Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to new adjuvants for vaccine compositions as well as said compositions comprising at least one antigen, in particular an antigen of viral, bacterial or parasitic origin and at least one adjuvant.
• injectable oils used as adjuvants in veterinary vaccines. They are very effective but they sometimes induce local reactions. They are used in admixture with the antigenic medium to form injectable fluid emulsions.
• vaccine compositions comprising, as an adjuvant, from 1 to 5% weight / volume of sodium alginate and of the insolubilization ions of the alginate, such as the calcium ion, the concentration of the sequestered ions of insolubilization of the alginate being less than the necessary concentration to form an amount of insoluble gel.
• the subject of the invention is a composition comprising a fatty phase and a non-zero quantity of an organometallic gel obtained by complexing an anionic polymer or a mixture of different anionic polymers, with a multivalent metal cation. or a mixture of different multivalent metal cations.
• the organometallic gel can be obtained by mixing a volume Vc of a suspension or a solution containing the multivalent cation salt or a mixture of multivalent cation salts, with a volume Vp of a solution or a suspension containing the anionic polymer or a mixture of anionic polymers in proportions sufficient to cause the phenomenon of gelation leading to the organometallic gel, with, if necessary, stirring of the resulting mixture.
• the fatty phase constituting the composition which is the subject of the present invention generally comprises one or more compounds chosen from oils of mineral, vegetable or animal origin, the alkyl esters of said oils, the alkyl esters of fatty acids or the ethers fatty acid alkyls, fatty acid esters and polyols or fatty alcohol ethers and polyols.
• oils of mineral origin there are oils of petroleum origin, such as white mineral oils such as MARCOL TM 52.
• oils of vegetable origin there is oil of peanut, olive oil, sesame oil, soybean oil, wheat germ oil, grape seed oil, sunflower oil, castor oil, l linseed oil, soybean oil, corn oil, coconut oil, palm oil, walnut oil, hazelnut oil, rapeseed oil or even squalane or olive squalene.
• oils of animal origin are spermaceti oil, tallow oil, squalane or squalene extracted from fish livers.
• alkyl esters of oils there are the methyl, ethyl, linear or branched propyl or linear or branched butyl esters of said oils.
• fatty acids suitable for the preparation of the esters mentioned above there are more particularly, those containing from 12 to 22 carbon atoms, such as for example, myristic acid, palmitic acid, oleic acid, ricinoleic acid or isostearic acid and advantageously a liquid fatty acid at 20 ° C.
• fatty acid esters are the 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, and more particularly, monoglycerides of fatty acids, diglycerides of fatty acids, triglycerides of fatty acids, esters of fatty acids with a polyglycerol or the fatty acid esters of propylene glycol, the fatty acid esters with a hexol, such as for example sorbitol or mannitol, the fatty acid esters with a hexol anhydride, such as sorbi - tane or mannitane.
• the fatty phase may comprise only one of the compounds mentioned above or else a mixture of several of the compounds mentioned above.
• composition which is the subject of the present invention generally comprises between approximately 5% and 70% by weight, and more particularly between 15% and 60% by weight of fatty phase.
• the multivalent metal cations liable to be complexed with the anionic polymer or the mixture of anionic polymers there are more particularly the divalent or trivalent metal cations and very particularly the divalent cations of calcium, magnesium, manganese or zinc or the trivalent cations of iron or aluminum.
• the concentration of metal cations [C] expressed in moles per liter of solution or suspension, is generally between approximately 10 ⁇ 3 moles per liter and 10 moles per liter, more particularly between 10 2 moles per liter and 5 moles per liter and very particularly between 0.1 moles per liter and 1 mole per liter
• cation salts are pharmaceutically acceptable, for example a hydroxide, a carbonate, a citrate, a gluconate, a glucoheptonate, a fructo- heptonate, lactate, acetate, propionate, salicylate, chloride or glycerophosphate.
• salts used in the preparation of the organometallic gel of the composition which is the subject of the present invention there are calcium hydroxide, magnesium carbonate, manganese carbonate, calcium gluconate, manganese gluconate , manganese glycerophosphate, zinc gluconate, calcium fructoheptonate, aluminum salicylate or aluminum acetate.
• the multivalent cation salt used is manganese glycerophosphate or a mixture of manganese glycerophosphate and manganese gluconate.
• anionic polymers capable of being complexed with multivalent metal cations there are more particularly sulfated polymers, dextran, carrageenans, carboxylic polymers, polyacrylates, pectins, alginates, natural gums, xanthan gum or guar gum.
• the anionic polymer used is a sodium alginate.
• the concentration of anionic polymers [P], expressed as a percentage by weight of the solution or of the suspension is generally between approximately 0.1% and 10% by weight, more particularly between 0.5% and 5% by weight and very particularly between 1% and 5% by weight.
• the proportions in suspension or solution of salt of the cation and in solution or suspension of anionic polymer to produce the mixture leading to the obtaining of the organic gel are chosen so that the ratio [P] / [C] is understood between 0.01 and 100, more particularly between 0.1 and 50 and very particularly between 1 and approximately 10.
• the solvents of said suspensions or solutions used to prepare the organometallic gel are generally polar solvents and preferably miscible with each other. It is preferably water or a pharmaceutically acceptable hydroalcoholic mixture.
• the organometallic gel can be obtained by mixing a suspension or an aqueous solution containing the multivalent cation salt or a mixture of multivalent cation salts, with an aqueous solution or suspension containing the anionic polymer or the mixture of anionic polymers, with stirring of the resulting mixture if necessary.
• the organometallic gel can be obtained by mixing an aqueous suspension or solution containing a salt of multivalent cation, with an aqueous solution or suspension containing an anionic polymer, with, if necessary, stirring of the resulting mixture.
• composition as defined above is preferably in the form of an emulsion and in particular in the form of an emulsion the continuous phase of which is the fatty phase and the dispersed phase the gelled anionic polymer - multivalent metal cation complex.
• composition as defined above can also comprise one or more pharmaceutically acceptable surfactants.
• nonionic surfactants for example, polyglycerol esters, sugar esters such as mannitane or sucrose sorbitan esters, esters of ethoxylated sugars, alkoxylated fatty alcohols, ethoxylated fatty acids, monoglycerides and diglycerides modified by reaction with acetic acid or lactic acid; mono glycerides, diglycerides or ethoxylated triglycerides, sugar ethers, such as glucose ethers, xylose ethers or lactitol ethers.
• nonionic surfactants for example, polyglycerol esters, sugar esters such as mannitane or sucrose sorbitan esters, esters of ethoxylated sugars, alkoxylated fatty alcohols, ethoxylated fatty acids, monoglycerides and diglycerides modified by reaction with acetic acid or lactic acid; mono glycerides, diglycerides or e
• the surfactants used are more particularly chosen so that the HLB of the mixture of surfactants is between 4 and 12 and preferably between 5 and 8.
• composition as defined above generally comprises between approximately 0.5% and 10% by weight and preferably between 1% and 5% by weight of surfactants.
• the subject of the invention is also a process for preparing the emulsion as defined above, comprising the following steps:
• Step (e) of the process generally consists of a mixture of a volume Vc of a suspension or of a solution of the salt of the cation with a volume Vp of a solution or of a suspension of anionic polymer, in a volume ratio Nc / Vp generally between 1/100 and 1/1 preferably between 1/50 and 1/10, either by pouring the suspension or the cation salt solution into the solution or the suspension of anionic polymer with, if necessary, stirring of the resulting mixture, either by pouring the suspension or solution of anionic polymer into the solution or suspension of cation salt with, if necessary, stirring of the resulting mixture.
• step (a) is preferably used, one or more salts chosen from calcium hydroxide, magnesium carbonate, manganese carbonate, calcium gluconate, manganese gluconate, glycerophosphate manganese, zinc gluconate, calcium fructoheptonate, aluminum salicylate or aluminum acetate.
• the emulsion obtained in step (e) is dissolved in a solvent for the fatty phase to obtain an organometallic gel suspension and the resulting suspension is subjected to centrifugation. tion to isolate said gel.
• This variant is used to prepare a composition with a low oil content.
• composition as defined above, as an adjuvant phase of a vaccine composition.
• the subject of the invention is also a method for preparing a vaccine comprising the addition as an immunity adjuvant, of an effective amount of the composition as defined above.
• composition as defined above can be used in combination with conventional oily adjuvants, known to those skilled in the art.
• the composition object of the present invention is mixed with the antigenic phase and then the whole is emulsified.
• its subject is a composition comprising at least one antigen or at least one in vivo generator of a compound comprising a sequence of amino acids and a non-zero amount of a composition as defined above.
• the term “antigen or at least one in vivo generator of a compound comprising an amino acid sequence” denotes either killed microorganisms, such as viruses, bacteria or parasites, or purified fractions of these microorganisms. , or living microorganisms whose pathogenic power has been reduced.
• viruses which can constitute an antigen according to the present invention there is the rabies virus, the herpes viruses, such as the Aujeszky's disease virus, the orthomixoviruses such as Influenzae, the picornaviruses. such as FMD virus or retroviruses such as HIV.
• the herpes viruses such as the Aujeszky's disease virus
• the orthomixoviruses such as Influenzae
• the picornaviruses. such as FMD virus or retroviruses
• retroviruses such as HIV.
• a microorganism of the bacterial type which can constitute an antigen according to the present invention mention may be made of E. Coli, and those of the genera Pasteurella, Furonculosis, Vibriosis, Staphylococcus and Streptococcus. Examples of parasites are those of the genera Trypanosoma, Plasmodium and Leishmania.
• Non-enveloped viruses such as adenoviruses, vaccinia virus, Canarypox virus, herpes viruses or baculoviruses.
• a live non-enveloped viral recombinant vector is also designated, the genome of which contains, preferably inserted into a part which is not essential for the replication of the corresponding enveloped virus, a sequence coding for an antigenic subunit inducing antibody synthesis and / or a protective effect against the above enveloped virus or pathogenic microorganism; these antigenic subunits can be, for example, a protein, a glycoprotein, a peptide or a peptide fraction and / or protective against infection by a living microorganism such as an enveloped virus, a bacterium or a parasite.
• the exogenous gene inserted into the microorganism can be, for example, from an Aujeszky or HIV virus.
• the aim of this last nucleotide sequence is to allow the expression of a compound comprising an amino acid sequence, this compound itself having the aim of triggering an immune reaction in a host organism.
• the term “in vivo” generator of a compound comprising an amino acid sequence means a whole biological product capable of expressing said compound in the host organism into which said generator has been introduced in vivo.
• the compound comprising the amino acid sequence can be a protein, a peptide or a glycoprotein.
• generators in vivo are generally obtained by processes resulting from genetic engineering. More particularly, they can consist of living microorganisms, generally a virus, playing the role of recombinant vector, into which is inserted a nucleotide sequence, in particular an exogenous gene. These compounds are known as such and used in particular as a recombinant unitary vaccine. In this regard, reference may be made to the article by M. ELOIT et al., Journal of virology (1990) 71, 2925-2431 and to international patent applications published under the numbers WO-A-91/00107 and WO-A-94/16681.
• the in vivo generators according to the invention can also consist of a recombinant plasmid comprising an exogenous nucleotide sequence, capable of expressing in a host organism a compound comprising an amino acid sequence.
• a recombinant plasmid comprising an exogenous nucleotide sequence, capable of expressing in a host organism a compound comprising an amino acid sequence.
• Such recombinant plasmids and their mode of administration in a host organism were described in 1990, by LIN et al., Circulation 82: 2217,2221; COX et al., J. of VIROL, Sept. 1993, 67, 9, 5664-5667 and in the international application published under the number WO 95/25542.
• the compound comprising the amino acid sequence which is expressed within the host organism can: (i) be an antigen, and allow the triggering of a immune reaction,
• (ii) have a curative action vis-à-vis a disease, essentially a functional disease, which is triggered in the host organism.
• the in vivo generator allows treatment of the host, of the gene therapy type.
• such a curative action may consist in a synthesis by the in vivo generator of cytokines, such as interleukins, in particular interleukin 2. These allow the triggering or the strengthening of an immune reaction aimed at selective elimination of cancer cells.
• cytokines such as interleukins, in particular interleukin 2.
• a composition according to the invention comprises an antigen concentration which depends on the nature of this antigen and on the nature of the subject treated. It is however particularly remarkable that an adjuvant according to the invention makes it possible to significantly reduce the usual dose of antigen required.
• the appropriate concentration of antigen can be determined conventionally by those skilled in the art. Generally, this dose is of the order of 0.1 ⁇ g / cm 3 to 1 g / cm 3 more generally between 1 ⁇ g / cm 3 and 100 mg / cm 3 .
• the concentration of said generator in vivo in the composition according to the invention depends, in particular, on the nature of said generator and on the host in which it is administered. This concentration can be easily determined by a person skilled in the art, on the basis of routine experience.
• the generator in vivo when the generator in vivo is a recombinant microorganism, its concentration in the composition according to the invention can be between 10 and 10 microorganisms / cm, preferably between 10 and 10 12 microorganisms / cm 3 .
• the generator in vivo is a recombinant plasmid, its concentration in the composition according to the invention can be between 0.01 g / dm 3 and 100 g / dm 3 .
• the vaccine as defined above is prepared by mixing the adjuvant phase and the antigenic phase, optionally adding water or a pharmaceutically acceptable diluent medium. The following examples illustrate the invention without, however, limiting it.
• a 1% solution of high viscosity, high guluronic acid sodium alginate (SATIALGINE TM SG800) is prepared.
• a 500 millimolar aqueous suspension of an insoluble salt of a salt insoluble in water, calcium hydroxide, is prepared. 1 ml of the suspension and 20 g of the sodium alginate solution are mixed. The mixture obtained is dispersed by means of a rapid stirrer in 100 g of a white mineral oil (MARCOL TM 52) containing 1% by weight of a lipophilic surfactant, sorbitan monoleate or MONT ANE TM 80, HLB number equal to about 4.3.
• MARCOL TM 52 white mineral oil
• An emulsion is obtained which is acidified with a few drops of concentrated acetic acid.
• This emulsion is in continuous oil phase; its dispersed phase consists of a stable gelled complex of calcium alginate.
• This calcium alginate emulsion constitutes an immunity adjuvant, which can be emulsified with an antigenic medium to form a vaccine W / O type, stable, with improved efficacy.
• This new immunity adjuvant can optionally be mixed with another oily adjuvant such as those of the MONTANIDE TM ISA family, sold by the company Seppic before manufacture of the final vaccine.
• Example 2
• a 3.5% solution of low viscosity, high guluronic acid sodium alginate (SATIALGINE TM S80) is prepared.
• An emulsion is obtained which is acidified with a few drops of concentrated acetic acid to dissolve the manganese carbonate.
• An immunity adjuvant is thus obtained, which is an emulsion which has a continuous oil phase and whose dispersed phase consists of a stable gelled manganese alginate complex.
• a 3.5% solution of low viscosity, high guluronic acid sodium alginate (SATIALGINE TM S80) is produced.
• a 500 millimolar suspension of a poorly soluble salt, manganese glycerophosphate, is prepared.
• the adjuvant thus obtained is an emulsion the continuous phase of which is the oil phase and the dispersed phase of which consists of a stable gelled complex of manganese alginate.
• the effectiveness of this adjuvant is evaluated in female OF1 strain mice weighing 20 grams, which are injected subcutaneously with 100 ⁇ l of vaccines containing ovalbumin grade V (OVA), as antigen (all preparations have been adjusted so that the dose of antigen administered per animal is constant and equal to 1 ⁇ g per injection).
• the vaccination schedule includes a booster 28 days after the first injection.
• a first group of mice receives a dose of OVA alone without adjuvant (control 1),
• a second group of mice receives a vaccine (A) of the W / O type (preparation A), consisting of a part of standard oily adjuvant (MONTANIDE TM ISA 564, sold by the company SEPPIC) and a part of 'OVA in physiological saline (composition according to the state of the art).
• a vaccine of the W / O type (preparation A)
• preparation A consisting of a part of standard oily adjuvant (MONTANIDE TM ISA 564, sold by the company SEPPIC) and a part of 'OVA in physiological saline (composition according to the state of the art).
• a third group of mice receives a preparation (B) consisting of three parts of vaccine (A) for 1 part of adjuvant containing a manganese alginate complex prepared as described above, (composition according to the invention)
• a fourth group of mice receives a preparation (C) consisting of a part of vaccine (A) for a part of adjuvant containing a manganese alginate complex prepared as described above (composition according to the invention).
• An immunity adjuvant consisting of a manganese alginate complex emulsified in mineral oil is prepared as in Example 3.
• a placebo emulsion (P) is prepared which consists of a part of standard MONTANIDE TM ISA 564 adjuvant and a part of physiological saline.
• This adjuvant is evaluated in female OF1 strain mice weighing 20 grams, which are injected subcutaneously with 100 ⁇ l of vaccines containing ovalbumin grade V (OVA), as antigen (all preparations have been adjusted so that the dose of antigen administered per animal is constant and equal to 1 ⁇ g per injection).
• the vaccination schedule includes a booster 28 days after the first injection.
• a first group of mice receives a dose of OVA alone without adjuvant (control 1),
• a second group of mice receives a vaccine (A) of W / O type (preparation A), consisting of a portion of MONTANIDE TM ISA 564 and part of OVA in physiological saline (composition according to the state of the art),
• a third group of mice receives a preparation (D) consisting of three parts of placebo (P) for a part of preparation (I) (composition according to the invention),
• a fourth group of mice receives a preparation (E) consisting of a part of placebo (P) for a part of preparation (I) (composition according to the invention).
• the emulsified manganese alginate complex obtained in Example 3 is half-diluted in an organic solvent (ether or isopropyl alcohol). Part of the mineral oil in the emulsion is dissolved and the alginate complex beads are isolated by centrifugation. The solvent residue is evaporated and an immunity adjuvant enriched in complex is obtained containing only about 5% of residual mineral oil.
• organic solvent ether or isopropyl alcohol
• This adjuvant is evaluated in female OF1 strain mice weighing 20 grams, which are injected subcutaneously with 100 ⁇ l of vaccines containing ovalbumin grade V (OVA), as antigen (all preparations have been adjusted so that the dose of antigen administered per animal is constant and equal to 1 ⁇ g per injection).
• the vaccination schedule includes a booster 28 days after the first injection.
• a first group of mice receives a dose of OVA alone without adjuvant (control 1),
• a second group of mice receives a vaccine containing, as an adjuvant, manganese glycerophosphate so that the concentration of Mn ++ cation is the same as that of preparation F and containing the same amount of OVA as preparation F (witness 2) (composition according to the state of the art),
• a third group of mice receives a preparation (F) consisting of the mixture of the adjuvant, enriched in manganese alginate complex with an antigenic solution of OVA to form a vaccine preparation (F) containing 10 ⁇ g / ml of albumin.
• a vaccine (G) is obtained consisting of the OVA antigen and an oily adjuvant composed of an oil and a manganese alginate complex.
• This adjuvant is evaluated in female OF1 strain mice weighing 20 grams, which are injected subcutaneously with 100 ⁇ l of vaccines containing ovalbumin grade V (OVA), as antigen (all preparations have been adjusted so that the dose of antigen administered per animal is constant and equal to 1 ⁇ g per injection).
• the vaccination schedule includes a booster 28 days after the first injection.
• a first group of mice receives a dose of OVA alone without adjuvant (control 1),
• a second group of mice receives a vaccine (A) of the W / O type (preparation A), consisting of a part of MONTANIDE TM ISA 564 and a part of OVA in physiological saline (composition according to the state technique),
• a third group of mice receives the vaccine (G) (composition according to the invention),
• a fourth group of mice receives a preparation (H) consisting of a part of the placebo (P) prepared in Example 4, and a part of vaccine (G) (composition according to the invention).
• Table 4 Vaccine G containing the emulsified complex as an adjuvant is more effective than standard vaccine A in the short term and of similar efficacy in the longer term.
• the H vaccine containing a mixture of two adjuvants is clearly more effective than the two vaccines with a single adjuvant, both in the short term and after 56 days. A synergy is therefore observed
• the emulsified manganese alginate complex obtained in Example 3 is taken up and half diluted in an organic solvent (ether or isopropyl alcohol). Part of the mineral oil in the emulsion is dissolved and the alginate complex beads can be isolated by centrifugation.
• organic solvent ether or isopropyl alcohol
• the solvent residue is evaporated and an immunity adjuvant enriched in complex is obtained containing only about 5% of residual mineral oil.
• the efficacy of this adjuvant is evaluated in female OF1 strain mice weighing 20 grams, which are injected subcutaneously with 100 ⁇ l of vaccines containing a parasitic antigen of Trichinella spiralis larvae (all preparations have been adjusted for that the dose of antigen administered per animal is constant and equal to 5 ⁇ g per injection).
• the vaccination schedule includes a booster 28 days after the first injection.
• a first group of mice receives a W / O type vaccine, consisting of part of
• mice receives a vaccine containing, as an adjuvant, manganese glycerophosphate so that the concentration of Mn ++ cation is the same as that of preparation J and containing the same quantity of parasitic antigen of larvae of. Trichinella spiralis as preparation J. (control 5),
• a third group of mice receives a preparation (J) containing 50 ⁇ g / ml of antigen consisting of the mixture of the adjuvant, enriched in manganese alginate complex with the antigenic solution of parasitic antigen of Trichinella spiralis larvae (compo - sition according to the invention).
• the vaccine according to the invention is as effective in the short term, as the vaccine containing the soluble salt and that it is more effective than the W / O type vaccine. In the long term it is almost as efficient as the oily W / O vaccine and more efficient than the soluble salt vaccine.
• the vaccines containing the various adjuvants described in examples 1 to 6 are injected subcutaneously into mice of OF1 strain (volume injected: 100 ⁇ l).
• the intensity of local reactions at the injection site is noted after seven days, on a numerical scale ranging from 0 (no reaction) to 5 (very strong reaction with tissue necrosis), after 7 days.
• the results recorded in the following table show that the vaccines containing the adjuvants according to the invention are well tolerated, the local reactions not exceeding that of control A

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