DD202623A5 - PROCESS FOR PREPARING SPORES POLYOSIDES - Google Patents

Abstract

1. Claims (for the Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE) Process for the production of immunogenic capsular polyosides from capsulated bacteriae containing same, characterised in that it consists in cultivating the bacteriae in a synthetic medium which may contain up to about 0.5% by weight of naturally occuring materials ; at the end of the growth stage of said culture removing the microbial bodies, submitting the liquid phase after removal of the microbial bodies to a filtration through a membrane which retains the molecules of a molecular weight of 100 000 daltons or more, to give a capsular polyosides-rich retained material ; and removing the proteins, the nucleic acids and the lipids from said retained material, to give a purified capsular polyoside. 1. Claims (for the Contracting State AT) Process for the production of immunogenic capsular polyosides from capsulated bacteriae containing same, characterised in that it consists in cultivating the bacteriae in a synthetic medium which may contain up to about 0.5% by weight of naturally occuring materials ; at the end of the growth stage of said culture removing the microbial bodies, submitting the liquid phase after removal of the microbial bodies to a filtration through a membrane which retains the molecules of a molecular weight of 100 000 daltons or more, to give a capsular polyosides-rich retained material ; and removing the proteins, the nucleic acids and the lipids from said retained material, to give a purified capsular polyoside.

Description

The invention relates to a process for the preparation of spore-polyosides, the polyosides obtained by this process and the use of these polyosides for the production of vaccines. «

Characteristic of the known technical solutions

It is known that the fight against infectious diseases can be carried out on two levels: either by a direct effect on the pathogen (antibiotics, antiseptics) or by an indirect effect by strengthening the body's defenses (vaccination, serum therapy, immunomodulation).

The use of direct-acting agents often encounters certain difficulties that may limit their effectiveness (especially toxicity of the molecule and resistance of the bacteria).

In contrast, strengthening the organism's defenses by acquiring the specific antibodies has certain advantages, particularly from the point of view of the term of protection and specificity. However, vaccination with whole microbes is not always without disadvantages: symptoms of hypersensitivity, poor tolerability or pyrexia are the most common · They are related to the presence of various antigens of the bacterium

Thus, the attention researchers place on the isolation of the responsible antigen of the protective antibodies is quite natural.

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In the case of bacterial spores, the spore polysides with little lift effects in humans prove to be immunizing.

A first application of these spore-polyosides in humans is the development of the yakzins antimeningococcus A and C and, more recently, the polyvalent vaccine containing the pure polyosides of 14 serological types of pneumococcus.

Several other bacterial spores play a role in various disease processes, such. B ,:

- Hemophilus influenzae, type b

- Klebsieila pneumoniae

- Escherichia coli

- Streptococcus, group B

and corresponding vaccines can be prepared from their spore polyosides ·

European Patent Application 0 002 404 has described a process for producing spore-polyosides from Streptococcus pneumoniae .

According to this method, the microorganism is grown in complex soil. After inactivation by means of phenol, without separating the microorganisms from the soil, a first addition of alcohol such as, for example, ethanol is carried out. Then separate the precipitated foreign matter and add a second MaJL alcohol to precipitate the polyoside ·. Thereafter, the impurities (proteins, nucleic acids) are removed from the resuspended polyoside by enzymatic treatment or by treatment with a cationic supra-active agent (citronium bromide), which can be supplemented by ultrafiltration.

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This procedure is not general, because the specific conditions of each stage change with the polyoside to be separated. Therefore, preliminary tests must be performed to determine the conditions of the different precipitations. In addition, this method may destroy polyosides due to the use of certain nutrient media Chains lead ·

Object of the invention

The object of the invention is to provide a more general, simple and less destructive method by which very high molecular mass polyols can be produced.

Explanation of the essence of the invention

The invention has for its object to find a new technology for the preparation of such polyosides.

According to the present invention, there is provided a process for producing spore immunosorbent polyosides from bacterial spores in which they are contained, characterized in that bacteria are grown in a synthetic or semi-synthetic nutrient medium at the end of the growth phase of said bacterial culture above floating phase, subjecting this top-floating phase to filtration through a membrane that holds molecules of molecular weight equal to or above 100,000 daltons, thereby recovering a spore-polyoside-rich retentate, and the proteins, nucleic acids, and nucleic acids Lipids are separated from this Hetentat, so that a pure spore-polyoside is obtained.

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Thus, according to a main characteristic of this invention, the culture of the bacteria is carried out on synthetic or semisynthetic medium. Synthetic soil is the name given to aqueous solutions of known low molecular weight chemical compounds which are present in certain proportions.

Semisynthetic Hährboden is referred to an analogous to the previous culture medium, but also contains a small proportion (generally less than 0.5 wt .-%) of natural substances such as protein hydrolysates.

In addition, according to the invention, the extraction of the polyosides takes place from the above-floating phase of the germ-free culture and not from the entire soil.

By filtration through a membrane whose porosity can retain molecules of molecular weight equal to or greater than 100,000 daltons, a retentate can be rapidly prepared which includes, in particular, the polyosides and, in minor amounts, proteins, nucleic acids and lipids.

The elimination of impurities is carried out favorably in the following way: the retentate is subjected to enzymatic hydrolysis, in particular by a mixture of pronase. RNase and DJKTase, add a butanol-chloroform mixture (preferably a 1: 1 volume mixture), separate the aqueous phase, dialyse the aqueous phase, and pass through a membrane containing the molecules of molecular weight equal to or greater than 100 000 daltons withhold, a filtration through.

The pure polyoside can be separated in a known manner from the retentate ^ and by precipitation by an alcohol such. B, ethanol »

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embodiment

The following examples illustrate this patent application j

example 1

Polyoside pneumococcus, type 3

It is based on a lyophilized strain Streptococcus pneuffioniae type 3,

a) Production of culture lots

The lyophilized strain is introduced into Broth T. The culture is carried out at 37 ^° C. for 16 hours. 5 ml of the culture obtained are mixed with 15 ml of sterilized skimmed milk. The whole is distributed on ampoules, in the ratio of 0.5 ml per ampoule, and lyophilized. The Loyphilisate be preserved at +4 ⁰ C.

b) preculture

Bringing a culture lot (ampoules lyophilisate) in a flask, a containing 200 ml of hylsynthetischen Mhrbodens given above, and the breeding leads 6 hours at 37 ⁰ C.

Hährboden:

Casein pancreatic peptone (IBP) 1500 mg

β-cystine 150 mg

dl-tryptophan 20 mg

Z, -tyrosine 200 mg

prim. Potassium phosphate 4960 mg

d-glucose, anhydrous 12500 mg

Magnesium sulfate, 7 HpO 500 mg

Bisenvitriol, 7H "0 5 mg

Zinc sulfate, 7H ₂ O 0.8 mg

Manganese sulfate, 7 H ₂ Q 0.3 mg

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Hydrochloric acid, r. ρ, smoking 17.8 mg

d-biotin 15 mg

nicotinic acid 1 mg

Pyridoxine 1 mg

Riboflavin 1 mg

Thiamine 1 mg

Calcium pentothenate 5 mg

Adenine HCl 10 mg

Urazil 10 mg

Choline chloride 10 mg

Asparagine 100 mg

distilled water, q. s, p. · 1000 ml

c) culture

The actual culture is carried out in a fermentation vessel containing 18 ml of the medium defined under b). The culture starting with 1% of the pre-culture, The culture is performed at 37 ⁰ C at pH 7.4, with stirring, generally about 16 hours until the end of the exponential Y / growth phase,

d) Separation of the above floating phase

At the end of the exponential phase, immediately the supernatant phase is separated by continuous centrifugation at 5000 U / min · The supernatant phase is a sterilizing filtration exposed through a millipore membrane 0.22 / U and used immediately or conserved at -25 ⁰ G ,

e) extraction

A volume of about 20 liters of the supernatant phase is concentrated by filtering through a Millipore membrane 10 capable of retaining molecules of molecular weight equal to or greater than 100,000 daltons. The resulting retentate is ultrafiltered through a Diaflon 10 membrane after 5 liters of sterile and pyrogen-free distilled water are added to complete the separation by molecular weight.

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Allow several subsequently performed Dltrafiltrationen to concentrate to about 350 ml, which are lyophilized in 1-liter flask · All these operations are at a temperature of at most 8 ⁰ C performed ·

f) cleaning

The lyophilized intermediate is dissolved in 900 ml of phosphate tampon 0.01 M pH7. Then 20 mg bladder, type 1, 20 mg pancreat DUase and 180 yul (20 units / ml) RNase T1 are added. This is followed by incubation at 37 ^° C. for 4 hours. It now is added 170 mg pronase, Type 8, 900 / ul pure phenol and 2 drops of toluene · Incubate the mixture at 37 ⁰ a laughing G. Thereafter, the reaction is stopped and an equal volume (900 ml) of a butanol - Chloroform - mixture 1: 1 added. After stirring for 1 hour, the two phases are decanted by centrifuging at 10,000 rpm for 30 minutes. The aqueous phase containing the polyoside is dialyzed for 60 hours through 20 liters of sterile distilled water. The dialysate is ultrafiltered through an Amicon 10 membrane. The retentate volume is 1140 ml. 16 ml of 40% sodium acetate (final concentration 5 %) and 3.9 liters of pure ethanol (3 volumes) are added. The precipitate is collected by centrifuging at 12,000 rpm for 30 minutes. This precipitate is dissolved in pyrogen-free distilled water. The solution is distributed to flasks of 4 ml volume each. × Vacuum lyophilization is carried out. 9 g of product are obtained. This is the pure polysaccharide that contains:

- Sugar 64 %

- proteins 0.8 %

Huulinic acid 0.8 %.

All operations (except the incubations) are carried out at a temperature between 0 and +4 ⁰ C.

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Example 2

Polyosid pneumococcus type 23

A lyophilized strain of Streptococcus pneumoniae type 23 is used. The culture and the separation of the above-floating phase are carried out under the conditions defined in Example 1 (stages a to d).

The extraction and purification of the polyoside are carried out as follows:

e) extraction

A volume of 15 liters of the supernatant phase is concentrated to a volume of 1.5 liters under the conditions defined in Example 1, 1. Three successive ultrafiltrations allow concentration followed by lyophilization. lan receives 1.8 g of product with the following composition:

- sugar 60 %

- Proteins 19%

Huulinic acid 3.6%.

f) cleaning

The lyophilized intermediate is mol with 200 ml phosphate buffer 0.01 - added to pH 7 · are added, 1 mg Rlfese, 1 mg DHase and 20 units RNase T1 / ml · Incubate 3 Ii at 37 ⁰ G. Hun Where added of pronase type 8 (180 μg / ml), 1 / μl / ml of phenol and one drop of toluene,

incubate for one night at 37 ⁰ C.

Successively carry out three extractions with the same volume of a mixture (200 ml) of butanol / chloroform 1: 1. The mixture is mechanically agitated.

The final aqueous phase is dialyzed for about 60 hours with 4 liters of sterile and pyrogen-free distilled water.

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The dialysate is ultrafiltered through a membrane Amicon 1080. It is concentrated to 50 ml "You now are 3 volumes of pure ethanol and latriumazetat (final concentration 5%) added · The whole is left to a Geesthacht at -20 ⁰ C rest ·

The precipitate is collected by centrifugation. It is dissolved in sterile pyrogen-free distilled water and then lyophilized.

This gives 1050 g of a product containing:

- sugar 75 %

- proteins 1.7%

- nucleic acid 0.7 %

All operations are carried out between 0 and +6 ⁰ G unless otherwise specified.

Example 3

Polyoside pneumococcus type 19

A lyophilized strain of Streptococcus pneumoniae type 19 is used. The culture and the separation of the above-floating phase are carried out under the conditions defined in Example 1 (stages a to d).

The extraction and purification of the polyoside are carried out as follows:

e) extraction

By proceeding as in Example 1, one concentrates a volume of 75 liters of top-floating phase through three ultrafiltrations to 2 liters. This gives 2.3 g with the following. Composition:

- sugar 30 %

- proteins 15 %

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Huulinic acid 3.4 %

f) consecration

The lyophilized intermediate is dissolved with 150 ml of phosphate buffer 0.01 mol - pH 7. Add 3 mg DUase, 3 mg RUase and 30 / Ul Rlase T1. It is then incubated for 4 h at 37 ⁰ C, then 62 mg Pronase, 150 / Ul phenol and a drop of toluene are added. The whole is allowed to a Geesthacht at 37 ⁰ G · react now be given of the deproteinization by the 1: 1 - chloroform-butanol mixture equal volume (3 extractions in succession). The aqueous phase is dialyzed for 60 hours with 4 liters of sterile and pyrogen-free distilled water. Ultrafiltration of the dialysate is performed through an Amicon XM 100 membrane. The retentate is precipitated by 3 volumes of ethanol containing 5% sodium acetate. After washing in the freezer, the precipitate is dissolved in sterile and pyrogen-free water and lyophilized. This gives 730 mg of product containing:

- sugar 73 %

- proteins 0.2 %

- Efucleic acid 0.5%.

Again, all operations (except incubations) are carried out at a temperature between 0 and +4 ⁰ C.

Example 4

Polyosid pneumococcus type 1

It is based on a lyophilized strain Streptococcus • pneumoniae type 1. The culture and separation of the supernatant phase are carried out under the conditions of Example 1.

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The extraction and purification of the polyoside are carried out as follows

e) extraction

100 l of the above-floating phase of the culture are ultrafiltered by PeI-licon cassettes (Millipore, membrane 10 *). The volume of the final retentate is 3 l. The lyophilization is carried out in a flask. 6.1 g of product are obtained which contains:

- galacturonic acid 46.5 %

Proteins 7,7 %

f) cleaning

Dissolve the product obtained in 800 ml of pyrogen-free EDS (sterile distilled water). The following are added:

- 16 mg RNase

- 16 mg Dlase

- 16O / Ul RNase T ₁ - '

Incubate 4 hours at 37 ⁰ C is then added 300 / ul phenol, 2! Drops of toluene and 150 mg pronase type 8 · added is incubated overnight at 37 ⁰ C. Then, the proteins or by Ghloroform-butanol mixture (1 : 1) of equal volume extracted. The aqueous phase is dialyzed for 60 hours. The ultrafiltration is done through a membrane Amicon XM 100 · The volume of the final retentate is 150 ml. The solution is dropped through 3 volumes of cold ethanol containing 5% 2-sodium acetate.

The residue is taken up again with 250 ml of pyrogen-free EDS. In glass bottles, the vacuum is lyophilised. 3.7 g of pure polyoside are obtained, which contains:

- galacturonic acid 55.5% (which corresponds to 85 % sugar) proteins 1 %

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The partition coefficient of the polyoside m gel IC _n is 0.05 ×

7 The molecular weight is at least 2 χ 10 »

Example 5

Polyoside of K · pneumoniae type 2

It is based on a strain Klebsiella pneumoniae type 2,

The culture of the microorganisms and the separation of the above floating phase under the conditions defined in Example 1.defined (stages a to d), but using as a nutrient medium, the following artificial Hährboden: trisodium citrate 0.85 g

Ammonium sulfate 0.17 g

Magnesium sulfate 0.17 g

Glutamic acid 0.17 g

d-glucose 16.70 g

sec. Potassium phosphate 10 g

prim. Potassium phosphate 6.66 g

distilled water, q.s.p. 1000 ml final pH: 6.8

The extraction and purification of the polyoside are carried out as follows:

e) extraction

It is used 90 l of the above-floating phase of the culture »It is ultrafiltered through a Pellicon cassette (2 cassettes, membrane 10 ^). Then, 3 rinses are made with 10 liters of water after concentrating to 1.5 liters. The final retentate is 2.6 liters. · One is lyophilized in flask and receives 6.1 g of product containing: - Sugar 50 %

Proteins 15 %

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f) cleaning

The product obtained is dissolved in 350 ml of water. »The following are added:

- 7 ml of phosphate buffer 0.5 mol, pH 7.0

- 7 mg RNase

- 7 mg DHase

- 70 / Ul RHase I ₁ .-

(previous filtration of the enzymes by 0.22 / u).

The mixture is incubated for 4 hours at 37 ⁰ C. Thereafter, 70 mg of protease type 8, 350 / Ul phenol and 1 drop of toluene added · incubate one night at 37 ⁰ C then the reaction is stopped and the deproteinization by 3 extractions with Butanol-chloroform (1: 1) is completed. The aqueous phase is dialyzed for 60 hours by pyrogen-free EDS. This is followed by ultrafiltration through the Amicon XM 100 membrane. The volume of the final retentate is 150 ml. The solution is precipitated by 3 volumes of cold pure ethanol containing 5% sodium acetate. The precipitate collected by centrifugation is dissolved in 250 ml of water, then vacuum lyophilised in glass bottles. A pure polyoside of the following composition is obtained:

- sugar 76 (hexoses / tetronic acid = 3: 1)

- proteins 3.8%

The distribution coefficient K _{n of} the obtained polyoside is

XJ η

0.1 · The molecular weight is thus over 10.

Example 6

Polyoside of H. influenzae type b

It is based on a lyophilized strain Hemophilus influenzae type b.

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The culture and separation of the supernatant phase are carried out under the conditions defined in example 1 (stages a to d), but the following semisynthetic bottom is used as the medium: proteose peptone 5 g

Sodium chloride 3> 5 g

d-glucose 4g

Prim, potassium phosphate 1.3 g

sec · Potassium phosphate 3.5 g

MD (Mycotinadenosine dinucleotide) 0.001 g Globuline extract 50 ml

distilled water q * s.p x 1000 ml

The extraction and purification of the polyoside are carried out as follows:

e) extraction

One uses 30 liters of floating phase of the culture · Ultrafiltration is carried out through a membrane Millipore 100 000 (2 cassettes) · The first concentration leads to a volume of 1 liter. It is rinsed 3 times with 10 liters of EDS. It follows a concentration of 1.3 liters. It is lyophilized in flask and finally receives 3.9 g of a product containing:

- Sugar 53 %

- Proteins 13 %

The product obtained is dissolved in 200 ml

Piltration by 0.22 / u) to:

4.5 ml phosphate buffer 0.5 mol, pH 7.0 4 mg bladder

4 mg DUase

40 / μl KHase T *

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Hun is incubated for 4 hours at 37 ⁰ C · Thereafter, 65 mg protease type 8, 200 / ul phenol and 2 drops of toluene are added, lan incubated a laugh at 37 ⁰ G. The reaction is carried out by 3 extractions with butanol-chloroform mixture (1: 1) stopped. The aqueous phase is collected by centrifugation at 12,000 rpm, 30 minutes at +4 ⁰ C. It is dialyzed for 60 hours with pyrogenic EDS (frequent changes of the dialysis medium). Concentration is then carried out through the Amicon XM 100 membrane (2 flushes 100 ml). The volume of the ultrafiltrate is 100 ml. The ultrafiltrate is precipitated by 3 volumes of cold pure ethanol containing 5% sodium acetate Centrifuged precipitate collected is taken up in 100 ml of pyrogen-free EDS. The solution is vacuum-lyophilised in glass bottles »

1.3 g of pure polyoside of the following composition are obtained:

- ^s sugar (PHP) 63 %

- Proteins 2 %

^s The ratio of the constituents of the polyoside is as follows: Eibose: Ribitol: Phosphorus = 0.9; 1; 0.98 (theoretical ratio = 1: 1: 1).

The. Partition coefficient Kg is close to 0.1, which corresponds to a molecular weight of about 10 '.

The immunogenic spore-polyosides prepared according to the invention can be used for the preparation of vaccines for preventive (or curative) use in humans and animals. The vaccines may be incorporated into physiologically acceptable carrier fluids such as a physiological solution or injection fluid.

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For this purpose, one or preferably several pure spore polyosides obtained according to the invention can be added from various serological basic forms. Furthermore, polyvalent vaccines can be prepared by adding two or more pure spore polyosides obtained from different microorganisms.

In the following, test results are given which show the immunizing effect of the polyoxides obtained according to the invention.

a) Titration of serum antibodies in mice

- Swiss mice of male sex of 20 g were distributed on lots of 10 animals:

1 Comparative lot was not subjected to any treatment.

Comparative lot was treated by multiple intraperitoneal administration of a dose of polyoside of Example 1 to each animal.

The titration of the agglutinating antibodies was carried out according to the techniques described in the literature, to which sheep's chromosome-sensitized red blood cells were used by the polyoside.

The results in Table I show an increase in the serum titer of the agglutinating antibodies in the animals treated with low doses compared to the control animals and a classic immunity paralysis at high doses *

Analogous experiments were carried out on muses with the polyoside of example 5 ( Klebsieila pneumoniae type 2 polyoside );

The results are listed in Table II.

1 128 4 7 ,8th O O O 64 6 7.5 4096 8th 13 , 9 2 128 + 1 7 , 84 O O 8th 3 1.85 2048 + 2 12 , 02 3 128 7 O O 512 9 128 7 4 16 4 O O 32 5 128 7 5 4 2 O O 512 9 256 8th 6 8th 3 O O 256 8th 128 7 7 64 6 O O 256 8th 256 8th 8th 64 6 O O 1024 10 64 6 9 O O O O 128 6 256 8th 10 64 6 O O 2048 11 4096 13 ±

Table 1 Mice - serum titers of agglutinating antibodies

Animal Animals Treated Animals Treated Animals Treated Animals CD

77 / Ug / mouse 0.77 / Ug / Maua 0.077 / Ug / mouse ^^

ρ 0.05 0.01

(1) agglutinating antibodies reverse to dilution f "" "*

(2) Agglutinating antibodies logg inverse to the dilution * _ω v>

Table II

Mr · Comparison Animals (1) (2)

Treated animals Treated animals / Ug / kg Treated animals / Ug / kg Treated animals / Ug / kg 1630 / Ug / kg 163 (2) 16.3 (2) 1.63 (2) (1) (2) (1) (1) 3 (1) O O 8th 4 O 4 O O 16 5 16 4 O O 32 4 16 5 O O 16 3 32 6 O O 3 8th 4 64 5 O 8th 16 4 32 4 O O 1 16 4 16 5 O 2 16 5 32 6 O O 32 6 64 4 O O 64 16

1 0 2 0 3 0 4 0 5 0 6 0 7 0 8th 0 9 0 10 0

cn

4.2 + 0.69 4.3 + 1.28

0.001 0.001

ro he »

(1) Agglutinating antibodies - inverse to dilution

(2) Agglutinating antibodies - log _{0 of} the reverse V / tert for dilution co -j

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b) Titration of serum antibodies in rats

The same examination was performed on male Sprague Dawly rats of 400-450 g.

The 10 animals were injected ip comprehensive lots of 2 and 20 ng polyoside of Streptococcus pneumoniae Type 3, per animal.

Titration of the agglutinating antibodies was done on 4, 6 and 14 days after administration.

The results are given in Table III.

In accordance with the literature, the titre gradually increases and reaches its maximum height on the 6th day.

Table III

Rats - serum titre - agglutinating antibodies log _{p of} the inverse of dilution

Ur ₄ , comparison animals

'14

1 O O O 2 O O O 3 O O O 4 O O O 5 O O / O 6 O O O 7 O O O 8th O O O 9 O O O

Treated animals 2 / Ug / batte

^J 4 ^J 6 ^J 14

7 7 6 5 7 5 4 6

7 8 6 7 6

5 6 6 8

5 7 3 6

4 2 5 3 7

5.7 6.55 4.66 + 0.89 + 0.82 +1.46 0.001 0.001 0.001 treated animals / ug / rat

5 6 8

9 10

6 10

5 6

7.22 +1.66 0.001

'14

0 0 2 0 3 0 3 0 0

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c) Specific protection

loose from 10 male mice of Swiss origin were immunized subcutaneously with Streptococcus pneumoniae type 1 polyoside obtained according to Example 4 (two injections at one week intervals); injected doses: 0.47 - 4.7 - 47 470 / ug / kg.

On the 10th day after the 2 nd immunization, the animals were subcutaneously infected with virulent pneumococcus type 1 (1250 germs / animal),

The mortality after ten days observation was as follows:

- untreated animals 100 %

- animals treated with 0.47 / μg / kg 30 %

- animals treated with 4.70 / ug / kg 10 %

- animals treated with 47.0 / ug / kg 30 %

- Animals treated with 470 / ug / kg 40 %

Lots of 10 male mice of Swiss origin were subcutaneously immunized with KP's polyoside (2 injections at one week intervals - injected doses: 53-> 533-5332 / Ug / Jsg),

On the 8th day after the last administration, the animals were injected with the strain of Klebsiella pneumoniae type 2 in the ratio of 650 germs per animal. The mortality after 10 days observation was as follows:

- untreated animals 100 %

- treated animals 53 / Ug / kg 0 %

- treated animals 533 / ug / kg 0 %

- treated animals 5333 / μg / kg 0 %

The polyosides obtained according to the invention can be used in particular for the preparation of the following vaccines:

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(a) Vaccines for the preventive and curative treatment of acute and chronic respiratory diseases, containing any of the pure polyosides corresponding to any of the following: Streptococcus pneumoniae Hemophilus influenzae . Type b Klebsieila pneumoniae Escherichia coli

As an example

1) Mixture of polyoxides of S · pneumoniae, types 1, 6, 14, 23 50 μug of polyoxide of K · pneumoniae, type 2 20 Polyoside of H. influenzae, type b 20 Final volume 0.5 ml of physiological water

2) Mixture of polyosides of S, pneumoniae, types 1, 6, 14, 23 25 / Ug Polyoside of K, pneumoniae, type 2 10 / Ug Polyoside of H · influenzae, type b 10

b) Yakzin antipneumococcus, which contains the pure polyosides of the types of Streptococcus pneumoniae :

1, 3, 4, 6A, 7P, 8, 91, 12P, 14, 181, 19F, 23, 2, 11A, 15i ".

As an an example;

Mixture a 50 / ug, in a volume of 0.5 ml of physiological water,

Mixture a 25 / Ug, in a volume of 0.5 ml physiological water ·

Claims (3)

invention claim A process for the preparation of immunizing spore polyosides from bacterial spores in which they are present, characterized in that a bacterial culture is cultivated in synthetic or semi-synthetic soil, at the end of the growth phase of this culture separates the microbes of a floating above phase, this floating phase above Subjecting filtration through a membrane which retains molecules of molecular mass equal to or greater than 100,000 daltons, thus obtaining a retentate rich in spore-polyosides, eliminating the proteins, nucleic acids and lipids from this retentate, eventually forming a receives pure spore polyoside, 2 "" method according to item 1, characterized in that for the elimination of the proteins, nucleic acids and lipids from the retentate this is subjected to enzymatic hydrolysis, adding a butanol-chloroform mixture, the aqueous phase is separated, subjected to dialysis and a Performs filtration through a membrane which retains molecules of molecular mass greater than 100,000 daltons. 3. Process according to item 1 or 2, characterized in that the culture is cultivated on semisynthetic soil containing less than 0.5% by weight of natural substances.