MXPA97004823A - Adjuvants for vacu - Google Patents

Abstract

The present invention relates to adjuvants for vaccines comprising a liquid medium containing polymers having anionic repeating constitutive units and hydrophobic repeating constituent units. Advantageously, the adjuvants are aqueous solutions of partially esterified polyacrylic acids. The novel adjuvants are highly stable, effective and with a relatively low level of local toxicity. In addition, the invention also relates to vaccines comprising such adjuvants, as well as to a process for their manufacture.

Description

ADJUVANTS FOR VACCINES DESCRIPTION OF THE INVENTION The present invention describes adjuvants for novel vaccines.

An antigen is defined as a foreign substance, which, when administered, for example, parenterally, induces an immune response, which includes the production of antibodies. Antibodies are substances contained in the blood and other fluids of the body, as well as in the tissues, and which bind to the antigen to make it harmless. Antibodies are one of the body's natural defense mechanisms. They are highly specific and can kill, agglutinate or render innocuous the antigen that has induced their formation.

The antigen, in contact with the immune system, thus activates a complex series of cellular interactions, to eliminate the antigen and / or reestablish the preceding equilibrium.

Two of the characteristic aspects of the antigens are their immunogenicity, that is, their capacity to induce an immune response in vivo (including the formation of specific antibodies), and their antigenicity, that is, their REF: 25059 capacity to be selectively recognized by the antibodies whose origins are the antigens. It is known that it is possible to deliberately stimulate the immune response by administering a specific antigen by means of a vaccine. This procedure allows the development in the organism of a state of immunological memory that ensures a faster and more effective response of the organism during the subsequent contact cor. the antigen. Sir. However, some antigens have only a weak immunogenicity, and induce an insufficient immune response to provide effective protection for the organism.

The immunogenicity of an antigen can be administered by administering it in a mixture with substances, called adjuvants, that increase the antigen response, either by acting directly on the immune system, or modifying the pharmacokinetic characteristics of the antigen, and thus increasing the time of antigen. interaction between the latter and the immune system.

The most widespread adjuvants are, on the one hand, Freund's adjuvant, an emulsion comprising dead mycobacteria in a saline solution in mineral oil and, on the other hand, incomplete Freund's adjuvant, which does not contain mycobacteria. These adjuvants are capable of either increasing the intensity of the immune response to the antigen, or of producing a specific activation of the immune system.

However, the use of these adjuvants comprises disadvantages such as the formation of irritation or abscesses er. the point of the injection. Furthermore, for these adjuvants to be effective, the concentration used must be greater than 50% of the volume injected, which limits the useful load of antigens that one can inject in a dose.

The high viscosity of these standard adjuvants based on oil and water makes their use impractical, because they are difficult to introduce into syringes and to inject them into animals.

Another type of adjuvant that has been described comprises a polyacrylic acid solution (Diamanstein et al., Z. Kiin, Chem. Klin. Biochem., Vol. 8, pp. 632-636 (1970) and Diamanstein et al., Eur. J. Immunol., Vol. 1, pp. 335-339 (1971)). The advantage of this type of adjuvant is that it is less viscous than conventional adjuvants based on mineral oil and water. This can therefore be manipulated and injected more easily. However, the efficacy of these adjuvants is not comparable to that of the water and mineral oil (W / O) based adjuvants.The purpose of the present invention is to propose a vaccine adjuvant that is effective in a small concentration, and without mineral oil This purpose is achieved by a vaccine adjuvant comprising an aqueous solution of polymers having anionic constituent repeating units and hydrophobic constitutive repeat units.

The term anionic constituent repeating units denotes, for the purposes of the present invention units of monomers constituting the polymer, containing groups capable of dissociating in water while forming anions. Examples of such monomer units which are useful in the present invention for forming the anionic constituent repeating units are (selected from) acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinyl sulfuric acid, styrenesulfonic acid, vinylphenylsulfuric acid, 2-methacryloyloxyethanesulfonic acid, 3-methacryloyloxy-2-hydroxypropanesulfonic acid, 2-acryl-2-methylpropanesulfonic acid, 3-acrylamido-3-methylbutanoic acid, 3-methacrylamido-3-methylbutanoic acid, vinylphosphoric acid, 4-vinylbenzoic acid, - 3-vinyloxypropane-1-sulphonic acid and N-vinylsuccimidic acid. Preferably, the monomer units of this type are selected from acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulphonic acid, vinyl sulfuric acid and styrenesulfonic acid.

It is preferred that the monomer units of this type are selected from acrylic acid, methacrylic acid, maleic acid and fumaric acid.

It is particularly preferred that the monomer units of this type are acrylic acid units.

The term "hydrophobic constituent repeating units" denotes, for the purposes of the present invention, monomer units that constitute the polymer that exclusively contain hydrophobic groups, also called lipophilic, which do not dissociate in water.

Examples of such monomer units that are useful in the present invention for forming the hydrophobic constitutive repeating units are (selected from) esters of the acids mentioned above (acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinylsulfuric acid) , styrenesulfonic acid, vinylphenylsulfuric acid, 2- methacryloyloxyethane sulfonic acid, 3-methacryloyloxy-2-hydroxypropanesulfonic acid, 2-acryl-2-methylpropanesulfonic acid, 3-acrylamido-3-methylbutanoic acid, 3-methacrylamido-3-methylbutanoic acid, vinylphosphoric acid , 4-vinylbenzoic acid, 3-vinyloxypropane-1-sulfonic acid and N-vinyl-succinic acid), and ethers (for example, methoxymethyl, ethoxyethyl, allyloxymethyl, 2-ethoxyethoxymethyl, benzyloxymethyl, cylcoxyethyl, 1-ethoxyethyl, 2-ethoxyethyl ethers) , 2-butoxyethyl, methoxymethoxyethyl, methoxyethoxyethyl, 1-butoxypropyl, 1-ethoxybutyl , tetrahydrofurfuryl or furfuryl). Preferably, the monomer units of this are selected from the alkyl esters of acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinyl sulfuric acid or styrene sulfonic acid. Preferably, the monomer units of this type are selected from the alkyl esters of acrylic acid, methacrylic acid, maleic acid or fumaric acid, which alkyl groups contain 4-8 carbon atoms. It is particularly preferred that the monomer units of this type are linear alkyl esters of acrylic acid, which alkyl group contains 4-8 carbon atoms. It is particularly preferred that the adjuvants used according to the present invention are aqueous solutions of polymers whose monomer units used to form the anionic constituent repeating units consist of acrylic acid, and whose monomer units used to form the hydrophobic constituent repeating units are selected of linear alkyl esters of acrylic acid whose alkyl groups contain 4-8 carbon atoms. The humoral response to vaccines comprising an aqueous solution of polymers having anionic constituent repeating units and hydrophobic constitutive repeat units is greater than the response induced by polymers having exclusively anionic constituent repeating units, such as, for example, polyacrylic acids. Indeed, the efficacy of the adjuvants according to the present invention is comparable to that of the standard adjuvants based on water in mineral oil, while, in general, their toxicity is much lower.

The adjuvants according to the present invention therefore do not pose any problem of instability, as do the standard adjuvants based on an oil in water (0 / W) or water in oil (W / O) emulsion, because the latter they are always sensitive to stabilizing factors such as salt concentration, temperature, etc., which is not the case for the adjuvants of the present invention. Its stability, in principle, corresponds to the stability of polymers containing exclusively anionic constituent repeating units, such as polyacrylic acids.

One of the advantages of the adjuvants according to the present invention is that it is effective at a low dose. Therefore it is possible to increase the charge of antigens per volume injected. In the W / O based vaccines, the mineral oil occupies approximately 50% of the volume of the vaccine, while the volume fraction occupied by the adjuvants according to the present invention (for example based on polyacrylic acids attached to hydrocarbon chains). ) approximately 10% of the volume of the vaccine can be decreased.

According to a first advantageous embodiment, the molecular weight of the polymers is between 10 and 10,000 kD. Advantageously, the molar ratio of hydrophobic constituent repeating units and anionic constituent repeating units is between 0.05 and 1.00, and preferably between 0.10 and 0.40.

Preferably, the solubility of the polymers in water is at least one g / 1.

According to another aspect of the present invention, a process for obtaining the polymer is described. The polymer can be obtained by one of the following processes: 1. copolymerization of anionic and hydrophobic monomers, 2. partial graft of polymers, 3. partial hydrolysis of polymers, and 4. by an intermediate anhydride.

According to another preferred embodiment, a vaccine with a polymer concentration of 1-40 mg / ml vaccine, preferably 4-24 mg / ml vaccine, and more preferably 8-16 mg / ml vaccine is proposed.

According to another aspect of the present invention, the vaccine comprises inactivated antigens of Newcastle disease virus (NDV) and / or infectious bronchitis virus (IBV) for the vaccination of domestic animals. Vaccines comprising an adjuvant based on polyacrylic acids bound to hydrocarbon chains are much more stable than vaccines comprising an adjuvant 'based on a W / O or 0 / W emulsion, or based on water in mineral oil in water (W / O / W), because the adjuvant is a solution.

According to yet another aspect of the present invention, it is proposed to use an aqueous solution of polymers which have anionic constituent repeating units and hydrophobic constitutive repeat units as adjuvants in vaccines.

According to another aspect the present invention provides a method for preparing a vaccine in solution, characterized in that an aqueous solution of an antigen and a polymer having anionic constituent repeating units and hydrophobic constitutive repeat units are mixed.

Example 1 Different water-soluble polymers according to the invention were synthesized by partial esterification of a polyacrylic acid with a molecular weight of 452,000 D (Carbopol 907 (PAA), Goodrich Cleveland, Ohio, U.S.A.). In this application, the term "PAA" refers to "Carbopol 907". This polyacrylic acid was esterified with different hydroxyalkanes according to the method described by Cenen, H. -L. in J. Poly. Sci., Vol. 14, pages 7-22 (1976. The resulting polymers, hereinafter referred to as "alkyl-PAA", (See Table I) contain acrylic acid monomer units and monomer units of the alkyl acrylate type. One gram of PAA was dissolved in 50 ml of the corresponding alkanol, and the solution was heated to 135 ° C. 50 μl of H2SO4 was added, and the reaction mixture was maintained at 135 ° C. The reaction was stopped by rapid cooling The pH of the solution was adjusted to pH = 6, and the solvents were evaporated at 80 ° C under reduced pressure (1019 x 10"6, 10" 6 b) The products obtained in this manner were dissolved in distilled water, dialyzed against distilled water, and then freeze-dried.The following compounds were synthesized, whose main properties are listed in table I: decyl-PAA (C10-PAA ), octyl-PAA (C8-PAA), butyl-PAA (C4-PAA), and methyl-PAA (Cl-PAA). of esterification of these compounds was determined by NMR analysis. It is expressed in molar%. The alkyl-PAAs were dissolved in a phosphate buffer (pH = 7.5), with slight heating if necessary, and a volume of the adjuvant solution was mixed with a volume of the solution containing the antigen.

Table I Main properties of the alkyl-PAAs synthesized.

NT = not transferred (not tested) Example 2 Chickens were immunized by intramuscular injection (IM) with 0.5 ml of vaccine comprising the NDV virus (Newcastle disease virus); inactivated (Kimber strain) and the inactivated IBV (infectious bronchitis virus) virus, which included strains M41 and D274, (iNDV / ilBV) without adjuvant, at the age of 4 weeks, and then at the age of 7 weeks , with the same antigens, this time with an adjuvant. The animals of the negative control group received buffered saline phosphate solution (PBS) instead of the adjuvant, and the animals of the positive control group were vaccinated with a vaccine comprising a standard adjuvant based on water in mineral oil. Three weeks after the second vaccination, blood was drawn to the hens, and in some experiments, blood samples were collected a second time several weeks later. The blood samples were incubated at room temperature, and after 2 hours, the blood clots were removed, and the remaining cells were removed by centrifugation (10 minutes at 2700 G), and serum samples were collected from each animal. and stored at -20 ° C until used. 96-well microtiter plates were saturated with purified NDV, the latter had been inactivated and diluted in a buffered carbonate solution (pH = 9.6, 0.1 M) for two hours at 37 ° C. The plates were saturated with 5% (weight / volume) of skimmed milk (SKM) in a carbonate buffer solution overnight at 4 ° C.

The serum of the hens was treated with kaolin, incubating a volume of serum with 4 volumes of kaolin % (weight / volume) of kaolin in a borate buffer (ICN Biomedicals, Inc., Costa Mesa, U.S.A.) for 30 minutes. The kaolin was then removed by centrifugation.

The hen serum was diluted 100-fold in PBS containing 1% (w / v) bovine serum albumin (PBS / BSA). The serum samples were serially diluted twice in the same solution in 96-well plates, and the plates were incubated for one to two hours at 37 ° C. Anti-chicken IgG, produced in goats and coupled to peroxidase, was added. , at a 1/1000 dilution in PVS / SKM, and the plates were incubated for 1-2 hours at 37 ° C. The amount of peroxidase in the plates was quantified by the addition of a substrate solution? e ABTS + H202 ( Kirkegaard? Perry Labs., U.S.A.) and the absorbance at 405 nm was measured using a Titertek of multiple sweeps. Antibody titers are expressed in the form of 2-log values of the regression coefficient of the optical density plot with respect to the reciprocal dilution factor. The antibody titer was also expressed by geometric averages (2-log +/- SEM value) and the antilog values of these averages (2 Promedi0). The activity of the adjuvant was expressed as a percentage of increase, calculated as follows: Percent increase = [antilog (sample) antilog (negative control)] / [antilog (positive control) - antilog (negative control)] * 100. They were taken performed Student's t tests to analyze the statistical significance of these results, and the value p > 0.05 was considered significant. - In four independent experiments, the adjuvant effect of one or more partially esterified polyacrylic acids was compared to the adjuvant effect of unesterified PAA, and to a negative control group without adjuvant (PBS) and to a positive control group with water in mineral oil ( W / O). Antibody titers were determined using the indirect ELISA method described above. The results of these experiments are listed in Table II.

Table II. Effects of different preparations of alkyl-PAA on the immune response against NDV / IBV, measured by indirect ELISA in hens n = number of hens per group. SEM = standard deviation of the average (standard error of the average). * = Tertiary C4 The positive and negative control groups produced reproducible antibody titers in the four independent experiments, and the positive control antibody titers were higher by 3 or 4 units 2-log (8-16 times) than those in the negative control group. The PAA (unmodified) increased the anti-iNDV antibody titre between 6% and 32% (Experiments I-IV), and it seems that this increase depends on the dose (Experiment IV). Optimal stimulation is obtained with a dose of 40 mg / ml (Experiment II).

The alkyl-PAA elicited significantly higher responses than the unmodified PAA. Octyl-PAA and butyl-PAA produced higher titers than decyl-PAA, t-butyl-PAA or methyl-PAA. The maximum responses were obtained with doses of 8-40 mg / ml octyl-PAA or butyl-PAA. The responses were as high as the responses obtained with a standard adjuvant based on oil and water, used as a positive control. Experiment IV has shown that the response depends on the dose of modified PAA used, at least as regards when butyl-PAA is used in the range of 1-16 mg / ml. At a very low dose - l-2mg / ml - butyl-PAA significantly increased the humoral response. Antibody titers comparable to those found with much higher doses of unmodified PAA were obtained. The humoral response increases with the dose of modified PAA used, and reaches a maximum that is equivalent to that caused by standard adjuvants based on water and oil. Doses of 24-40 mg / ml of butyl-PAA and octyl-PAA occasionally induced weaker responses than doses of 8 mg / ml, indicating the existence of an optimal concentration for these compounds, which is 8-24 mg / ml. Example 3 The anti-NDV antibody titers were also determined in the individual serum samples, using a commercially available anti-iNDV ELISA kit (Flockcheck Newcastle Disease antibody test kit); IDEXX Labs, Inc., Maine, U.S.A.) according to the protocol. 'The results of these analyzes are listed in Table III.

Table III. Effect of different PAA preparations on the anti-iNDV antibody titer measured by an IDEXX ELISA kit on chickens n = number of hens per group. SEM = standard deviation of the average (standard error of the average) NT = not tested * = tertiary C4 In the four experiments, the differences between the average values of the negative controls were small. In all four experiments, the difference in the average values of the positive controls was also small. The adjuvants of the modified PAAs, butyl-PAA and octyl-PAA were more effective than methyl-PAA or decyl-PAA.

The results obtained by this method confirm those of the first method, which was developed specifically for these tests. Example 4 In progressive 96-well plates progressive dilutions of the antiserum were incubated with 106 infectious particles (UPF) of a Kimber strain of NDV for 18 hours at 37 ° C. To each well were added 10 5 cells of the QT35 avian line, and They put on the plates. These plates were covered and incubated for an additional 48 hours at 37 ° C. The dilutions of serum that produced a 50% reduction in the number of infectious particles were considered as the antibody titer. The results of these experiments are listed in Table IV.

Table 4: Effects of different preparations of PAA on the titre of anti-iNDV antibodies measured by virus neutralization (VN) on n = number of hens per group SEM = standard deviation of the average (standard error of the average) * = tertiary C4 Animals from the positive and negative control groups produced reproducible antibody titers in the 4 independent experiments. The percentage of increase achieved by the unmodified PAA was 4-37% as a function of the dose of PAA used. All the alkyl-PAA induced higher responses than the unmodified PAA. The quality of the responses induced by the alkyl-PAA depended on the type of alkyl chain used and the degree of staphylaxis of the PAA. For butyl-PAA a close relationship was observed between the dose of adjuvants and the humoral response (Experiment IV), whereas for octyl-PAA, responses at a dose of 40 mg / ml did not always cause a higher response than the smaller doses, indicating that the optimal amount of octyl-PAA was between 24 mg / ml and 40 mg / ml. The evaluation of the biological function of the antibodies, performed by the virus neutralization test (NV), showed a close correlation between the 2 ELISA tests and the NV test. Example 5 The effect as an adjuvant of PAA was also tested in mice. Groups of 6 mice were vaccinated with 25 μl of a vaccine comprising one volume of an antigen solution consisting of 10 μg of inactivated influenza virus (strain MRC-11) and one mg of ovoaibumin (OVA) (SIGMA, USA) per ml and one volume of adjuvant. Three weeks after the injection, they were determined using the indirect ELISA method described in example 1. The mice serum was previously diluted in a 5% solution of skim milk in PBS (E3S / SKM). Serum samples were serially diluted twice in the same solution in 96-well plates, plates were incubated for 1-2 hours at 37 ° C. Anti-mouse IgG, produced in goats and coupled to peroxidase, was added. in a 1/300 dilution in PBS / BSA, and the plates were incubated for 1-2 hours at 37 ° C. The amount of peroxidase in the plates was quantified using a substrate solution of ABTS + H202 (Kirkegaard &Perry Labs ., USA) and the absorbance at 405 nm was measured using a Titertek of multiple scans. The results of these experiments are listed in Table V.

Table V. Effect as adjuvant of alkyl-PAA on mice 2-log of antibody titer against j Adjuvant, (mg / ml) MRC11 OVA average! SEM ipromedio 'SEM n = number of hens (sic; mice) per group SEM = Standard deviation of the average (standard error of the average).

The increase in the adjuvant effect of PAA by the addition of aliphatic chains was confirmed only partially in mice, because the two independent experiments produced different effects. Example 6 In addition to the adjuvant effect, other properties are important when evaluating a vaccine. They include the local reaction, which is an important aspect, although a certain level of reaction at the site of the injection is generally accepted by some animal species. Local toxicity was tested in vivo by monitoring the swelling of the paw pad of the mice after injection of the vaccine into the pad of the palm of the mouse paw. It has been shown that this method is very sensitive. 25 μl +/- 5 μl of a vaccine containing 1 volume of a diluted adjuvant was injected (subcutaneously) with 1 volume of an antigen solution containing 10 μg of MRC-11 and 1 mg of ovalbumin per ml of 0.9 NaCl % (weight / volume) in the plant of the left hind paw of groups of 6 mice.

The thickness of the paw was measured one day before the injection, at different intervals after injection, using a semi-electronic apparatus designed specifically for this purpose by the State University of Utrecht in the Netherlands. It has been shown that the accuracy of this device is up to about 0.02 mm. The swelling of the leg was calculated obtaining the difference between the thicknesses before and after the treatment expressed in 0.01 mm. The results of these experiments are listed in Table VI Table VI. Reactivity of PAA and alkyl-PAA in mice The local toxicity of PAA on mice is moderate; The swelling of the mice's paw reaches a maximum after a few days and then gradually decreases after 2 or 3 weeks. The addition of alkyl chains to PAA increases the local reaction which, however, disappears after 2-5 weeks, depending on the amount injected. The octyl groups cause a stronger reaction than the butyl groups. In contrast, the swelling caused by the injection of an adjuvant based on oil and water causes much larger swellings, which persist for more than 8 weeks.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (20)

1. CLAIMS 1. Adjuvants for vaccines, characterized in that they comprise an aqueous solution of a polymer having anionic constitutive repeating units and hydrophobic constitutive repeating units.
2. 2. Adjuvant according to claim 1, characterized in that the monomer units for forming the anionic constituent repeating units are selected eg acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinyl sulfuric acid, styrenesulfonic acid, vinylphenylsulfuric acid, 2-methacryloyloxyethanesulfonic acid, 3-methacryloyloxy-2-hydroxypropanesulfonic acid, 2-acryl-2-methylpropanesulfonic acid, 3-acrylamido-3-methylbutanoic acid, 3-methacrylamido-3-methylbutanoic acid, vinylphosphoric acid, 4-vinylbenzoic acid, acid 3-vinyloxypropane-1-sulfonic acid and N-vinyl-succinic acid. Adjuvant according to claim 2, characterized in that the monomer units for forming the anionic constituent repeating units are selected from acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinyl sulfuric acid and styrene sulfonic acid.
3. 3. Adjuvant according to claim 2, characterized in that the monomer units for forming the anionic constituent repeating units are selected from acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinyl sulfuric acid and styrenesulfonic acid.
4. 4. Adjuvant according to claim 3, characterized in that the monomer units for forming the anionic constituent repeating units are selected from acrylic acid, methacrylic acid, maleic acid and fumaric acid.
5. 5. Adjuvant according to claim 4, characterized in that the monomer units for forming the anionic constitutive repeat units are acrylic acid units.
6. 6. Adjuvant according to claim 1, characterized in that the monomer units for forming the hydrophobic constituent repeating units are selected from alkyl esters, cycloalkyl esters and hydroxyalkyl esters of acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinylsulfuric acid, styrenesulfonic acid, vinylphenylsulfuric acid, 2-methacryloyloxyethanesulfonic acid, 3-methacryloyloxy-2-hydroxypropanesulfonic acid, 2-acryl-2-methypropanesulfonic acid, 3-acrylamido-3-methylbutanoic acid, 3-methacrylamido-3-methylbutanoic acid, vinylphosphoric acid, 4-vinylbenzoic acid, 3-vinyloxypropan-1-sulfonic acid or N-vinyl-succinic acid), and the ethers.
7. 7. Adjuvant according to claim 6, characterized in that the monomer units for forming the hydrophobic constitutive repeating units are selected from alkyl esters of acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylene sulfonic acid, vinylsulfuric acid or styrenesulfonic acid.
8. 8. Adjuvant according to claim 7, characterized in that the monomer units for forming the hydrophobic constitutive repeating units are selected from alkyl esters of acrylic acid, methacrylic acid, maleic acid or fumaric acid whose alkyl groups contain 4-8 carbon atoms. carbon.
9. 9. Adjuvant according to claim 8, characterized in that the monomer units for forming the hydrophobic constitutive repeating units are selected from linear alkyl esters of cryic acid, which alkyl groups contain from 4-8 carbon atoms.
10. 10. Adjuvant according to any of claims 1-9, characterized in that the molar ratio of the constitutive hydrophobic repeating units to the constitutive anionic repeating units is between 0.05 and 1.00.
11. 11. Adjuvant according to any of claims 1-10, characterized in that the molar ratio of the hydrophobic constitutive repeating units to the constitutive anionic repeating units is 0.10 and 0.4C.
12. 12. Adjuvant according to claim 1, characterized in that the molecular weight of the polymers is 10-10,000 kD.
13. 13. Adjuvant according to any of claims 1-12, characterized in that the liquid medium is an aqueous solution, suspension or emulsion.
14. 14. Adjuvant according to any of claims 1-13, characterized in that the polymer is soluble in water.
15. 15. Vaccine characterized in that it comprises an immunogenic amount of an antigen and an adjuvant according to any of the preceding claims.
16. 16. Vaccine according to claim 15, characterized in that the concentration of adjuvants is 1-40 mg / ml, preferably 4-24 mg / ml.
17. 17. Vaccine according to claim 15, characterized in that, in addition to the adjuvant, it comprises inactivated antigens of the Ne castle disease virus (NDV) and / or the infectious bronchitis virus (IBV) for the vaccination of domestic animals.
18. 18. The use of a polymer having repeating constitutive hydrophobic units and constitutive anionic repeating units characterized in that it is as an adjuvant in vaccines.
19. 19. Use according to claim 18 characterized in that the polymer has hydrophobic constituent repeating units according to any of claims 2-5, and constitutive and anionic repeating units according to any of claims 6-9.
20. 20. Method for the preparation of a vaccine in solution, characterized in that an aqueous mixture of an antigen and a polymer having repeating constituent hydrophobic units and constitutive anionic constituent units are mixed. ADJUVANTS FOR VACCINES SUMMARY OF THE INVENTION The present invention describes adjuvants for vaccines comprising a liquid medium containing polymers having anionic repeating constitutive units and hydrophobic repeating constituents. Advantageously, the adjuvants are aqueous solutions of partially esterified polyacrylic acids. "Novel adjuvants are highly stable, effective and with a relatively low level of local toxicity, and the invention also relates to vaccines comprising such adjuvants, as well as to a process for their manufacture.