METHOD FOR PRODUCE A VACCINE COMPOSITION CONCERNING AT LEAST ONE ANTIGEN AND AT LEAST ONE ADJUVAN

Description

[0001] The present invention relates to a method for preparing a vaccine composition comprising at least one antigen, in particular an antigen of viral, bacterial or parasitic origin, and at least one adjuvant according to claim 1.

[0002] The development of inactivated vaccines or vaccines containing purified antigens is increasingly important, as it helps avoid undesirable side effects in the treated individual. However, improving the quality of the antigens comes at the expense of their immunogenicity. This is why they are combined with immunostimulating agents.

[0003] Immunity adjuvants are products that enhance the immune system's response when administered in the presence of viral, bacterial, or synthetic antigens. They induce a massive proliferation of macrophages at the injection site and subsequently in lymph nodes, increase the production of specific immunoglobulins (antibodies), and stimulate numerous cells involved in immune defense mechanisms.

[0004] These adjuvants are of various kinds. For example, they can consist of emulsions in the form of water-in-oil (W / O), oil-in-water (O / W), water-in-oil-in-water (O / W / W), or oil-in-water-in-oil (O / W / O).

[0005] Freund adjuvants are highly effective; they consist of a mineral oil and a mannitol ester, with or without a killed mycobacterium. Vaccines prepared by mixing equal parts of a Freund adjuvant with an aqueous antigenic medium remain the gold standard worldwide for laboratory studies. They are presented as water-in-oil (W / O) emulsions, meaning emulsions in which the continuous phase is an oil or a mixture of oils, and the dispersed phase is an aqueous phase that may include solubilizing excipients such as glycerol or dimethyl sulfoxide. The active ingredient is generally located in the aqueous phase, which is often a buffered saline solution. This phase is in the form of droplets separated by an oily film. This formulation allows for amplified and prolonged biological responses.

[0006] However, emulsions of this type (oil-in-water) are generally very viscous and therefore difficult to inject. They often require the use of syringes with a large needle diameter and can cause pain during injection and trauma at the injection site.

[0007] The aqueous phase content of injectable oil-in-water (O / W) emulsions is around 30% to 40% by mass. The maximum observed concentration is 50%. This limit is a significant obstacle, particularly for the development of polyvalent vaccines, which combine several antigens and for which it would be preferable to obtain O / W emulsions with aqueous phase contents exceeding 50%.

[0008] However, in a low-water-phase (w / o) emulsion, up to approximately 20% by mass, the emulsion's viscosity is very close to that of the oil. Increasing the mass proportion of the water phase increases its viscosity. Thus, an emulsion with 20% water by mass and a viscosity of 100 mPa·s, measured using a Brookfield LVT viscometer equipped with a No. 2 spindle rotating at 60 rpm, becomes a very difficult-to-inject cream when the water content is increased to 50% by mass.

[0009] Some commercial oily adjuvants in the form of W / O emulsions, such as MONTANIDE ™< ISA 70, allow for the production of injectable W / O emulsions containing approximately 30% by weight of aqueous phase, 70% by weight of oily phase, and having viscosities of the order of 50 to 100 mPa.s, measured using a BROOKFIELD LVT viscometer equipped with a No. 2 spindle rotating at a speed of 60 revolutions per minute. Other adjuvants, such as MONTANIDE ™< ISA 50V2, make it possible to obtain injectable W / O emulsions containing approximately 50% by weight of aqueous phase, 50% by weight of oily phase and having viscosities of less than 250 mPa.s measured using a BROOKFIELD LVT viscometer equipped with a No. 2 spindle rotating at a speed of 60 revolutions per minute.

[0010] The international patent application published under number WO 99 / 20305 discloses the use of mannitol oleate surfactants with mineral oils such as MARCOL ™< 52 to make emulsions, referred to as fluids, with viscosities of the order of 500 mPa.s measured using a BROOKFIELD LVT viscometer equipped with a No. 2 wheel rotating at a speed of 60 revolutions per minute.

[0011] The international patent application published under number WO 98 / 17310 describes adjuvant compositions based on water-soluble polyanionic polymers having anionic constitutional repeating units that may be identical or different, or polyanionic polymers having anionic constitutional repeating units (identical or different) and hydrophobic constitutional repeating units.

[0012] The patent application published under number US 2004 / 170640 A1 describes a process for preparing an adjuvant composition comprising a mixture of lecithin and an acrylic polymer or copolymer.

[0013] Patent application published under number WO 03 / 024354 describes adjuvant compositions comprising acrylic acid copolymers, more particularly a latex emulsion of an uncoalesced aqueous acrylic copolymer of acrylic acid, methacrylic acid mixed with styrene. Patent application published under number US 2001 / 028884 A1 describes the use of an acrylic acid or methacrylic acid polymer, a maleic anhydride polymer, and an alkenyl derivative for adjuvanting immunogenic compositions or vaccines, which may be formulated as an oil-in-water emulsion.

[0014] The concept of a fluid emulsion depends largely on the field of application. For injectable emulsions, the upper viscosity limit for a fluid emulsion is defined relative to the viscosity of a reference oil-in-water (O / W) type emulsion, which contains 50% by mass of aqueous phase and 50% by mass of Freund's incomplete adjuvant (IFA); this viscosity, measured using a Brookfield LVT viscometer equipped with a No. 3 spindle rotating at a speed of 30 revolutions per minute, is on the order of approximately 2,000 mPa·s. This emulsion is considered highly viscous.

[0015] An oil-in-water emulsion will be said to be fluid if its viscosity is less than 4 times that of this reference emulsion, i.e. less than 500 mPa.s at 25°C, measured using a BROOKFIELD LVT viscometer equipped with a No. 2 spindle and rotating at a speed of 30 revolutions per minute.

[0016] However, fluid emulsions are generally less stable than more viscous emulsions, since phase shifts at room temperature are observed only a few days after their preparation.

[0017] There are aqueous-phase thickening polymers available in powder form, such as homopolymers of acrylic acid in their sodium forms or copolymers based on acrylic acid and its esters. Examples include the polymers marketed by Noveon under the brand names CARBOPOL™ and PEMULEN™. They are described in particular in US patents 5,373,044 and 2,798,053, and in European patent EP 0 301 532. These polymers were originally developed as thickeners for formulations primarily intended for cosmetic applications and have been described and used for many years. These polymers are obtained from a monomer such as, for example, acrylic acid, methacrylic acid, esters of acrylic acid or methacrylic acid in solution in an organic solvent phase.During the polymerization reaction, achieved by adding various catalysts under specific temperature and pressure conditions, the polymer becomes insolubilized from the initial solvent phase and precipitates to the bottom of the reactor. This process is called "precipitation polymerization".

[0018] The polymers thus obtained, the best-known grades of which are CARBOPOL®, are highly effective thickeners widely used in cosmetics. These polymers are also used for a variety of pharmaceutical applications: Preparation of polymer matrices for delayed release effect, Preparation of polymer gels, and complexation of said polymers with proteins, Preparation of polymer films inducing protection upon contact with biological fluids, Preparation of bio-adhesive formulations ensuring greater persistence when applied to mucous membranes, Preparation of vaccine adjuvants.

[0019] The use as a vaccine adjuvant, with final polymer mass contents in the order of "percent", results in a fluid and translucent vaccine that is easily injectable.

[0020] The adjuvant properties of synthetic substances are closely linked to their physical forms at the time of administration. Thus, the particle size used to trigger a significant adjuvant effect is a major parameter that controls the potential for uptake by immunocompetent cells. Furthermore, depending on the particle size, it is also possible to direct the response towards antibody production (humoral response) or towards stimulating specific cells of the immune system (cellular response). The synthesis of microspheres of various polymers in solvent phase as dispersions is widely described in the literature. These microspheres can be used to encapsulate a biologically active agent (which may be an antigen in the case of vaccines) or as an adsorption support to deliver and present an active agent bound to the outer surface of the microspheres by interactions of varying strength.

[0021] Controlling the size of adjuvant particles is an ongoing challenge, as it must be accompanied by ensuring the stability of these particles over time. It is particularly important to avoid aggregation during storage or phase separation. Size control must also be accompanied by compositional stability after injection into the living organism, especially given the stresses associated with pH variations, the presence of enzymes, and temperature fluctuations.

[0022] Therefore, the applicant sought to develop an original polymer synthesis technology that would allow control over the size of polymer agglomerates (or "microgels"), thus ensuring excellent adjuvant performance. To this end, water-in-oil (W / O) emulsion polymerization technology, or inverse emulsion polymerization, is used to prepare these polymers – as disclosed in claim 1. Specifically, the size of the monomer solution droplets is controlled by using water-in-oil emulsifying surfactant systems adapted to W / O emulsion technologies. This generally involves coupling a lipophilic emulsifying surfactant, having, for example, an HLB between 2 and 7, with a hydrophilic emulsifying surfactant, having, for example, an HLB between 10 and 15, with a weighted average value ensuring a homogeneous W / O emulsion.The polymerization of monomers is ensured by the addition of catalysts and other crosslinking agents at the usual concentrations under pH and temperature conditions suitable for a complete reaction of the monomer to form polymers.

[0023] The size of adjuvant particles has a direct impact on the immunological properties of the formulations containing them. Furthermore, depending on the species to be vaccinated and the type of vaccine used, the optimal particle size may differ. Through emulsion formulation expertise (optimization of HLB, nature of surfactants, concentration, process), it is possible during the inverse emulsion synthesis process to control the droplet size distribution of the emulsion and thus the average size and particle size distribution of the agglomerates. Dispersion, depending on the surfactant content and the shear energy applied during the emulsification step, refers to heterogeneous systems that can be: suspensions, emulsions, microemulsions.

[0024] These different dispersed forms make it possible to control the size dispersion of microgels in ranges from 50 nm to 100 µm.

[0025] The chemical conditions of polymer synthesis (types of monomers, catalysts, initiators) allow control of the individual properties of the polymer obtained (average molar mass, crosslinking, anionic character).

[0026] The synthesized adjuvant is in the form of a "polymer droplets in oil" or "inverse latex" emulsion. When dispersed in an aqueous antigen solution containing a physiological buffer, this emulsion inverts and disperses to form a fluid, translucent dispersion of polymer microgels in water. For example, sodium polyacrylate microgel droplets can have an average size of between 0.5 and 5 micrometers, for example, approximately 2 micrometers.

[0027] The invention relates to a method for preparing a vaccine using a reverse latex. More particularly, the invention relates to a method for preparing a vaccine, comprising: a step a ) preparation of an adjuvant composition by dispersion in a physiologically acceptable aqueous solution, of at least one reverse latex or of a polymer powder resulting from the atomization of said reverse latex; step b ) of mixing the composition obtained in step a) in an antigenic medium, intended to form a vaccine composition.

[0028] Inverse latex is defined as a water-in-oil polymer emulsion in which the aqueous phase, dispersed within the continuous oil phase, contains the polymer. The inverse latex resulting from reverse emulsion polymerization is therefore in the form of a liquid emulsion. This reverse latex can be subsequently atomized, if necessary or desired, to form the polymer powder. Atomization is described, for example, in European patent application EP 1 496 081.

[0029] For the purposes of this invention, a physiologically acceptable aqueous solution means aqueous solutions suitable for use in the preparation of vaccines, such as water of a quality conforming to pharmacopoeias, particularly European or American ones, such as physiological sera, or saline and / or, where applicable, hydro-alcoholic solutions conforming to said pharmacopoeias.

[0030] Adjuvant composition refers to an adjuvant composition for immunity.

[0031] At least one hydrophilic surfactant may also be added prior to this step a) in order to improve the stability of such a dispersion over time.

[0032] Hydrophilic surfactants are defined as emulsifying surfactants with a sufficiently high HLB value, between 10 and 15, to provide stable oil-in-water emulsions, such as ethoxylated manithan esters, ethoxylated sorbitan esters such as, for example, ethoxylated sorbitan oleate with 20 moles of ethylene oxide, marketed by SEPPIC under the name MONTANOX™< 80.

[0033] An antigenic medium is defined as a medium comprising at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence. An antigen or at least one in vivo generator of a compound comprising an amino acid sequence is defined as either killed microorganisms, such as viruses, bacteria, or parasites, or purified fractions of these microorganisms, or live microorganisms whose pathogenicity has been attenuated. Examples of viruses that can constitute an antigen according to the present invention include the rabies virus, herpesviruses such as Aujeszky's disease virus, orthomixoviruses such as Influenzae, picornaviruses such as foot-and-mouth disease virus, or retroviruses such as HIV. An example of a bacterial microorganism that can constitute an antigen according to the present invention is E.E. coli, and those of the genera Pasteurella, Furunculosis, Vibriosis, Staphylococcus, and Streptococcus. Examples of parasites include those of the genera Trypanosoma, Plasmodium, and Leishmania. Recombinant viruses, particularly non-enveloped viruses such as adenoviruses, vaccinia virus, canarypox virus, herpesviruses, and baculoviruses, can also be mentioned.A live, non-enveloped recombinant viral vector is also defined as a vector whose genome contains, preferably inserted in a non-essential region for the replication of the corresponding enveloped virus, a sequence encoding an antigenic subunit that induces antibody synthesis and / or a protective effect against the aforementioned enveloped virus or pathogenic microorganism. These antigenic subunits may be, for example, a protein, a glycoprotein, a peptide, or a peptide fraction, and / or provide protection against infection by a live microorganism such as an enveloped virus, a bacterium, or a parasite. The exogenous gene inserted into the microorganism may, for example, originate from an Aujeszky's virus or HIV.

[0034] One example is a recombinant plasmid consisting of a nucleotide sequence into which an exogenous nucleotide sequence, originating from a pathogenic microorganism or virus, is inserted. This latter nucleotide sequence is designed to allow the expression of a compound comprising an amino acid sequence, this compound itself intended to trigger an immune response in a host organism.

[0035] An "in vivo" generator of a compound comprising an amino acid sequence refers to any biological product capable of expressing said compound in the host organism into which the generator has been introduced in vivo. The compound comprising the amino acid sequence may be a protein, a peptide, or a glycoprotein. These in vivo generators are generally obtained through genetic engineering processes. More specifically, they may consist of living microorganisms, usually a virus, acting as a recombinant vector into which a nucleotide sequence, notably an exogenous gene, is inserted. These compounds are known as such and used, in particular, as recombinant subunit vaccines. In this regard, see the article by M. ELOIT et al., *Journal of Virology* (1990) 71, 2925-2431, and the international patent applications published under numbers WO-A-91 / 00107 and WO-A-94 / 16681.The in vivo generators according to the invention may 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 method 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 the international application published under number WO 95 / 25542. Depending on the nature of the nucleotide sequence included in the in vivo generator, the compound comprising the amino acid sequence that is expressed within the host organism may: (i) to be an antigen, and to trigger an immune response, (ii) to have a curative effect on a disease, primarily a functional disorder, that has developed in the host organism. In this case, the in vivo generator allows for host treatment, of the gene therapy type.

[0036] As an example, such a curative action may consist of a synthesis by the in vivo generator of cytokines, such as interleukins, in particular interleukin 2. These allow the triggering or strengthening of an immune reaction aimed at the selective elimination of cancer cells.

[0037] The vaccine composition, as defined above, comprises an antigen concentration that depends on the nature of the antigen and the nature of the subject being treated. It is particularly noteworthy, however, that an adjuvant according to the invention makes it possible to significantly reduce the usual required antigen dose. The appropriate antigen concentration can be determined conventionally by those skilled in the art. Generally, this dose is in the range of 0.1 µg / cm³ to 1 g / cm³, more commonly between 1 µg / cm³ and 100 mg / cm³. The concentration of said generator in vivo in the composition according to the invention depends, again, in particular, on the nature of said generator and the host into which it is administered. This concentration can be easily determined by those skilled in the art, based on routine experience.As an indication, when the in vivo generator is a recombinant microorganism, its concentration in the composition according to the invention is generally between 10² and 10¹⁵ microorganisms / cm³ and preferably between 10⁵ and 10¹² microorganisms / cm³. When the in vivo generator is a recombinant plasmid, its concentration in the composition obtained according to the process of the invention may be between 0.01 g / dm³ and 100 g / dm³. The vaccine, as defined above, is prepared by mixing the adjuvant phase and the antigenic phase, optionally adding water or a pharmaceutically acceptable diluent.

[0038] The continuous oily phase, used to prepare the reverse latex implemented during step a) of the process of the present invention, comprises one or more compounds selected from oils of mineral, vegetable or animal origin, alkyl esters of said oils, alkyl esters of fatty acids or alkyl ethers of fatty acids, esters of fatty acids and polyols or ethers of fatty alcohols and polyols, synthetic oils.

[0039] A commercial mineral oil can also be used, that is to say a commercial mineral oil containing saturated hydrocarbons such as paraffins, isoparaffins, cycloparaffins, having at room temperature a density between 0.7 and 0.9 and a boiling point above 180°C, such as for example EXXSOL ™< D 100 S, or MARCOL ™< 52 marketed by the company EXXON CHEMICAL, isohexadecane or isododecane, or a mixture of several of these oils.

[0040] According to a preferred aspect of the present invention, the oil phase consists of MARCOL™< 52 or isohexadecane; isohexadecane, which is identified in Chemical Abstracts by the number RN = 93685-80-4, is a mixture of C12, C16, and C20 isoparaffins containing at least 97% C16 isoparaffins, the principal constituent of which is 2,2,4,4,6,8,8-heptamethyl nonane (RN = 4390-04-9). It is marketed in France by BAYER. MARCOL™< 52 is a commercial oil meeting the definition of petrolatum oils in the French Codex. It is a white mineral oil that complies with FDA regulations 21 CFR 172.878 and CFR 178.3620 (a) and is listed in the US Pharmacopoeia, US XXIII (1995) and the European Pharmacopoeia (1993).

[0041] Examples of oils of vegetable origin include peanut oil, olive oil, sesame oil, soybean oil, wheat germ oil, grapeseed oil, sunflower oil, castor oil, linseed oil, soybean oil, corn oil, coconut oil, palm oil, walnut oil, hazelnut oil, rapeseed oil, and squalene or squalane of vegetable origin marketed in France by the company SOPHIM, under the name PHYTOSQUALAN ™<, identified in Chemical Abstracts by the number RN = 111-01-3, and which consists of a mixture of hydrocarbons containing more than 80% by weight of 2,6,10,15,19,23-hexamethyl tetracosane.

[0042] Examples of oils of animal origin include spermaceti oil, tallow oil, squalane or squalene extracted from fish livers.

[0043] Examples of alkyl esters of oils include ethyl, linear or branched propyl, or linear or branched butyl esters of said oils.

[0044] As suitable fatty acids for the preparation of the esters mentioned above, there are in particular those comprising 12 to 22 carbon atoms, such as for example myristic acid, palmitic acid, oleic acid, ricinoleic acid or isostearic acid and advantageously a fatty acid liquid at 20°C.

[0045] Examples of fatty acid esters or fatty acid ethers include alkyl esters of fatty acids, such as ethyl oleate, methyl oleate, isopropyl myristate or octyl palmitate; esters of fatty acids and polyols or ethers of fatty alcohols and polyols, such as fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, fatty acid esters with a polyglycerol or fatty acid esters with propylene glycol, and especially fatty acid esters with a hexol, such as sorbitol or mannitol, fatty acid esters with a hexol anhydride, such as sorbitan or mannitol.

[0046] Examples of synthetic oil include hydrogenated polydecene or hydrogenated polyisobutene, marketed in France by the company Ets B. Rossow et Cie under the name PARLEAM - POLYSYNLANE ™<, which is cited by Michel and Irene Ash, "Thesaurus of Chemical Products", Chemical Publishing Co, Inc. 1986 Volume I, page 211 (ISBN 0 7131 3603 0).

[0047] Within the framework of the present invention, the continuous oily phase, used to prepare the reverse latex implemented during step a) of the process of the present invention, may comprise only one of the compounds mentioned above or a mixture of several of the compounds mentioned above.

[0048] The proportion of the oily phase in the reverse latex is between 10% and 50% by mass, and preferably between 15% and 25% by mass, of the total mass of the reverse latex.

[0049] Said at least one polymer obtained by reverse emulsion polymerization leading to the reverse latex at the basis of the preparation of the adjuvant composition prepared in step a) of the process of the invention is selected from anionic, branched or crosslinked polyelectrolytes selected from copolymers of acrylic acid and 2-methyl-[(1-oxo-2-propenyl)amino] 1-propanesulfonic acid (AMPS), copolymers of acrylamide and 2-methyl-[(1-oxo-2-propenyl)amino] 1-propanesulfonic acid, copolymers of 2-methyl-[(1-oxo-2-propenyl)amino] 1-propanesulfonic acid and (2-hydroxyethyl) acrylate, the homopolymer of 2-methyl-[(1-oxo-2-propenyl)amino] 1-propanesulfonic acid partially or totally salified, the partially or totally salified acrylic acid homopolymer, the partially or totally salified methacrylic acid homopolymer, the copolymers of acryloyl ethyl trimethyl ammonium chloride and acrylamide,copolymers of AMPS and vinylpyrolidone, copolymers of AMPS and Nm ethyl acrylamide, copolymers of AMPS and N,N-dimethyl acrylamide, copolymers of AMPS and methacrylamide, copolymers of AMPS and N-isopropylacrylamide, copolymers of AMPS and N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]propenamide [or tris(hydroxymethyl)acrylamidomethane or N-tris(hydroxymethyl)methyl acrylamide, also known as THAM], copolymers of acrylic acid and acrylamide, copolymers of acrylic acid and N-methyl acrylamide, copolymers of acrylic acid and methacrylamide, copolymers of acrylic acid and N-isopropylacrylamide, copolymers of acrylic acid and N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]propenamide [or tris(hydroxymethyl)acrilamidomethane or N-tris(hydroxymethyl)methylacrylamide also known as THAM],Copolymers of acrylic acid and alkyl acrylates with a carbon chain comprising between ten and thirty carbon atoms, and copolymers of AMPS and alkyl acrylates with a carbon chain comprising between ten and thirty carbon atoms.

[0050] A branched polymer is defined as a non-linear polymer that has dangling chains so that, when this polymer is dissolved in water, it becomes highly entangled, leading to very high viscosities at low velocity gradients.

[0051] A cross-linked polymer is a non-linear polymer that exists as a three-dimensional network insoluble in water, but swells in water and thus leads to the formation of a chemical gel.

[0052] When this polyelectrolyte is crosslinked, it is most particularly crosslinked with a diethylene or polyethylene compound in the molar proportion, expressed relative to the monomers used, of 0.005% to 1%, and preferably of 0.01% to 0.2%, and more particularly of 0.01% to 0.1%. Preferably, the crosslinking agent and / or the branching agent is chosen from ethylene glycol dimethacrylate, diethylene glycol diacrylate, sodium diallyloxyacetate, ethylene glycol diacrylate, diallyl urea, triallylamine, trimethylol propanetriacrylate, or methylene-bis-(acrylamide)₂, allylsucrose.

[0053] When reverse latex is used in the preparation step of the adjuvant composition prepared in step a) object of the process of the invention, it generally comprises 1% to 5% by mass of a water-in-oil (W / O) type emulsifying system.

[0054] In the preceding definition, a water-in-oil (W / O) type emulsifying system refers to either a single surfactant or a mixture of surfactants, provided that said mixture has a sufficiently low HLB value to induce water-in-oil emulsions. As a water-in-oil type emulsifying agent, there are, for example, sorbitan esters, such as sorbitan oleate, such as that marketed by the company SEPPIC under the name MONTANE™< 80, sorbitan isostearate, such as that marketed by the company SEPPIC under the name MONTANE™< 70 or sorbitan sesquioleate such as that marketed by the company SEPPIC under the name MONTANE™< 83. There are also certain polyethoxylated sorbitan esters, for example pentaethoxylated sorbitan monooleate such as that marketed by the company SEPPIC under the name MONTANOX™< 81 or pentaethoxylated sorbitan isostearate such as that marketed under the name MONTANOX™< 71 by the company SEPPIC.There is also diethoxylated oleocetyl alcohol such as that marketed under the name SlMULSOL ™< OC 72 by the company SEPPIC, polyesters with molecular weights between 1000 and 3000, products of the condensation between a poly(isobutenyl) succinic acid or its anhydride and diethanolamine such as HYPERMER ™< 2296 marketed by the company UNIQEMA or finally block copolymers with molecular weights between 2500 and 3500, such as HYPERMER ™< B246 marketed by the company UNIQEMA or SlMALlNE ™< IE 200 marketed by the company SEPPIC.

[0055] When reverse latex is used in the preparation step of the adjuvant composition prepared in step a) object of the process of the invention, it generally comprises 2% to 8% by mass of an oil-in-water (O / W) type emulsifying system.

[0056] In the preceding definition, an oil-in-water (O / W) type emulsifying system refers to either a single surfactant or a mixture of surfactants, provided that said mixture has a sufficiently high HLB value to induce oil-in-water emulsions.Examples of oil-in-water emulsifying agents include ethoxylated sorbitan esters such as polyethoxylated sorbitan oleate with 20 moles of ethylene oxide, marketed by SEPPIC as MONTANOX™< 80; polyethoxylated sorbitan laurate with 20 moles of ethylene oxide, marketed by SEPPIC as MONT ANOX TM< 20; polyethoxylated castor oil with 40 moles of ethylene oxide, marketed as SIMULSOL™< OL50; decaethoxylated oleodecyl alcohol, marketed by SEPPIC as SIMULSOL™< OC 710; heptaethoxylated lauryl alcohol, marketed as SIMULSOL™< P7; and monostearate of sorbitan polyethoxylated with 20 moles of ethylene oxide marketed by the company SEPPIC under the name MONTANOX ™< 60.

[0057] A polymer obtained by emulsion polymerization, leading to the inverse latex at the basis of the preparation of the adjuvant composition prepared during step a) of the process of the invention, advantageously used is chosen from the group consisting of the homopolymer of totally or partially salified acrylic acid, the homopolymer of partially or totally salified methacrylic acid, the copolymers of acrylic acid and 2-methyl-[(1-oxo-2-propenyl)amino]1-propanesulfonic acid, the copolymers of acrylamide and 2-methyl-[(1-oxo-2-propenyl)amino]1-propanesulfonic acid, the copolymers of 2-methyl-[(1-oxo-2-propenyl)amino]1-propanesulfonic acid and (2-hydroxyethyl) acrylate, and even more advantageously sodium polyacrylate.

[0058] The mass proportion of said at least one reverse latex in the adjuvant composition prepared in step a) of the process of the invention is between 0.5% and 30% of the total mass of the adjuvant composition, and preferably between 5% and 15% of the total mass of the adjuvant composition.

[0059] In step a) of the process of the present invention, an immunostimulating substance may be added. The immunostimulating substance is, for example, selected from one or more conventional immune-stimulating agents, such as Avridin™, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propanediamine, MDP (muramyl dipeptide) derivatives, in particular threonyl-MDP, mycolic acid derivatives, or Lipid A derivatives. Such a substance is more particularly an ionized substance. The ionized substance is, for example, selected from one or more organic salts of water-soluble metal cations, such as calcium gluconate, manganese gluconate, aluminum salicylate, or soluble aluminum acetate.When the adjuvant composition according to the invention comprises a pharmaceutically acceptable salt, it is at a concentration of 0.02 to 3000 mg / cm³, preferably 0.1 to 1000 mg / cm³, more preferably 0.1 to 150 mg / cm³. Insoluble salts commonly used as immune adjuvants, such as aluminum hydroxide or calcium phosphate, may also be used.

[0060] According to one aspect of the invention, in step a), at least one hydrophilic surfactant for stabilizing the emulsion is added. The HLB of said at least one hydrophilic surfactant is between 10 and 15.

[0061] The composition prepared in step a) comprises between 0.1% and 15% of said at least one surfactant and preferably between 0.1% and 5% of said at least one surfactant.

[0062] For the purposes of this invention, the HLB number of a surfactant is calculated using the formula HLB = 20 (1-Is / Ia), where Is represents the saponification value and Ia the acid value of the fatty acid used to prepare the surfactant. In the case of a mixture of surfactants, the HLB of the mixture is the weighted sum of the HLB values ​​of each individual surfactant. These two values, saponification and acid, are determined by methods described in the European Pharmacopoeia.

[0063] According to another particular aspect of the invention, the composition obtained during step a) of the process of the present invention has a mass content in oil phase of between 0.1% and 5% of the total mass of the composition obtained during step a) of the process of the present invention, and preferably between 0.2% and 1% by mass.

[0064] The hydrophilic surfactants used are generally chosen from among modified fats.

[0065] The modified fats used in the context of the present invention may be of mineral, vegetable, or animal origin. Examples of modified fats of mineral origin include petroleum-derived oils.

[0066] Modified vegetable fats include modified vegetable oils, for example, modified peanut, olive, sesame, soybean, wheat germ, grape seed, sunflower, castor, flaxseed, soybean, corn, coconut, palm, walnut, hazelnut or rapeseed oils.

[0067] Examples of modified animal fats include modified squalane, modified squalene, modified spermaceti oil, and modified tallow oil.

[0068] Modified fats include, in particular, alkoxylated derivatives of fats, and more specifically alkoxylated derivatives of oils or alkoxylated derivatives of alkyl esters of oils, and more specifically, ethoxylated and / or propoxylated derivatives of oils or ethoxylated and / or propoxylated derivatives of linear or branched methyl, ethyl, propyl, or linear or branched butyl esters of said oils. More specifically, the invention relates to a composition as defined above, in which the modified fat is selected from ethoxylated derivatives of oils having a number of moles of ethylene oxide between 1 and 10.

[0069] Modified fats also include esters of fatty acids and polyols or ethers of fatty alcohols and polyols, and more specifically, esters of fatty acids with a hexol, such as sorbitol or mannitol, or esters of fatty acids with a hexol anhydride, such as sorbitan or mannitan; alkoxylated derivatives of esters of fatty acids and polyols or alkoxylated derivatives of fatty alcohol ethers and polyols, such as alkoxylated fatty acid triglycerides; alkoxylated esters of polyglycerol fatty acids, and more specifically, alkoxylated esters of fatty acids with a hexol, such as sorbitol or mannitol, or alkoxylated esters of fatty acids with a hexol anhydride, such as sorbitan or mannitol. mannitane having a number of moles of ethylene oxide between 1 and 20.

[0070] In the context of this invention, fatty acid and polyol esters refer to fatty acid and polyol monoesters or fatty acid and polyol polyesters, such as fatty acid and polyol diesters or fatty acid and polyol triesters. The same applies to polyalkoxylated derivatives of said esters.

[0071] In the context of this invention, fatty acid and polyol ethers refer to fatty acid and polyol monoethers or fatty acid and polyol polyethers, such as fatty acid and polyol diethers or fatty acid and polyol triethers. The same applies to polyalkoxylated derivatives of said ethers.

[0072] Modified fats can be chosen from ethoxylated derivatives of fatty acid esters and polyols or ethoxylated derivatives of fatty alcohol ethers and polyols, and more particularly, ethoxylated esters of fatty acids with a hexol, such as for example sorbitol or mannitol, or ethoxylated esters of fatty acids with a hexol anhydride, such as sorbitan or mannitan, having a number of moles of ethylene oxide between 5 and 10.

[0073] Suitable fatty acids for the preparation of the modified fats described above include those with an average of 12 to 22 carbon atoms, such as, for example, those with 16 to 18 carbon atoms, such as oleic acid, ricinoleic acid or isostearic acid, and advantageously fatty acids that are liquid at 20 °C.

[0074] According to one aspect of the invention, in step b), at least one immunostimulant selected from saponins, animal and / or vegetable and / or mineral and / or synthetic oils, surfactants, aluminum hydroxide, lecithins and lecithin derivatives is added.

[0075] According to a particular aspect of the invention, the vaccine obtained in step b), comprises between 10% and 20% of the preparation obtained in step a) and between 80% and 90% of an antigenic medium.

[0076] The invention also relates to a vaccine composition comprising between 10% and 20% of a preparation obtained according to step a) of the process as described above and between 80% and 90% of an antigenic medium.

[0077] The vaccine composition as described above can be used as a preventive or curative drug. Depending on the nature of the antigen or in vivo generator, a composition according to the invention can be administered to fish, crustaceans such as shrimp, poultry, including geese, turkeys, pigeons, and chickens, canids such as dogs, felids such as cats, pigs, primates, bovines, ovines, horses, rodents such as rats, lagomorphs such as rabbits, caprines such as goats, and large mammals such as elephants. The composition according to the invention can also be administered to humans.

[0078] The composition can be administered conventionally via parenteral route, notably by subcutaneous, intramuscular, or intraperitoneal injection. It can also be administered orally, nasally, ocularly, by immersion, or by bathing.

[0079] The invention also relates to the use of an adjuvant composition obtained according to step a) of the process as described above for the preparation of a vaccine composition.

[0080] The percentages used throughout the application represent mass proportions.

[0081] Surprisingly, the comparison of the adjuvant properties of inverse latex dispersions with polymers of the same chemical composition but obtained by solvent-phase synthesis followed by precipitation demonstrates much superior adjuvant effects on both the humoral (diagram 1) and cellular response, accompanied by a high degree of safety (diagram 2).

[0082] The following examples illustrate the invention without, however, limiting it. Example 1: Adjuvant composition based on sodium polyacrylate obtained as a water-in-mineral-oil emulsion.

[0083] A fluid mineral oil, Marcol® < 52, supplied by Exxon, is used. The polymer emulsion obtained after polymerization is a white, viscous, oily gel. Its composition is 30% mineral oil (the continuous phase), 5% surfactant to stabilize the emulsion, 25% sodium polyacrylate, and 40% water. Upon contact with a 3% aqueous antigenic solution buffered at pH 7, the polymer-in-oil emulsion inverts to form a fluid polymer dispersion containing traces of oil. Example 2: Vaccine adjuvant based on sodium polyacrylate obtained by emulsion polymerization.

[0084] A dispersion in water containing 10% of the polymer described in Example 1 is prepared with sufficient mechanical stirring. The viscosity can be adjusted by adding inert ionized substances (salts) (e.g., sodium chloride) or immunostimulating substances (e.g., manganese gluconate). This ready-to-use gel can be redispersed at the required concentration in the intended antigenic medium. Additional surfactants can also be added to the gel to stabilize the oil-in-water dispersion. Example 3: Vaccine containing sodium polyacrylate obtained by emulsion polymerization.

[0085] A vaccine consists of 85% antigenic medium to which 15% of the preparation described in example 2 is added. The addition of other immunostimulants such as saponins, animal and / or vegetable and / or mineral and / or synthetic oils, surfactants, aluminum hydroxide, lecithins and lecithin derivatives, is possible.

Claims

1. A process for preparing a vaccine, comprising: - A step a) of dispersing, in a physiologically acceptable aqueous solution, an inverse latex of a partially or totally salified acrylic acid homopolymer in the form of a sodium salt, branched or cross-linked, as well as a hydrophilic surfactant, the HLB of which is between 10 and 15, to obtain an adjuvant composition comprising, for 100% of its weight, between 0.5% and 30% by weight of said inverse latex and between 0.1% and 15% of said hydrophilic surfactant; - A step b) of mixing the adjuvant composition obtained in step a) into an antigenic medium, to form said vaccine.

2. The process according to claim 1, wherein the adjuvant composition obtained in step a) comprises, for 100% of its weight, between 5% and 15% by weight of said inverse latex and between 0.1% and 5% of said hydrophilic surfactant.

3. The process according to any one of claims 1 or 2, wherein the adjuvant composition obtained in step a) exhibits an oil phase content by weight comprised between 0.1% and 5% by weight.

4. The process according to claim 3, wherein the adjuvant composition obtained in step a) exhibits an oil phase content by weight comprised between 0.2% and 1% by weight.

5. The process according to any one of claims 1 to 4, wherein at least one immunostimulating substance is added during step a).

6. The process according to any one of claims 1 to 5, wherein step b) consists of mixing 10% to 20% of the adjuvant composition obtained in step a) with 80% to 90% of an antigenic medium.

7. The process according to any one of claims 1 to 6, wherein at least one immunostimulating substance chosen from saponins, animal and / or vegetable and / or mineral and / or synthetic oils, surfactants, aluminum hydroxide, lecithins and lecithin derivatives is added during step b).