Vaccine adjuvant comprising reverse phase microlatex

Abstract

Disclosed is a vaccine adjuvant comprising at least one inverse microlatex comprising at least one oil, at least one surfactant, at least one polymer, such as, for example, a fully or partially neutralized polyacrylate in the form of an alkali metal salt or an ammonium salt, which can be completely sterilized by filtration or by heating in an autoclave and can be emulsified in one step with an aqueous phase comprising only the vaccine antigen.

Description

[0001] The present invention relates to a specific vaccine adjuvant, its preparation and a vaccine containing the vaccine adjuvant.

[0002] Vaccine compositions generally consist of an antigen, an immunogenic compound that induces protection against the disease of interest, and a vaccine adjuvant that is capable of amplifying the immune response of the vaccinated animal to the antigen. In particular, the use of an adjuvant in a vaccine composition can increase the intensity of the humoral or cellular immune response conferred by a dose of the vaccine, thereby ensuring a better level of protection; prolonging the protection period conferred by a dose of the vaccine; obtaining an efficacy comparable to that conferred by a full dose without the use of an adjuvant at a lower antigen dose; and reducing the number of immunizations required to ensure vaccine protection.

[0003] Various types of immunoadjuvants have been developed in the past. Among the prior art solutions for obtaining immunoadjuvants, mention may be made of emulsions comprising at least one oil phase and at least one aqueous phase (such as, for example, Freund's adjuvant), liposomes, synthetic immunostimulatory polymers, adjuvants of biological origin (saponins, chitosans, cytokines, oligonucleotides, etc.) or water-insoluble mineral salts (such as, for example, the very commonly used aluminum hydroxide).

[0004] Oily vaccine adjuvants are composed of oils and surfactants, and vaccines in the form of emulsions can be formulated, the aqueous phase of these emulsions containing vaccine antigens. Among the oils used, oils of plant origin, mineral oils, synthetic oils and oils of animal origin can be mentioned. The surfactant present in the oily adjuvant is an emulsifying surfactant with hydrophilic characteristics, characterized in that the hydrophilic-lipophilic balance (HLB) value is between 8 and 19, more particularly between 8 and 15. This hydrophilic surfactant can be composed of, for example, alkyl polyglycosides or mixtures of alkyl polyglycosides; saponins; lecithin; polyoxyethylated alkanols; polymers containing polyoxyethylene and polyoxypropylene blocks; esters obtained by condensation of fatty acids (advantageously liquid fatty acids at 20°C) with sugar polyols (such as, for example, sorbitol, mannitol or glycerol); esters obtained by condensation of fatty acids (advantageously liquid fatty acids at 20°C) with ethoxylated sugars.

[0005] The surfactant present in the oily adjuvant may also be an emulsifying surfactant of the "water-in-oil" type, meaning a surfactant having a sufficiently low HLB value (preferably greater than or equal to 1 and less than 8.0) for obtaining a water-in-oil emulsion in which an aqueous phase is dispersed in a lipophilic fatty phase. Among the water-in-oil surfactants, mention may be made of anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters.

[0006] The vaccine emulsion obtained can be of the water-in-oil type, the oil-in-water type or the water-in-oil-in-water type, depending in particular on the nature of the surfactant system used. In particular, adjuvants of the water-in-oil emulsion type can significantly increase the humoral and cellular responses to the vaccine antigens over an extended period of time compared to unadjuvanted vaccines or vaccines adjuvanted with aqueous adjuvants (e.g., aluminum hydroxide). This long-term response can reduce the number of vaccine injections. Adjuvants of the water-in-oil emulsion type are particularly useful for the preparation of vaccine compositions for the vaccination of cattle, sheep, goats, fish and poultry species against viral, bacterial or parasitic pathogens.

[0007] Some synthetic polymers also have immunostimulatory properties and have been used as vaccine adjuvants.

[0008] Among the immunostimulatory polymers used as adjuvants for veterinary vaccines, mention may be made in particular of block copolymers of polyoxyethylene and polyoxypropylene (POE-POP), polyethyleneimines, homopolymers of acrylic acid in sodium form, copolymers of acrylic acid and acrylic acid esters (also known as carbomers). The polymers obtained from acrylic acid, methacrylic acid, acrylic acid esters or methacrylic acid esters may be synthesized according to a precipitation polymerization method in a suitable solvent or by inverse emulsion polymerization, as described in the patent application published under number FR 2922767 A1. Among the acrylic acid polymers, mention will be made, for example, of the polyols marketed under the trade name CARBOPOL by the company Lubrizol. TM Commercial polymers are described, inter alia, in the US patents published under the numbers US 5373044, US 2798053 and in the European patent application EP 0301532 A2.

[0009] Carbomer (or acrylic acid polymer) is used as a vaccine adjuvant at a level of about one percent by weight, and its dilution is in the form of an easily injectable liquid and translucent vaccine.

[0010] These polymeric adjuvants have a very good safety profile and induce strong short-term responses against the relevant antigens and are used in particular for vaccination of pigs, as described, for example, in the patent application published under number WO 2007094893).

[0011] A promising research avenue consists in formulating these polymeric adjuvants in combination with emulsion-type oily adjuvants, with the goal of combining the immunostimulatory properties of both types of adjuvants to obtain an adjuvant with improved properties, as described, for example, in US Pat. No. 3,919,411.

[0012] However, combining these two technologies (i.e., oily adjuvants and polyacrylate gels) to obtain ready-to-use polymeric oily immunoadjuvants containing polyacrylates that are stable and can be directly emulsified by the user is a formidable challenge, especially in terms of adjuvant stability and sterilization.

[0013] For the purposes of the present invention, a "ready-to-use polymeric oily immunoadjuvant" is understood to mean a mixture consisting of an oil phase containing at least one surfactant and a polymer, which mixture has been sterilized and can be used immediately by mixing with an aqueous antigenic medium in an emulsification step. When the mixture is in contact with an aqueous phase (containing antigens and / or active ingredients), an emulsion is formed due to the use of a low shear or high shear stirring system. This type of adjuvant (hereinafter referred to as a "polymeric oily adjuvant") is used to obtain a preventive or therapeutic vaccine emulsion that is stable over time.

[0014] Several strategies can be considered and determined for the preparation of polymeric oil adjuvants, but each strategy presents some technical problems:

[0015] 1) A first method consists in adding to the oil phase at least one polyacrylic acid (a polymer in which the carboxyl groups are not salified and in the form of a powder). In this case, the powder is hardly stable in the oil in the form of a suspension and may present sedimentation problems over time. Moreover, the adjuvant obtained in this way leads to the production of an acidic emulsion, since the polymer is not neutralized and more particularly cannot be neutralized during the emulsification process. All these parameters mean that this technical solution is not satisfactory.

[0016] 2) A second method consists in adding an aqueous gel (formed beforehand by adding at least one polyacrylic acid to water) to an oily adjuvant. In this case, the dispersion of the aqueous gel in the oil phase has a first limitation, namely that the homogeneity of the mixture obtained at the end of this method is not guaranteed. In addition, this method has the major risk of causing phase separation of the dispersed phase and then of causing inhomogeneity of the desired product.

[0017] 3) A third approach is to disperse polymeric adjuvants such as polyacrylates in the form of an inverse latex (or a W / O emulsion whose dispersed aqueous phase comprises polyacrylates whose carboxyl functional groups have been previously neutralized in the form of alkali metal salts or ammonium salts) in an oily adjuvant. However, phase separation is observed over time, resulting in inhomogeneities in the oily adjuvant.

[0018] The difficulty in formulating a ready-to-use polymeric oil adjuvant is also related to the sterilization step of each compound incorporated into the vaccine composition. In particular, vaccine compositions intended for parenteral administration must be sterilized under aseptic conditions with the antigen before formulation. Among the sterilization methods that can be used, it can be noted that the product is sterilized by heating in an autoclave, followed by a step of sterilization by filtration on a filter with a pore size of 0.2 micrometers, or a step of irradiation with gamma rays.

[0019] Since the vaccine composition is in the form of an emulsion which cannot be sterilized, it is necessary to sterilize the oily adjuvant by filtration or by heating in an autoclave before the step of emulsification with the antigen medium. It should also be noted that the surfactants contained in the oily adjuvant are generally incompatible with irradiation sterilization.

[0020] As for the polymer vaccine adjuvants currently on the market, their cross-linked structure and thickening properties make it impossible to filter through a sterilizing filter with a pore size of 0.2 microns and to sterilize by irradiation. Therefore, heat exposure by autoclave is the only sterilization technology applicable to polymer vaccine adjuvants. Since this technology requires the preparation of a dilute solution of the polymer adjuvant in water, the polymer adjuvant cannot be sterilized by this approach when combined with an oily adjuvant.

[0021] From the above-noted elements, it can be seen that a sterile vaccine containing a combination of a polymeric adjuvant and an oily adjuvant needs to be prepared as follows:

[0022] - on the one hand, sterilizing the oily adjuvant by heating in an autoclave or by sterile filtration, and

[0023] - on the other hand, hydrating, diluting and sterilizing the polymer adjuvant in solution by heating in an autoclave, and

[0024] - the polymer adjuvant is aseptically mixed with the aqueous antigen medium, and

[0025] -The aqueous mixture of polymer adjuvant and antigen medium is aseptically emulsified with a sterile oil phase.

[0026] Therefore, the person skilled in the art considers this process to be expensive, since it involves many steps, consumes energy and does not allow for direct marketing of the mixture of the combination of polymeric adjuvant and oily adjuvant.

[0027] Therefore, a solution is needed which consists in providing a polymeric oily adjuvant containing at least one oil and at least one polymer such as, for example, a polyacrylate, which is stable over time for at least 1 year and more particularly for at least 2 years at 20° C. (“stable” is understood to mean the absence of phase separation, solidification of the polymer during storage), is easily sterilizable and allows for the realization of an emulsion which is stable over time for 1 year at +4° C. and for at least 1 month at +37° C. (i.e., without the occurrence of sedimentation or phase separation). The polymeric oily adjuvant according to the invention must make it possible to obtain a vaccine composition which is effective from an immunological point of view.

[0028] The solution of the present invention is a vaccine adjuvant comprising at least one inverse microlatex.

[0029] For the purposes of the present invention, an inverse microlatex denotes an inverse microemulsion comprising at least one polymer of the polyelectrolyte type.

[0030] For the purposes of the present invention, "microemulsion" means a mixture of two immiscible liquids that is thermodynamically stable (stabilized by the presence of a surfactant system comprising at least one emulsifying surfactant). Microemulsions are generally transparent because the droplet size of the dispersed phase is characterized by an average particle size of less than or equal to 200 nanometers, and preferably less than or equal to 100 nanometers.

[0031] For the purposes of the present invention, "reverse microemulsion" means a microemulsion as defined above, wherein the dispersed phase is an aqueous phase and the continuous phase is an oily phase.

[0032] For the purposes of the present invention, "polyelectrolyte polymers" means polymers in which all or some of the monomer units present in the polymer have ionizable chemical functional groups. Thus, anionic polyelectrolyte polymers predominantly comprise monomer units having anionic functional groups, and cationic polyelectrolyte polymers predominantly comprise monomer units having cationic functional groups.

[0033] For the purposes of the present invention, "anionic crosslinked polyelectrolyte polymer" means an anionic polyelectrolyte polymer as defined above and which, via its constituent monomer units, contains at least one monomer unit having at least two reactive functional groups which can be used during the polymerization reaction and can thus link at least two polymer chains together.

[0034] The inverse microlatex is prepared by implementing a method comprising the following steps:

[0035] - a step a) of preparing an aqueous solution containing the monomers and optionally various additives such as, for example, crosslinking monomers,

[0036] - a step b) of adding at least one oil, at least one surfactant to the aqueous phase obtained in step a), and mixing these various components,

[0037] - step c) of adding a free radical initiator to initiate a free radical polymerization reaction in the thermally insulating medium, and

[0038] - a step d) of homogenization by mechanical stirring of the reaction medium obtained during step c).

[0039] Such a process for preparing inverse microlatex is described in the European patent application published under the number EP 1 371 692 A1 , which is incorporated into the present patent application by reference.

[0040] Depending on the circumstances, the vaccine adjuvant according to the present invention may have one or more of the following characteristics:

[0041] - the inverse microlatex comprises an oil phase, an aqueous phase, at least one water-in-oil (W / O) surfactant, at least one oil-in-water (O / W) surfactant and an anionic crosslinked polyelectrolyte; wherein the anionic crosslinked polyelectrolyte comprises at least one crosslinking monomer and at least one hydrophilic monomer unit;

[0042] - the hydrophilic monomer units are derived from acrylic acid which is fully or partially salified with an alkali metal salt or an alkaline earth metal salt or an ammonium salt;

[0043] - complete or partial salification of acrylic acid with sodium or ammonium salts, preferably with sodium salts;

[0044] - Anionic crosslinked polyelectrolytes comprising monomer units having formula (1):

[0045]

[0046] wherein: R1 is selected from -H, -CH3, -C2H5 and -C3H7, preferably -CH3, n is between 0 and 50, and m is between 8 and 22;

[0047] - the adjuvant further comprises an oil (H1), at least one water-in-oil surfactant (E1) and at least one oil-in-water surfactant (E2);

[0048] - the adjuvant comprises between 1 and 10% by weight of a water-in-oil surfactant (E1), preferably from 3 to 8% by weight;

[0049] - the adjuvant comprises between 1 and 10% by weight of a water-in-oil surfactant (E2), preferably from 3 to 8% by weight;

[0050] - The adjuvant comprises per 100% by weight:

[0051] a) from 50 to 97.5% by weight of said oil (H1), preferably from 60 to 90%;

[0052] b) from 1 to 10% by weight of said water-in-oil surfactant (E1), preferably from 3 to 8%;

[0053] c) from 1 to 10% by weight of said oil-in-water surfactant (E2), preferably from 3 to 8%; and

[0054] d) from 0.5 to 30% by weight of at least one inverse microlatex, preferably from 1 to 10%, more preferentially between 1 and 10%,

[0055] It will be appreciated that the sum of the weight contents a)+b)+c)+d) equals 100%.

[0056] - The vaccine adjuvant according to the present invention is characterized in that the oil (H1) is white mineral oil. It is worth noting that the oil (H1) can also be a mineral oil, such as liquid paraffin, liquid petroleum jelly or isoparaffin.

[0057] Preferentially, the inverse microlatex included in the vaccine adjuvant according to the invention will contain, per 100% of its weight:

[0058] -a') from 10 to 40% by weight of water, preferably from 12 to 30% by weight,

[0059] - b') from 30 to 50% by weight of oil (H2), preferably from 38 to 50% by weight,

[0060] -c') from 5 to 30% by weight, preferably from 10 to 25% by weight, of a mixture of at least one water-in-oil surfactant (E'1) and at least one oil-in-water surfactant (E'2),

[0061] -d') from 5 to 35% by weight, preferably from 10 to 30% by weight, of said anionic crosslinked polyelectrolyte,

[0062] It will be appreciated that the sum of the weight contents a')+b')+c')+d') equals 100%.

[0063] The oil (H1) included in the vaccine adjuvant that is the subject of the present invention may be identical to or different from the oil (H2) included in the inverse microlatex.

[0064] According to a particular aspect, the oil (H1) included in the vaccine adjuvant that is the subject of the invention is identical to the oil (H2) included in the inverse microlatex.

[0065] Oil (H2) and oil (H1) are in particular selected from:

[0066] - oils of vegetable origin, such as sweet almond oil, coconut oil, monoi oil, castor oil, jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil, wheat germ oil, corn germ oil, soybean oil, cottonseed oil, alfalfa oil, poppy oil, red kuri squash oil, evening primrose oil, millet oil, barley oil, rye oil, safflower oil, kuju oil, passion fruit oil, hazelnut oil, palm oil, shea butter, almond oil, red calendula oil, garlic mustard oil, avocado oil, calendula oil;

[0067] - vegetable oils and their ethoxylated methyl esters;

[0068] - oils of animal origin, such as squalene or squalane;

[0069] - Synthetic oils, in particular fatty acid esters such as butyl myristate, propyl myristate, cetyl myristate, isopropyl palmitate, butyl stearate, cetyl stearate, isopropyl stearate, isocetyl stearate, lauryl oleate, hexyl laurate, propylene glycol dicaprylate, esters derived from lanolin acid (such as isopropyl lanolate, isocetyl lanolate), fatty acid mono-, di- and triglycerides (such as triheptanoin), alkyl benzoates, poly(α-olefins), polyolefins (such as poly(isobutane)), synthetic isoalkanes (such as isohexadecane, isododecane), and perfluorinated oils. Silicone oils can also be used in the context of the present invention.

[0070] Among the latter, mention may be made more particularly of polydimethylsiloxanes, polymethylphenylsiloxanes, silicones modified by amines, silicones modified by fatty acids, silicones modified by alcohols, silicones modified by alcohols and fatty acids, silicones modified by polyether groups, epoxy-modified silicones, silicones modified by fluorinated groups, cyclic silicones, and silicones modified by alkyl groups. However, for practical reasons, it may be desirable that the fatty phase does not comprise silicone oils;

[0071] - mineral oils, hydrocarbons obtained by petroleum distillation and subsequent treatment steps (e.g. desulfurization, deasphalting, extraction of aromatic compounds, extraction of waxes and other finishing treatment steps), such as liquid paraffin, liquid petroleum jelly, white mineral oil and isoparaffins. White mineral oil means a mineral oil that complies with the provisions of FDA 21 CFR 172.878 and CFR 178.3620 (a) as listed in the United States Pharmacopoeia US XXIII (1995) and meets the purity requirements of the European Pharmacopoeia (2008). For example, mention may be made of the product sold under the brand name Marcol TM 、Primol TM , Drakeol TM , Eolane TM 、Klearol TM 、Puretol TM Oil for sale;

[0072] - light oil. For the purposes of the present invention, "light oil" means an oil (H2) with a low boiling point (100° C. to 250° C. at atmospheric pressure) included in the fatty phase of the inverse microlatex, also consisting of at least one oil with a higher boiling point; said light oil is intended to be evaporated during the concentration step by distillation of the inverse microlatex formed to obtain a concentrated inverse microlatex. As a light oil meeting this definition, mention may be made of the oil sold under the trade name Isopar TM C. Isopar TM E. Isopar TM G. Isopar TM H. Isopar TM L and IsoparTM M isoalkanes containing 7 to 14 carbon atoms, which are marketed.

[0073] The water-in-oil surfactant (E1) included in the vaccine adjuvant that is the subject of the present invention is identical to or different from the water-in-oil surfactant (E'1) included in the inverse microlatex.

[0074] According to a particular aspect, the water-in-oil surfactant (E1) included in the vaccine adjuvant that is the subject of the invention is identical to the water-in-oil surfactant (E′1) included in the inverse microlatex.

[0075] For the purposes of the present invention, a "surfactant" is a compound that modifies the surface tension between two surfaces and is an amphiphilic molecule, that is, it has in its structure a lipophilic part and another hydrophilic part. Thus, a surfactant can dissolve and / or disperse a phase of certain polarity in another phase of different polarity.

[0076] The term "water-in-oil surfactant" means a surfactant having a sufficiently low HLB value (preferably greater than or equal to 1 and less than 8.0) for obtaining a water-in-oil emulsion in which an aqueous phase is dispersed in a lipophilic fatty phase.

[0077] Among the water-in-oil surfactants (E1) and (E′1), mention may be made of anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters.

[0078] The term "hexitol" refers to hexols derived from hexose, such as sorbitol, mannitol, dulcitol (also known as galactitol) or iditol.

[0079] The term "anhydrohexitol" means a product produced by the dehydration of a hexitol. Examples of anhydrohexitol include, for example, anhydrosorbitol, anhydromannitol, anhydrodulcitol or anhydroiditol. The term "anhydrohexitol" means a monoanhydrohexitol (such as, for example, sorbitan, mannide, dulcitan, iditan), optionally as a mixture with a dianhydrohexitol (such as isosorbide, isomannide, isodulcide, isoidide) obtained as a by-product during the same dehydration reaction.

[0080] The term "mixture of esters" denotes esters obtained from a single acid and a single hexitol, or from a single acid and from a mixture of several hexitols, or from a mixture of several acids and from a single hexitol, or from a mixture of several acids with several hexitols.

[0081] The expression "anhydrohexitol ester of a saturated or unsaturated, linear or branched aliphatic carboxylic acid containing 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups" denotes, for example, an ester of an acid selected from the group consisting of dodecanoic acid, dodecenoic acid, tetradecanoic acid, tetradecenoic acid, hexadecanoic acid, hexadecenoic acid, octadecenoic acid, octadecenoic acid, octadecadienoic acid, octadecatrienoic acid, octadecatetraenoic acid, eicosenoic acid, eicosenoic acid, eicosadienoic acid, docosanoic acid, docosenoic acid, hydroxyhexadecanoic acid, hydroxyoctadecanoic acid, dihydroxydocosanoic acid or dihydroxyoctadecanoic acid.

[0082] The expression "anhydrohexitol ester of a saturated or unsaturated, linear or branched aliphatic carboxylic acid containing 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups" denotes, for example, an ester of an acid selected from the group consisting of lauric acid, isolauric acid, 4-dodecenoic acid, 5-dodecenoic acid, myristic acid, palmitic acid, arachidic acid, stearic acid, isostearic acid, oleic acid, vaccenic acid, linoleic acid, isogeranic acid, linolenic acid, arachidic acid, 10,13-eicosadienoic acid, behenic acid, erucic acid, cetyl ester, citric acid, 3-hydroxyhexadecanoic acid, 4-hydroxyhexadecanoic acid, 11-hydroxyhexadecanoic acid, 16-hydroxyhexadecanoic acid, 12-hydroxystearic acid, basilic acid or 8,9-dihydroxystearic acid.

[0083] These esters are obtained by esterification of the corresponding acids and anhydrohexitols. The esterification reaction is known to the person skilled in the art; it is described in numerous patents and reference books.

[0084] Among the anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, mention may be made of sorbitan laurate (marketed under the trade name Montane TM 20), mannose laurate, dulracetam laurate, sorbitan isolaurate, mannose laurate, dulracetam isolaurate, sorbitan palmitate, mannose palmitate, dulracetam palmitate, sorbitan stearate (marketed under the trade name Montane TM 60), mannide stearate, dulracetam stearate, sorbitan isostearate (sold under the trade name Montane TM 70), mannide isostearate, dulracetam isostearate, sorbitan oleate (sold under the trade name Montane TM 80), mannide oleate (sold under the trade name Montanide TM 80), dulracetam oleate, sorbitan sesquioleate (sold under the trade name Montane TM83), mannide sesquioleate, sorbitan trioleate (sold under the trade name Montane TM 85 sales), mannose trioleate, sorbitan behenate, mannose behenate, sorbitan arachidate and mannose arachidate.

[0085] Among the water-in-oil surfactants (E1) and (E′1), mention may also be made of the surfactants marketed by the applicant under the name Montanox TM 81 Sorbitan oleate ethoxylated with 5 mol of ethylene oxide (5 EO) sold by the applicant under the name Simulsol TM OC72 is sold as diethoxylated (2 EO) oil cetyl alcohol.

[0086] The terms "oil-in-water surfactant" (E2) and (E'2) denote surfactants having a sufficiently high HLB value (preferably greater than or equal to 8.0 and less than or equal to 20, preferably greater than or equal to 8.0 and less than or equal to 15.0) for obtaining oil-in-water emulsions in which a lipophilic fatty phase is dispersed in an aqueous phase.

[0087] Among the oil-in-water surfactants (E2) and (E′2), mention may be made of dehydrated hexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters, which are subsequently subjected to a step of adding ethylene oxide to a variable extent ranging from 2 to 30 molar equivalents of ethylene oxide.

[0088] Among the anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups and ethoxylated, mention may in particular be made of the ethoxylated sorbitan esters and more particularly of the sorbitan esters disclosed by the Applicant under the name Montanox TM80 ethoxylated sorbitan oleate with 20 mol of ethylene oxide (20 EO), ethoxylated sorbitan oleate containing 15 mol of ethylene oxide (15 EO), ethoxylated sorbitan oleate with 10 mol of ethylene oxide (10 EO), ethoxylated sorbitan oleate with 5 mol of ethylene oxide (5 EO), ethoxylated mannide oleate with 20 mol of ethylene oxide (20 EO), ethoxylated mannide oleate with 15 mol of ethylene oxide (15 EO), ethoxylated mannide oleate with 10 mol of ethylene oxide (10 EO), ethoxylated mannide oleate with 5 mol of ethylene oxide (5 EO), ethoxylated mannide oleate with 20 mol of ethylene oxide (20 EO), ethoxylated mannide oleate with 15 mol of ethylene oxide (15 EO), ethoxylated mannide oleate with 10 mol of ethylene oxide (10 EO), ethoxylated mannide oleate with 5 mol of ethylene oxide (5 EO), ethoxylated sorbitan stearic acid with 20 mol of ethylene oxide (20 EO), ethoxylated mannide oleate with 10 mol of ethylene oxide (10 EO) ethoxylated sorbitan stearic acid, ethoxylated with 5 mol of ethylene oxide (5 EO), ethoxylated mannose stearic acid with 20 mol of ethylene oxide (20 EO), ethoxylated mannose stearic acid with 10 mol of ethylene oxide (10 EO), ethoxylated mannose stearic acid with 5 mol of ethylene oxide (5 EO), a methoxy ... TM 20 mol of sorbitan laurate ethoxylated with 20 mol of ethylene oxide (20 EO), sorbitan laurate ethoxylated with 10 mol of ethylene oxide (10 EO), sorbitan laurate ethoxylated with 5 mol of ethylene oxide (5 EO), mannide laurate ethoxylated with 20 mol of ethylene oxide (20 EO), mannide laurate ethoxylated with 10 mol of ethylene oxide (10 EO), mannide laurate ethoxylated with 5 mol of ethylene oxide (5 EO), sorbitan trioleate ethoxylated with 5 mol of ethylene oxide, sorbitan trioleate ethoxylated with 10 mol of ethylene oxide, sorbitan trioleate ethoxylated with 20 mol of ethylene oxide, marketed by the applicant under the name Montanox TM 85 sold as sorbitan trioleate ethoxylated with 25 mol of ethylene oxide, mannide trioleate ethoxylated with 5 mol of ethylene oxide, mannide trioleate ethoxylated with 10 mol of ethylene oxide, mannide trioleate ethoxylated with 20 mol of ethylene oxide, and mannide trioleate ethoxylated with 25 mol of ethylene oxide.

[0089] Among the oil-in-water surfactants (E2) and (E′2), mention may be made of ethoxylated vegetable oils such as, for example, those commercialized by the Applicant under the name Simulsol TMCastor oil ethoxylated with 25 EO sold in 1292, marketed by the applicant under the name Simulsol TM Castor oil ethoxylated with 40 EO sold as OL50, corn oil ethoxylated with 3 mol of ethylene oxide (3 EO), corn oil ethoxylated with 8 mol of ethylene oxide (8 EO), corn oil ethoxylated with 10 mol of ethylene oxide (10 EO), corn oil ethoxylated with 20 mol of ethylene oxide (20 EO), corn oil ethoxylated with 30 mol of ethylene oxide (30 EO), corn oil ethoxylated with 40 mol of ethylene oxide (40 EO), rapeseed oil ethoxylated with 3 mol of ethylene oxide (3 EO), rapeseed oil ethoxylated with 10 mol of ethylene oxide (10 EO), rapeseed oil ethoxylated with 20 mol of ethylene oxide (20 EO), rapeseed oil ethoxylated with 30 mol of ethylene oxide (30 EO), EO), rapeseed oil ethoxylated with 40 mol of ethylene oxide (40 EO), sunflower oil ethoxylated with 3 mol of ethylene oxide (3 EO), sunflower oil ethoxylated with 10 mol of ethylene oxide (10 EO), sunflower oil ethoxylated with 20 mol of ethylene oxide (20 EO), sunflower oil ethoxylated with 30 mol of ethylene oxide (30 EO), sunflower oil ethoxylated with 40 mol of ethylene oxide (40 EO).

[0090] Among the oil-in-water surfactants (E2) and (E'2), mention may be made of alkyl polyglycosides, more particularly alkyl polyglucose and alkyl polyxylose, or mixtures of alkyl polyglycosides, lecithin, saponin, polyoxyethylated alkanols, polymers comprising polyoxyethylene and polyoxypropylene blocks, the alkyl polyols commercially available from the Applicant under the name Silmulsol TM Lauryl alcohol ethoxylated with 7 mol of ethylene oxide (7EO) sold by P7, pentaethoxylated (5EO) cetyl alcohol, octaethoxylated (8EO) cetyl alcohol, sold by the applicant under the name Simulsol TM OC 710 deethoxylated (10EO) oil cetyl alcohol sold under the name G-1086 TM and G-1096 TM Polyethoxylated sorbitan hexaoleate is sold.

[0091] The crosslinking monomer unit refers to a unit derived from a monomer having at least two reactive functional groups, through which a covalent bond can be established between the extended polymer chain and the crosslinking monomer. For example, the crosslinking monomer unit can be derived from a monomer that can contain at least two vinyl functional groups in its structure, and when subjected to a free radical polymerization reaction with an acrylic monomer, the crosslinking monomer can covalently bond with two chains of the acrylic polymer during the diffusion step to obtain a crosslinked polymer.

[0092] Crosslinked polymers are understood to mean nonlinear polymers in the form of a three-dimensional network which is insoluble in water but can swell in water and thus results in a chemical gel.

[0093] A crosslinked polymer means a polymer composed of at least one crosslinking monomer unit and at least one other monomer unit, and more particularly a hydrophilic monomer unit. For the purposes of the present invention, a hydrophilic monomer unit is understood to mean a monomer unit resulting from a monomer that is soluble in water, and more particularly soluble in water at a temperature greater than or equal to 5° C., more particularly at a temperature greater than or equal to 10° C., more particularly at a temperature greater than or equal to 20° C.

[0094] The cross-linked polymer may comprise hydrophilic monomer units derived from:

[0095] 2-Methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid in free acid form or in partially or completely salified form; acrylic acid in free acid form or in partially or completely salified form, methacrylic acid in free acid form or in partially or completely salified form, itaconic acid in free acid form or in partially or completely salified form, 2-carboxyethylacrylic acid in free acid form or in partially or completely salified form, maleic acid in free acid form or in partially or completely salified form, acrylamide, N,N-dimethylacrylamide, methacrylamide, N-isopropylacrylamide, 2-hydroxyethyl acrylate, 2,3-dihydroxypropyl acrylate, 2-hydroxyethyl methacrylate, 2,3-dihydroxypropyl methacrylate, vinyl pyrrolidone.

[0096] The crosslinking monomer units are monomer units derived from diene or polyene monomers, in particular selected from ethylene glycol dimethacrylate, diethylene glycol diacrylate, ethylene glycol diacrylate, diallyl urea, triallylamine, trimethylolpropane triacrylate, methylenebis(acrylamide) or mixtures of these compounds, diallyloxyacetic acid or its salts (e.g. sodium diallyloxyacetate), or mixtures of these compounds.

[0097] The surfactants present in the adjuvant according to the invention are water-in-oil surfactants (E1) or water-in-oil surfactants (E′1) as defined and described above, having lipophilic properties (characterized by an HLB value greater than or equal to 1 and less than 8), and oil-in-water surfactants (E2) or oil-in-water surfactants (E′2) as defined and described above, having hydrophilic properties (characterized by an HLB value greater than or equal to 8 and less than or equal to 20).

[0098] Another subject of the invention is the use of the inverse microlatex as defined previously for the preparation of a vaccine adjuvant.

[0099] The method for preparing the vaccine adjuvant according to the present invention comprises the step of sterilizing the vaccine adjuvant by sterile filtration or autoclaving.

[0100] The filtration will preferably be carried out on a filter having pores with a mean diameter less than or equal to 0.22 micrometers (see standard ISO 13408-2:2018 (en)).

[0101] Before filtering, the adjuvant may be pre-filtered. For example, the adjuvant may be pre-filtered using a hydrophobic filter having pores with an average diameter of 0.45 μm. The pre-filtration and filtration steps may be performed in a single step involving the use of a double membrane hydrophobic filter, wherein the first membrane has pores with an average diameter of 0.45 μm and the second membrane has pores with an average diameter of 0.2 μm, or a combination of a first hydrophobic filter having pores with an average diameter of 0.45 μm and a second hydrophobic filter having pores with an average diameter of 0.2 μm. This means that the first membrane or first filter has larger pores than the second membrane or second filter. Ideally, the first membrane or first filter has pores with a diameter greater than or equal to 0.3 μm, preferably with a diameter less than or equal to 0.6 μm and more particularly equal to 0.45 μm. The second membrane has pores with a diameter less than or equal to 0.22 μm to obtain a sterilizing effect.

[0102] Filters and membranes used for filtering and / or pre-filtering adjuvants may consist of a polymer support of the PTFE (polytetrafluoroethylene) or PP (polypropylene) type.

[0103] Preferably, the method for preparing the adjuvant according to the invention comprises the following steps:

[0104] a) preparing, with mechanical stirring and at ambient temperature, an oil phase comprising at least one oil and an emulsified system comprising at least one water-in-oil surfactant (E1) and / or oil-in-water surfactant (E2);

[0105] b) adding at least one inverse microlatex at ambient temperature with mechanical stirring;

[0106] c) Maintaining the mechanical stirring at ambient temperature until a homogeneous mixture is obtained.

[0107] For the purposes of the present invention, ambient temperature is understood to mean a temperature higher than or equal to 15°C and lower than or equal to 30°C.

[0108] Another subject of the invention is a vaccine comprising an adjuvant according to the invention and at least one aqueous solution (S) of at least one antigen or at least one aqueous solution (S) of at least one in vivo generation agent of a compound comprising an amino acid sequence.

[0109] Preferably, the vaccine will contain:

[0110] - from 10 to 80% by weight of an adjuvant according to the invention, and

[0111] - From 20 to 90% by weight aqueous solution (S).

[0112] Preferably, the vaccine is in the form of a water-in-oil emulsion or an oil-in-water emulsion.

[0113] At least one in vivo generating agent of the antigen or the compound comprising the amino acid sequence means a killed microorganism (e.g., virus, bacteria or parasite), or a purified part of these microorganisms, or a live microorganism whose pathogenicity has been reduced. Examples of viruses that can constitute antigens according to the present invention include orthomyxoviruses such as influenza virus, paramyxoviruses such as Newcastle disease virus, coronaviruses such as infectious bronchitis virus, herpes viruses such as pseudorabies virus or Marek's disease virus. As microorganisms of the bacterial type that can constitute antigens according to the present invention, Escherichia coli, and microorganisms of the genus Pasteurella, the genus Avibacterium, the genus Staphylococcus and the genus Streptococcus can be mentioned. Examples of parasites include parasites of the genus Eimeria, the genus Trypanosoma and the genus Leishmania. Mention may also be made of recombinant viruses, in particular non-enveloped viruses, such as adenoviruses, vaccinia viruses, canarypox viruses, herpes viruses or baculoviruses. It also means a live, non-enveloped recombinant viral vector, the genome of which contains (preferably inserted into a part that is not essential for the replication of the corresponding enveloped virus) sequences encoding antigenic subunits that induce antibody synthesis and / or have a protective effect on the above-mentioned enveloped viruses or pathogenic microorganisms; these antigenic subunits may, for example, be proteins, glycoproteins, peptides or as peptide fractions and / or fractions that have a protective effect on infection by live microorganisms such as enveloped viruses, bacteria or parasites. The exogenous gene inserted into the microorganism may, for example, be derived from pseudorabies virus. Particular mention may be made of a recombinant plasmid consisting of a nucleotide sequence, into which an exogenous nucleotide sequence derived from a pathogenic microorganism or virus is inserted. The purpose of the latter nucleotide sequence is to allow the expression of a compound comprising an amino acid sequence, the purpose of which itself is to trigger an immune response in a host organism.

[0114] The concentration of antigen contained in a vaccine as defined above depends on the nature of the antigen and the nature of the individual to be treated. However, it is particularly noteworthy that the adjuvant according to the present invention can significantly reduce the usual antigen dosage required. The appropriate antigen concentration can be routinely determined by a person skilled in the art. Generally, the dosage is about 0.1 μg / cm 3 Up to 1 μg / cm 3 , more commonly at 1 μg / cm 3 and 100mg / cm 3 The concentration of the in vivo generating agent in the composition according to the invention also depends in particular on the nature of the generating agent and the host to which it is administered. This concentration can be easily determined by a person skilled in the art according to routine experiments. As an indication, when the in vivo generating agent is a recombinant microorganism, its concentration in the composition according to the invention is generally between 10 2and 10 15 Microorganisms / cm 3 between, and preferably between 10 5 and 10 12 Microorganisms / cm 3 When the in vivo generating agent is a recombinant plasmid, its concentration in the composition obtained by the method according to the subject matter of the invention may be between 0.01 g / dm 3 and 100g / dm 3 The vaccine as defined above is prepared by mixing the adjuvant phase and the antigen phase, optionally adding water or a pharmaceutically acceptable diluent medium.

[0115] The method for preparing a vaccine according to the present invention comprises the following steps:

[0116] a) preparing a vaccine adjuvant according to the present invention,

[0117] b) mixing the vaccine adjuvant obtained in step a) with the antigen medium.

[0118] Preferably, the antigenic medium is intended to form a vaccine and reference will be made to vaccine antigenic medium.

[0119] As mentioned above, antigenic medium means an aqueous medium comprising at least one antigen or at least one in vivo generating agent of a compound comprising an amino acid sequence.

[0120] Preferably, the mixture will be such that the vaccine will contain, per 100% of its weight, between 10 and 80% by weight of adjuvant and between 20 and 90% by weight of antigenic medium, preferably between 50 and 80% by weight of adjuvant and between 20 and 50% by weight of antigenic medium, and even more preferably between 50 and 70% by weight of adjuvant and between 30 and 50% by weight of antigenic medium.

[0121] During step b), an immunostimulant selected from saponins, animal and / or vegetable and / or mineral and / or synthetic oils, surfactants, aluminum hydroxide, lecithin and lecithin derivatives may optionally be added to the mixture.

[0122] Preferably, in step b), the antigenic medium is gradually added to the adjuvant under high shear stirring to form an emulsion.

[0123] At the end of the emulsification process, the vaccine is obtained in the form of a stable and homogeneous emulsion, preferably a water-in-oil vaccine.

[0124] The final vaccine can be administered immediately after manufacture and can be stored at a temperature of +4°C for at least 1 year (depending on the nature of the antigen or antigens present in the vaccine and their physicochemical stability over time).

[0125] Vaccines are intended for administration in human or veterinary therapy by injection and by oral, parenteral, mucosal or in ovo routes.

[0126] Examples of adjuvants according to the present invention are shown below.

[0127] Example 1: Preparation of inverse microlatex based on sodium polyacrylate

[0128] 1.1 Preparation of inverse microlatex (A), (B), (C) and (D)

[0129] The inverse micro-latex comprising the cross-linked sodium polyacrylate as a polymer is prepared according to the teaching content of the European patent disclosed with numbering 1 371 692 B1, which is incorporated herein by reference. More particularly, the teaching content of paragraphs

[0021] , paragraphs

[0025] and

[0026] , paragraphs

[0033] to

[0048] , even more particularly paragraphs

[0039] to

[0041] (Example 2) of the European patent disclosed with numbering 1 371 692 B1 is used to prepare the inverse micro-latex. For each in the micro-latex prepared, the liquid white mineral oil Marcol sold by Exxon Mobil (Exxon Mobil) company is used. TM 52. The same process for preparing the inverse microlatex is carried out in the presence of various weight concentrations of surfactants and makes it possible to obtain:

[0130] - inverse microlatex (A) when the weight of surfactant is equal to 14%,

[0131] - inverse microlatex (B) when the weight of surfactant is equal to 18%,

[0132] - inverse microlatex (C) when the weight of surfactant is equal to 22%,

[0133] - Inverse microlatex (D) when the weight of surfactant is equal to 25%.

[0134] The inverse microlattices (A), (B), (C) and (D) obtained after free radical polymerization are in the form of milky white to translucent oily compositions. These inverse microlattices contain 60% by weight of a mixture of an oil phase and a surfactant, 15% by weight of cross-linked sodium polyacrylate and 25% by weight of water.

[0135] 1.2 Preparation of inverse microlatex (E), (F), (G) and (H)

[0136] The method for preparing the inverse microlatex (A) was carried out using mannide oleate instead of sorbitan oleate as the water-in-oil surfactant to obtain the inverse microlatex (E).

[0137] The method for preparing the inverse microlatex (B) was carried out using mannide oleate instead of sorbitan oleate as the water-in-oil surfactant to obtain the inverse microlatex (F).

[0138] The method for preparing the inverse microlatex (C) was carried out using mannide oleate instead of sorbitan oleate as the water-in-oil surfactant to obtain the inverse microlatex (G).

[0139] The method for preparing the inverse microlatex (D) was carried out using mannide oleate instead of sorbitan oleate as the water-in-oil surfactant to obtain the inverse microlatex (H).

[0140] The inverse microlatexes (E), (F), (G) and (H) are in the form of milky white to translucent oily compositions containing 60% by weight of a mixture of an oil phase and a surfactant, 15% by weight of cross-linked sodium polyacrylate and 25% by weight of water.

[0141] Example 2: Preparation of inverse microlatex (A′), (B′), (C′) and (D′)

[0142] The method for preparing inverse microlatex (A), (B), (C) and (D) described in Example 1.1 is carried out in the presence of a mixture of acrylic acid and tetraethoxylated lauryl methacrylate (2 mol %) to obtain inverse microlatex (A′), (B′), (C′) and (D′), respectively.

[0143] The inverse microlatexes (A′), (B′), (C′) and (D′) are in the form of a milky white to translucent oily composition containing 60% by weight of a mixture of an oil phase and a surfactant, 15% by weight of a cross-linked copolymer of acrylic acid and tetraethoxylated lauryl methacrylate and 25% by weight of water.

[0144] Example 3: Preparation of inverse microlatex (B″) and (C″)

[0145] The method for preparing the inverse microlatex (B) of Example 1 is carried out in the presence of a relatively large amount of water to obtain an inverse microlatex (B″) in the form of a milky white to translucent oily composition containing 49% by weight of a mixture of an oil phase and a surfactant, 15% by weight of cross-linked sodium polyacrylate and 36% by weight of water.

[0146] The method for preparing the inverse microlatex (C) of Example 1 is carried out in the presence of a relatively large amount of water to obtain an inverse microlatex (C″) in the form of a milky white to translucent oily composition containing 49% by weight of a mixture of an oil phase and a surfactant, 15% by weight of cross-linked sodium polyacrylate and 36% by weight of water.

[0147] Example 4: Preparation of an adjuvant according to the present invention

[0148] The polymer oil adjuvant was prepared according to the following method:

[0149] a) preparing, with mechanical stirring and at ambient temperature, an oil phase comprising at least one oil and an emulsified system comprising at least one water-in-oil surfactant (E1) and / or oil-in-water surfactant (E2);

[0150] b) adding the inverse microlatex or inverse latex at ambient temperature with moderate mechanical stirring (50 to 150 rpm);

[0151] c) Maintain moderate mechanical stirring (50 to 150 rpm) at ambient temperature until a homogeneous mixture is obtained.

[0152] For the purposes of the present invention, ambient temperature is understood to mean a temperature higher than or equal to 15°C and lower than or equal to 30°C.

[0153] In this way, ADJ1, ADJ2, ADJ3, ADJ'1 adjuvants were prepared and characterized by the composition described in Table 1:

[0154] [Table 1]

[0155] ADJ 1 ADJ 2 ADJ 3 ADJ′1 <![CDATA[Marcol TM 52]]> 84％ 84％ 79.5％ 86％ Sorbitan oleate 6.5％ 6.5％ 7.5％ 6.5％ Polysorbate 80 4.5％ 4.5％ 3％ 5.5％ Microlatex (F) (Example 1) 5％ 0％ 10％ 0％ Microlatex (C') (Example 2) 0％ 5％ 0％ 0％ Inverse latex (1) 0％ 0％ 0％ 2％

[0156] Table 1: Adjuvants according to the invention and comparative adjuvants (composition in %)

[0157] (1): Sodium polyacrylate in the form of an inverse latex, the preparation of which is described in patent FR 2922767 B1.

[0158] Example 5: Evaluation of adjuvants according to the present invention and comparative adjuvants

[0159] 5.1 Filterability

[0160] The filterability of the adjuvants according to the invention and the comparative adjuvants were evaluated according to the following protocol:

[0161] - introduce 10 ml of adjuvant into a 2-piece 12 ml syringe,

[0162] - Connect a 0.22μm PTFE syringe filter with a diameter of 25mm,

[0163] - Applied weight of 3310g,

[0164] -Measure the quality of the filtration as a function of time.

[0165] The amount of adjuvant filtered was measured as a function of time and the results obtained for each adjuvant tested are reported in the following table:

[0166] [Table 2]

[0167]

[0168] Table 2: Kinetics of the amount of adjuvant ADJ1 according to the invention filtered

[0169] [Table 3]

[0170]

[0171] Table 3: Kinetics of the amount of adjuvant ADJ2 according to the invention filtered

[0172] [Table 4]

[0173]

[0174] Table 4: Kinetics of the amount of adjuvant ADJ3 according to the invention filtered

[0175] [Table 5]

[0176]

[0177] Table 5: Kinetics of the amount of adjuvant ADJ'1 according to the invention filtered

[0178] The results reported in Tables 2 to 5 show that on a hydrophobic filter made in particular of PTFE with an average pore diameter of 0.2 micrometer, the adjuvants ADJ1, ADJ2, ADJ3 according to the invention were filtered faster than the comparative adjuvant ADJ'1.

[0179] 5.2 Study on the stability of the adjuvant according to the present invention and the comparative adjuvant

[0180] The stability of the adjuvants ADJ1, ADJ2, ADJ3 according to the invention and the comparative adjuvant ADJ'1 were evaluated according to the following protocol:

[0181] i) An amount of 90 ml of the composition to be tested, contained in a 100 ml flask, is introduced into a climate chamber conditioned at 20° C. for a period of one year. The visual appearance of the tested compounds is evaluated before placing them in the chamber for stability testing and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).

[0182] ii) An amount of 90 ml of the composition to be tested, contained in a 100 ml flask, is introduced into a climate chamber conditioned at 37° C. for a period of one month (M1). The visual appearance of the composition tested is evaluated before placing it in the chamber for the stability test and after a period of one month.

[0183] Stability is understood to mean the absence of phase separation and / or the absence of observed sedimentation. The results of the observations are reported in Table 6 below.

[0184] [Table 6]

[0185]

[0186] Table 6: Stability results of adjuvants ADJ1, ADJ2, ADJ3 according to the present invention and comparative adjuvant ADJ'1

[0187] (Clear): Homogeneous and clear, single phase

[0188] (Inhomogeneous): Inhomogeneous, two or three phases are observed, and sediments are present.

[0189] The adjuvants ADJ1, ADJ2, ADJ3, ADJ'1 according to the invention had a homogeneous appearance under the storage conditions described below, whereas a heterogeneous appearance (phase separation and sedimentation) was observed with the comparative adjuvant ADJ'1 over time and at different temperatures.

[0190] 5.3. Characterization of the stability properties of vaccine compositions containing adjuvants according to the invention

[0191] The stability properties of placebo vaccine emulsions containing adjuvants ADJ1, ADJ2, ADJ3 according to the invention were evaluated in an amount of 200 g (prepared in a 250 ml low form beaker) using a Silverson L4 or L5 rotor-stator mixer equipped with a standard head providing a standard emulsion screen according to the following protocol:

[0192] i / Add 60 g of the aqueous phase to 140 g of the adjuvant using a Silverson L4 or L5 mixer at 1000 rpm with mechanical stirring (during this step, the stirring head should be placed 0.5 cm from the bottom of the beaker);

[0193] ii / An emulsion was produced by subjecting the mixture obtained in step i / to high shear (using a Silverson L4 or L5 mixer) at a rotation speed of 4000 rpm (or 7 m / s) for a period of 3 minutes.

[0194] The emulsion obtained at the end of step ii / is fluid, homogeneous and injectable. More particularly, the term "fluid" means a liquid emulsion, the dynamic viscosity of which is between 30 and 40 mPa.s measured at 20°C using a Brookfield LVDV1+ equipped with an M62 spindle at a rotation speed of 60 rpm.

[0195] The stability of the obtained emulsions was characterized as follows:

[0196] i) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climate chamber conditioned at 4° C. for a period of one year. The visual appearance of the tested compounds is evaluated before placing them in the chamber for stability testing and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).

[0197] ii) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climate chamber conditioned at 20° C. for a period of one year. The visual appearance of the tested compounds is evaluated before placing them in the chamber for stability testing and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).

[0198] iii) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climate chamber conditioned at 37° C. for a period of one month (M1). The visual appearance of the tested composition is evaluated before placing it in the chamber for the stability test and after a period of one month.

[0199] Stability is understood to mean the absence of phase separation and / or the absence of observed sedimentation. The results of the observations are reported in Table 7 below.

[0200] [Table 7]

[0201]

[0202] Table 7: Stability results of emulsions containing adjuvants ADJ1, ADJ2, ADJ3 according to the invention

[0203] (H): Homogeneous, only one phase observed

[0204] 5.4 Characterization of the immunological properties of vaccines containing adjuvants according to the invention

[0205] The adjuvant properties of the polymeric oil adjuvants ADJ1, ADJ2 and ADJ3 according to the invention and as described in the previous examples were characterized in several vaccine models and in several animal species.

[0206] During a first test, the adjuvant ADJ3 according to the invention was formulated with a solution of ovalbumin to obtain a vaccine intended for injection into mice.

[0207] In a second test, the adjuvant ADJ2 according to the invention was formulated with a bacterial antigenic medium consisting of killed Pasteurella multocida bacteria to obtain a vaccine intended for administration to avian species.

[0208] In a third test, the adjuvant ADJ1 according to the invention was used to formulate a viral vaccine intended for use in avian species against Newcastle disease and H9N2 influenza. These tests confirmed the vaccine adjuvant properties of the adjuvant according to the invention in several species and in several antigenic models.

[0209] The results obtained are shown below.

[0210] 5.4.1 Experiment 1: Experiment in mice using ovalbumin as an antigen

[0211] The test was performed in OF1 mice in a 90-day vaccination regimen with ovalbumin (OVA) as an antigen model. The vaccine was prepared from an antigen solution of OVA prepared at 10 mg / ml in physiological serum and sterilized by filtration on a 0.22 μm filter. The formulation of the vaccine comprising the OVA antigen and the ADJ3 adjuvant according to the invention was carried out by emulsification of the adjuvant ADJ3 / antigen medium according to the invention in a 70 / 30 (volume / volume) ratio via an i-linker.

[0212] [Table 8]

[0213]

[0214] Table 8: Composition of the vaccines tested

[0215] The safety of the vaccine was evaluated by observing local reactions at the injection site. Vaccine efficacy was evaluated by detecting IgG1 and IgG2a antibodies in the blood by ELISA. The test was performed on the day of vaccination ("primary vaccination" on D0), then 14 days later (D14), at booster vaccination on the 28th day (D28), then on the 42nd day (D42), then on the 56th day (D56), and then at euthanasia on the 90th day (D90).

[0216] No local reactions were observed in the members of the test group, thus indicating that the vaccine comprising the ADJ3 adjuvant according to the invention is well tolerated.

[0217] Figure 1 and 2 The determination of IgG1 and IgG2a antibodies at D14, D28, D42, D56 and D90 is shown.

[0218] [ Figure 1 ] Figure 1 is a graph showing IgG1 antibody responses to OVA antigen in mice for a vaccine comprising the ADJ3 adjuvant according to the present invention.

[0219] [ Figure 2 ] Figure 2is a graph showing IgG2a antibody responses to OVA antigen in mice for a vaccine comprising the ADJ3 adjuvant according to the present invention.

[0220] For both classes of antibodies, significantly higher antibody titers were observed for the vaccine comprising the ADJ3 adjuvant according to the present invention compared to the unadjuvanted vaccine comprising the antigens, confirming the vaccine adjuvant properties of the developed formulation.

[0221] 5.4.2 Trial 2: Vaccine trials in chickens using Pasteurella multocida bacterial antigens

[0222] The trial was conducted in chickens during a 42-day vaccination schedule against the bacterial pathogen Pasteurella multocida. The animals used in this experiment were red chickens that were 36 days old at the time of vaccination (D0). Each vaccine dose contained 1 dose (0.5 ml) = 0.5 x 10 8 CFU or 1x10 8 CFU / ml of killed Pasteurella multocida bacteria. The vaccine group consisted of 11 male and female chickens randomly distributed between the groups.

[0223] [Table 9]

[0224]

[0225] Table 9: Composition of the vaccines tested

[0226] Formulations containing the ADJ2 adjuvant according to the invention were prepared using Tube Drive emulsifier (sold by the company Ika) in a sterile DT50 tube with a 70 / 30 (weight / weight) ratio of ADJ2 adjuvant according to the invention / antigen medium: speed 3 for 2 minutes (1100 rpm) (for pre-emulsion) then speed 9 for 6 minutes (4000 rpm).

[0227] Animals were vaccinated on D0. Local reactions were observed at slaughter on D42. Blood samples will be collected on D0, D14, D42. Antibody levels in serum will be measured by antigen-specific ELISA assay using a commercial test kit (ID Screening Pasteurella multocida Chicken and Turkey Indirect Kit sold by ID-VEt).

[0228] The vaccine comprising the ADJ2 adjuvant according to the present invention was well tolerated in chickens, as no severe local reactions were observed at slaughter. Significantly higher antibody titers were also observed in chickens for the vaccine adjuvanted with the ADJ2 adjuvant compared to the non-adjuvanted vaccine, as shown in Table 10 below.

[0229] [Table 10]

[0230]

[0231] Table 10: IgY antibody responses against Pasteurella multocida in chickens for vaccines comprising adjuvant ADJ2 according to the invention.

[0232] 5.4.3 Trial 3: Vaccine trials in chickens using Newcastle disease / avian influenza virus antigens

[0233] The trial was conducted in chickens with a bivalent inactivated virus vaccine against Newcastle disease LaSota strain (NDV) and H9N2 avian influenza (AIV) in a 28-day vaccination schedule. The animals used in this experiment were SPF (specific pathogen-free) chickens that were 28 days old at the start of the experiment (D0). Vaccine groups were formed as shown in Table 11 below:

[0234] [Table 11]

[0235]

[0236] Table 11: Composition of vaccine groups tested

[0237] The test vaccine was formulated using the polymeric oil adjuvant ADJ1 according to the invention by emulsifying the ADJ1 adjuvant / antigenic medium according to the invention with an antigenic medium containing two valencies of inactivated AIV and NDV viruses at a ratio of 70 / 30 (w / w). The control group was not vaccinated.

[0238] The vaccine was injected intramuscularly at D0. Blood samples were collected at D0, D7, D14, D21 and D28 after vaccination and analyzed by hemagglutination inhibition test to determine the specific antibody titer for each valence (AIV and NDV). In the AIV group, a protective challenge was performed at D28 after vaccination to measure the viral load after challenge (2.10 in 0.2 ml intravenous injection of XZ strain H9N2 AIV virus). 6 EID 50 Oropharyngeal and cloacal swabs were collected 5 days after virus challenge and inoculated into SPF chicken embryos to measure virus presence after two generations of transmission.

[0239] After vaccination, strong antibody titers of both valencies were observed in the vaccinated group ( Figure 1 and Figure 2 After the virulent challenge, no mortality was observed and no viral load was observed in the swab samples in the vaccinated group, confirming complete protection against AIV viral challenge ( Figure 3 ).

[0240] [ Figure 3 ] Figure 3 It is a graph showing the change in antibody titer against Newcastle disease LaSota strain (NDV) of chickens from D0 to D28.

[0241] [ Figure 4 ] Figure 4 It is a graph showing the change in antibody titer against H9N2 avian influenza (AIV) from D0 to D28.

[0242] [ Figure 5 ] Figure 5 It is a graph showing the degree of protection obtained by the test vaccine group compared with the control group (number of protected animals / total number).

[0243] 5.5 Experimental Conclusion

[0244] The adjuvant according to the invention is characterized in that:

[0245] - filtration dynamics suitable for obtaining sterile adjuvants, in particular on hydrophobic filters (in particular made of polytetrafluoroethylene (PTFE)) with a mean pore size of 0.2 micrometers,

[0246] - stability over time at 20°C and 37°C, i.e. maintaining a homogeneous and clear appearance, showing no phase separation and / or sedimentation phenomena,

[0247] - obtaining a stable vaccine emulsion formed by emulsifying said adjuvant in the presence of an aqueous vaccine phase,

[0248] -Immune adjuvant effect in vaccine compositions in various animal species in the presence of various antigenic mediators.

Claims

1. A vaccine adjuvant, comprising at least a reverse microemulsion as a reverse microlatex, the reverse microemulsion comprising at least one polyelectrolyte polymer, wherein the vaccine adjuvant comprises, per 100% by weight: a) from 60 to 90% by weight of oil (H1); b) from 3 to 8% by weight of at least one water-in-oil surfactant (E1) selected from - anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters, and - diethoxylated cetyl alcohol oil; c) from 3 to 8% by weight of at least one oil-in-water surfactant (E2) selected from - anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters, which are subsequently subjected to a step of adding ethylene oxide to an extent ranging from 2 to 30 molar equivalents of ethylene oxide, - ethoxylated vegetable oils, and - alkyl polyglycosides, or mixtures of alkyl polyglycosides, lecithin, saponin, polyoxyethylated alkanols, polymers containing polyoxyethylene and polyoxypropylene blocks; and d) from 1 to 10% by weight of said inverse microlatex; in, The sum of the weight contents a)+b)+c)+d) equals 100%, and The reverse microlatex comprises an oil phase, an aqueous phase, at least one water-in-oil (W / O) surfactant, at least one oil-in-water (O / W) surfactant and an anionic crosslinked polyelectrolyte; wherein the anionic crosslinked polyelectrolyte comprises at least one crosslinking monomer and at least one hydrophilic monomer unit, the hydrophilic monomer unit being derived from acrylic acid fully or partially salified with an alkali metal salt or an alkaline earth metal salt or an ammonium salt, wherein the reverse microlatex comprises per 100% by weight: -a') from 10 to 40% by weight of water, -b') from 30 to 50% by weight of oil (H2), -c') from 5 to 30% by weight of a mixture of at least one water-in-oil surfactant (E'1) and at least one oil-in-water surfactant (E'2), wherein The at least one water-in-oil surfactant (E'1) is selected from - anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters, and - diethoxylated cetyl alcohol oil, The at least one oil-in-water surfactant (E'2) is selected from - anhydrohexitol esters of saturated or unsaturated, linear or branched aliphatic carboxylic acids containing from 12 to 22 carbon atoms, optionally substituted by one or more hydroxyl groups, or mixtures of these esters, which are subsequently subjected to a step of adding ethylene oxide to an extent ranging from 2 to 30 molar equivalents of ethylene oxide, - ethoxylated vegetable oils, and - alkyl polyglycosides, or mixtures of alkyl polyglycosides, lecithin, saponin, polyoxyethylated alkanols, polymers containing polyoxyethylene and polyoxypropylene blocks, and -d') from 5 to 35% by weight of said anionic crosslinked polyelectrolyte, The sum of the weight contents a')+b')+c')+d') is equal to 100%.

2. The vaccine adjuvant according to claim 1, characterized in that The alkyl polyglycosides are alkyl polyglucose and alkyl polyxyloside.

3. The vaccine adjuvant according to claim 1, characterized in that The inverse microlatex comprises a') from 12 to 30% by weight of water per 100% of its weight.

4. The vaccine adjuvant according to claim 2, characterized in that The inverse microlatex comprises a') from 12 to 30% by weight of water per 100% of its weight.

5. The vaccine adjuvant according to claim 3, characterized in that The inverse microlatex comprises b') from 38 to 50% by weight of oil (H2) per 100% of its weight.

6. The vaccine adjuvant according to claim 4, characterized in that The inverse microlatex comprises b') from 38 to 50% by weight of oil (H2) per 100% of its weight.

7. The vaccine adjuvant according to any one of claims 3 to 6, characterized in that The inverse microlatex comprises c') from 10% to 25% per 100% of its weight of a mixture of at least one water-in-oil surfactant (E'1) and at least one oil-in-water surfactant (E'2).

8. The vaccine adjuvant according to any one of claims 3 to 6, characterized in that The inverse microlatex comprises d′) from 10% to 30% of the anionic crosslinked polyelectrolyte per 100% of its weight.

9. The vaccine adjuvant according to claim 7, characterized in that The inverse microlatex comprises d′) from 10% to 30% of the anionic crosslinked polyelectrolyte per 100% of its weight.

10. The vaccine adjuvant according to any one of claims 1 to 6 and 9, characterized in that The acrylic acid from which the hydrophilic monomer units are derived is fully or partially salified with a sodium salt or an ammonium salt.

11. The vaccine adjuvant according to claim 10, characterized in that The acrylic acid from which the hydrophilic monomer units are derived is fully or partially salified with a sodium salt.

12. The vaccine adjuvant according to any one of claims 1 to 6, 9 and 11, characterized in that The anionic crosslinked polyelectrolyte comprises monomer units having formula (1): in: -R1 is selected from -H, -CH3, -C2H5 and -C3H7 -n is between 0 and 50, and -m is between 8 and 22.

13. The vaccine adjuvant according to claim 12, characterized in that R1 is -CH3.

14. The vaccine adjuvant according to any one of claims 1 to 6, 9, 11 and 13, characterized in that The oil (H1) is a white mineral oil.

15. A vaccine comprising an adjuvant as defined in any one of claims 1 to 14, and at least one aqueous solution (S) of at least one antigen.

16. The vaccine according to claim 15, characterized in that The vaccine contains: - from 10 to 80% by weight of an adjuvant as defined in any one of claims 1 to 14, and, - from 20 to 90% by weight of the aqueous solution (S).

17. The vaccine according to claim 15 or 16, characterized in that The vaccine is in the form of a water-in-oil emulsion or an oil-in-water emulsion.

18. A method for preparing a vaccine adjuvant as defined in any one of claims 1 to 14, characterized in that The method comprises the following steps: a) preparing, with mechanical stirring and at ambient temperature, an oil phase comprising at least one oil and an emulsified system comprising at least one water-in-oil surfactant (E1) and / or oil-in-water surfactant (E2); b) adding the at least one inverse microlatex at ambient temperature with mechanical stirring; c) Maintaining the mechanical stirring at ambient temperature until a homogeneous mixture is obtained.

19. A method as claimed in claim 18, comprising the step of sterilising the vaccine adjuvant by filtration or by autoclaving.

20. A method for preparing a vaccine, the method comprising the following steps: a) preparing a vaccine adjuvant by the method defined in claim 18 or 19, and b) mixing the vaccine adjuvant obtained in step a) with the antigen medium.

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