MXPA98007617A - Polysaccharide-peptide-conjugates - Google Patents

Abstract

The invention relates to a polysaccharide-peptide conjugate wherein the polysaccharide is advantageously immunogenic, which comprises a polysaccharide chain composed of repeat units and a plurality of peptide moieties, each moiety containing a cysteine residue and being covalently attached at random along the polysaccharide chain, through an indirect bound involving the thiol group of the cysteine residue and an amino, hydroxyl or carboxyl group of the polysaccharide, said indirect bound being achieved through either a linker or a spacer-linker moiety provided that the spacer entity of the spacer-linker moiety is linked to the amino, hydroxyl or carboxyl group of the polysaccharide. To this end, a useful linker may be e.g., N-(&ggr;-maleimidobutyryloxy) succinimide ester. Such a conjugate may typically exhibit a"Rake"configuration. Conjugation processes are also disclosed. Conjugates of the invention are in particular useful in the vaccinal field to elicita protective long term immune response against a pathogenic microorganism from which the immunogenic polysaccharide is derived.

Description

CONJUGATES OF PO ISACARID PEPTIDES DESCRIPTION OF THE INVENTION The present invention relates to a conjugate of polysaccharide peptide and a process for preparing it. In a In a particular embodiment of the invention, the conjugate uses bacterial and fungal polysaccharides and thus may be useful for vaccine purposes. Polysaccharides are a broad family of polymeric molecules which are useful in several fields technicians. In some cases, they need to be coupled to a polypeptide, e.g., protein or peptide. For example, polysaccharides are used in diagnostic or purification techniques as a matrix means for peptide reagents. Also useful are non-immunogenic polysaccharides such as dextran, to present small peptides, to the immune system as described in European Patent 326 111. Indeed the peptides have been linked to protein carriers or have to be administered with an adjuvant, the most commonly used adjuvants which are compounds from aluminum. However, small peptides mixed, adsorbed or precipitated with these adjuvants can be hindered by the aluminum gel and therefore, are not available to the immune system. To solve this problem, European Patent 326 111 teaches that the peptides can be conjugates to non-immunogenic polysaccharides. Such conjugates, in the presence of aluminum compounds, are capable of producing an immune response against the portion of the peptide. In the field of vaccines, it is also highly interesting to conjugate polypeptides for example, peptide or protein, to immunogenic polysaccharides, this once an immune response for the polysaccharides is sought. Indeed, the capsule and the cell wall of the bacteria (and also the fungal cell wall F) consist essentially of polysaccharides composed of very specific repeat units.

They carry motifs of epitopes that are not usually found in mammals and can mediate immunity. Therefore, polysaccharides for example, capsular polysaccharides have already been used as vaccines against bacterial diseases such as meningitis, pneumonia and typhoid fibre. 15 However, there is a major problem when using jrffe polysaccharides as vaccines. Although they have been immunogenically tested, that is, in other words, they produce an immune response when administered such as to a mammal, even if this response may be deficient, they are specific in that they belong to a small number of antigens that are able to induce the production of B cells without the help of T cells. Therefore, they are called T-independent. The immune response induced by antigens independent of T is characterized by a number of characteristics, among which: (i) the primary response is weaker and earlier than the response to T-dependent antigens; (ii) the antibody response does not mature in high IgG production, with an increase in affinity, as observed with T-dependent antigens; (iii) the immune memory that corresponds to the antigens independent of T is deficient and in this way, as the immune memory is the key to the secondary immune response that forms the basis of the principle of vaccination, an antigen independent of T is an antigen deficient to induce a long-term protective immune response; and (iv) infants are not able to respond to polysaccharides before one or two years of age. In order to induce a secondary immune response, the T-independent antigens need to be covalently coupled to a carrier protein such as diphtheria or toxoid toxin, which gives the antigen the T-dependent character. The conjugate thereof can then be supplemented with an adjuvant such as an aluminum compound or complete or incomplete Freund's adjuvant (these last two, exclusively for use in mammals other than humans), in such a way that the immune response increases (adjuvant effect). By the term "carrier" is meant a molecule which, when covalently linked to an antigen eg a polysaccharide, is capable of promoting a T-dependent response to the antigen. Such a response is shown after the vaccination schedule comprising at least two injections of the antigen-carrier conjugate, in separate days, weeks or months. * (first and reinforcement). After the first infection (first), a weak response of the * antibody is shown, whereas after the boost, the antibody response occurs at a high level. Such an increased response is not observed with the negative control constituted by the unconjugated antigen. Various methods for conjugates are already available in the art. The functional groups of polysaccharides that are commonly involved can be amino, carboxyl or hydroxyl groups located along the chain or aldehyde groups either at the terminal or along the chain. The groups Functional polypeptides which are usually involved can be amino or carboxyl groups, terminal or present on the amino side chains or even thiol groups. In a general form, polysaccharide conjugates can exhibit three types of structure depending on the location of functional groups of both the polysaccharide (either along the chain or at the end) and carrier, which are involved in the binding. These types of structures are called for ease of description, types "sun" or "" ear "," stick "and" network. "They are illustrated in Figure 1, where (A), (B) and (C) ) are found respectively for the types "sun" (neoglicoconjugado), "stick" and "network." In the type "sun", a polysaccharide is linked to a protein through a reactive group located exclusively at one end of the chain This usually involves a carbonyl group located at the reducing end of the polysaccharide chain.Several polysaccharide chains can be attached to the protein, the linkage usually involving an amino group carried for example, in a lysine residue. conjugates are also defined as neoglucoconjugates.As a matter of example, a conjugate of this type is achieved in Alonso de Velasco et al., Infect. Immun. (1995) 63: 961, Paradiso e t al, Vaccine Research (1993) 2 (4): 239, and Jennings' United States Patent No. 4,356,170. In the "stick" type, the peptides are joined along the polysaccharide chain. An example of this type is provided in Lett et al, Infect. Immun. (1994) 62: 785, and more appropriately, Lett et al, Infect. Immun (1995) 63: 2645 and Konenisoman et al, J. Immunol. (1995): 5977. The binding involves the amino group carried by the single lysine residue internal to the peptide sequence and / or the amino terminal group.

In the "red" type, the protein and the polysaccharide are cross-linked. This is made possible due to the fact that a protein is used rather than a peptide (usually amino or acid groups located along the protein) and to which the reactive groups located along the polysaccharide chain are involved. A conjugate of this type is described in Anderson's United States Patent No. 4,673,574. F Schneerson et al, J. Exp. Med. (1980) 152: 361 also describes a conjugation method leading to the "network" type. This uses CNBr and as a linker, adipic acid dihydrazide (ADH). The hydroxyl groups present throughout the length of the polysaccharide chain and amino groups of the side chain are involved. Each of these structures can be obtained according to a variety of conjugation processes. The link may be a direct link as in Anderson's United States Patent No. 4,673,574, Jennings' United States Patent No. 4,356,170, Lett et al or Kónenisoman et al. The link can also be a direct link in which a linker molecule is used as illustrated in Schneerson et al.

An additional spacer can also be used to the linker as described in Alonso de Valesco et al or Paradiso et al (for the polysaccharide for pneumococcus). Various functional groups present in the polysaccharide, protein, linker and optionally spacer may be involved. Some of the references of the prior art # cited above are presented with additional details as follows: In Alonso de Velasco et al, the carrier is a peptide of about 20 amino acid residues comprising a single residue of cysteine at either end. The 17F polysaccharide of Streptococcus pneumoniae is first derived at the reducing end by reductive amination with diaminopropane in the presence of NaCNBH = 3. Then the derivatized polysaccharide is bromoacetylated with N-succinimidyl bromoacetate corpo a The linker and the polysaccharide thus activated is * coupled to the thiol group of the unique C-terminal cysteine residue of the peptide. In this way the unique finished conjugate is obtained. In Lett et al (1994), the polysaccharide of S. mutans or Saccaromyces cerevisiae with periodate to create aldehyde groups throughout the length of the polysaccharide chain. Then the oxidized polysaccharide is coupled directly by reductive amination to a peptide, in the presence of NaCNBH3. 20 In Paradiso et al, two polysaccharides are used: a pneumococcal polysaccharide and the polyribityl phosphate (PRF) of Haemophilus influenzae. After the acid hydrolysis of the pneumococcal polysaccharide, an aldose group is first created at the end of the polysaccharide chain. They are added then the amino groups at the end of the chain after reductive amination with diaminomethane in the presence of pyridine and borane. The polysaccharide derivative is activated with diester succinimidyl of adipic acid and is coupled to amino groups of the protein or peptide: Returning to PRF, this 14lti.no is first subjected to oxidative cleavage using periodate. The aldehyde groups in this way are created at both ends. The oxidized PRF is then coupled to the amino groups of the protein and peptide. In both cases, a structure is created "Sun" . 10 In Kónenisoman et al, Vi polysaccharide and peptides (none of which contains a cysteine residue) or proteins are coupled together in the presence of 3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC): the carboxyl groups of the polysaccharide and amino groups are involved of the protein or peptides in the conjugation. As a result, a "network" structure is created when the protein is used. When the peptides are used, the structure depends on the number of amino acid residues that the amino groups carry. This can be either a simple "network" structure or a "stick" structure.

As can be easily understood, when the conjugation involves functional groups present throughout the length of the polysaccharide chain, this leads to a cross-linked conjugate if the carrier is a protein (several amino or carboxyl groups are available in the protein for binding). Yes the polypeptide carrier contains a single binding site (this situation is very frequent when it is sufficiently small), the conjugate thereof exhibits a "stick" structure (which can also be achieved if the polypeptide contains a few binding sites and the conjugation is carried out under closed control, not easy in such a way that a functional group reacts on the polypeptide). When the conjugation method uses carboxyl or amino groups of a naturally occurring polypeptide (or a fragment thereof), the latter must be very small in order to obtain a "stick" structure, since the carboxyl groups or amino are frequently found in the polypeptides. We have now found a novel conjugation method that can easily produce a "stick" structure, while using the thiol group of a cysteine residue. Since cysteine residues are less frequent than lysine and aspartic acid, this method is therefore suitable for conjugating larger peptides. As a carrier, peptides have some advantages over proteins since they can be easily purified when they are biologically produced, or synthesized and therefore, are more pure and more defined. Contrary to carrier proteins which may also have harmful properties (city), the peptides can be derived from those proteins to exhibit only the carrier property. However, conjugates of polysaccharide peptides # known in the art are less immunogenic than their counterparts polysaccharide proteine and definitely require adjuvants. __ Surprisingly, the polysaccharide peptide conjugate prepared by the novel method has good immunogenicity. One of the reasons for this lies in the fact that the peptide portion has a sufficient size. Therefore, the present invention relates to a peptide polysaccharide conjugate wherein the polysaccharide is advantageously immunogenic, which comprises: (i) A peptide portion having at least six amino acid residues, at least one of the which is a cysteine residue; (ii) A polysaccharide chain comprising at least four repeating units; and (iii) A linker moiety attached to a thiol group of the cysteine residue and linked to (a) amino, hydroxyl or carboxyl groups native to the polysaccharide chain or (b) amino groups created after hydrolysis of the N-acyl groups 20 native to the polysaccharide chain or (c) functional groups introduced into the polysaccharide chain after derivatization with a spacer moiety linked to the - ^ mino, hydroxyl or carboxyl groups native to the polysaccharide chain .

For use in the present invention, the peptide may contain one or more cysteine residues. The cysteine residues provide binding of the linker to the peptide. The use of a cysteine residue for coupling improves coupling selectivity as soon as the amount of the cysteine residues in a peptide is usually low. The cysteine residues may be located at either the peptide end or internal to the peptide chain, with the proviso that the binding at this site does not interfere with the structure and properties of the peptide. Regardless of the amount of the cysteine, it is preferred that a residuum of cysteine be located at the terminal N or C terminal. More preferably, the peptide contains two cysteine residues, each located at one end, or ur} alone cysteine residue located at either end; This last alternative is the most preferred. The entire amino acid sequence of the peptide can be found naturally as such or as part of a larger polypeptide. This can also be constituted by a naturally occurring sequence this is prolonged at the N or C terminal end or both ends by an additional cysteine residue. For use as a carrier in vacuo conjugate, the peptide advantageously contains at least one epitope dependent on T cells and therefore allow the development of a protective immune response against the polysaccharide - this is a T-dependent antigen, after administration of the conjugate to for example a mammal. For use in the present invention, the peptide can be chemically synthesized or produced by recombinant means. Either the method can be reached conventionally. A conjugate of the invention can comprise only one * peptide; in this case the peptide portions present in all along the polysaccharide chain are identical to each other. It can also comprise several peptides, for example, carrying different epitopes. However, a limited number of peptides are preferred; no more than six and more preferably 2 or 3. While a first peptide carries an epitope dependent on T, a second peptide can carry an epitope B. The polysaccharides used in the conjugates of the present invention can be of any type. In an embodiment of the invention, conjugates are prepared for purposes and therefore suitable polysaccharides include capsular polysaccharides, the derived polysaccharides form lipopolysaccharide (LPS or LOS) cell walls of Gram-negative bacteria, such as the O-specific side chain, and also fungal cell wall polysaccharides. For example, polysaccharides can be derived from bacteria including Pseudomonas, such as P. aeruginosa, Staphilococci, Streptoccocci, particularly S. pneumoniae, Klebsiellae, for example K. pneumoniae, Salmonellae, for example S. Typhi and for typhi, Escherichia coli Kl f K10o, 0157: H7, Neisseriae, example N. Meningi tidis, Shigellae, for example S. Dysenteriae, somnei and flexneri, Haemophilus, for example H. influenzae type b, and fungi such as Candida, Cryptococcus neoformans, and Hansenula. The polysaccharides are composed of repeated units. For use in conjugates of the invention, a polysaccharide comprises at least 4 repeating units, preferably up to 3,000. Especially for use as a vaccine ingredient, a polysaccharide is preferably composed of from 4 to 1,000 repeating units, more preferably from 7 to 700 repeating units, more preferably from 50 to 200 repeating units. A repeated unit is characteristic of a given polysaccharide and in this way the composition and molecular weight of the repeating unit varies greatly from one polysaccharide to another. For example, while the repeating unit of most capsular polysaccharides comprises hydroxyl and carboxyl groups, some of them contain amino groups (for example, Streptococcus pneumoniae serotype 1), others do not contain it (for example Streptococcus pneumoniae serotype 14), some contain β-acetyl (for example Streptococcus pneumoniae serotype 14), others do not (for example Streptococcus pneumoniae serotype 6B). Also as an example, the molecular weight of the capsular polysaccharides of Streptococcus pneumoniae types 3 and 4 are respectively 360 and 847. Thus, there is no general correspondence between the number of repeat units and the molecular weight of the polysaccharide, which can be applied globally, regardless of the composition of the polysaccharide. However, a person can independently indicate that a polysaccharide to be used in the present invention has a preferred molecular weight in the average range of 10,000 to 500,000. The molecular weight of a polysaccharide is always expressed as a mean value, since a polysaccharide is constituted by a population of molecules of heterogeneous size. The polysaccharides can be either chemically synthesized or purified from a natural source, if it exists, according to conventional methods. For example, in the case of bacterial and fungal polysaccharides, the latter can be extracted from microorganisms and treated to eliminate the toxic portions, if necessary. A particularly useful method is described by Gotschlich et al, J. Exp. Med. (1969) 129: 1349. The polysaccharides can be used as synthesized or purified. They can also be depolymerized before use. In effect, native capsular polysaccharides usually have a molecular weight greater than 500,000. When it is preferred to use the capsular polysaccharides of lower molecular weight, for example, 10,000 to 20,000 on average, the polysaccharides as purified can be subjected to fragmentation. For this purpose, conventional methods are available; for example, WO 93/7178 describes a method of reductive oxidation fragmentation. The hydroxyl, carboxyl or amino groups of the polysaccharide which are involved in the linkage can be native functional groups. Alternatively, they may have been artificially introduced by specific treatment. Amino groups have been created from controlled acidic or basic hydrolysis of native N-acyl groups for example N-acetyl groups. Functional groups may also be introduced which include hydroxyl, carboxyl, amino and other groups (although amino groups are preferred), after derivatisation with a spacer portion linked to the native anino, hydroxyl or carboxyl groups, the latter two being preferred. For this end. Typically, the spacer is a bifunctional molecule that is capable of reacting at one end with the hydroxyl, carboxyl or amino groups native to the polysaccharide and at the other end with the linker. In this way the spacer is provided for a functional group including, but not limited to, hydroxyl, carboxyl and amino groups. Another useful functional group that can also be introduced by the spacer is a thiol group, as is further detailed hereinafter. Functional groups 5 other than those already mentioned after the specific treatment can also be introduced. For example, aldehyde groups may have been introduced throughout the polysaccharide chain by treatment of periodate that cleaves a carbon-carbon bond between two carbon atoms bearing hydroxyl groups neighbors. A periodate treatment is preferably achieved in a polysaccharide, the chain of which is not susceptible to be cleaved by such treatment, for example, of S. Mu tans. When aldehyde groups are introduced along the length of the chain for conjugation purposes, the linker is bound, exhibits an amino group. The compounds to be used as spacers or ^^ linkers are further defined herein below. It is however correct now to indicate that the linker is a bifunctional molecule that has a length Suitable in such a way that the portions of peptides and polysaccharides do not interfere with each other for example, when presented to the immune system. The linker portion is advantageously linked to the cysteine residue of the peptide via a disulfide bridge or a thio ether bond and to groups The hydroxyl of the polysaccharide via an ether or ester bond, to amino groups via an amide or carbamate bond, to carboxyl groups through an ester, amide or carbamate linkage, or to aldehyde groups via a reduced imine bond. The nature and intensity of the immune response that can be elicited by a conjugate for vaccine of the invention, can be greatly influenced by the proportion of peptide: polysaccharide. It is essential that B-epitopes present in the polysaccharide and T-dependent epitopes carried by the peptide are available and presented correctly to the immune system. In this way, both types of epitopes must be present in sufficient and balanced amount to avoid for example spherical impedance of the epitopes of the polysaccharide by the peptide portions. In order to optimize the immune response, it is indicated that a ratio of 1 mole of peptide to 1 to 50 moles of repeating units is adequate. Preferably, this ratio is 1 mol of peptide per 3 to 30 mol of repeating units; more preferably, this ratio is 1 mole of peptide per 5 to 20 moles of repeating units. The conjugates of the invention are particularly useful in the field of vaccines to produce a long-term protective immune response against a pathogenic microorganism from which the immunogenic polysaccharide is derived.

Therefore, the invention also provides a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a conjugate of the invention, together with a pharmaceutically acceptable diluent or carrier. Such a composition can be prepared conventionally. This may also contain other ingredients such as an adjuvant, for example an aluminum compound. Suitable aluminum compounds include 'Aluminum hydroxide or aluminum phosphate. In a modality Preferred, it is not necessary to use an adjuvant to improve the immunogenicity of a conjugate of the invention. A composition according to the invention can be administered by any conventional route in use in the vaccine field. The choice of the administration route depends on the number of parameters such as the adjuvant used. A further aspect of the present invention is * relates a process to conjugate a peptide having at least six amino acid residues, at least one of which is a cysteine residue to a chain of polysaccharide, that is, an immunogenic polysaccharide chain, comprising at least four repeating units, which comprises coupling the peptide to a linker through a thiol group of the cysteine residue and coupling the polysaccharide to the linker through ( a) the amino groups, Hydroxyl, or carboxyl, native to the polysaccharide chain or (b) amino groups created after hydrolysis of the native N-acyl groups of the polysaccharide chain or (c) functional groups introduced into the polysaccharide chain after the derivatization with a spacer portion bond to the amino groups, hydroxyl, or carboxyl native to the polysaccharide chain It is preferred to first react the linker with the polysaccharide or a derivatized polysaccharide which is provided for an activated polysaccharide, i.e. a polysaccharide that carries the linker molecule that provides a functional group for the coupling to the peptide. In a second step, this activated polysaccharide is reacted with the peptide where the functional group of the linker, ie Rl t, reacts with the thiol group of a peptide cysteine residue. Stated another way, there is provided a process for conjugating a peptide containing a cysteine residue to a polysaccharide that is, an immunogenic polysaccharide, composed of at least four repeat units, the process comprising either: (i) Activating the polysaccharide with a bifunctional linker capable of reacting with a thiol group in such a way that the activated polysaccharide is obtained, wherein a plurality of linker portions are introduced randomly along the polysaccharide chain by covalent attachment, and (ii) ) Reacting the activated polysaccharide obtained in step (i) with the peptide in such a way that a conjugate is obtained, wherein the entidadeß of the The peptide is covalently linked to the linker portions through its cysteine residues; or (iii) Activating the peptide with a bifunctional linker capable of reacting with a thiol group, and (iv) Reacting the activated peptide obtained in (iii) with the polysaccharide in such a way that a conjugate is obtained, wherein they are introduced ithe randomly activated peptide entities to all along the polysaccharide chain by covalent attachment. A preferred process is according to steps (i) and (ü) An advantageous process according to the invention comprises reacting the polysaccharide with a bifunctional linker under conditions that allow the introduction of portions of the linker othe polysaccharide in a sufficient amount such that in step (ii) of the process, a conjugate is produced, which contains one mole of peptide per 1 to 50 moles of repeat units, preferably one mole of peptide per 3 to 30 moles of repeat units, more preferably one mole of peptide per 5 to 20 moles of repeat units . In a similar manner, the alternative process comprises reacting the polysaccharide with an activated peptide under conditions that allow the introduction of portions of peptide activated ithe polysaccharide in a sufficient amount such that in step (iv) of the process, a conjugate having the characteristics indicated hereinbefore. An advantageous linker is of the formula (I) R1-A-R2, wherein R1 is a functional group capable of reacting with a thiol group. A is an aromatic or preferably an aliphatic chain, for example, a carbon chain, substituted or not, and R2 is a functional group capable of reacting with a functional group of the polysaccharide. The A chain should not be too short (to avoid steric hindrance) or too long (to avoid interference with the immunogenic parts). In this way, chain A comprises from 1 to 12, preferably from 3 to 8 carbon atoms and is more preferably selected from C2-C8 alkylene, phenylene, C7-C2 aralkylene, C2-C8 alkyl, phenyl , C7-C12 aralkyl, alkanoyloxy and benzylcarbonyloxy of Cs, wherein alkyl, phenyl, alkylene and phenylene may or may not be substituted. R1 is preferably a thiol group; a carbonyl or imidyl, β-unsaturated group; an acylhalogen or an alkylhalide, wherein the halogen atoms are Br, Cl or I. In a more preferred embodiment, R 1 is a α, β-unsaturated carbonyl or imidyl group; especially a maleimidyl group, R2 is the functional group of the linker which is provided for linking to the polysaccharide. In this way, R2 is a group that can react with ie amino, carboxyl, hydroxyl or aldehyde groups. R 2 is preferably selected from amino, carbamoyl, amino carbamoyl, carboxyl, hydroxyl, succinimidyl, for example N-hydroxy succinimidyl and sulfosuccinimidyl for example N-hydroxy sulfosuccinimidyl and sulfosuccinimidyl for example N-hydroxy sulfosuccinimidyl. If the linker reacts with the hydroxyl, or carboxyl or aldehyde groups, R2 is preferably an amino group or a chemical moiety carrying an amino group, for example R2 is a hydrazide group ie NH2-NH-CO-. Compounds that are useful as a linker include succinimidyl-4- (N-maleiimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl-4- (4-maleimidephenyl) butyrate, N-succinimidyl-4-maleimido-butyrate, N-succinimidyl- 3-maleiimido-benzoate. As explained hereinabove, the polysaccharide can be derivatized with a spacer before conjugation. In this way, the polysaccharide can be reacted first with a spacer of the formula (II) R3-B-R4, wherein R3 is a functional group capable of reacting with amino, carboxyl or hydroxyl groups, B is an aromatic or aliphatic chain , and R4 is a functional group capable of reacting with the R2 group of the linker used in the additional conjugation step. Chain B can be a carbon chain, preferably carbonyl, Cl-C12 alkyl or alkylene or dicarbonyl. Preferably, R3 and R4 are independently an amino group or chemical moieties carrying an amino group, for example a hydrazide group, ie, NH2-NH-C0-. >; Compounds useful as a spacer in the present invention include cysteamine, cysteine, diamines, for example diaminohexane, adipic acid dihydrazide (ADH), urea, semicarbazide, and cystamine. When cysteine or cysteine is used as a spacer, the thiol groups are introduced into the polysaccharide. In this case, linkers useful to be used in combination include, for example, bismaleimidyl compounds such as bis maleimido hexane. In order to obtain a conjugate exhibiting an appropriate peptide: the proportion of polysaccharide as described hereinabove, it is within the knowledge of a person skilled in the art to test an adjustment in the reaction conditions for example, the concentration of the reagents involved in the derivation and / or activation stages. An additional step is offered as follows: When the amino groups of the polysaccharide react, the reaction is preferably achieved in the presence of a carbodiimide compound for example, (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC) in a pH from 4 to 7, with the proviso that the functional group of the linker that is involved in the reaction, is carboxyl. When the carboxyl groups of the polysaccharide react, the reaction is preferably achieved in the presence of a carbodiimide compound as above, with the proviso that the functional group of the linker or spacer that is involved in the reaction is an amino group. The molar ratio of repeating units of the polysaccharide: carbodiimide is advantageously in the range of 0.1 to 2, preferably in the range of 0.1 to 1, more preferably in the range of 0.2 to 0.6. as can be easily understood, by adjusting this ratio, the amount of the portions of the peptide can be controlled by repeated units. When the amino groups of the polysaccharide react with succinimidyl or sulfosuccinimidyl, the reaction is advantageously achieved at a pH of 6 to 9, preferably about 7.5. the succinimidyl groups react only with amino groups. When appropriate experimental conditions are used (e.g. excess of the linker), the reaction is almost immediate ie, within about 5 minutes. If the polysaccharide used in the reaction is a native polysaccharide that carries amino groups, the only possibility to control the amount of substitution by succinimidyl groups is to use adverse experimental conditions. (otherwise the reaction occurs immediately and all amino groups are substituted), such as an increased dilution of the reaction medium or a low amount of the linker. This allows the proportion of the peptide to be adjusted to repeat units in the appropriate range. If hydrolysis or derivatization is used to introduce amino groups into the polysaccharide, this proportion can be easily controlled by controlling the appearance of the amino groups in the polysaccharide. When the hydroxyl groups of the polysaccharide react, the reaction is preferably achieved in the presence of a cyanogen compound; at pH 8 to 12, if this compound is cyanogen bromide; or at pH from 6 to 10, preferably from 6 to 8, if the compound is l-cyano-4-dimethylaminopyridinium tetrafluoroborate. The molar ratio of repeating units of the polysaccharide: cyanogen compound is advantageously in the range of 0.1 to 3, preferably in the range of 0.1 to 2. When the aldehyde groups present along the polysaccharide chain react, it is carried out the reaction preferably in the presence of cyanoborohydride for example, NaCNBH3 at pH 6.5 to 8. By carrying out the method of the invention, a conjugate is obtained wherein the polysaccharide and the peptide portions are bound via a linker moiety or a combination of both the spacer and linker, wherein the linker or the linker / spacer portion has an optimal length and wherein the polysaccharide-peptide bond is stable. On the other hand, a conjugate is obtained, wherein the proportion of the peptide to repeated units of the polysaccharide is optimal for immunization processes. The invention is further illustrated as follows: EXAMPLE 1: Preparation of a synthetic peptide 105 that has the following sequences Cis Leu Tir Tir Lis Asn Tir Arg Tir Tir Ala Leu Lis Ser Gli Gli Ser Val Asn Ala Pro Met Pro Glu Asn Gli Gln Tre Glu Asn Asn Asp Trp lie Leu Met Gli Ser Tre Gln Glu Glu Ala Lis Lis Asn Ala Met Asn His Lis Asn Asn Gln Arg lie Ser Gli Fen Ser Gli Fen Fen Gli Glu Glu Asn Gli Lis Gli His Asn Gli Ala Leu Asn Leu Asn Fen Asn Gli Lis Be Ala Gln Asn Arg Fen Leu Leu Tre Gli Gli Tre Asn Leu Asn Gli Lis lie Ser Val Tre Gln Gli The peptide is synthesized using a fast Moc chemistry with an automatic peptide synthesizer (moedlo 431a, Applied Biosystems ). The solid phase is a Rink resin (0.13 mM TentaGel S RAM Spezial, 0.15 mM g "1, Rapp Plymore, Tübingen, Germany) which produces a peptide crowned in the C terminal. The synthesis uses Fmoc (9-fluorenylmethyloxycarbonyl) protected amino acids with Ot-butyl- (for the carboxyl or hydroxyl group of aspartic acid, glutamic acid, serine, threonine and tyrosine); trityl- (for the amino or imino group of histidine, asparagine and glutamine); t-butyloxycarbonyl (for the amino group of lysine); or PMC} (pentamethylchroman-6-sulfonyl) ((for the imino group of arginine) of lateral protection Activation and coupling is achieved in the presence of 2- (lH-benzotriazol-1-yl) hexafluorophosphate -3, 3, 3 -tetramethyluronium (HBTU) / diisopropylethylamine Double coupling is performed in cycles 1-2, 4, 10-13, 17, 27, 32, 49, 59, 66, 75-78, 84-85, 88, 96- 97 and 104-105 and free amino groups are blocked by acetylation with acetic anhydride After the last cycle, the peptide is deprotected with piperidine and the final product is acetylated at the N-terminal using acetic anhydride. the side chain and the cleavage of the resin support with 2.1% (v / v) of 1,2-ethanedithiol, 4.2% of (v / v) thionol, 4.2% (v / v) of water, 6.2% of phenol ( v / v) and 83% (v / v) of trifluoroacetic acid (ATF) for 3 hours at room temperature.The resin is removed by filtration and triethylsilane is added dropwise until it is the colorless solution. then the solution 3 hours at room temperature, 360 mg of peptide without 5 purifying is recovered after precipitation with t-butylmethylether followed by centrifugation and lyophilization. 130 mg of the unpurified peptide is dissolved in 40 ml of 50 mM ethylmorpholine, pH 8.3 containing 50 mM dithiothreitol and incubated overnight at room temperature. The pH is adjusted to 3.5 with 10% ATF and the peptide was purified by reverse phase HPLC (Pep-S, C2 / C18, pore size 100 A, 12 μm 22.5 mm × 25 cm, Pharmacia) using a gradient (25 to 45% (v / v) acetonitrile, 0.1% ATF (10 ml min "1, gradient of 0.33% min." 1. The peptide elutes as a peak at approximately 25% acetonitrile, and freeze-dried the peak (73 mg) before further use. An analysis by HPLC and mass spectrometry shows that more than 65% of the final product corresponds to the desired sequence. The N-terminal sequence is confirmed by N-terminal Edman sequencing of the sample removed before N-terminal acetylation. EXAMPLE 2: polysaccharide-peptide conjugate of serogroup C of N. Meningl tldls A dry powder of capsular polysaccharide of serogroup C is obtained for Neisseria meningi tidis subsequently in the present referred to as polysaccharide C, by a process of extraction as described by Gotschlich et al, J. Exp. Med. (1969) 129: 1349. One hundred mg of polysaccharide C is dissolved in 0.2 M NaCl for a final concentration of 11.1 mg / ml (solution A). In parallel, a solution of 0.2 M adipic acid dihydrazide (ADH) in 0.2 M NaCl (solution B) is prepared. A 0.5 M solution of ethyldimethylaminopropylcarbodiimine (EDAC) in 0.2 M NaCl (solution C) is also prepared. Nine ml of solution A, 10 ml of solution B and 1 ml of solution C are mixed together to give a preparation containing 5 mg / ml of polysaccharide C, ADH 0.125 M and EDAC 0.25 M. 0.1 M HCl is added to adjust the pH to 6.5; this pH is maintained during the total reaction period of 45 minutes. The temperature is approximately 20 ° C. The reaction is stopped with 40 μl of 0.1 N NaOH which raises the pH to 7.1. The reaction mixture is dialyzed against NaCl 0.5 M, 10 mM phosphate and then water and lyophilized subsequently. The size of the derived polysaccharide C is controlled in an HPLC exclusion column TSK 4000 (manufacturer Tosohaas). The results show that depolymerization does not occur in the course of the derivation. During derivation, approximately 3.4% of repeating units are derived with an NH2 group. The lyophilized product is dissolved in 0.02 M phosphate buffer, pH 7, at a concentration of 6.25 mg / ml and removes the gas. N- (? -maleimidobutyryloxy) succinimide ester (GBMS) is dissolved in dimethylsulfoxide (DMSO) under nitrogen at a concentration of 25 mg / ml and then added to the polysaccharide C derivative in the same amount. The reaction mixture is stirred for 90 minutes at room temperature under nitrogen. The activated polysaccharide C is purified by Sephadex G50 exclusion column chromatography. The excluded fraction is recovered and concentrated to approximately 7.5 mg / ml by ultrafiltration (Amicon 30K membrane). The gas is removed from the concentrated solution. x Twenty mg of the peptide is dissolved in Example 1 in water at a concentration of 10 mg / ml under nitrogen. 1.5 ml of the peptide solution is added to 1.2 ml of the preparation containing the activated polysaccharide C, such that the proportion (maleiimido residues) / (thiol residues) is 2. The reaction mixtures are maintained during the reaction. night under stirring at room temperature. The unreacted maleiimido residues are then inactivated by adding 0.010 ml of mercaptoethanol. The conjugate product is purified in a column of Sepharose 4BCL. The fractions eluted for the presence of saccharides (sialic acid) and peptides are tested. The fractions that respond positively in both trials are grouped. The amount of sialic acid residues is determined according to the dose method described in Svennerholm L., Biochem. Biophys. Acta (1957) 24: 604, and the amount of the peptide is determined according to the method of Lowry et al, J. Biol. Chem (1951) 193: 265. It is shown that the mole / mole ratio of (peptide) / (repeating units of polysaccharide C) is 1:18 5 (corresponding to a weight / weight ratio of 1.8: 1). EXAMPLE 3: Polysaccharide-peptide conjugate of S. pneumoniae The dry powder of capsular polysaccharide of # Streptococcus pneumoniae type 4, later in this as polysaccharide Pneumo 4, by an extraction process as described in the patent application WO 82/01995"It proceeds from purification of polyosides of Streptococcus pneumoniae et vaccins based on polyosides ainsi purifiés". One hundred mg of polysaccharide Pneumo 4 is dissolved in 0.2 M NaCl at a concentration final of 11.1 mg / ml (solution A). In parallel, a solution of adipic acid dihydrazide (ADH) in 0.2 M NaCl in a concentration of 0.25 M (solution B) is prepared. A solution of ethyldimethylaminopropylcarbodiimide (EDAC) in 0.2 M NaCl at a concentration of 0.5 M (solution C) is also prepared. HE mixed with each other nine ml of a solution A, 10 ml of solution B and 1 ml of solution C to give a preparation containing 5 mg / ml of polysaccharide Pneumo 4, ADH 0.125 M and EDAC 0.025 M. It is added 1 N HCl for pH 4.9; this pH is maintained during the total reaction period of 30 minutes. The temperature is approximately 25 ° C.

The reaction is stopped by 0.28 ml 1 N NaOH. The pH is increased to 7.5. The reaction mixture is dialyzed against 0.5 M NaCl and then water and lyophilized subsequently. The size of the polysaccharide Pneumo 4 derived in a HPLC exclusion column TSK 4000 (manufacturer Tosohaas) is controlled. De-polymerization does not occur in the course of derivation. During the derivation, approximately 8.2% of repeating units of the polymer Pneumo 4 are derived with an NH2 group. The lyophilized product is dissolved in 0.05 M NaCl, at a concentration of 2.76 mg / ml and the gas is removed. N- (? -maleimidobutyryloxy) succinimide ester (GBMS) is dissolved in dimethyl sulfoxide (DMSO) under nitrogen at a concentration of 25 mg / ml. 1.75 ml of the GMBS solution is added to 16 ml of the polysaccharide solution under nitrogen. The reaction mixture is left under stirring for 5 hours at room temperature under nitrogen. The activated Pneumo 4 polysaccharide is purified on Sephadex G50 exclusion column. The excluded fraction is recovered and concentrated to approximately 7 mg / ml in a 30K membrane (Amicon). The gas is removed from the concentrated solution. Twenty mg of the peptide obtained in the Example 1 in 0.1 M NaCl, 0.01 M phosphate buffer pH 7.5, at a concentration of 4.6 mg / ml under nitrogen. On the other hand, 2.2 ml of the peptide solution is added to 1.25 ml of the preparation containing the activated polysaccharide Pneumo 4, such that the proportion (maleiimido residues) / (thiol residues) equals 1 (conjugate of Pneumo 4-peptide-1). The reaction mixtures are kept for 6 hours under stirring at room temperature under nitrogen, then overnight at + 4 ° C. The unreacted maleiimide residues are then inactivated by adding 0.005 ml of mercaptoethanol to each reaction mixture. Are the conjugates purified in one? column of Sepharose 4BCL. The fractions eluted for the presence of sugars and peptides are tested. The fractions that respond positively in both trials are grouped. The amount of sugar is determined according to the dose method described in Dubois et al, Anal. Chem. (1956) 3: 350, and the amount of the peptide is determined according to the method of Lowry et al, J. Biol. Chem (1951) 193: 265. The mol / mol ratio (repeated units of (peptide / polysaccharide) is 1:30 for the conjugate Pn 4-peptide-l (corresponding to a weight / weight ratio of 0.4: 1). EXAMPLE 4: Polysaccharide-serogroup A peptide conjugate from N. meningi tidis The dry powder of capsular polysaccharide of serogroup A Neisseria meningi tidis, referred to as polysaccharide A in the following, is obtained by an extraction process as described in Gottschlich et al. al, J. Exp. Med. (1969) 129: 1349. One hundred mg of polysaccharide A are dissolved in water to a final concentration of 5 mg / ml (solution A). In parallel, a solution of cyanogen bromide (CNBr) in water is prepared in a concentration of 67 mg / ml (solution B). A solution of adipic acid dihydrazine (ADH) in 0.5 M NaHCO 3 at a concentration of 150 mg / ml (solution C) is also prepared. Twenty ml of a solution A and 0.75 ml of solution C are mixed together to give a preparation with a weight / weight ratio of polysaccharide / CNBr of. 0.1 N NaOH is added for pH'of 10.8; This pH is maintained during the total reaction period of 60 minutes. The temperature is approximately 20 ° C. The pH is then reduced to 8.5 by adding 0.15 ml of 0.1 N HCl. Seventeen ml of solution C is added in such a way that the ADH / polysaccharide weight / weight ratio is 3.5. The pH is maintained for 15 minutes. The reaction mixture is then left overnight under stirring at + 4 ° C. 1N HCl is added to lower the pH to 7. The reaction mixture is dialyzed against 0.5 M NaCl and then water and lyophilized subsequently. The size of the derivatized polysaccharide A is controlled on a TSK 4000 HPLC exclusion column (manufacturer Tosohaas). De-polymerization does not occur in the course of derivation. During the derivation, approximately 2.5% of repeating units of polymer A with an NH2 group are derived.

The process of Example 2 is then used to activate the derivatized polysaccharide A and conjugate the activated polysaccharide A to the peptide as obtained in Example 1. EXAMPLE 5: Immunogenicity studies with the serogroup C conjugate of N. meningi tidis as obtained in Example 2 The utility of the peptide of Example 1 as a carrier in a polysaccharide conjugate is shown as follows. NMRI mice of six weeks of age receive via the subcutaneous route one of the following compositions in a volume of 0.5 ml (each injection) and via the intraperitoneal route, adjuvant is used in the example: a) 5 μg of polysaccharide C (without peptide ) on days 1, 15 and 29, in the absence of the adjuvant; b) 5 μg of polysaccharide C (without peptide) together with Freund's complete adjuvant on day 1 and on days 15 and 29 together with incomplete Freund's adjuvant; c) 5 μg of polysaccharide C and 9 μg of the peptide together with Freund's complete adjuvant on day 1 and on days 15 and 29 together with incomplete Freund's adjuvant; d) the conjugate obtained in Example 2 containing 1 μg of polysaccharide C and 1.8 μg of the peptide on days 1, 15 and 29 in the absence of the adjuvant; e) the conjugate obtained in Example 2 containing 5 μg of polysaccharide C and 9 μg of the peptide on days 1, 15 and 29 in the absence of the adjuvant; f) the conjugate obtained in Example 2 containing 5 μg of polysaccharide C and 9 μg of the peptide together with incomplete Freund's adjuvant on day 1, and on days 15 and 29 the conjugate obtained in Example 2 with the adjuvant incomplete Freund; and g) a conjugate of 5 μg of polysaccharide C together with diphtheria anatoxin (DT). On days 15, 29 and 43 (calculated from the day of the first immunization), a blood sample is collected and anti-polysaccharide C antibodies are titrated by ELISA. The results are summarized in the following Table. Table 1 The antibody response to the unconjugated polysaccharide C is extremely weak at each step and does not increase over time, whereas the response to polysaccharide C conjugated to either DT or the peptide is satisfactory. With the conjugate of the present invention a reinforcing effect is obtained after the second injection, being an indication for persistent immune response. The # response of the polysaccharide C-peptide conjugate is equivalent to the response obtained with the polysaccharide C-DT conjugate. EXAMPLE 6: Immunogenicity studies with the S. Pneumonia conjugate as obtained in Example 3 The conjugate prepared in Example 3 is tested with a ratio (w / w) of peptide to polysaccharide of 0.4: 1 (corresponding to a ratio of peptide per repeated units of 1:30 (mol / mol)) in mice using the protocol of Example 5. This is immunogenic in mice in the presence of adjuvant and results in a reinforcement effect after the second injection. The results are subsequently observed in the present in Table 2. Table 2

Claims (19)

1. CLAIMS 1. A polysaccharide-peptide conjugate characterized in that the polysaccharide is immunogenic, which comprises: (i) A peptide portion having at least six amino acid residues, at least one of which is a cysteine residue; (ii) A polysaccharide chain comprising at least four repeating units; and (iii) A linker moiety linked to a tial group of the cysteine residue and linked to (a) the amino, hydroxyl or carboxyl groups native to the polysaccharide chain or (b) amino groups created after the hydrolysis of the groups N-acyl native to the polysaccharide chain or (c) functional groups introduced into the polysaccharide chain after derivatization with a spacer moiety linked to the amino, hydroxyl or carboxyl groups native to the polysaccharide chain.
2. 2. The conjugate in accordance with the claim 1, characterized in that the peptide contains from six to two hundred amino acid residues, including the cysteine residue.
3. 3. The conjugate according to claim 2, characterized in that the peptide contains from ten to one hundred and fifty amino acid residues, including the cysteine residue.
4. 4. The conjugate in accordance with the claim 3, characterized in that the peptide contains from fifteen to one hundred amino acid residues, including the cysteine residue.
5. 5. The conjugate in accordance with the claim 4, characterized in that the peptide contains from twenty to fifty amino acid residues, including the cysteine residue.
6. 6. The conjugate according to any of claims 1 to 5, characterized in that the cysteine residue is located at the C terminal end of the peptide portion.
7. The conjugate according to any of claims 1 to 6, characterized in that the peptide contains a T-dependent epitope.
8. The conjugate according to any of claims 1 7, characterized in that the polysaccharide is a native polysaccharide selected from the specific O chain of bacterial lipopolysaccharides, detoxified bacterial lipopolysaccharides and capsular polysaccharides.
9. 9. The conjugate according to any of claims 1 to 7, characterized in that the polysaccharide is derived from a native polysaccharide comprising N-acetyl groups by controlled basic or acid hydrolysis.
10. 10. The conjugate according to claim 9, characterized in that the polysaccharide is derived from a native selected from a capsular polysaccharide.
11. 11. The conjugate according to any of claims 1 to 10, characterized in that the polysaccharide is composed of 4 to 3000 repeated units.
12. 12. The conjugate according to claim 11, characterized in that the polysaccharide is composed of 4 to 1000 repeating units.
13. 13. The conjugate according to claim 1, characterized in that the polysaccharide is composed of 7 to 700 repeated units.
14. The conjugate according to any of claims 1 to 13, characterized in that it contains one mole of peptide per fifty moles of repeating units (1:50) to one mole of peptide per one mole of repeating units (1: 1)
15. 15. The conjugate according to claim 14, characterized in that it contains one mole of peptide per thirty moles of repeating units (1:30) to one mole of peptide per three moles of repeating units (1: 3).
16. 16. The conjugate according to claim 5, characterized in that it contains one mole of peptide per twenty moles of repeating units (1:20) to one mole of peptide per five moles of repeating units (1: 5). F
17. 17. A pharmaceutical composition characterized in that it comprises a conjugate according to any of claims 1 to 16, together with a pharmaceutically acceptable diluent or carrier.
18. 18. The composition according to claim 17, characterized in that it does not comprise adjuvant.
19. 19. The process for conjugating a peptide having flf by at least six amino acid residues, at least one of which is a cysteine residue to a chain of 10 polysaccharide ie, an immunogenic chain, comprising at least four repeating units, which comprise coupling the peptide to a linker through the thiol group of the cysteine residue and coupling the polysaccharide to a linker through (a) the native amino, hydroxyl or carboxyl groups 15 of the polysaccharide chain or (b) amino groups created fff after hydrolysis of the native N-acyl groups of the polysaccharide chain or (c) functional groups introduced into the polysaccharide chain after derivatization with a portion of spacer linked to the group the amino groups, 20 hydroxyl or carboxyl of the polysaccharide chain.