EP0946194A1

EP0946194A1 - Vaccine composition containing as additive a sympathomimetic compound

Info

Publication number: EP0946194A1
Application number: EP97943940A
Authority: EP
Inventors: Daniel Bout; Jérôme AUCOUTURIER; Vincent Ganne; Johan Hoebeke
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Societe dExploitation de Produits pour les Industries Chimiques SEPPIC SA
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Societe dExploitation de Produits pour les Industries Chimiques SEPPIC SA
Priority date: 1996-10-07
Filing date: 1997-10-03
Publication date: 1999-10-06
Also published as: AU4560097A; FR2754182B1; WO1998015288A1; FR2754182A1

Abstract

The invention concerns a composition containing: (i) at least one antigen or at least one generator in vivo of a compound comprising a sequence of amino acids, and (ii) at least one additive, characterised in that said additive contains at least one sympathomimetic compound. The invention also concerns their use as medicine.

Description

VACCINE COMPOSITION COMPRISING AS ADJUVANT A SYMPATHOMIMETIC COMPOUND

The present invention relates to new adjuvants for vaccine compositions as well as compositions comprising at least one antigen, in particular an antigen of viral, bacterial or parasitic origin and at least one adjuvant.

The use of adjuvants in the compositions has been known for a long time. The main purpose of these adjuvants is to increase the immune response. These adjuvants are of various natures. They may for example consist of liposomes, emulsions comprising at least one oil phase and at least one aqueous phase, of the Freund adjuvant type or, more commonly in mineral salts insoluble in water. These mineral salts can constitute, for example in aluminum hydroxide, a cerium nitrate, zinc sulphate, colloidal iron hydroxide or calcium chloride. Aluminum hydroxide is the most commonly used adjuvant. These adjuvants are described in particular in the article by Rajesh K. Gupta et al "Adjuvants, balance between toxicity and adjuvanticity", Vaccine, vol. 1 1, Issue 3, 1993, pages 993-306.

Local immunization is the most effective way to induce protection against pathogens that access the body through the mucous membranes. However, the weak immune response developed against antigens administered by this route requires the use of immunostimulatory agents. The adjuvant recognized as being the most powerful to stimulate the immunity of the mucous membranes is the cholera toxin, but the toxic effects which are associated with it restrict its use in humans. Its efficiency seems correlated with the ribosylation of ADP on an adenylate cyclase type enzyme leading to a continuous intracellular cAMP synthesis. These elements of the state of the art are known and disclosed in the following publications: RJ Jackson and Coll; Infection and Immunity, Oct 1993, 4272-4279; Shin-ichi Tamura and Coll; 1995, Vaccine 13, 339-341.

The Applicant has therefore sought to develop substances capable of modulating and / or transiently increasing the level of intracellular messengers, such as cAMP, in order to increase the immune response without thereby inducing toxicity vis-à-vis the subjects treated. The subject of the invention is a composition comprising: (i) at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence; and, (ii) at least one adjuvant, characterized in that said adjuvant comprises at least one sympathomimetic compound.

The term “antigen or at least one in vivo generator of a compound comprising an amino acid sequence” denotes either killed microorganisms, such as viruses, bacteria or parasites, or purified fractions of these microorganisms, or living microorganisms whose pathogenicity has been reduced; mention may in particular be made of a recombinant virus, in particular a non-enveloped virus such as adenoviruses, vaccinia virus, Canarypox virus, herpes viruses, baculoviruses; a non-enveloped living viral recombinant vector is also designated, the genome of which contains, preferably inserted into a part which is not essential for the replication of the corresponding enveloped virus, a sequence coding for an antigenic subunit inducing antibody synthesis and / or a protective effect against the above enveloped virus or pathogenic microorganism; these antigenic subunits can be, for example, a protein, a glycoprotein, a peptide, or a peptide and / or protective fraction against infection by a living microorganism such as an enveloped virus, a bacterium or a parasite. Mention may in particular be made of a recombinant plasmid consisting of a nucleotide sequence into which an exogenous nucleotide sequence is inserted, originating from a microorganism or a pathogenic virus. The aim of this last nucleotide sequence is to allow the expression of a compound comprising an amino acid sequence, this compound itself having the aim of triggering an immune reaction in a host organism;

By sympathomimetic compounds is meant in particular amphetamines, catecholamines, phenylisopropylamines or tyramine. As examples of such compounds, mention may be made in particular of isoproterenol, L-epinephrine, levarterenol, ephedrine, phenylephedrine or salbutamol.

In a first variant of the present invention, the latter also relates to a composition as defined above comprising, in addition, at least one pharmaceutically acceptable salt which is soluble in water, said salt consisting of an organic anion and of a metal cation. A water-soluble salt within the meaning of the present invention can be such that its solubility in water is greater than or equal to 10 g / l and, preferably, between 10 and 2000 g / l. The metal cation constituting said pharmaceutically acceptable salt is preferably a bivalent cation, advantageously this cation is a cation of a metal chosen from the group consisting of manganese, calcium and zinc. Manganese is a metal which is very particularly preferred in the context of the present invention. It has in fact been found that this pharmaceutically acceptable salt is soluble in water and allows the induction of a particularly important immune response while having a low toxicity. The organic anion constituting said pharmaceutically acceptable salt is advantageously an anion of a compound comprising at least one oxygen-containing functional group, preferably a phosphoric group or a carboxylic group. Glycerophosphoric acid is a group anion preferred phosphoric. The anions comprising at least one preferred carboxylic group are derived from compounds chosen from: acid oses, preferably acid oses having from 5 to 7 carbon atoms, more preferably those having 6 to 7 carbon atoms, mono or polycarboxylic acids, amino acids.

The preferred mono or polycarboxylic acids are fumaric acid or the compounds of formula (I)

R '

I

R- (CH ₂ ) s- (COH) t- (CH ₂ ) u-COOH (I)

where R represents COOH, CHaCO, CHa or CH2OH,

R 'represents H or COOH and s, t and u, which are identical or different, are between 0 and 3.

Preferred compounds of formula (I) are acetic acid, lactic acid, tartaric acid, malic acid, citric acid, pyruvic acid.

For the purposes of the present invention, an acidic ose consists of an ose comprising at least one carboxylic function, a ose being a carbohydrate constituted by the reducing sugars. These acid oses are advantageously derivatives of aldoses, obtained by oxidation of the primary alcohol function or of the aldehyde function into a carboxylic function. Such compounds may very particularly consist of gluconic acid, glucuronic acid, fructoheptonic acid, gluconoheptonic acid, glucoheptonic acid. When the organic anion is a derivative of an amino acid, this amino acid can be an α-amino acid such as glutamic acid, methionine and, very particularly, aspartic acid.

A pharmaceutically acceptable salt, soluble in water according to the invention can comprise a polyvalent cation, in particular divalent, associated with an organic anion or with several organic anions of nature. different. Thus, by way of example, a bivalent cation such as the calcium cation can be associated with an anion derived from gluconoheptonic acid.

A composition thus defined contains between 0.02 and 3000 mg / ml, preferably 0.1 and 1000 mg / ml of the mineral salt and, in particular, between 0.1 and 150 mg / ml.

In a second variant of the invention, the composition as defined above can also contain an oil. In such a case, the composition according to the invention is advantageously in the form of an emulsion associating at least one aqueous phase and at least one oil phase. This emulsion can be of the oil in water (O / W), water in oil (W / O), water in oil in water (W / O / W) or microemulsion type. However, the W / O / W and O / W emulsions are preferred. Such an emulsion can be prepared according to the conventional methods for preparing an emulsion, in particular according to the methods described in patent applications EP-A-489,181 and EP-A-481,982. Thus, the emulsion can be emulsified with stirring. constituent oil of the oil phase with the aqueous phase consisting of an aqueous solution or suspension containing the antigen.

An emulsion according to the invention can comprise, by weight, from 5 to 95% of oil phase for 95 to 5% of aqueous phase and, preferably, from 25 to 75% of oil phase for 75 to 25% of aqueous phase. The emulsion should preferably be stable for at least 12 months when it is stored at 4 ° C. The oils, consisting of said oily phase which can be used in the composition according to the invention are advantageously fluid oils chosen from natural or synthetic mineral oils, or non-mineral oils of vegetable, animal or synthetic origin known for their absence or their low toxicity.

They must be liquid at storage temperature (+ 4 ° C) or at least give liquid emulsions at this temperature. In particular, straight chain mineral oils having a number of carbon atoms preferably greater than 16 and free of compounds will be chosen. aromatic. Known examples are MARCOL 52 and DRAKEOL 6VR. Synthetic oils such as polyisobutenes or polyisopropenes can also be used. Among the vegetable oils, unsaturated oils rich in oleic acid which are biodegradable will be chosen, for example peanut, olive, sesame, soybean or wheat germ oils. For animal oils, the same criteria of tolerance and immune efficacy are required. By way of example as animal oils, mention may be made of squalane or spermaceti oil. Mention may also be made, as synthetic non-mineral oil, of esters of alcohols and of fatty acids such as, for example, ethyl oleate, isopropyl myristate, mono di or triglycerides, esters propylene glycol, partial glycerides, such as corn oil glycerides such as those sold under the name LANOL by the company SEPPIC, corn, or oleyl oleate. Furthermore, the composition according to the invention when it is in the form of an emulsion as defined above, can also advantageously comprise one or more emulsifying surfactants. The latter has a lipophilic or hydrophilic character characterized by an HLB (hydrophilic-lipophilic-balance) value of between 1 and 19. Such a surfactant can consist of:

an alkylpolyglycoside or a mixture of alkylpolyglycosides of formula Ra- (O) -Zn where Ra represents a saturated, linear or branched aliphatic radical, comprising from 4 to 24 carbon atoms, Z is the remainder of a sugar, preferably the glucose and n is between 1 and 5, preferably between 1, 1 and 2.

- saponins,

- lecithins,

- polyoxyethylated alkanols, such as those sold under the designation BRU by the company ICI, - polymers comprising polyoxyethylene and polyoxypropylene blocks, such as those sold under the designation PLURONICS by the company BASF.

Particularly preferred surfactants are polyethylene glycol esters, obtained by condensation of a fatty acid, in particular a liquid fatty acid at 20 ° C. with a polyethylene glycol of molecular weight between 80 and 200; such a surfactant is sold by the company SEPPIC under the brand SIMULSOL 2599.

Another preferred surfactant in the context of the present invention consists of an ester obtained by condensation of a fatty acid, advantageously a fatty acid which is liquid at 20 ° C. with a sugar, sorbitol or glycerol. Said sugar can consist of glucose, sucrose or, preferably, mannitol. As a particularly preferred mannitol ester, there may be mentioned mannitol oleates obtained by anhydrization of the polyhydroxylated carbon chain of mannitol which cyclizes in 1-4 or 2-6.

Derivatives of these polyethylene glycol, sorbitol or glycerol sugar esters can also be used. These derivatives exhibit modified hydrophilicity in particular by grafting hydrophilic functions such as alcohol, polyol, ethylene oxide, propylene oxide, carboxylic acid, amine or amide. Such derivatives may for example consist of fatty esters of polyoxyethylated sorbitan, such as TWEEN.

The other types of preferred surfactants consist of ethoxylated vegetable oils, such as, for example polyethoxylated castor oil, this oil being hydrogenated or not, or polyglycerol esters, in particular polyglycerol esters of natural fatty acids such as oleic acid, stearic acid, ricinoleic acid or isostearic acid and, in particular, ricinoleates or polyricinoleates of polyglycerol. A surfactant according to the invention is preferably pharmaceutically acceptable in the mucous membranes; it must in particular be free of heavy metals and have very low acid or peroxide indices. It is also desirable that it meets the standards of safety tests such as for example, those described by SS Berllin, Annales of Allergy, 1962, 20, 473 or the tests of abnormal toxicity described in the European pharmacopoeia. Preferably, the surfactant is combined with the oily adjuvant before the emulsion is formed. The concentration of surfactant in the composition as defined above can be between 0.01 and 500 mg / ml, and preferably between 0.1 and 200 mg / ml.

Oils associated with a surfactant (mannitol ester) which are very particularly suitable in the context of the present invention are those sold by the company SEPPIC under the brand MONTANIDE. The nature of these oils, the type of emulsion which they make it possible and the characteristics (viscosity and conductivity) of these emulsions are described in the international application WO94 / 1 6681, published on August 4, 1994 and, in particular, page 1 1, Table I. These compositions according to the invention can be used for the treatment of human or veterinary diseases. In particular, they make it possible to relieve infectious diseases with an intracellular agent, in particular respiratory diseases, opportunistic infectious diseases and, within the framework of veterinary medicine, infectious diseases of fish. The composition according to the invention can also contain substances which modulate immunity.

The oily adjuvant can also consist of a self-emulsifying oil, that is to say an oily preparation capable of forming a stable emulsion with an aqueous phase, practically without any energy supply, for example by dispersion in the aqueous phase by agitation slow mechanical. As such, there may be mentioned self-emulsifying oils as known in the European pharmacopoeia under the designations Labrafil and Simulsol. These oils are polyglycosylated glycerides.

Preferred self-emulsifiable oils are those described in French patent application No. 9500497 filed on January 18, 1995, in the name of the Applicant, entitled "use of ethoxylated fatty acid esters as self-emulsifiable components, in particular useful for the preparation of phytosanitary compositions or drugs for veterinary or human use ", the reference of which is incorporated into the present description. These oils consist of esters of ethoxylated fatty acids corresponding to one of the following formulas:

O

(II) Ri— ¹ C— r [O— CH ₂ -CH ₂ ] k— OR ₂

O O

"II

(III) R3— C— [O— CH ₂ -CH ₂ ] ι - R4O— [CH ₂ -CH2— θ] m - C-RB |

O O

(IV) R & - Il— [O— CH ₂ -CH ₂ ] - —R7- CH-R9-O— [CH2-CH2— 0] q - ^H C-R10

I

in which :

- R-), R3, R5, R6, Rδ ^{and R} 10 represent a hydrocarbon chain, linear or branched, saturated or unsaturated having from 5 to 30 carbon atoms; - R2 'F * 4' ^ 7 ^and ^ 9 represent a hydrocarbon chain, linear or branched, saturated or unsaturated having from 1 to 5 carbon atoms; the total number of ethylene oxide molecules respectively represented in the aforementioned formulas II, III and IV by k, l + m, n + p + q being an integer such as the HLB (hydrophilic-lipophilic balance) value of said said compounds is between about 4 and about 10, preferably between about 5 and about 9.

R- \ is preferably chosen from the residues of palmitic, stearic, ricinoleic, oleic, linoleic and linolenic acids and R2 represents a methyl radical and k is an integer between 1 and

5, preferably equal to 2, and moreover, the preferred ethoxylated fatty acid esters of formula III are those where:

(i) - Rβ, Rδ ^{and R} 10 represent hydrocarbon chains having from 16 to 22 carbon atoms corresponding in particular to the fatty chains of rapeseed oil, corn, soybean, peanut and apricot kernels;

- R7 and R9 represent a methylene group CH2;

- n, p, q represent whole numbers such that their sum is between 3 and 30, and preferably equal to 20; or

(ii) - Rg, Rβ and R10 represent hydrocarbon chains corresponding to the fatty chains of castor oil;

- R7 and R9 represent a methylene radical CH2;

- n, p, q represent whole numbers such that their sum is between 5 and 7.

The concentration of self-emulsifiable oil in the composition according to the invention can be between approximately 5 and 700 g / l, preferably between approximately 10 and 500 g / l.

In addition to the oil phase and the aqueous phase, the composition according to the invention can comprise a conventional immune stimulating agent. such as Avridine®, ie N, N-dioctadecyl-N ', N' - bis (2-hydroxyethyl) propanediamine, derivatives of MDP (muramyl dipeptide), in particular threonyl-MDP, derivatives of mycolic acid or Lipid A derivatives. The composition according to the invention may also comprise one or more surfactants, in the absence of any oily adjuvant.

The composition is then presented in the form of a micellar solution. This can be prepared by simple mixing of the surfactant with a dispersion in water of the antigen or of the antigen generator in vivo. The surfactant can be chosen from the surfactants described above, in combination with an oily adjuvant.

Said micellar solution can comprise from 0.5 to 500 mg / ml, preferably from 1 to 250 mg / ml as a surfactant.

A composition according to the present invention can comprise an antigen such as a virus, a microorganism, more particularly a bacterium or a parasite, or a compound comprising a peptide chain. Such a compound may consist of a protein or a glycoprotein, in particular a protein or a glycoprotein derived from a microorganism, a synthetic peptide or a protein or peptide derived from genetic engineering. Said viruses and microorganisms can be totally inactivated or live attenuated. As viruses which may constitute an antigen according to the present invention, mention may be made of the rabies virus, herpesviruses such as Aujeszky's disease virus, orthomixoviruses such as Influenzae, picornaviruses such as fever virus foot and mouth disease or retroviruses such as HIV. As a microorganism of the bacterial type which can constitute an antigen according to the present invention, mention may be made of E. coli, and those of the genera Pasteurella, Furonculosis, Vibriosis, Staphylococcus and Streptococcus. As a parasite, mention may be made of those of the genera Trypanosoma, Plasmodium and Leishmania. A composition according to the invention comprises an antigen concentration which depends on the nature of this antigen and on the nature of the subject treated. It is however particularly remarkable that an adjuvant according to the invention, associated or not with an oily adjuvant and / or a surfactant as defined above, makes it possible to significantly reduce the usual dose of antigen required. The appropriate concentration of antigen can be determined conventionally by those skilled in the art. Generally, this dose is of the order of 0.1 μg / ml to 1 g / ml more generally between 1 μg / ml and 100 mg / ml. A composition according to the invention can also comprise an in vivo generator of a compound comprising an amino acid sequence, that is to say a biological compound capable of expressing such a compound in the host organism in which one introduced said generator in vivo. The compound comprising the amino acid sequence can be a protein, a peptide or a glycoprotein.

These generators in vivo are generally obtained by processes resulting from genetic engineering.

More particularly, they can consist of living microorganisms, generally a virus, playing the role of recombinant vector, into which is inserted a nucleotide sequence, in particular an exogenous gene. These compounds are known as such and used in particular as a recombinant unitary vaccine.

In this regard, reference may be made to the article by M. ELOIT et al., Journal of virology (1990) 71 _. / 2925-2431 in international application WO-A-91/00107 or in international application WO-A- 94/16681.

Advantageously, the microorganism constituting a recombinant subunit vaccine is a non-enveloped recombinant virus, for example chosen from adenoviruses, vaccinia virus, canarypox virus, herpes viruses or baculoviruses. The exogenous gene inserted into the microorganism can be, for example, from an Aujeszky or HIV virus. The in vivo generators according to the invention can also consist of a recombinant plasmid comprising an exogenous nucleotide sequence, capable of expressing in a host organism a compound comprising an amino acid sequence. Such recombinant plasmids and their mode of administration in a host organism were described in 1990, by LIN et al., Circulation 82: 2217,2221; COX et al., J. of VIROL, Sept. 1993, 67, 9, 5664-5667 and in international application WO / FR 95/00345 of March 21, 1995, in the name of the Applicant, entitled "A composition comprising a plasmid and its uses as a vaccine and medicine ".

Depending on the nature of the nucleotide sequence included in the generator in vivo, the compound comprising the amino acid sequence which is expressed within the host organism, can:

(i) be an antigen, and allow the triggering of an immune reaction,

(ii) have a curative action vis-à-vis a disease essentially a functional disease, which is triggered in the host organism. In this case, the in vivo generator allows treatment of the host, of the gene therapy type. By way of example, such a curative action may consist in a synthesis by the in vivo cytokine generator, such as interleukins, in particular interleukin 2. These allow the triggering or the strengthening of an immune reaction aimed at the selective elimination of cancer cells. The concentration of said generator in vivo in the composition according to the invention again depends, in particular, on the nature of said generator and on the host in which it is administered. This concentration can be easily determined by a person skilled in the art, on the basis of routine experience. As an indication, it can however be specified that when the generator in vivo is a recombinant microorganism, its concentration in the composition according to the invention can be between 10 ² and 10 ¹⁵ microorganisms / ml, preferably between 10 ⁵ and 10 ¹² microorganisms / ml. When the in vivo generator is a recombinant plasmid, its concentration in the composition according to the invention can be between 0.01 and 100 g / l.

A composition according to the invention can be used as a preventive or curative medicament. Depending on the nature of the antigen or generator in vivo, a composition according to the invention can be administered to fish, crustaceans such as shrimps, poultry, in particular, geese, turkeys, pigeons and chickens, canines such as dogs, felines such as cats, pigs, primates, bovines, ovids and horses. The composition according to the invention comprising at least one sympathomimetic compound as defined above, can also be administered to humans. The administration of the composition can be carried out in a conventional manner by parenteral route, in particular by subcutaneous, intramuscular or intraperitoneal injection or by mucosal route in particular by oral route, rectal route, nasal route, vaginal route.

According to another aspect of the invention, it consists in the use of an adjuvant consisting of a sympathomimetic compound as defined above for the preparation of a vaccine intended for the prevention or treatment of a disease infectious, including an infectious disease caused by a virus or microorganism such as those mentioned above.

According to another aspect of the invention, it consists in the use of this adjuvant for the preparation of a composition intended to treat a functional disease, such as cancer or cystic fibrosis. In either of these uses, said adjuvant can be combined with a pharmaceutically acceptable salt, soluble in water and / or with an oily adjuvant, a surfactant or with an oily adjuvant itself associated with a surfactant; these oily adjuvants and surfactants being as defined above.

Adjuvant compositions comprising said sympathomimetic compound, said pharmaceutically acceptable salt and the oily adjuvant and / or the aforementioned surfactants constitute yet another aspect of the invention. Where appropriate, these adjuvant compositions comprising at least one aqueous phase.

In the latter case, the adjuvant compositions according to the invention, comprising at least one oily adjuvant, and, if necessary, a surfactant, can be in the form of an emulsion. The latter can be of the W / O, O / W, W / O / W or microemulsion type. These emulsions can comprise by weight, from 0.5% to 99.5

% of oil phase for 99.5% to 0.5% of aqueous phase, preferably from 5 to 95% of oil phase for 95 to 5% of aqueous phase and, more preferably, from 25 to 75% of oil phase for 75 to 25% of aqueous phase. If necessary, they can comprise from 0.01 to 500 mg / ml, preferably from 0.1 to 200 mg / ml of at least one surfactant.

When the adjuvant composition according to the invention comprises, in addition to the sympathomimetic compound, optionally the pharmaceutically acceptable salt and an aqueous phase, only one or more surfactants, it is then in the form of a micellar solution. The surfactant content of this micellar solution can be between 0.01 and 900 mg / ml, preferably between 1 and 250 mg / ml.

An adjuvant composition according to the invention usually comprises the sympathomimetic compound at a concentration of 10 ^"10 molar to 10 ^{" 2} molar, preferably from 10 ^"7 Molar to 10 ^{" 5} Molar. When it contains a pharmaceutically acceptable salt as defined above, it is at a concentration of 0.02 to 3000 mg / ml, preferably 0.1 to 1000 mg / ml, more preferably from 0.1 to 150 mg / ml.

These adjuvant compositions are useful for preparing the compositions according to the invention.

The latter can then be prepared by simple mixing of the adjuvant composition with a composition comprising an antigen or an in vivo generator of a compound comprising an amino acid sequence. The following example makes it possible to demonstrate the properties of the compositions according to the present invention.

Example: demonstration of the β-adrenergic receptor on the target cells of cholera toxin in the immune system of the mucous membranes. The first step before testing the adjuvant potential of β-adrenergic agonists was the demonstration of the receptor on cells involved in mucosal immunity, especially in cholera toxin target cells in the intestinal mucosa. of the mouse and the demonstration of the functional coupling of this receptor to its effector the enzyme adenylate cyclase. For this purpose, we have measured, in parallel, the production of cAMP by four cell populations after stimulation with different concentrations of cholera toxin and a β-adrenergic agonist, isoproterenol.

The cell populations studied were enterocytes (MODE-K cell line), PEYER plaque lymphocytes (LPP), mesenteric lymph nodes (LGM) and intraepithelial lymphocytes (LEL).

The results show that all the types studied respond more or less intensely to stimulation by cholera toxin (see FIG. 1), with indices of stimulation of cAMP production which range from 30 times for enterocytes to 2 times for PEYER plaque lymphocytes. This stimulation generally starts from 10 ng / ml.

With the exception of PEYER plate lymphocytes, all the cell types studied responded to β-adrenergic stimulation (Figure 1), with stimulation indexes ranging from 3 times for enterocytes to 1.5 times for lymphocytes from lymph nodes. mesenteric and intraepithelial lymphocytes. In all cases, stimulation with isoproterenol is observed from 100nM. The production of cAMP observed, is indeed due to the stimulation of the β-adrenergic receptor, because it is totally inhibited by propanolol, a specific antagonist.

It has thus been demonstrated the presence of functional β-adrenergic receptors on enterocytes, lymphocytes of the mesenteric nodes and intraepithelial lymphocytes. There is a co-localization of the cholera toxin receptor and the β-adrenergic receptor on three of the four cell populations studied, which led us to test the adjuvant potential of β-adrenergic agonists ultimately showing the advantages of compositions according to the present invention containing at least one adjuvant sympathomimetic compound.

Claims

1. Composition including:

(i) at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence; and, (ii) at least one adjuvant, characterized in that said adjuvant contains at least one sympathomimetic compound.

2. Composition according to claim 1, characterized in that the sympathomimetic compound is an in sympathomimetic such as a catecholamine, an amphetamine, a phenylisopropylamine or tyramine.

3. Composition according to claim 2, characterized in that the sympathomimetic compound is chosen from ephedrine or isoproterenol, L-epinephrine, levartererol, phenylephedrine or salbutamol.

4. Composition as defined in any one of claims 1 to 3, characterized in that the adjuvant further comprises at least one pharmaceutically acceptable salt and soluble in water, said salt consisting of an organic anion and of a metal cation.

5. Composition according to claim 4, characterized in that the metal cation is divalent and selected from the group consisting of cations of zinc, manganese and calcium.

6. Composition according to one of claims 4 or 5, characterized in that the organic anion is derived from a compound comprising at least one oxygenated functional group.

7. Composition according to claim 6, characterized in that the functional group is a carboxylic group or a phosphoric group.

8. Composition according to claim 7, characterized in that the organic anion is derived from a compound chosen from the group consisting by amino acids, acid oses comprising from 5 to 7 carbon atoms, and glycerophosphoric acid.

9. Composition according to claim 8, characterized in that the amino acid is aspartic acid.

10. Composition according to claim 9, characterized in that the acid dare is chosen from the group consisting of gluconic acid, fructoheptonic acid and glucoheptonic acid.

1 1. Composition according to Claim 10, characterized in that the pharmaceutically acceptable salt is manganese gluconate.

12. Composition according to one of claims 1 to 1 1, characterized in that it is in the form of an emulsion comprising at least one aqueous phase and at least one oil phase, the oil phase being constituted by an adjuvant .

13. Composition according to claim 12, characterized in that the emulsion is of the W / O / W, O / W or microemulsion type.

14. Composition according to one of claims 12 or 13, characterized in that the oily adjuvant is combined with a surfactant, preferably a surfactant chosen from an ester obtained by condensation of a fatty acid with a sugar, a polyethylene glycol, glycerol sorbitol, or a derivative of such an ester whose hydrophilicity has been modified, an ethoxylated vegetable oil or their mixtures.

15. Composition according to one of claims 12 or 13, characterized in that the oily adjuvant is combined with a surfactant chosen from polyglycerol esters, in particular a polyglycerol ester of natural fatty acids and, preferably, ricinoleates or polyricinoleates of polyglycerol or constituting in polyethoxylated castor oils hydrogenated or not.

16. Composition according to one of claims 12 or 13, characterized in that the oily adjuvant is a self-emulsifying oil.

17. Composition according to claim 16, characterized in that the oily adjuvant is an ester of ethoxylated fatty acids corresponding to one of the following formulas:

OO (III) R3— C— [O— CH2-CH ₂ ] ι - OR4O— [CH2-CH2— 0] m - CR _B |

O O

(IV) Re— C— [O— CH ₂ -CH ₂ ] - O— R7- CH-R9-O— [CH2-CH2— θ] _q - C-R10 I

in which :

- R-], R3, R5, Rβ, Rβ ^{and R} 10 represent a hydrocarbon chain, linear or branched, saturated or unsaturated having from 5 to

30 carbon atoms;

- R2, R4, R7 and Rg represent a hydrocarbon chain, linear or branched, saturated or unsaturated having from 1 to 5 carbon atoms; the total number of ethylene oxide molecules respectively represented in the aforementioned formulas II, III and IV by k, l + m, n + p + q being an integer such as the HLB (hydrophilic-lipophilic balance) value of said said compounds is between about 4 and about 10, preferably between about 5 and about 9.

18. Composition according to one of claims 1 to 1 1, characterized in that it further comprises a surfactant, said composition being in the form of a micellar solution.

19. Composition according to Claim 18, characterized in that the surfactant is either an ester obtained by condensation of a fatty acid with a sugar or glycerol, or a derivative of such an ester whose hydrophilicity has been modified or either an ethoxylated vegetable oil

20. Composition according to one of claims 1 to 19, further comprising an immune stimulant such as Avridine®, derivatives of MDP, derivatives of mycolic acid or derivatives of lipid A.

21. Composition according to one of claims 1 to 20, for the implementation of a method of treatment of the human or animal body.

22. The medicament as claimed in claim 21, intended to treat or prevent infectious and / or functional diseases and in particular diseases caused by a virus, a microorganism or a parasite.

23. The medicament of claim 21 for treating or preventing respiratory diseases.

24. Medicament according to claim 21 intended to treat Aujeszky's disease in pigs, ovine toxoplasmosis as well as fish diseases such as salmon lice or crustacean diseases.

25. Adjuvant as defined in one of claims 4 to 19.

26. Use of the adjuvant as defined in one of claims 1 to 19 for the manufacture of vaccines intended for mucosal vaccination, in particular oral, nasal, rectal or vaginal vaccination.

27. Adjuvant composition comprising an aqueous phase, an oily adjuvant, a surfactant, optionally a salt consisting of an organic anion and a metal cation, characterized in that it contains at least one sympathomimetric compound.

28. Composition according to claim 27 in the form of a W / O / W or O / W emulsion.