CH630806A5 - Method for preparing improved vaccines - Google Patents

Description

The present invention, carried out at the Pierre-Fabre Center for Immunology and Biology, relates to improved acellular vaccines.

It is already known, from French patents Nos. 73.43957, 75.10252 and 76.24124, to prepare acellular vaccines composed of ribosomes or ribonucleic acid (RNA) ribosomes associated with membrane polysaccharides and adjuvanted by membrane glycoproteins.

But it has now appeared that, for certain bacteria carrying capsules, the antigenic and therefore vaccine action of their ribosomes or RNA was strongly potentiated by the capsular polysaccharides corresponding to said bacteria.

This is why the present invention relates to a method for potentiating the action of acellular vaccines containing, as a vaccination principle, RNA or ribosomes of at least one bacterial strain with polysaccharide capsules, in which the vaccines are added to this vaccine. Capsular polysaccharides extracted from at least one of said bacterial strains with polysaccharide capsules.

This method is, in particular, usable for potentiating vaccines based on RNA or ribosomes of pneumococci such as Diplococcus, in particular D. pneumoniae as well as of Klebsiella pneumoniae and â'Hemophilus influenzae.

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

In particular, these vaccines may contain, in addition to the RNA or the ribosomes which constitute the actual vaccine fraction, membrane glycoproteins as an adjuvant and / or membrane polysaccharides.

The amount of capsular polysaccharides added can vary within wide limits but will preferably be included in a weight ratio of 2/1 to 1A relative to the RNA or ribosomes of the capsular bacteria of the vaccine.

To obtain a potentiated vaccine according to the invention, it is not essential to introduce capsular polysaccharides corresponding to 5 corresponding to all the strains of capsular bacteria used in the vaccine; it is sufficient that it comprises at least the capsular polysaccharides extracted from one of the capsulated strains whose RNA or ribosomes are used in the vaccine.

Examples of improved vaccines according to the present invention are given below:

1. Pneumococcal vaccine

Ribosomes of Diplococcus pneumoniae I 2 [ig

Ribosomes of Diplococcus pneumoniae II 2 (ig

Ribosomes of Diplococcus pneumoniae III 2 (ig

Diplococcus pneumoniae II 3 capsular polysaccharides (ig

Capsular polysaccharides of Diplococcus pneumoniae III 3 (ig

Membrane glycoproteins of Klebsiella pneumoniae 18 (ig

2. Pneumococcal RNA vaccine

20 Diplococcus pneumoniae I RNA 1.5 (ig

Diplococcus pneumoniae II RNA 1.5 [ig

Diplococcus pneumoniae III RNA 1.5 [ig

Diplococcus pneumoniae II 3 capsular polysaccharides [ig

Capsular polysaccharides of Diplococcus pneumoniae III 3 (ig

25 Klebsiella pneumoniae membrane glycoproteins 20 (ig

3. Anti-bronchial vaccine Ribosomes of Diplococcus pneumoniae I Ribosomes of Diplococcus pneumoniae II

m Ribosomes of Diplococcus pneumoniae III Ribosomes of Klebsiella pneumoniae Ribosomes â'Hemophilus influenzae Ribosomes of Streptococcuspyogenes A12

1! * G

2 (ig

1! * G

2 [ig l ^ g lf * g

Diplococcus pneumoniae II 2.5 capsular polysaccharides [ig Diplococcus pneumoniae III 2.5 capsular polysaccharides (ig Klebsiella pneumoniae 3 capsular polysaccharides (ig

Klebsiella pneumoniae 24 glycoproteins (ig

4. Anti-bronchial vaccine

Ribosomes of Diplococcus pneumoniae II 2 (ig

40 Ribosomes of Klebsiella pneumoniae 2 (ig

Ribosomes â'Hemophilus influenzae 1 (ig

Diplococcus pneumoniae II 2.5 capsular polysaccharides (ig

Capsular polysaccharides of Klebsiella pneumoniae 2,5 (ig

Capsular polysaccharides â'Hémophilus influenzae 1,5 [ig

45 Klebsiella pneumoniae membrane glycoproteins 17 [ig

5. Anti-bronchial vaccine

Diplococcus pneumoniae II RNA 1.5 [ig

RNA of Klebsiella pneumoniae 1.5 [ig n / a RNA â'Hemophilus influenzae 1 [ig

Capsular polysaccharides of Diplococcus pneumoniae II 2 (ig Capsular polysaccharides of Klebsiella pneumoniae 2 (ig

Capsular polysaccharides â'Hémophilus influenzae 1,5 (ig Membrane glycoproteins of Klebsiella pneumoniae 15 (ig 55 The various elements of these compositions can be assayed in particular by methods described in the French patents cited above.

The capsular polysaccharides can be prepared, in particular, from an aqueous solution of capsules by precipitation using cetylpyridinium chloride. The polysacchari-de / cetylpyridinium chloride complex is dissociated by an inorganic salt such as 2M sodium chloride; under these conditions, the Polysaccharide goes into solution and the cetylpyridinium chloride precipitates.

The Polysaccharides can then be recovered, for example by adding ethanol to the solution of Polysaccharides which precipitates. The Polysaccharides can then be washed and purified again. The Polysaccharides can be washed with a water / ethanol mixture in the presence of sodium acetate.

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If it is desired to purify the Polysaccharides to remove the proteins which are still present, these proteins can be extracted from an aqueous solution of Polysaccharides with phenol, for example phenol at 90% when hot.

The Polysaccharides contained in the aqueous phase are recovered by precipitation with ethanol.

The invention also relates to the vaccines obtained by the method described above.

Examples of the preparation of the various components of the vaccines according to the invention are given below, which in no way limit it.

General fermentation process

The bacterial strains are maintained on inclined agar tubes and the production of biomass is carried out in three successive stages, first in a 500 ml bottle static in the oven, then in a 201 fermenter which is seeded using 500 ml of the bottle and finally in an industrial fermenter of 6001 inoculated with the 201 of the previous fermenter.

For each germ, a pilot study on a 201 fermenter is carried out beforehand in order to determine the optimal development conditions with regard to temperature, pH, oxygen, the speed of agitation and the growth times. Each of these parameters being defined, its automatic control and regulation are ensured at the level of the fermenters of 20 and 6001.

The bacterial cells are collected at the end of the growth phase by continuous cold centrifugation and washed by dispersion in tris-HCl, MgCl2 buffer, pH 7 and concentrated by a new passage in continuous centrifugation. In the case of fragile germs which tend to self-lyse, the culture is rapidly cooled to around 8-10 ° C. before centrifugation.

The biomass thus obtained is kept frozen while waiting to be used. On a sample, a bacteriological control is carried out in order to verify the identity of the germ and the absence of contaminants. The dry extract content is also determined.

Process for the preparation of capsular polysaccharides

To obtain the capsular Polysaccharides, the washed cells are dispersed in a 0.01M tris-HCl buffer, pH 7, containing 0.10M NaCl and 0.01M MgCl2. The temperature of the suspension is brought to 40 ° C. and the capsules are separated by homogenization for a few minutes in a warring blendor at this temperature.

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

The pellet is kept for the preparation of ribosomes and ribosomal RNA. The supernatant containing the capsules is collected.

The capsular polysaccharides are first precipitated from the supernatant by the slow addition of an aqueous solution of cetylpyridinium chloride at 2%, until the end of flocculation. After standing for 1 h at 4 "C, the polysaccharide precipitate is collected by centrifugation and the supernatant discarded.

The precipitated polysaccharide / cetylpyridinium chloride complex is dissociated in 2M sodium chloride; under these conditions, the Polysaccharide goes into solution while the cetylpyridinium chloride is removed by centrifugation and the supernatant preserved.

Three volumes of cold ethyl alcohol are added to the supernatant to precipitate the Polysaccharide which is collected by centrifugation after 1 hour of rest at 4 C.

The precipitated polysaccharide is washed by dispersion in a water / ethanol mixture (30/70 v / v) containing 0.1 M CH3COONa, for 30 min at room temperature. The washing solution is removed by centrifugation and the pellet is taken up in a 0.1 M aqueous solution of CH3COONa.

In order to remove the proteins which may still contaminate the preparation at this stage, extraction is carried out for 10 min at 65 ° C. with a volume of 90% phenol in 1M sodium acetate. After vigorous stirring, 10 min at 65 ° C., the mixture is rapidly cooled in a displaced bath and the two phases are separated, by centrifugation, 5 min at 10,000 g and 0 ° C. The upper aqueous phase is gently removed. Three volumes of cold ethyl alcohol are added to the aqueous phase and the Polysaccharide is left in precipitation for 1 hour at 4 ° C.

The precipitate is collected by centrifugation and then dispersed in a water / ethanol mixture (30/70 v / v) containing 0.1 M CH3COONa for 30 min at room temperature. The washed precipitate is collected by centrifugation and taken up in distilled water, then sterilized by filtration on a 0.22 p membrane and lyophilized.

Process for the preparation of the ribosomal fraction

The ribosomal fraction is prepared either from the decapsulated cells obtained previously, in the case of ribosomes from encapsulated bacteria, or from the cells obtained in the general fermentation process after a possible washing. The cells are suspended in a tris-HCl, MgCl2 buffer, pH 7 and subjected to continuous grinding in a homogenizer equipped with a plate cooler under a pressure of 600 kg / cm2.

The suspension of bacterial ground material is then freed from cellular debris by centrifugation for 45 min at 30,000 g and 4 ° C; the clear supernatant, containing the ribosomes, is preserved.

The crude ribosomes are precipitated a first time from the preceding supernatant by the addition of 0.8 volume of ethyl alcohol at -20 ° C. After standing for 30 min at 4 ° C, the precipitate is collected by centrifugation and taken up in a solution at 0.5% sodiumdodecyl sulfate in the tris-HCl, MgCl2 buffer, pH 7.2, at room temperature with rapid stirring for 1 h.

The suspension obtained, containing the ribosomes, is precipitated by 0.7 volume of ethyl alcohol at - 20 ° C. After standing for 30 min at room temperature, the ribosomes are collected by centrifugation at 15 ° C and the pellet is taken up in tris-HCl, MgCl2 buffer, pH 7 at 4 ° C by slow stirring overnight at 4 ° C.

The suspension obtained is clarified by centrifugation at 30,000 g at 0 ° C for 45 min and the supernatant containing the purified ribosomes is stored.

At this stage, the solution can be stored frozen for the preparation of ribosomal RNA or else sterilized by filtration, then lyophilized.

Method for extracting ribosomal RNA

The ribosomal RNA is extracted from the preceding ribosomal fraction which is not lyophilized.

Mixing a volume of ribosome suspension and a volume of 80% phenol previously equilibrated with 0.01M tris-HCl buffer, pH 7, containing 0.001M EDTA (disodium salt) and 0.5% sodium dodecyl sulfate, then the temperature of the mixture is brought quickly to 65 ° C. and maintained for 10 min with stirring. The mixture is then quickly cooled in an ice bath.

The two phases are separated by centrifugation under 10,000 g for 5 min at 4 ° C and the upper aqueous phase is collected carefully.

The aqueous phase obtained is then reextracted twice under the same conditions with 0.5 volume of 80% phenol.

The final aqueous phase is collected and freed from the residual phenol by three successive extractions, each with a volume of ether, in a separatory funnel. A bubbling of nitrogen then makes it possible to remove the ether remaining in the aqueous phase.

Sodium chloride is then added to the aqueous phase to obtain a molarity of 0.1 M, the RNA is precipitated from this aqueous phase by the addition of two volumes of alcohol at -20 ° C. The precipitate is collected by centrifugation under 10,000 g for 10 min at 0 C. The RNA pellet is resuspended in 0.025M tris-HCl buffer, pH 8.1, containing 0.025M NaCl to have approximately 2 mg of RNA per milliliter of solution .

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Method for purifying ribosomal RNA

To the RNA solution obtained previously, a volume of a 2.5M solution of dipotassium phosphate and a volume of 2-methoxyethanol are added. After vigorous stirring for 2 to 3 min, the precipitate is removed by centrifugation at 10,000 g for 5 min at 4 C. The clear supernatant containing the RNA is collected and mixed with a volume of 0.2M sodium acetate. The RNA is then precipitated by the slow addition of 0.5 volume of an aqueous solution to

1% cetyltrimethylammonium bromide.

After standing for 5 min at 4 ° C, the RNA precipitate is collected by centrifugation under 10,000 g for 5 min at 4 ° C. The centrifuge tube is perfectly wiped and the precipitate washed twice with an excess of 70% alcohol containing 0.1 M CH3COONa.

The RNA precipitate is then redissolved in tris-HCl buffer, pH 7, and dialyzed overnight at 2 ° C. against 20 volumes of this buffer, then lyophilized after sterilization by filtration.

Process for the preparation of membrane glycoproteins

The membrane glycoproteins are prepared from the ground bacteria obtained, for example, by suspending the bacterial cells obtained after fermentation in a tris-HCl, MgCl2 buffer, pH 7, and continuous grinding in a homogenizer equipped with a plate cooler under a pressure of 600 kg / cm2. The ground material is centrifuged in the above buffer at 7,500 g for 15 min to remove a sedimentation pellet which contains the cellular debris, then the supernatant is centrifuged at 30,000 g for 15 min to sediment the membranes. The membrane fraction obtained, after optionally three cycles of washing by centrifugation in buffers of decreasing ionic strength, is suspended in distilled water and lyophilized.

The lyophilisate is then subjected to two successive extractions of

2 h at room temperature: the first with a chlorofor-me / methanol mixture (2/1), the second with an ethanol / ether mixture (3/1).

The degreased residue is dried and used to prepare the membrane glycoproteins.

After resuspension of the residue in a 2% aqueous solution of sodiumdodecylsulfate and clarification by centrifugation 15 min at 10,000 rpm, the glycoproteins are precipitated from the supernatant by the addition of 3 volumes of ethanol and a few milliliters of a concentrated solution sodium acetate.

After standing for 1 h at room temperature, the precipitate is collected by centrifugation and taken up in distilled water.

The recovery is clarified by centrifugation for 15 min at 10,000 rpm and the supernatant containing the glycoproteins is lyophilized.

These methods are used to prepare the components used in the experiments below.

Experiment 1:

In this experiment, the activity of the ribosome + capsular polysaccharide association of Diplococcus pneumoniae is tested.

4 groups of 10 mice are used.

Each mouse in group A is treated with 7 μg of D. pneumoniae ribosomes.

Each mouse of group B is treated with 10 μg of capsular polysaccharides of D. pneumoniae.

Each mouse of group C is treated with a combination of 7 μg of ribosomes and 10 μg of capsular polysaccharides of D. pneumoniae.

Group D is not treated.

Each indicated dose is injected 5 times subcutaneously at the rate of one injection every 3 d for 15 d.

Ten after the last injection, the animals are punctured by the retro-orbital sinuses and the serum is studied:

1. in immunodiffusion (Ouchterlony technique),

2. in immunoelectrodiffusion (Laurell technique),

in 1% agarose gel in veronal buffer, pH 8.6 against total D. pneumoniae antigen.

In immunodiffusion:

Against a total diplococcus pneumoniae antigen, the serum of group C treated mice shows precipitation arcs identical in number to a mouse serum vaccinated with the total diplococcus pneumoniae antigen.

In immunoelectric broadcasting:

Against a total diplococcus pneumoniae antigen, the serum of group C mice forms a specific immunoprecipitate in the form of a rocket whose height is much greater than that of rocket obtained with serum of group A or group B mice.

The height of the rockets can vary in proportions which range from double to five times the rate of specific antibodies, depending on the mouse, compared to the results obtained in group A.

Experiment 2:

The same experiments are carried out as above, replacing the 7 μg of D. pneumoniae ribosomes by 5 (μg of D. pneumoniae RNA.

In immunodiffusion:

Against a total diplococcus pneumoniae antigen, the serum of the mice treated with the association (group C) shows precipitation arcs identical in number to a serum of mice vaccinated with the total diplococcus pneumoniae antigen.

In immunoelectric broadcasting:

Against a total diplococcus pneumoniae antigen, the serum of group C mice forms a specific immunoprecipitate in the form of a rocket whose height is much greater than the height of the rocket formed by anti-RNA serum of Diplococcus pneumoniae (group A). Diplococcus pneumoniae capsular antipolysaccharide serum (group B) against a Diplococcus pneumoniae antigen also.

This height of rocket for group C can vary in proportions which range from double to five times the rate of specific antibodies according to the mice compared to the results obtained for group A.

Experiment 3:

The purpose of this experiment is to study a more complete vaccine than in Experiment 1.

To do this, 10 mice forming group E are used and these animals are injected with a combination of:

3.5 (ig of D. pneumoniae ribosomes,

5 [ig of capsular polysaccharides of D. pneumoniae,

12 (ig of membrane glycoproteins of Klebsiella pneumoniae, at the same rate as in experiment 1.

The serum of the punctured animals as above gives in immunoelectrodiffusion a specific immunoprecipitate in the form of rocket whose height is twice the results obtained on group C of experiment 1.

Experiment 4:

The procedure is as in experiment 2 to study a more complete vaccine than that of experiment 2.

To do this, 10 mice forming the group F are used and these animals are injected with a combination of:

2.5 [g of RNA from D. pneumoniae,

5 [ig of capsular polysaccharides of D. pneumoniae,

10 [ig of membrane glycoproteins of Klebsiella pneumoniae, at the same rate as in experiment 2.

The serum of these punctured animals as above gives in immunoelectrodiffusion a specific immunoprecipitate in the form of a rocket whose height is twice the results obtained for group C of experiment 2.

It can therefore be seen that the vaccines according to the present invention exhibit a vaccinating activity much superior to the vaccines of the prior art.

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Claims (7)

630 806 1. Method intended to potentiate the action of vaccines containing, as a vaccine principle, RNA or ribosomes of at least one bacterial strain with polysaccharide capsules, in which the capsular polysaccharides extracted from at least one are added to this vaccine one of said bacterial strains with polysaccharide capsules. 2. Method according to claim 1, characterized in that one adds to a vaccine containing RNA or ribosomes of at least one strain of pneumococcus of Klebsiella pneumoniae or â'Hemo-philus influenzae as a vaccine principle, the Polysaccharides capsular of at least one of said strains of pneumococcus of Klebsiella pneumoniae or â'Hemophilus influenzae. 2 3. Method according to claim 1, characterized in that one adds to a vaccine containing RNA or ribosomes Diplococ-cus of Klebsiella pneumoniae or â'Hemophilus influenzae as a vaccine principle, the capsular Polysaccharides of at least one of these strains of Diplococcus from Klebsiella pneumoniae or â'Hemophilus influenzae. 4. Method according to one of claims 1 to 3, characterized in that the capsular polysaccharides are prepared from bacterial capsules by precipitation with cetylpyridi-nium chloride, removal of the precipitate and decomposition of the cetylpyridinium / polysaccharide complex in a solution saline and recovery of the solution containing the capsular polysaccharides. 5. Method according to claim 4, characterized in that the saline solution is a saline solution of NaCl 2M. 6. Method according to one of claims 1 to 5, characterized in that the weight ratio between the capsular polysaccharides and the RNA or ribosomes of bacteria with polysaccharide capsules is between '/ 2 and 2/1. 7. Vaccine obtained by the method according to one of claims 1 to 6.