FR2888117A1 - Oil-in-water adjuvant emulsion, useful in immunogenic composition, comprises squalene, aqueous solvent, polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and hydrophobic nonionic surfactant - Google Patents

Abstract

An oil-in-water adjuvant emulsion comprises squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant. The emulsion is thermoreversible and 90% of the population by volume of the oil drops has a size less than 200 nm. Independent claims are also included for the following: (1) preparing an immunogenic composition comprises mixing at least one vaccine antigen with an oil-in-water emulsion, where the oil-in-water emulsion is obtained by a phase-inversion temperature process; and (2) an immunogenic composition comprising an antigen and the emulsion. ACTIVITY : Immunostimulant; Virucide; Anti-HIV. MECHANISM OF ACTION : Vaccine. A composition (C1) comprising: trans-acting transcription factor protein (20 mu g), squalene (5 mg), dehymuls SMO (0.75), eumulgin B1 (0.94) and mannitol (0.91 mg) was prepared. The composition (C1) was then evaluated for efficacy of immunogenicity against AIDS. Two groups of six 8-week-old female BALB/c mice were injected subcutaneously with the composition (C1) prepared, in a proportion of one dose of 200 mu l/mouse; and the injections were given on day zero and days 21. Blood samples were taken from the retro orbital sinus on days 14 in order to assess the primary response and on days 34 for the secondary response and the specific immunoglobulin 1 (IgG1) and immunoglobulin 2a (IgG2a) were determined. It was observed that the composition (C1) increased, overall, humoral response and promotes the type 1 T-helper response since the IgG2a response was increased more than the IgG1 response.

Description

The present invention relates to the field of vaccines; more

  particularly, the invention relates to the field of vaccines comprising an adjuvant.

  There are many vaccines in the prior art that contain one or more adjuvants. US Pat. No. 6,299,884 describes in particular an adjuvant formulation comprising an oil-in-water emulsion, the size of the oil droplets of which is between 100 and 1000 nm. This emulsion is obtained by means of a high pressure homogenizer (microfluidizer), during a manufacturing process using high mechanical energies in order to obtain shear forces large enough to reduce the size of oil drops. . According to this teaching, if the minimum value of the range of the drops obtained is 100 nm, the average value is much higher and is best at 170 nm, more generally around 500 nm.

  It is desirable to have an alternative formulation to that proposed in this patent, and especially that can be obtained by a simpler process (not requiring a particular shearing technology), and low energy while being reproducible and perfectly reliable; in addition, the adjuvant formulation must allow the vaccines to be effectively adjuvanted, in particular by making it possible to increase the immune response obtained or to reduce the dose of antigen present, while not exhibiting any sign of toxicity which would be detrimental to its administration. all security.

  To achieve this object, the subject of the present invention is a vaccine composition comprising at least one vaccine antigen and an oil-in-water emulsion comprising at least: squalene, a nonionic emulsifier comprising ethylene oxide groups, except for polyoxyethylene and polyoxypropylene copolymers; a hydrophobic nonionic emulsifier, characterized in that the emulsion is thermoreversible and in that 90% of the volume population of the oil drops is less than 200 nm in size.

  According to the invention, such an emulsion is obtained by a phase inversion method by temperature variation, which provides a very large advantage from an industrial point of view. Such a process has all the guarantees of safety and profitability necessary for the pharmaceutical industry. In addition, thanks to this process, it is possible to obtain a monodisperse emulsion whose oil droplet size is very small, which makes the emulsion thus obtained particularly stable and easily filterable by means of sterilizing filters whose threshold cutoff is 200 nm.

  According to a particular characteristic, 90% of the density population of the drops of oil has a size less than 160 nm, and even at 150 nm. In addition, thanks to the preparation method used, the dispersion of the droplets in terms of size is very small; indeed, when one realizes the curve of distribution of the sizes of drops obtained, one notices that one has a very tight Gaussian distribution; if the value of d90 is generally less than 150 nm, the value of d50 is less than 100 and even 90 nm.

  According to a particular embodiment of the invention, the emulsion according to the invention further comprises an alditol; this makes it possible to obtain a phase inversion at a temperature lower than that which would be necessary for the same composition not having alditol, which makes it possible to reduce the production costs as well as the risks of thermal denaturation of the constituents of the emulsion.

  According to a particularly advantageous embodiment, the hydrophobic nonionic surfactant of the invention is a surfactant based on sorbitan ester or mannide ester. Such surfactants have the advantage that they can be used safely in injectable solutions.

  According to one particular embodiment of the invention, the emulsion further comprises an alkylpolyglucoside and a cryoprotective agent such as a sugar, in particular dodecylmaltoside and sucrose.

  Thus, it is possible to obtain a freeze-dried emulsion which, after lyophilization and reconstitution, regains its properties, in particular granulometric properties, that is to say that the lyophilized and then reconstituted emulsion is always monodisperse and consists of droplets of oil in which 90% of the population density is less than 200nm. This is particularly important for the field of vaccines which sometimes have to be stored in freeze-dried form for reasons of stability (either of certain antigens or of certain adjuvants).

  The invention also relates to a method for preparing a vaccine composition comprising at least one vaccine antigen and an oil-in-water emulsion, characterized in that the emulsion is obtained by a phase inversion method by variation of the temperature.

  Thanks to such a process, which is very advantageous from an industrial point of view, a monodisperse emulsion is obtained, the size of the oil droplets of which is very small, which makes the emulsion thus obtained particularly stable and easily filterable by means of sterilizing filters.

  Many other advantages of the present invention will become apparent from the following description.

  For the purposes of the present invention, the term "vaccine composition" is intended to mean a composition that can be administered to humans or animals in order to induce an immune system response; this response of the immune system may result in antibody production or activation of certain cells, including antigen presenting cells (eg dendritic cells), T cells, B cells. The vaccine composition may be a prophylactic or therapeutic composition, or both. This cellular activation can be demonstrated by the presence of activation markers on the cell surface or by the release of cytokines. The vaccine composition according to the invention may be administered by any of the routes usually used or recommended for vaccines: parenteral route, mucosal route, and be in various forms, in particular liquid or freeze-dried form. It can be administered by means of a syringe or by means of a needleless injector for intramuscular, subcutaneous or intradermal injection, or by means of a nasal spray.

  For the purposes of the present invention, the term "antigen" is intended to mean any antigen that may be used in a vaccine, whether it be a whole seed or a subunit antigen, which is of little importance; the antigen may indeed be a peptide, a protein, a glycoprotein, a polysaccharide, a glycolipid, a lipopeptide, etc. The formulation according to the invention is particularly suitable for viral antigens; particularly good results have been obtained with antigens of human cytomegalovirus, human immunodeficiency virus, and influenza.

  With respect to influenza virus antigens, it is possible to use antigens from a single viral strain, or a mixture of different strains. It is possible to use antigens derived from viruses traditionally grown on eggs, or on cells. Thanks to the invention, it has been observed that it is possible, whether for a single strain or for a mixture of strains, to obtain a satisfactory response of the immune system while at the same time greatly reducing the quantity of antigens present in the vaccine dose. . This may be particularly important in the case of the preparation of a vaccine against an influenza pandemic, where it is necessary to produce in a very short time, very large amounts of vaccine doses.

  According to the invention, the oil-in-water emulsion comprises squalene which is an oil originally derived from shark liver; it is an oil whose chemical formula is C30HSO, comprising 6 double bonds, this oil is metabolizable and has qualities allowing its use in an injectable pharmaceutical product. There is also vegetable squalene extracted from olive oil. In particular, good results have been obtained using squalene supplied by Fluka, which is of animal origin. The amounts of squalene used for the preparation of a concentrated emulsion are advantageously between 5 and 45%; this concentrated emulsion is then diluted to prepare vaccine doses in which the amount of squalene is between 0.5 and 5%. . According to the invention, the emulsion comprises a nonionic hydrophilic emulsifier comprising ethylene oxide groups, with the exception of polyoxyethylene and polyoxyethylene copolymers which are unsuitable. A suitable emulsifier may especially be a surfactant consisting of an ether of polyoxyethylene glycols and fatty alcohols also called polyoxyethylenated alkyl ethers or macrogols ethers. Non-exhaustively, mention may be made as emulsifiers belonging to this category, which are known under the name BRIJ and which are marketed by ICI America's Inc., or some of the products known under the name EumulginTM marketed by the Company. Cognis. Thus, good results have been obtained with the product BRIJ 56. A compound that is particularly suitable and preferred because of its semisynthetic origin is the polyoxyethylene (12) cetostearyl ether, supplied by the company COGNIS under the name Eumulgin ™ B1.

  According to the invention, the vaccine composition also comprises a hydrophobic nonionic surfactant; it must be a surfactant that can be used in the pharmaceutical industry; suitable surfactants include sorbitan ester or mannide ester surfactants; the sorbitan ester surfactants are obtained by reaction of a fatty acid and a mixture of partial esters of sorbitol and its mono and di-anhydrous; it can be a mono, a di or a tri-ester, or even a mixture; they are hydrophobic surfactants whose hydrophilic-lipophilic balance (HLB) is less than 9. These include surfactants marketed by the company ICI America's Inc. under the name of SPAN, or by Cognis under the name of DehymulsTM , or by the company ICI under the name of ARLACELTM; Examples of surfactants that are particularly suitable are sorbitan monooleate, sold under the name Dehymuls SMO TM or SPAN 80. Among the surfactants based on mannide esters, mention may be made of the mannide monooleate marketed by the Company. SIGMA, or by the company SEPPIC under the name Montanide 80 ™ The quantities of squalene and each of the surfactants used are advantageously chosen so as to obtain a mixture whose phase diagram comprises a phase of zero mean curvature (microemulsion type or lamellar phase ) for which the interfacial tensions are extremely low.

  According to a particular embodiment of the invention, the vaccine composition further comprises an alditol such as in particular glycerol, erythritol, xylitol, sorbitol or mannitol. In particular, we obtained good results with the mannitol marketed by the company ROQUETTE Frères. The amounts of alditol used during the preparation process can vary between 1 and 10%, when it is desired to prepare an emulsion which will be preserved in the liquid state. However, in the case of an emulsion intended to be lyophilized, the amounts of alditol will be increased, and being about 20%.

  According to one characteristic of the invention, the vaccine composition further comprises a cryoprotectant which allows freeze-drying of the emulsion obtained; among the cryoprotectants, sugars are particularly preferred, and in particular sucrose. In addition, the emulsion according to the invention may comprise an alkyl polyglycoside which is a surfactant with a sugar head; it may especially be sodium decyl-D-galactoside uronate, or according to a preferred mode of dodecyl (3 maltoside available from the company Fluka.

  According to a particular embodiment of the invention, the aqueous phase of the emulsion is constituted by a buffer, such as phosphate-buffered Dulbecco's solutions (D-PBS, without calcium or magnesium).

  Thanks to the process for preparing the vaccine composition according to the invention by a phase inversion obtained by varying the temperature, very easily, in a very reproducible manner, a direct oil-in-water emulsion is obtained whose size of the oil droplets is very homogeneous; the value of d90 (by volume) is less than 200 nm, and generally close to 150 nm, at the same time as the value of d50 is less than 80 nm. It is thus possible, after it has been diluted, to carry out a sterilizing filtration of the emulsion obtained.

  Such emulsions whose drop size is homogeneous and very small, are stable over time. Thus, it has been noted that an emulsion prepared according to the invention and stored at 4 C, retained after 2 years, a monodisperse profile, with a d50 value of 90 nm and a d90 value of 116, which proves the very high stability of the emulsion.

  Measuring the size of the drops can be done by various means, and especially by LASER diffraction granulometers, such as Beckman Coulter LS range (including the LS230). The measurement principle of these devices is based on the analysis of the intensity of the light scattered by the particles according to the angle (detectors of large, medium and small angles) when the sample is illuminated by a beam LASER . This analysis is done using mathematical models chosen according to the size and nature of the material used. In the case of submicron particle size measurement, a special optical model (Mie theory) must be applied, taking into account the refractive indices of the sample (here 1.495 for squalene) and its medium (here 1.332 for the sample). water); it is also necessary to be able to detect the low intensities emitted by the very fine particles, which requires an additional detection cell for the measurement at large angles of the differential diffusion of the polarized intensities (PIDS system at Coulter which allows a measurement from 40 nm ). For the purpose of describing the invention, the parameters of d50 and d90 mentioned in the present patent application are volume values; the value of d50 means the value of 50% of the population density of the drops.

  The process according to the invention is carried out as follows: a concentrated oil / water emulsion is prepared by incorporating the aqueous phase (buffered polyol solution comprising the nonionic surfactant containing polyoxyethylene groups) into the oily phase ( squalene, and sorbitan ester surfactant). An oil-in-water emulsion is then obtained without calibration, which rapidly manifests its instability. This emulsion is heated until a phase inversion is obtained, that is to say obtaining a water-in-oil emulsion. The transition or phase inversion can be followed by conductimetry. The temperature at which the change in curvature resulting from the transition from one type of emulsion to another occurs is the phase inversion temperature. Once this phase inversion temperature is reached, and therefore in the presence of a water-in-oil emulsion, the heating is stopped and the mixture is cooled. The water / oil emulsion will then again be reversed to give rise again to an oil-in-water emulsion, the size of the oil droplets is this time very homogeneous and small; the emulsion obtained is then very stable. This concentrated emulsion can then be diluted with a solution comprising the vaccine antigen, and stored at +4 C. This emulsion is thermoreversible, which means that if it is brought to a temperature above the phase inversion temperature, it will become a water-in-oil emulsion.

  Advantageously according to the invention, the formulation of the emulsion is chosen to have a phase inversion temperature which is between 45 and 80 ° C., and more particularly between 50 ° and 65 ° C. Thus, it is avoided to heat too high temperatures which might induce chemical degradation of the constituents or evaporation of the aqueous phase, and it is also avoided to cause a new phase inversion during subsequent storage, ie during the preparation phase of the vaccine composition that is, before being incorporated into the antigenic solution, or later upon storage of the vaccine composition prior to use. Such a method can be implemented in different ways. Indeed, it is possible, as has just been described, to mix on one side squalene with the surfactant based on sorbitan ester and on the other hand the buffered solution of polyol with the surfactant containing oxides ethylene; homogenize each of the mixtures, and then combine them to obtain the crude emulsion which will then be heated and cooled. Alternatively, the separately prepared mixtures can be heated to a temperature slightly above the phase inversion temperature, before being combined to give a water-in-oil inverse emulsion which will be cooled until the submicron emulsion oil is obtained. water.

  These operations can be performed in separate tanks for batch preparation.

  It is also possible to use an online process.

  The process for producing the in-line emulsion consists of a hot mixing of the two aqueous and oily phases previously prepared separately, through a static thermostatic mixer followed by in-line cooling through a connected refrigerated heat exchanger. at the outlet of the static mixer, then the final recovery of the emulsion according to the invention in a suitable container (flask or reactor). A static mixer was used consisting of a succession of mixing elements composed of crossed blades and inclined with respect to the axis of the tube in which they are introduced. The energy required for the mixing is supplied by the pumps which convey the fluids and the mixture is produced without moving parts, through the mixing elements by the separation, the displacement and the successive meeting of the constituents of the mixture.

  The in-line manufacturing process is carried out as follows: the aqueous phase (buffered solution comprising the nonionic surfactant containing polyoxyethylene groups) and the oily phase (squalene and hydrophobic nonionic surfactant) are prepared separately in two flasks or reactors. The two phases are heated with stirring at a temperature slightly above the phase inversion temperature. The two phases are then introduced into a static thermostatic mixer by means of 2 pumps, the flow rates of which are regulated so as to obtain the composition of the emulsion according to the invention. The water-in-oil inverse emulsion is obtained during the passage of the two phases in the static mixer. The inverse emulsion is then cooled by passage in line through a refrigerated heat exchanger connected to the output of the static mixer. The water-in-oil emulsion will then be reversed through the refrigerated heat exchanger to give rise to an oil-in-water emulsion, which will be received in a flask or reactor and whose characteristics are identical to those of the emulsion obtained. by a batch process.

  The following examples illustrate various embodiments of the invention.

  Example 1: Preparation of a concentrated emulsion of squalene

  3.31 g of Eumulgin ™ B1 and 33.9 g of a 10% solution of mannitol in PBS buffer are mixed in a beaker, which is homogenized with stirring at approximately 30 ° C. In another container, 2 is mixed. 89g of DehymulsTM SMO and 19.5g of squalene which is stirred magnetically.

  When homogeneous phases are obtained in each of the containers, the aqueous phase is incorporated in the oily phase which is stirred at 30 ° C. When the incorporation is complete, the crude emulsion obtained is heated until the temperature is reached. reaches 58-60 C, while maintaining agitation.

  The heating is then stopped but stirring is maintained until the temperature reaches room temperature.

  We then have an oil-in-water emulsion, the size of the oil droplets of which is centered around 80 nm, and whose mass composition is as follows: 32.5% of squalene, 6.18% of polyoxyethylene (12%) cetostearyl ether, 4.82% sorbitan monooleate, 6% mannitol. Example 2: Vaccine composition against AIDS.

  Vaccine compositions comprising as antigen a detoxified TAT III B protein are prepared. The TAT protein was detoxified by an alkaline alkylation reaction using iodoacetamide under the following conditions: number of micromoles of iodoacetamide = 200 X number of micromoles of TAT + number of micromoles of DTT. This detoxified protein and its method of preparation are described in detail in the application WO99 / 33346 where it is identified by the term carboxymethyl TAT.

  This recombinant TAT antigen is stored in solution in the presence of 50 mM TRIS buffer, pH 7.5 at -70 ° C.

  The vaccine compositions to be administered are prepared from concentrated solutions, in order to obtain immunization doses of 200111 having the following quantitative compositions: for the antigen-only composition: 20 g TAT in 50mM TRIS buffer, NaCl 100 mM pH 7.5.

  for the composition according to the invention: E 20 g of TAT, E 5 mg of squalene 10- 0.75 mg of Dehymuls SMO, E 0.94 mg of Eumulgin B1, and 0.91 mg of mannitol.

  There are 2 groups of 6 BALB / c female mice, 8 weeks old, injected with one of the compositions prepared subcutaneously, at a dose of 20O 1 per mouse; the injections are performed at OJ and D21.

  Blood samples are taken from the retro-orbital sinus at day 14 for the assessment of the primary response and at day 34 for the secondary response. The determination of specific IgG1 and IgG2a levels is performed by standardized ELISA tests.

  The mice are sacrificed at J37; their spleen is removed and the splenocytes isolated.

  The results obtained with respect to the humoral responses are summarized in the table below, where the IgG levels are expressed in arbitrary ELISA units (log10).

  For each group of mice, the value indicated in the table is the average geometric title of the values obtained for each of the mice.

  Composition IgG1 IgG2a IgG1 IgG2a Immunization report at day 14 to day 14 at day 34 at day 34 IgG1 / IgG2a at day 34 Tat 2.6 1.2 4.4 3.0 Tat + PIT 3.5 2.5 5.7 5.0 5 The results obtained show that the emulsion according to the invention makes it possible to increase the overall humoral response and also tends to favor the T helper type 1 response because the IgG2a response is increased more than the IgG1 response.

  As regards the cellular response, it was possible to demonstrate, by ELISPOT assay after restimulation with recombinant TAT protein of the splenocytes removed, a clear increase in the number of cells producing interferon γ when the splenocytes come from mice immunized with a preparation according to the invention (486 spots per 106 cells against 39 by 106 for the preparation containing the antigen alone). Similarly, the cytokine assay in culture supernatants showed the highest secretion of both interferon γ (5028 μg / ml vs 1940 μg / ml) and Interleukin 5 (5365 μg / ml against 2394 μg / ml). ml). He-

  Example 3 Preparation of a vaccine composition against human cytomegalovirus infections.

  Vaccine compositions comprising, as vaccine antigen, recombinant protein derived from an envelope glycoprotein of the Cytomegalovirus Towne strain (CMV) designated gB, the nucleotide and protein sequences of which are described in USP Patent 5,834,307, are prepared. This recombinant protein is produced by a recombinant CHO line transfected with a plasmid called pPRgB27clv4 which contains a modified gB gene. Indeed, to facilitate the production of this recombinant protein by the CHO line, the gB gene has been modified beforehand by deleting the part of the gene which encodes the transmembrane region of the gB protein corresponding to the amino acid sequence included. between Valine 677 and Arginine 752 and introducing 3 point mutations so that the existing cleavage site in the native gB was deleted. Ultimately, the recombinant protein produced by the recombinant CHO line corresponds to a truncated gB protein lacking a cleavage site and a transmembrane region called gBdTM.

  The construction of the plasmid pPRgB27clv4 and the production of the truncated gBdTM protein by the recombinant CHO line are described in US 6,100,064. Purification of the truncated gB protein is performed on an immunoaffinity chromatographic column using the 15D8 monoclonal antibody described by Rasmussen L et al. J. Virol (1985) 55: 274280).

  From a stock composition at 0.975 mg / ml of gB antigen thus obtained and maintained in phosphate buffer, the emulsion according to the invention concentrated as described in Example 1, and an emulsion of the Prior art obtained by microfluidization, doses of 50 I immunizing compositions having the following compositions were prepared: 2 g g citrate buffer pH 6, (called gB group only) 2 g gB; 1. 075 mg of squalene; 0.133 mg of Montane VG 85 and 0.125 mg of TweenTM80 in citrate buffer at pH 6 (called group with emulsion of the prior art) - 2 g of gB; 1.25 mg of squalene; 0.185 mg of Dehymuls SMO; 0.235 mg of Eumulgin B1 and 0.230 mg of mannitol in PBS buffer at pH 7.4. (called group with emulsion of the invention) There are 3 groups of 10 female Outbred OF1 mice, aged 8 weeks which are immunized twice, at 0 and J21 by the subcutaneous route, with one of the indicated compositions. above (each group of mice receives the same 2 times the same composition) Samples are taken at J20 and J34 from the retro-orbital sinus which are used to determine the concentrations of IgGI and IgG2a antibodies specific for the antigen gB .

  Assays are performed using ELISA tests; the results obtained are collated in the table below, and are expressed in loglo of the ELISA titers. The values given are the average values obtained for each group of mice.

  J20 J34 Ratio at J34 Nature of IgGI IgG2a IgGI IgG2a IgG1 / IgG2a gB groups alone 2,474 2,094 3,801 2,941 137 gB + emulsion of the prior art 4,632,980 5,493 4,185 143 gB + emulsion according to the invention 4,615 3,914 5, 615 4,854 14 These results show the effectiveness of the emulsion according to the invention which allows a greater induction of antibodies both IgGI and IgG2a type, with, in addition, compared to the emulsion according to the prior art obtaining the desired result in the context of a vaccine against human cytomegalovirus, which is to direct the immune response towards a TH1 response (one indicator of which is the IgG2a level), while maintaining the TH2-type response (one indicator is the IgGI level) at a sufficient level.

  Example 4: Vaccine composition against influenza.

  Influenza virus antigens obtained according to the method described in the examples of the application WO9605294 are available, except that the viral strain used is the A / New Caledonia H1N1 strain.

  From this antigen preparation, the concentrated emulsion obtained in Example 1, and an aluminum suspension provided by REHEIS under the name A1O0H Rehydra, immunization doses of 500 are prepared. which have the composition indicated below, where the amounts of influenza antigens are expressed by weight of haemagglutinin HA.

  either 1 g of HA in PBS buffer, or 5 g of HA in PBS buffer, or 1 g of HA and 60 g of aluminum hydroxide, or 1 g of HA; 1.25 mg of squalene; 0.185 mg of DehymulsTM SMO; 0.235 mg of Eumulgin ™ B; 0.21 mg of mannitol; all in PBS buffer.

  There are 8 groups of 5 BALB / c female mice, 8 weeks old, to which the compositions prepared according to the following distribution are administered at 0: one group receives the composition having 1 g of HA subcutaneously, one group receives the same composition having 11.tg of HA by intradermal route, a group receives the composition having 5 g of HA subcutaneously, a group receives the same composition having 5 g of HA intradermally, a group receives the composition having 11 μg of HA and 60 g of aluminum subcutaneously, one group receiving the same composition having 1 g of HA and 60 g of aluminum intradermally, a group receiving the composition having 1 of HA and the emulsion according to the invention subcutaneously, a group receives the composition having 11.tg HA and the emulsion according to the invention 25 intradermally.

  Blood samples were taken from each of the mice at D7, D14, D27, D41, D56, D77 and D105.

  The sera of the immunized mice are tested on the one hand by the ELISA technique in order to evaluate their total antibody content induced against the trivalent vaccine influenza A / H1N1 strain. The antibody titers are expressed in arbitrary ELISA units in log10 value, with a detection threshold at 1.3 log10.

  These sera are further tested by the IHA (haemagglutination inhibition) technique to determine their functional antibody content against influenza A / H1N1 strain. The antibody titers are expressed in inverse dilution in arithmetic value, with a detection threshold of 5.

  The results obtained are given in the tables below, in which the values indicated represent the average of the titles of the mice of each group.

  ELISA Titers: Pathway group J14 J28 J41 J56 J105 immunization mice HA 1 g subcutaneous 2,838 3,288 3,556 3,581 3,535 HA 5 g subcutaneous 3, 203 3,642 3,836 3,732 3,844

J

  HA 1 g + subcutaneous 2,860 3,363 ^ 3,843 3,951 3,982 A1O0H HA 1 g + subcutaneous 4,043 4,511 4,753 4,723 4,681 emulsion HA 1 g intradermal 2,174 2,814 3,143 3,075 2,735 HA 5 g intradermal 1 2,839 3,297 3,496 3,549 3, 479 HA 1 g + intradermal 2,654 3,004 3,137 3,020 2,724 A1O0H HA 1 g + intradermal 4,134 4,692 4,895 4,911 4,823 emulsion IHA titres: Pathway group J14 J28 J41 J56 J105 immunization mice HA 1 g subcutaneous 5 6 5 40 23 HA 5 g subcutaneous HA 1 g + subcutaneously 5 1 1 2 1 1 1 + subcutaneous 6,100 368 422 485 intradermal HA 1 g emulsion 5 5 23 8 FIA 5 g intradermal 5 8 10 HA 1 g + intradermal These results show the interest of the present invention; indeed, aluminum hydroxide which is a well-known adjuvant and widely used in the prior art does not make it possible to increase the immunogenicity of influenza antigens with the same speed and strength as the formulation according to US Pat. 'invention; it is also found that the composition according to the invention is particularly effective, either subcutaneously or intradermally.

  Example 5: Influenza vaccine composition.

  It is desired to evaluate in mice, the interest of the present invention to reduce the amount of vaccine antigen when it is an influenza vaccine containing as antigens 3 strains of influenza virus, and which would be administered by intradermal route.

  For this purpose, the concentrated emulsion of Example 1 is diluted with PBS buffer in order to obtain a 5% squalene emulsion, which will be half diluted by a composition comprising the antigens.

  There is indeed a composition comprising influenza virus from 3 different viral strains obtained in the manner described in the patent application WO9605294, the 3 strains being here the strain A / New Caledonia (H1N1), the strain A / Wyoming (H3N2), strain B / Jiangsu. Such a trivalent vaccine composition is a conventional composition for an influenza vaccine and corresponds to the vaccine marketed in the Northern Hemisphere during the 2004 influenza campaign. The amounts of antigen of each of the viral strains are assessed by their quantity of hemagglutinins. HA.

  The prepared immunization doses, of volume 501.tl, have the compositions indicated below: 0.33 g of HA of each of the viral strains in PBS buffer at pH 7.4; 1.31 g of HA of each of the viral strains in PBS buffer at pH 7.4; 5.25 g HA of each of the viral strains in PBS buffer at pH 7.4; 10.5 g of HA of each of the viral strains in PBS buffer at pH 7.4; 21 g of HA of each of the viral strains in PBS buffer at pH 7.4; 0.33 g HA of each of the viral strains; 1.25 mg of squalene; 0.185 mg of DehymulsTM SMO; 0.235 mg of EumulginTM B1 and 0.230 mg of mannitol in PBS buffer at pH 7.4; 1.31 g HA of each of the viral strains; 1.25 mg of squalene; 0.185 mg of DehymulsTM SMO; 0.235 mg of EumulginTM B1 and 0.230 mg of mannitol in PBS buffer at pH 7.4; 5.25 g HA of each of the viral strains; 1.25 mg of squalene; 0.185 mg of DehymulsTM SMO; 0.235 mg of Eumulgin ™ B1 and 0, 230 mg of mannitol in PBS buffer at pH 7.4.

  There are 8 groups of 10 BALB / c female mice aged 6 to 8 weeks administered intradermally (inner side of the ear) one of the compositions prepared, one composition per group.

  3 weeks after immunization, blood samples are taken and ELISA for each of the groups, IgG induced against each of the viral strains; in addition, for each of the groups, a hemagglutinin inhibition test is carried out against each viral strain.

  The results obtained are shown in the table below as averages for each of the groups; the ELISA results are expressed in loglo of arbitrary units, and the IHA results are the arithmetic mean titers of the dilution inverses.

Nature H 1N 1 H3N2 B Composition

IHA ELISA ELISA IHA ELISA IHA

  0.33 g HA 3.51 4.38 243 4.28 25 1.31 g HA 3.96 65 4.74 640 4.35 32 5.25 g HA 4.16 92 5.05 970 4 62 53 10.5 g HA 4.38 197 5.18 1810 4.71 130 21 g HA 4.63 - 260 5.37 2389 4.98 184 0.33 g HA 4.27 226 5.20 2389 4, 91 149 + emulsion 1.31 g HA 4.46 279 5.37 3880 4.95 171 + emulsion 5.25 g HA 4.68 394 5.58 5487 5.12 22 + emulsion These results make it possible to highlight particular interest of the invention for reducing the amount of antigens; in fact, thanks to the emulsion according to the invention, it is possible for the same immune response to be induced to reduce very significantly the quantity of antigens present in the immunization dose.

  Example 6: Vaccine composition against trivalent influenza in non-naive individuals.

  It is desired to test the effectiveness of the emulsion of the invention in the case of an influenza vaccine that would be administered to persons whose bodies have already been in contact with antigens of the influenza virus, as well as this is often the case either because people have already been in contact with the influenza virus or because they have been previously vaccinated with an influenza vaccine.

  According to information published by C. W. Potter in Vaccine, 2003, 21: 940-5, it is possible to use BALB / c mice previously immunized intramuscularly with a trivalent vaccine as an animal model for this test. Thus, 50 l immunization doses comprising either PBS buffer or the 2004 campaign trivalent vaccine, that is, a vaccine comprising the strain A / New Caledonia (H 1N1), the strain, are prepared. A / Wyoming (H3N2), and the B / Jiangsu strain, at a rate of 5 g of HA of each of the strains, in PBS buffer at pH 7.4.

  There are 6 groups of 7 BALB / c mice; 3 groups are immunized with the doses comprising only buffer, and 3 others with the trivalent vaccine, intramuscularly.

  In addition, from the concentrated emulsion of Example 1, and a vaccine composition comprising the 3 viral strains of the 2004 campaign mentioned above, 30 l immunization doses with the following compositions are prepared. PBS buffer alone, 0.3 g HA of each of the virus strains in PBS buffer, 0.3 g HA of each of the viral strains; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of EumulginTM B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4.

  Each of the compositions thus prepared is used to immunize at J34 intradermally (in the inner side of the ear) both a group of mice that previously received only PBS buffer, and a group of mice that received a dose of trivalent vaccine.

  A sample of blood from each of the mice is taken at day 56, and the antibodies produced against the H1N1 strain (A / New Caledonia) are assayed by means of a hemagglutinin inhibition test.

  The results obtained are summarized in the table below and represent the average values obtained for each group of mice having followed the same immunization protocol.

  Identification Nature of the dose Type of dose Title IHA vis-à-vis Group of mice for priming im for the boost id of the H1N1 strain A PBS PBS 5 B PBS Trivalent vaccine at 59 0.3 g HA / strain C PBS Trivalent vaccine at 320 0.3 g HA / strain + emulsion D trivalent PBS vaccine 145 gg HA / strain E Trivalent vaccine with trivalent vaccine at 476 gg HA / strain 0.3 g HA / strain F Trivalent vaccine with trivalent 861 vaccine HA / strain 0.3 g HA / strain + emulsion These results show the whole point of the invention even in non-naive individuals vis-à-vis the administered antigen. Indeed, contrary to the observations of CW Potter during the use of this model which, he had been able to show a weak adjuvant effect of Iscoms when they were used to immunize pre-infected or pre-vaccinated mice by influenza antigens, here we see that the emulsion according to the invention makes it possible to significantly increase the induced response, that the immunized mice are naive mice, or mice that have already been immunized with influenza vaccine.

  Example 7: Influenza trivalent vaccine composition comprising low doses of antigens Prepared from the concentrated emulsion of Example 1, and a vaccine composition comprising the 3 viral strains of the 2004 campaign (strain A / New Caledonia (H1N1) strain, A / Wyoming strain (H3N2), and B / Jiangsu strain), 30 l immunization doses having the following compositions: 0.11 g HA of each of the viral strains in PBS buffer, 0.4 g of FIA of each of the viral strains in PBS buffer, 1.6 g of FIA of each of the viral strains in PBS buffer, 6.3 g of HA of each of the viral strains in PBS buffer, 0 1 g of HA; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of Eumulgin ™ B1 and 0.138 mg of mannitol in PBS buffer pH 7.4, 0.4 g of HA; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of EumulginTM B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4 There are 6 groups of 8 female BALB / c mice aged 8 weeks, administered intradermally by OJ (internal side of ear) a dose of 30 l of one of the compositions indicated above (1 composition per group).

  In each group, half of the mice on D29 were again administered intradermally, a second dose having the same nature as the first dose administered. Blood samples were taken on D22 and D43 to determine the amounts of antibody induced.

  The antibody assays are carried out by ELISA for the antibodies induced at J22 and at J43, with respect to all the strains administered: H1N1, H3N2 and B, and by IHA with respect to the H1N1 strain. only on both J22 and J43. The results obtained are summarized in the table below, where the titles expressed are the averages obtained for each group of mice. Regarding the results at D43, the averages were performed separately within the same group, for mice that received 2 doses of vaccine and those that received only one. -21 -

  Composition Title ELISA Title Title ELISA Title IHA Title IHA Vaccine H1N1 to J22 ELISA B to J22 H1N1 to J22 H1N1 to J43 H3N2 to J22 Mouse Mice Boosted unboosted 0.1 tg HA 3,467 4,131 4,063 57 95 80 0,4 g HA 3, 816 4,527 4,313 73,226 80 1,6 g HA 4,069 4,831 4,750 147 1280 135 6.3 g HA 4,534 5,298 4,947 320 1522 320 0,1 g HA 4,200 5,015 4,807 207 2153 538 + emulsion - 0.4 g HA 4,612 5,482 5,001 453 2560 640 + emulsion Composition Title ELISA Title ELISA Title Immunization ELISA HINI at J43 H3N2 at J43 B at J43 Mice Mice no Mice Mice no Mice Boosted non boosted mice Boosted boosted Boosted boosted 0.1 tg HA 3,833 3, 425 4,782 4,174 4,479 3,911 0.4 tg HA 4,385 3,599 5,250 4,354 5,053 4,079 1,6 g HA 4,906 3,876 5,526 4,779 5,458 4,487 6.3 gg HA 5,287 4,176 6,073 5,033 5,678 4,773 0.1.tg HA 5,210 4,523 5,990 5,264 5,723 4,943 + emulsion 0.4 HA 5,394 4,790 6,171 5,640 5,962 5,144 + emulsion These results show that thanks to the emulsion according to the invention, even with low doses of antigens, very large humoral responses are obtained. Thus, the results obtained with only 0.4 tg of HA of each of the viral strains, much better responses are obtained using the emulsion according to the invention than by using a dose of 6.3 g of HA alone. In addition, it is noted that even in individuals who have not received a booster dose, the immune system continues to induce antibodies while this is not the case for individuals who received the non-adjuvanted vaccine antigens.

  Example 8: Vaccine composition against trivalent influenza is prepared from the concentrated emulsion of Example 1, and a vaccine composition comprising the 3 strains of viral 2004 campaign (strain A / New Caledonia (H 1N 1 ), the A / Wyoming strain (H3N2), and the B / Jiangsu strain) of 30 l immunization doses having the following compositions: 0.1.tg of HA of each of the viral strains in PBS buffer, 0.4 g of HA of each of the viral strains in PBS buffer, 1.6 g of HA of each of the viral strains in PBS buffer, 6.3 g of HA of each of the viral strains in PBS buffer, 0.1 g of HA; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of Eumulgin ™ B1 and 0.138 mg of mannitol in PBS buffer pH 7.4, 0.4 g of HA; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of EumulginTM B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4.

  There are 6 groups of 8 female C57BL / 6J mice, aged 8 weeks, administered intradermally (inner side of the ear) by JO 30 l of one of the compositions indicated above (1 composition by group).

  Blood samples are taken on D23 to determine the amounts of antibody induced.

  The antibody assays are carried out by ELISA and by IHA for the antibodies induced vis-à-vis all the strains administered: H1N1, H3N2 and B. The results obtained are summarized in the table below, where the titers expressed are the averages obtained for each group of mice.

  - 23 - Composition ELISA ELISA ELISA IHA IHA IHA H1N1 H3N2 B H1N1 H3N2 B 0.1 g HA 2.924 3.506 3.920 26 174 95 0.4 g HA 3.673 4.227 4.431 135 269 147 1.6 g HA 3.948 4.593 4.786 160 381 190 6 , 3 g HA 4,446 5,106 5,190 587 1174 453 0.1 g HA 4,456 5,192 5,064 349 1974 320 + emulsion 0.4 g HA 4,659 5,337 5,174 761 2348 494 + emulsion Again, the results obtained show all the interest of the emulsion according to the invention by which it is possible to very strongly reduce the amounts of antigens present. Indeed, it can be generally considered that with only 0.11 g of HA adjuvanted by the emulsion according to the invention, we obtain as good results as with a quantity of 6.3.tg of HA.

  Example 9: Trivalent vaccine composition against influenza comprising an emulsion 10 according to the invention or according to the prior art.

  Is prepared from the concentrated emulsion of Example 1, and a vaccine composition comprising the 3 viral strains of the 2004 campaign (strain A / New Caledonia (H1N1), strain A / Wyoming (H3N2), and strain B / Jiangsu) 30 l immunization doses having the following compositions: 0.3 g HA of each of the viral strains in PBS buffer, 6.3 g HA of each of the viral strains in PBS buffer, 0.3 g HA; 0.21 mg squalene; 0.031 mg DehymulsTM SMO; 0.040 mg of Eumulgin ™ B1 and 0.039 mg of mannitol in PBS buffer pH 7.4 (0.7% emulsion), 0.3 g HA; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of Eumulgin ™ B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4 (2.5% emulsion) 0.3 g of HA; 0.645 mg of squalene; 0.075mg TweenTM 80; 0.075 mg of SpanTM 85 (emulsion according to the prior art obtained by microfluidization).

  There are 5 groups of 8 female BALB / c mice, 8 weeks old, administered intradermally (inner side of the ear) by JO 30 l of one of the compositions indicated above (1 composition by group).

  In order to evaluate the quantity of antibodies induced, blood samples are taken on day 21, on which IHA (inhibition of haemagglutination) is determined by the activity against the A / H1N1 strain, the A / H3N2 strain and the B strain. The results obtained for each group of mice are shown in the table below.

  Composition tested IHA against IHA against IHA against H1N1 H3N2 B 0.3 g HA 26 174 8 6.3 g HA 247 905 73 0.3 g HA + emulsion invention at 95 640 37 0.7% 0.3 g HA + emulsion invention to 269 1974 73 2.5% 0.3 g HA + 135 987 57 emulsion prior art These results show that with an emulsion obtained according to the invention thanks to a very simple process of preparation of phase inversion by means of At a temperature change, an adjuvant is obtained which is as good and even slightly better than the emulsion of the prior art obtained using very high shear rates.

  EXAMPLE 10 Trivalent Influenza Vaccine Composition Comprising an Emulsion According to the Invention at Different Concentrations One prepares from the concentrated emulsion of Example 1, and from a vaccine composition comprising the 3 viral strains of the invention. 2004 campaign (strain A / New Caledonia (H1N1), strain A / Wyoming (H3N2), and strain B / Jiangsu) of 30 l immunization doses having the following compositions: 0.3 g of HA each viral strains in PBS buffer, 6.3 g of HA of each of the viral strains in PBS buffer, 0.3 g of HA of each of the viral strains; 0.12 mg of squalene; 0.018 mg DehymulsTM SMO; 0.023 mg of EumulginTM B1 and 0.022 mg of mannitol in PBS buffer at pH 7.4 (0.4% emulsion), 0.3 g of HA of each of the viral strains; 0.299 mg of squalene; 0.044 mg DehymulsTM SMO; 0.057 mg of EumulginTM B1 and 0.055 mg of mannitol in PBS buffer at pH 7.4 (1% emulsion) 0.3 g of HA of each of the viral strains; 0.75 mg squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg of Eumulgin ™ B1 and 0.138 mg of mannitol in PBS buffer pH 7.4 (2.5% emulsion). There are 5 groups of 8 female BALB / c mice, 8 weeks old, administered JO intradermally (inner side of the ear) a dose of 30 l of one of the compositions indicated above (1 composition per group).

  To evaluate the amount of antibody induced, blood samples are taken at D21 on which the anti-H1N1 antibodies, the anti-H3N2 antibodies and the anti-B antibodies are determined by ELISA.

  The results obtained are indicated in loglo of arbitrary units in the table below, which indicates for each group of mice the average of the results obtained on each of the mice.

  Tested Composition Anti-HiN1 Anti-H3N2 Anti-B 0.3 g HA 3.864 4.594 4.406 6.3 g HA 4.478 5.041 5.053 0.3 g HA + 4.053 4.827 4.545 emulsion invention 0.4% 0.3 g HA + 4.3212 5.074 4,733 emulsion invention at 1% 0.3 g HA + 4,425 5,200 4,840 emulsion invention at 2,5% These results confirm again that, whatever the strain evaluated, the emulsion according to the invention allows with a very low dose of antigens, to have a very important immune system response.

  EXAMPLE 11 Preparation of a freeze-dried composition The procedure is as in Example 1, but using water instead of the buffer; the emulsion obtained is then diluted with an aqueous solution comprising mannitol, sucrose and dodecylmaltoside, in order to obtain an emulsion whose final composition is as follows: 5% squalene, 0.95% polyoxyethylene cetostearyl ether, 0 , 75% sorbitan monooleate 3% mannitol, 2% dodecylmaltoside, 6% sucrose This emulsion was lyophilized and stored for 3 months at 4 ° C .; then after reconstitution, it was found that its properties were preserved, especially its monodisperse emulsion qualities, with a value of d90 equal to that measured before lyophilization.

  This emulsion can be diluted in% 2 with a solution comprising vaccine antigens to give a vaccine composition according to the invention.

Claims (27)

  A vaccine composition comprising at least one vaccine antigen and an oil-in-water emulsion comprising at least: squalene, a nonionic surfactant comprising ethylene oxide groups, with the exception of polyoxyethylene and polyoxypropylene copolymers, a nonionic surfactant; ionic hydrophobic, characterized in that the emulsion is thermoreversible and in that 90% of the population density of oil drops has a size less than 200 nm.   2. Vaccine composition according to the preceding claim, characterized in that 90% of the volume population of oil drops has a size less than 160 nm.   3. Vaccine composition according to the preceding claim, characterized in that 90% of the volume population of oil drops has a size less than 150 nm.   4. Vaccine composition according to the preceding claim, characterized in that 50% of the volume population of oil drops has a size less than 100 nm.   5. Vaccine composition according to the preceding claim, characterized in that 50% of the population density of oil drops has a size less than 90 nm.   6. Vaccine composition according to one of the preceding claims, characterized in that it further comprises at least one alditol.   7. Composition according to Claim 1, characterized in that the hydrophobic nonionic surfactant is a surfactant based on sorbitan or mannide ester.   8. Vaccine composition according to one of the preceding claims, characterized in that the surfactant comprising ethylene oxide groups is polyoxyethylene (12) cetostearyl ether.   - 28 - 9. Vaccine composition according to one of the preceding claims, characterized in that the alditol is selected from glycerol, erythritol, xylitol, sorbitol, mannitol.   10. Vaccine composition according to one of the preceding claims, characterized in that the hydrophobic nonionic surfactant is sorbitan monooleate.   11. Vaccine composition according to one of the preceding claims, characterized in that the amount of squalene is between 0.5 and 5%.   12. Vaccine composition according to one of the preceding claims, characterized in that the amount of surfactant comprising ethylene oxide groups is between 0.02 and 0.95%.   13. Vaccine composition according to one of the preceding claims, characterized in that the amount of hydrophobic nonionic surfactant is between 0.015 and 0.75%.   14. Vaccine composition according to one of the preceding claims, characterized in that it comprises: 2.5% squalene, 0.47% polyoxyethylene (12) cetostearyl ether, 0.37% sorbitan monooleate, 0.46% of mannitol.   15. Vaccine composition according to one of the preceding claims, characterized in that it further comprises an alkyl glycoside.   16. Vaccine composition according to one of the preceding claims, characterized in that it further comprises a cryoprotectant.   17. Vaccine composition according to one of the preceding claims, characterized in that the vaccine antigen is a viral antigen.   - 29 - 18. Vaccine composition according to the preceding claim, characterized in that the vaccine antigen is an antigen influenza.   19. Vaccine composition according to the preceding claim, characterized in that the influenza vaccine antigen is a monovalent antigen.   20. Vaccine composition according to one of the preceding claims, characterized in that the vaccine antigen is a set of 3 strains of inactivated and fragmented influenza virus.   21. Use of a composition according to one of the preceding claims for the preparation of a vaccine intended to be administered intramuscularly.   22. Use of a composition according to one of claims 1 to 20 for the preparation of a vaccine for intradermal administration.   23. Use of a composition according to one of claims 1 to 20, for the preparation of a vaccine intended to be administered subcutaneously.   24. Use of a composition according to one of claims 1 to 20, for the preparation of a vaccine for administration against influenza.   25. Use of a composition according to one of claims 1 to 20, for the preparation of a vaccine to be administered against AIDS.   26. Use of a composition according to one of claims 1 to 20 for the preparation of a vaccine for administration against human cytomegalovirus pathologies.   27. Process for preparing a vaccine composition comprising at least one vaccine antigen and an oil-in-water emulsion, characterized in that the oil-in-water emulsion is obtained by a phase inversion method by varying the temperature.