DE2818922A1 - IMPROVED VACCINES WITH CAPSULAR POLYSACCHARIDES - Google Patents

Description

PIERRE FABRE S.A. P 729

DESCRIPTION

The present invention relates to improved acellular vaccines and methods for their preparation.

It is known from French patents 73 43 957, 75 10 252 and 76 24 124, acellular vaccines Manufacture from ribosomes or ribosomal ribonucleic acids in conjunction with membrane polysaccharides and membrane glycoproteins.

It has now been found that the antigenic and thus the vaccine effect for certain capsule bacteria their ribosomes or ribonucleic acid are considerably potentiated by capsular polysaccharides of the corresponding bacteria will.

The present invention accordingly relates to a method for significantly improving the effectiveness of acellular vaccines that act as the vaccine agent ribonucleic acids and / or ribosomes containing at least one bacterial strain with polysaccharide capsules, whereby the vaccine Capsular polysaccharide extracts at least one of the bacterial "strains with polysaccharide capsules added.

This method is particularly useful for significantly improving vaccines based on ribonucleic acids and / or ribosomes of pneumococci such as diplococcus, in particular D. pneumoniae as well as Klebsiella pneumoniae and Haemophilus influenzae are suitable.

Of course, acellular vaccines are known and described in the French patents cited.

In particular, these vaccines can, in addition to the ribonucleic acids and the ribosomes, which are the actual vaccine components form, contain membrane glycoproteins as adjuvant and / or membrane polysaccharides.

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The amount of capsule polysaccharides added is changeable within wide limits; it is preferably in weight ratios of 2/1 to 1/2, based on the ribonucleic acids or ribosomes of the capsular bacteria in the vaccine.

According to the invention, it is not absolutely necessary to use capsular polysaccharides, corresponding to all of them in the vaccine capsule bacteria strains used; it is enough that at least the capsular polysaccharide extracts one of the capsular bacterial strains contained in the vaccine, the ribonucleic acids or the ribosomes be used.

In the following some are improved according to the invention Vaccines tabulated:

I) anti-pneumococcal vaccine

Ribosomes of Diplococcus pneumoniae I 2 μg Ribosomes from Diplococcus pneumoniae II 2 μg ribosomes from Diplococcus pneumoniae III 2 [fig

Capsular polysaccharides from Diplococcus pneumoniae II 3 μg

Capsular polysaccharides from Diplococcus pneumoniae III 3 μg

Klebsiella pneumoniae membrane glycoproteins 18 µg

II) Anti-Pneumococcal Vaccine (RNS)

Diplococcus pneumoniae RNA I ■ 1.5 µg Diplococcus pneumoniae II RNA 1.5 μg Diplococcus pneumoniae III RNA 1.5 μg Diplococcus capsular polysaccharides

pneumoniae II 3 µg

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Diplococcus capsular polysaccharides
pneumoniae III 3 µg

Klebsieila membrane glycoproteins

pneumoniae 20 µg

III) Antibronchial Vaccine

Ribosomes of Diplococcus pneumoniae I 1 μg

Diplococcus pneumoniae II ribosomes 2 μg

Diplococcus pneumoniae III ribosomes 1 μg

Klebsiella pneumoniae ribosomes 2 μg

Hemophilus influenzae ribosomes 1 μg

Streptococcus pyogenes Aj ₂ 1 μg ribosomes

Diplococcus capsular polysaccharides
pneumoniae II 2, -5 µg

Diplococcus capsular polysaccharides

pneumoniae III 2.5 (ig

Capsular polysaccharides from Klebsiella

pneumoniae 3 µg

Glycoproteins from Klebsiella pneumoniae 24 μg

IV) Antibronchial Vaccine

Ribosomes of Diplicoccus pneumoniae II 2 μg Klebsiella pneumoniae ribosomes 2 μg

Hemophilus influenzae ribosomes 1 μg

Diplococcus capsular polysaccharides

pneumoniae II 2.5 µg

Capsular polysaccharides from Klebsiella

pneumoniae 2.5 µg

Hemophilus capsular polysaccharides

influenzae 1.5 µg

Klebsiella membrane glycoproteins

pneumoniae 17 µg

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V) antibronchial vaccine

RNA of Diplococcus pneumoniae II 1.5 ^ g

Klebsiella pneumoniae RNA 1.5 ng

Hemophilus influenzae RNA 1 ng

Diplococcus capsular polysaccharides

pneumoniae II 2 ng

Capsular polysaccharides from Klebsiella

pneumoniae 2 ng

Hemophilus influenzae capsular polysaccharides 1.5 ng

Klebsiella membrane glycoproteins

pneumoniae 15 ng

The different components of the compositions can be dosed according to the cited French patents.

The capsule polysaccharides can, starting from an aqueous solution of the capsules, by precipitation with cetylpyridinium chloride getting produced. The polysaccharide / cetylpyridinium chloride complex is dissociated by an inorganic salt such as sodium chloride (2 molar), whereby the polysaccharide goes into solution and the cetylpyridinium chloride fails.

The polysaccharides can then be precipitated, for example by adding ethanol.

The polysaccharides can then be washed and purified again. This can be done by washing the polysaccharides in a water / ethanol mixture in the presence of sodium acetate.

To the polysaccharides with separation of those still contained To purify proteins, you can use an aqueous solution of the polysaccharides,

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extract with phenol, for example with 90% phenol in the heat.

The polysaccharides contained in the aqueous pH phase are precipitated by ethanol.

The invention also relates to that according to the invention manufactured vaccines.

Exemplary methods of making the various components of the vaccines of the present invention are described below.
10

General fermentation process

The bacterial strains are placed in inclined tubes brought in; the production of the biomass is examined in three successive steps, namely first in a 500 ml flask statically in the heating cabinet, then in a fermentation device with one volume of 20 l inoculated with the contents of the 500 ml flask, and finally in an industrial fermentation device with a volume of 600 1, which with

inoculated into the contents of the 20 liter fermentation device - will.

For each germ, pilot tests were carried out with the 20 l fermentation device carried out in order to achieve the optimum development conditions in terms of temperature, des pH value, oxygen demand, stirring speed and growth time to be determined. After these parameters were defined, their control and automatic regulation in the fermentation devices with 20 l volume and 600 1 volume realized.

At the end of the growth phase, the bacterial cells are cooled by continuous centrifugation in Collected and dispersed in a state

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Buffer Tris-HCl, MgCl ₂ , washed at pH 7 and concentrated by repeated continuous centrifugation. In the case of sensitive strains with a tendency to autolysis, the culture is quickly cooled to 8 to 10 ° C before centrifugation.

The biomass obtained in this way is stored frozen until it is used. With a sampling a bacteriological control is carried out to determine the identity of the germ and its absence detect impurities. The dry extract content is also determined.

Process for the production of the capsular polysaccharides

In order to obtain the capsular polysaccharides, the washed cells are dispersed in a Tris-HCl buffer (0.01 M) at a pH of 7 containing 0.1 M NaCl and 0.01 M MgCl _2. The temperature of the suspension is brought to 40 ° C; the capsules are separated by homogenization for a few minutes in a Warring mixer at this temperature.

After cooling, the decapsulated cells are separated off by centrifugation at 30,000 g and 4 ° C. for 30 minutes .

The deposited product is preserved for the production of the ribosomes and the ribonucleic acids. Of the Supernatant containing the capsules is collected.

The capsule polysaccharides are first removed from the supernatant by slowly adding an aqueous 2% cetylperidinium chloride solution precipitated until the end of the flocculation. After an hour Allowing to stand at 4 ° C, the polysaccharide precipitate is collected by centrifugation and the supernatant product removed.

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The precipitated complex polysaccharide / cetylperidinium chloride is dissociated in a 2 molar sodium chloride solution, under these bonds the polysaccharide goes into solution, while the cetylpyridinium chloride passes through Centrifugation is removed; the excess product is kept.

3 volumes of cold ethyl alcohol are added to this supernatant added to the precipitation of the polysaccharide, which after One hour standing at 4 ° C is collected by centrifugation.

The precipitated polysaccharide is dispersed in a water / ethanol mixture (30:70 volume ratio) washed with 0.1 m CH3COO Na content at room temperature for 30 minutes. The washing solution is made by centrifugation separated; the deposited product is taken up in an aqueous solution which is 0.1 molar in CH3COO Na .

In order to remove the proteins, which may still contaminate the product at this stage, is a 10 minute extraction at 65 ° with a volume of 90% phenol in sodium acetate (1 m) carried out. After 10 minutes vigorous stirring at 65 ° C, the mixture is quickly cooled in an ice bath; the two phases are through Centrifugation for 5 minutes at 10,000 g and 0 ° C separately.

The above aqueous phase is carefully lifted off. 3 volumes of cold ethyl alcohol are added to the aqueous phase added, whereupon the polysaccharide can precipitate at 4 ° C over a period of one hour.

The precipitate is collected by centrifugation and then in a water / alcohol mixture (30:70 volume ratio) , which is 0.1 molar in CH3COO Na, dispersed for 30 minutes at room temperature. The washed precipitate is collected by centrifugation and resuspended in distilled water, and then by filtration sterilized through a membrane (0.22 μ) and freeze-dried.

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Process for the preparation of the ribosomal fraction

The ribosomal fraction is produced either from decapsulated cells, which are obtained as described above, if the ribosomes are from capsular bacteria, or from cells obtained according to the general fermentation process, optionally after washing. The cells are suspended in a buffer Tris-HCl, MgCl2 at pH and turned into a slurry ^{in a homogenizer with plate cooling under a pressure of 600 kg / cm 2.}

The suspension of the crushed bacteria is removed from the cell debris by centrifuging for 45 minutes 30,000 g and 4 ° C removed; the supernatant clear product containing the ribosomes is saved.

The crude ribosomes are first precipitated from this supernatant by adding 0.8 volume of ethyl alcohol at -20 ° C. After standing for 30 minutes at 4 ° C., the precipitate is collected by centrifugation and taken up in a 0.5% solution of sodium dodecyl sulfate in a buffer Tris-HCl, MgCl ₂ at pH 7.2 and room temperature with vigorous stirring for one hour .

The suspension obtained, which contains the ribosomes, is precipitated with 0.7 volumes of ethyl alcohol at -20 ° C.

After standing at room temperature for 30 minutes, the ribosomes are collected by centrifugation at 15 ° C; the deposited product is taken up in the buffer Tris-HCl, MgCl ₂ with a pH value of 7 at 4 ° C. with slow stirring and left to stand at 4 ° C. overnight.

The suspension obtained is clarified by centrifugation for 45 minutes at 30,000 g and 0 ° C .; the protruding Product contains the purified ribosomes and is stored.

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The solution can now be used for making the ribosomal Ribonucleic acids can be stored frozen or sterilized by filtration and then freeze-dried.

Method of extraction of ribosomal ribonucleic acids

The ribosomal ribonucleic acids are, starting from the previously described, not freeze-dried ribosomal fraction extracted.

One volume of the ribosome suspension and one volume of 80% phenol previously mixed with the Tris-HCl buffer (0.01 m) with the pH value 7 and content of ÄDTE (0.01 m) (disodium salt) and 0.5% sodium dodecyl sulfate was stabilized mixed; then the temperature of the mixture is quickly increased to 65 ° C and 10 minutes at this temperature below Leave stirring. Then the mixture is cooled very quickly in an ice bath.

The two phases are separated by centrifugation at 10,000 g for 5 minutes at 4 ° C. and the upper one aqueous phase carefully lifted off.

The aqueous phase obtained is then mixed twice under the same conditions with half a volume of 80% Phenol extracted.

The aqueous phase obtained is collected and residual phenol is extracted three times with removed one volume of ether at a time in a decanting flask. The remaining ether is taken from the watery one Phase driven out with nitrogen.

Sodium chloride is then added to the aqueous phase to a molarity of 0.1; the ribonucleic acids are precipitated from this aqueous phase by adding 2 volumes of alcohol at -20 ° C. The precipitation is centrifuged at 10,000 g for 10

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Collected at 0 ° C for minutes. The settled ribonucleic acids are in a buffer Tris-HCl (0.025 m) at a pH 8.1, which contains 0.025 M NaCl, resumed to a level of 2 mg RNA per milliliter of solution to obtain.

Purification procedure for the ribosomal RNA

The previously obtained RNA solution is 1 volume of a 2.5 molar dipotassium phosphate solution and 1 volume 2-methoxyethanol added. After two to three minutes vigorous stirring, the precipitate is separated off by centrifugation at 10,000 g for 5 minutes at 4 ° C. The protruding clear product containing the RNA is collected and mixed with a volume of 0.2M sodium acetate. The RNA is then removed by slowly adding 0.5 volume of an aqueous 1% cetyltrimethylammonium bromide solution failed.

After standing at 4 ° C for 5 minutes, the RNA is isolated by centrifugation at 10,000 g for 5 minutes at 4 ° C collected. The centrifuge tube is dried completely and the precipitate twice with an excess on 70% alcohol, which is 0.1 molar in CH3COO Na, washed.

The precipitated RNA is then dissolved in a Tris-HCl buffer at pH 7 and overnight at 2 ° C dialyzed against 20 volumes of the buffer and then, after sterilization by filtration, freeze-dried.

Process for the production of the membrane glycoproteins

The membrane glycoproteins are produced, for example, starting from the crushed bacteria,

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which the bacterial cells obtained after fermentation are _{suspended in a Tris-HCl, MgCl 2} buffer at pH 7 and continuously comminuted in a homogenizer equipped with cooling plates under a pressure of 600 kg / cm ² will. The comminuted product is centrifuged in the buffer at 7,500 g for 15 minutes to separate a deposit containing the cell debris; then the supernatant is centrifuged at 30,000 g for 15 minutes to separate the membranes. The membrane fraction obtained, which is optionally washed three times in a row by centrifugation in buffers of decreasing ionic strength, is suspended in distilled water and freeze-dried "

The freeze-dried product is then two consecutive Subjected to extractions of 2 hours at room temperature: the first extraction is carried out with a Mixture of chloroform / methanol (2: 1), the second carried out with a mixture of ethanol / ether (3: 1).

The purified (defatted) residue is dried and used for the production of the membrane glycoproteins.

After the residue is suspended in an aqueous solution that is 2% sodium dodecyl sulfate and is clarified by centrifugation at 10,000 rpm for 15 minutes, the glycoproteins are removed from the supernatant

precipitated by adding 3 volumes of ethanol and a few milliliters of a concentrated solution of sodium acetate. After standing for one hour at room temperature, the precipitate is collected by centrifugation and taken up in distilled water.

After clarification by centrifugation at 10,000 rpm for 15 minutes, the supernatant containing the glycoproteins is removed contains, freeze-dried.

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The products used in the following experiments were made according to the procedures previously described.

TRIAL 1

In this experiment the activity of the combined ribosomes + capsular polysaccharides of Diplococcus pneumoniae examined. There were 4 groups of 10 mice each used. Each mouse was given 7 μg of ribosomes D. pneumoniae treated.

Each group B mouse was given 10 μ9 of the capsular polysaccharides treated by D. pneumoniae.

Each mouse in group C was simultaneously treated with 7 μg of ribosomes and 10 μg of capsular polysaccharides D. pneumoniae treated.

Group D mice were not treated.

The indicated doses were given five times subcutaneously in the rhythm of one injection each over three days Injected for 15 days.

10 days after the last injection, the animals are punctured sinus-retro-orbitally; the serum is examined:

1) with regard to immunodiffusion (according to Ouchterlony)

2) regarding immunoelectrodiffusion (according to Laurell)

in a 1% agarose gel in a veronal buffer with the

pH 8.6 against the complete antigen of D. pneumoniae. During the immunodiffusion, the serum of the treated Animals of group C against a complete antigen Diplococcus pneumoniae occur precipitation arcs which are numerically identical to those of a serum from mice with the complete antigen Diplococcus pneumoniae have been vaccinated.

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During xmmunoelectrodiffusion, the serum of the mice of group C forms diplococcus against a complete antigen pneumoniae a specific immunoprecipitate in the form of "rockets", the height of which is considerably higher than that of the "rocket" obtained with the serum of the mice of group A or the Group B can be obtained.

The amount of "rocket" can be in the proportions from double to five times the content of the specific antibodies, according to the mice, in proportion to the results in group A.

TRIAL 2

The same results x <rerden achieved when the 7 μg of the ribosomes of D. pneumoniae by 5 μg of the RNA of D. pneumoniae to be replaced.

During the immunodiffusion, the serum of the mice treated with both products (group C) allows against a complete one Antigen Diplococcus pneumoniae precipitation arcs occur which are numerically identical to a serum of the Are mice that have been inoculated with the complete antigen Diplococcus "pneumoniae".

During immunoelectrodiffusion, the serum of group C mice forms diplococcus against a complete antigen Pneumoniae a specific immunoprecipitate in the form of "rocket", the height of which is considerably greater than that of the "rocket" produced by the anti-RNA serum of Diplococcus neumoniae (Group A) or by the anti-capsular polysaccharide serum of Diplococcus' neumoniae (Group B) against an antigen Diplococcus neumoniae is formed.

The height of the "rocket" for group C can be varied in proportions ranging from double to five times the content of specific antibodies, according to the

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Mice in relation to the results according to group A are sufficient.

TRIAL 3
5

In this experiment, a more complete one compared to Example 1 was completed Vaccine studied.

A group E of 10 mice were put together

3.5 µg of D. pneumoniae ribosomes

5 μg of capsular polysaccharides from D. pneumoniae

12 μg of membrane glycoproteins from Klebsiella pneumoniae

inoculated in the same rhythm as in Example 1. The serum of the punctured animals resulted in the immunoelectrodiffusion a specific immunoprecipitate in the form of "rocket", the height of which is twice that of group C at attempt 1 is.

TRIAL 4

According to Example 2, a vaccine that is completed compared to Example 2 is investigated.

A group F of 10 mice became 25 together

2.5 µg of D. pneumoniae RNA

5 μg of capsular polysaccharides from D. pneumoniae

10 μg of membrane glycoproteins from Klebsiella pneumoniae

injected in the same rhythm as in Example 2.

The serum of the punctured animals shows on immunoelectrodiffusion a specific immunoprecipitate in the form of "rocket", the height of which is twice that of the Group C according to Example 2 is.

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This makes it clear that the vaccines according to the invention are considerably more effective than the vaccine own known vaccines.

s *

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Claims (7)

DR. ROLF E. WILHELMS DR. HELMUT KILlAN QaSELSTRASSE β 80OO MUNICH TELEPHONE (OBQ) 4740 73 · TELEX 5234 07 <w «p-d) TELEGRAMS PATRANS MUNICH P 729 PIERRE FABRE S.A. PARIS, FRANKEICH Priority: April 29, 1977 - FRANCE - 77 13 009 Improved Vaccines Containing Capsular Polysaccharides 1. Process to improve the effectiveness of Vaccines containing ribonucleic acids as the active ingredient and / or contain ribosomes of at least one bacterial strain with polysaccharide capsules, thereby g e k e η η ζ e i c h η e t that the vaccine capsule polysaccha- 809 8 45/0 91 * ORIGINAL INSPECTED PIERRE FABRE S.A. P 729 ride extracts of at least one of the named bacterial strains with polysaccharide capsules added. 2. The method according to claim 1, characterized in that g e k e η η, that a vaccine containing ribonucleic acids and / or ribosomes at least one of the Pneumococcal strains of Klebsiella pneumoniae or of Haemophilus influenzae are the active ingredient components, the capsular polysaccharides at least one of the pneumococcal strains of Klebsiella pneumoniae or of Haemophilus influenzae clogs. 3. The method according to claim 1, characterized in that a vaccine containing Ribonucleic acid and / or diplococcus ribosomes from Klebsiella pneumoniae or from Haemophilus influenzae as active ingredient components the capsular polysaccharides of at least one of the diplococcus strains of Klebsiella pneumoniae or of Haemophilus influenzae clogs. 4. The method according to at least one of claims 1 to 3, characterized in that the Capsular polysaccharides, starting from bacterial capsules, by precipitation with cetylpyridinium chloride, separating the precipitate and decomposing the cetylpyridinium polysaccharide complex in saline and recovering the capsular polysaccharides therefrom containing solution. 5. The method according to claim 4, characterized in that g e k e η η, that a two-molar sodium chloride solution is used as the salt solution. 8098A5 / 09U PIERRE FABRE S.A. P. 6. The method according to at least one of claims to 5, characterized in that one at a weight ratio of capsular polysaccharides and ribonucleic acids or ribosomes of bacteria with 5 polysaccharide capsules from 1/2 to 2/1 works. 7. Vaccine according to at least one of the methods according to claim 1 to 8098A5 / 09H