WO1986007365A1

WO1986007365A1 - Products resulting from the conjugation of a macromolecular support, a hapten and a muramyl-peptide

Info

Publication number: WO1986007365A1
Application number: PCT/FR1986/000201
Authority: WO
Inventors: Michel Jolivet; Françoise Audibert; Louis Chedid; Pierre Lefrancier
Original assignee: Agence Nationale De La Valorisation De La Recherch
Priority date: 1985-06-10
Filing date: 1986-06-10
Publication date: 1986-12-18
Also published as: JPS63500515A; FR2583049B1; FR2583049A1; EP0224540A1

Abstract

Product resulting from the conjugation of a hapten and a muramyl-peptide to a macromolecular support, said product having selective immunogenic properties with respect to antigenes having in common an antigenic site or an epitope in common with the haptenic fraction which is part of the formation of said product. In the product according to the invention, the muramyl-peptide is bound to the macromolecular support through a chemical group and via its glucopyranosyle core, preferably in its position 6.

Description

Products resulting from the conjugation of a macromolecular support, a hapten and a muramyl-peptide via a group substituting the saccharide part of the muramyl-peptide and selectively immunogenic compositions containing these products.

The invention relates to products resulting from the conjugation of a macromolecular support, a hapten and a muramyl-peptide via a group substituting the saccharide part of the muramyl-peptide, these products having selective immunogenic properties of the haptenic part which enter into their constitution. It relates more particularly still to immunogenic compositions containing such products in association with an acceptable pharmaceutical vehicle, compositions which can be used for vaccination against antigens having an antigenic or epitope site in common with hapten. We know the interest in synthetic peptides, generally in low molecular weight haptens, to constitute the active principle of vaccines capable of protecting the host against hapten or more often against a pathogenic antigen. These haptens do not exhibit any sensitive immunogenic power when they are presented to the host organism. Various methods have already been proposed either to confer or reinforce the immunogenic properties of such haptens, or else to reinforce the immunogenicity of weak antigens, whether they are inherently weak antigens, or highly purified antigens of which a part of the immunogenic power has been lost, in particular during the operations necessary for their purification or of subunits derived from these antigens, these subunits however still carrying sites antigenic characteristics of these antigens.

It is understood that in the following, an extensive meaning will be given to the word "hapten" for the convenience of language. In the following, the word will designate not only the haptens themselves, having for example a molecular weight at most equal to 5000, but also the weak antigens of which the question above is concerned. One solution which has been envisaged and which leads to high immunogenicity consists in the fixing (conjugation or covalent coupling) of said haptens on macromolecular supports of high molecular weights. The conjugation or coupling products thus obtained often often exhibit increased immunogenicity capable of inducing in vivo an effective production of antibodies against the hapten present in the conjugation product or even against higher molecular weight antigens having a site. antigenic or an epitope in common with this hapten.

Although numerous macromolecular supports have already been proposed which are capable of being tolerated by the body, the conjugates thus produced nevertheless have a significant drawback. They not only induce an immune reaction in vivo against hapten, but also against the macromolecular support itself. This drawback may even be of an extremely serious nature, when the host has already been vaccinated against the macromolecular support. This case can arise, in particular when the macromolecular support is tetanus toxoid of which is known the significant amplifying effect with respect to the immunogenic reaction of haptens possibly grafted on this toxoid. It has also been demonstrated by tests carried out on animals, that immunization against a hapten coupled to a macromolecular support against which the animal had previously been immunized, could even lead to the suppression of the body's immune response capacity against hapten, instead of increasing it. It cannot therefore be envisaged to carry out vaccinations involving a macromolecular support against which the recipient is may already be protected. This would in particular be the case with conjugation products between a specific hapten and tetanus toxoid, since the latter is used for the vaccination of a large part of the population against tetanus. This is one of the reasons why the use for vaccination of active principles resulting from the conjugation between a hapten carrying an epitope specific for a pathogenic agent against which protection is sought, on the one hand, and a Macromolecular support of high molecular weight, on the other hand, has not been seriously considered in practice to date. This is also one of the reasons which prompted researchers to find other technical solutions to reveal or increase the immunogenic activity of such haptens, in proportions such that their use as a vaccinating principle could be envisaged. Even if positive results have been obtained, it must be recognized that the desired amplification capacity of the immunogenic properties in question is not always achieved. Even in the most favorable cases, this amplification does not reach the importance that we have already observed in formed conjugates, between a hapten and a macromolecular support of high molecular weight, such as tetanus toxoid or l diphtheria toxoid.

The object of the invention is to remedy at least in large part the difficulties which may be encountered in the use of conjugation products involving a hapten and a macromolecular support of the above-mentioned type. More particularly, the invention aims to make these conjugation products such that, while having an immunogenic effect characteristic of hapten, they are at the same time substantially devoid of this effect vis-à-vis the macromolecular support itself. In other words, the invention is intended to provide conjugation products of this type, in which this immunogenic macromolecular anti-support activity is practically suppressed.

The object of the invention can be achieved by using carrier molecules resulting from the conjugation of the macromolecular support chosen with one or more muramyl-peptide groups, via a chemical group playing the role of an arm, substituting the glucopyranosyl nucleus of the muramyl-peptide, preferably at position 6 of this nucleus.

The products of the invention therefore consist of conjugation products between the carrier molecules thus formed and the hapten - or haptens - themselves carrying one or more epitopes contained in a molecule, in particular an antigen against which protection can be wanted.

The product of the invention can therefore be characterized as containing three principles coupled together via covalent bonds, these three principles consisting respectively of a macromolecular support, a muramyl-peptide linked to this support by a chemical group via its nucleus glucopyranosyl and at least one hapten carrying at least one epitope responsible for the desired immunogenic activity. Preferably, the arm is chosen so as to confer, if necessary, amphiphilic properties on the arm-muramyl-peptide assembly.

Experience has shown that this tripleconjugation product benefits from a double amplification of the immunogenic activity of hapten, in other words amplifications respectively induced by the muramyl-peptide, the immuno-adjuvant activities of which are known, and by the carrier molecule, this triple conjugation product however being substantially, if not totally, devoid of immunogenic activity with respect to the macromolecular support or of the carrier molecule. This last effect (suppression or masking of the immunogenic properties specific to the carrier molecule or to the macromolecular support) is particularly remarkable, if one remembers the conjugation products described to date and formed between muramyl-peptides and macromolecular supports of high molecular weights, had immunogenic effects not only against the hapten possibly also present in the conjugation product, but also against the macromolecular support.

Muramyl peptides preferred for the desired modification of the macromolecular support are represented by the following general formula:

in which :

R ₁ is an OH group or an OC ₆ H ₄ -NH ₂ or O- (CL ₂ ) _m -R _{a group} , m being an integer from 1 to 10, and R _a being an amino, carboxyl or thiol function, hydroxyl or hydrogen,

- A is oxygen or an NH group;

R ₆ is a BR or CO-BR group in which B is a group or a linear or branched chain which can contain up to about 100 carbon atoms and R is a hydroxyl, carboxyl, amine, thiol or hydrogen, group B being optionally itself carrying one or more functional groups of the type of those designated above for R, or alternatively carbonyl, alkoxyl or acyl; - R ₂ is hydrogen or an alkyl group comprising from 1 to 4 carbon atoms, in particular methyl; - X is an alanyl, arginyl, lysyl, asparagyle, aspartyle, cystéinyle, glutaminyle, glutamyle, glycyle, histidyle, hydroxyprolyle, isoleucyle, leucyle, méthionyle, phenylalanyl, prolyl, seryle, threonyle, tryptophanyle or valyle residue;

Y is an OH, NH _{2 group} or, preferably, OR ₇ or NHR ₇ , R ₇ being a hydrocarbon group which may have from 1 to 20 carbon atoms or which may be an amino acid as indicated for X, such as alanyl, seryl, valyl or glycyl, said amino acid being free, amidated or esterified, the esterification group possibly comprising up to 4 carbon atoms;

Z is a carboxyl, carboxamide, ester group containing from 1 to 10 carbon atoms, or an alkyl group comprising from 1 to 4 carbon atoms, preferably -CH ₂ -CH ₃ or - (CH ₂ ) ₂ -CH ₃ ; it being understood that at least one of the groups R ₁ and R ₆ (and preferably R ₆ ) has a reactive function allowing its conjugation with a reactive function mentary carried by another molecule, in particular the macromolecular support.

Preferably, in the triple conjugate according to the invention, the muramyl-peptide on the one hand, the hapten on the other hand, are coupled to the macromolecular support via distinct functional groups initially present on or carried by the support macromolecular.

The "triple conjugates" thus obtained - preferred over the other couplings likely to be imagined - could then be represented by the global formula:]

[] _x [] _y

in which x and y represent the respective numbers of hapten groups and muramyl-peptide groups attached to the same macromolecular support, it being understood that the sum x + y cannot be higher than the total number of functional groups initially carried by the high molecular weight macromolecular support, examples of which will be given later. The sum x + y is for example between 2 and 100. This being so, it will be noted that the other types of conjugation which may be envisaged are not excluded from the protection. For example, it may be envisaged, if the muramyl-peptide group fixed on the macromolecular support still carries an appropriate free functional group, to also use it in a conjugation operation with the hapten. However, the preferred conjugates of the invention are those in which the muramyl peptide and the hapten are grafted separately from each other on the macromolecular support. A preferred group of muramyl-peptides used in the "triple conjugates" of the invention are characterized by the following formula:

:

vs

R ₁ is -OH or -OC ₆ H ₄ -NH ₂ or O- (CH ₂ ) _m -R with m from 1 to

10 and R _a being an amine, carboxyl, thiol, hydroxyl or hydrogen function; - R ₂ is -H or -CH ₃ ;

- R ₆ is -CO (CH ₂ ) _n -R with n from 1 to 10 and R being an amine, carboxyl, thiol, hydroxyl or hydrogen function;

- X is Ala, Gly, Ser or Val; Y is O (CH ₂ ) _p -H with p from 1 to 10;

- Z is CO-NH ₂ , COO (CH ₂ ) _r -H or (CH ₂ ) _r -CH ₃ with r from 1 to 4.

Preferably, the amino acid residues forming part of the group X are levogyres (except in the case where X is constituted by a glycyl residue).

The muramyl-peptides corresponding to derivatives of the 2- (2-acetamido-2-deoxy-3-OD-glucopyranosyl) - alkanoyl-peptides type are sufficiently known that it is unnecessary to insist on their methods of preparation. However, as we will have noticed on reading of the meanings of the groups belonging to the preferred muramyl-peptides which have been given above, the expression muramyl-peptides should be understood to also extend to muramyl-peptides of the 2- (2-acetamido-2-deoxy-) type 3-OD-glucopyranosyl) -alkanoyl-aminoacylnor Leucine or to derivatives of the latter muramyl-peptides.

Compounds of this type, which can be produced by processes entirely analogous to those which can be used in the preparation of known derivatives of the 2- (2-acetamido-2-deoxy-3-OD-gluco-) type. pyranosyl) -alkanoyl-peptides, have been more particularly described in French patent application 85 06596 filed on April 30, 1985. The content of this patent application must be considered as also forming an integral part of the present description, with regard to possible details relating to preferred methods of preparation of these compounds.

Preferred muramyl peptides belonging to the latter type of preferred compounds are those in which Y is an OR ₇ or NHR _{7 group} , R ₇ having the above-mentioned meaning, and in which the O CH group

is a derivative of a dextrorotatory nor Leucine group, more particularly of a D-nor Leucine group, in which

Z = -CH ₂ -CH ₃ .

Regardless of the type of muramyl peptide used, whether those in which group 2

is derived from glutamic acid or those in which this same group is derived from nor Leucine or its counterparts, it will also be observed that other categories of compounds which are particularly advantageous for the production of conjugations are those in which A is oxygen and R _{6 is} a group —CO- (CH ₂ ) _n -COOH such as the succinyl group. -CO- (CH ₂ ) ₂ -COOH, or the group -CO- (CH ₂ ) _n -NH ₂ , such as the epsilon-amino-caproyl group -CO- (CH ₂ ) ₆ -NH ₂ , n being a number from 1 to 10.

In other preferred compounds of the invention, the groups R ₆ contain, alone or in combination with the groups which have just been mentioned, linear or branched chains, the links of which consist of hydrocarbon chains, in which case where appropriate, some of the carbon atoms are replaced, from place to place, by nitrogen and / or oxygen atoms, with in particular the creation of ester, ether, amido, imido, imido, etc. functions.

Among the elements advantageously contained in the B chains, mention will be made of aminoacyl residues, in particular links containing from 1 to 3 successive aminoacyl residues, for example of the lysyl or alanyl type, (these being able moreover to be replaced by any other aminoacyl of the type indicated with respect to group X), or alternatively glyceryl elements: -O-CH ₂ -CH (O) -CH ₂ -O- or glycolyl: -O-CH ₂ -CH ₂ -O- or derived elements glycerol or glycol, glyceric or glycolic acids, for example those combining glyceryl or glycolyl elements, on the one hand, and aminocyl elements, on the other hand.

Other preferred groups B are formed by chains as defined above, themselves carrying ramifications, for example by acyl groups, in particular fatty acids, which respectively comprise in particular from 14 to 30 carbon atoms, by example palmitoyle groups. Thus, preferred groups B contain in particular of the terminal elements represented by the formula:

-OCH ₂ -CHO (R ₈ ) -CH ₂ O (R ₉ ) in which R ₈ and R ₉ are acyl groups comprising from 14 to 30, in particular from 16 to 24, carbon atoms.

As preferred muramyl-peptides capable of being used in the context of the present invention, mention will be made of those which may be designated by the following short formulas (the designation "MurNAc" relating to the N-acetylmuramic group):

- 6-O-epsilon-aminocaproyl-MurNAc-L-Ala-D-Gln-OnBu;

- 6-O-epsilon-aminocaproyl-MurNAc-L-Ala-D-Nle-OnBu;

- 6-O-succinyl-MurNAc-L-Ala-D-Gln-OnBu; - 6-O-succinyl-MurNAc-L-Ala-D-Nle-OnBu; or similar derivatives in which the n-butyl ester groups (OnBu) are replaced by methyl ester groups (OMe).

As the preferred macromolecular supports capable of being used in the "triple conjugates" of the invention, mention will be made of natural molecules of the human serum albumin type, toxoids or other carrier macromolecular supports having molecular weights of at least 50,000 , for example from 50,000 to 250,000 daltons. Synthetic peptide polymers can also be used, of the polylysine type or of the kind described in European patent 13 651.

In general, macromolecular supports are preferred, consisting of peptide chains whose sequence contains a large number of lateral functional groups allowing covalent coupling, with the particular muramyl-peptides used in the context of this invention and / or with haptens whose immunogenicity must be induced. In this In particular, tetanus and diphtheria toxins are particularly preferred. For example, the tetanus toxin, which has a molecular weight of the order of 160,000, carries around fifty free terminal NH ₂ groups making it possible to fix a sufficient number of hapten and muramyl-peptide groups. These toxins can, after fixing said muramyl peptides and / or haptens, then be easily detoxified, for example by a conventional treatment with formaldehyde. Conditions under which these conjugations can be carried out, then the detoxified toxins (if necessary) are illustrated below, in particular in relation to the attachment to the tetanus toxin of a peptide containing a surface element of the surface protein of the sporozoite of Plasmodium knowlesi and 6-O-epsilon-aminocaproyl-MurNAc-L-Ala-D-Gln-OnBu.

The invention finally applies to the production of "triple conjugates" involving any hapten comprising at least one "antigenic site" usable for the induction of antibodies in vivo, provided that it is conjugated to an amplifying molecule of its immunogenicity.

By "group comprising at least one antigenic site" is meant any group contained in an antigen in respect of which an in vivo production of antibodies is sought or any group equivalent on the immunogenic level. As haptens likely to be taken into account in the context of this patent application, mention will be made, for example, of those which meet the definition given by European patent application 89 290 filed by the National Agency Research Valuation (ANVAR). It is therefore not only haptens carrying an antigenic site which, when these haptens are coupled to a amplifying molecule, gives them the ability to induce protective antibodies against specific pathogens in vivo. The invention further extends to the manufacture of triple conjugates involving other haptens, such as those envisaged in European patent application 89 290, for example hormones.

Mention will be made, without limitation, among the haptens which can enter into the constitution of "triple conjugates" according to the invention, the luteotropin releasing factor, known under the designation LH-RH, peptide fragments, or synthetic peptides endowed with analogous properties, for example - decapeptides of the Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH _{2 type} or the corresponding free acid pGlu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-Gly; C-terminal peptides of human chorionic gonadotropin (hCG); - C-terminal peptides of the β subunit of hCG, in particular a triacontapeptide containing aminoacyl residues corresponding to those of hCG-β or of synthetic terminal derivatives of the genus described by S. Matsura et al. (publication already mentioned above), fragments of FSH, etc., or peptides consisting essentially of the sequences 109-145 and 111-145 of the C-terminal subunits of hCG-β described by Arthur CJ (Lee et al. in "Molecular Immunology", vol. 17, p. 749-756). The invention allows the coupling of such hCG-β fragments with tetanus toxoid, for the production of contraceptive vaccines for women.

The conjugation of the above hormones with the two other constituents as mentioned above, leads to immunogenic principles capable of very effectively induce in vivo the production of antibodies against the natural hormones in question.

Mention will also be made, by way of examples of haptens which can be used in the triple conjugates according to the invention, those which involve different synthetic peptides having aminoacyl residue sequences in common with the main peptides constituting the HBsAg antigen, in particular those whose end aminoacyles correspond to those which, in the sequence of HBsAg, occupy the following positions: 48-81; 2-16; 22-35; 95-100; 117-137; 122-137; 117-135 (according to the structures given by Pasek et al. And reproduced in the article by Lerner et al. (1981), Proc. Natl. Acad. Sci. USA, 28., N · 6, 3403-3407. will also mention as suitable haptens the vaccinating peptides carrying antigenic sites characteristic of diphtheria toxin, influenza viruses, herpes, polio, etc.

In addition, synthetic peptides of the type described by EH Beachey et al. which are capable, when coupled with a muramyl-peptide, of forming antibodies active against the protein M of the surface of Streptococcus pyogenes, this protein being designated later in Example II, under the abbreviation "M24". Reference may also be made to the peptide sequences defined in American patent 4,284,537, more particularly those defined under the abbreviations CB ₆ and CB ₇ .

Particularly preferred peptide haptens are one or more of those defined below:

The haptens involved were as follows: 1 ^. ) SODP: octodecapeptide containing a sequence of the "diphtheria loop" and of formula: H ₂ N-Ala-Ala-cys-Ala-Gly-Asn-Arg-Val-Arg-Arg-Ser-Val-Gly-Ser-Ser -Leu-Lys-Cys. 2 ^. ) SCB7: peptide containing an epitope characteristic of the streptococcus type M24 containing the sequence:

Asn-Phe-Ser-Thr-Ala-Asp-Ser-Ala-Lys-Ile-Lys-Thr-Leu-Glu- AlaGlu-Lys-Ala-Ala-Leu-Ala-Ala-Arg-Lys-Ala-Asp- Leu-Glu- Lys-Ala-Leu-Glu-Gly-Ala.

3 ^. ) HBs (peptide 99-121); peptide comprising an antigenic site of the envelope of the virus containing the sequence: Asp-tyr-Gln-Gly-Met-Leu-Pro-Val-Cys-Pro-Leu-Ile-Pro-Gly- 100 110

Ser-Ser-Thr-Thr-Ser-Thr-Gly-Pro-Cys.

120 4 ^. ) DDP Mal: peptide carrying an epitope characteristic of a protective antigen against Plasmodium knowlesi. agent of monkey malaria, described by Godson GN et al. (1983), Nature, 305 (29-33, constituted by the sequence:

Tyr-Gln-Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-Pro-Gln- Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-Pro- Cys.

It is also possible to use a peptide copying an element of the structure of the human parasite Plasmodium falciparum described by Enea et al. (Science, 1984, 225,

628) or by Dame et al. (Science, 1984, 225. 593. It is a tetrapeptide of the type (Asn-Ala-Asn-Pro) with n being from 2 to 8.

These examples are of course in no way limiting. In addition, the haptens that can be used cannot be limited to peptides. They are, for example, also saccharide or osidic haptens. Mention will be made, for example, of the blood groups A and B, the polysaccharide C of streptococcus C, the cellobiuronic acid (Pneumo type III), as described by WF Goebel, "J. Exp. Medicine", 1939, p. 353-363, or paraaminophenylcellobiuronic acid. Finally, the haptens capable of being used can consist of synthetic or semi-synthetic polypeptides containing several antigenic sites which are distinct from each other and capable of providing "polyvaccines". Such polyvaccines have been described in French patent application 84 13989 filed on September 12, 1984.

For the record, we recall some of the preferred techniques for achieving conjugations between the macromolecular support, on the one hand, and the muramylpeptide and hapten, on the other hand.

In accordance with a first technique, a coupling reaction can be used via glutaraldehyde, insofar as the macromolecular support, the hapten and the muramyl-peptide considered all contain amino functions. As regards the conjugation between the macromolecular support and the muramyl-peptide, one can have recourse to all forms of coupling involving determined positions on the glucopyranosyl cycle of the muramyl-peptide. In particular, these bonds will occur at the C ₁ position, preferably that at C ₆ of the glucopyranosyl ring of the muramyl peptide. As regards the substitutions involving the R ₆ group, recourse can be had to the coupling methods commonly used in peptide synthesis between the appropriate functions carried, on the one hand, by the muramyl-peptide and, on the other hand, by the macromolecular support, one carrying for example an amino function and the other a carboxyl, hydroxyl or sulfhydrile function or vice versa.

Of course, any other type of link can be used, bringing into play the combinations which may occur between a sulfhydride function carried by one of the partners that constitute the macromolecular support and the muramyl-peptide and a maleimide group carried by the other reagent, for example by making use of the technique described by T. Kitagawa and T. Aikagawa in "J. Biochem." 21 (1976), 233.

Conjugation can be done using conventional modes of diazotization or reaction involving an isothiocyanate, in particular when the muramylpeptide carries an amino aromatic function (in particular at the level of the R ₁ group) and when the macromolecular support carries an amino function capable of to intervene in this reaction. Such production techniques are described for example by BF Erlanger in "Pharmacol. Rev.", 25 (1973), 271. The coupling can also be carried out by the reduction of a Schiff base produced between an aldehyde function carried by the one of the reagents and an amino function carried by the other (GR Gray, "Arch. Biochem. Biophys. 163 (1974), 426). All these reactions are in themselves well known and the specialist will appreciate that many variants can be adapted to achieve the same results As regards more particularly covalent couplings which use the R ₁ group of the muramyl-peptide, it will also be noted that the conjugations can be carried out by means of bridging, the bridging agent consisting for example of a bifunctional reagent. Preferably, the bridging group between the muramyl-peptide and the macromolecular support in the final conjugate will not exceed the length of a corresponding sequence ndant to that of a p-decylic hydrocarbon chain.

Such bridging agents are for example mentioned in patent 78 16792 filed on June 5, 1978. In the foregoing, it was mainly mentioned the case of the coupling between the muramyl-peptide chosen and the macromolecular support.

It goes without saying that similar techniques can be used to couple the hapten to the macromolar support or to the carrier molecule.

Naturally, all of the above reactions assume that the active functions possibly carried by the partners and which must not participate in the coupling reaction are possibly protected. These are questions well known to specialists and there is no need to insist on them.

When the haptens and the muramyl-peptides are coupled separately to the macromolecular support, the relative proportions of hapten and muramylpeptides will be adjusted as a function of the number of free functional groups available on said macromolecular support. Experience and examples below show that it is not necessary to "saturate" the macromolecular support with muramyl-peptide groups of the category envisaged. It is indeed observed that the masking or the suppression of the immunogenic reaction specific to the macromolecular support itself is rapidly annihilated. On the other hand, it will generally be advantageous to fix on the macromolecular support - or on the chain - as many hapten groups as possible, in order to obtain a maximum immunogenic reaction specific to the hapten.

Advantageously, the number of muramylpeptide groups relative to the number of chains of the macromolecular support (numbers expressed in NH ₂ equivalents used in the reaction) will be in a 1: 1 ratio and the number of hapten groups relative to the number of macromolecular support chains will also be in a ratio at least equal, however preferably greater than 1, for example from 1 to 5.

In the foregoing, we have above all mentioned the case of separate fixations of hapten and muramylpeptide on the macromolecular support. One can naturally also envisage other combinations, for example those envisaged above, for example those where one fixes to the macromolecular support the muramylpeptide at the level of position 6 of its saccharide nucleus, then one fixes the hapten on the peptide part or, preferably, the position 1 of the saccharide nucleus of the muramyl-peptide. It is also possible to envisage first fixing the hapten on the muramyl-peptide, in particular at the level of position 1 of the saccharide cycle of the latter, before the fixation of the assembly on the raacromolecular support takes place, by l intermediate of the 6 position of the glucopyranosyl cycle of the muramyl-peptide previously modified. Other characteristics of the invention will become apparent during the description of the examples which follow. First, a particular example of production of a triple conjugate in accordance with the invention will be provided, involving 6-O-epsilon-aminocaproyl- MurNAc-L-Ala-D-Gln-OnBu (also referred to as abbreviation "MDP-6-O- (ε-aminocaproyl) -murabutide" in the following tables). The biological properties observed with the triple conjugate obtained will then be exposed. The properties observed with a "triple conjugate" obtained with a muramyl-peptide not falling within the category of muramyl-peptides used in the context of the invention will also be indicated by way of comparison, the N -acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-lysine (MDP-lysine). I - Process for obtaining the conjugate on tetanus toxin.

The following description applies to the manufacture of a conjugation product using particular proportions of the initial constituents indicated below. It goes without saying that the method is applicable in the same way to the production of triple conjugation products formed from different proportions of the initial constituents, for example the conjugates A to F mentioned below.

The coupling agent is glutaraldehyde used at the final concentration of 5.2 mM. Glutaraldehyde serves as both a coupling and a detoxifying agent when the carrier molecule is the non-detoxified toxin, which allows the availability of enough NH ₂ groups (fifty for a molecule having 160,000 molecular weight) to bind enough muramyl - adjuvant peptide and antigenic peptide (it goes without saying that "detoxification" is superfluous when the carrier molecule used is itself non-toxic).

The peptide is, in this example, a peptide copying a structural element of the surface protein of the sporozoite of Plasmodium knowlesi. Its structure is described in Godson et al. (Nature, 1983, 305. 29) and its immunogenicity was studied by gysin et al. (J. Exp. Mec, 1984, 160, 935).

The reaction is carried out in 0.1 m sodium carbonate-bicarbonate buffer, pH 8.5. Tetanus toxin (2.35 mg), sporozoite peptide (3.75 mg) and 6-O- (ε-amino-caproyl) -murabutide (2 mg) are dissolved and glutaraldehyde is added with stirring . The reaction lasts one week and the product is filtered on a molecular sieve, in particular that sold under the brand SEPHADEX-G25 so as to eliminate all molecules with molecular weights less than 20,000.

In the case of the peptide used, there is no possible reaction between the peptide coupled to the tetanus toxin and the muramyl-peptide, since the peptide only contains a reactive group at this pH. If the peptide and the adjuvant conjugate, the reaction product is removed by filtration; also, the only possible conjugate is the tryptic where the peptide (pep) and the adjuvant muramylpeptide (adj) are linked separately on the tetanus toxin (adj-support-pep). When the same process is implemented with peptides carrying several functional groups, the muramyl peptide alone can be reacted with the tetanus toxin for a certain time, before adding the peptide to the reaction medium, to promote the toxin binding tetanus adjuvant with respect to the peptide-adjuvant bond likely to come to conjugate in turn to the macromolecular support. The conjugate finally obtained is then subjected to the following tests: a) HPLC. The molecular weight of the conjugate sought corresponds to a monomer (between 150,000 and 180,000 daltons). The high molecular weight products, if they are formed, are eliminated by gel-filtration on a molecular sieve of crosslinked agarose, of the SEPHAROSE 6BCL type. b) Determination of the coupled glycopeptide. The assay is carried out by the method of Reissig (J. Biol. Chem., 1956, 217, 959). Among the reports tested, the most favorable was 2 μg of muramyl-peptide (expressed in CDM) per 100 μg of conjugate. Conjugates in which the dose of muramyl-peptide was 1 μg per 100 μg of conjugate made it possible to obtain similar results. c) Antigenicity of the tetanus toxin. This control is carried out by the ELISA method. The tetanus toxin and the conjugates are diluted so as to obtain 10 μg of tetanus antigen in 50 μl. Fifty microlitres of each dilution are deposited in the wells of the plate. After adequate treatment to obtain the fixation of the antigen on the plates, a fixed quantity of tetanus serum is calculated, calculated to have an optical density close to 1, after revelation by the anti-antibody system labeled with peroxidase. In the experiment implemented, the following figures were obtained:

D.O. obtained Tetanus toxin 10 μg 0.715 Conjugate containing 10 μg tetanus toxin 0.145 Background noise 0.120

This experiment shows that the conjugation of the muramyl-peptide and the peptide to the tetanus toxin modified the antigenicity of the latter. The experiments described below show that this phenomenon also modified its immunogenicity, without affecting its macromolecular support properties. d) Absence of toxicity. Two tests were performed. One of these tests has shown that the tetanus toxin has lost its toxic activity. Ten mice each received an amount of conjugate corresponding to 10 lethal doses of tetanus toxin: they were observed one week without showing any clinical sign of tetanus.

The second test consisted of a pyrogenic test; it showed that 1.5 μg of muramyl-peptide conjugate did not induce, in any of the three rabbits tested, a rise in temperature greater than or equal to 0.5ºC. Remember that 0.8 μg of a conjugate of MDP-Lys and toxin tetanus, obtained by coupling under the same conditions, caused temperature increases ranging from 1.6ºC to 2ºC. It will be noted that in this last test, the conjugation was, by necessity, carried out at the level of the peptide group of the muramyl-peptide!

II - Biological tests and comparative properties of triple conjugates in accordance with the invention and of triple conjugates using the same hapten and the same macromolecular support, on the one hand, and MDP-lvsine on the other hand.

By the method of production of covalent bonds involving glutaraldehyde under the conditions which have been indicated above, triple conjugates were produced using variable initial proportions of the peptide, of the tetanus toxin and of 6-O -ε-aminocaproyl-murabutide (conjugates D, E and F), or MDP-lysine (conjugates A, B and C). The amounts of muramyl-peptide will be expressed in MDP- (N-acetyl-muramyl) -L-alanyl-D-isoglutamine equivalents). In the following, reference will also be made to the NH ₂ equivalents, for the assessment of the ratios of the various constituents entering into the triple conjugates considered below.

Conjugates A and D were obtained from 2.35 mg of tetanus toxin, 3.75 mg of the malaric peptide and 2 mg of MDP; conjugates B and E were obtained from 2.35 mg of TT; 7.5 mg PEP and 1 mg MDP; finally, conjugates C and F were obtained from 11.7 mg of TT, 18.75 mg of PEP and 0.5 mg of MDP. Finally, in particular for the purposes of comparisons, a PEP-TT conjugate was obtained by proceeding as described above, by the conjugation of PEP and tetanus toxin. The toxin was used as a carrier because it contained more reactive groups than toxoid. The detoxification of the conjugates obtained was carried out by adding sufficient quantities of glutaraldehyde, then by incubation for 5 days. The conjugates obtained and the relative proportions of their constituents appear in Table I. The complete detoxification of each of the conjugates was checked before use, by subcutaneous injection into 3 mice of a dose of conjugate corresponding to the double dose of TT resulting in the death of animals. Detoxification was noted due to the absence of any sign of paralysis in the mice used in this test or in those which were the subject of the tests which follow.

Groups each comprising 6 adult BALB / c female mice were immunized subcutaneously with 50 μg of each of the AC conjugates (Table II) administered in the form of saline solution. Another group received 50 μg of PEP-TT in a mixture with 1.00 μg of MDP-lysine in a saline solution. 6 control mice received 50 μg of PEP-TT in saline solution in the absence of adjuvant. The animals were boosted with the same antigens 3 weeks later and then bled either 7 days or 14 days after the boost. In a second series of experiments, the animals were immunized with 50 μg of the DF conjugates (table I) in a saline solution or with 50 μg of PEP-TT mixed with 100 μg of 6-O-aminocaproyl- murabutide in a saline solution. Control mice received 50 μg of PEP-TT without the adjuvant. The animals were bled 2 weeks after the primary immunizations. On day 25, they received a booster injection. Finally, blood samples were taken after delays of 7, 14 and 123 days respectively after the recall. The sera collected from animals in each of the groups considered were pooled and tested for their ability to induce in vivo production of anti-peptide and anti-tetanus antibodies in diagnostic tests involving the ELISA method. The native protein was recognized by anti-peptide antibodies in radioimmunoassays (from the radioimmunoassay: RIA) using sporozoite extracts fixed on plastic plates, according to the method of Vanderberg et al. ., 1969, and by tests involving antibodies and indirect fluorescence (indirect fluorescence antibody test: IFA) according to the method Gvadz et al., 1979. The reaction of circumsporozoites (CSP) according to Nordin et al., 1979, was used to test the biological activity of anti-PEP antibodies. Antibody production in vitro.

Spleen cells were obtained from mice. These cells, obtained under aseptic conditions, were washed and suspended at a concentration of 10 ⁷ cells / ml in the medium known under the designation RPMI 1640 manufactured by the company SEROMED, containing 2 g / 1 of NaHCO ₃ of glutamine ( 2 mM), 100 units / ml of penicillin, 100 μg / ml of streptomycin, 5% fetal calf serum and 2-mercaptoethanol (2x10 -5M), the suspensions were divided into aliquots of 100 μl. These were placed in the wells of 96-well tissue culture plates (Costar). 20 μl of TT or PEP-TT were added to each of the wells at a rate of 0.1 μg / ml. The final volumes in each of the wells were adjusted to 0.2 ml. The control wells did not receive antigen. The plates were incubated in an atmosphere containing 10% CO ₂ and 90% oxygen at 37ºC. On the 4th day, the plates were washed to remove the antigen. 6 days later, the supernatants were collected and tested for their antibody content by the ELISA method. The following results were thus observed.

In vivo response expressed in quantity of antibodies produced to conjugates before variable substitution rates for PEP, TT and MDP-lysine.

The results in Table II show that the antibody titers produced with respect to both the peptide and TT vary in certain proportions according to the levels of peptide and adjuvant fixed on TT. It was the mice that were immunized with the conjugate that contained the highest peptide content that exhibited the strongest anti-PEP response. The increase in MDP-lysine substitution rate was not manifested by a higher concomitant anti-PEEP response; on the other hand, the increase in the MDP-lysine substitution rate was manifested by a high anti-TT response. Mixtures of MDP-lysine with PEP-TT resulted in a slight increase in the anti-PEP antibody response against the controls; a strong increase in the antibody response was however observed when the adjuvant was conjugated to TT. Higher anti-TT titers were also observed than in controls in those groups who had been treated with MDP-lysine (group 3). This result clearly means that the conjugation of the adjuvant to PEP-TT is manifested by an increase in the responses both with regard to the peptide and to the support. In vivo antibody response to conjugates containing 6-O-ε-amino-caproyl-murabutide. Two tests were carried out with these latter types of conjugates. Representative results of these tests are shown in Table III. It is noted that all the groups which received the adjuvants manifested approximately equivalent immune responses 15 days after the recall, and this independently of the conjugation or non-conjugation of the adjuvant with PEP-TT. This equivalence was observed despite the fact that the DF conjugates contained respectively only 1.0 μg, 0.6 μg and 0.3 μg of adjuvant per 50 μg of conjugate. It follows from the observations which have been made that conjugates containing 100 times less of the adjuvant induce anti-peptide antibody responses of the same order of magnitude as the mixtures using the muramyl-peptide not covalently combined with the antigen . A clear contrast was observed, however, in the primary anti-tetanus responses. Those of animals which had received the conjugates led only to very weak responses (titers lower than 400) while those which had received PEP-TT alone or in mixture with the adjuvant led to the observation of titers 8-40 times higher.

The differences between the anti-support responses that could be observed between groups 1-2 on the one hand, and groups 3-5 on the other hand were even more pronounced after the reminders (on days 32 and 39). Of the groups treated with the conjugate, it was group 5 which had been treated with the conjugate containing the lowest proportions of adjuvant which exhibited the highest anti-TT antibody responses. The production of strongly favored anti-TT antibodies in animals which had received the adjuvant in the form associated (and not conjugated) with the other constituents of the conjugate could still be observed 123 days after the booster injection. The following observations were made at the level of the anti-peptide responses. Slightly higher responses were observed in animals treated with conjugates D and E than those observed in animals treated with PEP-TT in admixture. An activity was found relatively weak adjuvant in those of the animals which had received the F conjugate, therefore the one whose adjuvant contents were initially low. These results seem to indicate that the dose of adjuvant used (0.3 μg / mouse / injection) was at the limit of the minimum quantities necessary to induce an anti-peptide response.

All of these results nevertheless show that the amphiphilic adjuvant groups exposed on the surface of the support, and this after conjugation of the carrier molecule thus formed with the peptide, play an important role in terms of the induction of the production of antibodies. anti-PEP and anti-TT respectively.

Similar observations were made on day 32 in those of animals whose reactivity was tested by RIA using the natural circum-sporozoite protein, in the plastic plates coated with extracts of sporozoites and incomplete Freund's adjuvant . Biological activities, measured by CSP were also measured. The results obtained are shown in Table IV. High titers were observed in sera from all groups, either in those who had received PEP-TT in admixture with the adjuvant or in those who had been treated with the triple conjugate. However, it was the animals treated with conjugate D which led to the highest titers (both in the IFA and CSP tests). These results demonstrate that it is the conjugates which not only recognize the parasite in its three-dimensional form, but also cause a regression of the circumsporozoite protein of living parasites.

Antibody production in vitro by mouse splenic cells, in contact with a PEP-TT-6-O- (ε-amino-caproyl) -murabutide conjugate. The ability of mouse spleen cells to produce anti-TT antibodies in vitro following stimulation with PEP-TT or free TT was tested, due to the particularly low level of circulating anti-TT antibodies in these animals, when these had been treated with conjugates containing murabutide. The results are shown in Table V. The supernatants of these cultures were tested in the ELISA system. They reveal that only splenic cells of groups 4 and 5 produced anti-TT antibodies, after being stimulated either with TT or with PEP-TT in vitro. On the contrary, all the experimental groups were found to produce anti-peptide antibodies, when they were stimulated in vitro with PEP-TT. Anti-PEP antibodies were not detected in supernatants of spleen cells from control animals (group 1), possibly because the population of cells producing anti-PEP antibodies in the spleen of animals n had not received the adjuvant was too weak to be detected. These results demonstrate that the conjugation of 6-O- (ε-amino-caproyl) -murabutide to PEP-TT leads to less effective stimulation with regard to the macromolecular TT support than does a mixture of the macromolecular support acting as than antigen with the adjuvant. This difference may be due to changes introduced in the determinants of TT or to the fact that these determinants have been made inaccessible to the immune system, hence the reduced immunogenicity observed. The foregoing tests call for the following observations.

Considerable differences have been observed in in vivo adjuvanticity tests with triple conjugates containing the same carriers macrόmoleculars and peptides, on the other hand, different muramyl peptides, in particular 6-O- (ε-amino-caproyl) - murabutide and MDP-lysine respectively. In the case of the last adjuvant, the increase in the proportion of adjuvant in the conjugate also resulted in an increase in the macromolecular anti-support response. On the contrary, when the muramyl-peptide entering into the constitution of the conjugate was 6-O- (ε-amino-caproyl) -amphiphilic murabutide, the response in anti-TT antibodies was hardly increased. However, sera from mice treated with the conjugate containing the most adjuvant also gave the highest titers of antibodies recognizing the peptide and the native protein. These latter types of conjugates also caused a CSP reaction. This is correlated to the degree of protection that can be observed in mice and monkeys. Consequently, the low doses of PEP-TT-conjugated 6-O- (ε-amino-caproyl) -murabutide have been shown to induce a significantly higher protective antibody response than that which can be obtained with doses of the same adjuvant. the absence of conjugation or with MDP-lysine.

Sera from mice treated with murabutide-containing conjugates were found to have much lower anti-TT antibody titers than sera from animals that had received PEP-TT (in the absence of the muramyl peptide). Several hypotheses can be formulated to explain this reduced response. According to a first hypothesis, the use of particular muramylpeptides falling within the scope of the invention has introduced conformational changes in the macromolecular support on the occasion of its conjugation with the peptide and the adjuvant. According to a second hypothesis, the antigenicity of the macromolecular support has been modified by masking its determinants, these then being made inaccessible to cells of the immune system.

The coupling of 6-O- (ε-amino-caproyl) -murabutide with tetanus toxin may make the toxoid sufficiently hydrophobic for it to be slightly masked with respect to the immune system, while on the contrary the peptide is more exposed and sensitive to the adjuvant action of muramyl-peptide. It is perhaps because of the hydrophilic nature of MDP-lysine that the adjuvant effect with respect to the toxin is added to the observable adjuvant effect with respect to the peptide.

Finally, in vitro antibody production assays were chosen to determine whether mouse cells which had been stimulated with the conjugates could also be stimulated in vitro by PEP-TT or TT and therefore to determine to what extent the determinants of tetanus toxoid were expressed within the conjugates. The results clearly show that the cells of the groups treated with the conjugate give only weak responses, if not an absence of response to TT, whether the latter is used in the free state or in the conjugated state to the peptide. These results support the hypothesis that the antigenicity of TT within triple conjugates has been modified. In contrast, the anti-peptide responses in vivo are approximately the same in groups 1-4 (Table II), slightly weaker for animals in group 5. In addition, cells from all these groups formed anti-peptide antibodies. peptide in vitro, after stimulation with PEP-TT. Taken as a whole, these results suggest that the absence of anti-TT antibodies in vitro produced by cells of groups 1-3, was due to an alteration of the antigenic determinants of the macromolecular support or to their masking, during the stimulation in vivo. Finally, the tests also demonstrate that the production of anti-peptide antibodies can be strongly stimulated by the use of conjugates in accordance with the invention, even when these contain only relatively small proportions of the particular muramyl-peptides envisaged.

It follows from the foregoing tests that the triple conjugates according to the invention have an immunogenicity specifically directed against hapten. The invention therefore also relates to pharmaceutical compositions in which the above-defined triple conjugates are associated with a physiologically acceptable vehicle.

Advantageous pharmaceutical compositions consist of injectable solutions containing an effective dose of at least one triple conjugate according to the invention. Preferably, these solutions are produced in an isotonic sterile aqueous phase, preferably saline or glucose. The invention also relates to compositions in which these triple conjugates are encapsulated in liposomes capable of forming injectable suspensions.

The invention relates more particularly to such solutions or suspensions which are capable of being administered by intradermal, intramuscular or subcutaneous, intravenous injections or even by scarification.

The invention also relates to pharmaceutical compositions which can be administered by other routes, in particular by the oral or rectal route, or also in forms intended to come into contact with mucous membranes, in particular the ocular, nasal, pulmonary or vaginal mucous membranes.

Consequently, it relates to pharmaceutical compositions in which at least one of the triple conjugates according to the invention is associated with pharmaceutically acceptable excipients, solid or liquid, suitable for the constitution of oral, ocular or nasal administration forms, or with excipients adapted for the constitution of rectal administration forms. It also relates to compositions intended for the pulmonary route, in particular solutions prepared for administration by means of a conventional aerosol device. By way of examples of doses which can be administered, in order to stimulate the immune responses of the host against determined antigens, mention will be made of doses, in micrograms per kg of body, for example from 50 to 1000, preferably from 100 300, μg / kg, when administered parenterally, or 0.5-10 mg / kg orally.

Preferred vaccine compositions according to the invention further contain a vehicle, such as polyvinylpyrrolidone, facilitating the administration of the vaccine. In place of polyvinylpyrrolidone, one can use any other type of adjuvant in the conventional sense that was formerly given to this expression, that is to say a substance allowing the easier absorption of a drug. or facilitating its action in the body. By way of examples of other adjuvants of the latter type, mention will also be made of carboxymethylcellulose, aluminum hydroxides and phosphates or any other adjuvants of this type, well known to those skilled in the art. The pharmaceutical compositions according to the invention are therefore essentially intended to induce in the host an immune reaction, allowing him to produce antibodies or cell-mediated immunity with respect to an antigen containing the antigenic site specifically carried by the hapten entering the triple conjugate according to the invention.

The invention also relates more particularly to another type of pharmaceutical composition useful in human or veterinary therapy, whenever a change in the evolution or the behavior of the human or animal organism is sought, for example at the level of fertility. of the species or of the directed orientation of its metabolism towards the suppression of certain hormones. In preferred cases of the latter type, the hapten entering the conjugate used consists of the hormone itself or a fragment thereof.

In the veterinary field, mention will also be made of the increased production of certain constituents, to the detriment of other constituents. Mention will be made, for example, of the applications of veterinary vaccines administered with the aim of increasing the production of meat in animals by immunological castration.

The subject of the invention is in particular veterinary compositions containing effective doses of the above triple conjugates, in which the hapten itself consists either of the hormone itself, or of a peptide sequence having a common part with the hormone , the hormone belonging to the class of peptide or glycopeptide hormones. A particularly interesting hormone in this regard is the release factor of the luteostimulating hormone LH-RH.

Effective unit doses of these conjugates are, for example, between approximately 50 and 1000, preferably 100 to 300, μg / kg of body, when administered parenterally, and between approximately 0.5 and 10 mg / kg when administered orally. The invention also also relates to biological reagents essentially formed from these triple conjugates. In particular, the haptenic part can consist of an active principle of medicament or drug (for example morphine) or also the expression product, of an oncogene. These biological reagents can then be used to make antibodies, in particular monovalent or monoclonal antibodies. These antibodies are then in their turn useful for the constitution of reagents allowing for example the study of the action of drugs in question on the living host. In particular, these antibodies will allow the detection of cellular receptors, or the study of the metabolism and the mode of action of the drugs in question.

As goes without saying, and as follows from the above, the invention is in no way limited to those of its more specially indicated embodiments, on the contrary, it embraces all variants, in particular those where muramyl- peptide entering into the triple conjugates of the invention would carry substituents distinct from those which have been indicated, insofar as these substituents would not however cause any modification of the above-mentioned behavior of the muramyl-peptide thus modified inside the triple conjugates here considered.

It will also be noted that the contents of the documents and patents mentioned in the present application are considered to belong to the description of the latter.

∞

Claims

CLAIMS 1. Immunogenic product resulting from the conjugation of three principles between them, these three principles consisting respectively of a macromolecular support, at least one muramylpeptide and at least one hapten carrying at least one epitope responsible for the desired immunogenic activity, carterized in that the muramylpeptide is linked via its glucopyranosyl nucleus to this support by means of chemical groups forming between them a link arm.

2. Product according to claim 1, characterized in that the muramyl-peptide on the one hand, the hapten on the other hand, are coupled to the macromolecular support by means of distinct functional groups initially present on or carried by the support macromolecular.

3. Product according to claim 1 or 2, characterized in that the muramyl-peptide forming part of this product can be represented by the following general formula:

in which :

R ₁ is an OH group or an OC ₆ H ₄ -NH _{2 group} or O- (CH ₂ ) _m -R _a , m being an integer from 1 to 10, and R _a being an amine, carboxyl, thiol, hydroxyl or hydrogen function;

- A is oxygen or an NH group; - R ₆ is a group BR or CO-BR in which B is a group or a linear or branched chain which can contain up to approximately 100 carbon atoms and R is a hydroxyl, carboxyl, amino, thiol or hydrogen, group B optionally itself carrying one or more functional groups of the type of those designated above for R, or alternatively carbonyl, alkoxyl or acyl;

- R ₂ is hydrogen or an alkyl group comprising from 1 to 4 carbon atoms, in particular methyl; - X is an alanyl, arginyl, lysyle, asparagyle, aspartyle, cystéinyle, glutaminyle, glutamyle, glycyle, histidyle, hydroxyprolyle, isoleucyle, leucyle, méthionyle, phenylalanyl, prolyle, seryle, threonyle, tryptophanyle or valyle residue; - Y is an OH, NH _{2 group} or, preferably, OR ₇ or NHR ₇ , R ₇ being a hydrocarbon group which may have from 1 to 20 carbon atoms or which may be an amino acid as indicated for X, such that alanyl, seryl, valyl or glycyl, said amino acid being free, amidated or esterified, the esterification group possibly comprising up to 4 carbon atoms;

Z is a carboxyl, carboxamide, ester group containing from 1 to 10 carbon atoms, or an alkyl group comprising from 1 to 4 carbon atoms, preferably -CH ₂ -CH ₃ or - (CH ₂ ) ₂ -CH ₃ ; it being understood that at least one of the groups R ₁ and R ₆ (and preferably R ₆ ) has a reactive function allowing its conjugation with a complementary reactive function carried by another molecule, in particular the macromolecular support.

4. Product according to any one of claims 1 to 3, characterized by the following overall structure:

[]

in which: the "macromolecular support" comprises at least two functional groups allowing the covalent coupling of other molecules to this macromolecular support, the lines in dashed lines symbolize x covalent conjugation bonds between the hapten and either the macromolecular support or position 1 or position 6 on the glucopyranosyl nucleus of the muramylpeptide, with the index x being at least equal to 1 in the first case and 'equal to 1 in the second case, the solid line symbolizes y covalent bonds between the position 6 or position 1 on the glucopyranosyl nucleus of the muramyl peptide or, when the hapten is conjugated directly to the macromolecular support, the two positions at the same time, the index y being at least equal to 1,

- the sum x + y is at least equal to 2 and at most equal to the maximum number of functional groups carried by the macromolecular support.

5. Product according to claim 3 or 4, characterized by the following formula:

y

- R ₁ is -OH or -OC ₆ H ₄ -NH ₂ or O- (CH _n ) _m -R with m from 1 to

10 and R _a being an amine, carboxyl, thiol, hydroxyl or hydrogen function;

- R ₂ is -H or -CH ₃ ;

- X is Ala, Gly, Ser or Val;

- Y is O (CH ₂ ) _p -H with p from 1 to 10; Z is CO-NH ₂ , COO (CH ₂ ) _r -H or (CH ₂ ) _r -CH ₃ with r from 1 to 4.

6. Product according to any one of claims 1 to 5, characterized in that the amino-acyl residues forming part of the group X are levogyres (except in the case where X is constituted by a glycyl residue).

7. Product according to any one of claims 1 to 6, characterized in that the muramyl-peptide used in the constitution of said product is of the type 2- (2-acetamido-2-deoxy-3-OD-glucopyranosyl) -alkanoyl- peptide.

8. Product according to any one of claims 1 to 6, characterized in that the muramyl-peptide used in making up said product is of the 2- (2-acetamido-2-deoxy-3-OD-glucopyranosyl) -alkanoyl type - aminoacyl-nor Leucine.

9. Product according to any one of claims 1 to 8, in which Y is an OR ₇ group, with R ₇ comprising from 1 to 10 carbon atoms, and Z is a -CONH ₂ group.

10. Product according to any one of claims 1 to 8, in which Y is an OR ₇ group, with R ₇ comprising from 1 to 10 carbon atoms, and Z is a -CH ₂ -CH _{3 group} .

11. Product according to any one of claims 3 to 10, characterized in that A is oxygen and R ₆ a group -CO- (CH ₂ ) -COOH such as the succinyl group -CO- (CH ₂ ) ₂ -COOH, or the group -CO- (CH ₂ ) _n -NH ₂ , such as the epsilon-amino-caproyl group: -CO- (CH ₂ ) ₆ -NH ₂ , n being a number from 1 to 10.

12. Product according to any one of claims 1 to 11 thereof, the muramyl-peptide part of which is derived from one of the following muramyl-peptides: - 6-O-epsilon-aminocaproyl-MurNAc-L-Ala-D-Gln -OnBu;

- 6-O-epsilon-aminocaproyl-MurNAc-L-Ala-D-Nle-OnBu;

- 6-O-succinyl-MurNAc-L-Ala-D-Gln-OnBu;

- 6-O-succinyl-MurNAc-L-Ala-D-Nle-OnBu; or also previous muramyl-peptides in which the N-butyl ester groups (OnBu) are replaced by methyl ester groups (OMe).

13. Product according to any one of claims 1 to 11, characterized in that the macromolecular support has a molecular weight of at least 50,000, in particular between 50,000 and 250,000 daltons.

14. Product according to claim 13, characterized in that the macromolecular support is derived from tetanus toxoid.

15. Product according to any one of claims 1 to 14, characterized in that the hapten part entering said product has a molecular weight at most equal to 5000.

16. Product according to claim 15, characterized in that the hapten part is a polypeptide group.

17. Pharmaceutical composition having in particular selective immunogenic properties inducing protective antibodies against a determined antigen, characterized in that the active principle of this composition conforms to any one of claims 1 to 16 and in that the hapten constituent of this product contains an antigenic site or an epitope common with the antigen against which protection is sought.