Synthesis and Immunological Properties of Conjugates Composed of ...

Vol. 58, No. 3

INFECTION AND IMMUNITY, Mar. 1990, p. 687-694

0019-9567/90/030687-08$02.00/0 Copyright C 1990, American Society for Microbiology

Synthesis and Immunological Properties of Conjugates Composed of Group B Streptococcus Type III Capsular Polysaccharide Covalently Bound to Tetanus Toxoid TERESA 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 adipic acid dihydrazide as a spacer, is described. Type Ill alone or as a conjugate with TT was injected subcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by these immunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after each immunization than did the type HI alone. These levels were related to the dosage of the conjugate, enhanced by 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/c mice. Type Ill-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type HI antibodies were not detected. The first two immunizations with the conjugates elicited type HI antibodies in the IgGl and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injection of type Ill-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type III organisms and did not react with the structurally related pneumococcus type 14. TT alone or as a component of 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 of and conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TT component. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type III antibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women of child-bearing age is planned.

Lancefield, in 1933, classified hemolytic streptococci into (39). At that time, almost all severe streptococcal infections of humans were due to group A. Lancefield noted that group B streptococci (GBS) from parturient women were associated with mild infections (42). In 1938, Fry reported three cases of fatal puerperal fever due to GBS (29). It was an unexpected phenomenon when GBS were reported to be the most frequent cause of neonatal sepsis at the Boston City Hospital during 1961 to 1963 (22). This apparent increase of neonatal sepsis caused by GBS was confirmed (2, 9, 28). GBS remain a frequent cause of serious infections in neonates: about two-thirds of strains from patients are of type III (3, 11, 18, 26). GBS are now also a major cause of puerperal infections (3, 25). Lancefield showed that passive protection of mice by rabbit hyperimmune sera was type specific (41, 43). The structure of these type antigens, shown to be capsular polysaccharides (CP), has been elucidated (34, 36, 37, 57, 58). The virulence of GBS is due to their ability to invade the blood stream and multiply. This property of invasiveness is related to the anti-phagocytic properties conferred to GBS by its CP (8, 16, 20, 45, 55). The protective activity of human antibodies has been demonstrated by in vitro assays and in vivo animal models and by the correlation between the susceptibility of neonates to invasive GBS diseases with the absence of placentally transmitted IgG type III antibodies (4-7, 16, 21, 23, 55).

The structure of type III and its immunologic properties in adults 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 -Dgalactose to form the type-specific site (35, 58). Without this NeuNAc moiety, the structure of type III is identical to that of the pneumococcus type 14 CP (27, 35, 38). Antibodies elicited by type III have secondary biological properties that have been correlated with protective immunity (4-8, 11, 16, 20, 45, 55). Active immunization of newborns is not practical, since most cases occur within the first month of life (3). Type III as a vaccine for maternal-fetal passive immunization is limited because it induces protective levels of IgG antibodies in only about 60% of antibody-negative women of child-bearing age (5, 6). We used the approach, reported by Avery and Goebel in 1931, of binding type III to a protein in order to increase its immunogenicity (1, 30). A synthetic scheme for binding Haemophilus influenzae type b CP to proteins resulted in increased immunogenicity and T-cell dependent properties to this CP (12, 13, 50). In contrast to the age-related and the T-independent properties of the CP alone, a H. influenzae type b CP conjugate elicited booster responses, comprised mostly of IgG, and protected .12month-old children against meningitis caused by this pathogen (24). Here, we describe the synthesis of a GBS-tetanus toxoid (TT) conjugate (type III-TT) and some of its immunologic properties in mice.

Corresponding author. t Present address: Department of Medical Microbiology, University of Goteborg, Goteborg, Sweden.

MATERIALS AND METHODS Reagents. Yeast extract and tryptic soy broth were from Difco Laboratories, Detroit, Mich.; mutanolysin, DNase,

groups

*

687

688

LAGERGARD ET AL.

INFECT. IMMUN.

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

4

I

-D-Ga lp

3

I

2

O(-D-NeuNAc p FIG. 1. The structure of the GBS type III capsular polysaccharide as described by Wessels et al. (58).

RNase, pronase, bovine serum albumin, adipic acid dihydrazide (ADH), avidin, agarose, and thimerosal were from Sigma 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 pyrogen-free saline were from Travenol Laboratories, Lincoln, Ill.; cyanogen bromide and trinitrotoluene benzene sulfonic acid were from Eastman Chemical Products, Inc., Rochester, N.Y.; 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide was from Calbiochem-Behring, Fullerton, Calif.; and Nhydroxysuccinimidobiotin was from Pierce Chemical Co., Rockford, Ill. TT was kindly donated by C.-Y.Chu, Shanghai Institute of Biological Products. The TT was concentrated and passed through an S-300 Sephacryl column (5 by 90 cm) (12). Fractions, corresponding to a molecular mass of -150 kilodaltons, were concentrated by vacuum dialysis to 41 mg/ml, sterile filtered (0.4-nm membrane; Nalgene Labware Div., Rochester, N.Y.), and stored at 3 to 8°C. The Kd of this preparation, through CL-4B Sepharose, was 0.71. Group B-specific and type-specific antisera, prepared by Rebecca Lancefield, were donated by Emil C. Gotschlich, The Rockefeller University, N.Y. Another GBS type III antiserum and a pneumococcus type 14 typing antiserum were donated by J0rgen Henrichsen, Statens Seruminstitut, Copenhagen, Denmark. Bacterial growth. GBS type III, strain 110, was kindly provided by Stephen Mattingly, University of Texas Health Science Center, San Antonio, Texas. This strain was cultivated on tryptic soy broth agarose overnight at 37°C. Several colonies were inoculated into 2.8-liter baffled Fernbach flasks (Bellco Glass, Inc., Vineland, N.J.) containing 1.0 liter of media (1.0% yeast extract dialysate, 1% dextrose, 0.1 M sodium phosphate; final pH 7.2). The flasks were placed on a rotary shaker and incubated at 37°C at 100 rpm for 18 h. These flasks were then delivered to 20-liter carboys containing the same growth media (10% inoculum) and incubated for about 18 h at 37°C. The bacteria were separated by centrifugation and stored at -20°C. The culture supernatant was passed through a 0.4-nm filter and concentrated by ultrafiltration (10,000-molecular-weight-cutoff membrane; Pellicon Cassette System, Bedford, Mass.) to -10% of its original volume. Purification of type III. The CP from the bacterial pellet and the culture supernatant were treated separately. The bacterial pellet was treated with 5,000 U of mutanolysin per 60 to 80 g of bacteria (wet weight), as described previously (10, 17, 19, 54, 60). The cells were then removed by centrifugation. The supernatant from the enzyme-treated bacteria and from the culture supernatant were treated sequentially with DNase, RNase, and pronase and then extracted with cold phenol (50). The products from the supernatant were passed through 4B-CL Sepharose equilibrated in 0.2 M NaCl to remove low-molecular-weight material (.0.6 Kd). The higher-molecular-weight fractions from the supernatant and the products from the bacteria

were fractionated by anion-exchange chromatography through DEAE-Sephacel by linear NaCl gradient elution (10). Fractions containing type III, as tested by double immunodiffusion, were dialyzed extensively against sterile pyrogen-free water, freeze-dried, and stored at -20°C. The yields of type III from 500 g (wet weight) of bacterial pellet were about 300 mg, and those from the culture supernatant were about 130 mg. Analysis. Type III was measured by the anthrone reaction by using the purified CP as a standard (56). Protein was measured by the method of Lowry et al., by using bovine serum albumin as a standard (59). The adipic acid hydrazide (AH) content of the derivatized type III was measured by the trinitrotoluene benzene sulfonic acid reaction by using ADH as a standard (33). The partition coefficients (Kd) through CL-4B Sepharose were calculated as previously described (59). Gas-liquid chromatography was kindly performed by Willie F. Vann, Office of Biologics, Research and Review, Food and Drug Administration, to measure the content of rhamnose (group B cell wall polysaccharide) (14, 32). The molecular weight was estimated by end group analysis by using the Park-Johnson reaction, with dextrose as the standard (46). Synthesis of type III-TT conjugates. The synthesis followed closely the scheme used for H. influenzae type b CP conjugates (12). Briefly, type III was activated with cyanogen bromide at pH 10.5 for 6 min at 4°C in a pH stat (Radiometer, Copenhagen, Denmark). The weight ratio of cyanogen bromide-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 final concentration of 0.25 M, pH 8.5. After tumbling for 18 h at 3 to 8°C, the reaction mixture was dialyzed against 0.2 M NaCl at 3 to 8°C and passed through a 4B-CL Sepharose column. The CP-containing fractions were pooled, dialyzed against sterile pyrogen-free water, and freeze-dried. A solution containing 10 mg each of type III-AH and TT per ml was brought to pH 5.6 with 0.1 N HCl. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide was added to a final concentration of 0.05 M, and the pH was maintained at 5.6 with 0.1 N NaOH for 3 h at room temperature. The reaction mixture was dialyzed against 0.2 M NaCl at 3 to 8°C and was passed through a 4B-CL Sepharose column (5 by 95 cm) equilibrated in 0.2 M NaCl. The void volume fractions were stored in 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 injected subcutaneously with 2.5 ,uig of the type III alone or as a component of a conjugate (12). For the dosage-response experiment, mice were injected with 0.5, 2.5, or 5.0 ,ug of the type III-TT, on the basis of its CP content. Separate groups were injected 1, 2, or 3 times at 2-week intervals and were bled 2 weeks after the first injection and 1 week after the second and third injections. Controls were mice which were bled before the first immunization or those injected with saline and bled after the last injection. One group of mice was injected with 5.0 ,ug of the type III-TT in complete Freund adjuvant (FA) and reinjected 4 weeks later with the same conjugate in incomplete FA. Two weeks later, these mice were bled and their pooled sera, assigned a value of 1,000 U, were used as a reference standard. Serology. Double immunodiffusion and capillary precipitation were performed with rabbit typing antisera to GBS types Ia, Ib, II, and III and to pneumococcus type 14. Enzyme-linked immunosorbent assay (ELISA) was performed with avidin-treated plates and biotinylated type III as the coating antigen (53). Plates were coated with 4.0 pLg of

689

GROUP B STREPTOCOCCUS TYPE III-YT CONJUGATES

VOL. 58, 1990 TABLE 1. Composition of GBS type III CP-TT conjugates

CONJUGATE No.2

Conjugate

% Yield of CP

(%AH/CP [wt/wt])

CP/protein (wt/wt)

1 2 3

11.0 32.0 36.0

1.0 1.3 1.8

0.13 0.17 0.20

0.4 t

Kd tetanus toxoid

0

0.3

E

avidin per ml and 4.0 pg of biotinylated type III per ml. Eight threefold dilutions of reference and test sera were added. Horseradish peroxidase conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Copenhagen, Denmark) was added and read at 450 nm. For assay of the isotype composition of GBS type III and TT antibodies, alkaline phosphatasecoupled goat anti-murine immunoglobulin class and IgG subclass antibodies were obtained from Southern Biotechnology Association, Birmingham, Ala. The specificity of these antibody reagents has been reported (31). The optimal concentration of each antiglobulin reagent was determined by checkerboard titrations. The immunoglobulin class and IgG subclass antibody content was expressed as the reciprocal of the serum dilution giving an absorbance of 0.2 optical density above the background level; values less than this level at a serum dilution of 1:10 were considered as being negative (