JPH05502157A - Enzymatic production and recovery of group B Streptococcus type 3 capsular oligosaccharides - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Enzymatic production and recovery of group B Streptococcus type NI capsular oligosaccharides I1 standing 1 ken The government has certain rights in the invention.

The general field of the invention is Group B Streptococcus To produce antibodies useful for type III (CBS-III) polysaccharide capsule (PC) antigens and methods for obtaining these antigens.

CBS-III multilayer capsules can be recovered from cultures of GBS-III streptococci. and that it can generate an antibody response that protects against GBS-III infection. For example, Kasper U.S. Pat. No. 4,207,414 and 43 No. 67223 and Reissue Patent No. 31672 involve culturing streptococci under specific conditions. By doing so, a large (0,8~6XI O'Dalton) is characterized by the recovery of purified polysaccharide antibodies. polysaccharide capsule is sufficient It has been characterized that D-N-acetylglucosamine (pyranose form) is 3 bonds to D-galactose (pyranose form), and the latter is further linked to β1- D-glucose (in the form of pyranose) is linked by 4 bonds to form a main chain. It consists of hundreds of repeating units arranged in a uniform pattern. Glucose connects to the βt-s bond is linked to a D-N-acetylglucosamine residue in the next repeating unit. . Each repeating unit connects to D-N-acetylglucosamine in the main chain through 1-4 bonds. with attached side chains. This side m is D- attached to the main chain as shown in Figure 1. It consists of a terminal sialic acid residue bound to Gakudose through 2-3 bonds. Figure 1 is In Figure 1, which shows this repeating unit, GlcNAcp is an N-acetyl group. Cosamine (pyranose form), GaLp represents galactose (pyranose form) , Glcp represents glucose (pyranose form), α-D-NANA represents sialic acid.

l Mitate 11 We present a polysaccharide fragment that retains the repeating unit of the GBS-III polysaccharide capsule intact. fragment (see Figure 1) is immunologically useful and It has been found that the diameter can be immunologically produced from the capsule. We use the term “ The polysaccharide capsule is a naturally occurring GBS-I that generally contains several hundred repeating units. II is used to describe the polysaccharide capsule. We also use the term “polysaccharide fragment” or ``oligotang'' of GBS-III polysaccharide capsule produced according to the present invention. fragments (preferably 1 to 150 units, most preferably 50 units or less) Used to clarify.

One aspect of the invention is that group B Streptococcus I Type II (CBS-III) polysaccharide capsule M (PC) is selectively digested to intact CBS- Features a method for producing a fragment of a polysaccharide capsule consisting of rrlPc repeating units . In this method, GBS-IIIPC is prepared, and the main chain of GBS-111PC is specific for the galactose-glucose gluzocoside bond (see arrow in Figure 1). and other CBS-IIIPC glycosidic bonds that do not substantially hydrolyze By reacting endo-β-galactosidase with the GBS-IrIPC There is no need to digest the GBS-111PC and then collect the resulting fragments. In a preferred embodiment of the first aspect of the invention, the polysaccharide fragment is CBS-II For example, they are substantially less than 150 (most preferably contains up to 50 repeating units). In addition, they can be separated from large polysaccharide components. be separated. Also preferably, the endo-β-galactosidase contains an enzyme substrate (e.g. bacteria that can be induced to produce enzymes by exposure to degradable mucins It is something that can be obtained from Citrobacterium froinsii (Citro) bacter freundii) can be induced to make enzymes, Such bacteria are particularly preferred bacterial sources. More preferably, CBS-II Digestion of IPC was carried out using endo-β-galactosidase sterile filtered polysaccharide capsules. At least 10 hours at a temperature below the denaturation temperature of the formulation and enzyme (e.g. below 37°C) This is accomplished by incubating for up to 5 days.

A first aspect of the invention provides anti-G allow the formation of immunogenic complexes that give rise to BSS-III antibodies. (e.g. in active or passive vaccination). Especially for protein components. Conjugation is desirable to improve or generate immunogenicity of multiple such conjugates. It is advantageous to use these fragments as sugar components. Because, Specifically, the coupling method couples the protein to the entire naturally occurring polysaccharide capsule. This is because coupling proteins to polysaccharide fragments is more reliable than coupling proteins to polysaccharide fragments. , such fragments also allow for better control of the coupling method and the resulting conjugate. This is because it improves the yield of.

A second aspect of the invention generally relates to galactose- GBS-TIIP specific for glucose glycosidic bonds and other Production of endo-β-galactosidase that does not substantially hydrolyze C-glycosidic bonds It features a method of The method comprises: i) a liquid aqueous phase; ii) a water-insoluble and a second phase in contact with the liquid aqueous phase at its interface; and 11]) the aqueous phase; bacterial cells generally disposed in the liquid aqueous phase at the interface between the liquid aqueous phase and the second phase; Use a multiphase medium containing Endo-β-galactosidase is extracted from the liquid aqueous phase. It will be collected.

In a preferred embodiment of the second aspect of the invention, endo-β-galactosidase enzymes are microorganisms (e.g. Citrobacterium froinsii) that produce enzymes. enzymes such as mucins that have been chemically degraded (e.g. by oxidation and acid hydrolysis) This is what can be obtained from those that are induced in response to the presence of a substrate for.

The enzyme substrate is preferably included in the second phase. Also preferably, the second phase is an aqueous absorbent polymer or an aqueous gel.

The second aspect of the invention allows for high cell densities and high yields of enzyme recovery.

A third aspect of the invention comprises an intact repeating unit of GBS-III multilayer capsule. The present invention is characterized by substantially isolated GBS-[1 multicapsule fragments comprising: "fruit ``Qualitatively isolated'' means GBS-III polycapsules and other reaction components. means that it is effectively separated from the However, small amounts of these or other Preferably, the fragments are between 1 and 1, without excluding the possibility that foreign bodies may be present. Consisting of 50 (most preferably 1 to 50) GBS-III PC repeat units.

Other features and advantages of the invention will be apparent from the following description of exemplary embodiments and from the claims. It will become clear.

Good JL Chief” 2A Engineer Dan Yu II I FIG. 1 is the formula of the repeating unit of the GBS-III polysaccharide capsule.

FIG. 2 is a diagram of the culture apparatus.

GBS New 11J1 Yu I” Substantially isolated G B suitable for use as a starting material in the present invention Methods for obtaining S-III capsular membranes are generally well known. Especially Mr. Kasper's U.S. Reissue Patent No. 31,672 describes the cultivation of streptococci and 1@Discloses the acquisition of polysaccharides.

Endo-galactosidase ′ The endo-β-galactosidase used in the present invention has one repeating unit in the main chain. specifically cleaved the GBS-III polysaccharide capsule at only one location to produce an oligomer that leaves the side chains intact and contains a complete repeat of the capsular polysaccharide unit. It must be something that can be done.

A preferred bacterial source of suitable endo-β-galactosidase is Citrobacterium Citrobacter freundii (formerly known as Citrobacter freundii) It is Escherichia freundii. So, this is Mr. Nakayo et al.'s 1"J, Biol, Chem, J 255:5955 -5959 (1980), “Journal of the Japanese Fisheries Society” by Mr. Kitamikado et al., DanJpn, Soc. , Sci, Fish, ] 36; 592-596 (1970) and Kitamikado. It is reported in another article, ``Journal of the Japanese Society of Fisheries Science, J 36: 592 (1970). You can get it like this. In addition, Citrobacterium froinsii was described by these authors. It can also be obtained from.

These cells are specifically designed to produce large amounts of endo-β-galactosidase. Although not limiting the invention, , one particular growth medium that can be used in the present invention is 25% (w/v) heart influenza. Fusion Broth (Difco), 02% (w/v) Smith's Decomposed Pork Stomach Chin and 2.5% (w/v) purified agar.

Porcine gastric mucin (Sigma Chemical Co., Ltd.) is described, for example, by Fukuda (1981) and Slott, cited above. Mr. Miani et al. rJ, Bi.

1, described in Chem, Co. 247, 5062-5070 (1972) It can be manufactured by the general technique of.

First, crude mucin (500 g) was dissolved in water (2 liters), and the pH was adjusted to 1% NaO Adjust to 8.0 with H. Add pronase (0.2 g) and Incubate for 1 day at 7 °C. Digest with 6 Bronase every 24 hours. g of pronase and continue for 3 days. The pH was then adjusted to 4.0 with glacial acetic acid. Condition and remove insoluble material by centrifugation. Add 3 volumes of 95% ethanol to the supernatant liquid. Add mol. The precipitated mucin is collected and dried.

Add the above growth medium (1200 ml) to a 4 liter Erlenmeyer flask. Then autoclave and solidify. Sterile dHxO (300ml ) is added as a liquid top layer and equilibrated at 30° C. overnight. Mucin is included in aqueous soil and stone. 2 ml cultures grown for 2 days at 30°C in heart infusion broth with Inoculate with Trobacterium freundschii cells. Contain cells and medium on day 3 The liquid phase is removed and replaced with 300 ml of sterile water. Incubate for another 3 days After harvesting, you will get a second crop.

In the above system, the solid phase mainly composed of agar contains nutrients and enzymes for cell growth. Contains a substrate (Smith's degraded porcine stomach mucin) to induce the production of mucin. liquid The aqueous phase is sterile water.

Figure 2 schematically shows the equipment for achieving a two-phase culture, flask E is suitable for Broth medium or water diffuser, including cotton and gauze pads to provide adequate air supply The liquid layer (H, O) of is shown as layer B, the solid agar medium is shown as [C, the depression D Retain the agar base and prevent its collapse when incubating on a shaker.

After 24 hours at 30°C, cells grown in the above system are grown in batch or broth culture. much higher than that typically achieved by nutrition.

Endo--galactosidase.・　.

Endo-β-galactosidase is generally described by Lee et al. In, Enzymology J1, 61.0-625 (1982). from the liquid aqueous phase. Centrifuge the culture solution (16,300X G, 45 min, 4°C) and purified the protein at 4°C with (NH,) SO, 75% Precipitate by adding to saturation. Centrifuge the precipitate! 11 (18,300 XG, 15 min, 4°C), suspended in distilled water, and placed in a dialysis tube (spectral bottle). Rule 1, Spectrum Medical Industries, Inc., Los Angeles, CA. ) and dialyzed against dH20 at 4°C. 50mM sodium acetate buffer Following dialysis against pH 5.5, the protein was incubated with QAE-separate equilibrated in the same buffer. Fadex A-50' (Pharmacia LKB, Sweden) column (2,6 x 25 cm) and column fractions were analyzed for endo-β-galactosidase activity. Test. Enzyme activity was determined by comparing the 20 μm column fraction to 60 μm from group B streptococci. By incubating with μg of purified type III Tatsu at 37°C overnight. On the first day of demonstration, the digestion mixture was reduced in volume to dryness and dissolved in 5 μm dHao. and apply to thin 1 chromatography (TLC') plates. sample Separation is carried out in a solvent consisting of 1-butanol:acetic acid/water in a volume ratio of 2:1:1. charcoal Make the hydrate visible using diphenylamine spray. End- β-galactosidase activity was determined on TLC by the presence of a single repeating pentasaccharide of type III polysaccharide. Detected by presence. Column fractions containing endo-β-galactosidase activity are pooled. It was used to digest a large amount of type III polysaccharide. Pollution Noiramini QAE is produced by passing the substance through a column of Sephadex G-100. - Separated from Sephadex-purified endo-β-galactosidase.

Otsudo Kogyo 2nd Station The natural type III capsular polysaccharide of Geloop B Streptococcus was used as described above (rCa n, J. Biochem,] 58.1.12-120 (1980)). Purify from the culture supernatant. Type III polysaccharide (120m, g) 5-10 Suspended in ml of dHzo and filtered with a 0.45 μm syringe filter (MiI 10 ml of sterile digestive fluid under aseptic conditions using buffer solution (50mM sodium acetate, 10mM CaC1z - pH 5,5) and Filter into a container containing 50mM dH20. In addition, condition the enzyme preparation in advance. (1 mg/ml of bovine serum albumin in dHa○ solution 1 ml), then Pass d Hz O3ml through the filter) 0.45μm syringe filter Filter aseptically using a filter and add to the polysaccharide. The final concentration of polysaccharide is Lmg/ml It is. Digestion is allowed to occur for 2-5 days at 37°C. Digestion daily using TLC Monitor and ensure that the density of the single repeating polysaccharide band is the same as the density of the native band. Stop when you think so. Lyophilize the digest and suspend in 2 ml of dH*O let Add (NH-)x SO4 to the digestate at 4°C to a saturation of 75% (W/v). Precipitate the protein by adding up to 2 hours. trr-type capsular oligosaccharide and The supernatant containing polysaccharides was clarified by centrifugation and then poured into 10 ml Tris buffer. Pour into a G-75 (1.6 x 80 cm) gel filtration column equilibrated with buffer pH 7.0. Am to monitor the 6 fractions. 81111) + Bed volumetric peak prior to salt elution. TL for the column fraction corresponding to the A2゜6□ peak that elutes first before A single polysaccharide repeat unit (lru) and a pentasaccharide or two repeat units Check the exact location of (2r　u). Oligotang molecules eluted in early fractions Dimensions to elution volumes of Iru oligosaccharides, 2ru oligosaccharides, and dextran aiming samples. It is calculated by calculating the basis <K.

The resulting polysaccharide fragments create immunogens that bind to proteins and induce protective antibodies. can be done. The protein portion of the complex is an inert carrier protein or CBS-III protein or tetanus or diphtheria toxin (CRIM) may be proteins that provide other protection such as toxoid proteins from other bacteria such as , techniques for conjugating polysaccharide fragments to proteins are known6. Ng U.S. Pat. No. 4,356,120 or Malberg U.S. Pat. No. 46,956. The resulting conjugate, see No. 24, can be passively or actively processed according to standard methods. For example, the conjugate produced as described above can be used as a vaccine. The bodies were recovered according to the technique described in Jenning's US patent and placed in a normal saline solution. Vaccine suspension as described in Kasper U.S. Reissue Patent No. 31,672. It can be injected as an injection. Also, Mr. Kasper's reissued patent No. 3167 Achieving passive vaccine uptake using globulin fractions as described in No. 2 I can do it.

The above description is for the purpose of illustrating the present invention and is not intended to limit the present invention. do not have. Other embodiments are also within the scope of the claims. 〇-Galactosidase was sent to Osaka Fabric Fermentation Research Institute (IFO) IFO No. 14 Cytophaga keratricii deposited as 087 eratolytica) (formerly known as Flavobacterium ceratolytica) (Flavobacterium keratolyticus)) However, the production of enzymes by these bacteria The endo-β-galactosidase does not require induction (unlike its production from A). A suitable general procedure for recovering the enzyme is described by Kitamikado et al., rJ, Biol, Ch. em, J256; 3906-3909 (1981) and Nakazawa et al. rJ, Bi ol, Chem, J 250: 912-917 (1975). There is. Other aspects of endo-β-galactosidase of Citrobacter freundii inducing agents include keratin sulfate or other suitable enzymatic agents.

β-D-Galp international search report

Claims (15)

[Claims] 1. Group B Streptococcus type III (GBS-I) II) Selective digestion of capsular polysaccharide (PC) to recover intact CBS-III polysaccharide capsule In producing a GBS-III PC fragment consisting of a return unit, GBC-I II capsular polysaccharide is prepared, Galactose-glucose glycosidic linkages in the GBC-III capsular polysaccharide backbone and substantially hydrolyzes other GBS-III PC glycosidic bonds. The GBC-IIIPC is reacted with bacterial endo-β-galactosidase that does not digest. digest the GBC-IIIPC by The resulting oligosaccharides are then recovered. A method for selectively digesting group B Streptococcus type III polysaccharide, comprising: 2. the fragment generally contains no more than 150 GBS-III PC repeat units; Recycling occurs by separating oligosaccharides from larger oligosaccharide and polysaccharide components. The method according to claim 1, wherein the method comprises: 3. the fragment generally contains no more than 50 GBS-III PC repeat units, and Recovery by separating oligosaccharides from larger oligosaccharide and polysaccharide components 2. The method according to claim 1. 4. Bacterial endo-β-galactosidase produces endo-β-galactosidase The substrate for endo-β-galactosidase induces the bacteria to The method according to claim 1, which is obtained by exposing the bacteria. 5. The enzyme is Citrobacter freundii. 2. The method according to claim 1, wherein the endo-β-galactosidase of dii) is an endo-β-galactosidase. 6. The enzyme is Bacteroides fragilis. is) or Cytophagakeratoly tica) endo-β-galactosidase. 7. Digest GBS-III PC with endo-β-galactosidase and sterile filtration. Incubate the prepared preparation at a humidity below the denaturation temperature of the endo-β-galactosidase. 2. The method of claim 1, wherein the method is achieved by: 8. Streptococcus croup - type III (GBC-III) in the polysaccharide capsule (PC) backbone specific for galactose-glucose glycosidic bonds and other Endo-β-galactin that does not substantially hydrolyze GBS-IIIPC glycosidic bonds In producing tosidase, a) i) liquid aqueous character, ii) water-insoluble and a second phase in contact with the liquid aqueous phase at its interface; and iii) the aqueous phase. bacterial cells generally disposed in the liquid aqueous phase at the interface between the liquid aqueous phase and the second phase; Prepare a multiphase medium containing b) Recovering endo-β-galactosidase from the liquid aqueous phase A method for producing endo-β-galactosidase, comprising: 9. 9. The method of claim 8, wherein the second phase is an aqueous absorbent polymer. 10. 9. The method of claim 8, wherein the second phase is an aqueous gel. 11. Because bacterial cells contain endo-β-galactosidase in the second phase is induced to produce endo-β-galactosidase in response to the presence of a substrate. 9. The method according to claim 8, wherein: 12. The substrate for endo-β-galactosidase included in the second phase is degraded. 12. The method according to claim 11, wherein the mucin is mucin. 13. A substantially isolated fragment of a group BIII streptococcal polysaccharide capsule, Fragment containing intact GBS-III PC unit. 14. 14. The fragment comprises no more than 150 GBS-III PC repeat units. Substantially isolated fragments of 15. 14. The fragment comprises 50 or fewer GBS-III PC repeat units. A substantially isolated fragment of.