INFECTION AND IMMUNITY, Mar. 1990, p. 687-6940019-9567/90/030687-08$02.00/0Copyright C 1990, American Society for Microbiology

Synthesis and Immunological Properties of Conjugates Composed ofGroup B Streptococcus Type III Capsular Polysaccharide

Covalently Bound to Tetanus ToxoidTERESA LAGERGARD,lt* JOSEPH SHILOACH,2 JOHN B. ROBBINS,' AND RACHEL SCHNEERSON'

National Institute of Child Health and Human Development' and National Institute of Diabetes,Digestive, and Kidney Diseases,2 Bethesda, Maryland 20892

Received 1 December 1989/Accepted 6 December 1989

A synthetic scheme for covalently binding group B streptococcus type III to tetanus toxoid (TT), using adipicacid dihydrazide as a spacer, is described. Type Ill alone or as a conjugate with TT was injectedsubcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by theseimmunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after eachimmunization than did the type HI alone. These levels were related to the dosage of the conjugate, enhancedby Freund adjuvant, and exhibited booster responses. Type III alone elicited only immunoglobulin M (IgM)antibodies in Swiss albino mice and mostly IgM and low levels of IgG antibodies of the IgG3 subclass in BALB/cmice. Type Ill-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type HIantibodies were not detected. The first two immunizations with the conjugates elicited type HI antibodies in theIgGl and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injectionof type Ill-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type IIIorganisms and did not react with the structurally related pneumococcus type 14. TT alone or as a componentof type IH-TT induced mostly antibodies of the IgG class; IgGl levels were the highest of the four subclasses.No IgA TT antibodies were detected. The conjugation procedure, therefore, enhanced the immunogenicity ofand conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TTcomponent. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type IIIantibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women ofchild-bearing age is planned.

Lancefield, in 1933, classified hemolytic streptococci intogroups (39). At that time, almost all severe streptococcalinfections of humans were due to group A. Lancefield notedthat group B streptococci (GBS) from parturient womenwere associated with mild infections (42). In 1938, Fryreported three cases of fatal puerperal fever due to GBS (29).It was an unexpected phenomenon when GBS were reportedto be the most frequent cause of neonatal sepsis at theBoston City Hospital during 1961 to 1963 (22). This apparentincrease of neonatal sepsis caused by GBS was confirmed (2,9, 28). GBS remain a frequent cause of serious infections inneonates: about two-thirds of strains from patients are oftype III (3, 11, 18, 26). GBS are now also a major cause ofpuerperal infections (3, 25).

Lancefield showed that passive protection of mice byrabbit hyperimmune sera was type specific (41, 43). Thestructure of these type antigens, shown to be capsularpolysaccharides (CP), has been elucidated (34, 36, 37, 57,58). The virulence ofGBS is due to their ability to invade theblood stream and multiply. This property of invasiveness isrelated to the anti-phagocytic properties conferred to GBSby its CP (8, 16, 20, 45, 55). The protective activity of humanantibodies has been demonstrated by in vitro assays and invivo animal models and by the correlation between thesusceptibility of neonates to invasive GBS diseases with theabsence of placentally transmitted IgG type III antibodies(4-7, 16, 21, 23, 55).

* Corresponding author.t Present address: Department of Medical Microbiology, Univer-

sity of Goteborg, Goteborg, Sweden.

The structure of type III and its immunologic properties inadults have been characterized (Fig. 1) (4, 5, 21, 23, 27, 35,38, 58). Jennings et al. proposed that the carboxyl of the-D-NeuNAcp was linked to the 0-3 of the adjacent -D-galactose to form the type-specific site (35, 58). Without thisNeuNAc moiety, the structure of type III is identical to thatof the pneumococcus type 14 CP (27, 35, 38). Antibodieselicited by type III have secondary biological properties thathave been correlated with protective immunity (4-8, 11, 16,20, 45, 55). Active immunization of newborns is not practi-cal, since most cases occur within the first month of life (3).Type III as a vaccine for maternal-fetal passive immuniza-tion is limited because it induces protective levels of IgGantibodies in only about 60% of antibody-negative women ofchild-bearing age (5, 6). We used the approach, reported byAvery and Goebel in 1931, of binding type III to a protein inorder to increase its immunogenicity (1, 30). A syntheticscheme for binding Haemophilus influenzae type b CP toproteins resulted in increased immunogenicity and T-celldependent properties to this CP (12, 13, 50). In contrast tothe age-related and the T-independent properties of the CPalone, a H. influenzae type b CP conjugate elicited boosterresponses, comprised mostly of IgG, and protected .12-month-old children against meningitis caused by this patho-gen (24). Here, we describe the synthesis of a GBS-tetanustoxoid (TT) conjugate (type III-TT) and some of its immu-nologic properties in mice.

MATERIALS AND METHODS

Reagents. Yeast extract and tryptic soy broth were fromDifco Laboratories, Detroit, Mich.; mutanolysin, DNase,

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688 LAGERGARD ET AL.

->3 if-D-Galp-( I->4 ) --D-Glep-( 1->6 ) -J-D-GlcNAcp-( I-n4I

-D-Ga lp3I2

O(-D-NeuNAc p

FIG. 1. The structure of the GBS type III capsular polysaccha-ride as described by Wessels et al. (58).

RNase, pronase, bovine serum albumin, adipic acid dihy-drazide (ADH), avidin, agarose, and thimerosal were fromSigma Chemical Co., St Louis, Mo.; 4B-CL Sepharose,S-300 Sephacryl, and DEAE Sephacel were from Pharmacia,Inc., Piscataway, N.J.; sterile pyrogen-free water and pyro-gen-free saline were from Travenol Laboratories, Lincoln,Ill.; cyanogen bromide and trinitrotoluene benzene sulfonicacid were from Eastman Chemical Products, Inc., Roches-ter, N.Y.; 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidewas from Calbiochem-Behring, Fullerton, Calif.; and N-hydroxysuccinimidobiotin was from Pierce Chemical Co.,Rockford, Ill. TT was kindly donated by C.-Y.Chu, Shang-hai Institute of Biological Products. The TT was concen-trated and passed through an S-300 Sephacryl column (5 by90 cm) (12). Fractions, corresponding to a molecular mass of-150 kilodaltons, were concentrated by vacuum dialysis to41 mg/ml, sterile filtered (0.4-nm membrane; Nalgene Lab-ware Div., Rochester, N.Y.), and stored at 3 to 8°C. The Kdof this preparation, through CL-4B Sepharose, was 0.71.Group B-specific and type-specific antisera, prepared byRebecca Lancefield, were donated by Emil C. Gotschlich,The Rockefeller University, N.Y. Another GBS type IIIantiserum and a pneumococcus type 14 typing antiserumwere donated by J0rgen Henrichsen, Statens Seruminstitut,Copenhagen, Denmark.

Bacterial growth. GBS type III, strain 110, was kindlyprovided by Stephen Mattingly, University of Texas HealthScience Center, San Antonio, Texas. This strain was culti-vated on tryptic soy broth agarose overnight at 37°C. Severalcolonies were inoculated into 2.8-liter baffled Fernbachflasks (Bellco Glass, Inc., Vineland, N.J.) containing 1.0 literof media (1.0% yeast extract dialysate, 1% dextrose, 0.1 Msodium phosphate; final pH 7.2). The flasks were placed ona rotary shaker and incubated at 37°C at 100 rpm for 18 h.These flasks were then delivered to 20-liter carboys contain-ing the same growth media (10% inoculum) and incubated forabout 18 h at 37°C. The bacteria were separated by centrif-ugation and stored at -20°C. The culture supernatant waspassed through a 0.4-nm filter and concentrated by ultrafil-tration (10,000-molecular-weight-cutoff membrane; PelliconCassette System, Bedford, Mass.) to -10% of its originalvolume.

Purification of type III. The CP from the bacterial pelletand the culture supernatant were treated separately. Thebacterial pellet was treated with 5,000 U of mutanolysin per60 to 80 g of bacteria (wet weight), as described previously(10, 17, 19, 54, 60). The cells were then removed bycentrifugation. The supernatant from the enzyme-treatedbacteria and from the culture supernatant were treatedsequentially with DNase, RNase, and pronase and thenextracted with cold phenol (50). The products from thesupernatant were passed through 4B-CL Sepharose equili-brated in 0.2 M NaCl to remove low-molecular-weightmaterial (.0.6 Kd). The higher-molecular-weight fractionsfrom the supernatant and the products from the bacteria

were fractionated by anion-exchange chromatographythrough DEAE-Sephacel by linear NaCl gradient elution(10). Fractions containing type III, as tested by doubleimmunodiffusion, were dialyzed extensively against sterilepyrogen-free water, freeze-dried, and stored at -20°C. Theyields of type III from 500 g (wet weight) of bacterial pelletwere about 300 mg, and those from the culture supernatantwere about 130 mg.

Analysis. Type III was measured by the anthrone reactionby using the purified CP as a standard (56). Protein wasmeasured by the method of Lowry et al., by using bovineserum albumin as a standard (59). The adipic acid hydrazide(AH) content of the derivatized type III was measured by thetrinitrotoluene benzene sulfonic acid reaction by using ADHas a standard (33). The partition coefficients (Kd) throughCL-4B Sepharose were calculated as previously described(59). Gas-liquid chromatography was kindly performed byWillie F. Vann, Office of Biologics, Research and Review,Food and Drug Administration, to measure the content ofrhamnose (group B cell wall polysaccharide) (14, 32). Themolecular weight was estimated by end group analysis byusing the Park-Johnson reaction, with dextrose as the stan-dard (46).

Synthesis of type III-TT conjugates. The synthesis followedclosely the scheme used for H. influenzae type b CP conju-gates (12). Briefly, type III was activated with cyanogenbromide at pH 10.5 for 6 min at 4°C in a pH stat (Radiometer,Copenhagen, Denmark). The weight ratio of cyanogen bro-mide-CP was 0.5, 1.0, and 1.5 for conjugates 1, 2, and 3,respectively. ADH was added in 0.5 M NaHCO3 to a finalconcentration of 0.25 M, pH 8.5. After tumbling for 18 h at3 to 8°C, the reaction mixture was dialyzed against 0.2 MNaCl at 3 to 8°C and passed through a 4B-CL Sepharosecolumn. The CP-containing fractions were pooled, dialyzedagainst sterile pyrogen-free water, and freeze-dried. A solu-tion containing 10 mg each of type III-AH and TT per ml wasbrought to pH 5.6 with 0.1 N HCl. 1-Ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide was added to a final concentra-tion of 0.05 M, and the pH was maintained at 5.6 with 0.1 NNaOH for 3 h at room temperature. The reaction mixturewas dialyzed against 0.2 M NaCl at 3 to 8°C and was passedthrough a 4B-CL Sepharose column (5 by 95 cm) equili-brated in 0.2 M NaCl. The void volume fractions were storedin 0.01% thimerosal at 3 to 8°C.Immunization of mice. General purpose, Swiss albino, or

BALB/c mice, 6 weeks old, 8 to 10 per group, were injectedsubcutaneously with 2.5 ,uig of the type III alone or as acomponent of a conjugate (12). For the dosage-responseexperiment, mice were injected with 0.5, 2.5, or 5.0 ,ug of thetype III-TT, on the basis of its CP content. Separate groupswere injected 1, 2, or 3 times at 2-week intervals and werebled 2 weeks after the first injection and 1 week after thesecond and third injections. Controls were mice which werebled before the first immunization or those injected withsaline and bled after the last injection. One group of micewas injected with 5.0 ,ug of the type III-TT in completeFreund adjuvant (FA) and reinjected 4 weeks later with thesame conjugate in incomplete FA. Two weeks later, thesemice were bled and their pooled sera, assigned a value of1,000 U, were used as a reference standard.

Serology. Double immunodiffusion and capillary precipita-tion were performed with rabbit typing antisera to GBStypes Ia, Ib, II, and III and to pneumococcus type 14.Enzyme-linked immunosorbent assay (ELISA) was per-formed with avidin-treated plates and biotinylated type III asthe coating antigen (53). Plates were coated with 4.0 pLg of

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GROUP B STREPTOCOCCUS TYPE III-YT CONJUGATES

TABLE 1. Composition of GBS type III CP-TT conjugates

Conjugate % Yield AH/CP CP/proteinof CP (% [wt/wt]) (wt/wt)

1 11.0 1.0 0.132 32.0 1.3 0.173 36.0 1.8 0.20

avidin per ml and 4.0 pg of biotinylated type III per ml. Eightthreefold dilutions of reference and test sera were added.Horseradish peroxidase conjugated rabbit anti-mouse immu-noglobulin (Dakopatts, Copenhagen, Denmark) was addedand read at 450 nm. For assay of the isotype composition ofGBS type III and TT antibodies, alkaline phosphatase-coupled goat anti-murine immunoglobulin class and IgGsubclass antibodies were obtained from Southern Biotech-nology Association, Birmingham, Ala. The specificity ofthese antibody reagents has been reported (31). The optimalconcentration of each antiglobulin reagent was determinedby checkerboard titrations. The immunoglobulin class andIgG subclass antibody content was expressed as the recip-rocal of the serum dilution giving an absorbance of 0.2optical density above the background level; values less thanthis level at a serum dilution of 1:10 were considered as beingnegative (<10). TT antibodies were measured by ELISA aspreviously described (12).

Total type III and TT antibody levels were calculated byparallel line analysis compared with the hyperimmune refer-ence serum and were expressed in ELISA units (44).

Opsonization of GBS type III organisms was assayed aspreviously described (8, 15). Guinea pig plasma and periph-eral blood leukocytes were used. Leukocytes were sepa-rated by using gradient centrifugation in sodium metrizoate(Nyegaard Co., Oslo, Norway) and methylcellulose (FlukaChemical Corp., Ronkonkona, N.Y.). Bacteria (1 x 108/ml)were mixed with the test sera or saline and with 1 x 106leukocytes per ml in the proportions 1:1:2. Three test sera,with antibody levels close to the geometric mean (GM), were

used for each group. The reaction mixtures were incubatedfor 30 min and stained with either Giemsa stain or Loeffler'smethylene blue. One hundred granulocytes were examinedon each stained smear, and the results were expressed as thenumber of bacteria per granulocyte minus the level observedwith saline (phagocytic index) and the standard deviation.Pneumococcus type 14 antibodies were measured by

Gerald Schiffman, Downstate Medical School, SUNY,Brooklyn, N.Y. (49).

E

CONJUGATE No.2

tKd tetanustoxoid

0.4

0

0.3

:30.2

0.1

10 20fV

30 40 50 60fV

70

°0 N E Vt

FRACTION NUMBER

FIG. 2. Gel filtration of GBS type III CP-TT conjugate number 2through a CL-4B Sepharose column (2.5 by 90 cm). The opticaldensity of the fractions at 280 nm is shown on the left axis and theoptical density of the fractions, as measured by the anthronereaction (optical density at 620 nm), is shown on the right axis. Theeluting solvent was 0.2 M NaCl, and the fraction size was 5.0 ml.

Statistical analysis. The unpaired t test was used to calcu-late p values between experimental groups.

RESULTS

Characterization of the type III-TT conjugates (Table 1).Type III, prepared from either the bacteria or the culturesupernatants, had similar compositions (less than 1% proteinor nucleic acids, no detectable mannose, and 5.0% rham-nose) and molecular size characteristics; their molecularweight, as estimated by reducing end group analysis, was

-30,000. The Kd of both these preparations, through CL-4BSepharose, was 0.46. Derivatized type III preparations hadfrom 1.0 to 1.8% (wt/wt) AH; the Kd of the AH derivativeswas 0.46, as was the Kd native CP.Both the native and AH-derivatized type III reacted with

the homologous antisera by immunodiffusion; neither re-

acted with the pneumococcus type 14 antisera (data notshown). Figure 2 shows a representative elution profile ofthe reaction mixture (conjugate number 2) following theconjugation. The void volume contained the conjugate.

TABLE 2. Comparative levels of type-specific antibodies elicited in mice by GBS type III CP (type III CP) alone or conjugated to TTa

Type III CP antibodies (ELISA units), GM (range)Immunogen

Post-injection 1 Post-injection 2 Post-injection 3

Experiment lbCP (bacteria) 3.8 (0.5-11.0)* 3.6 (0.5-10.0)* Not doneCP (supematant) 2.8 (0.5-9.0)* 3.2 (1.0-8.6)* Not doneConjugate 1 8.7 (3.6-17.7)** 20.7 (9.0-46.5)t 21.1 (9.1-48.8)tConjugate 2 10.7 (2.5-31.1)** 49.4 (29.1-151.6)t 73.3 (15.9-328)4

Experiment 2CCP (supernatant) Not done 6.7 (2.0-32.5)§ Not doneConjugate 2 15.9 (4.0-33.1)§ 92.4 (38.9-311)11 Not doneConjugate 3 17.5 (7.2-48.4)§ 116.7 (37.4-250)I Not done

a The sera from controls (saline injected) and from nonimmune mice had 0.6 (0.4 to 1.1) U of type III CP antibody.b For general purpose mice, n = 10 for each bleeding. ** versus *, P < 0.05; t versus *, P < 0.001; t or 11 versus *, P = 0.001; : versus t, P < 0.001.' For the Swiss albino mice, n = 8 for each bleeding. 11 versus §, P < 0.001.

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TABLE 3. Dosage-antibody response elicited by GBS type III CP-TT conjugate 2 in micea

p.g of CP in Type III CP antibodies (ELISA units), GM (range)'type III CP-TT Post-injection 1 Post-injection 2 Post-injection 3

0.5 5.0 (1.3-12.0)* 6.0 (2.011.1)* 5.6 (2.0-9.0)*2.5 10.7 (2.5-31.1)* 43.1 (29.4.152.6)** 72.5 (15.0-328)**5.0 20.4 (6.9-53.6)* 74.2 (17.0-104.1)** Not done

a General purpose mice were injected with various dosages of type III-TT, subcutaneously, as described in Materials and Methods.b** versus *, P < 0.001.

About 50% of the type III remained unbound. We could notdetect unbound TT. No unbound type III was detected byimrmunodiffusion or gel filtration through CL-4B Sepharosein the three preparations of conjugates (data not shown).Increasing derivatization of the type III to 1.8% AH wasrelated to both a higher yield (percent CP recovered in theconjugate) and to a higher CP/protein ratio.

Immunogenicity in mice (Table 2). Only low levels of typeIII antibodies were detected in preimmunization sera or fromthe control mice. Immunization with the type III CP fromeither the bacteria or culture supernatant elicited low butsignificant antibody rises (P < 0.001); there was no boostereffect and no significant difference in the levels elicited byeither preparation.

Conjugates 1 and 2 were tested in general purpose mice,add conjugates 2 and 3 were tested in Swiss albino mice. Allthree conjugates were more immunogenic than the type IIIalone. After the first injection, the levels elicited by the threeconjugates were higher than those elicited by type III alone(P < 0.05). The difference between the conjugates and thetype III alone after the second injection was P < 0.001.There was no significant difference between the type IIIantibody levels elicited by the conjugates after the firstinjection; after the second injection, the difference betweenconjugate 1 and type III was P = 0.001 and the differencebetween conjugates 2 and 3 and type III was P < 0.001.Higher levels of type III antibodies were achieved in Swissalbino than in general purpose mice with conjugate 2 (P <0.05). There was, however, no statistical difference betweenthe antibody levels elicited by conjugates 2 and 3 in Swissalbino mice after two injections.There were no rises of pneumococcus type 14 antibody

levels in the postimmunization sera.Dosage-antibody response (Table 3). The 0.5-,ug dosage of

conjugate 2 elicited only a slight antibody response after thefirst injection; this low level did not increase after the secondand third injections. Increasing the dosage to 2.5 ,ug of

conjugate resulted in greater antibody synthesis, withbooster effects after injections 2 and 3 (P < 0.01). Doublingthis dosage resulted in still higher antibody levels.Immunoglobulin class composition of type III antibodies

(Table 4). The unconjugated type III elicited only low levelsof IgM antibodies in the Swiss albino mice. BALB/c miceresponded with high levels of IgM and low levels of IgG typeIII antibodies. The type III-TT conjugate, in contrast, elic-ited both IgM and IgG antibodies in the Swiss albino miceafter one injection. A second injection of type III-TT eliciteda 10- to 40-fold rise of IgG and only a 4- to 7-fold rise of IgMantibodies in the two strains of mice. A third injection oftype III-TT did not elicit a booster response of IgM or IgGantibodies in Swiss albino mice. Two injections of typeIII-TT in FA elicited the highest levels of both IgM and IgGantibodies (P = 0.001).IgA type III antibodies were not detected in any of the

experimental groups (data not shown).IgG subclass composition of type III antibodies (Table 5).

The low levels of IgG type III antibodies elicited by the CPalone in the BALB/c mice comprised IgG3 only. One injec-tion of type III-TT elicited low levels of IgGl in Swiss albinomice; reinjection increased IgGl and induced detectableIgG3 antibodies. The levels of these two IgG antibodies werenot increased by a third injection of the type III-TT conju-gate, but low levels of IgG2a antibodies were detected.Similar antibody responses were obtained in BALB/c mice(data not shown).Type III-TT in FA injected into Swiss albino mice elicited

high levels of IgGl, IgG2a, and IgG3 type III antibodiescompared with those elicited by the conjugate in saline (P <0.001). Also, low levels of IgG2b type III antibodies weredetected in the mice injected with III-TT in FA.

Opsonization by conjugate-induced type III antibodies (Ta-ble 6). A low level of phagocytosis was observed with threesera from nonimmune mice (mean index, 4.1). The referenceserum had a phagocytic index of 2,500. There was a consis-

TABLE 4. IgM and IgG composition of CP antibodies in mice injected with GBS type III alone or conjugated to TT (type III-TT)

ugg of Mouse GM antibody titer (range)CP/mouse strain IgG gM

Type III 2.5 10 SAa <10 50 (15-79)Type III 2.5 5 BALB/c 63 (25-316) 794 (251-3162)Type III-TT x 1 2.5 10 SA 63 (10-2512) 40 (10-398)Type III-TT x 2 2.5 8 SA 2,405 (199-31622) 316 (199-631)Type III-TT x 3 2.5 10 SA 1,584 (100-63095) 365 (63-1000)Type III-TT x 1 5.0 10 SA 45 (16-199) 125 (25-794)Type III-TT x 2 5.0 10 SA 1,000 (50-3980) 631 (25-1995)Type III-TT x 1 2.5 5 BALB/c 100 (31-1000) 100 (20-1259)Type III-TT x 2 2.5 5 BALB/c 1,000 (79-2512) 398 (316-631)Type III-TT + FAb 5.0 10 SA 3.98 x 104 5,011

a SA, Swiss albino mice.bTen mice were injected with 5.0 ,ug of CP as type III-TT with FA (see Materials and Methods) and their sera pooled.

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GROUP B STREPTOCOCCUS TYPE III-TT CONJUGATES

TABLE 5. Immunoglobulin G subclass composition of CP antibodies in mice injected with GBS type IIIalone or conjugated to TT (type III-TT)

ugg of Mouse GM antibody titer (range)Immunogen CP/mouse No. strain IgG1 IgG2a IgG2b IgG3

Type III x 2 2.5 5 BALB/c <10 <10 <10 16 (10-32)Type III x 2 2.5 8 SAa <10 <10 <10 <10Type III-TT x 2 2.5 7 SA 10 (<10-199) <10 <10 <10Type III-TT x 2 2.5 8 SA 204 (25-7942) <10 <10 63 (10-501)Type III-TT x 3 2.5 7 SA 251 (40-6309) 10 (<10-251) <10 63 (10-1000)Type III-TT x 1 5.0 10 SA 10 (<10-80) <10 <10 <10Type III-TT x 2 5.0 10 SA 316 (25-5012) <10 <10 79 (12-251)Type III-TT + FA 5.0 10 SA 3.16 x 104b 1,259 316 1.26 x 104

a SA Swiss albino mice.b Mice were injected with 5.0 ,ug of CP as type III-TT with FA. Their sera were pooled and used as a reference standard.

tent rise in the phagocytic index of the three mice injectedwith conjugates 1 and 2 after each injection. The phagocyticindexes of these immune sera correlated with their type IIIantibody levels measured by ELISA.TT antibodies (Table 7). Antibodies to TT were not detect-

able in the preimmune or in the control sera. All miceresponded with TT antibodies after the first injection andshowed a booster response after the second injection (P =0.0001). The third injection increased the TT antibody levelsonly slightly. No differences in the TT antibody responseselicited by the two conjugates were observed.Immunoglobulin composition of TT antibodies (Table 8).

Injection of the type III-TT in saline did not elicit detectableTT antibodies of the IgM class. IgM TT antibodies, how-ever, were detected in the pooled sera of Swiss albino miceinjected with the type III-TT in FA (data not shown). IgA TTantibodies were not detected in any of the mouse sera.One injection of the type III-TT elicited TT antibodies

composed of all four IgG subclasses. A second injectionelicited increases in the levels of all IgG subclasses of TTantibodies; these levels increased further after a third injec-tion. A composition of IgG subclasses similar to that elicitedby the conjugate was elicited by injection of TT followed byone injection of the conjugate. The type III-TT in FA elicitedthe highest levels of IgG TT antibodies, composed of all IgGsubclasses.As expected, the type III alone did not elicit TT antibod-

ies.

DISCUSSION

There is convincing evidence from in vitro assays, in vivoanimal models, and clinical studies that the CP of GBS are

TABLE 6. Opsonization activity of type-specific antibodieselicited in mice by injection of GBS type III CP-TT conjugatesa

No. of Mean phagocytic index (- SD)bAntisera serum

specimens Conjugate 1 Conjugate 2

Post-injection 1 3 7.2 ± 2.1 11.0 + 2.3Post-injection 2 3 14.0 ± 2.8 20.0 ± 2.5Post-injection 3 3 20.3 + 3.5 30.2 ± 4.1

a Opsonization of GBS type III organisms, using guinea pig plasma ascomplement and their peripheral blood leukocytes for granulocytes, wasassayed in sera from mice injected 1, 2, or 3 times with conjugate numbers 1and 2. The reference antiserum (Materials and Methods) had a phagocyticindex of 2,500 and three nonimmune mouse sera had a phagocytic index of4.1.

b Number bacteria per granulocyte with test sera minus saline control.

both virulence factors and protective antigens (4-8, 16, 21,27, 38, 41, 43). Their immunopathogenic properties aresimilar to those of CP of other capsulated pathogens, such asmeningococci, pneumococci, H. influenzae type b, andSalmonella typhi that cause invasive diseases in otherwisehealthy children and adults and Escherichia coli Kl thatcause invasive diseases in neonates (48). Most diseasescaused by GBS, including type III, occur either during lateintrauterine life or during the first month of life (3). Accord-ingly, prevention of neonatal diseases due to GBS willrequire immunization of women in order to provide protec-tive levels of placentally acquired IgG type-specific antibod-ies.GBS CP have been purified, and their immunogenicity in

young adult humans has been studied (4-8, 16, 21, 23, 35).All GBS CP studied, including type III, were less immuno-genic than other CP vaccines, such as polyvalent meningo-coccus and pneumococcus and H. influenzae type b CPvaccines, probably due to their lower molecular weights, animportant determinant of immunogenicity (48, 59). Thislimits their use as vaccines, because the type III does notinduce a sufficiently high rate of IgG antibody responsive-ness in seronegative women of child-bearing age (6). Wesynthesized conjugates with TT with the hope of increasingthe percentage of women responding with type III antibod-ies. TT was chosen because it is an effective carrier protein,it elicits high antitoxin levels in adults, and because mater-nally derived 1T antitoxin prevents tetanus neonatorum, afrequent cause of death of newborns in many developingcountries (51).The immunoglobulin class and IgG subclass composition

of antibodies elicited by the type III were similar to thosereported for polysaccharides in mice (47). The unconjugatedtype III elicited mostly IgM antibodies; IgG type III anti-bodies were not detected in Swiss albino mice and weredetected in low levels in BALB/c mice. Type III CP-TT

TABLE 7. TT antibodies elicited in mice by GBS type IIICP-TT conjugates (type III-TT)a

TT antibodies (U/ml) GM (range)bConjugate

Post-injection 1 Post-injection 2 Post-injection 3

1 7.9 (1.2-25.0)* 58.1 (24.4-136.0)** 47.6 (20.0-95.7)t2 14.2 (3.0-77.6)* 44.5 (12.6-102.3)** 61.0 (10.0-183)4

a Groups of general purpose mice were injected with 2.5 ,ug of theconjugates (weight of CP) and bled 7 days after each injection. Mice injectedwith saline had less than 0.2 TT ELISA U.

b **, t, or t versus *, P = 0.0001; t versus t, P < 0.05.

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TABLE 8. Immunoglobulin class and IgG subclass composition of TT antibodies in Swiss albino mice injected withTT, GBS type III alone, or type III conjugated to TT (type III-TT)

Lg of GM TT antibody titer (range)Immunogen TI/mouse No.

IgG (total) IgGl IgG2a IgG2b IgG3

Type III x 1 0 7 <10 <10 <10 <10 <10Type 11-TI x 1 12.5 7 158 (20-1995) 100 (16-100) 63 (<10-631) 10 (<10-63) 25 (<10-199)Type III-TT x 2 12.5 10 1,000 (631-2541) 1,000 (158-3162) 126 (10-3981) 158 (16-794) 398 (63-6309)Type III-TT x 3 12.5 10 1,585 (79-5011) 1,585 (199-10000) 794 (50-3981) 316 (31-1259) 794 (251-63096)TT x 1 12.5 10 199 (50-15850) 1,945 (63-25119) 126 (10-3981) 316 (16-3162) 398 (63-6309)Type III-TI + FAI 25.0 10 107b 3.16 x 104 1.0 X 104 2,512 1,585

a Mice were injected with type III-TT, containing 5.0 ,ug of CP and 25 ,ug of TT, in FA (see Materials and Methods).b Sera from the 10 mice were pooled for use as a standard.

conjugates were evaluated by using the dosage and immuni-zation schedule in mice shown to be predictive of theusefulness of H. influenzae type b CP-TT in human infants(50, 52). In this model, type III-TT was shown to be bothmore immunogenic and to have more T-dependent proper-ties compared with the type III alone. There was a relationbetween the dosage of type III CP-TT injected and the levelsof conjugate-induced antibodies similar to that observedwith H. influenzae type b CP-TT conjugates in laboratorymice (12). Other T-dependent properties of type III-ITconjugates were a booster effect comprised mostly of IgGantibodies, enhancement by FA, and IgGl as the majorsubclass.Type III antibodies elicited by the conjugates had in vitro

opsonic activities proposed to be a correlate of protectiveimmunity (6, 8, 16). The structure of the CP was retained,since the conjugate elicited type III antibodies but not a risein pneumococcus type 14 antibodies (38). Our syntheticscheme was easily standardized and reproducible. Types Ia,lb, II, and IV CP have similar repeat units (34-37, 57, 58). Itis probable that our synthesis used for the type III will beapplicable to the other GBS CP. Murine TT antibodies wereof the IgG class, predominantly with antibodies in all foursubclasses. TT alone or as a component of type III conju-gates elicited a similar response. Humans respond to injec-tion of IT similarly; most serum antibodies are IgG, with thefour IgG subclasses represented and with the IgGl subclasspredominant. The type III-TT conjugates elicited IgG re-sponses in all mice, which were boostered following reinjec-tion. Since there is a correlation between the immunologicproperties of other CP and their protein conjugates in miceand in humans, we feel that our results provide a basis forclinical evaluation of the type III-TT conjugate as a potentialvaccine for prevention of neonatal GBS diseases.GBS is also a frequent cause of bovine mastitis, hence its

designation as Streptococcus agalactiae (40). Our conjugatevaccines may also serve to prevent infections in cattle due toGBS.

ACKNOWLEDGMENTS

This work was supported in part by the Swedish Medical Society.We are grateful to Dolores Bryla for the statistical analysis of thesedata. Toby Eisenstein provided us with a preparation of type IIIpolysaccharide and helpful advice at the beginning of the study.

LITERATURE CITED1. Avery, 0. T., and W. F. Goebel. 1931. Chemo-immunological

studies on conjugated carbohydrate-proteins. V. The immuno-logical specificity of an antigen prepared by combining thecapsular polysaccharide of type III Pneumococcus with foreignprotein. J. Exp. Med. 54:437-447.

2. Baker, C. J., F. F. Barrett, R. C. Gordon, and M. D. Yow. 1973.Suppurative meningitis due to streptococci of Lancefield groupBr. J. Pediatr. 82:724-729.

3. Baker, C. J. 1980. Group B streptococcal infections. Adv.Intern. Med. 25:475-501.

4. Baker, C. J., and D. L. Kasper. 1976. Correlation of maternalantibody deficiency and susceptibility to neonatal group Bstreptococcal infection. N. Engl. J. Med. 294:753-756.

5. Baker, C. J., D. L. Kasper, and M. S. Edwards. 1978. Immuno-genicity of polysaccharides from type III group B Streptococ-cus. J. Clin. Invest. 61:1107-1100.

6. Baker, C. J., M. Rench, M. S. Edwards, R. J. Carpenter, B. MHays, and D. L. Kasper. 1988. Immunization of pregnant womenwith a polysaccharide vaccine of group B Streptococcus. N.Engl. J. Med. 319:1180-1185.

7. Baltimore, R. S., C. J. Baker, and D. L. Kasper. 1981. Antibodyto a group B streptococcus type III in human sera measured bya mouse protection test. Infect. Immun. 32:56-61.

8. Baltimore, R. S., D. L. Kasper, C. J. Baker, and D. K. Gorott.1977. Antigenic specificity of opsonophagocytic antibodies inrabbit antisera to group B streptococci. J. Immunol. 118:673-678.

9. Barton, L. L., R. D. Feigin, and R. Lins. 1973. Group Bbeta-hemolytic streptococcal meningitis in infants. J. Pediatr.82:719-723.

10. Carey, R. B., T. Eisenstein, G. Shockman, T. Greber, and R.Swenson. 1980. Soluble group- and type-specific antigens fromtype III group B streptococcus. Infect. Immun. 28:195-203.

11. Christensen, K. K., P. Christensen, J. Hagerstrand, V. Linden,F. Nordbring, and N. Svenningsen. 1982. The clinical signifi-cance of group B Streptococci. J. Perinat. Med. 10:133-146.

12. Chu, C. Y., R. Schneerson, J. B. Robbins, and S. Rastogi. 1983.Further studies on the immunogenicity of Haemophilus influ-enzae type b and pneumococcal type 6A polysaccharide-proteinconjugates. Infect. Immun. 40:245-256.

13. Claesson, B. A., R. Schneerson, J. B. Robbins, J. Johansson, T.Lagergard, J. Taranger, D. Bryla, L. Levi, T. Cramton, and B.Trollfors. 1989. Protective levels of serum antibodies stimulatedin infants by two injections of Haemophilus influenzae capsularpolysaccharide-tetanus toxoid conjugate. J. Pediatr. 114:97-100.

14. Curtis, S. N., and R. M. Krause. 1964. Antigenic relationshipsbetween groups B and G streptococci. J. Exp. Med. 120:629-637.

15. Dahlberg-Lagergard, T. 1982. Target antigens for bactericidaland opsonizing antibodies to Haemophilus influenzae. ActaPathol. Microbiol. Immunol. Scand. Sect C 90:209-216.

16. De Cueninck, B. J., T. K. Eisenstein, T. S. McIntosh, G. D.Shockman, and R. M. Swenson. 1983. Quantitation of in vitroopsonic activity of human antibody induced by a vaccineconsisting of the type III-specific polysaccharide of group Bstreptocococcus. Infect. Immun. 39:1155-1160.

17. De Cueninck, B. J., G. D. Shockman, and R. M. Swenson. 1982.Group B, type III streptococcal cell wall: composition andstructural aspects revealed through endo-N-acetylmuramidase-catalyzed hydrolysis. Infect. Immun. 35:572-582.

INFECT. IMMUN.

on April 15, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

GROUP B STREPTOCOCCUS TYPE III-TT CONJUGATES

18. Dillon, H. C. Jr., S. Khare, and B. M. Gray. 1987. Group Bstreptococcal carriage and disease: a 6 year prospective study.J. Pediatr. 110:31-36.

19. Doran, T. I., and S. J. Mattingly. 1982. Association of type- andgroup-specific antigens with the cell wall of serotype III group BStreptococcus. Infect. Immun. 36:1115-1122.

20. Edwards, M. S., C. J. Baker, and D. L. Kasper. 1979. Opsonicspecificity of human antibody to the type III polysaccharide ofgroup B Streptococcus. J. Infect. Dis. 140:1004-1008.

21. Edwards, M. S., P. A. Fuselier, M. A. Rench, D. L. Kasper, andC. J. Baker. 1984. Class specificity of naturally acquired andvaccine-induced antibody to type III group B streptococcalcapsular polysaccharide determination with a radioimmunopre-cipitin assay. Infect. Immun. 44:257-261.

22. Eickhoff, T. C., J. 0. Klein, A. K. Daly, D. Ingall, and M.Finland. 1964. Neonatal sepsis and other infections due to groupB beta-hemolytic streptococci. N. Engl. J. Med. 271:1221-1228.

23. Eisenstein, T. K., B. De Cueninck, R. Resavy, G. D. Shockman,R. B. Carey, and R. M. Swenson. 1983. Quantitative determina-tion in human sera of vaccine-induced antibody to type-specificpolysaccharides of group B Streptococci using an enzyme-linked immunosorbent assay. J. Infect. Dis. 147:847-856.

24. Eskola, J., H. Peltola, A. H. Takala, H. Kayhty, M. Hakulinen, V.Karanko, E. Kela, P. Rekola, P.-R. Ronnberg, J. S. Samuelson,L. K. Gordon, and P. H. Makela. 1987. Efficacy of Haemophilusinfluenzae type b polysaccharide-diphtheria toxoid conjugatevaccine in infancy. N. Engl. J. Med. 317:717-722.

25. Faro, S. 1981. Group B beta-hemolytic streptococci and puerp-eral infections. Am. J. Obstet. Gynecol. 139:686-689.

26. Faxelius, G., and S. Ringertz. 1987. Neonatal septicemia inStockholm. Eur. J. Clin. Microbiol. 6:262-265.

27. Fisher, G. W., G. H. Lowell, M. H. Crumrine, and J. W. Bass.1978. Demonstration of opsonic activity and in vivo protectionagainst group B Streptococcus type III by Streptococcus pneu-moniae type 14 antisera. J. Exp. Med. 147:776-786.

28. Franciosi, L. L., J. D. Knostman, and R. A. Zimmerman. 1973.Group B streptococcal neonatal and infant infections. J. Pediatr.82:707-718.

29. Fry, R. M. 1938. Fatal infections by hemolytic streptococcus ofgroup B. Lancet i:199-201.

30. Goebel, W. F., and 0. T. Avery. 1931. Chemo-immunologicalstudies on conjugated carbohydrate-proteins. IV. The synthesisof the p-aminobenzyl ether of the soluble specific substance oftype III Pneumococcus and its coupling with protein. J. Exp.Med. 54:431-436.

31. Harriman, G. R., D. Y. Kunimoto, J. F. Eliott, V. Paetkau, andW. Strober. 1988. The role of IL-5 in IgA B cell differentiation.J. Immunol. 140:3033-3039.

32. Henry, R. J., A. B. Blakeney, P. S. Harris, and B. A. Stone.1983. Detection of neutral and amino sugars from glycoproteinsand polysaccharides as their alditol acetates. J. Chromatogr.256:419-427.

33. Inman, J. K., and H. M. Dintzis. 1969. The derivatization ofcross-linked polyacrylamide beads. Controlled induction offunctional groups for the purpose of special-purpose biochemi-cal absorbents. Biochemistry 4:4074-4080.

34. Jennings, H. J., E. Katzenellenbogen, C. Lugowski, and D. L.Kasper. 1983. Structure of native polysaccharide antigens oftype la and type lb group B Streptococcus. Biochemistry22:1258-1264.

35. Jennings, H. J., C. Lugowski, and D. L. Kasper. 1981. Confor-mation aspects critical to the immunospecificity of the type IIIgroup B streptococcal polysaccharide. Biochemistry 20:4514-4518.

36. Jennings, H. J., K.-G. Rosell, and D. L. Kasper. 1980. Structureand serology and the native polysaccharide antigen of type lagroup B Streptococcus. Proc. Natl. Acad. Sci. USA 77:2931-2935.

37. Jennings, H. J., K.-G. Rosell, E. Katzenellenbogen, and D. L.Kasper. 1983. Structural determination of the capsular polysac-charide antigen of type II group B Streptococcus. J. Biol. Chem.258:1793-1798.

38. Kasper, D. L., C. J. Baker, R. S. Baltimore, J. H. Crabb, G.

Schiffman, and H. Jennings. 1979. Immunoderminant specificityof human immunity to type III group B Streptococcus. J. Exp.Med. 149:327-339.

39. Lancefield, R. C. 1933. A serological differentiation of humanand other groups of hemolytic streptococci. J. Exp. Med.57:571-595.

40. Lancefield, R. C. 1934. A serologic differentiation of specifictypes of bovine hemolytic streptococci (group B). J. Exp. Med.59:441-458.

41. Lancefield, R. C. 1974. Antigens of group B streptococci.Relation to mouse-protective antibodies and immunity, p. 145-151. In Proceedings of the Symposium on New Approaches forInducing Natural Immunity to Pyogenic Organisms. U. S.Department of Health, Education, and Welfare, publication no.(NIH) 74-553. National Institutes of Health, Bethesda, Md.

42. Lancefield, R. C., and R. Hare. 1935. The serological differen-tiation of pathogenic and non-pathogenic strains of hemolyticstreptococci from parturient women. J. Exp. Med. 61:335-349.

43. Lancefield, R. C., M. McCarty, and W. N. Everly. 1975. Multiplemouse-protective antibodies directed against group B Strepto-cocci. J. Exp. Med. 142:165-179.

44. Manclark, C. R., B. D. Meade, and D. G. Burstyn. 1986.Serological response to Bordetella pertussis, p. 388-394. InN. R. Rose, H. Friedman, and J. L. Fahey. (ed.), Manual ofclinical laboratory immunology, 3rd ed. American Society forMicrobiology. Washington, D.C.

45. Markham, R. B., A. Nicholson-Weller, G. Schiffman, and D. L.Kasper. 1982. The presence of sialic acid on two relatedbacterial polysaccharides determines the site of the primaryimmune response and the effect of complement depletion on theresponse in mice. J. Immunol. 128:2731-2733.

46. Park, J. T., and M. J. Johnson. 1949. A submicro determinationof glucose. J. Biol. Chem. 181:149-151.

47. Perlmutter R. M., D. Hansburg, D. E. Briles, R. A. Nicolotti, andJ. M. Davie. 1978. Subclass restriction of murine anti-carbohy-drate antibodies. J. Immunol. 121:566-572.

48. Robbins, J. B., R. Schneerson, W. B. Egan, W. Vann, and D. T.Liu. 1980. Virulence properties of bacterial capsular polysac-charides-unanswered questions, p. 115-132. In H. Smith, J. J.Skehel, and M. J. Turner (ed.), The molecular basis of microbialpathogenicity. Verlag Chemie GmbH, Weinheim, Federal Re-public of Germany.

49. Schiffman, G., R. M. Douglas, M. J. Bonner, M. Robbins, and R.Austrian. 1980. Radioimmunoassay for immunologic phenom-ena in pneumococcal disease and for the antibody response topneumococcal vaccine. I. Method for the radioimmunoassay ofanticapsular antibodies and comparison with other techniques.J. Immunol. Methods 33:133-144.

50. Schneerson, R., 0. Barrera, A. Sutton, and J. B. Robbins. 1980.Preparation, characterization and immunogenicity of Haemoph-ilus influenzae type b polysaccharide-protein conjugates. J.Exp. Med. 152:361-376.

51. Schneerson, R., J. B. Robbins, J. C. Parke, Jr., A. Sutton, A.Wang, J. J. Schlesselman, G. Schiffman, C. Bell, A. Karpas, andM. C. Hardegree. 1986. Quantitative and qualitative analyses ofserum Haemophilus influenzae type b, pneumococcus type 6Aand tetanus toxin antibodies elicited by polysaccharide-proteinconjugates in adult volunteers. Infect. Immun. 52:519-528.

52. Schneerson, R., J. B. Robbins, S. C. Szu, and Y. Yang. 1987.Vaccines composed of polysaccharide-protein conjugates: cur-rent status, unanswered questions, and prospects for the future,p. 307-327. In R. Bell and G. Torrigiani (ed.), Towards bettercarbohydrate vaccines. John Wiley & Sons Ltd., United King-dom.

53. Sutton, A., W. Vann, A. Karpas, K. E. Stein, and R. Schneerson.1985. An avidin-biotin based ELISA for quantitation of anti-body to bacterial polysaccharides. J. Immunol. Methods 82:215-224.

54. Tai, J. Y., E. C. Gotschllch, and R. Lancefield. 1979. Isolation oftype-specific polysaccharide from group B type lb streptococci.J. Exp. Med. 149:58-66.

55. Tieffenberg, J., L. Vogel, R. R. Kretschmer, D. Padnos, and S. P.Gotoff. 1978. Chicken embryo model for type III group B

VOL. 58, 1990 693

on April 15, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

INFECT. IMMUN.

beta-hemolytic streptococcal septicemia. Infect. Immun. 19:481-485.

56. Trevelyan, W. E., and J. S. Harrison. 1952. Studies on yeastmetabolism. I. Fractionation and microdetermination of cellcarbohydrates. Biochem. J. 50:298-303.

57. Wessels, M. R., V.-J. Bened, H. J. Jennings, F. Michon, J. L.DiFabio, and D. L. Kasper. 1989. Isolation and characterizationof type IV group B Streptococcus capsular polysaccharide.Infect. Immun. 57:1089-1094.

58. Wessels, M. R., V. Pozsgay, D. L. Kasper, and H. J. Jennings.1987. Structure and immunochemistry of an oligosaccharide

repeating unit of the capsular polysaccharide of type III group BStreptococcus. A revised structure for the type III group Bstreptococcal polysaccharide antigen. J. Biol. Chem. 262:8262-8267.

59. World Health Organization Expert Committee on BiologicalStandardization. 1977. Technical report series no. 610. WorldHealth Organization, Geneva.

60. Yeung, M. K., and S. L. Mattingly. 1986. Covalent linkage of thetype- and group-specific antigens to the peptide moiety of thepeptidoglycan of serotype III group B Streptococcus. Curr.Microbiol. 14:205-211.

694 LAGERGARD ET AL.

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nloaded from