BACTERIAL PROTEOGLYCANES PURIFY, PROCEEDED FOR THEIR PREPARATION AND VACCINE THE CONTAINER
the present invention relates to purified bacterial membrane protéoglyeanes and detoxified and a method for their preparation and their use as immunity ajuvant in vaccines. It is recall that the vaccines are, in general, one or more immunogenic elements, i.e. which might reveal an immune response. In some cases, however, the immunogenic elements are unable to develop their immunogenic alone or does develop to a degree that the extremely low, it takes the need for products called "immunity adjuvant". the immunity adjuvant are Conventionally, the products which have immunostimulatory activity without being immunogenic by themselves; for example include incomplete Freund's adjuvant (IFA) which is composed of 85,5 " paraffin and 15 $naphthalene. Da, the present invention provides immunity adjuvant useful in vaccines and more particularly in vaccines or said ribosomal RNA-based. Ribosomal This type of vaccines has been described in particular in the French patent no. 73,43957. S ^ It acts of vaccines containing as immunogenic compound, or bacterial ribosome, RNA from either bacterial pathogenic bacteria against which immunity is desired. However, as described in the previous Patent, these immunogenic compounds cannot completely that in the presence of immunity adjuvant protéoglyeanes constituted by the membrane extracts of bacteria membranes. However, the currently known methods for preparation of these protéoglyeanes do not allow for complete purification and detoxify these protéoglyeanes which can result in some cases, to vaccine reactions or pyrogenic reactions that could be of interest to be able to avoid. In particular, the present invention relates to a method for purifying the protéoglyeanes to obtain bacterial membrane protéoglyeanes water soluble which is a considerable advantage because the protéoglyeanes thus obtained can be sterilized by filtration and therefore without the need to heat that heretofore could denature properties. Therefore, in the frame of the present invention, is used to hearing by protéoglyeanes protéoglyeanes purified water soluble. Therefore the present invention relates to a process for purifying and detoxifying proteoglycans bacterial membrane, purified membrane proteoglycans and their application as adjuvant of immunity in vaccines, the present invention relates to a method for preparing purified soluble bacterial membrane proteoglycans, characterized in that it comprises at least one step of treating raw proteoglycans in an aqueous medium by a base or hypobromite followed by removal of excess reagent and the insoluble residue, proteoglycans purified in aqueous solution. In one embodiment of the method according to the present invention, raw proteoglycans are processed using an alkali metal hydroxide, in particular soda, in water at a molarity of between 0.05 and 2 M, preferably 0.1 M, the treatment is continued e.g. hours 24 hours at temperature close to room temperature, for example 25 °C, and preferably under stirring. The treatment has poùr effect of hydrolyzing controlled proteoglycans to extract the soluble fraction. In this embodiment, to remove excess reagent, , l.' hydrolysate is neutralized by an acid such as hydrochloric acid and the salt thus formed is removed e.g. by dialysis against distilled water. Then a Ilreste that the insoluble residue can be removed by precipitation, in particular by centrifugation, for example 45 minutes to 30,000 g and 4 °C. the supernatant constitutes the purified soluble proteoglycans. In a second embodiment of the process according to the present invention, raw proteoglycans in water are processed by a alkali hypobromite for a period of the order of a few tens of minutes, preferably at room temperature and under stirring, and 1' hypobromite excess can be eliminated in particular by dialysis of 24 hours against water and current against distilled water. The insoluble residue can be removed as aforesaid. The method of the present invention includes, further, preferably a step of removing the lipid A by acid hydrolysis in an aqueous medium, preferably at a temperature comprised between 70 and 100 °C in the presence of about 1 The present invention also relates to as an adjuvant of immunity in vaccines, the bacterial membrane proteoglycans water soluble, purified, characterized in that they have the following composition by weight: 24-42 31-55 12-18. 2-6 56' 0,5 0,2 * 0,001 Among such proteoglycans, to include in particular proteoglycans having the following composition: Hexoses Proteins Lipids Hexosaminea RNA, less than DNA less than LPS less than Hexoses' 37+5% Proteins '-36.5 + 5% Lipids 15 + 3%. Hexosamines [5+ RNA < 0.5% DNA " 0, 2%. LPS < 0,001% which can be obtained, in particular by implementing the above method wherein alkali metal hydroxides are used. It should be also be mentioned among the proteoglycans proteoglycans of the invention having the following composition: which can be obtained, in particular by implementing the above method using a hypobromite. In order to characterise the different fractions, can be used the following analytical methods: Hexoses : by the reaction of anthrone-SCOTT (t. A.), MELVIN (E. II.) 1953, Anaïyt. Proteins: by reaction of the according to GORNALL Biuret (A. g.), (C.A.) BARDAXILL DAVID (V.V.), 1949, J. Biol. Chem ., 177.751 '' Lipids : by a method based on a gravimetric overall extraction by organic solvents. Hexosamines ; by their reaction with the e. dimé thylaminobenzaldéhyde MORGAN of after (S. W. t.), RONDEL (C. J.M.), 1955, Bioehem J. 61. RNA r (ribonueléique acid): by direct speetropliotométrie u. V. to 260 nm and by the reaction? i the oreinol according to LUSENA (C. V.), 1951, Canad, J. Biochejn., 29, 107-108 DNA (deoxyribonucleic acid) by the color reaction according to the clipliénylamine BURTON (K.), 1956, J. 62,315 Bioehem. LPS (Lipopolysaccharides), by the réaetio' rï Coloured earboeyanine described ia • parJANDA (J.), WORK (E.), 1971, FEBS Letters, vol.l6, ' No. 4 p. 343-345 Residual water : by the reaction of Karl FISCHER.. In general, the raw membrane protéoglyeanes can be prepared by any method known, in particular by. the method described in the mentioned patent or fractionated by centrifugation from a bacterial biomass crushed to successively settle cellular debris in a acceleration preferably between 7,000 and 8,000 g for a few minutes, and the membranes in an acceleration of 20,000 to 40,000 g for a few tens of minutes, and separation from proteoglycans of the membranes by centrifugation and fractionated into saline solution in water. be following scheme provided an example of obtaining of proteoglycans purified from a bacterial biomass. Schematic representation of the preparation of the proteoglycans Biomass Grinding 7.500 centrifugation g -10 mins. Centrifugation g -45 30,000 min Na Cl 0.15 M Resumption Φ 7.500 centrifugation g -10 mins. 4 Centrifugation 30,000 min * g -45. Resumption H_0 -' Centrifugation 7.500 non-g -10. Centrifugation 30. 000 g -45 min Resumption H-O Hundred ri 7.500 ugatïon g -10 mins. \ Hydrolyze by NaOH 0.1 N Neutralisation Dialysis Centrifugation 30,000 g 4 Hydrolyze by BrONa i Dialysis- Centrifugation 30,000 g ^* Soluble Protéoglycanes Hydrolyze CILs CO01i 1% by >1, Ψ Dialysis Sterilizing Purified Protéoglycanes , détoxi The proteoglycan membrane of the present invention, which are particularly useful are extracted from the membranes of gram negative bacteria, particularly of the genus Klebsiella, Serratia and any Escherichia and j particularly Klebsiella pneumoniae Serratia marcescens Escherichia coli. The present invention also relates to vaccines containing membrane proteoglycans 3 previously described and in particular vaccines containing as immunogenic fraction the ribosomes or the RNA extracted of pathogenic bacteria against which immunity is desired. The ribosomes and The RNA for use in vaccines ? according to the present invention can be prepared by known methods as soon in particular, the methods described in the French patents 73,43957, and 76,24124 75,10252. In one embodiment of this aspect of the invention, it is provided that the weight ratio between the 0. immunogenic fraction and proteoglycans of the invention is between 1.4 and 1.6 and preferably 1.5. Include particularly the following vaccines: 1) Vaccines B r oncho -ORL 5 has) Association Ribosomes -proteoglycans of-ribosomes. Klebsiella pneumoniae ribosomes of Streptococcus pneumoniae •-.. -ribosomes of Streptococcus pyogenes ribosomes of IIemopbilus -influenzae 0- Protéoglycanes of Klebsiella pneumoniae 3,5 3,0 3, 0 0,5 jig 15,0 ng b) Association- protéoglycanes_ RNA-ribosomal -Ribosomal RNA of Klebsiella pneumoniae 2.45 Ribosomal RNA of IIcmopMlus -influenzae 0, 35^Ig -" Do Klebsiella pneumoniae Protéoglycanes. 10.50 ^ Ig 2) Vaccines antî - pyorrhciquea a) Association Ribosomes -proteoglycans rothia dentocariosus -ribosomes of... 1.2 -. ribosomes ^ Ig of Streptococcus mutans 0.8 -the ibosomes of Streptococcus salivarius. 2.0 ~ ^ Ig-ribosomes of Lactobacillus casei 1.2 ^ Ig - Protéoglycanes of Klebsiella pneumoniae... 9, b) Association RNA-ribosomal P rotated ogly canes Rothia dentocariosus -ribosomal RNA of... 0, 84 -Ribosomal RNA of Actinomyces viscosus... 0.84 Ig ^. -Ribosomal RNA of Streptococcus mutans... 0.56 jîg -Ribosomal RNA of Streptococcus salivarius... 1.40 -Ribosomal RNA of Lactobacillus casei 0.84 - Protéoglycanes of Klebsiella pneumoniae... G,72 jxg 3)- Dermatological Vaccine, -anti-acne a) ribosomes- Protéoglycanes Association -ribosomes of Corynebacterium acncs ytg • 2 corynebacterium parvum-ribosomes of ' 2 ribosomes of Staphylococcus epidermidis 2- - Protéoglycanes Seri of ' atia rca rces cens 12 ng b) AEN Association ribosomal- Protéoglycanes Ribosomal-AEN Corynebacterium acnes... Ribosomal-AEN Corynebacterium parvum.. AEN ribosomal-Streptococcus pyogenes... Ribosomal-AEN of Staphylococcus epidermidis - Protéoglycanes of Serratia marcescens -8.4 jig 4) Gynecological Vaccine a) Association rîbosomes -proteoglycans ribosomes of EschericMa coîi 2- s- ribosorœ of Streptococcus faecaîis D 2 ribosomes of Staphylococcus epidermidis 2- t of ribosomes-Candida albicans- · 2} *£ Protéoglycanes -dl Escherichia coli 15 . b) ribosomal RNA Protaoglycanes Association -Ribosomal RNA of Escherichia coli 1.4 ^ tg -Ribosomal RNA of Streptococcus faecalis D-... 1.4 jig. ~ ribosomal RNA of Streptococcus H 1.4 ^ Ig •-Ribosomal RNA of Etaphyloeoecus.-1.4 epidermidis. - Protéoglycanes Escheriehia of coli. 10,5 jig 5) Intestinal Vaccine, anti-typhoid a) Association Ribosomes - Prôtéoglyeanes coli ribosomes Bacterium of 2~ -ribosomes of Salmonella paratyphi B... jig 2 ribosomes of Shigella dysenteria -2- ribosomes-d1 Enterococcus 2 - Protéoglycanes of Serratia mareescens ... 15 b) ribosomal RNA- Protéoglycanes Association Ribosomal RNA of Bacterium -coli... 1.4 -Ribosomal RNA of Salmonella pai ' atÿphi B jig. 1.4. ' Protein preparation raw pçlycanesmembranaires âe K. pneumoniae. the strain of K. pneumoniae is cultured on a medium containing per liter: Meat extract 5 g Yeast extract 5 g Sucrose 2 g Phosphate disodiqûe 2.5 g The cells are then separated from the culture medium by centrifugation followed by washing with a saline solution (NaCl) and dried and optionally stored at -20 °C. The biomass obtained by fermentation is concentrated and washed by continuous centrifugation. The bacterial concentrate thus obtained is dispersed in saline to have a final concentrationf in dry cells of 50 g per liter (spectrophotometric measurement). The bacterial suspension to cell by passing through a Gaulin homogenizer APV-Manton equipped with special valves disintegration. During this operation, the temperature of the suspension is maintained below 10 °C. Canes The protéogly a centrifugation -10 g and minutes to 7.500 + 4 °C to remove cells and unground cellular debris. The supernatant is then subjected to centrifugation of 45 minutes to 30,000 + 4 g ande C for sêdimenter raw the membrane fraction. • •-• • • -the base 30 g. 000 is dispersed by means of a homogenizer in v. a- '- lume of Na Cl 0.15 M cold and then subjected to the same cycle to 7.500 centrifugations g and 30 g as before.. 000'. 30,000-• the base g is resumed this time in a body of water and dispersed to distillée_ perfect homogeneity. -'. •-the suspension from the distilled water is again subjected to a cycle to 7.500 centrifugations 30,000 g and g and the residue is placed again this time in 1/4 of the initial volume of distilled water. The suspension is centrifuged 10 minutes to 7.500 g and the supernatant containing proteoglycans raw is maintained. EXAMPLE 2 -At supernatant obtained in the previous example 1 containing proteoglycans raw, is added 1 ml of sodium hydroxide (10 N) for 100 ml of supernatant to obtain a final molarity of 0.1 M in NaOH. To 25 °C-On incubated for 24 hours with moderate agitation to extract controlled hydrolysis the soluble fraction of the proteoglycans. -The hydrolysate is then neutralized with a solution (3 N) hydrochloric acid and sodium chloride formed is removed by dialysis against distilled water. -The insoluble residue is removed after dialysis by centrifuging 45 minutes to 30,000 g and 4 °C, the supernatant containing the soluble proteoglycans is maintained. EXAMPLE 3 -At supernatant obtained from the example 1 containing raw proteoglycans is added 2 ml sodium hypobromite (10 ml bromine + 60 ml concentrated sodium hydroxide) to 100 ml of supernatant. -The suspension is kept for 30 minutes at room temperature under stirring. Hypobromite-The excess is then removed by a dialysis of 24 hours against water and current against distilled water. -The insoluble residue is removed by centrifugation after dialysis to 4 °C during 45 minutes to 30,000 g and the supernatant containing soluble proteoglycans is maintained. EXAMPLE 4 The proteoglycan prepared to the example 2 are not fully detoxifies. They still contain small quantities of lipopolysaccharides hyperthermisant having an effect. Therefore, processes the proteoglycans of the example 2 as follows: -to 100 ml of dialyzed supernatant containing the soluble proteoglycans is added 1 ml of glacial acetic acid, and then door during 90 minutes at a temperature ' C of 90°. * The separates the lipid hydrolysis quantitatively A of the LPS * and the cooling in ice causes it to precipitation. Lipid A The precipitate is removed by centrifugation 20 minutes g and 0 to 30,000 ° C. •... • The supernatant is collected and dialyzed 24 hours against distilled water for * removing acetic acid. •. The dialysate is sterilized by membrane filtration 0.22 and the filtrate is stored frozen or lyophilized. Eroism Its composition is as follows Hexoses 3? + 5% Proteins 36.5 + 5% 15 +3% Lipids Hexosamines' .5 + 2% RNA < 0.5% DNA '" 0, 2%. LPS < 0,001% EXAMPLE 5 Incorporating as in the example 4 from proteoglycans purified example 3j proteoglycans is obtained the following composition: Hexoses Proteins Lipids Hexosamines RNA DNA LPS- 29 + 5% 48 + 5% 15 ± 3% 4. •± 2% <0,5% <0001% Of course, in the same manner from Serratia marcescens and of Escherichia coli are obtained purified detoxifies proteoglycans and that can be used in 1 ε ℮ compositions described previously. The vaccine formulations according to the invention can be packaged with the holders and excipients known in the art according to their mode of application, the compositions which have been previously are preferably injectable forms. The vaccines of the present invention have been tested for their immunogenic activity and it has been found a substantial better than that the vaccines described to patent 73 43957 due to the replacement membrane proteoglycans by purified membrane proteoglycans and detoxified. The most advantageous aspect But vaccines according to the present invention is their absence of pyrogenic effect. A the effect pyrogen has been conducted as follows: Is injected intravenously in a dose of vaccine 1 ml of water per rabbit 2 to 2.5 kg, and observing the rectal temperature by thermocouple probe, the results observed are given in the table I. Novel vaccines of the present invention are free of pyrogenic effect. GÎT 153-Vaccines antipyorrhéiques 2a, ' proteoglycans, are prepared by the method of the example 4, GÎT 156-Same vaccines, but the proteoglycans are prepared by the process of example 5, GÎT 155-Same vaccines that previously but proteoglycans which has not undergone total that delipidation chloroform. This invention relates to a process for the preparation of purified bacterial membranal proteoglycans. The process comprises at least one step in which crude proteoglycans are treated in aqueous medium with a base or a hypobromite, followed by elimination of the excess reactant and the insoluble residue, the purified proteoglycans being present in aqueous solution. The thus obtained purified proteoglycans may be used as immunity adjuvants in vaccines without causing side-effects such as pyrogenic reactions. RBVÆNJICATIOBS 1) A process for the preparation of purified bacterial membrane proteoglycans, characterized in that it comprises at least one step of treating raw proteoglycans in an aqueous medium by a base or hypobromite followed by removal of excess reagent and the insoluble residue, proteoglycans purified in aqueous solution. 2) The method of claim 1, wherein that the base used is an alkali hydroxide with a molarity of between 0.05 and 2 M and that the treatment time is a few hours at a temperature close to the room temperature. 5} The method of claim 1, wherein that 1' hypobromite used is a alkali hypobromite and that the treatment time is a few tens of minutes at room temperature. 4) Method according to one of claims 1 to 3, characterized in that the excess of reagent is removed by dialysis and the insoluble residue is removed by centrifugation. 5) Method according to one of claims 1 to 4, > being cai in that the method of preparing includes at least one step of hydrolysis with acetic acid in medium, d. aqueous membrane proteoglycans at a temperature of between 70 and 100 °C and removal of the insoluble fraction, 6) A method according to claim 5" wherein the acid hydrolysis is conducted using glacial acetic acid to 1 $by volume. 7) Method according to one of claims 1 to 6, characterized in that the proteoglycans are extracted from gram negative bacteria. 8) The method of claim 7, wherein the gram negative bacteria are selected from: Klebsiella pneumoniae Serratia mareescens Escherichia coli. 9) As an immunity adjuvant for vaccines, proteoglycans Purified by implementing the method according to one of claims 1 to 8. 10) As an immunity adjuvant for vaccines, proteoglycans water-soluble extracts of bacterial membranes having the following composition by weight Hexoses Proteins 31-53 24-42* lipid 12-18 DNA less than 0.2 11) As of auxiliaries according to proteoglycans having the following composition by weight Hexoses Proteins lipid Hexosamines RNA DNA IPS. 37 ± 5 J6 36,5 ± 5 15 5 ± 2 < f < 0.2 56 < 0,001 # 12) As of auxiliaries according to claim 10, the proteoglycans having the following composition by weight: 29 i 5 48 ± 5£ 15 5δ i 3 4 ± 2$ <0,5 56 " ≠ 0.2 <0,001 5É 13) vaccine ribosome and/or bacterial RNA as immunogen containing as immune adjuvant an adjuvant according to any of claims 9 to 12. 14) Vaccine according to claim 13 having the following composition: 1) Vaccines B r oncho -ORL Hexoses Proteins lipid Hexosamines RNA DNA IPS a) Association Ribosomcs -proteoglycans ribosomes of Klebsiella pneumoniae-... ■-ribosomes of Streptococcus pneumoniae -ribosomes of Streptococcus pyogenes A -ribosomes of Hemophilus influenzae. 12 3,5 3, 0 b) RNA-ribosomal Association-proteoglycans. -Ribosomal RNA of Klebsiella pneumoniae... 2.45 Ribosomal RNA of IIemophiius -influenzae -' Protéoglycanes of Klebsiella pneumoniae... ., . 10.50 a) Association Ribosomes -proteoglycans rothia dcntocario -ribosomes of '^ Ig addition. 1.2' -ribosomes of Acünomyces 1.2 jag viscosus. streptococcus mutans-. ribosomes of 0, 8 -ribosomes of Streptococcus salivarius 2.0 ^ Ig. --ribosomes of Lactobacillus casei... ' 1.2 - Protéoglycanes of Klebsiella pneumoniae... b) Association Protéoglycanes RNA-ribosomal Rothia dentocariosus -ribosomal RNA of... -Ribosomal RNA of Actinomyces viscosus... -Ribosomal RNA of Streptococcus mutans... -Ribosomal RNA of Streptococcus salivarius... -Ribosomal RNA of Lactobacillus casei... - Protéoglycanes of Klebsiella pneumoniae... 0,84 jïg 0.84 a) ribosomes- Protéoglyeanes Association corynebacterium acnes • 2-ribosomes of jig ribosomes of Corynebacterium parvum 2- ribosomes of Staphylococcus epidermidis 2- - Protéoglyeaues serratia rces cens 12 im b) ribosomal RNA- Protéoglyeanes Association -Ribosomal RNA of Corynebacterium acnes... l, ^ Ig 4 -Ribosomal RNA of Corynebacterium parvum..' 1.4 -Ribosomal RNA of Staphylococcus epidermidis .1,4 - Protéoglyeanes of Serratia marcescens...-8.4 jig 4) Gynecological Vaccine a) Association rîbosorncs -proteoglycans ribosomes-d1 Escherichia eoli 2 ribosomes of Streptococcus-H... ' ^ Jg -2 -ribosomes of Staphylococcus epidermidis... 2. ^ ig ribosomes of Candida albicans-2-. - Protéoglyeanes Eseheriehia eoli of 15 . b) ribosomal RNA Protaoglycancs Association Ribosomal RNA of Escherichia coli -1.4 -Ribosomal RNA of Streptococcus faecalis D-... 1.4 jig. " Ribosomal RNA of Streptococcus II l, ^4 tg Epidermidis •-ribosomal RNA of Etaphylococciuj.. •1,4 p - Proteoglycan is Escherichia coli a) Association Ribosomes - Protéoglycanes ** coli ribosomes of Bacterium 2 -ribosomes of Salmonella paratyphi A .2 •-ribosomes of Salmonella paratyphi B 2 ribosomes of Shigella dysenteria -2. •••• .- ribosom Entex is' ococcus ... 2 - Protéoglycanes of Serraüa marcescens... " 15 b) ribosomal RNA- Protéoglycanes Association Ribosomal RNA of Bacterium -coli... 1.4 jxg Ribosomal RNA-Salmonella paratyphi A jrg. 1.4. -Ribosomal RNA of Salmonella paratyphi B. 1.4 ^ Ig. Ribosomal RNA of Shigella dysenteriae -1.4 Ribosomal RNA of Enterococcus 1.4- - Protéoglycanes of Serratia rra rcescens ... 10.5 g^ Hexoses 29 + 5% Proteins Lipids 15 Hexosamines ÂRN <0,5% DNA LPS <0) 101% Supernatant: raw proteoglycans
Supernatant
•Supernatant
Proteoglycans purified preparation
Proteoglycans purified preparation
i i i i I 4J 3 s 30' '' 45' ' 1 h 1' h 15 1 h 30' ! h 45' 2 h 2' h 30 3 h 3 h 30' 4 h 4' h 30 5' h 5 h 30'. GN.153 0.03 0' 0 -0.01- j0,05 -0.03 -0.03 -0.03 -0.10 -0.10 0 -0.16 -0.15 -0.16 -0,181 Gn 155 + 0.06 • + 0.50 1.16+ 1.16+ 1.11+ + 1.06 + 1.43 + 1.66 + 1.90 + 1.48 + .M + 0.71 ' + 0.36 GN 156 + 0.06 -0.04 0.16 0.06-J '-0.20 -0.26 -0.26 -0.30 -0.40 -0.46 -0.20 -0.50 -0.44 -0.46 1-0.46.