Oil adjuvant compositions and vaccine emulsions containing such oil adjuvant compositions

Abstract

The present invention relates to an oil adjuvant composition comprising at least one oil, at least one nonionic lipophilic surfactant which is a sorbitan ester or a mannitan ester, and at least one nonionic hydrophilic surfactant which is an ethoxylated fatty alcohol. The present invention also relates to adjuvant oil-in-water emulsions and vaccine oil-in-water emulsions obtained from the oil adjuvant compositions of the present invention, and the use of such vaccine oil-in-water emulsions for the treatment of porcine circovirus type 2 disease and / or porcine epizootic pneumonia.

Description

[Technical Field]

[0001] The present invention relates to oil adjuvant compositions containing specific surfactants that are particularly suitable for formulating vaccine oil-in-water emulsions. [Background technology]

[0002] The use of adjuvants in pharmaceutical compositions, such as vaccine compositions, is well known in the art. Generally, emulsions used in vaccine formulations comprise a mixture of a fatty phase, an aqueous phase, and at least one surfactant.

[0003] When formulating pharmaceutical compositions, especially vaccine emulsions, the two main criteria for selecting an appropriate adjuvant are stability and safety, especially in the presence of bacterial and / or viral suspensions. For example, the stability of an adjuvant may be affected due to interactions between the emulsion components and the enzyme activity of lipases or esterases present in the antigen suspension of the vaccine. This problem may be particularly noticeable when the vaccine is based on an inactivated or attenuated pathogen or a combination of pathogens. In addition, the toxicity of some surfactants present in adjuvants may cause safety issues.

[0004] WO 2005 / 009462 discloses an oil-in-water emulsion containing an aqueous phase containing an immunogen and an oil phase containing at least three surfactants (i.e., a non-ionic lipophilic surfactant that is a fatty acid ester of sorbitan, a non-ionic hydrophilic surfactant that is an ethoxylated sorbitan fatty acid monoester, and a non-ionic hydrophilic surfactant that is a sorbitan fatty acid triester). The adjuvant composition is disclosed to be suitable for forming vaccine compositions with increased safety and stability. However, the exclusive use of sorbitan ester surfactants can affect the stability of the oil-in-water emulsion, especially when the adjuvant composition is used with antigens produced from cell lines containing enzymes such as lipases or esterases. For example, cell lines used to produce Mycoplasma hyopneumoniae antigens may contain enzymes such as esterases that can destabilize emulsions made with adjuvant compositions containing only ester surfactants, especially ethoxylated ester surfactants.

[0005] WO 2006 / 113373 discloses an oil-in-water adjuvant formulation containing two surfactants, one nonionic lipophilic ethoxylated fatty alcohol, and one nonionic hydrophilic ethoxylated fatty alcohol. This formulation exclusively uses ethoxylated fatty alcohol surfactants, which may result in reduced formulation stability and raise more safety concerns due to the toxicity and ecotoxicity of this emulsifier family. In fact, ethoxylated fatty alcohol surfactants contain ether functional groups that cannot be reversed and decomposed by enzymes such as esterases. Therefore, these surfactants have poor biocompatibility with enzymes and / or pH changes, which contributes to their toxicity (see, for example, Oda et al. Research in Veterinary Science 81 (2006), pp. 51-57). Therefore, the exclusive use of such surfactants in the composition increases safety concerns in terms of the toxicity of the composition. WO 2006 / 113373 discloses that the claimed formulations exhibit increased safety and stability, however, no stability results are presented in this patent application.

[0006] Therefore, there remains a need to provide new adjuvant compositions with increased stability and safety, even when the antigen may contain additional components such as enzymes. Summary of the Invention

[0007] In this regard, the present inventors have unexpectedly demonstrated that the use of a particular combination of surfactants comprising at least one non-ionic lipophilic surfactant that is a sorbitan ester or a mannitan ester and at least one non-ionic hydrophilic surfactant that is an ethoxylated fatty alcohol results in an oil adjuvant composition with increased stability and safety, even when used in vaccine emulsions with antigens that include additional components such as enzymes.

[0008] Therefore, the present invention first relates to an oil adjuvant composition, which comprises: - at least one oil, - at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester; at least one non-ionic hydrophilic surfactant which is an ethoxylated fatty alcohol.

[0009] In one embodiment, the oil comprises at least one mineral oil, in particular at least one light paraffin oil, or a combination of at least two mineral oils.

[0010] In one embodiment, the mineral oil is selected from the group consisting of paraffinic oils, such as isoparaffinic oils, and / or naphthenic oils, pristane, and any mixtures thereof.

[0011] In one embodiment, each sorbitan ester or mannitan ester is a sorbitan ester, preferably selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, sorbitan monoisostearate, and sorbitan isostearate, more preferably sorbitan monooleate.

[0012] In another embodiment, each sorbitan ester or mannitan ester is a mannitan ester, preferably selected from the group consisting of mannitan oleate, mannitan monooleate, mannitan dioleate, mannitan trioleate, and mannitan tetraoleate.

[0013] In one embodiment, the ethoxylated fatty alcohol is selected from the group consisting of polyoxyethylene (23) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) oleyl ether, polyoxyethylene (20) oleyl ether, polyoxyethylene (21) stearyl ether, polyoxyethylene lauryl ether, eicosaethylene glycol octadecyl ether, triethylene glycol oleyl ether, and any mixture thereof, preferably polyoxyethylene oleyl ether, in particular polyoxyethylene (10) oleyl ether.

[0014] In one embodiment, the oil adjuvant composition comprises: - 76% to 92% oil, - 1% to 7% of at least one nonionic lipophilic surfactant which is a sorbitan ester or a mannitan ester; - 7% to 17% of at least one nonionic hydrophilic surfactant which is an ethoxylated fatty alcohol; Percentages are expressed by weight relative to the total weight of the oil adjuvant composition.

[0015] In one embodiment, the oil adjuvant composition comprises: about 82% oil, preferably mineral oil, - about 4% of at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester, preferably sorbitan monooleate; - about 14% of at least one non-ionic hydrophilic surfactant, preferably polyoxyethylene oleyl ether, which is an ethoxylated fatty alcohol; Percentages are expressed by weight relative to the total weight of the oil adjuvant composition.

[0016] The present invention also relates to an adjuvant oil-in-water emulsion, the adjuvant oil-in-water emulsion comprising 40% to 70% of an oil adjuvant composition according to the present invention and 30% to 60% of an aqueous solvent, the oil adjuvant composition being dispersed in the aqueous solvent, the percentages being expressed by volume relative to the total volume of the oil adjuvant composition.

[0017] The present invention further relates to a vaccine oil-in-water emulsion comprising an adjuvant oil-in-water emulsion according to the invention, at least one antigen, and a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier.

[0018] In one embodiment, the vaccine oil-in-water emulsion comprises 10-80% adjuvant oil-in-water emulsion and 20%-90% pharmaceutically acceptable carrier; The percentages are expressed relative to the total amount of vaccine oil-in-water emulsion.

[0019] In one embodiment, the at least one antigen is selected from the group consisting of an inactivated porcine circovirus type 2 (PCV2) antigen, a Mycoplasma hyopneumoniae antigen, a Streptococcus suis antigen, and combinations thereof.

[0020] The present invention further relates to a vaccine oil-in-water emulsion according to the invention for use as a medicament, preferably for use as a vaccine.

[0021] A final object of the invention is a vaccine oil-in-water emulsion according to the invention for use in the treatment of Streptococcus suis infections such as porcine circovirus type 2 disease, porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death, and / or porcine epizootic pneumonia.

[0022] In one embodiment, the vaccine oil-in-water emulsion is administered, preferably by injection, to an animal that is a pig or piglet. [Brief explanation of the drawings]

[0023] [Figure 1] Photographs of the appearance of the composition tested in Example 2 after one day (a), one week (b), and one month (c) are shown. [Figure 2] Graphs of particle size distribution over time in the compositions tested in Example 2 are shown: FIG. 2 for composition A, FIG. 3 for comparative composition B, FIG. 4 for composition LR3, and FIG. 5 for composition LR4. [Figure 3] Graphs of particle size distribution over time in the compositions tested in Example 2 are shown: FIG. 2 for composition A, FIG. 3 for comparative composition B, FIG. 4 for composition LR3, and FIG. 5 for composition LR4. [Figure 4] Graphs of particle size distribution over time in the compositions tested in Example 2 are shown: FIG. 2 for composition A, FIG. 3 for comparative composition B, FIG. 4 for composition LR3, and FIG. 5 for composition LR4. [Figure 5] Graphs of particle size distribution over time in the compositions tested in Example 2 are shown: FIG. 2 for composition A, FIG. 3 for comparative composition B, FIG. 4 for composition LR3, and FIG. 5 for composition LR4. [Figure 6] 1 is a graph comparing the temperature rise in the four groups of the safety study of Example 4. [Figure 7] For the study of Example 5, histograms of viremia for each group are shown on days 14 (A), 21 (B), and 28 (C) post-challenge. [Figure 8] 1 shows histograms of the logarithm of nasal swabs on days 7 (A), 14 (B), 21 (C), and 28 (D) for the study in Example 5. [Figure 9] 1 shows histograms of nasal swabs on days 7 (A), 14 (B), 21 (C), and 28 (D) for the study of Example 5. [Figure 10] Histograms of log quantification of virus at day 28 for inguinal lymph nodes (A), mesenteric lymph nodes (B), lungs (C), mediastinal lymph nodes (D), and tonsillar lymph nodes (E) are shown for the study in Example 5. [Figure 11]1 is a graph showing the percentage of local reactions per day for the study of Example 6. [Figure 12] Graphs showing antibody development by group before the first vaccination (B0), between the first and second vaccination (B1), and two weeks after the second vaccination (B2) in the study of Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition "About" is used herein to mean approximately, roughly, around, or within a range thereof. When the term "about" is used in conjunction with a numerical range or value, it modifies that range or value by extending the boundaries above and below that numerical range or value by 10%, preferably 5%, and more preferably 1%.

[0025] An "adjuvant composition" is a composition suitable for forming a pharmaceutical composition, e.g., a vaccine composition, when combined with at least one active agent, e.g., an antigen. The adjuvant composition enhances the immune response to the vaccine antigen compared to the response induced by the vaccine antigen alone. In some other, less preferred, embodiments, the adjuvant composition does not at least reduce the immune response to the vaccine antigen compared to the response induced by the vaccine antigen alone.

[0026] The term "animal" includes all vertebrates, including humans. In particular, the term "vertebrate" includes humans, canines (such as dogs), felines (such as cats), equines (such as horses), bovines (such as cattle), porcines (such as pigs, boars, or sows), ovines (such as sheep), caprines (such as goats), and avians (such as chickens).

[0027] "Antigen" refers to a substance that induces a specific immune response in a host animal. Antigens can be inactivated, attenuated, or genetically modified epitopes, immunogenic fragments, or whole organisms (bacteria, viruses).

[0028] "Emulsion" refers to a colloidal system made of two immiscible components, such as oil and water, in which one component (the dispersed phase) exists in the form of droplets dispersed in the other component, which constitutes the continuous phase. "Oil-in-water emulsion" refers to an emulsion in which the dispersed phase is a fatty phase, such as oil, and the continuous phase is an aqueous phase, such as water.

[0029] The term "ethoxylated" when used to qualify a compound containing a labile hydrogen atom, such as the hydrogen atom of a hydroxy group, means that the hydrogen labile group is -(C2H4O) n refers to compounds substituted with -H moieties, where n is 1 or greater, and n typically ranges from 1 to 20. The term ethoxylated can be used, for example, to modify fatty alcohols (ethoxylated fatty alcohols) or sorbitan esters or mannitan esters (ethoxylated sorbitan esters or ethoxylated mannitan esters).

[0030] The term "fatty", when used to qualify a compound containing a hydrocarbon chain, refers to a compound containing an aliphatic hydrocarbon chain containing 4 to 36 carbon atoms, preferably 4 to 28 carbon atoms, more preferably 8 to 20 carbon atoms, and especially 12 to 18 carbon atoms. The aliphatic hydrocarbon chain may be saturated or unsaturated. The term fatty may be used to qualify, for example, alcohols (fatty alcohols), esters (fatty acid esters), and / or acids (fatty acids).

[0031] "Mannitan esters" are derivatives obtained by esterification of at least one of the alcohol and / or phenol functional groups of mannitan.

[0032] "Mineral oil" is any of various colorless, odorless, light mixtures of higher alkanes from mineral sources, especially distillates of petroleum.

[0033] An "oil" is a fatty substance that is fluid, preferably liquid, at room temperature (25-35° C.) and insoluble in water. Oils may be synthetic or of vegetable, animal, or mineral origin.

[0034] The term "pharmaceutically acceptable carrier" refers to a fluid vehicle for containing a pharmaceutically active agent, such as an antigen, that can be administered to an animal, preferably injected, without significant adverse effects. A pharmaceutically acceptable carrier does not interact with the pharmaceutically active agent and preferably does not affect its pharmaceutical efficiency. Suitable pharmaceutically acceptable carriers known in the art include sterile water, saline, glucose, dextrose, and buffers. Carriers may include auxiliary agents such as diluents, stabilizers, sugars, amino acids, preservatives, wetting agents, emulsifiers, pH buffers, viscosity-enhancing additives, coloring agents, or any mixture thereof.

[0035] The terms "swine", "pig" and "piglet" refer to animals of porcine origin.

[0036] "Sorbitan esters" are derivatives obtained by esterification of at least one of the alcohol and / or phenol functional groups of sorbitan.

[0037] "Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (reduce) the targeted pathological condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to having the disorder or those in whom the disorder is to be prevented. A subject or animal has been successfully treated for an infection if, after receiving a therapeutic amount of a composition according to the present invention, the subject shows an observable and / or measurable reduction or absence of one or more of the following: a reduction in the number of pathogenic cells, a reduction in the percentage of total cells that are pathogenic, and / or an alleviation to some extent of one or more symptoms associated with the particular disease or condition, a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in disease can be readily measured by routine procedures familiar to physicians.

[0038] Oil adjuvant composition The first object of the present invention is an oil adjuvant composition, which comprises: - at least one oil, - at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester; at least one non-ionic hydrophilic surfactant which is an ethoxylated fatty alcohol.

[0039] The present inventors have demonstrated that the oil adjuvant composition and vaccine oil-in-water emulsion according to the present invention are very stable and exhibit high safety, even when the antigen may contain additional elements such as enzymes. The stability of the oil adjuvant composition and vaccine oil-in-water emulsion has further been found to be higher than that of formulations disclosed in the prior art as being highly stable.

[0040] Oils are known to be immunostimulants and therefore suitable adjuvants for active agents. The oil composition according to the present invention is particularly suitable as an adjuvant for veterinary compositions. Furthermore, oil-in-water emulsions are known to be particularly suitable as adjuvants for veterinary compositions, as disclosed in publications such as Burakova et al., Viral Immunology 31, 1, 2018, pp. 11-22.

[0041] The oil adjuvant composition according to the present invention comprises at least one nonionic hydrophilic surfactant which is an ethoxylated fatty alcohol and at least one nonionic lipophilic surfactant which is a sorbitan ester or a mannitan ester.

[0042] In some embodiments, the at least one nonionic hydrophilic surfactant and the at least one nonionic lipophilic surfactant are the only surfactants included in the oil adjuvant composition according to the present invention. In some embodiments, the oil adjuvant composition according to the present invention comprises one ethoxylated fatty alcohol and one sorbitan ester or mannitan ester as the only surfactants.

[0043] The hydrophilic ethoxylated fatty alcohol may contain 1 to 50, preferably 5 to 30, ethylene oxide moieties. The fatty alcohol may be a C4 to C36 fatty alcohol, preferably a C9 to C22 fatty alcohol, advantageously selected from the group consisting of oleyl, cetyl, stearyl, isostearyl, lauryl, and octadecyl alcohol, and combinations thereof, advantageously oleyl alcohol. More advantageously, the ethoxylated fatty alcohol is oleyl alcohol having 3 to 20 ethylene oxide moieties.

[0044] The ethoxylated fatty alcohol may be selected from the group consisting of, for example, polyoxyethylene (23) lauryl ether such as Brij 76®, polyoxyethylene (20) cetyl ether such as Brij 56® or Brij 58®, polyoxyethylene (10) oleyl ether such as Brij 96 / 97® or Brij O10®, polyoxyethylene (20) oleyl ether such as Brij 98® or Brij O20®, polyoxyethylene (21) stearyl ether such as Brij 721®, polyoxyethylene lauryl ether such as Brij 35®, eicosaethylene glycol octadecyl ether such as Brij 78®, triethylene glycol oleyl ether such as Volpo N5®, and any mixtures thereof.

[0045] In some embodiments, the ethoxylated fatty alcohols are used as a mixture.

[0046] In some embodiments, the ethoxylated fatty alcohols are selected from the following brand names: Ceteareth-6® (68439-49-6), Ceteareth-12® (68439-49-6), Ceteareth-20® (68439-49-6), Ceteareth-25® (68439-49-6), Ceteareth-23® (68439-49-6), Laureth-9®, Laureth-21®, Laureth-25® ), Ceteth-6®, Ceteth-7®, Ceteth-15®, Ceteth-23®, Ceteth-25®, C12-14 Pareth-5®, C12-14 Pareth-7®, C12-14 Pareth-9®, C12-14 Pareth-12®, C12-15 Pareth-10®, Oleth-7®, Oleth-15®, Oleth-50®, Beheneth-20® (Nikko Chemicals), and any combination thereof.

[0047] The ethoxylated fatty alcohols preferably exhibit a hydrophilic-lipophilic balance (HLB) of 10-18, preferably 12-18.

[0048] The surfactants of the present invention may comprise fatty alcohols of animal or vegetable origin.

[0049] The amount of at least one ethoxylated fatty alcohol in the oil adjuvant composition according to the present invention can vary within a wide range and can be determined by a person skilled in the art depending, inter alia, on the chemical nature of the at least one ethoxylated fatty alcohol, the nature of the other components (oil and / or sorbitan ester or mannitan ester), and / or the nature of the pharmaceutically active agent, in particular the antigen, present in the vaccine oil-in-water emulsion comprising the oil adjuvant composition.

[0050] The ethoxylated fatty alcohol may be present in the oil adjuvant composition in an amount ranging from about 5 to about 50% of the oil adjuvant composition, preferably from about 7 to about 17% of the oil adjuvant composition, percentages expressed by weight relative to the total amount of the oil adjuvant composition.

[0051] The nonionic lipophilic surfactants are non-ethoxylated sorbitan esters or non-ethoxylated mannitan esters. Ethoxylated sorbitan esters and mannitan esters containing more than six ethylene oxide moieties are hydrophilic.

[0052] Nonionic lipophilic surfactant is sorbitan or mannitan ester, preferably sorbitan or mannitan fatty acid ester.In some embodiments, each nonionic lipophilic surfactant is sorbitan ester, preferably sorbitan fatty acid ester, more preferably sorbitan fatty acid monoester or sorbitan fatty acid triester.In other embodiments, each nonionic lipophilic surfactant is mannitan ester, preferably mannitan fatty acid ester.

[0053] Examples of fatty acid esters of sorbitan include sorbitan monolaurate such as Span 20®, sorbitan monopalmitate such as Span 40®, sorbitan monostearate such as Span 60®, sorbitan tristearate such as Span 65®, sorbitan monooleate such as Span 80®, sorbitan trioleate such as Span 85®, sorbitan monoisostearate such as Arlacel 987®, and sorbitan isostearate such as Crill 6®.

[0054] Examples of fatty acid esters of mannitan include mannitan oleates such as Montanide 80®, mannitan monooleates such as Arlacel A®, mannitan dioleate, mannitan trioleate, and mannitan tetraoleate.

[0055] The fatty acids used to form the fatty alcohols and / or fatty acid esters practiced in the present invention are preferably selected from the group consisting of oleic acid, palmitic acid, stearic acid, isostearic acid, lauric acid, and combinations thereof.

[0056] The amount of at least one sorbitan ester or mannitan ester in the oil adjuvant composition according to the present invention can vary within a wide range and can be determined by a person skilled in the art depending, inter alia, on the chemical nature of the at least one sorbitan ester or mannitan ester, the nature of the other ingredients (oil and / or ethoxylated fatty alcohol), and / or the nature of the pharmaceutically active agent, in particular the antigen, present in the vaccine oil-in-water emulsion comprising the oil adjuvant composition.

[0057] The at least one sorbitan ester or mannitan ester may be present in the oil adjuvant composition in an amount ranging from about 0.1 to about 25% of the oil adjuvant composition, preferably from about 1 to about 7% of the oil adjuvant composition, the percentages being expressed by weight relative to the total amount of the oil adjuvant composition.

[0058] The oil may be synthetic or of vegetable, animal, or mineral origin.

[0059] The oil may be a mineral oil, including, but not limited to, paraffinic oil, such as isoparaffinic oil, and / or naphthenic oil, pristane, and any mixture thereof. One advantageous mineral oil useful in the present invention may be an oil containing a linear or branched carbon chain having more than 15 carbon atoms, preferably 15 to 32 carbon atoms, and containing no aromatic compounds. The oil may be, for example, one of the oils commercially available under the names MARCOL 52® or MARCOL 82® (manufactured by Esso, France), DRAKEOL 6VR® (manufactured by Penreco, USA), or EOLANE 150®, EOLANE 170®, or EOLANE 130® (manufactured by Total Energies). The mineral oil may be selected from the group consisting of CAS number 8042-47-5 oil (white mineral oil), CAS number 8012-95-1 oil (light liquid paraffin), CAS number 8020-83-5 oil (mineral oil), and any mixture thereof.

[0060] The oil may be a synthetic oil, including, but not limited to, squalane, polyisobutene oil, hydrogenated polyisobutene oil, polydecene oil, polyisoprene oil, polyisopropene oil, and the like, and any mixtures thereof.

[0061] In one embodiment, the oil comprised in the oil adjuvant composition according to the invention comprises at least one mineral oil, in particular at least one light paraffin oil, or a combination of at least two mineral oils.

[0062] In one embodiment, the oil comprised in the oil adjuvant composition according to the invention consists of at least one mineral oil, in particular at least one light paraffin oil, or a combination of at least two mineral oils.

[0063] The oil may also be a mixture of oils containing at least two oils selected from those described herein in any proportion.

[0064] The amount of oil in the oil adjuvant composition according to the present invention can vary within a wide range and can be determined by one skilled in the art depending, inter alia, on the chemical nature of the oil, the nature of the other components (sorbitan esters or mannitan esters and / or ethoxylated fatty alcohols), and / or the nature of the pharmaceutically active agent, in particular the antigen, present in the vaccine oil-in-water emulsion comprising the oil adjuvant composition.

[0065] The oil may be present in the oil adjuvant composition in an amount ranging from about 50 to about 95% of the oil adjuvant composition, preferably from about 76 to about 92% of the oil adjuvant composition, percentages expressed by weight relative to the total amount of the oil adjuvant composition.

[0066] In some embodiments, an oil adjuvant composition according to the present invention comprises a mixture of a liquid saturated hydrocarbon, preferably MARCOL 52® oil, sorbitan monooleate, and polyoxyethylene (10) oleyl ether.

[0067] In some embodiments, the oil adjuvant composition according to the present invention comprises: 76% to 92%, preferably about 82%, of an oil, preferably a mineral oil, - about 1 to 7%, preferably about 4%, of at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester, preferably sorbitan monooleate; - about 7 to 17%, preferably about 14%, of at least one non-ionic hydrophilic surfactant, which is an ethoxylated fatty alcohol, preferably polyoxyethylene oleyl ether; Percentages are expressed by weight relative to the total weight of the oil adjuvant composition.

[0068] The oil adjuvant composition according to the present invention may be prepared by any suitable method known in the art, for example by using a mixer or an emulsifier.

[0069] As an example, the following method (Preparation Method A) may be used to prepare an oil adjuvant composition according to the present invention, preferably an oil adjuvant composition comprising 76-92% light liquid paraffin (CAS 8012-95-1), 1-7% sorbitan monooleate (CAS 338-43-8), 7-17% polyoxyethylene 10 oleyl ether (CAS 9004-98-2), and sufficient water to obtain a clear composition.

[0070] Preparation method A comprises the following steps: i. measuring appropriate amounts of an oil, preferably light liquid paraffin, a sorbitan ester or mannitan ester, preferably sorbitan monooleate, and an ethoxylated fatty alcohol, preferably polyoxyethylene 10 oleyl ether; ii. Mixing thoroughly in a suitable mixer; iii. adding water until the mixture becomes clear; iv. autoclaving the mixture of step iii in a closed vessel at a temperature comprised between 110°C and 130°C, for example 121°C, for a period comprised between 30 minutes and 1 hour, for example 40 minutes; or iv. filtering the mixture of step iii through a 0.22 μm filter.

[0071] Adjuvant oil-in-water emulsion A second object of the invention is an adjuvant oil-in-water emulsion, the oil phase of which comprises, preferably consists of, an oil adjuvant composition according to the invention.

[0072] In one embodiment, the adjuvant oil-in-water emulsion according to the present invention comprises 40% to 70%, preferably 40% to 60%, of the oil adjuvant composition according to the present invention and 30% to 60%, preferably 40% to 60%, of an aqueous solvent. The oil adjuvant composition is dispersed in the aqueous solvent. The percentages are expressed by volume relative to the total volume of the adjuvant oil-in-water emulsion.

[0073] The aqueous solvent of the adjuvant oil-in-water emulsion may be selected from the group consisting of, for example, water, physiological solutions such as 0.9% NaCl in water, and aqueous buffers such as PBS (phosphate buffered saline) buffer. In some embodiments, the aqueous solvent is water.

[0074] Adjuvant oil-in-water emulsions according to the present invention may be prepared by any suitable method known in the art.

[0075] As an example, the following method (Preparation Method B) may be used to prepare an adjuvant oil-in-water emulsion according to the present invention.

[0076] Preparation method B comprises the following steps: i') preparing an oil adjuvant composition according to the present invention, preferably by preparation method A detailed above; ii') measuring a portion (50% by volume) of the oil adjuvant composition and a portion (50% by volume) of an aqueous solvent, preferably sterile PBS, under sterile conditions; iii') heating both phases, for example at 70°C; iv') adding the water phase to the oil phase with stirring; v') gradually cooling the emulsion while continuing to stir; and vi') storing the resulting emulsion at 2°C to 8°C until use;

[0077] Unless otherwise stated, in this application percentages are expressed as volume relative to the total volume of the adjuvant oil-in-water emulsion.

[0078] The present invention also relates to a diluted oil-in-water emulsion comprising an adjuvant oil-in-water emulsion according to the invention and a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier, which may be used to contact an active agent, such as an antigen, or a composition comprising such an active agent, preferably an oil-in-water emulsion.

[0079] Vaccine oil-in-water emulsion A third object of the present invention is an oil-in-water emulsion, preferably a vaccine oil-in-water emulsion, comprising an adjuvant oil-in-water emulsion according to the invention, a further pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier, and at least one active agent, such as an antigen.

[0080] Although the oil-in-water emulsions of the present invention are particularly suitable for use with antigens to form vaccine emulsions, they may also exhibit advantages when used with different active agents.

[0081] Antigens may include killed, attenuated, or live whole organisms, subunits or parts of organisms, recombinant vectors containing inserts with immune properties, pieces or fragments of DNA capable of inducing an immune response upon presentation to a host animal, proteins, polypeptides, peptides, epitopes, haptens, or any combination thereof. Alternatively, antigens may include toxins or antitoxins. Antigens may be inactivated.

[0082] In some embodiments, the antigen is an antigen from a pathogen associated with swine disease, preferably selected from viral and bacterial antigens associated with swine disease.

[0083] The viral antigen derived from a virus associated with a swine disease may be selected from the group consisting of classical swine fever virus antigens, swine influenza virus antigens, porcine reproductive and respiratory syndrome (PRRS) virus antigens, porcine respiratory coronaviruses such as porcine epidemic diarrhea virus (PEDV) antigens, porcine parvovirus antigens, porcine rotavirus antigens, and porcine circovirus antigens such as porcine circovirus type 2 (PCV2). Preferably, the antigen derived from a virus associated with a swine disease is a porcine circovirus type 2 (PCV2) antigen, such as ORF2 PCV2 antigen.

[0084] The bacterial antigen from a bacterium associated with swine disease may be selected from the group consisting of Actinobacillus pleuropneumoniae antigen, Mycobacterium spp. antigen, preferably Mycoplasma hyopneumoniae (M hyo) antigen, Streptococcus suis antigen, preferably IgM protease antibody, more preferably IdeSsuis antigen, Lawsonia intracellularis antigen, Escherichia coli antigen, Eperythrozoonosis suis antigen, Erysipelothrix rhusiopathiae antigen, Bordetella bronchiseptica antigen, Pasteurella multocida antigen, Salmonella antigen, Haemophilus parasuis antigen, Clostridium spp., e.g., Clostridium perfringens antigen, and Leptospira spp. antigen. Preferably, the antigen from a bacterium associated with swine disease is IdeSsuis antigen, Lawsonia intracellularis antigen, or Mycoplasma hyopneumoniae (M hyo) antigen.

[0085] The bacterial antigen from a bacterium associated with swine disease may be selected from the group consisting of Actinobacillus pleuropneumoniae antigen, Mycobacterium spp. antigen, preferably Mycoplasma hyopneumoniae (M hyo) antigen, Escherichia coli antigen, Erysipelothrix suis antigen, Erysipelothrix rhusiopathiae antigen, Bordetella bronchiseptica antigen, Pasteurella multocida antigen, Salmonella antigen, Haemophilus parasuis antigen, Clostridium spp., e.g., Clostridium perfringens antigen, and Leptospira spp. antigen. Preferably, the antigen from a bacterium associated with swine disease is Mycoplasma hyopneumoniae (M hyo) antigen.

[0086] In one embodiment, the at least one antigen comprised in the vaccine oil-in-water emulsion according to the invention is selected from the group consisting of a Porcine Circovirus Type 2 (PCV2) antigen, a Streptococcus suis antigen, a Mycoplasma hyopneumoniae antigen, and combinations thereof. More preferably, the at least one antigen comprised in the vaccine oil-in-water emulsion according to the invention is selected from the group consisting of a Porcine Circovirus Type 2 (PCV2) antigen, a Mycoplasma hyopneumoniae antigen, and combinations thereof.

[0087] In one embodiment, the at least one antigen comprised in a vaccine oil-in-water emulsion according to the invention is a combination of a porcine circovirus type 2 (PCV2) antigen, such as an ORF2 PCV2 antigen, a Mycoplasma hyopneumoniae antigen, and a Lawsonia intracellularis antigen.

[0088] A vaccine oil-in-water emulsion according to the present invention may comprise 10-80% adjuvant oil-in-water emulsion and 20%-90% pharmaceutically acceptable carrier, the percentages being expressed by volume relative to the total volume of the vaccine oil-in-water emulsion.

[0089] In some embodiments, the pharmaceutically acceptable carrier in a vaccine oil-in-water emulsion according to the invention is the same as the aqueous solvent in the adjuvant oil-in-water emulsion, hi a preferred embodiment, the pharmaceutically acceptable carrier in a vaccine oil-in-water emulsion according to the invention is water.

[0090] In some embodiments, a vaccine oil-in-water emulsion according to the present invention is injectable, meaning that its components and physical properties, such as its viscosity, are suitable for injection.

[0091] The vaccine oil-in-water emulsion according to the invention may comprise further components, such as components classically used in the formulation of vaccine oil-in-water emulsions, preferably which do not affect the safety or stability of the vaccine oil-in-water emulsion.

[0092] The vaccine oil-in-water emulsion according to the invention may comprise any further compound suitable for increasing and / or accelerating the immune response to the antigen in an animal.

[0093] In one embodiment, the vaccine oil-in-water emulsion further comprises an immunostimulant, which may be selected from the group consisting of dimethyloctadecylammonium (DDA), saponin, vitamin E, and immunostimulant polymers such as polyacrylic acid polymers. The amount of immunostimulant may comprise 0.5% to 5%, preferably about 1%, of the vaccine oil-in-water emulsion by weight relative to the total volume of the vaccine oil-in-water emulsion.

[0094] Use of vaccine oil-in-water emulsions A final object of the present invention is a vaccine oil-in-water emulsion according to the invention for use as a medicament, preferably for use as a vaccine, preferably for the treatment of Streptococcus suis infections such as porcine circovirus type 2 disease, porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death, and / or porcine epizootic pneumonia, more preferably porcine circovirus type 2 disease and / or porcine epizootic pneumonia.

[0095] Preferably, the vaccine oil-in-water emulsion according to the invention is used in a non-human mammal, more preferably a porcine mammal, in particular a pig or piglet.

[0096] In some embodiments, the treatment of Streptococcus suis infections such as porcine circovirus type 2 disease, porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis, or porcine sudden death, and / or porcine epidemic pneumonia is prophylactic or preventative treatment.

[0097] The vaccine oil-in-water emulsion according to the present invention can be administered to animals by any suitable route. For example, the vaccine oil-in-water emulsion according to the present invention can be administered to animals via transdermal administration, via mucosal administration, via oral administration, or via injection, for example intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal injection. Preferably, the vaccine oil-in-water emulsion according to the present invention is administered to animals by injection.

[0098] Those skilled in the art can adapt the dose and / or frequency of the active agent, preferably the antigen, administered to the animal depending, inter alia, on the age, sex, weight, species and condition of the animal, the route of administration and the properties of the active agent. Administration can be carried out in one or several doses, preferably 1 to 3 doses. In a preferred embodiment, administration is carried out in a single dose or in two doses.

[0099] Preferably, the vaccine oil-in-water emulsion according to the present invention is sterile.

[0100] The present invention also relates to a method for treating Streptococcus suis infections such as porcine circovirus type 2 disease, porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death, and / or porcine epizootic pneumonia, which method of treatment comprises administering to a non-human mammal, preferably a porcine mammal, an effective dose of a vaccine oil-in-water emulsion according to the present invention.

[0101] The present invention also relates to the use of a vaccine oil-in-water emulsion according to the invention for the preparation of a medicament, preferably a vaccine, in particular a vaccine for the treatment of Streptococcus suis infections, such as porcine circovirus type 2 disease, porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death, and / or porcine epizootic pneumonia.

[0102] The present invention also relates to a method for inducing an immune response in an animal, preferably a pig or piglet, which method comprises administering to the animal a vaccine oil-in-water emulsion according to the present invention.

[0103] The present invention also relates to a method for immunizing an animal against, or preventing or alleviating symptoms caused by, a pathogenic infection (e.g., a viral, bacterial, fungal, or protozoan infection), preferably wherein the pathogenic bacteria is porcine circovirus type 2, Streptococcus suis, or Mycoplasma hyopneumoniae, more preferably porcine circovirus type 2 or Mycoplasma hyopneumoniae.

[0104] Method for producing vaccine oil-in-water emulsion The vaccine oil-in-water emulsion according to the present invention can be prepared by any suitable method known in the art, hi one embodiment, the vaccine oil-in-water emulsion is obtained by diluting an adjuvant oil-in-water emulsion with a solution of the antigen in a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier.

[0105] The active agent, preferably the antigen, may be incorporated into the emulsion during its formation or may be incorporated into the emulsion at a later time, hi some embodiments, the active agent is incorporated into the vaccine oil-in-water emulsion immediately prior to use of the emulsion.

[0106] The size of the droplets in the emulsion can be from about 10 nm to about 5 μm, hi one embodiment, the average size of the droplets in the emulsion is about 60 nm.

[0107] kit The present invention also relates to a kit, the kit comprising: - an active agent as defined above, preferably an antigen, an adjuvant oil-in-water emulsion or a diluted adjuvant oil-in-water emulsion according to the invention.

[0108] The kit may further comprise a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier, and / or instructions on how to prepare a vaccine oil-in-water emulsion according to the invention.

[0109] As used herein, the term "comprises" and its derivatives should be understood as not limiting the presence of other components. In some embodiments, the term "comprises" and its derivatives can be understood to be more restrictive and mean "consisting essentially of," or even "consisting of."

[0110] The present invention is also described in further detail in the following examples, which are not intended to limit the scope of the invention as defined by the appended claims. [Example]

[0111] Example 1: Composition according to the invention The following oil adjuvant compositions were prepared by conventional methods: Composition A is an oil adjuvant composition according to the invention, and Composition B is a comparative composition.

[0112] [Table 1]

[0113] Marcol™ 52 is a refined mixture of liquid saturated hydrocarbons.

[0114] TEGO® SMO V is a non-ionic lipophilic surfactant containing sorbitan oleate.

[0115] BRIJ O10 is a nonionic hydrophilic surfactant containing polyoxyethylene (10) oleyl ether.

[0116] Simulsol 2599 PHA VG is a hydrophilic nonionic surfactant containing polyethylene glycol oleate (10).

[0117] Example 2: Stability testing A comparative stability study was performed on samples of Hyogen® vaccine formulated with different adjuvants and stored for one month at 2–8°C. The antigen contained in the emulsion tested was the Mycoplasma hyopneumoniae (M HYO) antigen.

[0118] Each test formulation contained the Hyogen® vaccine adjuvant: composition A, - comparative composition B, - the comparative adjuvant composition LR3 oil phase disclosed in the prior art WO 2006 / 113373, and - Comparative adjuvant composition LR4, disclosed in prior art WO 2006 / 113373, in which one of the oil phases was replaced by an oil-in-water emulsion.

[0119] The oil phase of LR3 contains Brij® 92 (polyoxyethylene (2) oleyl ether), Brij® 96 (polyoxyethylene (10) oleyl ether), paraffin oil (Marcol® 82), and preservatives.

[0120] The oil phase of LR4 contains Brij® 92 (polyoxyethylene (2) oleyl ether), Volpo® N5 (polyoxyethylene (5) oleyl ether), paraffin oil (Marcol® 82), and preservatives.

[0121] FIG. 1 shows the appearance of the test composition after one day (a), one week (b), and one month (c).

[0122] (a) After 1 day, the compositions appear homogeneous overall, with slight creaming observed only in the LR3-based vaccine compositions. (b) After one week, the same creaming is still observed in the LR3-based vaccine composition. The vaccine composition based on comparative adjuvant composition B has separated into two phases. (c) After one month, the LR3-based composition and comparative composition B separate almost completely into two phases. A slight creaming is observed on top of the LR4-based composition.

[0123] The particle size distribution in each composition was measured by laser diffraction using a Mastersizer 3000 particle size analyzer at day 1, week 1, and month 1. The results are shown in Table 1 below.

[0124] [Table 2]

[0125] Figures 2 to 5 show the evolution of the particle size distribution for each composition from one day to one week.

[0126] Figure 2: Composition A. The emulsion is completely stable with respect to particle size. The distribution is Gaussian, with a mean value well below 1 μm, and does not evolve over time.

[0127] Figure 3: Comparative composition B: Particle size increased from 60 nm to 100 μm after one week at 4°C. Particle size correlates closely with visual aspects. The emulsion was already broken after one week and further broken after one month.

[0128] Figure 4: Composition LR3: Two particle populations are present, the larger one being above 1 μm. The particle size correlates closely with the visual aspect. The particle size distribution deteriorated over 1 month.

[0129] Figure 5: Composition LR4: Particle sizes above 1 μm are present. This results in a non-uniform particle size distribution. For this reason, good stability over time is not predicted. The non-uniform particle size distribution remains over a month.

[0130] In summary, the vaccine composition based on composition A according to the present invention is the only oil-in-water emulsion that is completely stable in particle size and appearance after storage for 1 month at 4° C. This vaccine composition is expected to be stable for 1-2 years at 4° C.

[0131] Example 3: Safety test of the Hygen vaccine according to the present invention The safety of Hyogen® vaccine formulated with oil-in-water emulsion adjuvant composition A according to the present invention was evaluated in a field trial conducted on three farms in different provinces of China.

[0132] The pigs' reactions after administration of the formulations were assessed and scored according to the severity scoring scale detailed in Table 2 below.

[0133] [Table 3]

[0134] Table 3 below shows the safety results of the formulations.

[0135] [Table 4]

[0136] Of the 665 pigs tested, none showed any reaction after administration of the vaccine emulsion according to the invention.

[0137] This demonstrates that the vaccine emulsion according to the present invention is highly safe, especially when used in conjunction with Mycoplasma hyopneumoniae antigens.

[0138] Example 4: Safety test of PCV2 vaccine according to the present invention The safety of a PCV2 vaccine formulated using an oil-in-water emulsion adjuvant composition according to the present invention (composition A in Example 1) was evaluated in a field trial.

[0139] The safety of overdosing three batches of PCV2 vaccine oil-in-water emulsion according to the present invention was investigated compared with a mock vaccine using a total of 20 PRRS-negative, PCV2-negative piglets aged 2 to 3 weeks. Prior to vaccination, the animals were randomly assigned to four groups, A to D (five animals per group). Three of the groups (groups A to C) were injected with 4 mL of the vaccine oil-in-water emulsion according to the present invention, and one group (group D) was injected with an equivalent dose of mock vaccine. To assess safety performance, each animal's temperature was collected daily from three days before vaccination through six days after vaccination. Temperatures were also collected immediately before vaccination and four hours after vaccination. Clinical observations were conducted daily from days 1 to 14 after vaccination, with attention paid to any abnormal local or systemic reactions.

[0140] The results showed that a transient rise in body temperature was observed within the first day after vaccination, after which all animals recovered. The maximum temperature rise in each group did not exceed 1.5°C, with an average of 0.8-0.9°C. Figure 6 and Table 4 below show the temperature data for each group. No significant differences were observed between groups. Apart from the transient temperature rise, no systemic or local adverse reactions were observed during the study. The safety of the three batches of vaccine oil-in-water emulsions according to the present invention was confirmed.

[0141] The maximum body temperature rise of each animal was calculated for ANOVA test. No significant difference was found between the groups (p=0.701).

[0142] [Table 5]

[0143] Example 5: Efficacy of adjuvant compositions according to the present invention A comparative efficacy study was conducted in piglets to test PCV2 vaccine formulations containing different adjuvants.

[0144] Eighty purebred (Large Yorkshire, Landrace, Genesus) piglets were divided into four groups of 20 pigs.

[0145] - Group A was injected with the reference product Circoflex (porcine circovirus type 2 ORF2 protein with carbomer as adjuvant) and challenged with PCV2 strain SEVC1702 21 days after vaccination.

[0146] Group B was injected with a placebo (oil-in-water emulsion adjuvant of the invention obtained from oil composition A of Example 1 + phosphate buffered saline PBS) and challenged with PCV2 strain SEVC1702 21 days after vaccination.

[0147] - Group C was injected with a PCV2 vaccine comprising the porcine circovirus type 2 ORF2 protein and an oil-in-water emulsion adjuvant according to the invention and challenged with PCV2 strain SEVC1702 21 days after vaccination.

[0148] Group D was injected with a PCV2 vaccine comprising the porcine circovirus type 2 ORF2 protein and an oil-in-water emulsion adjuvant according to the invention and challenged with PCV2 strain SEVC1702 14 days after vaccination.

[0149] The piglets in each group were necropsied 28 days after challenge (Groups A, B and C on day 49, and Group D on day 42).

[0150] Figures 7A, 7B, and 7C show viremia in each group on days 14, 21, and 28 post-challenge, respectively. Each group had significantly reduced viremia compared to placebo (group B).

[0151] The adjuvants according to the invention (groups C and D) have a significantly lower viremia than the reference product Circoflex.

[0152] For qPCR, nasal swabs were collected after tapping on days 7, 14, 21, and 28. Test results were log-transformed and analyzed.

[0153] Figures 8A, 8B, and 8C show the logarithms of nasal swabs on days 7, 14, 21, and 28, respectively. Group D shed significantly less virus through the nasal passages than Group A.

[0154] For qPCR assay, anal swabs were collected after tapping on days 7, 14, 21, and 28. Test results were log-transformed and analyzed.

[0155] Figures 9A, 9B, 9C, and 9D show the logarithms of nasal swabs on days 7, 14, 21, and 28, respectively. Virus shed via feces was significantly lower in Group D than in Group A on days 14, 21, and 28 post-challenge.

[0156] All animals were euthanized 28 days post-challenge (DPC28), and the tonsils, mediastinal lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, and lungs were collected by dissection. Each tissue was cut into small pieces about the size of a grain of rice, and total DNA was extracted from the tissue using a genome extraction kit. Viral nucleic acid was then quantified using a qPCR kit. Data were log-transformed and intergroup differences were analyzed using the Kruskal-Wallis test, followed by a 2-to-2 comparison of intergroup differences using the Wilcoxon rank-sum test.

[0157] Figures 10A, 10B, 10C, 10D, and 10E show the log quantitation of virus on day 28 for inguinal, mesenteric, lung, mediastinal, and tonsillar lymph nodes, respectively.

[0158] Both groups A and C were able to significantly inhibit the viral load in lymph node tissue, lungs, and tonsils compared with the placebo group (group B). Between the groups, groups C and D suppressed the viral load in lymph nodes and tonsils better than group A, and the difference was significant.

[0159] Finally, PCV2-specific antibody levels were assessed using a commercially available ELISA kit.

[0160] The results showed that all vaccine immunization groups, except for the placebo group, could induce the production of specific antibodies in the animals. The animals in the placebo group did not show antibody turnover before tapping. Compared with the placebo group, higher antibody levels could be stimulated in the immunization group after tapping in a shorter period of time.

[0161] Conclusion: The results showed that both PCV2 vaccines containing the adjuvant of the present invention were able to significantly reduce the amount of virus in the blood, the amount of virus excreted through the nose and anus, and the PCV2 virus levels in the lymph nodes, tonsils, and lungs compared to the placebo. The two batches of vaccines containing the adjuvant of the present invention used in this study significantly increased the inhibition levels of nasal and fecal excretion and the inhibition levels of virus load in the lymph nodes and tonsils compared to the reference product, indicating that the vaccine containing the adjuvant of the present invention provides excellent protection against the 2d genotype PCV2 strain.

[0162] The clinical safety of the two laboratory vaccine batches was also observed in this study: after immunization, neither vaccine batch caused any severe acute reactions, and no local adverse reactions at the injection site were observed.

[0163] Example 6: Vaccine composition comprising an IdeSsuis antigen and an adjuvant composition according to the invention Streptococcus suis (S. suis) is a major swine pathogen that causes meningitis, arthritis, and several other pathologies. IDesusis, the immunoglobulin M-degrading enzyme of S. suis, is a highly protective antigen against S. suis infection.

[0164] One hundred piglets aged 7-10 weeks were divided into five groups.

[0165] Piglets in groups 1-5 were vaccinated twice (on D0 and D21) with recombinant IdeSsuis IgM protease protein as described in the test items listed in Table 5 below. Control group 6 was treated with physiological NaCl solution. The O / W adjuvant of the present invention corresponds to an O / W emulsion obtained from composition A, the oil composition of Example 1, and phosphate buffered saline (PBS).

[0166] [Table 6]

[0167] Local and systemic reactions were assessed on D7, D10, D14, D24, D27, D31 and D35. Clinical health was monitored daily.

[0168] Blood samples were collected from all animals on study days -1, 21, and 35. The study was terminated on study day 35.

[0169] result 1. Local tolerance Local tolerance was observed (by palpation) on D7, D10, D14, D24, D27, D31 and D35.

[0170] The percentage of local reactions per day is shown in FIG.

[0171] Groups 3 and 4, which were vaccinated with the adjuvant emulsion of the present invention, tolerated the vaccine better or equally well than the other groups on all days.

[0172] Between D24 and D31, groups 3 and 4 showed better local reaction results than any other group.

[0173] Groups 1 and 2 showed significant differences in local reactions depending on the adjuvant concentration. Such differences are not seen with the adjuvants of the present invention.

[0174] In conclusion, the adjuvants of the present invention show fewer local reactions than the reference adjuvants and exhibit a better safety profile due to the specific formulation used.

[0175] 2. Antibody titer Antibody titers against the antigens in the vaccine preparation were determined from serum samples using ELISA. The results are shown in Figure 12. B0 corresponds to before the first vaccination, B1 corresponds to between the first and second vaccinations, and B2 corresponds to two weeks after the second vaccination.

[0176] result Use of the adjuvants of the present invention to prepare a vaccine against S. suis showed similar antibody development as use of the reference adjuvant.

Claims

1. 1. An oil adjuvant composition comprising: - at least one oil, at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester, - at least one non-ionic hydrophilic surfactant which is an ethoxylated fatty alcohol.

2. 2. The oil adjuvant composition of claim 1, wherein the oil comprises at least one mineral oil, in particular at least one light paraffin oil, or a combination of at least two mineral oils.

3. 3. The oil adjuvant composition of claim 2, wherein the at least one mineral oil is selected from the group consisting of paraffin oils, such as isoparaffin oils, and / or naphthenic oils, pristane, and any mixture thereof.

4. 4. The oil adjuvant composition according to claim 1, wherein each sorbitan ester or mannitan ester is a sorbitan ester, and the sorbitan ester is preferably selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, sorbitan monoisostearate, and sorbitan isostearate, and is more preferably sorbitan monooleate.

5. The oil adjuvant composition according to any one of claims 1 to 3, wherein each sorbitan ester or mannitan ester is a mannitan ester, preferably selected from the group consisting of mannitan oleate, mannitan monooleate, mannitan dioleate, mannitan trioleate, and mannitan tetraoleate.

6. 6. The oil adjuvant composition according to any one of claims 1 to 5, wherein the ethoxylated fatty alcohol is selected from the group consisting of polyoxyethylene (23) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) oleyl ether, polyoxyethylene (20) oleyl ether, polyoxyethylene (21) stearyl ether, polyoxyethylene lauryl ether, eicosaethylene glycol octadecyl ether, triethylene glycol oleyl ether, and any mixture thereof, preferably polyoxyethylene oleyl ether, in particular polyoxyethylene (10) oleyl ether.

7. The oil adjuvant composition comprises: - 76% to 92% oil, - 1% to 7% of at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester; - 7% to 17% of at least one non-ionic hydrophilic surfactant which is an ethoxylated fatty alcohol; The oil adjuvant composition according to any one of claims 1 to 6, wherein the percentage is expressed by weight relative to the total amount of the oil adjuvant composition.

8. The oil adjuvant composition comprises: about 82% of oil, preferably mineral oil, about 4% of at least one non-ionic lipophilic surfactant which is a sorbitan ester or a mannitan ester, preferably sorbitan monooleate; about 14% of at least one non-ionic hydrophilic surfactant, which is an ethoxylated fatty alcohol, preferably polyoxyethylene oleyl ether; 8. The oil adjuvant composition of claim 7, wherein the percentage is expressed by weight relative to the total amount of the oil adjuvant composition.

9. An adjuvant oil-in-water emulsion comprising 40% to 70% of an oil adjuvant composition according to any one of claims 1 to 8 and 30% to 60% of an aqueous solvent, preferably water; The oil adjuvant composition is dispersed in an aqueous solvent, The percentages are expressed by volume relative to the total volume of the adjuvant oil-in-water emulsion.

10. 10. A vaccine oil-in-water emulsion comprising the adjuvant oil-in-water emulsion of claim 9, at least one antigen, and a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier.

11. the vaccine oil-in-water emulsion comprises 10-80% of the adjuvant oil-in-water emulsion and 20-90% of a pharmaceutically acceptable carrier; 11. The vaccine oil-in-water emulsion of claim 10, wherein said percentage is expressed by volume relative to the total volume of said vaccine oil-in-water emulsion.

12. 12. The vaccine oil-in-water emulsion of claim 10 or claim 11, wherein the at least one antigen is selected from the group consisting of a Porcine Circovirus Type 2 (PCV2) antigen, a Streptococcus suis antigen, a Mycoplasma hyopneumoniae antigen, and combinations thereof.

13. A vaccine oil-in-water emulsion according to any one of claims 10 to 12 for use as a medicament, preferably for use as a vaccine.

14. 13. The vaccine oil-in-water emulsion of any one of claims 10 to 12 for use in the treatment of porcine circovirus type 2 disease, Streptococcus suis infection, such as porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death, and / or porcine epizootic pneumonia.

15. 15. A vaccine oil-in-water emulsion for use according to claim 13 or 14, wherein said vaccine oil-in-water emulsion is administered, preferably injected, to an animal which is a pig or piglet.

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