MXPA97009011A - Vaccination composition comprising polyribosil phosphate ribitol and its manufacture procedure - Google Patents

Abstract

The present invention relates to a stable vaccine composition during storage comprising at least one antigen consisting of the polysaccharide type b Haemophilus influenza type bo phosphate poliribosilribitol high molecular weight coupled to tetanus toxoid as well as an auxiliary or added based on aluminum, presents by nature or after the addition of anions a point of zero charge less than about 7.2. The subject of the invention is also a process for the preparation of such a vacu composition.

Description

COMPOSITION OF VACCINE COMPRISING POLYBRYOSILRIBITOL PHOSPHATE AND ITS MANUFACTURING PROCEDURE DESCRIPTION OF THE INVENTION The invention concerns the field of vaccine compositions and more particularly, vaccine compositions comprising at least one antigen consisting of the capsular polysaccharide of Haemophilus type b, or high molecular weight polyribosylribitol phosphate coupled to the toxoid. tetanic It is known in the prior art and especially by the article "Quantitative and Qualitative Analyzes of Serum Antibodies Elicited in Adults by Haemophilus Influenzae Type b, and Pneumococcus Type 6A Capsular Polysaccharide Tetanus Toxoid Conjugates" Rachel Schnerson et al, Infect. Immun May 1986, an antigen that can be used for vaccination purposes in man in order to protect them from infections caused by Haemophilus influenzae Type b. This antigen is constituted by a capsular polysaccharide of the bacteria, the phosphate Poliribosilribitol (or PRP), which becomes T-dependent, thanks to a coupling with a carrier protein, tetanus toxoid. Tests carried out in small rhesus have shown that in relation to this article the immunological response was both more important and precocious, if the antigen was associated with aluminum hydroxide. However, as mentioned in another article entitled "Clinical and Immune responses to the capsular polysaccharide of Haemophilus influenzae type b, alone or conjugated to tetanus toxid in 18-23 month-old-childrenw, Bo A. Cleasson and al, The Journal of Pediatrics, May 1988, it has been noted that this antigen adsorbed on aluminum hydroxide was after storage less immunological than the antigen maintained in saline, which may be due to a degradation of the polysaccharide. problem, stability of the PRP-T, it has been proposed in the prior art to lyophilize it. This solution, while allowing the antigen to retain its immunogenic character over time, nevertheless presents drawbacks, especially at the manufacturing level; freeze-drying and the particular conditioning operations that it requires, complicate the manufacturing process, which increases the cost. In addition, at the time of administration it is necessary to carry out a repeated taking of the lyophilisate, which means that it is necessary to also dispose of lyophilisate of a retake liquid of that lyophilisate; this operation represents a supplementary problem for the practitioner and presents as any manipulation the risk of performing poorly. In addition, a number of combinations of liquid vaccines possess antigens adsorbed on an aluminum-based auxiliary and it would be advantageous to be able without loss of immunity to add the antigen constituted by the PRP-T. Indeed, the solution proposed by the prior art and consisting of a particular syringe with two compartments (a first compartment containing the PRP-T, in lyophilized form and a second compartment containing the other antigens in aqueous suspension), where the content is mixed untimely at the time of administration, it is not satisfactory, both for the level of manufacturing costs and for the level of operations that the user must perform. Then, it is desirable to be able to have a liquid vaccine composition comprising the antigen constituted by the PRP-T, having a good immunological character conserved over time, and where the manufacturing conditions allow the production at a minimum cost. In order to achieve these objectives, the invention relates to a vaccine composition comprising at least one antigen consisting of the capsule polysaccharide of Haemophilus influenzae type bo phosphate polyribosilbibitol, of high molecular weight coupled to tetanus toxoid, as well as an auxiliary based on aluminum, characterized in that the assistant or assistant based on aluminum has a point of zero charge, less than about 7.2. It has thus been noted that, surprisingly, under these conditions, PRP-T retains its good immunogenic character over time in a liquid medium. According to the particular feature of the invention, the aluminum-based auxiliary comprises aluminum hydroxides to which anions have been added. Thus, it is possible to use a perfectly qualified auxiliary for a vaccine use, preserved the PRP-T, in a liquid medium with a good in ungenicity. According to another feature of the invention, the anions are selected from the phosphates or citrates. Thus the composition obtained presents all the security guarantees necessary for the administration of a vaccine. According to a particular embodiment, the vaccine composition further comprises one or several vaccine valencies chosen from diphtheria, tetanus, pertussis, hepatitis B, polio. Thus, stable liquid vaccine combinations are available in which each antigen retains its immunogenicity, which allows the user, without additional manipulation, to vaccinate simultaneously against several diseases.; this allows reducing costs both in terms of products and in terms of the number of visits that must be made. Another object of the invention is a method for manufacturing a vaccine composition comprising at least one antigen consisting of the capsule polysaccharide of Haemophilus type bo phosphate polyribosylribitol of high molecular weight coupled to the tetanus toxoid, characterized in that it consists in adding to the vaccine composition at least one suspension of aluminum complexes having a zero charge point less than about 7.2. The present invention will be better understood upon reading the following detailed description. The antigen constituted by the capsular polysaccharide of Haemophilus influenzae type b, is a linear polymer consisting of ribose, ribitol and phosphoric acid whose monomeric structure is as follows: OH The number of monomers of this type is higher than 100, which leads to a polysaccharide whose molecular weight is of the order of 500 thousand to 1 million. In order to induce an immune response of T cells, in young infants, this antigen is conjugated with a carrier protein constituted by tetanus toxoid. Such an antigen can, for example, be obtained according to the method described in "Quantitative and Qualitative analysis of serum antibodies elicited in adults by Haemophilus influenzae type b, and Pneumococcus type 6A, capsular polysaccharide tetanus toxoid conjugates" Schnerson et al, Infect. Imun 52: 519 (1986). The characteristics of this antigen: high number of monomers of the polysaccharide, nature of the carrier protein, nature of the link between the polysaccharide and the carrier protein, give it particular qualities and especially a very good immunogenicity. In order to preserve these qualities in the liquid medium, over the course of time, it has now been found that aluminum complexes can be used where the zero charge point is less than 7.2. Indeed, it has been found that surprisingly when the PRP-T, is associated with aluminum complexes, its very good immunogenic character is preserved in the course of time in the liquid medium, and this whatever the degree of binding to the aluminum complexes. The zero charge point of the alurainyl complexes is the equivalent of the isoelectric point of the proteins: it is the pH at which the charge on the surface of two aluminum complexes is zero. In fact, this point of zero charge is approximated by measurements of zeta potential, which can be performed under different techniques, with the base method being electrophoresis. It is possible to make the measurements by means of an apparatus, such as DELSA 440 from Coulters Electronics, Hialeah, Fl, USA. The methods of measuring devices may be different, the results obtained may vary equally. The aluminum complexes suitable for the purposes of the invention are those for which that point of zero charge is less than about 7.2, this value can not be more than an approximate value. Aluminum complexes suitable for the purposes of the invention are those that by nature have a zero load point lower than 7.2, or those that can be modified in order to lower their zero load point. Among the aluminum complexes that naturally have a zero charge point lower than 7.2, one can cite those commonly called aluminum phosphates, in the field of vaccine auxiliaries, even though from a chemical point of view, they possess other salts that the aluminum phosphates. This is, for example, aluminum phosphate ADJUFOS (registered trademark), supplied by the company SUPERFOS BIOSECTOR. It can also be aluminum complexes obtained by the reaction of sodium carbonate in PBS buffer, on potassium and aluminum sulfates. With such complexes, although the PRP-T is completely or partially bound to the aluminum complexes, its immunogenicity is preserved over time. Alternatively, it is possible according to the invention to use aluminum complexes which by nature have a zero charge point higher than 7.2, and which are modified to lower that loading point. These are especially known aluminum complexes in the field of vaccine auxiliaries such as aluminum hydroxides, even if, from a chemical point of view, they are not exclusively constituted by aluminum hydroxide. It is possible especially to treat ALHYDROGEL aluminum hydroxide (registered trademark), supplied by the company SUPERFOS BIOSECTOR, of the product used as an auxiliary in the D.T. Coq. (registered trademark), marketed by the company PASTEUR MERIEUX S & V, or also of the product used as auxiliary in the Recombivax (registered trademark), marketed by the company MERCK. According to the invention, the modification of these complexes consists in the addition of anions. The aggregated anions can be of different kinds, provided that they present all the necessary safety guarantees for their use for vaccine purposes.

It has been noted that the addition of citrate or phosphate ions agree particularly well for the purposes of the invention. The phosphate ions can be provided by a solution containing monopotassium phosphate, disodium phosphate and sodium chloride. It is also possible to use a combination of different anions, for example, a combination of phosphate and carbonate ions. It is possible that the anions are added to the suspension of aluminum complexes before adding the PRP-T, or that the anions are added to the PRP-T, prior to its contact with the aluminum complexes. For convenience it is preferred to put the PRP-T, in suspension in a solution containing the selected anions before bringing it into contact with the auxiliary. The quantity of anions added is calculated to reduce the zero load point of the aluminum complexes used, to a value lower than approximately 7.2. This amount then varies depending on the nature of the aluminum complexes used as well as on the amount of anions optionally provided by the buffer substances used, their determination depends on the technician. Thus, a stable vaccine composition in the liquid state is obtained, that is, in which the PRP-T retains its good immunogenic character.

It has been noted that, thanks to the vision of anions, the fixation of PRP-T is reduced to aluminum complexes, which should contribute to maintaining its integrity and then its immunogenicity. Although this is not preferred in the sense of the invention, it is also possible to add anions to aluminum complexes which have a zero charge point by nature, and at least about 7.2; in this case, it has been noted that the zero load point can be lowered, and that also the fixation of the PRP-T, to the aluminum complexes is reduced. By binding, it means any form of binding that makes the PRP-T inaccessible when the supernatant is collected after centrifugation. The vaccine compositions according to the invention comprise the vaccine antigen constituted by the PRP-T, but may also comprise other vaccine antigens and especially those intended to protect against diphtheria, tetanus, whooping cough (cellular or non-cellular) , poliomyelitis, hepatitis A, B, etc. In fact, all vaccine antigens compatible with PRP-T and aluminum complexes can enter the vaccine composition according to the invention. Thus, it is possible to have liquid vaccine combinations that allow, with a single administration, to vaccinate against several diseases. The vaccine compositions according to the invention are particularly adapted for administration to young children. Furthermore, it has been noted that the use of such composition of liquid vaccine, allows in the place of the repeated vaccination carried out of infants of 12 months who have received injections at 2, 4 and 6 months, increasing the production of antibodies, anti-PRP-T, particularly acute with respect to a repeat performed under the same conditions with the TETRAct-HIB vaccine, marketed by the company PASTEUR MERIEUX. Example 1. A vaccine composition is manufactured from the following elements: - purified tetanus toxoid (ATP) 1 u.vaccine - purified diphtheria toxoid (ADP) "- Pertussis 150U u.opacimetric cell mixtures - PRP-T (weight of PRP) 12μg - mentolato 43.75μ - aluminum hydroxide 0.3 mg (expressed Al) as presented in D.T.Coq. - phosphates 30 μmoles - buffer tris lOmmolar with 0.125 ml sucrose at 8.5% - water to complete 0.5 ml the phosphate ions are provided by a solution containing monopotassium phosphate, disodium phosphate and sodium chloride. Example 2. The immunogenicity of the vaccine composition obtained according to example 1 has been tested in young children compared to a vaccine of the prior art, constituted by the vaccine marketed under the TETRAct-HIB brand., and which has the same valency of vaccine, but where the PRP-T valence, preserved freeze-dried, is reconstituted just before the injection by the vaccine composition containing the diphtheria, tetanus toxoids, as well as the Pertussis cell mixture. This trial was carried out in a group of 262 infants, of whom 130 received the formulation according to example 1, and 132 received the commercial vaccine TETRAct-HIB. The administration of the vaccines has been carried out at 2, 6, and 12 months intramuscularly. Before the immunization the GMT, antibody, anti-PRP titer was 0.2 μg / ml, in the two groups; was 1.9 and 1.4 after the second injection and 5.9 and 5.8 after the third injection respectively for the vaccine according to the invention and the vaccine of the prior art. After the second injection, 98% (with the vaccine according to the injection), and 93% (with the vaccine according to the prior art), of the infants had an anti-PRP antibody rate, higher than 0.15 μg / ml; this rate was reached after the third injection in 100% of the infants who had received the vaccine according to the invention and 99% of the infants who had received the vaccine according to the prior art. After the third injection, the quantities of antibodies directed against each of the vaccine valencies were, on average, the following: Vacuna tec.ant. Vacuna inv Diphtheria 1.35 1.56 IU / L Tetanus 5.1 4.9 IU / ml Tit. 597 601 Agl.Pertussis GMT PRP 5.8 5.9 μg / ml After the repeat injection at 12 months, the amounts; of antibodies were this time: Vac.tec.ant. Vac. inv. Diphtheria 3.2 4.5 IU / ml Tetanus 12.0 11.5 IU / ml Tit. 2447 2560 Agl.Pertussis GMT PRP 19.4 32.6 μg / ml These results show that the vaccine combination obtained according to the invention is stable; In fact, the repeat injection in 12-month-old infants was carried out with a vaccine that had been manufactured for 12 months, where the results show that the immunogenicity of each of the antigens in the combination had been preserved. In addition, a particularly superior repetition effect with the vaccine composition according to the invention is surprisingly obtained in relation to the repeat effect obtained with a vaccine of the prior art possessing the same vaccine valencies. Example 3. Dose of vaccine composition as described in example 1, is maintained at + 4 ° C, for 18 to 24 months, subsequently used in a clinical trial including 104 infants. The titles obtained for each of the vaccine valencies are indicated in the following table: Before vac. After vac Diphtheria 0.013 0.736 G.M.T.

Tetanus 0.181 3.831 G.M.T. PRP G.M.T. μg / ml 0.22 6.40 It can thus be seen that even after a long storage at + 4 * C, the vaccine composition of the invention retains its immunogenic character, both as regards the PRP-T, as other vaccinated antigens. Example 4. A vaccine composition is prepared, each comprising PRP-T, in a concentration of 20 μg, of PRP / ml, in the presence of aluminum complexes of different classes and zero charge points (PCZ), the amount of aluminum complexes is such that the concentration in aluminum in the composition is 0.6g / l. Composition 1: aluminum complex constituted by aluminum hydroxide as used in the D.T.Coq. Vaccine marketed by PMsv. PCZ = 11.3. Composition 2: aluminum complex constituted by aluminum hydroxide as used in the Reco bivax vaccine marketed by Mere. PCZ = 7.4. Composition 3: aluminum complex consisting of aluminum phosphate obtained by mixing sodium chloride and trisodium phosphate. PCZ = 6.2. Composition 4: aluminum complex constituted by the product called Alum, and obtained by reaction of sodium carbonate in buffer, PVS on potassium and aluminum sulfates. PCZ = 5.4. The vaccine compositions are obtained by simple mixing of the suspensions containing the aluminum complexes and the PRP-T. Example 5. Immunogenicity is tested in mice with the different compositions obtained. In order to verify the immunological stability, the obtained compositions are subjected to accelerated aging conditions, that is, they are kept for two weeks at 37 ° C. The immunogenicity test is carried out in mice of 22-24g, to which they are administered. subcutaneously 0.5ml dose, each containing 2.5μg of PRP. Administrations are made to JO and J14; blood is taken from the mice to J14, and J21, and the rate of antibodies is determined by dosage, immunological radio, the number of mice inoculated for each vaccine composition, is 8. The result is considered satisfactory if: It is had when minus 75% of the mice in J21, which have a titer greater than or equal to 0.5; There is a significant difference between the results obtained in J14, and those obtained in J21. N.T. J = day. It is considered that the vaccine composition is stable if the results obtained after aging are satisfactory. The results obtained for the compositions tested are indicated in the following table: PCZ Inm. Cl 11.3 does not satisfy C2 7.4 does not satisfy C3 6.2 satisfac. C4 5.4 satisfac. It is thus seen that when the zero charge point of the aluminum complexes is an acid pH, the vaccine composition is stable. Example 6. The percentage of PRP-T, fixed to the aluminum complexes, is verified for each of the compositions of Example 4. For this, each of the compositions is centrifuged; the supernatant is collected in which the amount of PRP-T, not fixed, is determined by ELISA or by RIA. The difference between the concentration of PRP-T, in the starting composition and the quantity dosed in the supernatant, allows to determine the fixed percentage of PRP-T. The results obtained are the following: Cl: 100% C2: 100% C3: 100% C4: 70% Example 7. Composition 1 is modified, adding phosphate ions in order to obtain a concentration of 50mMol / l.

The immunogenicity test performed on mice after accelerated aging of the solution then leads to a satisfactory result. The determination of the percentage, of fixation of PRP-T, in the aluminum complexes, shows that under these conditions only 20% of the PRP-T has been fixed. Example 8. Composition 1 is modified, this time adding citrate ions, in order to obtain a concentration of 200mMoles / l. The immunogenicity test performed on the mice, after accelerated aging of the solution, then leads to a satisfactory result. The determination of the percentage of fixation of PRP-T, to the aluminum complexes shows that under these conditions the PRP-T, has not been fixed. Example 9. Composition 2 is modified, adding phosphate ions to it in order to obtain a concentration of 20mMol / l. The immunogenicity test performed on mice after accelerated aging of the solution then leads to a satisfactory result. The determination of the percentage of fixation of the PRP-T, to the aluminum complexes, shows that under these conditions the PRP-T, has not been fixed. Example 10. Composition 3 is modified, adding phosphate ions in different amounts and determining as percent of fixing of PRP-T, to aluminum complexes. If the amount of added phosphate ions is such that the concentration of phosphates in the composition is 2mMol / l, the percentage of PRP-T, fixed is reduced to 10%. If the amount of phosphate ions added is such that the concentration of phosphates in the composition is 4mMol / l, the PRP-T has not been fixed. The immunogenicity tests performed on the mice after the accelerated aging of the solution are satisfactory. Example 11 The composition 4 is modified by adding phosphate ions to reach a concentration of 60 mMol / 1. the determination of the percentage of PRP-T fixed to the aluminum complexes shows that under these conditions, only 30% of the PRP-T is fixed. The immunogenicity test performed on the mice after the accelerated aging of the solution leads to a satisfactory result. Example 12 A vaccine composition is prepared from the following elements: Aluminum hydroxide (expressed in Al) 0.25mg PRP-T (expressed by weight of PRP) lOμg ADP 1 dose vac, ATP 1 dose vac. Phosphates 15 μ Moles Antigens polio type I 40 U type II 8 U type III 32 U Anatoxin Pertussis 25 μg F-HA Pertussis 25 μg buffer tris 50 mmolar 0.125 ml comprising 42.5% sucrose Water to complete 0.5 ml Immunogenicity tests relative to PRP-T performed in the mice with a solution thus prepared, as well as with a solution stored for 1 month at 37 ° C, a solution stored for 2 months at 25 °, and a solution stored for 6 months at 4 °, all of them leading to results satisfactory, and that show the stability of PRP = T in such an environment. EXAMPLE 13 A vaccine composition is prepared with the following elements: Aluminum hydroxide (expressed in Al) 0.3 mg PRP-T (expressed by weight of PRP) lOμg ADP 1 dose vac. ATP 1 empty dose Anatoxin Pertussis 25μg F-HA 25μg Hbs protein (as used in the GenHevac B Pasteur vaccine) 20 μg Polio antigens type I 40 U type II 8 U type III 32 U phosphates 20 μ moles carbonates 5 μ moles Tris cushion 50 m molar 0.125 ml comprising 52.5% sucrose water to complete 0.5 ml The stability of the solution thus prepared is checked by submitting it 2 weeks to 37 * and then carrying out an immunogenicity test of the PRP-T in the mice as described in the example 5. The results obtained are satisfactory. An immunogenicity test of the Hbs protein is performed; This test is performed in mice and consists of dosing the anti-Hbs antibodies by ELISA, after determining the 50% effective dose that must be lower than 0. 970 μg of Hbs protein, to be satisfactory. The results obtained with the solution prepared as indicated above and maintained 2 weeks at 37 * before being tested, have been satisfactory. Example 14 A vaccine composition is prepared with the following elements: ATP 1 dose vac. Anatole Pertussis 10 μg F-HA 5 μg Fimbriae 5 μg Perctactin 3 μg Protein Hbs (such as 20 MG present in GenHevac PASTEUR vaccine) polio antigens type I 40 U type II 8 U type III 32 U phosphates 20 μ moles carbonates 10 μ moles MgCl2 5 μmol Tris 50m mole cushion comprising 42.5% sucrose 0.125 ml water to complete 0.5 ml The immunogenicity tests relative to PRP-T performed as described in example 5, and those related to protein Hbs made as described in example 13, all of them have led to satisfactory results showing that the stability of the vaccine composition according to the present invention.

Claims (8)

1. REVINDI CAC IONS 1.-Vaccine composition comprising at least one antigen consisting of the capsule polysaccharide of Haemophilus influenza type bo. High molecular weight Poliribosilribitol phosphate coupled to the tetanus toxoid as well as an aluminum-based auxiliary, characterized in that the auxiliary aluminum base has a zero charge point less than about 7.2.
2. 2. Vaccine composition according to claim 1, characterized in that the aluminum-based auxiliary comprises aluminum hydroxides to which anions are added.
3. 3. Vaccine composition according to claim 2, characterized in that the anions are selected from phosphates and citrates.
4. 4. Vaccine composition according to one of the preceding claims, characterized in that the aluminum-based auxiliary comprises aluminum phosphates.
5. 5. Vaccine composition according to one of the preceding claims, characterized in that the aluminum-based auxiliary comprises potassium and aluminum sulfates.
6. 6. Vaccine composition according to one of the preceding claims, characterized in that it also comprises one or more valencies of vaccines selected from: diphtheria, tetanus, whooping cough, hepatitis B, poliomyelitis.
7. 7. - Method of manufacturing a vaccine composition comprising at least one antigen consisting of the capsular polysaccharide of Haemophilus influenza type bo phosphate polyribosilbitol coupled to the tetanus toxoid, characterized in that it consists of adding the vaccine composition by means of a suspension of complexes of aluminum that has a zero charge point less than approximately 7.2
8. 8. - Process according to claim 7, characterized in that it consists in adding to the aluminum complexes anions selected from phosphates and citrates. SUMMARY The invention relates to a vaccine composition comprising at least one antigen consisting of the polysaccharide of type b Haemophilus influenza type bo phosphate polyribosylribitol of high molecular weight coupled to the tetanus toxoid as well as an auxiliary or added based on aluminum, in which the aluminum-based aggregate has, by its nature or after the addition of anions, a zero charge point less than about 7.2. The subject of the invention is also a process for the preparation of such a vaccine composition.