IE53582B1 - Process for the production of capsular polyosides,capsular polyosides obtained thereby,and their application to the preparation of vaccines - 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

This invention relates to a process for the production of capsular polyosides, to the polyosides obtained by said process, and the application of said polyosides to the preparation of vaccines.

It is known that it is possible to combat infectious diseases at two different levels: either by direct action against the pathogenic germ (antibiotics, antiseptics) , or by an indirect action via the reinforcement of the systemic defences (vaccination, serotherapy, im10 munomodulation).

The use of agents likely to exert a direct action frequently comes up against certain difficulties which may limit their action (particularly: toxicity of the molecule and bacterial resistance).

In contrast, the reinforcement of the systemic defences via the acquisition of specific antibodies has definite advantages, particularly from the standpoint of the duration of the protection and of the specificity. However, vaccination with whole microbial bodies is not always free from drawbacks: hypersensitization phenomena, poor tolerance or pyrogenioity are among the most frequent. They are correlated to the presence of multiple antigens of the bacteria.

It is therefore quite naturally that searchers have specifically directed their efforts to the isolation of the antigen responsible for the protective antibodies.

In the case of capsulated bacteriae, the capsular polyosides are found to be immunogenic in humans, with little side-reactions.

A first application in humans of such capsular polyosides is the development of antimeningococcic vaccine A and C and, quite recently, of a polyvalent vaccine containing the purified polyosides of fourteen sero5 logic types of pneumococcus.

Several other capsulated bacteriae are involved in a variety of pathological processes, such as: - Hemophilus influenzae, type b - Klebsiella pneumoniae, - Escherichia coli, or - Streptococci, group B, and corresponding vaccines may be prepared from their capsular polyosides.

In European patent application 0 002 404 is descri15 bed a process for the production of capsular polyosides from Streptococcus pneumoniae.

According to this process, the microorganism is cultivated with complex culture media. Then, after inactivation with phenol, and without separating the microor20 ganisms from the culture medium, a first addition of an alcohol such as ethanol is effected, the impurities which have precipitated out are separated, and a second addition of alcohol is then effected to precipitate out the polyoside. The contaminants (proteins, nucleic acids) are then removed from the re-suspended polyoside, by an enzyme treatment or by treatment with a cationic surfactant (cetrimonium bromide), which treatments may be completed by a diafiltration.

In fact, this process is not generally applicable, because the specific conditions concerning each step vary with the polyoside to be separated. Thus, it is nesessary to effect pre-tests to determine the conditions of the various precipitations. In addition, due to the use of certain media, this process may cause a degrada35 tion of the polyoside chains.

The object of this invention is to provide a more generally applicable, simpler and less degradating process for the production of polyosides of very high molecular weight.

Therefore, this invention relates to a process for the production of immunogenic capsular polyosides from capsular bacteria containing same, comprising cultivating the bacteria in synthetic medium, which may contain up to about 0.5% by weight of naturally occuring materials, at the end of the growth step of this culture separating the microbial bodies from a supernatant phase, after separation of said microbial bodies, submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight equal to 100,000 daltons or more, to give a capsular polyoside-rich retained material, and then removing the proteins, the nucleic acids and the lipids from this retained material, to give a purified capsular polyoside.

Thus, according to an essential feature of the present invention, the bacterial culture is effected in synthetic medium. By synthetic medium are meant aqueous solutions of known chemicals, of low molecular weight, which are present in definite amounts which may contain up to about 0.5% of naturally occurring materials such as protein hydrolysates. Moreover, according to the present invention, the extraction of the polyosides is effected on the supernatant of the culture after removal of the microbial bodies, and not on the overall culture medium.

Filtration through a membrane whose porosity is capable of retaining molecules of a molecular weight of 100,000 daltons or more, makes it possible to obtain rapidly a retained material comprising predominantly the polyosides, together with small amounts of proteins, nucleic acids and lipids.

Removal of the contaminants is advantageously effected in the following manner: the retained material is submitted to an enzyme hydrolysis, typically with a mixture of Pronase, RNase and DNase, a butanol-chloro5 form mixture (preferably a 1:1 mixture, by volume) is added thereto, the aqueous phase is separated, the aqueous phase is submitted to a dialysis after which the resulting material is filtered through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more.

The purified polyoside may be separated, in known manner, from the retained material, by precipitation with an alcohol such as ethanol.

The following non-limiting Examples illustrate the present invention.

EXAMPLE 1 Pneumococcic polyoside, type 3 A freeze-dried strain of Streptococcus pneumoniae, type 3, is used as starting material. a) Preparation of seeding materials The freeze-dried strain is added to T-broth. Cultivation is effected for 16 hours at 37°C. ml of the resulting culture are mixed with 15 ml sterilized skim milk. The material is filled into ampoules in an amount of 0.5 ml/ampoule and is then freeze-dried. The freeze-dried materials are stored at +4°C. b) Pre-culture A seeding material (freeze-dried material stored in an ampoule) is added to a flask containing 200 ml of the semi-synthetic medium described below, and cultivation is effected at 37°C for 6 hours.

Culture medium: Pancreatic peptone from casein (IBF) 1500 mg ^-cystine 150 mg 53583 dl-Tryptophan 20 mg 5--Tyrosine 200 mg Dipotassium phosphate 4960 mg d-Glucose, anhydrous 12500 mg 5 Magnesium sulfate. 7H2O 500 mg Ferrous sulfate . 7H2O 5 mg Zinc sulfate . 7H2O 0.8 mg Manganese sulfate . 7H2O 0.3 mg Fuming hydrochloric acid RP 17.8 mg 10 d-Biotine 15 mg Nicotinic acid 1 mg Pyridoxine HC1 1 mg Riboflavin 1 mg Thiamine 1 mg 15 Calcium pantothenate 5 mg Adenine HCl 10 mg Uracil 10 mg Choline chloride 10 mg Asparagine 100 mg 20 Distilled water, sufficient to make 1000 ml c) Culture The culture itself is effected in a fermenter containing 18 litres of the culture medium defined in b). Seeding is effected in an amount of 1% with the pre25 culture.

The culture is effected at 37°C, at pH 7.4, with stirring for a period of time of generally about 16 hrs, to the end of the exponential growth phase. d) Separation of the supernatant 30 At the end of the exponential phase, the supernatant is immediately separated by continuous centrifugation at 5000 rpm. The supernatant is submitted to a sterilizing filtration through Millipore membrane 0.22pm and is used immediately or is stored at -25°C. e) Extraction Λ volume of supernatant of about 20 litres is concentrated to a volume of 2 litres by filtration through a Millipore (trade mark) 10 nenbrane capable of retaining the mole5 cules of a molecular weight of 100,000 daltons or more.

The resulting retained material is filtered through a Diaflo (trada mark) 10® nenbrane after addition of 5 litres apyrogenic sterile distilled water to complete the separation according to the molecular weight.

Several successive diafiltrations permit a concentration to about 350 ml which are then lyophilized in 1 litre flasks.

All the above operations are effected at a temperature of up to 8°C maximum. f) Purification The lyophilized intermediate product is dissolved in 900 ml phosphate buffer 0.01 M - pH 7. mg RNase type 1 , 20 mg pancreatic DNase and 180 jil (20 units/ml) RNase T1 are added thereto. The material is incubated for 4 hrs at 37°C, after which 170 mg Pronase type 9, 800 jil pure phenol and 2 drops toluene are added thereto.

The mixture is incubated overnight at 37°C. The reaction is terminated and an equal volume (900 ml) of butanol-chloroform (1:1) is added thereto. The resulting material is stirred for 1 hour. The two phases are decanted by centrifugation at 10,000 r.p.m. for 30 min.

The aqueous phase which contains the polyoside is dialyzed against 20 litres apyrogenic sterile distilled water for a period of time of about 60 hrs.

The dialysate is submitted to a diafiltration through an Amicon (trade mark) 10 membrane. The volume of the retained material is 1140 ml. 160 ml 40% sodium acetate (final concentration 5%) and 3,9 litres absolute ethanol (3 volumes) are then added. 53583 The precipitate is collected by centrifugation at 12,000 r.p.m. for 30 mn. Said precipitate is dissolved in apyrogenic distilled water. The resulting solution is filled in vials containing 4 ml solution each. The material is freeze-dried in vacuo, to give 9 g of the desired product. Said product is the purified polysaccharide which contains: - sugars 64% - proteins 0.8% - nucleic acid /0.8% All the above steps (apart from the incubations) are conducted between 0 and +4°C.

EXAMPLE 2 Pneumococcic polyoside type 23 15 A freeze-dried strain of Streptococcus pneumoniae type 23 is used as starting material.

The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d).

Extraction and purification of the polyoside are effected in the following manner: e) Extraction A 15 litre volume of supernatant is concentrated to a volume of 1.5 litre under the conditions defined in Example 1-e.

Three successive diafiltrations make it possible to concentrate, and then to freeze-dry, to give 1.8 g of a material of the following composition: - sugars 60% - proteins 19% - nucleic acid 3.6% f) Purification The freeze-dried intermediate product is taken up into 200 ml 0.01M phosphate buffer (pH 7). 1 mg RNase, 1 mg DNase and 20 units RNase Tl/ml are then added. The resulting material is incubated at 37°C for 3 hrs, after which Pronase type 8 (180 pg/ml), phenol(1 jil/ml) and toluene (1 drop) are added thereto. The whole is incubated overnight at 37°C.

Three successive extractions are effected with an equal volume (200 ml) of butanol-chloroform 1:1. The resulting mixture is then stirred mechanically.

The final aqueous phase is dialyzed for about 60 hrs against 4 litres apyrogenio sterile distilled water.

The dialyzate is filtered through Amioon 105 membrane. The material is concentrated to 50 ml. Absolute ethanol (3 volumes) and sodium acetate (final concentration 5%) are added thereto and the mixture is left aside overnight at -20°C.

The resulting precipitate is collected by centrifugation. Said precipitate is dissolved in apyrogenic sterile distilled water, and is then freeze-dried, to give 1050 g of a product which contains: - sugars 75% - proteins 1.7% - nucleic acid 0.7% All the above steps (except otherwise indicated) are effected between 0 and +6°C.

EXAMPLE 3 Pneumococcic polyoside type 19 A freeze-dried strain of Streptococcus pneumoniae type 19 is used as starting material.

The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d).

Extraction and purification of the polyoside are effected in the following manner: e) Extraction Using the procedure of Example 1-e, a 75 litre vo35 lume of supernatant is concentrated to 2 litres by three diafiltrations, to give 2,3 g having the following composition: - sugars 30% - proteins 15% - nucleic acid 3.4% f) Purification The freeze-dried intermediate product is dissolved with 150 ml 0.01M phosphate buffer (pH 7). DNase (3 mg), RNase (3 mg) and RNase T1 (30 pi) are added thereto, and the resulting material is then incubated at 37°C for 4 hrs, after which Pronase (62 mg), phenol (150 jil) and toluene (1 drop) are added. The whole is allowed to react overnight at 37°C.

Deproteination is effected with an equal volume (3 successive extractions) of chloroform-butanol 1:1.

The aqueous phase is dialyzed for 60 hrs against 4 litres apyrdgenic sterile distilled water. The dialyzate is submitted to a diafiltration through Amicon XM 100 membrane.

The retained material is precipitated with 3 volumes ethanol containing 5% sodium acetate.

After overnight storage in a freezer, the precipitate is dissolved in apyrogenic sterile distilled water, and freeze-dried, to give 730 mg of product which con25 tains: - sugars 73% - proteins 0.2% - nucleic acid 0.5%.

Here, again, all the above steps (aside from the 30 incubations) are effected between 0 and +4°C.

EXAMPLE 4 Pneumococcic polyoside type 1 A freeze-dried strain of Streptococcus pneumoniae type 1 is used as starting material. The culture and the separation of the supernatant are effected under the conditions defined in Example 1.

Extraction and purification of the polyoside are effected in the following manner: e) Extraction 100 Litres of the culture supernatant are submitted to a diafiltration through Pellicon (trade mark) cassettes (Millipore 105 membrane). The volume of the final retained material is 3 litres which are freeze-dried in flasks, to give 6.1 g of product which contains: ~ galacturonic acid 46.5% - proteins 7.7% f) Purification The resulting product is dissolved in 800 ml apyrogenic sterile distilled water. To the solution are added - 16 mg RNase - 16 mg DNase - 160 pi RNase Tj.

The material is incubated at 3 7 °C for 4 hrs, after which phenol (300 pi), toluene (2 drops) and Pronase type 8 (150 mg) are added thereto and the whole is then incubated overnight at 37°C. The proteins are extracted with an equal volume of chloroform-butanol (1:1).

The aqueous phase is dialyzed for 60 hrs. Diafiltration is effected through Amicon XM 100 membrane The volume of the final retained material is 150 ml. The solution is precipitated with 3 volumes cold ethanol containing 5% sodium acetate.

The plug is taken up into 250 ml apyrogenic sterile distilled water and freeze-dried in flasks in vacuo, to give 3.7 g purified polyoside which contains: - galacturonic acid 55.5% (which corresponds to 85% sugars) - proteins 1% The diffusion coefficient in the Κβ gel of the polyoside is 0.05. Therefore, the molecular weight is 3 582 at least 2 x 10 EXAMPLE 5 Polyoside from K.pneumoniae, type 2 A strain of Klebsiella pneumoniae type 2 is used as starting material.

The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d), but using as culture medium the following synthetic culture medium: 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 Dipotassium phosphate 10 g Monopotassium phosphate 6.66 g Distilled water, sufficient, to make 1000 ml final pH : 6.8 Extraction and purification of the polyoside are effected in the following manner: e) Extraction litres of the culture supernatant are used. Diafiltration is effected through. Pellicon cassettes (2 cassettes, 10 membrane). After concentration to 1.5 litre, the material is washed 3 times with 10 litres water. The final retained material is 2.6 litres. The material is freeze-dried in flasks , to give 6.1 g of product which contains: - sugars 50% - proteins 15% f) Purification The resulting product is dissolved in 350 ml water. There are added thereto: - 7 ml 0.5M phosphate buffer, pH 7.0 - 7 mg RNase - 7 mg DNase - 70 pi RNase Tj (prior filtration of the enzymes through 0.22 μ).

The whole is incubated at 37°C for 4 hrs, after which protease type 8 (70 mg), phenol (350 pi) and toluene (1 drop) are added thereto, and the mixture is incubated overnight at 37°C. The reaction is terminated and deproteination is completed by 3 extractions with butanol-chloroform (1:1). The aqueous phase is dialyzed for 60 hrs against apyrogenic sterile distilled water. Diafiltration is effected through Amicon XM 100 membrane. The volume of the final retained material is 150 ml. The solution is precipitated with 3 volumes cold absolute ethanol containing 5% sodium acetate. The precipitate is collected by centrifugation, dissolved in 250 ml water and then freeze-dried in flasks, in vacuo. The pure polyoside thus obtained contains: - sugars 76% (Hexoses/uronic acid = 3:1) - proteins 3.8% The K& of the polyoside obtained is 0.1. Thus, the molecular weight is in excess of 107.

EXAMPLE 6 Polyoside from H. influenza, type b A freeze-dried strain of Hemophilus influenzae type b is used as starting material.

The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d), but using as culture medium the following semi-synthetic culture medium: Proteose peptone 5 9 Sodium chloride 3.5 g d-Glucose 4 g Monopotassium phosphate 1.3 g Dipotassium phosphate 3.5 g NAD (nicotine adenosine dinucleotide) 0.001 g Globular extract 50 ml Distilled water, sufficient, to make 1000 ml Extraction and purification of the polyoside are effected in the following manner: e) Extraction Litres of the culture supernatant are used. Diafiltration is effected through Millipore 100,000 membrane (2 cassettes ). The first concentration gives a volume of 1 litre. The material is washed with 3 x 10 litres sterile distilled water. It is concentrated to 1.3 litre and lyophilized in flasks, to give 3.9 g of a product which contains: - sugars 53% - proteins 13% The product thus obtained is dissolved in 200 ml water.

After filtration through 0.22 p, the following materials are added: 4.5 ml 0.5M phosphate buffer, pH 7 mg RNase mg DNase pi RNase Τχ The whole is incubated at 37°C for 4 hrs, after which protease type 8 (65 mg), phenol (200 pi) and toluene (2 drops) are added and the resulting material is incubated overnight at 37°C. The reaction is terminated with 3 extractions with butanol-chloroform (1:1). The aqueous phase is collected by centrifugation at 12,000 r.p.m. at +4°C for 30 minutes. It is dialyzed for 60 hrs against apyrogenic sterile distilled water (frequent changes of the dialysis medium). Concentration is effected through Amicon XM 100 membrane ( 2 washings x 100 ml). The volume of the diafiltrate is 100 ml. The diafiltrate is precipitated with 3 volumes cold absolute ethanol containing 5% sodium acetate. The precipitate, collected by centrifugation, is taken up into 100 ml apyrogenic sterile distilled water. The solution is freeze-dried in flasks, in vacuo, to give 1.3 g pure polyoside which contains: - S sugars (PRP) 63% - proteins 2% e The ratio of the polyoside components is the following: Ribose: Ribitol: Phosphorus : 0.9 : 1 : 0.98 (theoretical ratio: 1:1:1).

The KD is close to 0.1, which corresponds to a molecular weight of about 107.

The purified immunogenic capsular polyosides obtained according to the present invention may be used for the preparation of vaccines intended to be administered to humans and to animals for preventive (or curative) purposes. The vaccines may be prepared by incorporation of the purified capsular polyosides obtained according to the present invention in a physiologically acceptable liquid carrier such a physiologic solution or water for injection.

For this purpose, one or - preferably - more purified capsular polyosides obtained according to the present invention and from different serologic types may be incorporated. In addition, polyvalent vaccines may be prepared by incorporation of two or more purified capsular polyosides obtained from different germs.

The results of tests which demonstrate the immunogenic activity of the polyosides produced according to the present invention are given hereinafter. a) Titration of the serum antibodies in mice a) Male Swiss mice of 20 g body weight were used in groups of 10 animals each: reference group was not given any treatment. group was treated by the I.P. route at the rate of several doses of the polyoside of Example 1 per animal.

The titration of the hemagglutinating antibodies was effected according to the techniques described in the literature, using the red blood corpuscle of sheep sensitized by the polyoside by means of chromium chloride.

The results reported in Table I demonstrate an increase of the serum level of hemagglutinating antibodies with respect to the reference animals, together with a conventional immunitary paralysis at high dosages . (5) - Analogous tests were conducted in mice with the polyoside of Example 5 (polyoside from Klebsiella pneumoniae, type 2).

The results obtained are given in Table XI. b) Titration of serum antibodies in rats.

The same study was conducted with male Sprague Dawley rats of 400-450 g body weight.

The groups of 10 animals were administered an I.P. injection of 2 and 20 pg polyoside from Streptococcus pneumoniae, type 3, per animal.

Titration of thehemagglutinating antibodies was effected on the 4th, 6th and 14th day after administration.

The results obtained are given in Table III.

Consistent with the literature, the sarum antibody level increases gradually to reach a maximum level on the 6th day. c) Specific protection Groups of 10 male Swiss strain mice were immunized with the polyoside from Streptococcus pneumoniae type 1, obtained according to the procedure of Example 4, by the sub-cutaneous route (two injections at a one week interval); dosages injected; 0.47 - 4.7 - 47 and 470 ug/kg.

On the 10th day following the 2nd immunization, H| Wl ml rfl HI (0 ffl η ω (8 3 g 9 H g c \ n) tn Φ r•P rnJ o Φ · d o e-i Γφ Or 0) it P > (0 C* Φ > d · &H O mrM>r-oor-co1 CM Λ A tocooiinoicooooio, «tfCOCMCMCMKOlO'N’OOCO 10 r-fOt-tniflCMCN·^· m in cm cm ο «- o r- CM oooooooooo OOOOOOOOOO CM σι o CO CM + 1 in in co +1 in 3 o id ♦ c •d o 0 Ό 1 •d »d 3 d «—I U 1 •d 0 1 Ό d tb rd »d 1 d 0 1 0 Φ >4 0 d Λ •d o 0 a) d CP 0 id io to Φ Φ •d •d »0 Ό 0 0 Λ Λ •d •d -P +» c fi d d X^h.«4tCMC*)lOlOOkO COCOCOlO’ffCOM'^t'O’N' CM CM CM 10 io 10 T-cMro'N'inior^cooio i *«* J 1 ( bi tn 00 CO I 1 c c • * 1 1 •d •d r- | 1 +) +) + 1 1 1 d nJ ----1 C a I 1 Ή •d 1 1 +) +J 1 1 3 1 1 rd id 1 1 fn m 1 1 CP rp 1 I d d g g 1 1 Φ Φ 1 I m m 1 & 1 1 I t 1 T— CM 1 1 ·—» ·«»* ----1- t 1---Γ I 03 1 1 1 rH 1 1 1 nJ Cn 1 fcs.2 1 CM 1 1 •H 1 £ Cn 1 I 1 I <0^ i i | 1 1 Ό o 1 G) <0 1 -P · 1 r- 1 I rd 1 a) 1 1 1 M 1 1 1 I H i i 1 1 1 1" 1 1 T"—}"~ I W I i-1 1 1 I nJ tJ> 1 I 1 S 1 •H \ 1 CM I 1 C Cn 1 1 1 I « 1 1 1 Ό o ,---1-- 1 Φ . 1 •P <0 1 <- 1 1 nJ 1 Φ 1 1 I M I 1 Eh 1 I i i I 1 - 1 I »---4— I 03 1 1 1 I—) 1 1 1 nJ 1 t H| 1 ε cn 1 •h -X 1 CM 1 H| 1 G^ 1 nJ Cn I 1 1 1 Ό 1 1 1___ Wl 1 φ o 1 4J IO i i 1 nJ T- ml 1 Φ 1 1 1 I I 1 1 I ml 1 i i P- "1---p- I 03 1 1 -Cl 1 r—I 1 1 1 nJ 1 1 I ε Cn Hl 1 •H 34 1 CM I 1 G \ 1 (tf Cn 1 J 1 I a 1 1 1 Φ O 1 •P O 1 T- I 1 ns © 1 I 1 Φ r- 1 1 1 M I 1 1 Eh I 1 Γ "I---Γ" 1 03 1 I 1 »—1 1 | 1 nJ ε 1 CM 1 I •H 1 1 1 G 1 I J nJ 1 J 1 Φ i—-r- 1 0 1 1 1 G 1 Φ i *" i 1 i ** i 1 Φ 1 1 1 Ψ) I 1 1 Φ 1 | 1 Pi I I 1- 1 ----1-- 1 ©©lOCM^CMlOCMM*© -cnkOlOr-rOkDrO’S’LO'tfO’^’^'^’LOlD COMDCMkOCOlDVOkOCNv? • O *— r- «— O 10 ©©©©©COOCMO© oooooooooo oooooooooo + 1 o M· o + 1 CM c •H •P a r4 •rl »G Π----Ί----1 -f----1----1 1____I___J r-—r ·ΓΜθτριοιοΓ^οοσ\ο i—! rd o o M ft ϋ Φ M φ •H »ϋ o Λ Ή 4J a nJ tn c •H +J nJ G •H •P G r4 tn Cn nJ ε Φ S nJ o o M ft •H υ Φ M CM Cn H 0) a) •H Ό Λ •H •P G nJ Cn G Ή P nJ G •H +» G H tn Cn nJ ε V w M* oocMocnocnoo ω H| H| H| HI HI CQl <1 H| w H flj E +> •d (Π G M cn M Ό Φ H Q =c () +> (U (0 o H Q) CM Eh 25 £h cn K H H s «3 o a H H H H 0 0 H J H in cn Q r4 § rt H b R «! •d w V G +) Λ a nJ os nJ 1 λ u H *s. cn o Ό t7 u ω w ¢5 0 +> > (0 W Pm Φ CM H O M g r> CM CD w <> cn ω H H M* V" b CO rt ε •r| G nJ 0) G 0) μ φ »w mcooooiocoomco OO^COOOMOO mr-foiDM'CMuiforr-OOCOf^COincOCOCO Γ-Γ*-ΙΟΙΠΓ*ΙΛΜ*ΙΟΙΛ οοσσοοοοο ooooooooo ooooooooo •ΓΊίΌΜ’ίΛΙΟΓ-ΟΟσ» CM <0 CM CO co co O co M1 O • • • r— Q 4* 1 V in CM o in co o « • co o o + 1 V x— cn O r* 00 O in o o + 1 V the animals were infected with the virulent pneumococcus (type 1) , by the sub-cutaneous route (1250 germs/animal).

After ten days observation, the death rate was as follows: - untreated animals - treated animals (0.47 pg/kg) (4.7 pg/kg) 100% % % % 40% - treated animals - treated animals (47 pg/kg) - treated animals (470 pg/kg) P - Groups of 10 male Swiss mice were immunized with the KP2 polyoside by the sub-cutaneous route (2 injections at a weekly interval; dosages injected: 53 - 533 5332 pg/kg).

On the 8th day after the last treatment, Klebsiella pneumoniae type 2 strain was injected to the animals at a rate of 650 germs/animal. After 10 days observation, the death rate was found to be as follows: - untreated animals - treated animals (53 pg/kg) - treated animals - treated animals 100% 0% 0% 0% (533 jig/kg) (5333 pg/kg) The polyosides produced according to the present invention are typically useful for the preparation of the following vaccines: a) vaccines for the preventive and curative treatment of acute and chronic respiratory diseases, comprising one of the purified polyosides corresponding to each of the following germs: Streptococcus pneumoniae Hemophilus influenzae, type b Klebsiella pneumoniae Escherichia coli For example: 1) mixture of polyosides from S. pneumoniae types 1, 6, 14, 23 ............... 50 jug polyoside from K. pneumoniae, type 2 . . 20 jug polyoside from H. influenzae, type b . . 20 jug final volume: 0.5 ml physiological solution 2) mixture of polyosides from S. pneumoniae types 1, 6, 14, 23 ............... 25 pg polyoside from K.pneumoniae, type 2 . . 10 jug polyoside from H.influenzae, type b . . 10 jug b) Antipneumococcic vaccine comprising the purified polyosides from the following types of Streptococcus pneumoniae: 1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18L, 19F, 23, 2, 11A, 15F.

For example: - mixture comprising 50 jUg active material in a volume of 0.5 ml physiological solution, - mixture comprising 25 pg active material in a volume of 0.5 ml physiological solution.

Claims (15)

1. CLAIMS:1. A process for the production of immunogenic capsular polyosides from capsulated bacteria containing same, comprising cultivating the bacteria in a synthetic medium which may 5 contain up to about 0.5% hy weight of naturally occurring materials; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase after separation of said microbial bodies to a filtration through a membrane which retains 10 the molecules of a molecular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material; and removing the proteins, the nucleic acids and the lipids from said retained material, to give a purified capsular polyoside.

2. A process as claimed in claim 1, wherein, to remove 15 the proteins, the nucleic acids and the lipids from the retained material, said retained material is submitted to an enzyme hydrolysis, a butanol-chloroform mixture is added thereto, the aqueous phase is separated, said aqueous phase is submitted to a dialysis and then to a filtration through a 20 membrane which retains the molecules having a molecular weight in excess of 100,000 daltons.

3. A process as claimed in claim 1 or 2, wherein the culture is effected in a synthetic medium comprising less than 0.5 wt% protein hydrolysate. 25

4. A process as claimed in claim 3 for the production of capsular polyosides from Streptococcus pneumoniae, wherein as culture medium there is used a medium having substantially the following composition Pancreatic peptone from casein (IBF) 1,500 mg 1-cystine 150 mg dl-Tryptophane 20 mg 1-Tyrosine 200 mg Dipotassium phosphate 4,960 mg d-Glucose, anhydrous 12,500 mg Magnesium sulfate. 7H 2 <3 500 mg Ferrous sulfate. 7H 2 O 5 mg Zinc sulfate. 7Η 2 <3 0.8 mg Manganese sulfate. 7^0 Fuming hydrochloric acid RP 0.3 mg 17.8 mg d-Biotine 15 mg Nicotinic acid 1 mg Pyridoxine HC1 1 mg Riboflavin 1 mg Thiamine 1 mg Calcium pantothenate 5 mg Adenine HC1 10 mg Uracil 10 mg Choline chloride 10 mg Asparagine 100 mg Distilled water, sufficient to make 1,000 ml

5. A process as claimed in claim 1, for the production of 15 capsular polyosides from Klebsiella pneumoniae, wherein as culture medium there is used a medium having substantially the following composition Trisodium citrate 0.85 g Ammonium sulfate 0.17 g 20 Magnesium sulfate 0.17 g Glutamic acid 0.17 g d-Gluoose 16.70 g Dipotassium phosphate 10 g Monopotassium phosphate 6.66 g 25 Distilled water, sufficient to make 10,000 ml

6. A process as claimed in claim 1 for the preparation of capsular polyosides from Hemophilus influenzae, wherein as culture medium there is used a medium having substantially the following composition 30 Proteose peptone 5 g Sodium chloride 3.5 g d-Glucose 4 g Monopotassium phosphate 1.3 g Dipotassium phosphate 3.5 g 35 NAD (nicotine adenosine dinucleotide) O.OOlg Globular extract 50 ml Distilled water, sufficient to make 1,000 ml 53583

7. A process as claimed in any one of claims 1-3 wherein capsular polyosides are obtained from Streptococcus pneumoniae, Hemophilus influenzae, Klebsiella pneumoniae or Escherichia coli. 5

8. , A capsular polyoside whenever produced by a process as claimed in any one of claims 1-7.

9. A capsular polyoside as claimed in claim 8, which is obtained from Streptococcus pneumoniae. Hemophilus influenzae, Klebsiella pneumoniae or Escherichia coli.

10. 10. A capsular polyoside as claimed in claim 8, which is obtained from Streptococcus pneumoniae.

11. A vaccine comprising at least one capsular polyoside as claimed in any one of claims 8-10.

12. A process according to claim 1 for the production of 15 immunogenic capsular polyosides from capsulated bacteria containing same, substantially as hereinbefore described with particular reference to the accompanying Examples.

13. An immunogenic capsular polyoside whenever produced by a process claimed in a preceding claim. 20

14. A capsular polyoside according to claim 8, substantially as hereinbefore described with particular reference to the accompanying Examples.

15. A vaccine according to claim 11, substantially as hereinbefore described with particular reference to the 25 accompanying Examples.