VACCINE ADJUVANT COMPRISING AN INVERSE MICROLATEX

Abstract

Disclosed is a vaccine adjuvant including at least one inverse microlatex, the inverse microlatex including at least one oil, at least one surfactant, at least one polymer such as, for example, a polyacrylate that is totally or partially neutralized in the form of alkali metal salts or ammonium salt, the vaccine adjuvant being entirely sterilizable by filtration or by passing through the heat of an autoclave and emulsifiable in one step with the aqueous phase including only a vaccine antigen.

Claims

1. A vaccine adjuvant comprising at least, as inverse microlatex, an inverse microemulsion comprising at least one polyelectrolyte-type polymer.

2. The vaccine adjuvant as claimed in claim 1, wherein the inverse microlatex comprises an oily 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 and crosslinked polyelectrolyte; with said anionic and crosslinked polyelectrolyte comprising at least one crosslinking monomer and at least one hydrophilic monomer unit.

3. The vaccine adjuvant as claimed in claim 2, wherein the monomer unit originates from acrylic acid that is completely or partially salified with an alkali or alkaline-earth metal salt or an ammonium salt.

4. The vaccine adjuvant as claimed in claim 3, wherein the acrylic acid is totally or partially salified with a sodium salt or an ammonium salt.

5. The vaccine adjuvant as claimed in claim 2, wherein the anionic and crosslinked polyelectrolyte comprises a monomer unit of formula (1): ##STR00002## with: R1 chosen from —H, —CH.sub.3, —C.sub.2H.sub.5 and —C.sub.3H.sub.7, n between 0 and 50, and m between 8 and 22.

6. The vaccine adjuvant as claimed in claim 1, wherein said adjuvant further comprises an oil (H.sub.1), at least one water-in-oil surfactant (E.sub.1) and at least one oil-in-water surfactant (E.sub.2).

7. The vaccine adjuvant as claimed in claim 6, further comprising between 1% and 10% by weight of water-in-oil surfactant (E.sub.1).

8. The vaccine adjuvant as claimed in claim 6, further comprising between 1% and 10% by weight of water-in-oil surfactant (E.sub.2).

9. The vaccine adjuvant as claimed in claim 6, comprising, per 100% of its weight: a) from 50% to 97.5% by weight of said oil (H.sub.1); b) from 1% to 10% by weight of said water-in-oil surfactant (E.sub.1); c) from 1% to 10% by weight of said oil-in-water surfactant (E.sub.2); and d) from 0.5% to 30% by weight of at least one inverse microlatex; it being understood that the sum of the weight contents a)+b)+c)+d) is equal to 100%.

10. The vaccine adjuvant as claimed in claim 6, wherein the oil (H.sub.1) is a white mineral oil.

11. (canceled)

12. A vaccine comprising the adjuvant as defined in claim 1 and also at least one aqueous solution (S) of at least one antigen or of at least one in vivo generator of a compound comprising an amino acid sequence.

13. A vaccine comprising the adjuvant as defined in claim 1 and also at least one aqueous solution (S) of at least one antigen or of at least one in vivo generator of a compound comprising an amino acid sequence, wherein the vaccine comprises: from 10% to 80% by weight of the adjuvant, and, from 20% to 90% by weight of the aqueous solution (S).

14. The vaccine as claimed in claim 12, wherein said vaccine is in the form of a water-in-oil emulsion or an oil-in-water emulsion.

15. A process for preparing a vaccine adjuvant as defined in claim 1, comprising a step of sterilizing the vaccine adjuvant by filtration or by autoclaving.

16. The process as claimed in claim 15, wherein said process comprises the following steps: a) preparing, with mechanical stirring and at ambient temperature, an oily phase comprising at least one oil and an emulsifying system comprising at least one water-in-oil surfactant (E.sub.1) and/or oil-in-water surfactant (E.sub.2); b) adding the at least one inverse microlatex with mechanical stirring at ambient temperature; c) maintaining the mechanical stirring, at ambient temperature, until a homogeneous mixture is obtained.

17. A process for preparing a vaccine, comprising the following steps: a) preparing the vaccine adjuvant as claimed in the process as defined in claim 15, and b) mixing the vaccine adjuvant obtained in step a) with an antigen medium.

18. The vaccine of claim 5, wherein R1 is CH.sub.3.

19. The vaccine adjuvant of claim 7, comprising from 3% to 8% by weight of water-in-oil surfactant.

20. The vaccine adjuvant as claimed in claim 6, comprising, per 100% of its weight: a) from 60% to 90% by weight of said oil (H.sub.1); b) from 3% to 8% by weight of said water-in-oil surfactant (E.sub.1); c) from 3% to 8% by weight of said oil-in-water surfactant (E.sub.2); and d) from 1% to 10% by weight of at least one inverse microlatex; it being understood that the sum of the weight contents a)+b)+c)+d) is equal to 100%.

21. The vaccine adjuvant as claimed in claim 3, wherein the anionic and crosslinked polyelectrolyte comprises a monomer unit of formula (1): ##STR00003## with: R1 chosen from —H, —CH.sub.3, —C.sub.2H.sub.5 and —C.sub.3H.sub.7, n between 0 and 50, and m between 8 and 22.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0121] FIG. 1 is a graph representing the response of the IgG1 antibody against the OVA antigen in mice for a vaccine comprising the ADJ3 adjuvant according to the invention.

[0122] FIG. 2 is a graph representing the response of the IgG2a antibody against the OVA antigen in mice for a vaccine comprising the ADJ3 adjuvant according to the invention.

[0123] FIG. 3 is a graph representing the change in the titre of antibodies against Newcastle disease LaSota strain (NDV) from DO to D28.

[0124] FIG. 4 is a graph representing the change in the titre of antibodies against H9N2 avian influenza (AIV) from DO to D28.

[0125] FIG. 5 is a graph representing the degree of protection (number of animals protected/total number) obtained by the test vaccine group in comparison with the control group.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

Example 1: Preparation of Inverse Microlatexes Based on Sodium Polyacrylate

[0127] 1.1 Preparation of Inverse Microlatexes (A), (B), (C) and (D)

[0128] Inverse microlatexes comprising, as polymer, a crosslinked sodium polyacrylate, are prepared according to the teachings of the European patent published under the number 1 371 692 B1, which is incorporated here by reference. More particularly the teachings of paragraph [0021], paragraphs [0025] and [0026], paragraphs [0033] to [0048], and even more particularly paragraphs [0039] to [0041] (example 2) of the European patent published under the number 1 371 692 B1, are used to prepare the inverse microlatexes. For each of the microlatexes prepared, a fluid white mineral oil, Marcol™52 sold by the company Exxon Mobil, is used. The same process for preparing inverse microlatexes is carried out in the presence of various weight concentrations of surfactants, and makes it possible to obtain: [0129] the inverse microlatex (A) when the weight amount of surfactants is equal to 14%, [0130] the inverse microlatex (B) when the weight amount of surfactants is equal to 18%, [0131] the inverse microlatex (C) when the weight amount of surfactants is equal to 22%, [0132] the inverse microlatex (D) when the weight amount of surfactants is equal to 25%.

[0133] The inverse microlatexes (A), (B), (C) and (D), obtained after radical polymerization, are in the form of opalescent to translucent oily compositions. These inverse microlatexes contain 60% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 25% by weight of water.

[0134] 1.2 Preparation of Inverse Microlatexes (E), (F), (G) and (H)

[0135] The process for preparing the inverse microlatex (A) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (E).

[0136] The process for preparing the inverse microlatex (B) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (F).

[0137] The process for preparing the inverse microlatex (C) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (G).

[0138] The process for preparing the inverse microlatex (D) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (H).

[0139] The inverse microlatexes (E), (F), (G) and (H) are in the form of an opalescent to translucent oily composition, containing 60% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 25% by weight of water.

Example 2: 1.1 Preparation of Inverse Microlatexes (A′), (B′), (C′) and (D′)

[0140] The process for preparing the inverse microlatexes (A), (B), (C), and (D), described in example 1.1, is carried out in the presence of a mixture of acrylic acid and tetraethoxylated lauroyl methacrylate (2 mol %), in order to obtain respectively the inverse microlatexes (A′), (B′), (C′), and (D′).

[0141] The inverse microlatexes (A′), (B′), (C′), and (D′) are in the form of opalescent to translucent oily compositions, containing 60% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked copolymer of acrylic acid and tetraethoxylated lauroyl methacrylate and 25% by weight of water.

Example 3: Preparation of Inverse Microlatexes (B″) and (C″)

[0142] The process for preparing the inverse microlatex (B) of example 1 is carried out in the presence of a larger amount of water, so as to obtain an inverse microlatex (B″) that is in the form of opalescent to translucent oily compositions, containing 49% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 36% by weight of water.

[0143] The process for preparing the inverse microlatex (C) of example 1 is carried out in the presence of a larger amount of water, so as to obtain an inverse microlatex (C″) that is in the form of opalescent to translucent oily compositions, containing 49% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 36% by weight of water.

Example 4: Preparation of Adjuvants According to the Invention

[0144] The polymeric oily adjuvants are prepared according to the following process: [0145] a) preparing, with mechanical stirring and at ambient temperature, an oily phase comprising at least one oil and an emulsifying system comprising at least one water-in-oil surfactant (E.sub.1) and/or oil-in-water surfactant (E.sub.2); [0146] b) adding the inverse microlatex or inverse latex, with moderate mechanical stirring (50 to 150 rpm) at ambient temperature; [0147] c) maintaining the moderate mechanical stirring (50 to 150 rpm), at ambient temperature, until a homogeneous mixture is obtained.

[0148] For the purposes of the present invention, ambient temperature is understood to mean a temperature above or equal to 15° C. and below or equal to 30° C.

[0149] In this way, the ADJ1, ADJ2, ADJ3, ADJ′1 adjuvants are prepared and are characterized by the compositions described in table 1:

TABLE-US-00001 TABLE 1 adjuvants according to the invention and comparative adjuvants (composition in %) ADJ 1 ADJ 2 ADJ 3 ADJ′1 Marcol ™ 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 .sup.(1)   0%   0%   0%   2%

[0150] (1): Sodium polyacrylate that is in the form of an inverse latex, the preparation of which is described in patent FR2922767 B1.

Example 5: Evaluation of the Adjuvants According to the Invention and of the Comparative Adjuvants

[0151] 5.1 Filterability

[0152] The filterability of the adjuvants according to the invention and of the comparative adjuvants is evaluated according to the following protocol: [0153] introduce 10 ml of adjuvant into a 2-piece 12 ml syringe, [0154] connect a 0.22 μm PTFE syringe filter with a diameter of 25 mm, [0155] apply a weight of 3310 g, [0156] measure the mass filtered as a function of time.

[0157] The amount of adjuvant filtered is measured as a function of time and the results obtained for each adjuvant tested are recorded in the tables below:

TABLE-US-00002 TABLE 2 kinetics of the amount of filtered adjuvant ADJ1 according to the invention Time (s) 10 20 30 60 90 120 Mass 0.48 1.12 1.68 3.27 4.85 6.34 filtered (g)

TABLE-US-00003 TABLE 3 kinetics of the amount of filtered adjuvant ADJ2 according to the invention Time (s) 10 20 30 60 90 120 Mass 0.69 1.39 2.14 4.38 6.74 9.03 filtered (g)

TABLE-US-00004 TABLE 4 kinetics of the amount of filtered adjuvant ADJ3 according to the invention Time (s) 10 20 30 60 90 120 Mass 0.43 0.85 1.28 2.56 3.83 5.11 filtered (g)

TABLE-US-00005 TABLE 5 kinetics of the amount of filtered adjuvant ADJ′1 according to the invention Time (s) 10 20 30 60 90 120 Mass 0 0 0 0 0 0 filtered (g)

[0158] The results recorded in tables 2 to 5 show that the filtration of the adjuvants according to the invention ADJ1, ADJ2, ADJ3 is faster than that of the comparative adjuvant ADJ′1 on a hydrophobic filter, in particular made of PTFE, with a mean pore diameter of 0.2 micrometers.

[0159] 5.2 Study of the Stability of the Adjuvants According to the Invention and of the Comparative Adjuvants

[0160] The stability of the adjuvants according to the invention ADJ1, ADJ2, ADJ3 and of the comparative adjuvant ADJ′1 is evaluated according to the following protocol:

[0161] i) An amount of 90 ml of the composition to be tested, contained in a 100 ml flask, is introduced into a climatic chamber regulated at 20° C., for a period of one year. The visual appearance of the compound tested is evaluated before being placed in the stability test in the chamber and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).

[0162] ii) An amount of 90 ml of the composition to be tested, contained in a 100 ml flask, is introduced into a climatic chamber regulated at 37° C., for a period of one month (M1). The visual appearance of the composition tested is evaluated before being placed in the stability test in the chamber and after a period of one month.

[0163] Stability is understood to mean the absence of phase separation and/or observed sedimentation. The results of the observations are recorded in table 6 below.

TABLE-US-00006 TABLE 6 stability results of the adjuvants ADJ1, ADJ2, ADJ3 according to the invention, and of the comparative adjuvant ADJ′1 ADJ 1 ADJ 2 ADJ 3 ADJ′1 Stability at 20° C. 1 month (M1) Clear Clear Clear Heterogenous 2 months (M3) Clear Clear Clear Heterogenous 6 months (M6) Clear Clear Clear Heterogenous 1 year (Y1) Clear Clear Clear Heterogenous Stability at 37° C. 1 month (M1) Clear Clear Clear Heterogenous

[0164] (Clear): homogeneous and clear, a single phase

[0165] (Heterogeneous): heterogeneous, two or three phases observed, presence of deposit.

[0166] The adjuvants ADJ1, ADJ2, ADJ3, ADJ′1 according to the invention have homogeneous appearances under the storage conditions described below, whereas heterogeneous appearances (phase separation and sedimentation) are observed over time and at various temperatures for the comparative adjuvant ADJ′1.

[0167] 5.3. Characterization of the Stability Properties of Vaccine Compositions Containing Adjuvants According to the Invention

[0168] The stability properties of the placebo vaccine emulsions containing the adjuvants according to the invention ADJ1, ADJ2, ADJ3, are evaluated on an amount of 200 grams (prepared in a 250 ml low form beaker) according to the following protocol, using a Silverson L4 or L5 rotor-stator mixer fitted with its standard head provided with a standard emulsion screen:

[0169] i/ 60 grams of aqueous phase are added to 140 grams of adjuvant with mechanical stirring using a Silverson L4 or L5 mixer at a rotational speed of 1000 rpm (during this step the stirring head should be placed 0.5 cm from the bottom of the beaker);

[0170] ii/ the emulsion is produced by subjecting the mixture obtained in step i/ to high shear (with the Silverson L4 or L5 mixer) for a period of 3 minutes at a rotational speed of 4000 rpm (or 7 m/s).

[0171] The emulsions obtained at the end of step ii/ are fluid, homogeneous and injectable. The term “fluid” more particularly means a liquid emulsion, the dynamic viscosity of which, measured using a Brookfield LVDV1+ equipped with an M62 spindle at a rotational speed of 60 rpm, at 20° C., is between 30 and 40 mPa.Math.s.

[0172] The stability of the emulsions obtained is characterized as follows:

[0173] i) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climatic chamber regulated at 4° C., for a period of one year. The visual appearance of the compound tested is evaluated before being placed in the stability test in the chamber and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).

[0174] ii) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climatic chamber regulated at 20° C., for a period of one year. The visual appearance of the compound tested is evaluated before being placed in the stability test in the chamber and after a period of one month (M1), three months (M3), six months (M6) and one year (A1).

[0175] iii) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climatic chamber regulated at 37° C., for a period of one month (M1). The visual appearance of the composition tested is evaluated before being placed in the stability test in the chamber and after a period of one month.

[0176] Stability is understood to mean the absence of phase separation and/or observed sedimentation. The results of the observations are recorded in table 7 below.

TABLE-US-00007 TABLE 7 stability results of the emulsions containing the adjuvants ADJ1, ADJ2, ADJ3 according to the invention Emulsion Emulsion Emulsion ADJ 1 ADJ 2 ADJ 3 Stability at 4° C. 1 month (M1) H H H 2 months (M3) H H H 6 months (M6) H H H 1 year (Y1) H H H Stability at 20° C. 1 month (M1) H H H 2 months (M3) H H H 6 months (M6) H H H 1 year (Y1) H H H Stability at 37° C. 1 month (M1) H H H (H): homogeneous, only one phase observed

[0177] 5.4 Characterization of the Immunological Properties of Vaccines Containing Adjuvants According to the Invention

[0178] The adjuvant properties of the polymeric oily adjuvants ADJ1, ADJ2 and ADJ3 according to the invention and as described in the preceding examples were characterized on several vaccine models and several animal species.

[0179] During a first test, the adjuvant according to the invention ADJ3 was formulated with a solution of ovalbumin to obtain a vaccine intended to be injected into mice.

[0180] In a second test, the adjuvant according to the invention ADJ2 was formulated with a bacterial antigen medium consisting of inactivated Pasteurella multocida bacteria to obtain a vaccine intended to be administered to avian species.

[0181] In a third test, the adjuvant according to the invention ADJ1 was used for the formulation of a viral vaccine intended for avian species against Newcastle disease and H9N2 influenza. These tests demonstrate the vaccine adjuvant properties of the adjuvants according to the invention in several species and several antigen models.

[0182] The results obtained are presented below.

[0183] 5.4.1 Trial 1: Trial in Mice, with Ovalbumin as Antigen

[0184] The trial is carried out on OF1 mice in a 90-day vaccination protocol with ovalbumin (OVA) as an antigen model. The vaccines were prepared from an antigen solution of OVA prepared in physiological serum at 10 mg/ml, 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 is carried out by emulsification through an i-connector in an adjuvant according to the invention ADJ3/antigen medium ratio of 70/30 (volume/volume).

TABLE-US-00008 TABLE 8 composition of the vaccines tested ADJ 3 Physiological OVA 10 mg/ml Groups μl Serum μl μl Test Vaccine Group 1400  580 20 Antigen Control Vaccine Group 1980 20

[0185] The safety of the vaccines is assessed by observing local reactions at the injection sites. Vaccine efficacy is assessed by detection of IgG1 and IgG2a antibodies in the blood by the ELISA method. This detection is carried out on the day of vaccination (“primo-vaccination” on DO), then after 14 days (D14), at the time of the booster vaccination on the twenty-eighth day (D28), then on the forty-second day (D42), then on the fifty-sixth day (D56), then on the ninetieth day (D90) at the time of euthanasia.

[0186] No local reaction was observed among the members of the test group, thus signifying that the vaccine comprising the ADJ3 adjuvant according to the invention was well tolerated.

[0187] FIGS. 1 and 2 show the assay of the IgG1 and IgG2a antibodies on D14, D28, D42, D56 and D90.

[0188] FIG. 1 is a graph representing the response of the IgG1 antibody against the OVA antigen in mice for a vaccine comprising the ADJ3 adjuvant according to the invention.

[0189] FIG. 2 is a graph representing the response of the IgG2a antibody against the OVA antigen in mice for a vaccine comprising the ADJ3 adjuvant according to the invention.

[0190] Significantly higher antibody titers are observed for the vaccine comprising the ADJ3 adjuvant according to the invention compared to the non-adjuvanted vaccine comprising the antigen, for the two classes of antibodies, which confirms the vaccine adjuvant properties of the formula developed.

[0191] 5.4.2 Trial 2: Vaccine Trial in Chickens, Pasteurella multocida Bacterial Antigen

[0192] This trial was carried out on chickens in a 42-day vaccination protocol against the Pasteurella multocida bacterial pathogen. The animals used in this experiment are red chickens aged 36 days at the time of vaccination (DO). Each vaccine dose contains 1 dose (0.5 ml)=0.5×10.sup.8 CFU or 1.10.sup.8 CFU/ml of inactivated Pasteurella multocida bacteria. The vaccine groups consist of 11 male and female chickens randomly distributed between the groups.

TABLE-US-00009 TABLE 9 composition of the vaccines tested Adjuvant Antigen Phys. Group Antigen dose ADJ 2 (g) (ml) Serum (g) Test Vaccine 0.5 × 10.sup.8 UFC 21 1.5  7.5 g 0.5 ml Antigen 0.5 × 10.sup.8 UFC 1.5 28.5 g Control 0.5 ml

[0193] The formulation containing the ADJ2 adjuvant according to the invention is prepared using a Tube Drive emulsifier (sold by the company Ika) in sterile DT50 tubes in an ADJ2 adjuvant according to the invention/antigen medium ratio of 70/30 (weight/weight): speed 3 for 2 minutes (1100 rpm) for the pre-emulsion then speed 9 for 6 minutes (4000 rpm).

[0194] The animals are vaccinated on DO. Local reactions are observed at slaughter on D42. Blood samples will be taken on DO, D14, D42. Antigen-specific ELISA assays will be performed for the detection of antibody levels in serums using a commercial detection kit (ID Screen Pasteurella multocida chicken and turkey indirect kit sold by ID-VEt).

[0195] The vaccine comprising the ADJ2 adjuvant according to the invention is well tolerated in chickens, since no critical local reaction is observed at slaughter. Significantly higher antibody titers are also observed for the vaccine adjuvanted with the ADJ2 adjuvant compared with the non-adjuvanted vaccine in chickens, as shown in table 10 below.

TABLE-US-00010 TABLE 10 IgY antibody response against Pasteurella multocida in chickens for a vaccine comprising the adjuvant ADJ2 according to the invention. D0 D14 D42 Test Vaccine Group 213 7506 6176 IgY titer (in AU) Antigen Control 225  277  791 Vaccine Group IgY titer (in AU)

[0196] 5.4.3 Trial 3: Vaccine Trial in Chickens, Newcastle Disease/Avian Influenza Viral Antigen

[0197] This trial was carried out on chickens in a 28-day vaccination protocol with a bivalent inactivated viral vaccine against Newcastle disease LaSota strain (NDV) and H9N2 avian influenza (AIV). The animals used in this experiment are SPF (specific pathogen free) chickens aged 28 days at the start of the experiment (DO). The vaccine groups are formed as follows in table 11:

TABLE-US-00011 TABLE 11 composition of the vaccine groups tested Groups No. of animals AI-ND vaccine dose Valency tested Test vaccine 10  0.3 ml AIV 10 0.02 ml NDV Unvaccinated  5 / AIV control group  5 / NDV

[0198] The test vaccine is formulated using the polymeric oily adjuvant ADJ1 according to the invention by emulsification in an ADJ1 adjuvant according to the invention/antigen medium ratio of 70/30 (weight/weight) with the antigen medium containing the two AIV and NDV inactivated viral valencies. The control group is not vaccinated.

[0199] The vaccines are injected intramuscularly on DO. Blood samples are taken on D0, D7, D14, D21 and D28 after vaccination and analyzed by hemagglutination inhibition test to determine the specific antibody titers against each valency (AIV and NDV). In the AIV group, a protection challenge is carried out on D28 after vaccination to measure the viral load after challenge (2.10.sup.6 EID.sub.50 in 0.2 ml intravenously of XZ strain H9N2 AIV virus). Oropharyngeal and cloacal swabs are collected 5 days after viral challenge and inoculated into SPF chicken embryos to measure viral presence after two generations of transmission.

[0200] Strong antibody titers are observed in the vaccinated group after vaccination for the two valencies (FIG. 1 and FIG. 2). After virulent challenge, no mortality was observed, and in the vaccinated group there was also no viral load observed in the swab samples, which demonstrates complete protection against the AIV viral challenge (FIG. 3).

[0201] FIG. 3 is a graph representing the change in the titre of antibodies against Newcastle disease LaSota strain (NDV) from DO to D28.

[0202] FIG. 4 is a graph representing the change in the titre of antibodies against H9N2 avian influenza (AIV) from DO to D28.

[0203] FIG. 5 is a graph representing the degree of protection (number of animals protected/total number) obtained by the test vaccine group in comparison with the control group.

[0204] 5.5 Experimental Conclusion

[0205] The adjuvants according to the invention are characterized by: [0206] filtration kinetics, in particular on hydrophobic filters (in particular made of polytetrafluoroethylene (PTFE)) with a mean pore diameter of 0.2 micrometers, suitable for obtaining a sterile adjuvant, [0207] stability over time at 20° C. and 37° C., i.e. retaining a homogeneous and clear appearance, without displaying phase separation and/or sedimentation phenomena, [0208] obtaining stable vaccine emulsions, formed by emulsification of said adjuvants in the presence of vaccine aqueous phase, [0209] an adjuvant effect of immunity in various animal species in vaccine compositions in the presence of various antigen media.