EP0493521A1 - Enzymatic generation and recovery of group b streptococcus type iii capsular oligosaccharides - Google Patents
Enzymatic generation and recovery of group b streptococcus type iii capsular oligosaccharidesInfo
- Publication number
- EP0493521A1 EP0493521A1 EP90915363A EP90915363A EP0493521A1 EP 0493521 A1 EP0493521 A1 EP 0493521A1 EP 90915363 A EP90915363 A EP 90915363A EP 90915363 A EP90915363 A EP 90915363A EP 0493521 A1 EP0493521 A1 EP 0493521A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- iii
- gbs
- galactosidase
- endo
- phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
Definitions
- 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.
- GBS-III Group B Streptococcus type III
- PC polysaccharide capsule
- 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.
- 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 ⁇ 1-3 linkage to D-galactose (in the pyranose form) which in turn is connected by a ⁇ 1 ⁇ 4 linkage to D-glucose (in the pyranose form) .
- the glucose is connected by a ⁇ 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 ⁇ 2 ⁇ 3 linkage to D-galactose which in turn is linked to the backbone as described above.
- Fig. 1 shows the repeating unit.
- GlcNAcp stands for N-acetyl glucosamine (pyranose form)
- Galp stands for galactose (pyranose form)
- Glcp stands for glucose (pyranose form)
- ⁇ -D-NANA stands for sialic acid.
- polysaccharide fragments which preserve intact the repeating unit of the GBS-III polysaccharide capsule are immunologically useful and can be generated enzymatically from the GBS-III polysaccharide capsule.
- polysaccharide capsule to describe the naturally occurring GBS-III polysaccharide capsule, which generally contains several hundred repeating units.
- 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.
- 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.
- 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.
- 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.
- a temperature e.g. below 37°C
- 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) .
- 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.
- 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.
- the second phase is an aqueous absorbing polymer or an aqueous gel.
- 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.
- the fragments consist of from 1-150 (most preferably 1-50) GBS-III PC repeating units.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ m 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 ⁇ m 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 ⁇ 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.
- 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.
- 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.
- IFO Institute of Fermentation
- a suitable general procedure for recovering that endo-J-galactosidase is disclosed in Kitamikado et al. J ⁇ _ Biol. Chem.
- C t . freundii endo-j8-galactosidase production include keratin sulfate or other suitable enzyme substrates.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
L'invention concerne la digestion sélective de capsules de polysaccharides (PC) de streptocoques du groupe B de type III (GBS-III) pour obtenir des fragments consistant en unités répétitives de capsules de polysaccharides de GBS-III intacts. La capsule de polysaccharide de GBS-III est digérée en la faisant réagir avec une endo-beta-galactosidase bactérienne qui est spécifique pour la liaison galactose-glucose glycosidique dans l'ossature GBS-III CP, et qui n'hydrolyse pratiquement pas d'autres liaisons glycosidiques GBS-III CP. Les fragments de polysaccharide obtenus sont récupérés et sont utiles à des fins immunologiques. L'endo-beta-galactosidase est produite dans une culture multiphase qui comprend: i) une phase liquide aqueuse; ii) une seconde phase qui est une phase aqueuse insoluble et en contact avec la phase liquide aqueuse au niveau d'une interface entre les deux; iii) des cellules bactériennes positionnées généralement dans la phase liquide aqueuse et au niveau de l'interface entre la phase liquide aqueuse et la seconde phase. L'endo-beta-galactosidase est récupérée de la phase liquide aqueuse.The invention relates to the selective digestion of polysaccharide (PC) capsules of group B type III streptococci (GBS-III) to obtain fragments consisting of repeating units of intact GBS-III polysaccharide capsules. The GBS-III polysaccharide capsule is digested by reacting it with a bacterial endo-beta-galactosidase which is specific for the glycosidic galactose-glucose bond in the GBS-III CP framework, and which practically does not hydrolyze other glycosidic linkages GBS-III CP. The polysaccharide fragments obtained are recovered and are useful for immunological purposes. Endo-beta-galactosidase is produced in a multiphase culture which comprises: i) an aqueous liquid phase; ii) a second phase which is an insoluble aqueous phase and in contact with the aqueous liquid phase at an interface between the two; iii) bacterial cells generally positioned in the aqueous liquid phase and at the interface between the aqueous liquid phase and the second phase. Endo-beta-galactosidase is recovered from the aqueous liquid phase.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US40867089A | 1989-09-18 | 1989-09-18 | |
US408670 | 1989-09-18 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0493521A1 true EP0493521A1 (en) | 1992-07-08 |
EP0493521A4 EP0493521A4 (en) | 1993-05-05 |
Family
ID=23617253
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900915363 Withdrawn EP0493521A4 (en) | 1989-09-18 | 1990-09-14 | Enzymatic generation and recovery of group b streptococcus type iii capsular oligosaccharides |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0493521A4 (en) |
JP (1) | JPH05502157A (en) |
AU (1) | AU6538490A (en) |
CA (1) | CA2066036A1 (en) |
WO (1) | WO1991004335A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6284884B1 (en) | 1995-06-07 | 2001-09-04 | North American Vaccine, Inc. | Antigenic group B streptococcus type II and type III polysaccharide fragments having a 2,5-anhydro-D-mannose terminal structure and conjugate vaccine thereof |
US7060483B1 (en) * | 1999-10-22 | 2006-06-13 | Seikagaku Corporation | DNA encoding endo-β-galactosidase |
WO2002016438A2 (en) * | 2000-08-22 | 2002-02-28 | National Research Council Of Canada | Synthesis of complex carbohydrates |
US11612664B2 (en) | 2016-04-05 | 2023-03-28 | Gsk Vaccines S.R.L. | Immunogenic compositions |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1987006267A1 (en) * | 1986-04-16 | 1987-10-22 | The Brigham And Women's Hospital, Inc. | Bacterial antigens, antibodies, vaccines, and methods of manufacture |
-
1990
- 1990-09-14 JP JP2514324A patent/JPH05502157A/en active Pending
- 1990-09-14 EP EP19900915363 patent/EP0493521A4/en not_active Withdrawn
- 1990-09-14 AU AU65384/90A patent/AU6538490A/en not_active Abandoned
- 1990-09-14 WO PCT/US1990/005244 patent/WO1991004335A1/en not_active Application Discontinuation
- 1990-09-14 CA CA002066036A patent/CA2066036A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1987006267A1 (en) * | 1986-04-16 | 1987-10-22 | The Brigham And Women's Hospital, Inc. | Bacterial antigens, antibodies, vaccines, and methods of manufacture |
Non-Patent Citations (4)
Title |
---|
JOURNAL OF BACTERIOLOGY vol. 75, no. 1, January 1958, BALTIMORE, USA pages 1 - 4 TYRREL, E.A. ET AL 'Biphasic system for growing bacteria in concentrated culture' * |
JOURNAL OF BIOLOGICAL CHEMISTRY vol. 256, no. 8, 25 April 1981, BALTIMORE, MD US pages 3900 - 3905 FUKUDA, M.N. ET AL 'Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin' * |
JOURNAL OF BIOLOGICAL CHEMISTRY vol. 265, no. 30, 25 October 1990, BALTIMORE, MD US pages 18278 - 18283 PAOLETTI, L.C. ET AL 'An oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus' * |
See also references of WO9104335A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA2066036A1 (en) | 1991-03-19 |
WO1991004335A1 (en) | 1991-04-04 |
EP0493521A4 (en) | 1993-05-05 |
JPH05502157A (en) | 1993-04-22 |
AU6538490A (en) | 1991-04-18 |
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