Oil adjuvant compositions and vaccine emulsions comprising such oil adjuvant compositions

Abstract

The present invention relates to an oil adjuvant composition comprising at least one oil; at least one non-ionic lipophilic surfactant, wherein the at least one non-ionic lipophilic surfactant is dehydrated sorbitol ester or dehydrated mannitol ester; and at least one nonionic hydrophilic surfactant, the at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol. The invention also relates to an oil-in-water adjuvant emulsion and an oil-in-water vaccine emulsion obtained from the oil adjuvant composition of the invention, and to the use of such an oil-in-water vaccine emulsion for the treatment of porcine circovirus type 2 disease and / or porcine endemic pneumonia.

Description

Technical Field

[0001] The present invention relates to an oil adjuvant composition comprising a specific surfactant, which is particularly suitable for formulating oil-in-water vaccine emulsions. Background Art

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

[0003] When preparing pharmaceutical compositions, especially vaccine emulsions, two main criteria for selecting suitable adjuvants are stability and safety, especially in the presence of bacteria and / or virus suspensions. For example, due to the interaction of the lipase or esterase enzymatic activity present in the components of the emulsion and the antigen suspension of the vaccine, the stability of the adjuvant may be affected. Especially, when the vaccine is based on inactivated or attenuated pathogens or based on the combination of pathogens, this problem can be observed. In addition, due to the toxicity of some surfactants present in the adjuvant, safety issues may occur.

[0004] WO 2005 / 009462 discloses an oil-in-water emulsion comprising an aqueous phase containing an immunogen and an oil phase comprising at least three surfactants: a nonionic lipophilic surfactant that is a fatty ester of sorbitan; a nonionic hydrophilic surfactant that is an ethoxylated sorbitan fatty acid monoester; and a nonionic hydrophilic surfactant that is a sorbitan fatty acid triester. The adjuvant composition is disclosed as being suitable for forming a vaccine composition with enhanced safety and stability. However, the use of only sorbitan ester surfactants can affect the stability of the oil-in-water emulsion, particularly when the adjuvant composition is used with an antigen produced by a cell line containing an enzyme, such as a lipase or esterase. For example, a cell line used to produce a Mycoplasma hyopneumoniae antigen may contain an enzyme, such as an esterase, that can destabilize an emulsion prepared using an adjuvant composition containing only an ester surfactant, particularly an ethoxylated ester surfactant.

[0005] WO 2006 / 113373 discloses an oil-in-water adjuvant formulation comprising two surfactants, one a nonionic lipophilic ethoxylated fatty alcohol and the other a nonionic hydrophilic ethoxylated fatty alcohol. The formulation utilizes only ethoxylated fatty alcohol surfactants, which, due to the toxicity and ecotoxicity of this family of emulsifiers, can lead to reduced formulation stability and increased safety concerns. In fact, ethoxylated fatty alcohol surfactants contain ether functional groups that cannot be reversed and degraded by enzymes (such as esterases). Consequently, 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, using only these surfactants in a composition increases safety concerns regarding the toxicity of the composition. WO 2006 / 113373 discloses that the claimed formulation exhibits 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 comprise additional components, such as enzymes. Summary of the Invention

[0007] In this regard, the inventors have surprisingly demonstrated that the use of a specific combination of surfactants comprising at least one nonionic lipophilic surfactant which is a sorbitan ester or a mannitol ester and at least one nonionic hydrophilic surfactant which is an ethoxylated fatty alcohol provides an oil adjuvant composition having increased stability and safety even when used in vaccine emulsions with antigens comprising additional components such as enzymes.

[0008] Therefore, the present invention first relates to an oil adjuvant composition, which comprises:

[0009] - at least one oil,

[0010] - at least one nonionic lipophilic surfactant, the at least one nonionic lipophilic surfactant being a sorbitan ester or a mannitol ester, and

[0011] - at least one nonionic hydrophilic surfactant, the at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol.

[0012] 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.

[0013] 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.

[0014] In one embodiment, each sorbitan ester or anhydromannitol ester is a sorbitan ester, wherein 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, more preferably sorbitan monooleate.

[0015] In another embodiment, each sorbitan ester or anhydromannitol ester is an anhydromannitol ester, preferably selected from the group consisting of anhydromannitol oleate, anhydromannitol monooleate, anhydromannitol dioleate, anhydromannitol trioleate and anhydromannitol tetraoleate.

[0016] 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, eicosanyl glycol stearyl ether, triethylene glycol oleyl ether and any mixture thereof, preferably polyoxyethylene oleyl ether, in particular polyoxyethylene (10) oleyl ether.

[0017] In one embodiment, the oil adjuvant composition comprises:

[0018] -76% to 92% oil,

[0019] - 1% to 7% of at least one nonionic lipophilic surfactant, the at least one nonionic lipophilic surfactant being a sorbitan ester or a mannitol ester, and

[0020] - 7% to 17% of at least one nonionic hydrophilic surfactant, the at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol,

[0021] The percentages are expressed by weight relative to the total volume of the oil adjuvant composition.

[0022] In one embodiment, the oil adjuvant composition comprises:

[0023] - about 82% of oil, preferably mineral oil,

[0024] - about 4% of at least one nonionic lipophilic surfactant, the at least one nonionic lipophilic surfactant being a sorbitan ester or anhydromannitol ester, preferably sorbitan monooleate, and

[0025] - about 14% of at least one nonionic hydrophilic surfactant which is an ethoxylated fatty alcohol, preferably polyoxyethylene oleyl ether, wherein said percentages are expressed by weight relative to the total volume of said oil adjuvant composition.

[0026] The present invention also relates to an oil-in-water adjuvant emulsion comprising 40% to 70% of an oil adjuvant composition according to the present invention and 30% to 60% of an aqueous solvent, wherein the oil adjuvant composition is dispersed in the aqueous solvent, wherein the percentages are expressed by volume relative to the total volume of the oil-in-water adjuvant emulsion.

[0027] The present invention further relates to an oil-in-water vaccine emulsion comprising the oil-in-water adjuvant emulsion according to the present invention, at least one antigen and a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier.

[0028] In one embodiment, the oil-in-water vaccine emulsion comprises 10% to 80% of an oil-in-water adjuvant emulsion and 20% to 90% of a pharmaceutically acceptable carrier.

[0029] The percentages are expressed by volume relative to the total volume of the oil-in-water vaccine emulsion.

[0030] 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.

[0031] The present invention further relates to the oil-in-water vaccine emulsion according to the invention for use as a medicament, preferably as a vaccine.

[0032] A final object of the present invention is an oil-in-water vaccine emulsion according to the present invention for use in the treatment of porcine circovirus disease type 2, Streptococcus suis infections such as porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death and / or porcine enzootic pneumonia.

[0033] In one embodiment, the oil-in-water vaccine emulsion is administered, preferably injected, to an animal, which is a pig or piglet. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Photographs of the visual appearance of the composition studied in Example 2 after 1 day (a), after 1 week (b) and after 1 month (c) are presented.

[0035] Figures 2 to 5 A graph of the size distribution of particles in the composition studied in Example 2 over time is presented. Figure 2 : Composition A; Figure 3 : Comparative composition B; Figure 4 : Composition LR3, and Figure 5 : Composition LR4.

[0036] Figure 6 4 is a graph comparing the temperature increases in the four groups of the safety study of Example 4.

[0037] Figure 7 Histograms of viremia for each group at day 14 (A), day 21 (B), and day 28 (C) post-challenge for the study of Example 5 are presented.

[0038] Figure 8 Histograms of the logarithm of nasal swabs at day 7 (A), day 14 (B), day 21 (C), and day 28 (D) of the study of Example 5 are presented.

[0039] Figure 9 Histograms of nasal swabs on Day 7 (A), Day 14 (B), Day 21 (C), and Day 28 (D) of the Example 5 study are presented.

[0040] Figure 10 Histograms of the logarithmic quantification of virus at day 28 for the inguinal lymph nodes (A), mesenteric lymph nodes (B), lung (C), mediastinal lymph nodes (D), and tonsillar lymph nodes (E), respectively, from the study of Example 5 are presented.

[0041] Figure 11 is a graph presenting the percentage of local reactions per day for the study of Example 6.

[0042] Figure 12 is a graph presenting the antibody development of each group in the study of Example 6 before the first vaccination (B0), between the first and second vaccinations (B1), and 2 weeks after the second vaccination (B2). DETAILED DESCRIPTION

[0043] definition

[0044] "About" is used herein to mean approximately, roughly, around, or in the region of... When the term "about" is used in conjunction with a numerical range or value, it modifies the range or value by extending the boundaries above and below the numerical range or value by up to 10%, preferably 5%, more preferably 1%.

[0045] An "adjuvant composition" is a composition that, when combined with at least one active agent (such as an antigen), is suitable for forming a pharmaceutical composition (such as a vaccine composition). The adjuvant composition increases the immune response to the vaccine antigen when compared to the response induced by the vaccine antigen alone. In some other non-preferred embodiments, the adjuvant composition at least does not decrease the immune response to the vaccine antigen when compared to the response induced by the vaccine antigen alone.

[0046] The term "animal" encompasses 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 cows), porcines (such as pigs, domestic pigs, or sows), ovines (such as sheep), caprines (such as goats), and avian animals (such as chickens).

[0047] "Antigen" refers to a substance that induces a specific immune response in a host animal. Antigens can be epitopes, immunogenic fragments, or whole organisms (bacteria, viruses) that have been inactivated, attenuated, or genetically modified.

[0048] An "emulsion" is a colloidal system made of two immiscible elements (e.g., oil and water), one of which (the dispersed phase) exists as droplets dispersed in the other, which constitutes the continuous phase. An "oil-in-water emulsion" is 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).

[0049] The term "ethoxylated" when used to qualify compounds containing labile hydrogen atoms, such as hydrogen atoms of hydroxy groups, means compounds wherein the hydrogen-labile groups have been replaced by -(C2H4O) n -H moiety substituted, n is 1 or greater. n is generally in the range of 1 to 20. The term ethoxylated can be used, for example, to define fatty alcohols (ethoxylated fatty alcohols) or sorbitan or mannitol esters (ethoxylated sorbitan esters or ethoxylated mannitol esters).

[0050] The term "fat," when used to define compounds containing hydrocarbon chains, refers to compounds containing aliphatic hydrocarbon chains 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 can be saturated or unsaturated. The term fat can be used, for example, to define alcohols (fatty alcohols), esters (fatty esters), and / or acids (fatty acids).

[0051] "Anhydromannitol esters" are derivatives obtained by esterification of at least one of the alcohol and / or phenol functions of anhydromannitol.

[0052] "Mineral oil" is any of various colorless, odorless, light mixtures of higher alkanes derived from mineral sources, especially petroleum distillates.

[0053] An "oil" is a fatty substance that is fluid, preferably liquid, at room temperature (25° C. to 35° C.) and insoluble in water. It may be synthetic or of vegetable, animal or mineral origin.

[0054] The term "pharmaceutically acceptable carrier" refers to a fluid medium for containing a pharmaceutically active agent (such as an antigen), and can be administered to an animal, preferably injected into an animal, without significant adverse effects. Pharmaceutically acceptable carriers do not interact with the pharmaceutically active agent, and preferably do not affect its drug efficiency. Suitable pharmaceutically acceptable carriers known in the art include sterile water, saline, glucose, dextrose, and buffered solutions. Carriers may include adjuvants, such as diluents, stabilizers, sugars, amino acids, preservatives, wetting agents, emulsifiers, pH buffers, viscosity enhancing additives, coloring agents, or any mixture thereof.

[0055] The terms "domestic pig," "pig," and "piglet" refer to animals of Suidae origin.

[0056] "Sorbitan esters" are derivatives obtained by esterification of at least one of the alcohol and / or phenol functions of sorbitan.

[0057] "Treating" or "treatment" refers to therapeutic treatment and prophylactic or preventative measures, wherein the purpose is to prevent or slow down (mitigate) the targeted pathological condition or disorder. Those in need of treatment include those already suffering from the disorder, as well as those susceptible to the disorder or those to be prevented from the disorder. A subject or animal is successfully "treated" for 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 decrease in the number of pathogenic cells; a decrease in the percentage of total pathogenic cells; and / or relief to some extent of one or more of the symptoms associated with a particular disease or disorder; reduced morbidity and mortality, and improved quality of life issues. The above parameters for assessing successful treatment and improvement of the disease can be easily measured by routine procedures familiar to physicians.

[0058] Oil adjuvant composition

[0059] The first object of the present invention is an oil adjuvant composition, which comprises

[0060] - at least one oil,

[0061] - at least one nonionic lipophilic surfactant, the at least one nonionic lipophilic surfactant being a sorbitan ester or a mannitol ester, and

[0062] - at least one nonionic hydrophilic surfactant, the at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol.

[0063] The inventors have shown that the oil adjuvant composition and the oil-in-water vaccine emulsion according to the present invention are highly stable and exhibit a high safety profile, even when the antigen may contain additional elements (such as enzymes). The stability of the oil adjuvant composition and the oil-in-water vaccine emulsion has even been shown to be higher than that of the formulations disclosed in the prior art and to be highly stable.

[0064] Oils are known to be suitable adjuvants for active agents because they are immunostimulants. The oil compositions according to the present invention are suitable as adjuvants, particularly for veterinary compositions. Furthermore, oil-in-water emulsions are known to be suitable as adjuvants, particularly for veterinary compositions, as disclosed in publications such as Burakova et al., Viral Immunology 31, No. 1, 2018, pp. 11-22.

[0065] 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 dehydrated mannitol ester.

[0066] In some embodiments, at least one nonionic hydrophilic surfactant and at least one nonionic lipophilic surfactant are the only surfactants contained in the oil adjuvant composition according to the present invention. In some embodiments, the oil adjuvant composition according to the present invention comprises an ethoxylated fatty alcohol and a sorbitan ester or anhydromannitol ester as the only surfactants.

[0067] 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, and is advantageously selected from the group consisting of oleyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, lauryl alcohol and stearyl alcohol, and combinations thereof, advantageously oleyl alcohol. More advantageously, the ethoxylated fatty alcohol is oleyl alcohol having 3 to 20 ethylene oxide moieties.

[0068] The ethoxylated fatty alcohols may for example be selected from the group consisting of polyoxyethylene (23) lauryl ether (such as Brij ), polyoxyethylene (20) cetyl ether (such as Brij or Brij ), polyoxyethylene (10) oleyl ether (such as Brij or Brij ), polyoxyethylene (20) oleyl ether (such as Brij or Brij ), polyoxyethylene (21) stearyl ether (such as Brij ), polyoxyethylene lauryl ether (such as Brij ), Eicosanyl glycol octadecyl ether (such as Brij ), triethylene glycol oleyl ether (such as Volpo ) and any mixture thereof.

[0069] In some embodiments, the ethoxylated fatty alcohols are used as a mixture.

[0070] In some embodiments, the ethoxylated fatty alcohols are selected from those having the following brand names: (68439-49-6), (68439-49-6), (68439-49-6), (68439-49-6), (68439-49-6), C12-14 C12-14 C12-14 C12-14 C12-15 (Nikko Chemicals) and any combination thereof.

[0071] The ethoxylated fatty alcohols preferably exhibit a hydrophilic-lipophilic balance (HLB) value of between 10 and 18, preferably between 12 and 18.

[0072] The surfactants of the present invention may comprise fatty alcohols of animal or vegetable origin.

[0073] The amount of the at least one ethoxylated fatty alcohol in the oil adjuvant composition according to the invention can vary within wide ranges 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 or anhydromannitol ester) and / or the nature of the pharmaceutically active agent (in particular the antigen) to be present in the oil-in-water vaccine emulsion comprising the oil adjuvant composition.

[0074] 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, the percentage being expressed by weight relative to the total volume of the oil adjuvant composition.

[0075] The nonionic lipophilic surfactant is a non-ethoxylated sorbitan ester or a non-ethoxylated anhydromannitol ester. Ethoxylated sorbitan and anhydromannitol esters containing more than six ethylene oxide moieties are hydrophilic.

[0076] The nonionic lipophilic surfactant is an ester of sorbitan or an ester of mannitol, preferably a fatty ester of sorbitan or mannitol. In some embodiments, each nonionic lipophilic surfactant is an ester of sorbitan, preferably a fatty ester of sorbitan, more preferably a fatty acid monoester of sorbitan or a fatty acid triester of sorbitan. In other embodiments, each nonionic lipophilic surfactant is an ester of mannitol, preferably a fatty ester of mannitol.

[0077] Examples of fatty esters of sorbitan include sorbitan monolaurate (such as Span ), sorbitan monopalmitate (such as Span ), sorbitan monostearate (such as Span ), sorbitan tristearate (such as Span ), sorbitan monooleate (such as Span ), sorbitan trioleate (such as Span ), sorbitan monoisostearate (such as Arlacel ) and sorbitan isostearate (such as Crill ).

[0078] Examples of fatty esters of anhydromannitol include anhydromannitol oleate (such as Montanide ), anhydromannitol monooleate (such as Arlacel ), dehydromannitol dioleate, dehydromannitol trioleate and dehydromannitol tetraoleate.

[0079] The fatty acids used to form the fatty alcohols and / or fatty 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.

[0080] The amount of the at least one sorbitan ester or anhydromannitol ester in the oil adjuvant composition according to the invention can vary within wide ranges and can be determined by a person skilled in the art, depending inter alia on the chemical nature of the at least one sorbitan or anhydromannitol ester, the nature of the other components (oil and / or ethoxylated fatty alcohol) and / or the nature of the pharmaceutically active agent (in particular the antigen) to be present in the oil-in-water vaccine emulsion comprising the oil adjuvant composition.

[0081] The at least one sorbitan ester or anhydromannitol 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 percentage being expressed by weight relative to the total volume of the oil adjuvant composition.

[0082] The oils may be synthetic, or of vegetable, animal or mineral origin.

[0083] The oil may be a mineral oil, including but not limited to paraffinic oils, such as isoparaffinic oils and / or naphthenic oils, pristane, and any mixtures thereof. An advantageous mineral oil that may be used in the present invention may include an oil comprising a linear or branched carbon chain having a carbon number greater than 15, preferably 15 to 32, and containing no aromatic compounds. The oil may be, for example, a petrolatum oil sold under the name MARCOL®. or MARCOL (produced by Esso, France), DRAKEOL (produced by Penreco, USA) or EOLANE EOLANE or Eolane The mineral oil may be selected from the group consisting of oil with CAS number 8042-47-5 (white mineral oil), oil with CAS number 8012-95-1 (light liquid paraffin), oil with CAS number 8020-83-5 (mineral oil) and any mixture thereof.

[0084] The oil may be a synthetic oil including, but not limited to, squalane, polyisobutylene oil, hydrogenated polyisobutylene oil, polydecene oil, polyisoprene oil, polyisopropylene oil, and the like, and any mixtures thereof.

[0085] 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.

[0086] 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.

[0087] The oil may also be a mixture of oils comprising at least two oils selected from the oils described herein in any proportion.

[0088] The amount of oil in the oil adjuvant composition according to the invention may vary within wide limits and can be determined by a person skilled in the art, depending inter alia on the chemical nature of the oil, the nature of the other components (sorbitan or anhydromannitol esters and / or ethoxylated fatty alcohols) and / or the nature of the pharmaceutically active agent (especially the antigen) to be present in the oil-in-water vaccine emulsion comprising the oil adjuvant composition.

[0089] 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, the percentages being expressed by weight relative to the total volume of the oil adjuvant composition.

[0090] In some embodiments, the oil adjuvant composition according to the present invention comprises a liquid saturated hydrocarbon (preferably MARCOL Oil), sorbitan monooleate and polyoxyethylene (10) oleyl ether.

[0091] In some embodiments, the oil adjuvant composition according to the present invention comprises

[0092] - 76% to 92%, preferably about 82%, of oil, preferably mineral oil,

[0093] - 1% to 7%, preferably about 4%, of at least one nonionic lipophilic surfactant, the at least one nonionic lipophilic surfactant being a sorbitan ester or anhydromannitol ester, preferably sorbitan monooleate, and

[0094] - 7% to 17%, preferably about 14%, of at least one nonionic hydrophilic surfactant, the at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol, preferably polyoxyethylene oleyl ether,

[0095] The percentages are expressed by weight relative to the total volume of the oil adjuvant composition.

[0096] 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.

[0097] As an example, the following method (Preparation Method A) can be used to prepare an oil adjuvant composition according to the present invention, preferably an oil adjuvant composition comprising 76% to 92% light liquid paraffin (CAS 8012-95-1), 1% to 7% sorbitan monooleate (CAS 338-43-8), 7% to 17% polyoxyethylene 10 oleyl ether (CAS 9004-98-2) and sufficient water to obtain a clear composition.

[0098] Preparation method A comprises the following steps:

[0099] i. Measure appropriate amounts of oil (preferably light liquid paraffin), sorbitan or mannitol ester (preferably sorbitan monooleate) and ethoxylated fatty alcohol (preferably polyoxyethylene 10 oleyl ether);

[0100] ii. Mix thoroughly with a suitable mixer;

[0101] iii. Add water until the mixture is clear;

[0102] iv. in a closed container at a temperature between 110°C and 130°C, for example at 121°C

[0103] autoclaving the mixture of step iii for a duration between 30 minutes and 1 hour, for example 40 minutes; or

[0104] iv. Filter the mixture from step iii through a 0.22 μm filter.

[0105] Oil-in-water adjuvant emulsion

[0106] A second object of the present invention is an oil-in-water adjuvant emulsion, the oil phase of which comprises, preferably consists of, the oil adjuvant composition according to the present invention.

[0107] In one embodiment, the oil-in-water adjuvant 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 oil-in-water adjuvant emulsion.

[0108] The aqueous solvent of the oil-in-water adjuvant emulsion can, for example, be selected from the group consisting of water, physiological solutions (such as 0.9% NaCl aqueous solution) and aqueous buffers (such as PBS (phosphate buffered saline) buffer). In some embodiments, the aqueous solvent is water.

[0109] The oil-in-water adjuvant emulsion according to the present invention can be prepared by any suitable method known in the art.

[0110] As an example, the following method (Preparation Method B) can be used to prepare the oil-in-water adjuvant emulsion according to the present invention.

[0111] Preparation method B comprises the following steps:

[0112] i') preparing the oil adjuvant composition according to the present invention, preferably by the preparation method A described in detail above;

[0113] ii') measuring under sterile conditions one part oil adjuvant composition (50% vol) and one part aqueous solvent (50% vol) (preferably sterile PBS);

[0114] iii') heating the two phases, for example at 70°C;

[0115] iv') adding the aqueous phase to the oil phase while stirring;

[0116] v') gradually cooling the emulsion while continuing to stir; and

[0117] vi') The obtained emulsion was stored between 2°C and 8°C until use.

[0118] Unless otherwise stated, in this application percentages are expressed by volume relative to the total volume of the oil-in-water adjuvant emulsion.

[0119] The present invention also relates to a diluted oil-in-water emulsion comprising an oil-in-water adjuvant emulsion according to the present invention and a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier. The diluted oil-in-water emulsion can be used in contact with an active agent (such as an antigen) or with a composition (preferably an oil-in-water emulsion) comprising such an active agent.

[0120] Oil-in-water vaccine emulsion

[0121] A third object of the present invention is an oil-in-water emulsion, preferably an oil-in-water vaccine emulsion, which comprises the oil-in-water adjuvant emulsion according to the present invention, a further pharmaceutically acceptable carrier (preferably an aqueous pharmaceutically acceptable carrier) and at least one active agent (such as an antigen).

[0122] Even though the oil-in-water emulsions of the present invention are particularly suitable for use with antigens to form vaccine emulsions, they may also present advantages when used with different active agents.

[0123] Antigens may comprise killed, attenuated, or living whole organisms; subunits or parts of organisms; recombinant vectors containing inserts having immunogenic properties; DNA fragments or segments capable of inducing an immune response when presented to a host animal; proteins, polypeptides; peptides; epitopes; haptens, or any combination thereof. Alternatively, antigens may comprise toxins or antitoxins. Antigens may be inactivated.

[0124] In some embodiments, the antigen is an antigen from a pathogen associated with a domestic pig disease, preferably selected from antigens of a virus and a bacterium associated with a domestic pig disease.

[0125] The viral antigens from viruses associated with domestic pig diseases 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 coronavirus (such as porcine epidemic diarrhea virus (PEDV)) antigens, porcine parvovirus antigens, porcine rotavirus antigens, and porcine circovirus (such as porcine circovirus type 2 (PCV2)) antigens. Preferably, the antigens from viruses associated with domestic pig diseases are porcine circovirus type 2 (PCV2) antigens, such as ORF2 PCV2 antigens.

[0126] Bacterial antigens from bacteria associated with domestic pig diseases may be selected from the group consisting of: Actinobacillus pleuropneumoniae antigens, Mycobacterium spp. antigens (preferably Mycoplasma hyopneumoniae, My hyo) antigens, Streptococcus suis antigens (preferably IgM protease antigens, more preferably IdeSsuis antigens), Lawsonia intracellularis antigens, Escherichia coli antigens, Eperythrozoonosis suis antigens, Erysipelothrix rhsiopathiae antigens, Bordetellabronchiseptica antigens, Pasteurella multocida antigens, Salmonella spp.) antigens, Haemophilus parasuis antigens, Clostridium spp. (such as Clostridium perfringens) antigens and Leptospira spp. antigens. Preferably, the antigen from a bacterium associated with domestic pig diseases is an IdeSsuis antigen, a Lawsonia intracellularis antigen or a Mycoplasma hyopneumoniae (My hyo) antigen.

[0127] The bacterial antigens from bacteria associated with domestic pig diseases can be selected from the group consisting of: Actinobacillus pleuropneumoniae antigens, Mycobacterium antigens (preferably Mycoplasma hyopneumoniae (M hyo) antigens), Escherichia coli antigens, Eperythrozoon suis antigens, Erysipelothrix rhizogenes antigens, Bordetella bronchiseptica antigens, Pasteurella multocida antigens, Salmonella antigens, Haemophilus parasuis antigens, Clostridium antigens (such as Clostridium perfringens antigens) and Leptospira antigens. Preferably, the antigens from bacteria associated with domestic pig diseases are Mycoplasma hyopneumoniae (M hyo) antigens.

[0128] In one embodiment, the at least one antigen contained in the oil-in-water vaccine emulsion according to the present invention is selected from the group consisting of: porcine circovirus type 2 (PCV2) antigens, Streptococcus suis antigens, Mycoplasma hyopneumoniae antigens, and combinations thereof. More preferably, the at least one antigen contained in the oil-in-water vaccine emulsion according to the present invention is selected from the group consisting of: porcine circovirus type 2 (PCV2) antigens, Mycoplasma hyopneumoniae antigens, and combinations thereof.

[0129] In one embodiment, the at least one antigen comprised in the oil-in-water vaccine emulsion according to the invention is a combination of porcine circovirus type 2 (PCV2) antigen (such as ORF2 PCV2 antigen), Mycoplasma hyopneumoniae antigen and Lawsonia intracellularis antigen.

[0130] The oil-in-water vaccine emulsion according to the present invention may comprise 10% to 80% of the oil-in-water adjuvant emulsion and 20% to 90% of a pharmaceutically acceptable carrier, wherein the percentages are expressed by volume relative to the total volume of the oil-in-water vaccine emulsion.

[0131] In some embodiments, the pharmaceutically acceptable carrier contained in the oil-in-water vaccine emulsion according to the present invention is the same as the aqueous solvent of the oil-in-water adjuvant emulsion. In a preferred embodiment, the pharmaceutically acceptable carrier contained in the oil-in-water vaccine emulsion according to the present invention is water.

[0132] In some embodiments, the oil-in-water vaccine emulsion according to the invention is injectable, meaning that its composition and physical properties (such as its viscosity) are suitable for injection.

[0133] The oil-in-water vaccine emulsion according to the present invention may comprise additional components, such as those classically used in the formulation of oil-in-water vaccine emulsions. Preferably, the additional components do not affect the safety of the oil-in-water vaccine emulsion, nor its stability.

[0134] The oil-in-water vaccine emulsion according to the invention may contain any additional compound suitable for increasing and / or accelerating the immune response elicited by the antigen in an animal.

[0135] In one embodiment, the oil-in-water vaccine emulsion further comprises an immunostimulant. The immunostimulant can be selected from the group consisting of dimethyloctadecyl ammonium (DDA), saponin, vitamin E, and an immunostimulatory polymer (such as a polyacrylic acid polymer). The amount of the immunostimulant can be between 0.5% and 5%, preferably about 1%, of the oil-in-water vaccine emulsion, based on the weight of the total volume of the oil-in-water vaccine emulsion.

[0136] Uses of oil-in-water vaccine emulsions

[0137] A final object of the present invention is an oil-in-water vaccine emulsion according to the present invention for use as a medicament, preferably as a vaccine. The oil-in-water vaccine emulsion according to the present invention is preferably used to treat porcine circovirus disease type 2, Streptococcus suis infections such as porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death and / or porcine enzootic pneumonia, more preferably porcine circovirus disease type 2 and / or porcine enzootic pneumonia.

[0138] Preferably, the oil-in-water vaccine emulsion according to the invention is used in non-human mammals, more preferably in porcine mammals, in particular in pigs or piglets.

[0139] In some embodiments, 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 enzootic pneumonia is a prophylactic or preventative treatment.

[0140] The oil-in-water vaccine emulsion according to the present invention can be administered to animals by any suitable route. For example, the oil-in-water vaccine emulsion according to the present invention can be administered to animals via transdermal administration, via mucosal administration, via oral administration, or via injection, such as intradermal injection, intramuscular injection, subcutaneous injection, intravenous injection, or intraperitoneal injection. Preferably, the oil-in-water vaccine emulsion according to the present invention is administered to animals by injection.

[0141] Those skilled in the art will be able to adjust the dosage and / or frequency of administration of the active agent, preferably an antigen, to be administered to an animal, depending on, inter alia, the age, sex, weight, species and condition of the animal, the route of administration and the nature of the active agent. Administration can be performed in one or more doses, preferably between one and three doses. In a preferred embodiment, administration is performed in a single dose or two doses.

[0142] Preferably, the oil-in-water vaccine emulsion according to the present invention is sterile.

[0143] The present invention also relates to a method for treating porcine circovirus type 2 disease, porcine Streptococcus suis infection such as porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death and / or porcine enzootic pneumonia, which comprises administering an effective dose of the oil-in-water vaccine emulsion according to the present invention to a non-human mammal, preferably a porcine mammal.

[0144] The present invention also relates to the use of the oil-in-water vaccine emulsion according to the present invention for the preparation of a medicament, preferably a vaccine, in particular a vaccine for the treatment of porcine circovirus disease type 2, Streptococcus suis infection such as porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death and / or porcine enzootic pneumonia.

[0145] The present invention also relates to a method for inducing an immune response in an animal, preferably a pig or a piglet, which method comprises administering to the animal the oil-in-water vaccine emulsion according to the invention.

[0146] The present invention also relates to a method for immunizing an animal against infection with a pathogenic organism (e.g., an infection with a virus, bacteria, fungus, or protozoan parasite) or for preventing or alleviating the symptoms caused by the infection. Preferably, the pathogenic organism is porcine circovirus type 2, Streptococcus suis bacteria, or Mycoplasma hyopneumoniae bacteria, more preferably porcine circovirus type 2 or Mycoplasma hyopneumoniae bacteria.

[0147] Method for producing oil-in-water vaccine emulsion

[0148] The oil-in-water vaccine emulsion according to the present invention can be prepared by any suitable method known in the art. In one embodiment, the oil-in-water vaccine emulsion is obtained by diluting an oil-in-water adjuvant emulsion with a solution of an antigen in a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier.

[0149] The active agent, preferably an antigen, can be incorporated during the formation of the emulsion, or incorporated into the emulsion at a later time. In some embodiments, the active agent is incorporated into the oil-in-water vaccine emulsion just prior to use.

[0150] The size of the droplets in the emulsion may range from about 10 nm to about 5 μm. In one embodiment, the average size of the droplets in the emulsion is about 60 nm.

[0151] Reagent test kit

[0152] The present invention also relates to a kit comprising:

[0153] - an active agent as defined above, preferably an antigen, and

[0154] - an oil-in-water adjuvant emulsion or a diluted oil-in-water adjuvant emulsion according to the invention.

[0155] The kit may further comprise a pharmaceutically acceptable carrier, preferably an aqueous pharmaceutically acceptable carrier, and / or instructions for a method for making the oil-in-water vaccine emulsion according to the present invention.

[0156] As used herein, the term "comprising" and its derivatives must be understood as not limiting the presence of other components. In some embodiments, the term "comprising" and its derivatives may be understood as being more restrictive and meaning "consisting essentially of" or even "consisting of."

[0157] The present invention will be further described in detail in the following examples, which are not intended to limit the scope of the invention, which is defined by the appended claims.

[0158] Example

[0159] Example 1: Composition according to the invention

[0160] The following oil adjuvant compositions were prepared by conventional methods: Composition A is an oil adjuvant composition according to the present invention, and Composition B is a comparative composition.

[0161]

[0162] Marcol TM 52 is a purified mixture of liquid saturated hydrocarbons.

[0163] SMO V is a nonionic lipophilic surfactant comprising sorbitan oleate.

[0164] BRIJ O10 is a nonionic hydrophilic surfactant containing polyoxyethylene (10) oleyl ether.

[0165] Simulsol 2599 PHA VG is a hydrophilic nonionic surfactant containing polyethylene glycol oleate (10).

[0166] Example 2: Stability Study

[0167] Formulated with different adjuvants The vaccine samples were subjected to a comparative stability study and stored for one month at 2° C. to 8° C. The antigen contained in the emulsions studied was a Mycoplasma hyopneumoniae (M HYO) antigen.

[0168] By replacing the oil-in-water emulsion with one of the following Vaccine adjuvants were used to obtain each tested formulation:

[0169] - Composition A,

[0170] - Comparative composition B,

[0171] - the comparative adjuvant composition LR3 oil phase disclosed in prior art WO2006 / 113373, and

[0172] - Comparative adjuvant composition LR4 oil phase disclosed in prior art WO2006 / 113373.

[0173] The oil phase of LR3 contains 92 (polyoxyethylene (2) oleyl ether), 96 (polyoxyethylene (10) oleyl ether), paraffin oil ( 82) and preservatives.

[0174] The oil phase of LR4 contains 92 (polyoxyethylene (2) oleyl ether), N5 (polyoxyethylene (5) oleyl ether), paraffin oil ( 82) and preservatives.

[0175] Figure 1 The visual appearance of the investigated compositions after 1 day (a), 1 week (b) and 1 month (c) is presented.

[0176] (a) After 1 day, the composition appeared homogeneous overall, with only slight creaming observed for the LR3-based vaccine composition.

[0177] (b) After 1 week, the same creaming is still visible on the LR3-based vaccine composition. The vaccine composition based on comparative adjuvant composition B has separated into two phases.

[0178] (c) After 1 month, the LR3-based composition and the comparative composition B have almost completely separated into 2 phases. A slight creaming is now visible on top of the LR4-based composition.

[0179] At 1 day, 1 week and 1 month, the size distribution of the particles in each composition was measured by laser diffraction using a Mastersizer 3000 particle size analyzer. The results are presented in Table 1 below.

[0180]

[0181] Table 1: Size distribution of each composition after 1 day, 1 week and 1 month

[0182] Figures 2 to 5 The evolution of the size distribution from 1 day to 1 week is presented for each composition.

[0183] 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.

[0184] Figure 3 Comparative composition B: After one week at 4°C, the particle size increased from 60 nm to 100 μm. The size correlated well with the visual aspect. The emulsion broke after one week, and the breaking worsened after one month.

[0185] Figure 4 Composition LR3: Two particle populations are present, with the larger one larger than 1 μm. Size closely correlates with visual characteristics. Size distribution deteriorates within one month.

[0186] Figure 5 : Composition LR4: Particles with a size greater than 1 μm are present, which makes the particle size distribution non-uniform. This does not predict good stability over time. The non-uniform size distribution persists for more than one month.

[0187] In summary, the vaccine composition based on composition A according to the present invention is the only oil-in-water emulsion whose particle size and visual appearance are completely stable after one month of storage at 4 degrees. This vaccine composition is expected to be stable at 4 degrees for 1 to 2 years.

[0188] Example 3: Safety study of the Hyogen vaccine according to the present invention

[0189] The field study conducted on three farms in different provinces of China evaluated the The safety of vaccines.

[0190] The pigs' reactions to the formulations were assessed and scored according to the severity scoring scale detailed in Table 2 below.

[0191]

[0192] Table 2: Rating Scale for Safety Studies Table 3 below presents the safety results of the formulations.

[0193]

[0194]

[0195] Table 3: Results of safety studies

[0196] Out of 665 studied pigs, none showed a reaction after administration of the vaccine emulsion according to the invention.

[0197] This demonstrates that the vaccine emulsion according to the present invention is highly safe, especially when used together with M. hyopneumoniae antigen.

[0198] Example 4 Safety study of the PCV2 vaccine according to the present invention

[0199] The safety of a PCV2 vaccine formulated with an oil-in-water emulsion adjuvant composition according to the present invention (Composition A of Example 1) was evaluated in a field study.

[0200] A total of 20 2- to 3-week-old PRRS-negative PCV2-negative piglets were used to investigate the overdose safety of three batches of PCV2 oil-in-water vaccine emulsions according to the present invention compared to a mock vaccine. Prior to vaccination, the animals were randomly assigned to four groups A to D (5 animals each), with three groups (Groups A to C) receiving 4 mL of the oil-in-water vaccine emulsion according to the present invention and one group (Group D) receiving an equivalent dose of the mock vaccine. To investigate the safety profile, body temperature was collected daily from three days before vaccination to six days after vaccination. In addition, temperatures were collected immediately before vaccination and four hours after vaccination. Clinical observations were performed daily, noting abnormal local and general reactions from day one to day 14 after vaccination.

[0201] The results showed that a transient increase in body temperature was observed within the first day after vaccination, and all animals subsequently recovered. The maximum temperature increase in each group did not exceed 1.5°C, with an average value between 0.8°C and 0.9°C. Figure 6Table 4 below presents the temperature data for each group. No significant differences were found between the groups. Except for the transient temperature increase, no overall or local adverse reactions were observed during the study. The safety of the three batches of oil-in-water vaccine emulsions according to the present invention was confirmed.

[0202] For the ANOVA test, the maximum temperature increase was calculated for each animal.No significant differences were found between the groups (p=0.701).

[0203]

[0204]

[0205] Table 4: Summary data of temperature increase monitoring in each group

[0206] Example 5: Efficacy of the adjuvant composition according to the present invention

[0207] A comparative efficacy study was conducted in piglets, testing PCV2 vaccine formulations containing different adjuvants.

[0208] 80 purebred (largewhite, landrace, Genesus) piglets were divided into 4 groups of 20 animals each.

[0209] - Group A was injected with the reference product Circoflex (porcine circovirus type 2 ORF2 protein, adjuvanted with carbomer) and challenged with PCV2 strain SEVC1702 21 days after vaccination;

[0210] - Group B was injected with a placebo (an oil-in-water emulsion adjuvant of the invention obtained from the oil composition Composition A of Example 1 + phosphate buffered saline (PBS)) and challenged with PCV2 strain SEVC1702 21 days after vaccination;

[0211] - Group C was injected with the PCV2 vaccine according to the present invention comprising the porcine circovirus type 2 ORF2 protein and an oil-in-water emulsion adjuvant and challenged with the PCV2 strain SEVC1702 21 days after vaccination

[0212] - Group D was injected with the PCV2 vaccine according to the present invention comprising the porcine circovirus type 2 ORF2 protein and an oil-in-water emulsion adjuvant and challenged with the PCV2 strain SEVC1702 14 days after vaccination.

[0213] Piglets from each group were necropsied 28 days after challenge (day 49 for Groups A, B, and C, and day 42 for Group D).

[0214] Figure 7 A. Figure 7 B and Figure 7 C shows the viremia of each group on day 14, day 21, and day 28 after challenge, respectively. Compared with placebo (Group B), the viremia of each group was significantly reduced.

[0215] The adjuvants according to the invention (Group C and Group D) had significantly lower viremia than the reference product Circoflex.

[0216] Nasal swabs were collected for qPCR on days 7, 14, 21, and 28 after the experimental intervention (tapping). The test results were logarithmically transformed and analyzed.

[0217] Figure 8 A. Figure 8 B and Figure 8 C shows the logarithm of nasal swabs on days 7, 14, 21, and 28. Group D secreted significantly less virus through the nasal passages than Group A.

[0218] Anal swabs were collected for qPCR assays on days 7, 14, 21, and 28 after the experimental intervention. The test results were logarithmically transformed and analyzed.

[0219] Figure 9 A. Figure 9 B. Figure 9 C and Figure 9 D shows the logarithm of nasal swabs on days 7, 14, 21, and 28, respectively. On days 14, 21, and 28 after challenge, virus excretion via feces in group D was significantly lower than that in group A.

[0220] After attacking the 28th day (DPC28) to all animals being euthanized, tonsils, mediastinal lymph nodes, mesenteric lymph nodes, inguinal lymph nodes and lungs were collected by dissection. The small pieces of every kind of tissue of about rice size were cut, and the total DNA of tissue was directly extracted with genome extraction kit. Subsequently, the quantitative of viral nucleic acid was carried out with qPCR test kit. Test data were logarithmically converted, and the Kruskal-Wallis test was used to analyze the difference between the groups, and then the Wilcoxon rank sum test was used to carry out the pairwise comparison of the difference between the groups.

[0221] Figure 10 A. Figure 10 B. Figure 10 C. Figure 10 D and Figure 10 E shows the log quantification of virus in inguinal lymph nodes, mesenteric lymph nodes, lung, mediastinal lymph nodes, and tonsillar lymph nodes, respectively, on day 28.

[0222] Compared with the placebo group (Group B), Groups A and C were able to significantly suppress the viral load in lymph node tissue, lungs and tonsils. Among the groups, Groups C and D suppressed the viral load in lymph nodes and tonsils better than Group A, and the differences were significant.

[0223] Finally, the PCV2-specific antibody levels were assessed using a commercially available ELISA kit.

[0224] The results showed that all vaccine-immunized groups, except the placebo group, induced the production of specific antibodies in the animals. Animals in the placebo group did not show any antibody turnover before the experimental intervention. Compared to the placebo group, higher antibody levels were stimulated in the immunized animals within a shorter period of time after the experimental intervention.

[0225] in conclusion :

[0226] The results showed that both PCV2 vaccines containing the adjuvant of the present invention significantly reduced viral load in the blood (i.e., the amount of virus excreted through the nose and anus), as well as PCV2 virus levels in the lymph nodes, tonsils, and lungs, compared to placebo. Compared to the reference product, both vaccine batches containing the adjuvant of the present invention used in this study significantly increased the inhibition of nasal and fecal elimination, as well as the inhibition of viral load in lymph nodes and tonsils, demonstrating that vaccines containing the adjuvant of the present invention provide superior protection against genotype 2d PCV2 strains.

[0227] The clinical safety of both laboratory vaccine batches was also observed in the study. After immunization, none of the vaccine batches produced severe acute reactions, and no local adverse reactions at the injection site were observed.

[0228] Example 6: Vaccine composition comprising IdeSsuis antigen and an adjuvant composition according to the present invention

[0229] Streptococcus suis (S. suis) is a major swine pathogen that causes meningitis, arthritis, and several other pathologies. The immunoglobulin M-degrading enzyme IdeS suis of S. suis is a highly protective antigen against S. suis infection.

[0230] One hundred piglets aged 7 to 10 weeks were divided into 5 groups.

[0231] Piglets from Groups 1 to 5 were vaccinated twice (on D0 and D21) with the recombinant IdeSsuis IgM protease proteins listed 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 the oil composition Composition A of Example 1 and phosphate-buffered saline (PBS).

[0232]

[0233] Local and systemic reactions were evaluated on D7, D10, D14, D24, D27, D31, and D35. Clinical health was checked daily.

[0234] Blood samples were collected from all animals on study days -1, 21, and 35. The study was terminated on study day 35.

[0235] result

[0236] 1. Local tolerance

[0237] Local tolerance (by palpation) was observed on D7, D10, D14, D24, D27, D31 and D35.

[0238] The percentage of local reactions per day is expressed in Figure 11 middle.

[0239] Groups 3 and 4 vaccinated with the adjuvant emulsion according to the present invention showed better or equal tolerability than the other groups on any day.

[0240] Between D24 and D31, Groups 3 and 4 showed better local response results than any other groups.

[0241] Groups 1 and 2 showed important differences in local reactions depending on the adjuvant concentration. With the adjuvants of the invention, such differences were not visible.

[0242] In conclusion, the adjuvants of the present invention showed fewer local reactions than the reference adjuvants and displayed a better safety profile due to the specific formulation used.

[0243] 2. Antibody titer

[0244] Antibody titers against antigens in vaccine preparations were determined from serum samples using ELISA. The results are presented in Figure 12 B0 corresponds to before the first vaccination, B1 corresponds to between the first and second vaccinations, and B2 corresponds to 2 weeks after the second vaccination.

[0245] result

[0246] The production of vaccines against S. suis using the adjuvants of the present invention showed similar antibody development as using the reference adjuvant.

Claims

1. An oil adjuvant composition, comprising: - at least one oil, - at least one nonionic lipophilic surfactant, said at least one nonionic lipophilic surfactant being a sorbitan ester or a mannitol ester, and - at least one nonionic hydrophilic surfactant, said at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol.

2. The oil adjuvant composition according to 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. The oil adjuvant composition according to claim 2, wherein the at least one mineral oil is selected from the group consisting of paraffinic oils such as isoparaffinic oils and / or naphthenic oils, pristane and any mixtures thereof.

4. The oil adjuvant composition according to any one of claims 1 to 3, wherein each sorbitan ester or anhydromannitol ester is a sorbitan ester, wherein 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, more preferably sorbitan monooleate.

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

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, eicosanyl glycol stearyl ether, triethylene glycol oleyl ether and any mixture thereof, preferably polyoxyethylene oleyl ether, especially polyoxyethylene (10) oleyl ether.

7. The oil adjuvant composition according to any one of claims 1 to 6, wherein the oil adjuvant composition comprises: -76% to 92% oil, - 1% to 7% of at least one nonionic lipophilic surfactant, said at least one nonionic lipophilic surfactant being a sorbitan ester or a mannitol ester, and - 7% to 17% of at least one nonionic hydrophilic surfactant, said at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol, The percentages are expressed by weight relative to the total volume of the oil adjuvant composition.

8. The oil adjuvant composition according to claim 7, wherein the oil adjuvant composition comprises: - about 82% of oil, preferably mineral oil, - about 4% of at least one nonionic lipophilic surfactant, said nonionic lipophilic surfactant being a sorbitan ester or anhydromannitol ester, preferably sorbitan monooleate, and - about 14% of at least one nonionic hydrophilic surfactant, said at least one nonionic hydrophilic surfactant being an ethoxylated fatty alcohol, preferably polyoxyethylene oleyl ether, The percentages are expressed by weight relative to the total volume of the oil adjuvant composition.

9. An oil-in-water adjuvant emulsion comprising 40% to 70% of the 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 the aqueous solvent, The percentages are expressed by volume relative to the total volume of the oil-in-water adjuvant emulsion.

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

11. The oil-in-water vaccine emulsion according to claim 10, wherein the oil-in-water vaccine emulsion comprises 10% to 80% of the oil-in-water adjuvant emulsion and 20% to 90% of a pharmaceutically acceptable carrier, The percentages are expressed in volume relative to the total volume of the oil-in-water vaccine emulsion.

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

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

14. The oil-in-water vaccine emulsion according to any one of claims 10 to 12, which is used to treat porcine circovirus disease type 2, Streptococcus suis infection such as porcine meningitis, porcine septicemia, porcine arthritis, porcine endocarditis or porcine sudden death and / or porcine enzootic pneumonia.

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

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