ENZYMATIC GENERATION AND RECOVERY OF GROUP B STREPTOCOCCUS TYPE III CAPSULAR OLIGOSACCHARIDES

Abstract: Selectively digesting the Group B Streptococcus type III (GBS-III) polysaccharide capsule (PC) to yield fragments consisting of intact GBS-III polysaccharide capsule repeating units. GBS-III polysaccharide capsule is digested by reacting it with a bacterial endo- beta -galactosidase that is specific for the galactose-glucose glycosidic linkage in the GBS-III CP backbone, and that substantially does not hydrolyse any other GBS-III CP glycosidic linkages. The resulting polysaccharide fragments are recovered and are immunologically useful. The endo- beta -galactosidase is produced in a multiphase culture that comprises: i) a liquid aqueous phase; ii) a second phase that is aqueous insoluble and in contact with the liquid aqueous phase at an interface therebetween, and iii) bacterial cells positioned generally in the liquid aqueous phase and at the interface between the liquid aqueous phase and the second phase. The endo- beta -galactosidase is recovered from the liquid aqueous phase.

Description: ENZYMATIC GENERATION AND RECOVERY OF GROUP B STREPTOCOCCUS TYPE III CAPSULAR OLIGOSACCHARIDES Background of the Invention This invention was made at least in part with funds provided by the U.S. Government, and the Government has certain rights in the invention. The general field of this invention is antigens for generating useful antibodies to the Group B Streptococcus type III (GBS-III) polysaccharide capsule (PC) , and methods for obtaining those antigens. It is known that the GBS-III polysaccharide capsule can be recovered from cultures of GBS-III Streptococcus. and that it can generate an antibody response that protects against GBS-III infection. For example, Kasper U.S. Patents 4,207,414; 4,367,223; and Re 31,672 feature recovering large (0.8-6 x 10 daltons) purified polysaccharide antigen specific for GBS-III, by culturing Streptococcus under specific conditions. The polysaccharide capsule is well characterized and consists of several hundred repeating units arranged to form a backbone of D-N-acetyl glucosamine (in the pyranose form) connected by a β 1-3 linkage to D-galactose (in the pyranose form) which in turn is connected by a β 1→4 linkage to D-glucose (in the pyranose form) . The glucose is connected by a β 1→6 linkage to the D-N-acetyl glucosamine residue in the next repeating unit. Each repeating unit has a side chain attached by a 1→4 linkage to the backbone D-N-acetyl glucosamine. The side chain consists of a terminal sialic acid residue linked by an α 2→3 linkage to D-galactose which in turn is linked to the backbone as described above. Fig. 1 shows the repeating unit. In Fig. 1: GlcNAcp stands for N-acetyl glucosamine (pyranose form) ; Galp stands for galactose (pyranose form) ; Glcp stands for glucose (pyranose form) ; and α-D-NANA stands for sialic acid. Summary of the Invention We have found that polysaccharide fragments which preserve intact the repeating unit of the GBS-III polysaccharide capsule (see Fig. 1) are immunologically useful and can be generated enzymatically from the GBS-III polysaccharide capsule. We use the term polysaccharide capsule to describe the naturally occurring GBS-III polysaccharide capsule, which generally contains several hundred repeating units. We use the term polysaccharide fragments or oligosaccharides to describe fragments (preferably 1-150 units and most preferably less than 50 units) of the GBS-III polysaccharide capsule generated according to the invention. One aspect of the invention features a method for selectively digesting the Group B Streptococcus type III (GBS-III) polysaccharide capsule (PC) to yield polysaccharide capsule fragments consisting of intact GBS-III PC repeating units. The method comprises the steps of providing GBS-III PC and then digesting that GBS-III PC by reacting it with a bacterial endo-0-galactosidase that is specific for the galactose-glucose glycosidic linkage in the GBS-III PC backbone (see the arrow in Fig. 1) , and that substantially does not hydrolyze any other GBS-III PC glycosidic linkages. The resulting fragments are recovered. In preferred embodiments of the first aspect of the invention, the polysaccharde fragments are substantially smaller than the GBS-III PC—e.g. they contain less than 150 (and most preferably less than 50) repeating units—and they are separated from larger polysaccharide components. Also preferably, the endo-?-galactosidase is one that is obtainable from a bacterium that can be induced to produce the enzyme by exposure to an enzyme substrate (e.g. degraded mucin) . Citrobacter freundii can be induced to make the enzyme and such bacteria are a particularly preferred source of it. Also preferably, the GBS-III PC digestion is accomplished by incubating the polysaccharide capsule with a sterile filtered preparation of the endo-3-galactosidase at a temperature (e.g. below 37°C) below denaturing temperatures for the enzyme, for at least 10 hours and not more than 5 days. The first aspect of the invention permits the use of fragments of the GBS-III polysaccharide capsule to form immunogenic conjugates that raise anti GBS-III antibodies (e.g. in active or passive vaccination) . Specifically, conjugation to a protein component is desirable to improve or generate immunogenicity. The use of these fragments as the polysaccharide component of such conjugates is advantageous, inter alia because coupling procedures are more reliable for coupling protein to the polysaccharide fragments than for coupling protein to the entire naturally occurring polysaccharide capsule, and because such fragments provide better control of the coupling procedure and better yield of the resulting conjugate. A second aspect of the invention generally features a method for producing endo-j8-galactosidase that is specific for the galactose-glucose glycosidic linkage in the GBS-III PC backbone and that substantially does not hydrolyze any other GBS-III PC glycosidic linkages. The method uses a multiphase culture that comprises: i) a liquid aqueous phase; ii) a second phase that is aqueous insoluble, and in contact with the liquid aqueous phase at an interface therebetween, and iii) bacterial cells positioned generally in the liquid aqueous phase and at the interface between the liquid aqueous phase and the second phase. The endo-0-galactosidase is recovered from the liquid aqueous phase. In preferred embodiments of the second aspect of the invention the endo-0-galactosidase is one that is obtainable from an organism (such as C^. freundii) whose enzyme production is induced in response to the presence of a substrate for the enzyme, such as a chemically degraded mucin (e.g. by oxidation and acid hydrolysis) . The enzyme substrate is preferably included in the second phase. Also preferably, the second phase is an aqueous absorbing polymer or an aqueous gel. The second aspect of the invention permits high cell densities and recovery of high yields of enzyme. A third aspect of the invention features substantially isolated GBS-III polysaccharide capsule fragments comprising intact repeating units of GBS-III polysaccharide capsule. "Substantially isolated" means effectively separated from the GBS-III polysaccharide capsule and other reaction components. It does not exclude the possibility that small amounts of these or other foreign substances may be present. Preferably the fragments consist of from 1-150 (most preferably 1-50) GBS-III PC repeating units. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims. Description of the Preferred Embodiment Drawings Fig. 1 is a formula for the repeating unit of the GBS-III polysaccharide capsule. Fig. 2 is a diagram of a culture system. Obtaining GBS-III Capsular Polysaccharide It is generally well known how to obtain substantially isolated GBS-III capsular polysaccharide suitable for use as a starting material in this invention. In particular, Kasper U.S. Re 31,672, which is hereby incorporated by reference, discloses culturing Streptococcus and recovering GBS-III capsular polysaccharide. Obtaining Endo-g-Galactosidase The endo-j3-galactosidase used in the invention should be one that specifically cleaves the GBS-III polysaccharide capsule at one and only one site per repeating unit in the backbone, leaving the side chain intact and yielding oligosaccharides which contain repeats of the complete capsular polysaccharide units. A preferred source of suitable endo-3-galactosidase is Citrobacter freundii (previously named Escherichia freundii) which can be obtained as reported by Nakagawa et al. Jj. Biol. Chem. 2j>5:5955-5959 (1980), and by Kita ikado et al., Nippon Suisan Gakkaishi 3>:H"75 (1970), and Kitamikado et al. Bull. Jon. Soc. Sci. Fish. 3.6:592-596 (1970), and Kitamikado et al. Nippon Suisan Gakkaishi 3J>:592 (1970) . C. freundii are also obtainable from those authors. These cells are grown in a biphasic culture with a polysaccharide inducer to produce particularly large amounts of endo-j3-galactosidase. Without limiting the invention, we note that one specific growth medium that can be used is composed of 2.5% (w/v) heart infusion broth (Difco) , 0.2% (w/v) Smith-degraded hog gastric mucin and 2.5% (w/v) purified agar. Hog gastric mucin (Sigma Chemical) can be prepared as by the general technique of Fukuda (1981) cited above, and Slomiany et al. Jj_ Biol. Chem. 247:5062-5070 (1972). Crude mucin (500 g) is dissolved in water (21) and pH is adjusted to 8.0 with 1% NaOH. Pronase (0.2g) is added and incubated at 37°C for 1 day under toluene. Pronase digestion is continued for 3 days with addition of 0.2g pronase every 24 hours. Then the pH is adjusted to 4.0 with glacial acetic acid and insoluble material is removed by centrifugation. To the supernatant, 3 volumes of 95% ethanol are added. Precipitated mucin is collected and dried. The growth medium described above (1200 ml) is placed in a 4 liter Erlenmeyer flask, autoclaved and allowed to solidify. Sterile dH 2 0 (300 ml) is added as the liquid overlay and allowed to equilibrate overnight at 30°C. The aqueous overlay is seeded with 2 ml of C. freundii cells grown for 2 days at 30°C in heart infusion broth containing mucin. On the third day, the liquid phase containing cells and media are removed and replaced with 300 ml of sterile water. A second harvest is obtained after an additional 3 days of incubation. In the above system, the agar based solid phase contains the nutrients for cell growth, and the substrate (Smith-degraded hog gastric mucin) for inducing enzyme production. The liquid aqueous phase is sterile water. Fig. 2 generally depicts a set-up for performing the biphasic culture. Flask E includes a cotton and guaze pad to provide adequate air supply. The liquid (H 2 0) layer of broth medium or water diffusate is shown as layer B. The solid agar medium is shown as layer C. Indentations D hold the agar base and prevent its breakup if incubated on a shaking machine. After 24 hours at 30°C, the cells grown in the above system can generally reach a density of 2.5 x 10 9 cfu/ml, far higher than generally achieved by batch or broth cultures. Partial Purification of Endo-β-Galactosidase Endo-j3-galactosidase is isolated from the above-described liquid aqueous phase generally as described by Li et al. Methods in Enzvmology 83:610-625 (1982) . Culture fluids are clarified by centrifugation (16,300 x g, 45 min, 4°C) and proteins precipitated at 4°C by the addition of (NH 4 ) 2 S0 4 to 75% of saturation. Precipitate is collected by centrifugation (16,300 x g, 15 min, 4°C), suspended in distilled water, placed in dialysis tubing (Spectropor 1, Spectrum Medical Industries, Los Angeles, CA) and dialysed against dH 2 0 at 4°C. Following dialysis against 50 mM sodium acetate buffer pH 5.5, proteins are placed on a QAE-Sephadex A-50 (Pharmacia LKB, Sweden) column (2.6 x 25 cm) equilibrated with the same buffer. Column fractions are tested for endo-β-galactosidase activity. Enzyme activity is demonstrated by incubating 20 μl of column fraction with 60 μg of purified type III polysaccharide from group Bj. Streptococcus overnight at 37°C. The following day, the digest mixture is reduced to dryness, suspended in 5 μl dH 2 0 and applied to a thin layer chromatographic (TLC) plate. Samples are separated in a solvent composed of 1-butanol:acetic acid:water in a 2:1:1 volume ratio. Carbohydrates are visualized using a diphenylamine spray. Endo- / 8-galactosidase activity is detected by the presence on TLC of the single repeating pentasaccharide of type III polysaccharide. Column fractions containing endo-0-galactosidase activity were pooled and used to digest large quantities of type III polysaccharide. Contaminating neuraminidase is separated from the QAE-Sephadex-purified endo-jS-galactosidase by passage of the material over a column of Sephadex G-100. Generation of Oligosaccharides Native type III capsule polysaccharide of group B Streptococcus are purified from culture supernatant fluids as previously described (Jennings et al., Can. J. Biochem. 58.:112-120 (1980)). Type III polysaccharide (120 mg) is suspended in 5-10 ml dH 2 0 and filtered sterile using a 0.45 μm syringe filter (Millex HA, Millipore) into a container containing 10 ml sterile digestion buffer (50 mM sodium acetate, 10 mM CaCl 2 pH 5.5) and 50 ml sterile dH 2 0. Enzyme preparations are also filtered sterile using a preconditioned (1 ml of 1 mg/ l bovine serum albumin in dH 2 0, followed by 3 ml dH 2 0 passed through the filter) 0.45 μm syringe filter and added to the polysaccharide. The final concentration of polysaccharide is 1 mg/ml. Digestion is allowed to proceed for 2-5 days at 37°C. Digestion is monitored daily using TLC and stopped when the density of the single repeat pentasaccharide band appears to be equal to that of the native band. The digest is lyophilized to dryness and suspended in 2 ml dH 2 0. Proteins are precipitated from the digest by the addition of (NH 4 ) 2 S0 4 to 75% (w/v) of saturation at 4°C for 2 hours. The supernatant fluid, containing type III capsular oligo- and polysaccharides, is clarified by centrifugation and placed on a G-75 (1.6 x 80 cm) gel filtration column equilibrated with 10 mM Tris pH 7.0. Fractions are monitored (^o β n*) and TLC is performed on the column fractions corresponding to a peak in A 206nm eluting first before the bed volume peak prior to salt elution to confirm the exact locations of the single pentassacharide repeating unit (1 ru) and the decasaccharide or two repeating unit (2 ru) . Molecular size of oligosaccharides eluted in earlier fractions are estimated by calculations of K based on elution volumes of the 1 ru oligosaccharide, 2 ru oligosaccharide and dextran standards. The resulting polysaccharide fragments can be conjugated to a protein to create an immunogen which elicts protective antibodies. The protein part of the conjugate can be an inert carrier protein or a protein offering other protection such as a GBS-III protein or a toxoid protein from another bacterium such as tetnus or diphtheria toxin (CRIM) . Techniques for conjugating the polysaccharide fragments to a protein are known. See, e.g. Jennings U.S. Pat. 4,356,120 or Marburg et al. U.S. Pat. 4,695,624. The resulting conjugates can be used as a passive or active vaccine according to standard techniques. For example, conjugates produced as described above can be recovered according to the technique described by Jennings '120 and suspended in a normal saline solution and injected as a vaccine, as described in Kasper Re 31,672. Passive vaccination can also be accomplished using the globulin fraction, as described in Kasper Re 31,672. The foregoing description is intended to exemplify and not to limit the invention. Other embodiments are within the claims. For example, suitable endo- / 9-galactosidase also can be obtained from C tophaga keratolytica (previously named Flavobacterium keratolyticus) deposited at the Institute of Fermentation (IFO) Osaka, Japan under designation IFO #14087, although production of enzyme by those organisms (unlike production from ( _ freundii) does not require induction. A suitable general procedure for recovering that endo-J-galactosidase is disclosed in Kitamikado et al. J\_ Biol. Chem. 256:3906-3909 (1981) and Nakazawa et al. J_ 5 _ Biol. Chem. 250:912-917 (1975). Other inducers of C t . freundii endo-j8-galactosidase production include keratin sulfate or other suitable enzyme substrates.

Claims: Claims 1. A method for selectively digesting the Group B Streptococcus type III (GBS-III) capsular polysaccharide (PC) to yield GBS-III PC fragments consisting of intact GBS-III polysaccharide capsule repeating units, which method comprises, providing GBS-III capsular polysaccharide; and digesting the GBS-III capsular polysaccharide by reacting it with a bacterial endo-,3-galactosidase that is specific for the galactose-glucose glycosidic linkage in the GBS-III PC backbone, and that substantially does not hydrolyze any other GBS-III PC glycosidic linkages, and recovering the resulting oligosaccharides. 2. The method of claim 1 in which the fragments generally comprise less than 150 GBS-III PC repeating units, and the oligosaccharides are recovered by separating them from larger oligosaccharide and polysaccharide components. 3. The method of claim 1 in which the fragments generally comprise less than 50 GBS-III PC repeating units, and the oligosaccharides are recovered by separating them from larger oligosaccharide and polysaccharide components. 4. The method of claim 1 in which the bacterial endo-0-galactosidase is obtainable from a bacterium by exposing the bacterium to a substrate for the endo-J-galactosidase which induces the bacterium to produce the endo-3-galactosidase. 5. The method of claim 1 in which the enzyme is an endo-/3-galactosidase of Citrobacter freundii. 6. The method of claim 1 in which the enzyme is an endo-j9-galactosidase of Bacteroides fragilis or Cvtophaga keratolytica. 7. The method of claim 1 in which said digesting is accomplished by incubating the GBS-III PC with a sterile filtered preparation of the endo-jS-galactosidase at a temperature below denaturing temperatures for the endo-j8-galactosidase. 8. A method for producing an endo-0-galactosidase that is specific for the galactose-glucose glycosidic linkage in the Group B Streptococcus-type III (GBS-III) polysaccharide capsule (PC) backbone, and that substantially does not hydrolyze any other GBS-III PC glycosidic linkages, comprising, a) providing a multi-phase culture comprising: i) a liquid aqueous phase; ii) a second phase that is aqueous insoluble, and in contact with the liquid aqueous phase at an interface therebetween, and iii) bacterial cells positioned generally in the liquid aqueous phase and at the interface between the liquid aqueous phase and the second phase; and b) recovering the endo- / 3-galactosidase from the liquid aqueous phase. 9. The method of claim 8 in which the second phase is an aqueous absorbing polymer. 10. The method of claim 8 in which the second phase is an aqueous gel. 11. The method of claim 8 in which the bacterial cells can be induced to produce the endo-/8-galactosidase responsive to the presence of a substrate for the endo-ø-galactosidase which is included in the second phase. 12. The method of claim 11 in which the substrate for the endo-jS-galactosidase included in the second phase is degraded mucin. 13. Substantially isolated fragments of the Group B type-Ill Streptococcus polysaccharide capsule, the fragments comprising intact GBS-III PC units. 14. The substantially isolated fragments of claim 13, wherein said fragments comprise fewer than 150 GBS-III PC repeating units. 15. The substantially isolated fragments of claim 13, wherein said fragments comprise fewer than 50 GBS-III PC repeating units.

Inventors: KASPER DENNIS L, PAOLETTI LAWRENCE C, WESSELS MICHAEL, KASPER, DENNIS, L., PAOLETTI, LAWRENCE, C., WESSELS, MICHAEL
Applicants: BRIGHAM & WOMENS HOSPITAL, BRIGHAM AND WOMEN'S HOSPITAL
Application year: 1990
Main classification: C12P19
IPC codes: C08B37, C12N9, C12P19