GB1590144A

GB1590144A - Vaccines based on ribosome fractions

Info

Publication number: GB1590144A
Application number: GB31746/77A
Authority: GB
Original assignee: Pierre Fabre SA
Current assignee: Pierre Fabre SA
Priority date: 1976-08-06
Filing date: 1977-07-28
Publication date: 1981-05-28
Also published as: ES461415A2; FR2360314A2; CA1087093A; ZA774733B; JPS5320418A; AU511331B2; DE2735411C2; AU2753877A; FR2360314B2; DE2735411A1; BE857014R

Description

(54) NEW IMPROVED VACCINES BASED ON RIBOSOME FRACTIONS (71) We, PIERRE FABRE S.A. of 125 rue de la Faisanderie, 75016 Paris, France, a body corporate organised under the laws of France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to new vaccines of the type described in the main patent No.

1543962 entitled: "acellular vaccines".

The invention relates more particularly to new vaccines based on ribosome fractions combined with the saccharide fractions of cell membranes, consisting of membrane lipopolysaccharides and polysaccharides.

The old vaccines, consisting of attenuated dead bacteria or of microbial lysates, are inferior in their vaccinating power to vaccines of living germs.

Attempts have therefore been made to find new vaccines with a more powerful antigenic activity than that of the old vaccines.

The present invention is the outcome of research showing that the ribosomes of bacterial cells carry the vaccinating activity and that this activity manifests itself only in the presence of an immunity adjuvant. In the present case, this soluble adjuvant is extracted from the cell membranes of bacteria in the form of a saccharide fraction.

The present invention thus proposes an acellular vaccine, which comprises, incorporated in an acceptable pharmaceutical vehicle, a combination of: (a) the ribosome fraction of at least one strain of pathogenic bacteria corresponding to the disease which is to be prevented, and (b) the saccharide fraction extracted from the membranes of bacteria of at least one bacterial strain selected from the genera Klebsiella, Serratia and Corynebacterium.

The present invention relates in particular to a vaccine in which the saccharide fraction is extracted from the bacterial membranes of at least one bacterial strain selected from the following species: Klebsiella pneumoniae Klebsiella rhinoscleromatis Serratia corallina Serratia indica Serratia keilensis Serratia kiliensis Serratia marcescens Serratia plymuthica Corynebacterium avidum Corynebacterium bovis Corynebacterium enzymicum Corynebacterium equi Corynebacterium fascians Corynebacterium flaccumfaciens Corynebacterium flavidum Corynebacterium fusiforme Corynebacterium granulosum Corynebacterium helvolum Corynebacterium hypertrophicans Corynebacterium insidiosum Corynebacterium liquefaciens Corynebacterium parvum Corynebacterium parvum infectiosum Corynebacterium paurometabolum Corynebacterium pyogenes Corynebacterium tumescens Corynebacterium xerosis In a preferred method of carrying out the process according to the present invention, the saccharide fraction used as adjuvant is extracted from the membranes of Klebsiella pneumoniae.

In one particular embodiment of the invention, a membrane saccharide fraction extracted from the bacteria corresponding to the ribosomes used is employed as adjuvant in addition to the above mentioned membrane saccharide fraction.

In the vaccines according to the present invention, the proportion by weight of ribosome fraction to saccharide fraction is preferably between 0.5 and 1.

It should be understood, however, that the unit dose of vaccine may be varied according to the requirements of the treatment or the method of administration.

Below are given specific examples of doses which may be used according to the invention but which should be regarded as non-limiting: A broncho-otolaryngeal vaccine having the following composition: Ribosome fractions Ribosomes of Klebsiella pneumoniae 3.5 Rg Ribosomes of Diplococcus pneumoniae 3.0 llg Ribosomes of group A12 Streptococcus pyogenes 3.0 Rg Ribosomes of Haemophilus influenza 0.5 llg 10.0 llg Soluble adjuvant Membrane saccharide fraction of Klebsiella pneumoniae 15.0 llg 1 dose 25.0 Rg of pure active constituent.

A dental vaccine having the following composition: Ribosome fractions Ribosomes of Actinomyces viscosus 1.2 jig Ribosomes of Rothia dentocariosus 1.2 llg Ribosomes of Streptococcus mutans 0.8 Fg Ribosomes of Strep to ccocus salivarius 1.0 Fg Ribosomes of Lactobacillus casei 1.2 Rg 5.4 Fg Soluble adjuvant Membrane saccharide fraction of Klebsiella pneumoniae 9.6 Fg 1 dose 15.0 Rg of pure active constituent.

A veterinary vaccine having the following composition: Ribosome fractions Ribosomes of Pasteurella haemolytica H1 3.0 Rg Ribosomes of Pasteurella multocida Al - 1048 3.0 Fg Ribosomes of Pasteurella multocida A3 - A13 3.0 g 9.0 g Soluble adjuvant Membrane saccharide fraction of Klebsiella pneumoniae 13.5 Rg Membrane saccharide fraction of Pasteurella haemolytica H1 1.0 llg Membrane saccharide fraction of Pasteurella multocida A1 - 1048 1.0 Fg Membrane saccharide fraction of Pasteurella multocida A3 - A13 1.0 llg 16.5 llg 1 dose 25.5 llg of pure active constituent.

In the vaccines according to the present invention, the saccharide fraction may be obtained, for example, by extracting an aqueous suspension of membranes with phenol, the saccharides being isolated from the aqueous phase after removal of the phenolic phase. This phenolic extraction is preferably carried out at a temperature of between 60 and 70"C, more preferably at 650C, with a 90% phenol solution.

The aqueous suspension of membranes used for carrying out the extraction described above may be obtained, as has been described in the main patent No. 1543962 from a bacterial culture which is crushed and then centrifuged to bring about selective sedimentation of the membranes which are then again suspended in an aqueous solution.

Sedimentation of the membranes may be carried out, for example, by centrifuging at an acceleration of substantially 30,000 g after removal of the cellular debris.

The ribosome fraction may be prepared by extracting crushed bacterial cells with buffer containing hydrochloric acid, MgCl2, NaCl and sodium dodecyl sulphate. An aqueous solution is thereby obtained which is precipitated with polyethylene glycol and ethyl alcohol, the ribosome fraction being contained in the precipitate. In order to obtain a more highly purified product, the precipitate is preferably taken up in a buffer containing hydrochloric acid, MgCl2, NaCl and sodium dodecyl sulphate, and this aqueous phase is reprecipitated with ethyl alcohol, preferably 95% ethyl alcohol.

The precipitate obtained in this way may be used as the basis for the vaccines according to the invention by mixing it with the above-mentioned saccharide fraction in the proportions which have already been defined above and which will become clearer in the course of the following description.

It is to be understood that the vaccines according to the present invention may be used according to their constituents for the treatment or for the prevention of various diseases, as is well known in the field of vaccines. Thus, the bronchootolaryngeal vaccine defined earlier may be used for the treatment or the prevention of the various forms of chronic rhinotracheobronchitis, of asthma complicated with bronchitis, of bronchiectasis, and of viral pneumopathy with secondary infections as well as for the treatment of the various forms of sinusitis and of rhinopharyngolaryngitis.

The dental vaccine can be used for preventing the major common infections of the mouth.

The vaccines according to the present invention are preferably presented in the form of aerosols but they may also be provided in other medicinal forms. Some of the vaccines according to the invention may be prepared fro parenteral administration, for example, by injection.

It is not necessary at this point to provide a list of components which may enter into the composition of the excipient for the vaccine according to the invention since they are well known in the art of preparing vaccines or pharmaceutical preparations.

The two components of the vaccine are generally kept in the form of a solution but they may be freeze dried when preparing the vaccine, and all that is then necessary is to mix the various components of the vaccine in the required proportions.

The analytical standards of the product obtained may be determined as follows: The concentration of ribosomes of the ribosome fraction is calculated from the RNA concentration as follows: concentration of RNA x 100 Ribosome concentration = concentration of RNA X 100 70 The RNA concentration is in turn measured by spectrophotometric determination of the phosphomolybdic complex compared with an RNA sample of Klebsiella of known concentration measured by determination of the phosphorus.

The concentration of proteins in the ribosome fraction is determined colorimetrically with biuret by comparison with a range of standard samples of albumen.

The percentage of RNA in the sum of RNA + proteins varies approximately from 55 to 65%.

The concentration of saccharides in the adjuvant is determined from the glucose concentration measured colorimetrically with anthrone, as follows: concentration of saccharides = concentration of glucose x 100 50 A standard preparation of highly purified saccharide was used to determine the glucose content of the saccharides, which is 50% by this method of determination.

The preferred method of carrying out the process of the present invention to prepare vaccines according to the present invention is described below by way of example.

Preparation of an aqueous solution of ribosomes The various strains from which it is desired to extract the ribosomes are cultivated in known manner on suitable growth media in fermentation apparatus.

After cultivation, the bacterial cells are collected in a Westfalia or Sharples centrifuge and washed by centrifuging with cold physiological serum at a temperature of substantially 4"C.

The bacterial concentrate obtained in this way may be stored frozen at -200C if it is not required for immediate use.

As in the case of the extraction of saccharides, extraction of the ribosomes may be carried out on a mixture of various strains but it is preferable to treat each strain separately so that the product obtained can be more easily controlled; in that case, the ribosomes and/or saccharides are mixed at the end of extraction to constitute the vaccine.

A sample is removed from the bacterial concentrate obtained to determine the dry weight of the cells after stove drying (the ratio of dry cells to wet cells various between 20 and 25%).

Another sample is removed to test the purity of the culture and to identify the strain.

The bacterial concentrate obtained as described above is then disintegrated as follows: If only a small quantity of wet cells is present (less than 50 g), the cells are dispersed in a proportion of 1 g (moist weight) in 4 ml of the following buffer: tris-HCl M/100, pH 7.2, MgC12, 6 H2O 0.01 M containing: NaC1 9 g/l sodium dodecyl sulphate 5 g/l When the suspension has been thoroughly homogenised it is disintegrated by ultrasonic treatment for three periods of 10 minutes each while it is kept in an ice bath. An ultrasonic apparatus Sonnimasse 500 T, (trade mark) for example, may be used.

If larger quantities of cells are to be treated (more than 50 g), the defrosted cells are dispersed in the same buffer as used above and in the same proportions, and are then homogenised by vigorous stirring for 10 minutes in a high-speed reactor.

They are then crushed in the cold in a Manton-Gaulin mill (5 cycles for each strain). The coarsest cellular debris is eliminated by passing the sample over a Sharples apparatus, and the supernatant layer is then centrifuged at 4"C for 30 minutes and 30,000 g (Beckman 521-B (trade mark) centrifuge with rotor 6 x 250 ml JA 14).

The sediment formed by centrifuging is discarded and the supernatant layer containing the ribosomes is stored at 40C.

Extraction of the ribosomes 100 g/l of polyethylene glycol 4000 (PEG 4000 - Merck) are added to the aforementioned supernatant layer and, after complete solution, 1 volume of 95% ethyl alcohol is added at -20 C.

The mixture is left to stand in the cold for 20 minutes and the precipitate is then collected on a Sharples apparatus at 40C (the supernatant layer is discarded).

The precipitate is then redissolved in the same volume of buffer as that used for crushing (tris-HCl, MgCl2, NaCl, SDS 0.5%), by agitation in the reactor at room temperature for 30 to 60 minutes (the purpose of washing at room temperature is to remove non-ribosome proteins).

0.8 Volume of 95% ethyl alcohol at -200C is then added and the substance is left to precipitate at 100C for 30 minutes.

The ribosome precipitate is collected on the Sharples apparatus and the supernatant layer is discarded (without refrigeration in order not to precipitate the SDS). The sediment of ribosomes is taken up in buffer (MgCl2 0.02 M; KCl 0.02 M; pH 7) at 40C in a proportion of 2 ml for 1 gram of moist cells originally used (stirring on average for 2 to 4 hours).

The sediment taken up in the buffer is then centrifuged at 30,000 g for 45 minutes at 0 C (removal of residual SDS).

The sediment is discarded and the supernatant layer is kept.

A sample (1 to 2 ml) is removed to determine the RNA concentration, the concentration of proteins and the number of doses per ml.

The ribosomes are stored in sterile plasma bottles at -200C until required for use.

SDS = sodium dodecyl sulphate Preparation of an aqueous suspension of membranes After culture, the cells are collected by centrifuging in a Westfalia or Sharples apparatus and then washed by centrifuging with cold physiological serum. The bacterial concentrate obtained may be stored at -200C.

The dry residue is determined on a sample by drying it in a stove and the identity and purity of the sample are controlled bacteriologically.

The defrosted cells are suspended in cold buffer in a proportion of 1 g of cells to 4 ml of buffer.

The buffer has the following composition: tris-HCl 0.01 M, pH 7 NaCl 0 g/l MgCl2, 6 H2O 0.01M The cells are crushed in a Manton-Gaulin apparatus (3 cycles at maximum pressure).

Both the intact cells and the cellular debris are removed in a Sharples apparatus and the supernatant layer is stored at 40C.

The membranes are then collected by centrifuging for 45 minutes at 30,000 g and 40C (Beckman J 21 - B, rotor JA 14).

The supernatant layer is discarded (a portion may be kept back for the preparation of a ribosome RNA standard for analysis). The sediment is kept to extract the soluble adjuvant from it. The various sediments are resuspended in distilled water used in an amount of 2.5 ml for each gram of wet cells used as starting material (homogenisation in a Turrax (trade mark) apparatus for 1 to 2 minutes).

Extraction of the saccharides One volume of aqueous phenol (90% phenol) which has previously been adjusted to a temperature of approximately 80"C is added to the aqueous suspension obtained as described above, and the mixture is heated to 650C for 5 minutes with vigorous stirring.

This is immediately followed by rapid cooling to 0 C.

In the case of small volumes, the aqueous phase is separated by centrifuging at 5000 revs/min at a temperature of 0 C for 5 minutes in stainless steel containers. The upper, aqueous phase is then collected.

For larger volumes, the aqueous phase is separated by passing the sample over a liquid-liquid Westfalia or Sharples separator.

The phenolic phase is discarded.

The residual phenol in the aqueous phase is removed by dialysis against running water for 24 to 48 hours, either in a dialysis tube if the volumes are small, or on a plate dialyser (Sartorius type) in the case of larger volumes.

The slight precipitates which form during dialysis are then removed by centrifuging at 10,000 g and 0 C for 30 minutes.

The supernatant layer is kept in sterile plasma bottles at -20 C.

A sample of 1 or 2 ml is removed for analytical control.

The vaccines according to the present invention may be used both in human and in veterinary medicine.

1) Investigation of the immunogenic power of the veterinary vaccine mentioned above This investigation is carried out for the purpose of checking the appearance and persistence of specific antibodies in the serum of vaccinated mice.

Female mice of Swiss breed weighing 20rut2 g are used for the investigation. They are kept in cages of 10 in a room kept at a substantially constant temperature of 22+1 C 10C and a relative humidity of 40 to 60% and given food in the form of pellets (UAR mouse diet) and drinking water.

UAR = Usine d'Alimentation Rationelle, France.

Immunisation of the animals Each animal is injected subcutaneously with the dose of vaccine made up to a volume of 0.2 ml.

The injections are carried out at the following intervals: 5 inoculations in 15 days (i.e. one injection every 3 days), 1 week's rest 2 inoculations in 8 days (booster dose), puncture through the tetro-orbital sinuses, at least 10 days and not more than 15 days after the last booster injection.

Method of detecting the antibodies The serum of each vaccinated mouse is studied by Laurell's technique of immunoelectro diffusion.

Each animal serum is studied against the Pasteurella antigen from which the membrane saccharides were obtained: Pasteurella haemolytica H1 and Pasteurella multocida A3.

Each serum is then studied in the same manner against all the Pasteurellae together from which the ribosomes of the preparation were extracted.

The chosen antigen is incorporated in a 1% agar gel buffered with veronal at pH 8.6. This gel is poured over a glass plate measuring 1.5-x 90 x 110 mm.

7 pL of the serum trom each mouse are deposited in pits previously formed in the gel.

Deposition takes place at the cathode. Migration takes place for 9 hours under a continuous current with a potential difference of from 2 to 3 volts/cm.

At the end of this migration, the plates are washed and dried and the immunoprecipitates are shown up by colouration with Coomassie Brilliant Blue.

Results The presence of specific immunoprecipitates between the antibodies of the serum of treated mice and the detector antigen is seen in the form of a "rocket", the height of which represents the quantity of antibodies contained in the serum of the vaccinated animals.

Highly significant results are seen on all the plates with the serum of vaccinated mice compared with the sera of control mice, thus confirming the high vaccinating power of the compositions according to this invention.

2) Investigation of the immunogenic power of the dental vaccine mentioned above Female mice of Swiss breed weighing 20+2 g, kept in cages of 5 and given food in the form of pellets (UAR mouse diet) and drinking water in an animal room kept at a constant temperature of 22+1"C and a humidity of 40 to 60% are used in the same way as under (1) above.

Immunisation of the animals Each animal is injected subcutaneously with the dose of vaccine in a volume of 0.3 ml (it should be noted that tests carried out with 1:1, 1:1.5 and 1:2 ratios of ribosome fraction to saccharide fraction showed that the best results are obtained with the 1:1.5 composition mentioned above).

The injections were carried out at the following intervals: 5 inoculations in 15 days (one injection every 3 days, 1 week's rest, 2 inoculations in 8 days (booster injection), puncture through the retroorbitol sinuses at least 10 days and not more than 15 days after the last booster injection.

Method of detecting the antibodies The serum of each vaccinated mouse is studied by Laurell's immunoelectrodiffusion technique.

The antigen corresponding to each type of bacteria from which the ribosomes are obtained is embodied in a 1% agar gel buffered with veronal at pH 8.6. This gel is poured over a glass plate measuring 1.5 x 90 x 110 mm.

7 Fl of the serum from each mouse are deposited in the pits previously hollowed out of the gel.

Deposition takes place at the cathode. Migration lasts 9 hours under a continuous current with a potential difference of 2 to 3 volts/cm.

At the end of the migration, the plates are washed and dried and the immunoprecipitates are shown up by colouration with Coomassie Brilliant Blue.

Results The presence of specific immunoprecipitate between the antibodies of the serum of the treated mice and the detector antigen is seen in the form of a "rocket" the height of which represents the quantity of antibodies contained in the serum of the vaccinated animals.

Highly significant results are observed on all the plates with the serum of the vaccinated mice, compared with sera from control mice, thus demonstrating the high vaccinating power of the vaccines according to the invention.

WHAT WE CLAIM IS: 1. An acellular vaccine, which comprises a combination of: (a) the ribosome fraction of at least one strain of pathogenic bacteria corresponding to the disease which is to be treated or prevented, and (b) the saccharide fraction extracted from the membranes of bacteria of at least one bacterial strain of the genera Klebsiella, Serratia and Corynebacterium.

2. An acellular vaccine according to claim 1, in which the saccharide fraction - is extracted from the membranes of at least one bacterial strain chosen from among the following: Klebsiella pneumoniae Klebsiella rhinoscleromatis Serratia corallina Serratia indica Serratia keiliensis Serratia marcescens Serratia plymuthica Corynebacterium avidum Corynebacterium bovis Corynebacterium enzymicum Corynebacterium equi Corynebacterium fascians Corynebacterium flaccumfaciens Corynebacterium flavidum Corynebacterium fusiforme Corynebacterium granulosum Corynebacterium helvolum Corynebacterium hypertrophicans Corynebacterium insidiosum Corynebacterium liquefaciens Corynebacterium parvum Corynebacterium parvum infectiosum Corynebacterium paurometabolum Corynebacterium pyogenes Corynebacterium tumescens Corynebacterium xerosis.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. saccharide fraction showed that the best results are obtained with the 1:1.5 composition mentioned above). The injections were carried out at the following intervals: 5 inoculations in 15 days (one injection every 3 days,

1 week's rest,

2 inoculations in 8 days (booster injection), puncture through the retroorbitol sinuses at least 10 days and not more than 15 days after the last booster injection.

3. A vaccine according to claim 1 or 2, which contains, in addition, the saccharide

fraction extracted from the membranes of the bacteria from which the ribosomes are extracted.

4. A vaccine according to any of claims 1 to 3, in which the proportion by weight of the ribosome fraction relative to the saccharide fraction is within the range of from 0.5:1 to 1:1.

5. A vaccine according to any of claims 1 to 4, in which the saccharide fraction is extracted from the membranes of Klebsiella pneumoniae.

6. A vaccine according to claim 5, which is a bronchootolaryngeal vaccine containing the ribosome fraction extracted from the following: Klebsiella pneumoniae, Dip lo coccus pneumoniae, Group A12 Streptococcus pyogenes and Haemophilus influenzae.

7. A vaccine according to claim 5, which is a dental vaccine in which the ribosome fractions are extracted from the following: Actinomvces viscosus Rothia dentocariosus Streptococcus mutans Streptococcus salivarious and Lactobacillus casei.

8. A vaccine according to claim 4 or 5, which is a veterinary vaccine containing as ribosome fraction, the ribosomes extracted from the following: Pasteurella haemolytica H1 Pasteurella multocida A1 - 1048 and Pasteurella multocida A3 - A13.

9. A vaccine according to any of claims 1 to 8, in which the saccharide fraction is obtained by extraction of an aqueous suspension of membranes by means of phenol, the saccharides being isolated from the aqueous phase after removal of the phenolic phase.

10. A vaccine according to claim 9, in which extraction is carried out at temperature within the range of from 60 to 70"C.

11. A vaccine according to any of claims 1 to 10, in which the ribosome fraction is prepared by extraction of crushed bacterial cells with a buffer containing HCI, MgCl2, NaCl and sodium dodecyl sulphate, and precipitation of the resulting homogeneous aqueous phase with polyethylene glycol and ethyl alcohol, the ribosomes being contained in the precipitate.

12. A vaccine according to claim 11, in which the buffer comprises M/100 hydrochloric acid, 0.01 M MgC12, 9 g/l of NaCl and 5 g/l of sodium dodecyl sulphate.

13. A vaccine according to claim 11 or 12, in which the precipitate obtained is taken up in a buffer containing HC1, MgCl2, NaCl and sodium dodecyl sulphate, and is then reprecipitated with ethyl alcohol.

14. An acellular vaccine substantially as herein described with reference to any of the specific examples.