgG class, but was found in the IgM class in at least one instance. Although maternal immune factors play an important role in protecting some neonates from GBBH streptococcal disease, our results indicate that the presence of maternal antibody cannot invariably be relied upon to provide protection of the human newborn against GBBH streptococcal infection. If antibody is important in the newborn infant's resistance to infection with GBBH strepto-



13. 14.

15. 16.



tive against protein antigens. J. Exp, Med. 142:165179,1975. Deutsch, H. F., Morton, J. I. Dissociation of human serum macroglobulins. Science 125:600-601, 1957. Quie, P. G., White, J. G., Holmes, B., Good, R. A. In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood. J. Clin. Invest. 46:668-679, 1967. Siegel, S. Nonparametric statistics for behavioral sciences. John Wiley and Sons, New York, 1956, p. 116. Lancefield, R. C., Freimer, E. H. Type-specific polysaccha"ride antigens of group B streptococci. J. Hyg. 64:191-203,1966. Klesius, P. H., Zimmerman, R. A., Mathews, J. H., Krushak, D. H. Cellular and humoral immune response to group B streptococci. J. Pediatr. 83:926932,1973. Mathews, J. H., Klesius, P. H., Zimmerman, R. A. Opsonin system of the group B streptococcus. Infec. Immun, 10:1315-1320, 1974. Hemming, V. G., Hall, R. T., Rhodes, P. G., Shigeoka,






A. 0., Hill, H. R. Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence. J. Clin. Invest. 58:13791387,1976. Anthony, B. F. Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Ib, and Ie. J. Exp. Med. 143:1186-1198, 1976. Baltimore, R. S., Kasper, D. L., Baker, C. J., Goroff, D. K. Antigenic specificity of opsonophagocytic antibodies in rabbit antisera to group B streptococci. J. Immunol. 118:673-678, 1977. Stewardson-Krieger, P., Albrandt, K., Kretschmer, R. R., Gotoff, S. P. Group B streptococcal long chain reaction. Infec. Immun., 1977 (in press). Stollerman, G. H., Siegel, A. C., Johnson, E. E. Evaluation of the "long chain reaction" as a means for detecting type-specific antibody to group A streptococci in human sera. J. Exp. Med. 110:887-897, 1959. Janeway, C. A., Rosen, F. S. The gamma globulins. IV. Therapeutic uses of gamma globulin. N. Engl. J. Med.275:826-831,1966.

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Stewardson-Krieger et al.

Perinatal immunity to group B beta-hemolytic streptococcus type Ia.

THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136. NO.5. NOVEMBER 1977 © 1977 by the University of Chicago. All rights reserved. Perinatal Immunity to Gr...
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