Stable emulsion of antigen

Abstract

The present invention relates to the field of vaccinology, more particularly to the field of veterinary vaccinology. In particular, the present invention relates to an adjuvant composition comprising an emulsion of water, tocopherol or a pharma- ceutically acceptable ester thereof, and polyethoxyethylene cetostearyl ether as an emulsifier. This adjuvant composition can be used to formulate a vaccine, in particular an emulsion vaccine, comprising a bacterial, parasitic or viral antigen. The resulting vaccine composition can be used in a method for protecting a human or animal subject from infection and / or disease caused by a pathogen, in particular caused by a bacterium, a parasite or a virus. The present invention further relates to a method for producing such an adjuvant composition and a method for producing such a vaccine composition.

Description

[Technical field]

[0001] The present invention relates to the field of vaccinology, more particularly to the field of veterinary vaccinology. In particular, the present invention relates to an adjuvant composition comprising an emulsion of water, tocopherol or a pharma- ceutically acceptable ester thereof, and polyethoxyethylene cetostearyl ether as an emulsifier. This adjuvant composition can be used to formulate a vaccine, in particular an emulsion vaccine, comprising a bacterial, parasitic and / or viral antigen. The resulting vaccine composition can be used in a method for protecting a human or animal subject from infection and / or disease caused by a pathogen, in particular caused by a bacterium, a parasite or a virus. The present invention further relates to a method for producing such an adjuvant composition and a method for producing such a vaccine composition. [Background technology]

[0002] Introduction Infections by pathogenic bacteria, parasites or viruses and their resulting diseases have been known since the beginning of humanity and affect both humans and animals with often serious consequences for health and well-being.

[0003] Since the middle of the 20th century, bacterial infections can be effectively treated with antibiotic drugs, but the accumulation of resistance is a constant threat. The use of antibiotics in animal husbandry in the agricultural sector is a special situation. On the one hand, treatment is urgently needed due to the high infection pressure resulting from the manner and conditions in which animals are often placed. On the other hand, the generalized non-therapeutic use of antibiotics, for example in animal feed, is now recognized as one of the causes of the increase in bacterial resistance, which is also relevant for human health. As a result, such generalized use of antibiotics is being phased out in an increasing number of countries.

[0004] A similar situation applies to the prophylactic use of antiparasitic drugs.

[0005] In the case of viral infections, the situation is even more difficult, as the use of antiviral drugs is often too expensive for veterinary purposes.

[0006] Next to improved farm management, the best alternative to combat pathogenic infections and diseases in a sustainable way is vaccination.Bacterial, parasitic and viral vaccines, both for humans and animals, have been known for over a century and are available in many forms.Such vaccines may be live, i.e., contain replicating (attenuated) bacteria, parasites or viruses, or non-live, i.e., contain inactivated bacteria, parasites or viruses, or one or more of their components.

[0007] Vaccines that contain non-live (i.e., non-replicating) antigens often require an adjuvant to provide immune stimulation against the non-live antigen. In veterinary vaccines, a wide variety of compounds can be used as adjuvants, including mineral oils such as Bayol™ or Markol™, Montanide™ or paraffin oil; non-mineral oils such as squalene, squalane, or vegetable oils such as ethyl oleate; aluminum salts such as aluminum hydroxide or aluminum phosphate; peptides such as dimethylglycine or tuftsin; bacterial cell wall components such as lipid A and muramyl dipeptide; (synthetic) polymers such as pluronics, dextran, carbomer, pyran or saponin; cytokines; and stimulators of toll-like receptors, such as immunostimulatory oligodeoxynucleotides containing unmethylated CpG groups.

[0008] To facilitate administration and to enhance the adjuvant effect, oil adjuvants can be emulsified with antigens in an aqueous phase to form emulsions, which can be used in the preparation of vaccines. In such emulsions, one liquid phase is dispersed in another, typically as a water-in-oil (W / O) or oil-in-water (O / W) emulsion. The choice of one or the other type of emulsion can be based, for example, on the type of immune response desired.

[0009] Creating and maintaining such emulsions requires the input of both mechanical and chemical energy, with separate liquids being mixed in suitable equipment using specific levels of shear, pressure, and temperature to disperse one phase into another. The chemical energy input is provided by the use of emulsifiers (also surfactants), which stabilize the dispersed phase by taking up a position at the water-oil interface. Vaccine emulsions can be composed of one or more adjuvants with one or more emulsifiers.

[0010] A large number of emulsifying agents are available for use in emulsion vaccines, and more are constantly being developed.

[0011] Examples of adjuvant and emulsifier combinations used in commercial veterinary vaccines include Amphigen® (Zoetis), which contains light mineral oil with lecithin as the emulsifier; Xsolve® (formerly known as Microsol-Diluvac Forte®, MSD Animal health), which contains a combination of the adjuvant light mineral oil and vitamin E-acetate with the emulsifier Tween® 80 (polysorbate 80 or polyoxyethylene sorbitan monooleate); MetaStim® (Zoetis), which contains squalane, Pluronic® (a nonionic triblock copolymer of blocks of polyoxyethylene and polyoxypropylene), and Tween 80; Fortasol® (also known as Diluvac), which contains vitamin E acetate and Tween 80. Forte®); SP-oil™ (Pluronic™, squalane and Tween); AS03™ (GSK) (containing 2.1% w / v squalene and 2.4% w / v vitamin E with 1.0% w / v Tween 80 as an emulsifier).

[0012] Emulsions for use as vaccines should be stable rather than "break", meaning that the type, size and number of droplets of the dispersed phase should not change too much over time, which could eventually lead to a decrease in dispersion and ultimately complete phase separation.

[0013] Maintaining emulsion stability is important to ensure the use and efficacy of an emulsion vaccine during its registered shelf life. Gravitational effects over time, such as settling or creaming, are harmless and can be easily reversed by shaking by hand. However, the actual breakdown of the emulsion is irreversible, and the resulting suboptimal distribution of phases can lead to incorrect dosing, safety issues, and can affect the immunological potency of the vaccine antigen(s).

[0014] In addition to the overriding requirement that a vaccine be safe and effective, there are some special requirements for vaccines used in animal husbandry. These relate to aspects of ease of use, especially cost, since the production of animal proteins is typically a high-volume, low-margin business. For these reasons, veterinary vaccines are often directed against several diseases or pathogens at once, by containing several different antigens in a single vaccine formulation. This is advantageous, since it not only reduces the stress of the target animal by preventing the need for repeated treatments, but also reduces the labor costs for administration.

[0015] Oil-adjuvanted emulsion vaccines using bacterial, parasitic and / or viral antigens are currently commercially available for many pathogens and for all major animal target groups: pigs, cattle, sheep, poultry, companion animals (e.g., cats, dogs, horses) and fish.

[0016] However, not all desired selections of antigens can be formulated into stable emulsion vaccines. This is a further consequence of the need to make vaccines affordable for agricultural use, namely, that such vaccines usually do not contain expensive and pure components (e.g. recombinantly expressed subunits) and cannot be produced using sophisticated purification techniques (e.g. column chromatography). In practice, this means that the non-live antigens contained in such vaccines are typically of relatively impure and somewhat undefined composition. This is especially true when compared to more purified vaccine antigens in vaccine products with higher market value, such as those for use in companion animals (cats, dogs, and horses) or for use in humans. Alternatively, the specific protective antigen may not be known, so crude antigen preparations are the only way to include the required antigen.

[0017] Thus, non-live bacterial, parasitic or viral vaccines for agricultural use typically contain little purified antigens, e.g. derived from inactivated bacterial, parasitic or viral cultures, or extracts or fractions of such cultures. Such rather crude antigens may be based on inactivated bacteria or parasites or inactivated viruses, possibly washed or concentrated once, or on bacterial, parasitic or viral fractions, such as lysed or destroyed bacteria, parasites or viruses. As a result, these crude antigen preparations may contain undefined or unintended impurities that may affect the safety, efficacy or stability of the (combination) vaccine. For this reason, as part of their development process, vaccines must undergo rigorous testing for safety, efficacy and stability before they can be marketed as commercial products, receiving marketing approval from governmental or regulatory authorities.

[0018] Unspecified components of non-live antigen preparations are not necessarily desirable because they may act as additional adjuvants and thus provide a specific boost to the immune response. Potential interfering factors of biological nature are also expected to be inactivated during the preparation of non-live bacterial, parasitic or viral antigens. Nevertheless, undesirable effects on vaccine safety, efficacy or stability can be observed during the development of emulsion-based vaccines that contain crude antigen preparations.

[0019] Another obstacle to overcome in the creation of adjuvanted vaccines is to prevent interactions between various vaccine components that may adversely affect immune response or vaccine safety or stability. Such interactions may occur, for example, between antigens themselves, for example, because some are very crude products. Also, adjuvants may interfere with or damage vaccine antigens. Therefore, it is difficult to develop adjuvanted vaccines that induce effective immune responses against one or more antigens, especially against complex combinations that involve antigens from multiple pathogens.

[0020] Furthermore, adjuvanted vaccines should be safe when used in animals, i.e., they should not cause significant side reactions such as fever, local swelling, loss of appetite, etc. More practical properties are also relevant, and emulsion vaccines should ideally be economical to produce, sufficiently stable during formulation and storage, and allow for methods of potency testing of each antigen in the presence of other antigens.

[0021] Some of the most prominent diseases affecting young pigs are caused by bacteria such as Mycoplasma hyopneumoniae and Lawsonia intracellularis, and viruses such as porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV).

[0022] Mycoplasma hyopneumoniae (Mhyo) is the primary agent causing epizootic pneumonia, a chronic respiratory disease of pigs occurring worldwide. Young piglets are particularly susceptible to this highly contagious disease. The bacterium is relatively small, lacks a cell wall, and belongs to the Mollicutes genus. These bacteria live a parasitic lifestyle on or within the host cells.

[0023] Mhyo-derived lung diseases are mainly immune-mediated pathologies leading to consolidative pneumonia. The bacteria colonize and damage the ciliated epithelium of the lungs, resulting in the loss of ciliary activity. Depending on housing conditions and environmental stresses, the most problematic consequence of the disease is the predisposition to different secondary infections of the pig respiratory system, for example by other bacterial and viral pathogens. This leads to the so-called Porcine Respiratory Disease Syndrome (PRDC), which presents with severe lung lesions. Next to the discomfort of the animals, enzootic pneumonia and PRDC cause significant economic losses to the pig industry due to reduced performance in growth rate and feed conversion, as well as through the costs for veterinary care and antibiotic use.

[0024] Lawsonia intracellularis causes proliferative enteritis, also known as ileitis, a common intestinal disease of postweaned pigs worldwide. The characteristic lesion is the proliferation of immature enterocytes in the ileal intestinal crypts, which contain the causative bacteria. Exclusion of the bacteria from the enterocytes results in the resolution of the associated proliferative lesions. Histological lesions can be confirmed as Lawsonia positive by visualization of 1.5-2.5 μm long Vibrio-like shaped bacteria within enterocytes as well as intestinal macrophages. The bacteria can be detected by PCR in clinical or subclinical cases. Clinical cases are usually present during the grow-finish stage.

[0025] The L. intracellularis bacterium is an obligate intracellular non-motile Gram-negative rod from the family Desulfovibrionaceae.

[0026] Porcine circovirus type 2 (PCV2) is associated with the postweaning multisystemic wasting syndrome (PMWS) observed in young pigs. Clinical signs and pathology were described in 1996 and include progressive weakness, dyspnea, tachypnea, and occasionally icterus and jaundice. This novel agent was designated PCV2 as distinct from known PCVs that are natural contaminants of PK-15 cells.

[0027] PCV2 is a very small, non-enveloped virus of the genus Circovirus. It contains a circular, single-stranded DNA genome with two major genes. The ORF2 gene encodes the viral capsid protein of approximately 233 amino acids. Recombinantly expressed PCV2 ORF2 protein forms virus-like particles that are highly effective as subunit vaccines.

[0028] Porcine reproductive and respiratory syndrome virus (PRRSV) was first described in 1987 and became a pandemic in the early 1990s. It is a small enveloped RNA virus of the Arterivirus genus that contains a single-stranded positive-sense RNA genome. The virus causes significant losses in the pig industry due to reproductive failure and growth retardation. Like Mhyo, PRRSV plays an important role in multifactorial PRDC. Clinical signs are abortion and stillborn or mummified fetuses, as well as cyanosis of the ears and vulva. In newborn pigs, the virus causes respiratory distress and increased susceptibility to secondary respiratory infections such as Glaesser's disease (caused by Haemophilus parasuis). However, subclinical infections are also common. The virus is highly diverse, and after the European (type 1) and North American (type 2) types, there is now a third genotype, a highly pathogenic variant that emerged in China in 2000 and is now causing severe disease in pigs in Asia.

[0029] There are commercially available vaccines for each of these pathogens.

[0030] To limit stress to the animals and expense and effort for breeders, some swine vaccines are prepared as combination vaccines.

[0031] WO 2018 / 115435 describes a combination vaccine for pigs containing a non-live antigen from PCV2 and live PRRSV. The vaccine is an oil-in-water emulsion containing squalane and vitamin E-acetate.

[0032] WO 2021 / 048338 describes a combination vaccine for pigs to protect against pathogenic infection with PCV2 and Mhyo, comprising a non-live immunogen for PCV2 and a non-live immunogen for Mhyo. The vaccine is an oil-in-water emulsion containing squalane, vitamin E-acetate and silica.

[0033] There remains interest in the field of veterinary vaccinology for effective vaccines, particularly those that are safe and effective across species.

[0034] FMD is a highly contagious and destructive disease of cloven-hoofed animals worldwide that has had a major economic impact. The disease is characterized by fever, lameness, lymphopenia and the appearance of vesicular lesions on the mouth, tongue, nose, feet and teats and is controlled by inhibiting the movement of susceptible animals, slaughtering infected animals and vaccination. Most countries immunize animals with inactivated whole virus vaccines to control the spread of foot-and-mouth disease virus (FMDV). However, the vaccine technology used is fraught with efficacy concerns, especially with regard to cell-mediated immunity. To address these concerns, alternative vaccines such as recombinant protein and peptide vaccines, empty capsid vaccines and genetically engineered inactivated vaccines are currently being developed. Several types of vaccine adjuvants have been studied for their efficacy in promoting immune responses to FMDV vaccines. These adjuvants include mineral oil, saponin (Quil-A), Toll-like receptor (TLR) ligands, cytokines, liposomes, etc. (Y. Cao, 2014, Expert Rev. Vaccines, vol. 13, p. 1377-1385).

[0035] To address this problem, much effort is currently being put into the development of effective vaccines by combining the application of protective antigens with the search for effective adjuvants that maximize immunogenicity for the desired immune response.

[0036] Cao (supra) provides a review of the mechanisms of action and immune stimulatory effects of both traditional and currently under development adjuvants for FMD vaccines. Below, we briefly describe some of the most promising approaches.

[0037] Conventional FMD inactivated whole virus vaccines are often formulated with aqueous Al(OH) 3 and saponin, or oil adjuvants. Of these, Al(OH) 3 / Saponin-based vaccines are not ideal for use in pigs as they provide low protective efficacy in this species.

[0038] In contrast, W / O / W emulsion vaccines based on Montanide ISA-206 oil adjuvant (a mineral oil-based adjuvant containing esters of octadecenoic acid and anhydromannitol in an oily solution) containing inactivated antigens are preferred for FMD prevention because they can be used to protect all susceptible species and are ideal for emergency vaccination. Oil-adjuvanted vaccines also contain Al(OH) 3 It produces a higher and more durable immune response than adjuvanted vaccines. A newly developed mineral oil-based adjuvant, Montanide ISA-201, has been suggested as an alternative because it appears to induce earlier and higher neutralizing antibody responses, higher cell-mediated immunity and protective efficacy in cattle compared to ISA-206.

[0039] The real challenge for FMD vaccines is to find adjuvants that work across species. For ruminants, most adjuvanted vaccines use W / O emulsions, and for pigs, typically O / W emulsions. The current gold standard in FMDV vaccines is a W / O / W emulsion vaccine based on Montanide ISA-206. However, it only offers a partial and complex solution to the problem of cross-species FMDV vaccine composition, and as "double emulsions", or more precisely as reversible emulsions, these have several drawbacks. An important one is the fact that the emulsion inverts above 32°C, which means that the vaccine should be kept refrigerated as much as possible and temperatures above 32°C should be avoided. This can be a challenge in countries where "cold chain" logistics cannot be maintained all the time. Another drawback for use is that this emulsion is difficult to inject (low injectability) due to its composition. A further concern is the susceptibility of the formulation to high antigen loads that can occur with vaccines containing multiple FMDV antigens. It may be necessary to cover all strains circulating in the field, but has been observed to disrupt multivalent FMDV vaccine compositions based on Montanide ISA-206 emulsion.

[0040] As a further alternative, immune stimulating complexes (ISCOMs) composed of saponins (such as Quil-A), cholesterol, phospholipids and antigens have been described. ISCOMs have been shown to induce high titer, long-lasting antibodies as well as strong helper and cytotoxic T lymphocyte responses. ISCOMs have been described as adjuvants for FMD recombinant protein (the C-terminal half of the VP1 protein) vaccines. It has been found that a combination of recombinant protein in ISCOMs with Montanide ISA-206 can achieve early protective titers and longer-lasting immunity in guinea pigs, but it remains to be seen whether FMD vaccines formulated in ISCOMs will be as successful in the target species. Also, the technology is rather expensive.

[0041] The adjuvant effect of synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODN) on FMD vaccine was also evaluated. This study demonstrated that the combination of CpG ODN with ISA-206 could promote recombinant FMDV vaccine A7 (containing multiple B and T cell epitopes) to induce vigorous and long-lasting specific antibody responses in mice and cattle. However, CpG combined with FMD inactivated vaccine did not promote protection (Cao, supra). (Alves et al., 2009, Pigs. Clin. Vaccine Immunol., vol. 16, p. 1151-1157).

[0042] In view of the above, to overcome the shortcomings of currently available adjuvants in FMDV emulsion vaccines, there is an ongoing need for FMD vaccine adjuvants suitable to induce desired immunological enhancements, such as strong and long-lasting humoral and cellular immunity, while adhering to the general requirements of safety, stability and ease of use.

[0043] However, because vaccine development is a highly empirical process and different types of responses are required to protect against FMDV, there is little basis to conclude that one particular adjuvant will be significantly more useful than other available alternatives (Cao, supra, p. 1381 right column).

[0044] In view of the above, there is a continuing need in the field of veterinary vaccinology to provide affordable methods and materials that enable the formulation of safe, stable and effective adjuvanted vaccines containing bacterial, parasitic and / or viral antigens. [Prior art documents] [Patent documents]

[0045] [Patent Document 1] International Publication No. 2018 / 115435 [Patent Document 2] International Publication No. 2021 / 048338 [Non-patent literature]

[0046] [Non-Patent Document 1] Y.Cao,2014,Expert Rev.Vaccines,vol.13,p.1377-1385 [Non-Patent Document 2] Alves et al.,2009,Pigs.Clin.Vaccine Immunol.,vol.16,p.1151-1157 Summary of the Invention [Problem to be solved by the invention]

[0047] It is therefore an object of the present invention to overcome the shortcomings of the prior art and address this need in the field by providing stable adjuvanted emulsions of bacterial, parasitic and / or viral antigens that can be used as effective vaccines. [Means for solving the problem]

[0048] The present invention provides an adjuvant composition comprising an emulsion of water, tocopherol or a pharma- ceutically acceptable ester thereof, and polyethoxyethylene cetostearyl ether as an emulsifier, which can be advantageously used in the formulation of emulsion vaccines.

[0049] The present invention further provides a vaccine composition comprising this adjuvant composition and an antigen.

[0050] The present invention also provides a method for producing a vaccine composition comprising the claimed adjuvant composition and an antigen, comprising the steps of: a) preparing an aqueous phase containing an antigen; b) mixing the aqueous phase with the claimed adjuvant composition; The present invention provides a method comprising:

[0051] Furthermore, the present invention provides the claimed vaccine compositions for use in a method for protecting a human or animal subject from infection and / or disease caused by a pathogen.

[0052] Details and selection of adjuvant compositions according to the invention and vaccine compositions incorporating same are described herein below. [Brief description of the drawings]

[0053] [Figure 1]Figure 1A is a micrograph of sample IP.1 (Eumulgin in the aqueous phase) at t=0, Figure 1B is a micrograph of sample IP.1 (Eumulgin in the aqueous phase) after 10 days at 37° C., and Figure 1C is a micrograph of sample IP.1 (Eumulgin in the aqueous phase) after 3 weeks at 37° C. Figure 1D is a micrograph of sample IP.2 (Eumulgin in the oil phase) at t=0, Figure 1E is a micrograph of sample IP.2 (Eumulgin in the oil phase) after 10 days at 37° C., and Figure 1F is a micrograph of sample IP.2 (Eumulgin in the oil phase) after 3 weeks at 37° C. FIG. 1G is a micrograph of sample IP.3 (polysorbate 80 in the aqueous phase) at t=0, FIG. 1H is a micrograph of sample IP.3 (polysorbate 80 in the aqueous phase) after 10 days at 37° C., and FIG. 1I is a micrograph of sample IP.3 (polysorbate 80 in the aqueous phase) after 3 weeks at 37° C. [Diagram 2] Vaccination of cattle with the Asia1 / Shamir SVEA-E / FMDV vaccine composition resulted in high virus neutralization titers (Panel 2A) and also provided complete protection against FMDV challenge (Panel 2B), as detailed in Example 8. [Diagram 3] FIG. 1 shows the duration of an immunization study in cattle using a SVEA-E adjuvanted FMD vaccine composition containing O / TUR / 5 / 2009 antigen given as a prime and boost, compared to the corresponding Montanide ISA206 adjuvanted vaccine composition. [Figure 4] FIG. 1 shows the duration of an immunization study in pigs using a SVEA-E adjuvanted FMD vaccine composition containing the O / TUR / 5 / 2009 antigen given as a single immunization and compared to the corresponding Montanide ISA206 adjuvanted vaccine composition. [Diagram 5] FIG. 1 shows the duration of an immunization study in goats using a SVEA-E adjuvanted FMD vaccine composition with the O / TUR / 5 / 2009 antigen given as a single immunization and compared to the corresponding Montanide ISA206 adjuvanted vaccine composition. [Figure 6]FIG. 1 shows a study of the stability of FMDV capsid over time at 4° C. when included in a vaccine composition formulated with SVEA-E adjuvant. Details are in Example 8. [Figure 7] 1 is a graphical representation of the Mastersizer results for each of Samples IP.1, IP.2, and IP.3 (top to bottom) of Example 4, measured after 10 days at 37° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] During the study of oil-adjuvanted emulsion vaccines with multiple bacterial antigens, we observed occasional failures in emulsion stability. Emulsions were found to break down (i.e., showed loss of dispersion and increased phase separation) already after a few weeks in stability tests, rendering the vaccine ineffective. Obvious possible causes, such as poor batch quality of the mineral oil used or the emulsifiers used (Polysorbate 80 [Tween] and Sorbitan Monooleate [Span]), were quickly ruled out, and the problem remained unresolved. Without any indication of where to start, we began a lengthy investigation of the observed emulsion breakdowns, both for factors affected and for factors causing that effect.

[0055] It has surprisingly been found that this objective can be met, and thus one or more of the shortcomings of the prior art can be overcome, by an adjuvant composition comprising an emulsion of water, tocopherol or a pharma- ceutically acceptable ester thereof, and polyethoxyethylene cetostearyl ether, which has been found to overcome the occurrence of emulsion instability, even with complex mixtures and when using relatively crude preparations of antigens.

[0056] This finding is very beneficial for the development of vaccines based on water and oil emulsions.In particular, it overcomes the need to modify the quality or composition of non-live bacterial, parasitic and / or viral antigens used in such emulsion vaccines, making it possible to use relatively impure non-live bacterial, parasitic and / or viral antigen preparations in emulsion vaccines.Indeed, antigens can now be used at lower purity and / or even higher concentrations in vaccines than before, but there is no need to establish the level of impurities, and antigens of all purity levels can be formulated into the stable emulsion vaccines of the present invention using routine skills.

[0057] In particular, it is not known exactly how the adjuvant composition comprising an emulsion of water, tocopherol or its pharma- ceutically acceptable esters, and polyethoxyethylene cetostearyl ether as emulsifiers, confers stability to oil and water emulsions containing non-live antigens. The inventors do not wish to be bound by any theory or model that may explain these findings, but speculate that the sorbitan-based emulsifiers used in the prior art, especially the polysorbates, degraded over time, leading to a loss of emulsifying capacity and subsequent emulsion destruction. The inventors also speculate that the factor that degraded the prior art emulsifiers was the enzymes present in the crude preparations of non-live antigens. Of the various enzymes present, namely proteases, esterases, carbohydrases and nucleases, esterases may have been responsible for degrading the emulsifiers, considering that it can be observed that in emulsions that showed destruction, an increase in the free fatty acid level correlates with a decrease in the concentration of the prior art emulsifiers.

[0058] Such esterase enzymes are produced by many of the known bacterial and parasitic genera and may act in vivo as virulence factors and / or to mobilize nutritional components. All major families of bacteria, both Gram-positive and Gram-negative, are involved, as well as both benign or pathogenic strains of the genera Bacillus, Staphylococcus, Pseudomonas, Salmonella, etc. As a result, such enzymes may in principle be present in any preparation of non-live bacterial antigens. Similar considerations apply to crude preparations of non-live parasitic antigens.

[0059] In view of the above, it is particularly surprising that, among the large variety of possible emulsifiers that can replace the sorbitan-based emulsifiers used in the prior art, such as polysorbates, a particular adjuvant composition according to the invention comprising tocopherol or a pharma- ceutically acceptable ester thereof and polyethoxyethylene cetostearyl ether can be used as an effective emulsifier that has been found to be insensitive to the effects of esterases.

[0060] This was not at all obvious from any disclosure in the prior art, since emulsion vaccines are complex formulations containing a large number of biological and chemical compounds, and when emulsion instability is observed, it is very difficult to determine which factors are affected and which components of the composition are responsible.

[0061] For example, a typical emulsion vaccine having non-live bacterial, parasitic and / or viral antigens has many potential components, any one or more of which may adversely affect one of the other components:

[0062] - components of culture media used to grow bacterial cultures: for example, animal sera, broths or extracts of animal tissues, peptones or hydrolysates of animal or vegetable origin, lipids, sugars, amino acids, vitamins and minerals, antifoam agents, antifungals, etc.

[0063] - (Traces of) inactivating agents: e.g. thiomersal, benzalkonium chloride, formaldehyde, beta-propiolactone, or detergents.

[0064] -Stabilizers: dextran, glycerol, gelatin, amino acids, buffers, etc.

[0065] - Preservatives: thiomersal, phenoxyethanol, formaldehyde, antibiotics (e.g. gentamicin), -Components of microorganisms: cell walls, internal organelles, fragments of nucleic acids; external bacterial organelles such as pili, fimbriae, or flagella; bacterial proteins, lipids, and carbohydrates, e.g. toxins, lipopolysaccharides, various enzymes, etc.

[0066] - one or more adjuvants, emulsifier(s) and their trace elements or impurities.

[0067] Furthermore, emulsion stability may be affected by one or more effects from physical factors in vaccine preparation, purification, formulation, emulsification, filling, shipping and storage.

[0068] Finally, the inventors had no clue about the selection of a suitable replacement for polysorbate emulsifiers, especially in that the emulsifier to be replaced must provide an emulsion with the necessary stability in relation to the various other components of the emulsion vaccine, such as the oil-based adjuvant and the antigen used.

[0069] Thus, in a first aspect, the present invention relates to an adjuvant composition comprising an emulsion of water, tocopherol or a pharma- ceutically acceptable ester thereof, and polyethoxyethylene cetostearyl ether.

[0070] In this composition, tocopherol or a pharma- ceutically acceptable ester thereof acts as an oily adjuvant and polyethoxyethylene cetostearyl ether acts as an emulsifier.

[0071] The water used in the adjuvant composition according to the invention is preferably water of high purity, of pharmaceutical quality and suitable for parenteral injection. Such good quality water is typically sterile and essentially free of pyrogens, for example (multiple) distilled water, reverse osmosis water or water for injection (WFI).

[0072] An "adjuvant" is a substance that increases or modulates the immune response, for example to a vaccine. In particular, adjuvants provide immune stimulation to antigens that are otherwise not sufficiently immunogenic, particularly non-live antigens. This triggers different pathways in the immune system, but the underlying mechanisms of this process are not fully understood.

[0073] An "emulsion" is a mixture of at least two immiscible liquids, whereby one is dispersed in the other. Typically, the droplets of the dispersed phase are very small, no more than a few micrometers in diameter.

[0074] The emulsifiers of the present invention are molecules that have amphiphilic properties and have both hydrophobic and hydrophilic sides. Many emulsifiers are known in the art for their various properties. Most are readily available commercially and vary in purity.

[0075] As used herein, the term "polyethoxyethylene cetostearyl ether" refers to a class of hydrophilic nonionic emulsifiers used in the preparation of various O / W emulsions. This term is used interchangeably with the term "polyoxyethylene cetostearyl ether" in this application. Polyoxyethylene cetostearyl ether is an ether of cetostearyl alcohol. Cetostearyl alcohol, cetearyl alcohol or cetylstearyl alcohol is a mixture of fatty alcohols consisting mainly of cetyl (16C) and stearyl alcohol (18C) and is classified as a fatty alcohol. In this application, "polyethoxyethylene cetostearyl ether" is synonymous with "a mixture containing polyethoxyethylene cetyl ether and polyethoxyethylene stearyl ether."

[0076] Specific examples include polyethoxyethylene 12 cetostearyl ether (INCI: Ceteareth-12, CAS number: 68439-49-6, Ph.Eur. Macrogol cetostearyl ether 12), polyethoxyethylene 20 cetostearyl ether (Ceteareth-20, CAS number: 68439-49-6, Macrogol cetostearyl ether 20) and polyethoxyethylene 30 cetostearyl ether (Ceteareth-30, CAS number: 68439-49-6).

[0077] Polyethoxyethylene 12-cetostearyl ether has an HLB (Hydrophilic-Lipophilic Balance) value of 14 and is a mixture of the following components:

[0078] Polyethoxyethylene 12 Cetyl Ether (Cetyl = C16): [ka] n=12

[0079] Polyethoxyethylene 12 stearyl ether (stearyl = C18): [ka] n=12

[0080] Polyethoxyethylene 12 cetostearyl ether is commercially available under various trade names, such as Eumulgin® B1, Kolliphor® CS12, Volpo® CS12, Cremophor® A25, Simulsol™ or Brij CS12®.

[0081] Polyethoxyethylene 12-cetostearyl ether finds application in antiperspirants and deodorants, sunscreens (after-sun, self-tanning, sun protection), body, color and face care products. It is also used in baby care and cleansing, facial cleansing, hair coloring and conditioning formulations. Additionally, it is used as a mild non-ionic emulsifier for pharmaceutical oil-in-water emulsions to provide good sensory properties during product application.

[0082] Polyethoxyethylene 12 cetostearyl ether is a component of the adjuvant known as AF03, which also includes squalene (note: not squalane), Eumulgin® B1 (polyethoxyethylene 12 cetostearyl ether), Montane™ 80 (sorbitan oleate), mannitol, and phosphate buffered saline.

[0083] "Tocopherol," as used herein, refers to a class of organic chemical compounds that have vitamin E activity. Tocopherols include α-tocopherol (CAS Number: 10191-41-0), β-tocopherol (CAS Number: 148-03-8), γ-tocopherol (CAS Number: 54-28-4) and δ-tocopherol (CAS Number: 119-13-1).

[0084] Pharmaceutically acceptable esters of tocopherol (vitamin E) include, in particular, α-tocopheryl acetate, also called tocopheryl acetate or vitamin E acetate, which has the CAS number 58-95-7. α-tocopheryl acetate can be derived from plant materials such as seeds, nuts, fruits or leaves, or from fatty meats, but can also be synthetically produced. Thus, included in the definition of vitamin E acetate are natural, synthetic or semi-synthetic forms, or mixtures thereof. Vitamin E acetate is commercially available in various purities.

[0085] The alpha-tocopheryl-acetate for the adjuvant composition according to the invention is preferably DL-alpha-tocopherol acetate, which is the racemic form of the chemical substance with CAS number: 7695-91-2.

[0086] As used herein, the term "comprising" (and variations thereof, such as "comprise," "comprises," and "comprised") refers to all possible elements and any possible combinations of the invention covered or included by the text section, paragraph, claim, etc. in which the term is used, and does not refer to the exclusion of any of such element(s) or combination, even if such element or combination is not explicitly recited. Thus, any such text section, paragraph, claim, etc. can also relate to one or more embodiments in which the term "comprises" (or variations thereof) is replaced with terms such as "consist of," "consisting of," or "consist essentially of."

[0087] The adjuvant composition may further comprise a pharma- ceutically acceptable carrier.

[0088] The adjuvant composition can be the same as the vaccine composition described herein, but lacks the antigen.

[0089] A "pharmaceutically acceptable carrier" according to the present invention is a highly pure, preferably sterile, aqueous liquid, such as water, saline, or phosphate-buffered saline. The carrier may contain additional additives, such as stabilizers or preservatives.

[0090] In a preferred embodiment of the adjuvant composition according to the invention, the polyethoxyethylene cetostearyl ether is selected from polyethoxyethylene 12 cetostearyl ether, polyethoxyethylene 20 cetostearyl ether, and polyethoxyethylene 30 cetostearyl ether. Even more preferably, the polyethoxyethylene cetostearyl ether is polyethoxyethylene 12 cetostearyl ether.

[0091] In another embodiment of the adjuvant composition according to the invention, the pharma- ceutically acceptable ester of tocopherol is alpha-tocopheryl acetate.

[0092] In a preferred embodiment, the adjuvant composition according to the invention comprises polyethoxyethylene 12-cetostearyl ether and α-tocopheryl acetate.

[0093] In a preferred embodiment, the adjuvant composition according to the invention does not contain an ester surfactant, such as an ester of a fatty acid, particularly an ester formed from a sugar alcohol or its derivatives and a fatty acid, such as a sorbitan or mannide ester. Such esters are commonly used as surfactants in oil-based adjuvant compositions. Sorbitan esters are also known as polysorbates, Tweens or Span. Particular examples of esters include polyoxyethylene sorbitan monooleate (Polysorbate 80 or Tween® 80), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), and sorbitan monolaurate (Span 20). Other examples include esters of octadecanoic acid and anhydromannitol (contained in Montanide ISA-206).

[0094] Also preferably, the vaccine composition according to the invention does not contain esters of fatty acids.

[0095] In another embodiment, the adjuvant composition according to the invention further comprises squalane.

[0096] "Squalane" is a well-known compound, preferably referring to the compound with CAS number 111-01-3. Some alternative names are hydrogenated shark liver oil, hexamethyltetracosane or perhydrosqualene. It should not be confused with squalene (CAS number 111-02-4), which is a polyunsaturated C30 oil and can be metabolized as a compound in the cholesterol pathway. However, squalane is a fully hydrogenated form of squalene and is therefore not susceptible to oxidation. Thus, squalane is a non-mineral oil and is transported away from the injection site but is non-metabolizable.

[0097] Originally, the precursor of squalane was obtained from shark liver, but environmental concerns have led to a shift to other natural sources, such as olive oil, or chemical synthesis. Thus, the definition of squalane includes natural, synthetic or semi-synthetic forms, or mixtures thereof. Squalane is commercially available in various purities, for example from vegetable sources, Worlee (squalane, vegetable), or Croda (Pripure Squalane), or synthetic, for example Kuraray (squalane-PE). In the present invention, high purity squalane is preferred, preferably with a purity of more than 75%, more preferably with a purity of more than 80%, more preferably with a purity of more than 90%, or even with a purity of more than 95%, in order of preference.

[0098] In one embodiment, the adjuvant composition according to the invention further comprises mineral oil.

[0099] Examples of mineral oils suitable for use in the adjuvant compositions include, for example, Bayol™ or Markol™, Montanide™, or light (or white) liquid paraffin oils such as Marcol® 52 (Exxon Mobile) or Drakeol® 6VR (Penreco) or Klearol® (Sonneborn).

[0100] In one embodiment, the adjuvant composition according to the invention further comprises a non-mineral oil.

[0101] Non-mineral oils can be synthetic, animal or vegetable origin. For use in the adjuvant composition of the present invention, non-mineral oils are preferably biodegradable (metabolizable) and biocompatible. Examples of synthetic oils suitable for use in the adjuvant composition include, for example, Shell Ondina® oil, such as Shell Ondina X420. Examples of animal oils suitable for use in the adjuvant composition include, for example, fish oil. Examples of vegetable oils suitable for use in the adjuvant composition include oils from nuts, seeds and grains, such as peanut oil, soybean oil, palm oil and olive oil; jojoba oil; safflower oil, cottonseed oil, sunflower seed oil, sesame oil; corn oil, or other grain oils, such as wheat, oat, rye, rice, teff and triticale oil.

[0102] In a particularly preferred embodiment, the adjuvant composition according to the invention comprises polyethoxyethylene 12-cetostearyl ether, α-tocopheryl acetate and squalane.

[0103] In another particularly preferred embodiment, the adjuvant composition according to the invention comprises polyethoxyethylene 12-cetostearyl ether, α-tocopheryl acetate and a non-mineral oil. Preferably, the non-mineral oil is a synthetic oil, such as Shell Ondina X GTL based medicinal white oil.

[0104] In another particularly preferred embodiment, the adjuvant composition according to the invention comprises polyethoxyethylene 12-cetostearyl ether, α-tocopheryl acetate and mineral oil. Preferably, the mineral oil is light liquid paraffin oil.

[0105] In one embodiment, the adjuvant composition according to the invention is an oil-in-water emulsion.

[0106] "Oil-in-water emulsions" are well-known compositions that comprise an outer aqueous phase containing an inner dispersed oily phase; in the present invention, the dispersed phase is formed by droplets of an oily adjuvant (tocopherol or a pharma- ceutically acceptable ester thereof).

[0107] Such emulsions can be formed by selecting the appropriate type and concentration of emulsifier(s). Procedures and equipment for the preparation of oil-in-water or water-in-oil emulsions for use as vaccines are well known in the art and described in handbooks known to those skilled in the art.

[0108] An "oil phase" (or "oily phase") is an oil-based liquid. "Oil" is used herein in its general sense to refer to a non-polar chemical that is hydrophobic and lipophilic and has a high hydrocarbon content. Oils can be of mineral origin or non-mineral, e.g., synthetic, animal or vegetable origin. Some non-mineral oils are metabolizable.

[0109] The oil phase may contain excipients such as emulsifiers. In the present invention, the oil phase is the dispersed phase, typical of an O / W emulsion. When formulated into a vaccine, the oil phase acts as an adjuvant. A mineral oil adjuvant often used in veterinary vaccines is light liquid paraffin oil, such as Marcol® (Exxon Mobile) or Drakeol® (Penreco). Common non-mineral oil adjuvants are squalene and squalane (shark liver oil) and tocopherol (vitamin E).

[0110] An "aqueous phase" is a liquid based on water. The aqueous phase may contain, for example, a buffer or saline, and one or more excipients, such as emulsifiers or stabilizers.

[0111] The particle size of the dispersed phase of the adjuvant composition of the invention is preferably very small. When the average diameter of the dispersed phase particles is less than about 1 micrometer, such an emulsion is commonly referred to as a "submicron emulsion."

[0112] In one embodiment of the O / W emulsion of the adjuvant composition according to the invention, the emulsion is a submicron emulsion.

[0113] Equipment for measuring particle sizes below 1 micrometer is commonly available, for example by laser diffraction measurement. Typically, particle size is expressed in nanometers (nm) and is expressed as the average particle size, also known as the median size, and as the D50 of the cumulative particle size distribution.

[0114] In the present invention, particle size is expressed in nm of D50 determined using a Mastersizer™ (Malvern Instruments). Particle size measurements can be performed in (concentrated) oil emulsions constituting the adjuvant or vaccine composition. The particle refractive index of the oil phase of the present invention is 1.48. Malvern Mastersizer size analysis reports present D50 as D(0.50).

[0115] There are many methods available for producing such submicron emulsions, typically by the use of high energy emulsification processes using, for example, high pressure homogenizers, rotor-stator devices, blenders, ultrasound, microporous membranes, or microchanneling devices.

[0116] A preferred method for high energy emulsification to prepare adjuvant compositions according to the invention is the use of a high pressure homogenizer, preferably a Microfluidiser™ (Microfluidics). Typically, 1 to 3 passes at a pressure of 500 to 1500 bar (i.e. 7000 to 22000 psi) are sufficient.

[0117] Emulsions prepared in this way typically have dispersed phase particles with a D50 of 500 nm or less and a narrow size distribution. Such emulsions are called nanoemulsions.

[0118] Typically, emulsions having such very finely sized particles of the dispersed phase are prepared in several successive steps, in which an initial relatively coarse oil-based emulsion is prepared by low energy mixing, followed by one or more subsequent high energy treatments to achieve further reduction in particle size.

[0119] The "microfluidized" oil emulsion containing the adjuvant(s) and emulsifier in water is then combined with an aqueous phase with or without antigen to prepare the adjuvant composition, vaccine composition, respectively, according to the invention.

[0120] Thus, in one embodiment of the submicron oil-in-water emulsion of the adjuvant or vaccine composition according to the invention, the oil droplets, i.e. (emulsion) particles, have a D50 of 500 nm or less, preferably a D50 of 250 nm or less. More preferably, the D50 is 150 nm or less. Particularly preferably, the emulsion particles have a particle size D50 of about 80 to about 200 nm, more preferably about 40 to about 100 nm.

[0121] For reasons of product consistency and quality, it is advantageous to monitor and control not only the median particle size, but also the particle size spread, also known as particle size distribution. The size distribution of the oil droplets in the submicron oil-in-water emulsion of the adjuvant composition or vaccine composition according to the present invention is preferably relatively narrow. An indicator of particle size distribution is the D90 of the cumulative particle size distribution.

[0122] Thus, in one embodiment of the submicron oil-in-water emulsion of the adjuvant or vaccine composition according to the invention, the oil droplets have a D90 of less than 900 nm, more preferably a D90 of less than 500 nm, less than 400 nm, or even less than 300 nm, in that order of preference. The most preferred D90 is about 250 nm or less.

[0123] One advantage of emulsions with such small particle size and distribution is that they can be sterilized by filtration without significant loss of material, since typical sterilizing filters have pore sizes of about 0.2 micrometers. Such filter sterilization overcomes the need for other sterilization methods, such as heat, chemicals, or irradiation, which can damage the quality of the oil emulsion's components.

[0124] Thus, in one embodiment, the adjuvant or vaccine composition according to the invention comprises emulsion particles having a particle size D50 of about 80 to about 200 nm, preferably about 40 to about 100 nm.

[0125] In the adjuvant composition according to the invention, the polyethoxyethylene cetostearyl ether emulsifier is present in an amount of about 1 to about 9% by weight based on the weight (w / w) of the adjuvant composition. Preferably, the polyethoxyethylene cetostearyl ether is present in an amount of about 2 to about 7% w / w of the adjuvant composition.

[0126] In a particularly preferred embodiment, the polyethoxyethylene cetostearyl ether is present in an amount of about 6 to about 7% w / w of the adjuvant composition.

[0127] In another particularly preferred embodiment, the polyethoxyethylene cetostearyl ether is present in an amount of about 2 to about 4% w / w of the adjuvant composition.

[0128] For the present invention, "about" indicates that a number may vary between ±25% around its indicated value. Preferably, "about" means ±20% around its value, more preferably, "about" means ±15, 12, 10, 8, 6, 5, 4, 3, 2% around its value, or even "about" means ±1% around its value.

[0129] In the adjuvant composition according to the present invention, tocopherol or a pharma- ceutically acceptable ester thereof is present in an amount of about 1 to about 20% (by weight) based on the weight of the adjuvant composition. Preferably, tocopherol or a pharma-ceutically acceptable ester thereof is present in an amount of about 6 to about 16% w / w of the adjuvant composition.

[0130] In a particularly preferred embodiment, the tocopherol or a pharma- ceutically acceptable ester thereof is present in an amount of about 14 to about 16% w / w of the adjuvant composition.

[0131] In another particularly preferred embodiment, the tocopherol or a pharma- ceutically acceptable ester thereof is present in an amount of about 2 to about 8% w / w of the adjuvant composition.Most preferably, the tocopherol or a pharma- ceutically acceptable ester thereof, particularly α-tocopheryl acetate, is present in an amount of about 15% w / w of the adjuvant composition.

[0132] In the adjuvant composition according to the invention, squalane is present in an amount of about 5 to about 20% (by weight) based on the weight of the adjuvant composition. Preferably, squalane is present in an amount of about 6 to about 15% w / w of the adjuvant composition. In a particularly preferred embodiment, squalane is present in an amount of about 13 to about 14% w / w of the adjuvant composition.

[0133] In the adjuvant composition according to the invention, the mineral oil is present in an amount of about 5 to about 40% (by weight) based on the weight of the adjuvant composition, hi a particularly preferred embodiment, the mineral oil is present in an amount of about 6 to about 7% w / w of the adjuvant composition.

[0134] In another particularly preferred embodiment, the mineral oil is present in an amount of about 30 to about 40% w / v of the adjuvant composition, preferably about 35% w / v of the adjuvant composition.

[0135] In the adjuvant composition according to the invention, the non-mineral oil is present in an amount of about 5 to about 40% (by weight) based on the weight of the adjuvant composition, hi a particularly preferred embodiment, the non-mineral oil is present in an amount of about 6 to about 7% w / w of the adjuvant composition.

[0136] In another particularly preferred embodiment, the non-mineral oil is present in an amount of about 30 to about 40% w / v of the adjuvant composition, preferably about 35% w / v of the adjuvant composition.

[0137] The adjuvant composition according to the invention can be prepared as a concentrate. The concentrate can be, for example, a 2x w / v concentrate, meaning that the concentrate represents 50% of the final volume of the vaccine. Similarly, if the concentrate is, for example, a 4x w / w concentrate, meaning that the concentrate represents 25% of the weight of the final vaccine, and so on.

[0138] In a particularly preferred embodiment, the adjuvant composition comprises about 5 to about 7% w / w, preferably about 6% w / w, of polyethoxyethylene 12-cetostearyl ether and about 14 to about 16% w / w, preferably about 15% w / w, of α-tocopheryl acetate. This adjuvant composition is referred to herein as Fortasol-E. Fortasol-E is a 2x w / v concentrate of the final vaccine, meaning that the concentrate represents 50% of the volume of the final vaccine. The Fortasol-E formulation is a nanoemulsion.

[0139] In a particularly preferred embodiment, the adjuvant composition according to the present invention comprises about 6 to about 7% w / w polyethoxyethylene 12-cetostearyl ether, about 15 to about 16% w / w α-tocopheryl acetate, and about 13 to about 14% w / w squalane. This adjuvant composition is referred to herein as SVEA-E. SVEA-E is a 4x w / v concentrate and is included as 25% of the final vaccine volume. The SVEA-E adjuvant composition is a nanoemulsion.

[0140] In another preferred embodiment, the adjuvant composition according to the present invention comprises about 3 to about 4% w / w polyethoxyethylene 12-cetostearyl ether, about 7 to about 8% w / w α-tocopheryl acetate, and about 6 to about 7% w / w non-mineral oil. This adjuvant composition is referred to herein as EVO420. EVO420 is a 2x w / v concentrate and is included as 50% of the final vaccine volume. The EVO420 adjuvant composition is a nanoemulsion.

[0141] Surprisingly, a vaccine composition according to the invention comprising the SVEA-E adjuvant composition and further comprising the Orf2 antigen of PCV2 and the crude bacterin antigen of Mhyo was found to be stable after 27 months at 4° C. A similar vaccine composition comprising SVEA-E, but containing FMDV empty capsid as antigen, was found to be stable for up to 8 months at 4° C.

[0142] In one embodiment, the adjuvant composition according to the invention further comprises silica.

[0143] Preferably, the silica is “fumed silica.” More preferably, the fumed silica is present in an amount of about 0.3% w / w to about 0.5% w / w of the adjuvant composition, more preferably in an amount of about 0.4% w / w.

[0144] "Fumed silica" as used herein refers to silicon dioxide having an average particle size of about 6 to about 13 nm. Fumed silica is commercially available under the trade name Aerosil 380 (average particle size 7 nm). Pharmaceutical quality grade fumed silica is commercially available as Aerosil 300 (average particle size 7 nm with less surface area compared to Aerosil 380) or Aerosil 200 (average particle size 12 nm).

[0145] In a further preferred embodiment, the adjuvant composition according to the present invention comprises about 6 to about 7% w / w polyethoxyethylene 12-cetostearyl ether, about 15 to about 17% w / w α-tocopheryl acetate, about 13 to about 14% w / w squalane, and about 0.3 to 0.5% w / w fumed silica. This adjuvant composition is referred to herein as SVEA-E double plus. SVEA-E double plus is a 2x w / w concentrate and is included as 50% of the final vaccine weight. The SVEA-E double plus adjuvant composition is a nanoemulsion.

[0146] In a further preferred embodiment, the adjuvant composition according to the invention contains only 1 / 3 of the amount of emulsifier in the SVEA-E composition, i.e. about 2 to about 2.3% w / w polyethoxyethylene-12-cetostearyl ether. This adjuvant composition is referred to herein as microSVEA-E. The microSVEA-E adjuvant composition represents a microemulsion since it has emulsion particles in the micrometer range.

[0147] Similarly, microSVEA-E double plus contains only 1 / 3 of the amount of emulsifier in the SVEA-E double plus composition, i.e., about 2 to about 2.3% w / w polyethoxyethylene 12-cetostearyl ether. This adjuvant composition is referred to herein as microSVEA-E double plus. The microSVEA-E double plus adjuvant composition has emulsion particles in the micrometer range, and thus represents a microemulsion.

[0148] In a particularly preferred embodiment, the adjuvant composition according to the present invention comprises about 2 to about 3% w / w polyethoxyethylene 12-cetostearyl ether, about 2 to about 3% w / w α-tocopheryl acetate, and about 30 to about 40% w / v, preferably about 35%, non-mineral oil. This adjuvant composition is referred to herein as Xsolve 2.0. The Xsolve 2.0 adjuvant composition is a nanoemulsion.

[0149] The adjuvant composition according to the invention may be used to formulate an emulsion vaccine, such as a vaccine composition according to the invention.

[0150] In the context of the present invention, "formulation" relates to the preparation of a vaccine composition according to the invention by mixing an aqueous phase comprising one or more antigens as described herein with an adjuvant composition according to the invention.

[0151] Adjuvant compositions according to the invention may be used to formulate non-live vaccines, for example vaccine compositions according to the invention which comprise a non-live antigen as described herein.

[0152] In one embodiment, the adjuvant composition according to the invention does not contain (i.e. is free of) emulsifiers susceptible to degradation resulting from crude bacterial or parasitic antigens as defined for the invention, preferably susceptible to degradation by esterases or lipases as defined herein. More preferably, the emulsion does not contain a sorbate-based emulsifier or does not contain polysorbates. Even more preferably, it does not contain polysorbates and sorbitan monooleate.

[0153] In a further aspect, the present invention relates to a vaccine composition comprising the adjuvant composition according to the invention and an antigen.

[0154] An "antigen" is a substance capable of inducing an immunological response in a human or animal subject, possibly with the aid of an immunostimulatory compound such as an adjuvant. Antigens can be synthetically prepared or derived from biological sources, for example, a microorganism (replicable or not), or a part thereof, such as a protein, lipid, carbohydrate, or nucleic acid, or a combination thereof, such as a peptidoglycan, lipoglycan, lipopeptide, or lipopolysaccharide.

[0155] In the context of the present invention, non-live (non-replicating) antigens relate to molecules such as proteins, carbohydrates, lipids or nucleic acids, or are more or less pure complex combinations thereof. When prepared from a microorganism, non-live antigens can refer to the killed (i.e. non-replicating) microorganism or can be a part thereof, such as an extract, fraction, homogenate or sonicate. Non-live antigens can also be nucleic acid-based or recombinant products, such as expression vectors or expressed proteins, or products of in vitro expression systems, all of which are well known in the art.

[0156] "Live" antigen refers to a live (i.e. replicating) bacterium, parasite, or virus suitable for use as a vaccine component, i.e. with a reduced level of pathogenicity, also known as attenuated or modified live.

[0157] As used herein, "attenuated" is defined as causing lower levels of pathology and / or having a reduced rate of infection or replication, all when compared to the unmodified or "wild-type" bacterium, parasite or virus.

[0158] Attenuation of a microorganism can be obtained in vitro, for example by passage through laboratory animals, or by cell culture and selection, or through recombinant DNA techniques, all of which are well known in the art.

[0159] Although it is biologically incorrect to call a virus "live," this is a common way of referring to a virus that is replicable and has not been inactivated. Thus, for the present invention, the term "live" with respect to a virus refers to a virus that is capable of replicating under appropriate conditions, for example in a suitable host cell or animal.

[0160] The vaccine composition according to the present invention comprises a bacterial, parasitic and / or viral antigen. The antigen is preferably a non-live antigen. The vaccine composition may comprise one or more additional antigens, for example from bacteria, parasites, viruses, fungi, ectoparasites, etc. The additional antigens may be live or non-live.

[0161] Thus, in a preferred embodiment of the vaccine composition according to the invention, the vaccine composition comprises one or more antigens selected from bacterial, parasitic and viral non-live antigens. Preferably, the vaccine composition contains multiple (two or more) antigens, more preferably two or more bacterial antigens.

[0162] A "bacterial antigen" is an antigen that is derived from, based on, or obtained from bacteria. In the context of the present invention, bacteria are prokaryotic microorganisms currently classified within the taxonomic kingdom Bacteria.

[0163] A "parasite antigen" is derived from, based on, or obtained from a parasite. In the present case, a parasite is a eukaryotic microorganism, for example one currently classified in the Apicomplexa clade or Nematoda phylum.

[0164] Ectoparasites are, for example, flies, fleas, mites or ticks.

[0165] The fungus is, for example, Aspergillus.

[0166] A "viral antigen" is an antigen derived from, based on, or obtained from a virus. The antigens included in the vaccine composition according to the invention are derived from microorganisms that are pathogenic for the subject species to be protected from infection and / or disease by the vaccine.

[0167] Examples of microorganisms pathogenic for porcine animals are:

[0168] Viruses: Porcine Circovirus Type 2, Porcine Parvovirus, African Swine Fever Virus, Swine Influenza Virus, Porcine Respiratory Coronavirus, Porcine Cytomegalovirus, Porcine Rotavirus, Swine Pox Virus, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Pseudorabies Virus, Hog Cholera Virus, Swine Influenza Virus, Foot and Mouth Disease Virus, Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus and Vesicular Stomatitis Virus.

[0169] Bacteria: Mycoplasma, Lawsonia, Escherichia, Brachyspira, Streptococcus, Salmonella, Clostridium, Actinobacillus, Pasteurella, Haemophilus, Erysipelothrix, Leptospira, Burkholderia, Enterococcus, Mycobacterium, and Bordetella.

[0170] Parasites: Toxoplasma, Isospora and Trichinella.

[0171] Examples of microorganisms that are pathogenic for ruminants are:

[0172] For cattle: Neospora, Dictyocaulus, Cryptosporidium, Ostertagia, Bovine Rotavirus, Bovine Viral Diarrhea Virus, Bovine Coronavirus, Infectious Bovine Rhinotracheitis Virus (Bovine Herpesvirus 1), Bovine Paramyxovirus, Bovine Parainfluenza Virus, Bovine Respiratory Syncytial Virus, Rabies Virus, Bluetongue Virus, Escherichia, Salmonella, Staphylococcus, Streptococcus, Mycobacterium, Brucella, Clostridium, Pasteurella, Yersinia Mannheimia Mannheimia, Haemophilus, Leptospira, Bacillus, Moraxella, Francisella, Histophilus, Trueperella, Fusobacterium, Actinomyces, Coxiella, Campylobacter, Erysipelothrix, Listeria, Burkholderia, Nocardia, Mycoplasma, Bacteroides, and Chlamydia.

[0173] For sheep and goats: Toxoplasma gondii, Peste des petit ruminant virus, Bluetongue virus, Schmallenberg virus, Mycobacterium, Brucella, Clostridia, Coxiella, Escherichia, Chlamydia, Clostridium, Pasteurella and Mannheimia.

[0174] In deer: interzoan hemorrhagic disease virus, bluetongue virus, papillomavirus, Borrelia burgdorferi, Mycobacterium bovis and Trueperella pyogenes.

[0175] Particularly advantageously, the vaccine composition according to the invention may comprise a crude preparation of bacterial or parasitic antigens: due to the emulsion stabilizing effect provided by the components of the adjuvant composition according to the invention, elaborate and expensive purification of the bacterial and / or parasitic antigens is not required.

[0176] The term "crude" as used herein refers to antigens from bacteria, parasites or viruses that have been harvested from in vitro culture and prepared with little or no further purification after they have been inactivated and / or lysed or destroyed. Such crude antigen preparations still contain undefined or unintended impurities that may affect the stability of vaccines based on oil and water emulsions.

[0177] The antigen contained in the vaccine composition according to the invention is typically contained in a liquid, such as an aqueous buffer.

[0178] As stated, the advantages of the present invention apply particularly to the use of such vaccine compositions for the agricultural sector.

[0179] In one embodiment of a vaccine composition according to the invention, the antigen is a non-live bacterial antigen, preferably the non-live bacterial antigen is an inactivated whole cell culture (i.e. a bacterin) or is part of such a culture.

[0180] Preferably, the portion of the inactivated whole bacterial culture is selected from pellets, supernatants, concentrates, dialysates, extracts, sonicates, lysates, and fractions of such cultures.

[0181] What constitutes a "bacterial culture" or "portion thereof" is well known to those skilled in the art and is described in handbooks and manuals such as "Veterinary Vaccinology" (ibid.) In the present invention, inactivated bacterial cultures are used in their entirety, i.e., as the complete contents of a particular culture vessel, or as a portion thereof.

[0182] Methods and materials for preparing such inactivated cultures or for preparing such parts are generally known and available on any scale. For example, inactivation of bacteria can be performed using chemical or physical means. Physical means are, for example, heating, irradiation, or very high pressure. Chemical means are, for example, incubation with merthiolate, formalin, diethylamine, binary ethyleneamine, β-propiolactone, benzalkonium chloride, or glutaraldehyde.

[0183] The supernatant or pellet can be prepared by centrifugation.

[0184] The concentrate or dialysate can be prepared, for example, by the method of cross-flow filtration.

[0185] Extracts can be made, for example, by washing or incubation with a solvent or detergent solution. The solvent can be a liquid or a gas, and the liquid can be aqueous, for example, water or a buffer, or an organic solvent, such as alcohol, acetone, ether, etc. The extract is the portion removed with the solvent, and is often recovered from the solvent in a subsequent process.

[0186] The sonicate can be prepared using a sonication device, such as a flow-through sonication cell.

[0187] The lysate can be prepared by physical or (bio)chemical means, for example using a French press or using enzymatic treatment.

[0188] A fraction is that part of the whole that is purified from the remainder, e.g., the filtrate or precipitate, whereby the fraction is the retentate.

[0189] Most used in bacterial vaccines for agricultural applications are non-live bacterial antigens that contain inactivated bacterial cells. Generally, such antigen preparations of killed bacterial cells are called bacterins.

[0190] Inactivated bacterial cells may be in any form, either intact or damaged. Inactivated bacterial cells may be of any level of purity, for example with or without the bacterial culture medium in which they were fermented, for example resulting from sedimentation, centrifugation or concentration.

[0191] Thus, in one embodiment of a vaccine composition according to the invention, the non-live bacterial antigen comprises inactivated bacterial cells.

[0192] The bacterium against which the non-live antigen is prepared may be any bacterium of human medical and / or veterinary relevance, for example any bacterium that is a (potential) pathogen, either as a primary or secondary (opportunistic) pathogen.

[0193] By way of example, suitable bacterial antigens include Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae (Mhyo), Lawsonia intracellularis, Erysipelothrix rhusiopathiae, and Leptospira, or any portion thereof, alone or in any combination.

[0194] Clearly, combinations of non-live bacterial antigens derived from two or more bacteria may also be prepared and used, and combinations with antigens derived from viral or parasitic pathogens may be desirable.

[0195] The bacterial antigen is preferably a crude antigen as described herein. More preferably, the crude bacterial antigen is a bacterin from Mhyo or from L intracellularis.

[0196] In the present invention, viral antigens are preferably derived from in vitro cultures of viruses and host cells. The viruses can be obtained from culture supernatants, from pellets, or contained within the host cells. A wide variety of pathogenic viruses are known to be relevant to veterinary medicine.

[0197] Exemplary viral antigens include PCV2, PRRSV and FMDV, or any portion thereof, alone or in any combination.

[0198] The antigens of the invention may also be included in the vaccine composition according to the invention by means of a recombinant vector. Such a vector may be a nucleic acid or a replicon particle (RP), the nucleic acid being preferably a eukaryotic expression plasmid or an RNA molecule, and the RP being preferably an alphavirus RP.

[0199] In one embodiment of the vaccine composition according to the invention, the antigen comprises a viral antigen.Preferably, the viral antigen comprises a non-live antigen of FMDV, preferably a virus-like particle of FMDV, such as a recombinantly expressed virus-like particle of FMDV.The FMDV antigen is particularly preferably the empty capsid of recombinantly expressed FMDV.

[0200] In one embodiment of the vaccine composition according to the invention, the non-live antigen is selected from one or more of Porcine Circovirus Type 2, Mycoplasma hyopneumoniae and Foot and Mouth Disease Virus. Preferably, one or more additional non-live antigens selected from PRRSV or L. intracellularis may be included.

[0201] A "vaccine" is a well-known medically effective composition that includes an immunologically active ingredient and a pharma- ceutically acceptable carrier. The aqueous phase or the adjuvant composition according to the present invention itself serves as the "carrier" of the present invention. The "immunologically active ingredient" of the present invention is an antigen derived from a bacterium, a parasite or a virus, or a combination of antigens derived from one or more of these microorganisms.

[0202] The vaccine composition according to the invention stimulates the immune system of a human or animal subject to induce a protective immune response, which may be derived from the subject's innate and / or adaptive immune system and may be of the cellular and / or humoral type.

[0203] A vaccine provides protection "against infection and / or disease" in a vaccinated subject by, for example, reducing the severity of infection by reducing the number of pathogens or by shortening the period of replication of the pathogen in the subject, and by reducing the number, intensity or severity of lesions caused by infection with a pathogen. Additionally or as a result, the vaccine is effective in reducing or ameliorating (clinical) symptoms of disease that may be caused by such infection or replication or by the subject's response to said infection or replication. A reference for such diseases and clinical signs is "The Merck veterinary manual" (supra). Such vaccines are colloquially referred to as vaccines "against" a particular pathogen, e.g., "Mhyo vaccine" or "FMDV vaccine".

[0204] To be immunologically effective, a vaccine must contain a sufficient amount of antigen, which may already be known from the relevant vaccine or may be readily determined, for example, by monitoring the immunological response after vaccination and (in the case of animal subjects) challenge infection, e.g., by monitoring the subject for signs of disease, clinical scores, or reisolation of the pathogen, and comparing these results with the vaccination-challenge response seen in sham-vaccinated animals.

[0205] The amount of bacterial, parasitic or viral antigen for the vaccine composition according to the invention can be expressed in different ways depending on the type of antigen used. For example, the antigen dose can be expressed as the number of bacterial cells, parasites or virions counted before inactivation. Alternatively, the antigen can be quantified by serological or biochemical tests such as ELISA or AlphaLisa™ and expressed in relative units compared to an appropriate reference standard. All of these are well known in the art.

[0206] The vaccine composition according to the invention can be used as a prophylactic, metaphylactic or therapeutic treatment.

[0207] The vaccine composition according to the invention may serve as an effective priming vaccination, which may be followed up and amplified later by booster vaccinations with the same or a different vaccine.

[0208] The vaccine composition according to the invention may further comprise other compounds such as further antigens or microorganisms, cytokines or immunostimulatory nucleic acids containing unmethylated CpGs, or the vaccine composition according to the invention may itself be added to the vaccine.

[0209] As mentioned, the vaccine composition according to the invention is of particular relevance in the field of animal husbandry.

[0210] Consequently, for different groups of animal subjects, the non-live bacterial antigens of the invention are derived from bacteria of the following bacterial families, for example:

[0211] For vaccination of ruminants, the bacterium is selected from one of Pasteurella, Escherichia, Salmonella, Yersinia, Staphylococcus, Streptococcus, Mycobacterium, Moraxella, Bacillus, Brucella, Clostridium, Mannheimia, Haemophilus, Francisella, Fusobacterium, Histophilus, Fusobacterium, Treperella (Alkanobacterium), Actinomyces, Clostridium, Coxiella, Campylobacter, Erysipelothrix, Leptospira, Listeria, Burkholderia, Nocardia, Mycoplasma, Bacteroides and Chlamydia.

[0212] For vaccination of pigs, the bacteria is selected from one of Mycoplasma, Lawsonia, Escherichia, Brachyspira, Streptococcus, Salmonella, Clostridium, Actinobacillus, Pasteurella, Haemophilus, Erysipelothrix, Leptospira, Burkholderia, Enterococcus, Mycobacterium, and Bordetella.

[0213] In an even more preferred embodiment of the vaccine composition according to the invention, the non-live antigen is derived from Porcine circovirus type 2, Mycoplasma hyopneumoniae and / or Foot and Mouth Disease virus.

[0214] Further optimization of the vaccine composition according to the present invention is well within the reach of a person skilled in the art.In general, this involves fine-tuning the efficacy of the vaccine to further improve the immune protection provided.This can be done by adapting the dose, volume, adjuvant or antigen content of the vaccine, or by application via different routes, methods or regimes.All of this is within the scope of the present invention.

[0215] In the vaccine composition according to the present invention, the emulsifier is present in an amount of about 1 to about 5% (by weight) based on the volume (w / v) of the vaccine composition. Preferably, the emulsifier is present in an amount of about 1 to about 4% w / v of the vaccine composition. In a particularly preferred embodiment, the emulsifier is present in an amount of about 1 to about 2% w / v of the vaccine composition. In another particularly preferred embodiment, the emulsifier is present in an amount of about 2 to about 3% w / v of the vaccine composition. In another particularly preferred embodiment, the emulsifier is present in an amount of about 3 to about 4% w / v of the vaccine composition. In another particularly preferred embodiment, the emulsifier is present in an amount of about 0.3 to about 1.2% w / v of the vaccine composition.

[0216] In the vaccine composition according to the present invention, the tocopherol or its pharma- ceutically acceptable ester is present in an amount of about 2 to about 9% (by weight) based on the volume of the vaccine composition. Preferably, the tocopherol or its pharma- ceutically acceptable ester is present in an amount of about 3 to about 8% w / v of the vaccine composition. In a particularly preferred embodiment, the tocopherol or its pharma- ceutically acceptable ester is present in an amount of about 2 to about 3% w / v of the vaccine composition. In another particularly preferred embodiment, the tocopherol or its pharma- ceutically acceptable ester is present in an amount of about 3 to about 4% w / v of the vaccine composition. In another particularly preferred embodiment, the tocopherol or its pharma- ceutically acceptable ester is present in an amount of about 7 to about 8% w / v of the vaccine composition.

[0217] In the vaccine composition according to the present invention, squalane is present in an amount of about 3 to about 7% (by weight) based on the volume of the vaccine composition. Preferably, squalane is present in an amount of about 3 to about 4% w / v of the vaccine composition. Also preferably, squalane is present in an amount of about 6 to about 7% w / v of the vaccine composition.

[0218] In the vaccine composition according to the present invention, the mineral oil is present in an amount of about 3 to about 40% (by weight) based on the volume of the vaccine composition. In a preferred embodiment, the mineral oil is present in an amount of about 3 to about 4% w / v of the vaccine composition. In another preferred embodiment, the mineral oil is present in an amount of about 30 to about 40% w / v of the vaccine composition, preferably about 35% w / v of the vaccine composition.

[0219] In the vaccine composition according to the present invention, the non-mineral oil is present in an amount of about 3 to about 40% (by weight) based on the volume of the vaccine composition. In a preferred embodiment, the non-mineral oil is present in an amount of about 3 to about 4% w / v of the vaccine composition. In another preferred embodiment, the non-mineral oil is present in an amount of about 30 to about 40% w / v of the vaccine composition, preferably about 35% w / v of the vaccine composition.

[0220] In a particularly preferred embodiment, the vaccine composition is based on the Fortasol-E adjuvant composition and comprises about 5 to about 7% w / v, preferably about 6% w / v, of polyethoxyethylene 12-cetostearyl ether and about 14 to about 16% w / v, preferably about 15% w / v, of α-tocopheryl acetate. The density of the vaccine composition based on the Fortasol-E adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0221] In a particularly preferred embodiment, the vaccine composition is based on the SVEA-E adjuvant composition and comprises about 1 to about 2% w / v polyethoxyethylene 12-cetostearyl ether, about 3 to about 4% w / v α-tocopheryl acetate, and about 3 to about 4% w / v squalane. The density of the vaccine composition based on the SVEA-E adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0222] Vaccines based on the SVEA-E double plus adjuvant composition are particularly suitable for intradermal administration, for example by the IDAL® device, particularly for intradermal administration to pigs.

[0223] In a particularly preferred embodiment, the vaccine composition is based on the EVO420 adjuvant composition and comprises about 1 to about 2% w / v polyethoxyethylene 12-cetostearyl ether, about 3 to about 4% w / v α-tocopheryl acetate, and about 3 to about 4% w / v squalane. The density of the vaccine composition based on the EVO420 adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0224] In a particularly preferred embodiment, the vaccine composition is based on the SVEA-E double plus adjuvant composition and comprises about 3 to about 4% w / v polyethoxyethylene 12-cetostearyl ether, about 7 to about 8% w / v α-tocopheryl acetate, about 6 to about 7% w / v squalane, and about 0.1 to 0.3% w / v fumed silica. The density of the vaccine composition based on the SVEA-E double plus adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0225] In a particularly preferred embodiment, the vaccine composition is based on the microSVEA-E adjuvant composition and comprises about 0.3 to about 0.7% w / v polyethoxyethylene 12-cetostearyl ether, about 3 to about 4% w / v α-tocopheryl acetate, and about 3 to about 4% w / v squalane. The density of the vaccine composition based on the microSVEA-E adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0226] In a particularly preferred embodiment, the vaccine composition is based on the microSVEA-E double plus adjuvant composition and comprises about 0.8 to about 1.2% w / v polyethoxyethylene 12-cetostearyl ether, about 7 to about 8% w / v α-tocopheryl acetate, about 6 to about 7% w / v squalane, and about 0.1 to 0.3% w / v fumed silica. The density of the vaccine composition based on the microSVEA-E double plus adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0227] A vaccine composition according to the invention based on microSVEA-E or microSVEA-E double plus as the adjuvant composition according to the invention is particularly suitable for intramuscular administration.

[0228] Such vaccine compositions based on microSVEA-E or microSVEA-E double plus are both microemulsions, so they have dispersed particles in the emulsion in the micrometer range. However, surprisingly, these vaccine compositions did not show any stability problems, such as the destruction of the emulsion. Thus, by using an adjuvant composition that is a microSVEA-E or microSVEA-E double microemulsion, it is possible to obtain a vaccine composition with larger emulsion particles in the micrometer range, but nevertheless with good safety and no stability problems. This can be used advantageously in terms of protection against certain pathogens and diseases, since a vaccine with a larger dispersed particle size can be expected to induce a different type of immune response (i.e. less Th1-oriented) than a vaccine with a nanoemulsion.

[0229] In a particularly preferred embodiment, the vaccine composition is based on the Xsolve 2.0 adjuvant composition and comprises about 2 to about 3% w / w polyethoxyethylene 12-cetostearyl ether, about 2 to about 3% w / w α-tocopheryl acetate, and about 30 to about 40% w / v, preferably about 35%, non-mineral oil. The density of the vaccine composition based on the Xsolve 2.0 adjuvant composition is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0230] In one embodiment, the vaccine composition has a high loading of antigen(s). The total antigen concentration is preferably at least 20% v / v, preferably at least 25% v / v, more preferably at least 30% v / v of the vaccine composition.

[0231] The vaccine composition according to the invention is preferably an emulsion vaccine.

[0232] The vaccine composition according to the present invention has been found to be highly effective, safe and stable when formulated as an oil-in-water emulsion.

[0233] Thus, in a preferred embodiment, the vaccine composition according to the invention is an oil-in-water emulsion vaccine.

[0234] Where the vaccine composition according to the invention is an oil-in-water emulsion vaccine, aqueous antigens can be combined with the adjuvant composition using low shear mixing techniques, as the adjuvant composition is itself already emulsified, potentially involving high shear mixing.

[0235] The outer aqueous phase preferably comprises the antigen in a pharma- ceutically acceptable carrier.The oil phase comprises alpha-tocopheryl acetate as the oil adjuvant(s) and may comprise squalane.

[0236] The O / W emulsion according to the invention can itself be used for the formulation of further emulsions, such as W / O / W or O / W / O emulsions. This may require the use of additional emulsifiers. The selection and optimization of such conditions is within the capabilities of the skilled person.

[0237] As detailed above, it has surprisingly been found that the adjuvant compositions of the present invention are suitable for formulating vaccines containing crude antigens, i.e. antigens that are scarcely purified, e.g. derived from inactivated bacterial or parasitic cultures or from extracts or fractions of such cultures.

[0238] Thus, in one embodiment of the vaccine composition according to the invention, the antigen comprises a crude antigen.

[0239] Since elaborate and expensive purification of bacterial and / or parasitic antigens is not required, the vaccine compositions according to the invention are cost-effective. Furthermore, reduced batch-to-batch variability has been observed, along with the suitability of the compositions of the invention for large-scale production.

[0240] Furthermore, the properties of the adjuvant composition according to the invention make it particularly useful in combination with antigens containing enzymes with esterase activity, especially non-live bacterial or parasitic antigens, which can degrade the emulsifiers used in prior art oil and water phase emulsions and destroy the emulsion.

[0241] Thus, in one embodiment of the vaccine composition according to the invention, the antigen comprises an esterase, preferably a lipase.

[0242] As used herein, the term "esterase" refers to enzymes having esterase activity, particularly esterases and lipases, which are characterized by EC 3.1.1.1 or EC 3.1.1.3, respectively, in the IUBMB (International Union of Biochemistry and Molecular Biology) enzyme classification system.

[0243] Tests to determine whether an antigen of the present invention "contains esterase" refer to tests for esterase activity, which exist in a wide variety of forms and types of assays and are commercially available.

[0244] Direct detection of esterase activity is by spectrophotometric assay by detecting yellow coloration from the release of nitrophenols from p-nitrophenyl esters by active esterases. The paper by De Yan et al. (2013, Biotechn. & Appl. Biochem., vol. 60, p. 343-347) lists several variations of this type of assay in its references 2-16. All of these are variations of the basic assay as described by De Caro et al. (1986, Eur. J. of Biochem., vol. 158, p. 601-607).

[0245] In the present invention, to establish whether an antigen contains an esterase, a detection test is carried out according to De Caro et al. (supra) using p-nitrophenyl-butyrate as substrate. Those skilled in the art are fully capable of setting up and carrying out such a test.

[0246] Any level of esterase activity in an antigen may be detrimental to the stability of prior art emulsions, especially upon long-term storage. Thus, the presence of any detectable level of esterase activity, based on appropriate positive and negative reference standards, qualifies an antigen as "containing esterase" according to the present invention.

[0247] The adjuvant composition according to the invention, and in particular a vaccine composition comprising this adjuvant composition in combination with an antigen, surprisingly exhibits several further advantages.

[0248] In particular, although it is not easy to find adjuvants that work well across species, both the adjuvant composition and the vaccine composition according to the invention have been successfully used in pigs and ruminants, preferably in pigs, cattle, sheep and goats.

[0249] Furthermore, the adjuvant composition and the vaccine compositions derived therefrom have a lower density compared to prior art adjuvant compositions, making them easier to administer. Preferably, the density of the adjuvant is about 0.9 to about 1.1 g / ml, preferably about 0.9 to about 1.0 g / ml.

[0250] Furthermore, the adjuvant composition and the vaccine compositions derived therefrom are safe as no serious adverse events were observed.

[0251] A favorable feature of the O / W emulsion is that it is possible to determine the antigen content in the vaccine composition according to the invention without directly destroying the vaccine composition in an ELISA plate.

[0252] Vaccine compositions containing high antigen amounts, such as multivalent or combination vaccines, are considerably more stable compared to prior art compositions.

[0253] The adjuvant compositions and vaccine compositions derived therefrom are able to withstand higher temperatures compared to prior art compositions, such as Montanide ISA206, which undergoes phase inversion above 32°C.

[0254] Quite surprisingly, the vaccine composition according to the invention provides a rapid onset of immunity (OOI).

[0255] Even more surprisingly, the vaccine composition according to the invention provides a longer duration of immunity (DOI).

[0256] The vaccine composition according to the present invention can be prepared using well-known methods and materials. The details of these procedures depend on the type of emulsion to be prepared. For example, as an O / W emulsion, the emulsification of the oil phase and the water phase (without antigen) can be carried out separately (e.g., to constitute the adjuvant composition according to the present invention), and then the antigen can be mixed to prepare the emulsion vaccine according to the present invention.

[0257] In some cases, it may be necessary to apply some heat during preparation of the adjuvant composition to completely dissolve the emulsifier, for example to 70-90°C. Additional emulsifiers can be included in the oil and / or water phases as needed. In general, the water and oil phases can be emulsified using suitable equipment such as ultrasonic or rotor-stator type mixing.

[0258] The inventors further discovered that the polyethoxyethylene cetostearyl ether of the adjuvant composition according to the invention is surprisingly soluble in the oil phase, which was unexpected, especially for an emulsifier with a high HLB number (e.g., polyethoxyethylene 12 cetostearyl ether has an HLB of 14), since polysorbate 80, which has approximately the same HLB number (15), is not very soluble in the oil phase.

[0259] This allows for the use of emulsification methods that require lower shear and less power input, resulting in various savings and operational advantages.

[0260] Alternatively, the use of high intensity emulsification of water and oil phases to create O / W emulsions where the antigen is typically not present during the initial emulsification of the oil phase, emulsifier and water is a further option, for example by using microfluidization.

[0261] Therefore, in a further aspect, the present invention relates to a method for producing a vaccine composition according to the invention, said method comprising the steps of: a) preparing an aqueous phase containing an antigen; b) mixing the aqueous phase with an adjuvant composition according to the invention; Includes.

[0262] With respect to the method of producing a vaccine according to the present invention, each of the aqueous phase, the oil phase, the adjuvant composition and the antigen is as defined herein.

[0263] In an alternative embodiment, an aqueous phase can be prepared that contains the antigen and an emulsifier (i.e., polyethoxyethylene cetostearyl ether). Separately, to obtain the vaccine composition of the invention, an oil phase that contains tocopherol or a pharma- ceutically acceptable ester thereof (and may contain squalane, mineral oil and / or non-mineral oil) is prepared before mixing both phases.

[0264] Preferably, the manufacturing method according to the present invention is carried out in a manner that allows the medical use of the vaccine composition. In general, this concerns the use of equipment and excipients that are pharma- ceutically acceptable and comply with quality regulations such as good manufacturing practices. All of these are well known to those skilled in the art and are specified in government regulations such as pharmacopoeias and handbooks such as Remington and Pastoret (both cited above). Typically, such manufacturing is carried out aseptically.

[0265] As mentioned, the vaccine composition according to the invention is particularly advantageous when applied as a vaccine against bacterial, parasitic and / or viral diseases, all as described herein.

[0266] Thus, in a further aspect, the present invention relates to a vaccine composition according to the invention for use in a method for protecting a human or animal subject against infection and / or disease caused by a pathogen.

[0267] The selection of the target for the protection method according to the present invention is mainly determined by the host range of the pathogen to be protected, for humans, animals, or both.Alternatively, the pathogen may be pathogenic for humans, but not pathogenic for the animals that carry it.In that case, it is still reasonable to apply animal vaccination to prevent zoonotic and food-borne diseases in humans who may consume infected animal products, such as meat or milk.

[0268] The age, weight, sex, immunological status, and other parameters of the subject to be vaccinated are not critical, although it is clearly preferable to vaccinate healthy, uninfected subjects, and to vaccinate as soon as possible.

[0269] An "animal" according to the present invention is any animal of veterinary relevance, such as a cow, pig, goat, sheep, deer, dog, cat, horse, bird, fish, or shrimp.

[0270] In one embodiment of a vaccine composition for use in a method of protection according to the invention, the animal subject is a pig or a ruminant.

[0271] The term "swine" refers to animals of the Suidae family, preferably of the genus Sus, also called porcine. Examples are wild or domestic pig, hog, boar, babirusa, or warthog. It also includes pigs designated by any name, referring to their sex or age, such as sow, queen, boar, barrow, hog, gilt, weaner, or piglet. Furthermore, the term swine refers to any type of swine animal, such as breeding or fattening type, and parental lines of any of these types.

[0272] The term "ruminant" refers to large, ungulate, herbivorous or browsing mammals that can obtain nutrients from plant-based foods by fermenting them in a specialized stomach prior to digestion, primarily through microbial action. Ruminant mammals include cattle, all domestic and wild bovids, goats, sheep, giraffes, deer, gazelles, and antelopes.

[0273] In a preferred embodiment of the vaccine composition for use in the method for protecting an animal subject, the subject is a ruminant, which is a livestock animal, in particular selected from cattle, bovines, sheep and goats.

[0274] In a particularly preferred embodiment of the vaccine composition for use in the method for protecting an animal subject, the subject is a pig, which is a sow or a juvenile pig.

[0275] A "young pig" according to the present invention is a pig under 6 months of age, preferably under 5, 4, 3, 2 months of age, or even under 1 month of age, in that order of preference.

[0276] In one embodiment of a vaccine composition for use in a method of protection according to the invention, the pathogen is selected from bacteria, parasites, and viruses, all as described herein.

[0277] Preferably, the pathogen is selected from one or more of porcine circovirus type 2, Mycoplasma hyopneumoniae and foot and mouth disease virus.

[0278] In one embodiment of the vaccine composition for use in the method of protection according to the invention, the vaccine composition is administered by the intradermal or intramuscular route.

[0279] In a further aspect, the present invention relates to a vaccine composition according to the invention for use in vaccinating a human or animal subject against infection and / or disease caused by a pathogen.

[0280] In one embodiment of the vaccine composition for use in vaccination according to the invention, the animal subject is a ruminant or a pig, and / or the pathogen is a bacterium, a parasite or a virus, all as described herein.

[0281] As the skilled person is well aware, the vaccine composition according to the invention can be applied "for use in a protection method" or "for use in vaccination" in various ways. For example, the vaccine composition itself can be applied as a vaccine. Alternatively, the vaccine composition can be used as a component in further processing, for example into a W / O / W or O / W / O emulsion, which can then be applied as a vaccine. The use as a vaccine can also include the mixing or inclusion of certain further components, for example stabilizers or preservatives. Preservatives are for example thiomersal, phenoxyethanol, formalin, antibiotics (for example gentamicin). Stabilizers are for example dextran, glycerol, gelatin, amino acids or buffers. Depending on the type of emulsion, further components may be added during or after the preparation of the vaccine composition according to the invention.

[0282] In a further aspect, the present invention relates to the use of an adjuvant composition according to the invention for the manufacture of a vaccine composition for protecting a human or animal subject against infection and / or disease caused by a pathogen.

[0283] In one embodiment of the use according to the invention, the animal subject is a ruminant or a pig, and / or the pathogen is a bacterium, a parasite or a virus, all as described herein.

[0284] The vaccine composition according to the invention must be administered to a human or animal subject to achieve its beneficial immunogenic effect.

[0285] Thus, in a further aspect, the present invention relates to a method for vaccination of a human or animal subject against infection and / or disease caused by a pathogen, the method comprising administration to the subject of a vaccine composition according to the invention.

[0286] In one embodiment of the method for vaccination according to the invention, the animal subject is a ruminant or a pig, and / or the pathogen is a bacterium, a parasite or a virus, all as described herein.

[0287] The "administration" of the vaccine composition according to the invention to a human or animal subject can be carried out using any feasible method and route. Typically, the optimal route and method of administration is determined by the type of vaccine applied, as well as the characteristics of the subject and the disease it is intended to protect against. Various administration techniques can be applied. For example, as an O / W emulsion, the vaccine composition according to the invention is characteristically aqueous and can therefore be administered by the enteral or mucosal route, i.e. via eye drops, nasal drops, oral, enteric, oral nasal drops, spray. Other possibilities are by methods of mass administration, by drinking water, coarse spray, atomization, on-feed, etc. The skilled person is fully capable of selecting and optimizing such routes and methods of administration.

[0288] The preferred method of administration for the method of vaccination according to the invention is by the parenteral route.

[0289] "Parenteral" refers to administration through the skin, for example, by intramuscular, intraperitoneal, intradermal, submucosal or subcutaneous routes.

[0290] In one embodiment of the method for vaccination according to the invention, the vaccine composition is administered by the intradermal or intramuscular route.

[0291] The volume of a dose of the vaccine composition according to the present invention, for example when administered parenterally, is a volume that is tolerated by a human or animal subject, and may be, for example, about 0.1 to about 10 ml. Preferably, a single dose has a volume of 0.1 to 5 ml, more preferably a single dose has a volume of 0.2 to 3 ml.

[0292] When administered by the intramuscular route, the volume of a single dose is preferably about 0.5 to about 3 ml, more preferably 1 to 2 ml.

[0293] When administered via the intradermal route, the volume of a single dose is preferably about 0.1 to about 0.5 ml, more preferably about 0.2 ml.

[0294] The method, timing and amount of administration of the vaccine composition according to the invention is preferably integrated into existing vaccination schedules of other vaccines that the human or animal subject may require, in order to reduce stress on the subject and to reduce labor costs. These other vaccines may be administered in a simultaneous, parallel or sequential manner, in a manner compatible with their registered use.

[0295] The adjuvant composition according to the invention can be marketed as an independent product. This allows it to be used by qualified personnel to prepare O / W emulsion vaccines containing the preferred antigens just prior to administration. Such on-site preparation, also called field-side mixing, allows flexibility in the choice and combination of antigens to be mixed. However, vaccine compositions prepared in this way do not have the guaranteed characteristics of quantity, quality and sterility that extemporaneous mixed vaccine compositions prepared in the pharmaceutical plants of registered manufacturers.

[0296] An alternative is the provision of the adjuvant composition according to the invention in a kit of parts. Such a kit is typically a package comprising two or more containers, the contents of which can be mixed to prepare the vaccine composition according to the invention just before administration. In the present case, the kit of parts may comprise one container with the adjuvant composition according to the invention and one or more further containers containing one or more antigens. The one or more further antigens may be in liquid form.

[0297] Alternatively, and advantageously, the one or more further antigens may be in dried or lyophilized form. This utilizes the advantageous properties of the adjuvant composition of the invention as an O / W emulsion, i.e., it behaves as an aqueous composition, and can be used as a diluent to reconstitute dried or lyophilized antigens. The lyophilized antigen may be a live or killed microorganism, for example, a lyophilized preparation of live PRRSV or a lyophilized preparation of non-live L. intracellularis.

[0298] Thus, in a further aspect, the present invention relates to a kit of parts, the kit comprising at least two containers, one container comprising the adjuvant composition according to the invention and one container comprising an antigen.

[0299] In one embodiment of the kit of parts according to the invention, the antigen is lyophilized live PRRSV or a lyophilized preparation of non-live L. intracellularis.

[0300] A kit of parts according to the invention may comprise a box having two or more containers and may comprise instructions for use, which may, for example, be written on the box containing the components of the kit, may appear in, on or in a leaflet included in the box, or may be viewable on or downloadable from an internet website of the kit manufacturer or distributor, etc.

[0301] The invention will now be further illustrated by the following non-limiting examples.

[0302] [Example] [Example 1] Fortasol-E Fortasol-E is a 2x w / v concentrate. As a reference, a similar composition was prepared with Polysorbate 80 (PS80) instead of Eumulgin B1 as the emulsifier.

[0303] Composition per 100g: Oil Phase DL-α-Tocopherol Acetate 15.00g Eumulgin B1 6.00g

[0304] aqueous phase WFI 79.00g

[0305] Preparation: - Heat the oil and water phase to 75-85°C. - Slowly add the oil phase to the water phase while stirring with a visible vortex -Continue stirring for 1 minute after addition. - Filter through a 0.2μm filter Mix 1:1 with Mhyo antigen for stability at -37°C

[0306] The Mhyo antigen used was a crude antigen preparation of Mhyo bacterin grown in culture with pig blood in medium.

[0307] result: - The oil phase containing Eumulgin B1 was transparent, while the oil phase containing Polysorbate 80 was cloudy. - Both adjuvant compositions are easily filterable through 0.2 μm nylon filters -The adjuvant composition containing Eumulgin showed some slight clustering of visible particles Macroscopically, both adjuvant compositions appear as nanoemulsions with a blue glow

[0308] Mastersizer results are in μm.

[0309] [Table 1]

[0310] Conclusion: -It was possible to prepare Fortasol 2x concentrate using Eumulgin B1 instead of Polysorbate 80 as an emulsifier.

[0311] - Fortasol-E was stable at a 1:1 ratio with Mhyo antigen even after 55 days at 37°C, while the reference with polysorbate 80 showed disruption and increased particle size

[0312] [Example 2] SVEA-E SVEA-E is a 4x w / v concentrate that formed 25% w / v of the final vaccine.

[0313] The final vaccine contained the following ingredients and strengths: -Eumulgin B1(0.941gr / ml):1.62%w / v -Squalane (d=0.81gr / ml): 3.375%w / v -Vitamin E Acetate (0.953gr / ml): 3.97% w / v Final vaccine density = 0.9892gr / ml

[0314] O / W emulsions were prepared as 4x w / v concentrates. WFI was used as the aqueous phase. High pressure homogenization was used.

[0315] Composition 4x w / v concentrate: Per 100g (104.36ml)

[0316] Oil Phase Eumulgin B1 6.48g Squalane-PE (Kuraray) 13.50g DI-α Tocopherol Acetate 15.88g (purity corrected)

[0317] aqueous phase WFI ad 100.00g

[0318] Preparation: Oil Phase 1. Weigh out the amount of squalane. 2. Add amount of Vitamin E Acetate and homogenize until a clear mixture is obtained. 3. Add the amount of Eumulgin B1 and homogenize at 70 ° C. A clear mixture appears. 4. The mixture is sterilized by filtration using a 0.2 μm membrane filter in a double jacketed filter house at a temperature of 70° C., maintaining the temperature at 70° C. during filtration. 5. Cool to 55-65℃

[0319] aqueous phase 6. Use sterilized (autoclaved) water for injection. Heat to 55-65℃.

[0320] final product 7. Prepare a premix by adding the oil phase to the water phase while mixing with a magnetic or overhead stirrer at 900 rpm. 8. Continue mixing for 5 minutes. 9. Process the premix by passing it three times through a (sterile) microfluidizer at 800 bar (temperature 55-75 °C).

[0321] The SVEA-E 4x concentrate appeared microscopically as an O / W lysate with several droplets of 1-5 μm.

[0322] [Example 3] SVEA-E Double Plus SVEA-E Double Plus is a 2x w / w concentrate.

[0323] The densities of the concentrate and the final vaccine were determined: Concentrate density=0.965g / ml Final vaccine density = 0.986g / ml

[0324] The final vaccine contained the following ingredients and strengths: - Squalane (d=0.81g / ml): 6.77% w / v - Vitamin E Acetate (0.953g / ml): 7.97% w / v -Macrogol cetastearyl ether NS (0.941g / ml): 3.25% w / v -Aerosil 300 pharma: 0.20% w / v

[0325] composition: Per 100g of concentrate (= 50% w / w of final vaccine) Squalane 13.74g Vitamin E Acetate (Purity Corrected) 16.17g Macrogol cetostearyl ether 12NS 6.59g Aerosil 300 pharma 0.41g Water for injection ad 100.00g

[0326] Preparation: 1. Weigh out WFI, Aerosil 300 and Eumulgin B1 (=aqueous phase) and homogenize at 40-50°C until Eumulgin B1 is dissolved. Sterilize at 121°C for 20 minutes while stirring. 2. Weigh out squalane and Vitamin E acetate (=oil phase) and homogenize until a clear composition appears. Sterilize at 70°C by 0.2 μm filtration. 3. Add the oil phase to the aqueous phase while stirring at 55-65 °C with a visible vortex using a magnetic stirrer. 4. Continue stirring at 55-65°C for 5 minutes. 5. Process the premix through a microfluidizer at 800 bar for three passes at 55-75 °C.

[0327] [Example 4] Mhyo antigen in SVEA-E Double Plus The final vaccine contained the following SVEA-E double plus composition: 3.25% w / v polyethoxyethylene 12-cetostearyl ether; 7.97% w / v α-tocopheryl acetate; 6.77% w / v squalane; and 0.20% w / v fumed silica.

[0328] The SVEA double plus reference composition contained in the final vaccine was: 3.24% w / v polysorbate 80; 7.94% w / v α-tocopheryl acetate; 6.75% w / v squalane; and 0.20% w / v fumed silica.

[0329] [Table 2]

[0330] Micrographs of samples IP.1, IP.2 and IP.3 are shown in Figure 1: t=0, after 10 days at 37°C, after 3 weeks at 37°C. [Table 3]

[0331] A graphical representation of the Mastersizer results for the three samples after 10 days at 37° C. is shown in FIG.

[0332] The test sample results reveal a breakdown of the reference adjuvant composition IP.3 after 10 days at 37° C., with Figure 7 showing the appearance of a large peak centred at 10 μm, whereas adjuvant compositions IP.1 and IP.2, both containing the SVEA-E double plus adjuvant, maintain an intact emulsion over time.

[0333] Conclusion: An emulsion vaccine containing a crude Mhyo antigen preparation and an adjuvant composition with Eumulgin B1 as the emulsifier is clearly more stable than a reference emulsion vaccine with the same antigen but with polysorbate 80 as the emulsifier. The reference is destroyed within 10 days at 37°C, whereas the Eumulgin emulsion shows no deterioration in emulsion quality even after 3 weeks at 37°C.

[0334] [Example 5] EVO420 EVO420 is a 2x w / v concentrate containing Vitamin E-Acetate, Shell Ondina X420 and Eumulgin B1. Density is equal to SVEA.

[0335] The final vaccine contained the following ingredients and strengths: -Eumulgin B1: 1.62%w / v -Ondina X420(d=0.81gr / ml):3.375%w / v -Vitamin E Acetate (0.953gr / ml): 3.97% w / v Final vaccine density = 0.9913g / ml

[0336] The O / W emulsion was prepared as a 2x concentrate. WFI was used as the aqueous phase. High pressure homogenization was used.

[0337] Composition of 2x concentrate: Per 100g (101.73ml):

[0338] Oil Phase Eumulgin B1 3.24gr Ondina X420(Shell) 6.75gr DL-α Tocopherol Acetate 7.94gr (purity corrected)

[0339] aqueous phase WFI ad 100.00gr

[0340] Preparation: Oil Phase 1.Weigh out the amount of Ondina X420. 2. Add amount of Vitamin E Acetate and homogenize until a clear mixture is obtained. 3. After adding 100 ml of Eumulgin B1 and homogenizing at 70°C, a clear solution appeared. 4. Heat the mixture to 70°C. 5. Sterilize the mixture by filtration using a nylon 66 or PVDF 0.2 μm membrane filter in a double jacketed filter house at a temperature of 70° C. and maintain the temperature at 70° C. during filtration. 6. Cool to 55-65℃

[0341] aqueous phase 7. Use sterile (autoclaved) WFI heated to 55-65°C.

[0342] final product 8. Prepare a premix by adding the oil phase to the water phase with high shear mixing. 9. Continue mixing for 5 minutes.

[0343] Process the premix by passing it three times through a (sterile) microfluidizer at 800 bar (temperature 55-75 °C).

[0344] Microscopically, a 2x w / v concentrate of EVO420 appears as an O / W nanoemulsion with several droplets of 1 μm to 5 μm.

[0345] [Example 6] Xsolve 2.0 The concentrate is based on Xsolve adjuvant except that mineral oil is replaced by Shell Ondina X GTL based medicated white oil by volume and polysorbate 80 is replaced by Eumulgin B1 by weight.

[0346] Ratio of Microsol-E concentrate:Fortasol-E concentrate in Xsolve 2.0 = 5:1 v / v

[0347] 1. Medicinal white oil concentrate based on Microsol-E and Shell Ondina X GTL: Formulation per 100ml (calculated density = 0.8925g / ml): -Shell Ondina X GTL based medicinal white oil 39.25g -Eumulgin B1 2.00g -WFI 48.00g

[0348] Preparation: aqueous phase 1. Weigh out the amount of WFI 2. After adding 200 ml of Eumulgin B1 and homogenizing at 50°C, a clear solution appeared. 3. Sterilize in an autoclave at 121°C for 20 minutes. 4. Cool to 55-65℃

[0349] Oil Phase 5. Use sterile (0.2 μm filtered) Shell Ondina X GTL based medicinal white oil. Heat to 55-65°C.

[0350] final product 6. Prepare a premix by adding the oil phase to the water phase with high shear mixing 7. Continue mixing for 5 minutes. 8. Process the premix by passing it three times through a (sterile) microfluidizer at 800 bar (temperature 45-55 °C).

[0351] A microemulsion with 99% <1 μm oil droplets should appear.

[0352] Microscopically, the Microsol-E / Shell Ondina X GTL based medicinal white oil adjuvant composition appears as a homogenous O / W emulsion with some droplets of 1 μm to 5 μm.

[0353] 2.Fortasol-E concentrate 2x: Formulation per 100ml (density = 1.00g / ml) -DL-α-tocopherol acetate 15.00g (purity corrected) -Eumulgin B1 6.00g -WFI ad 100.00g

[0354] Preparation: Oil Phase 1. Weigh out DL-α-tocopherol acetate 2. After adding 200 ml of Eumulgin B1 and homogenizing at 50°C, a clear solution appeared. 3. The mixture is sterilized by 0.2 μm filtration at 50°C. 4. Heat to 75-85℃ aqueous phase 5. Use sterile (autoclaved) water for injection. 6. Heat to 75-85℃

[0355] Final product: 1. Slowly add the oil phase to the water phase at 75-85°C while stirring with a visible vortex. 2. After addition, continue stirring for 1 minute. 3. Check microscopy and you should see a nanoemulsion with almost no visible particles.

[0356] Microscopically, the Fortasol-E concentrate appears as a homogeneous O / W emulsion with several droplets of 1 μm to 5 μm.

[0357] Final vaccine formulation and calculated density: Xsolve50 2.0 (0.9552g / ml) per 100ml Microsol Eumulgin B1 / Shell Ondina X GTL-based medicinal white oil concentrate: 37.19g Fortasol-E concentrate 2x: 8.33g Antigen + buffer 50.00g

[0358] Xsolve30 2.0 (0.9731g / ml) per 100ml Microsol Eumulgin B1 / Shell Ondina X GTL-based medicinal white oil concentrate: 22.31g Fortasol-E concentrate 2x: 5.00g Antigen + buffer 70.00g

[0359] Xsolve12 2.0 (0.9893g / ml) per 100ml Microsol Eumulgin B1 / Shell Ondina X GTL-based medicinal white oil concentrate: 8.93g Fortasol-E concentrate 2x: 2.00g Antigen + buffer 88.00g

[0360] [Example 7] Stability test using Xsolve2.0 (Eumulgin B1 / Marcol 52) Polysorbate 80 was replaced by Eumulgin B1 by weight in the commercially available Xsolve oil-in-water concentrate to give Xsolve 2.0 which contains: [Table 4]

[0361] Note: For the purposes of these comparative experiments, these vaccine compositions contained crude Mhyo bacterin antigen in amounts far in excess of those present in the comparable commercial Mhyo O / W vaccines, in order to force the appearance of the effect of the crude antigen on the PS80 emulsifier and, correspondingly, on the stability of the emulsion.

[0362] The adjuvant compositions were subjected to stability testing and the appearance (by optical microscope viewing) and particle size (by Mastersizer measurement) were determined.

[0363] Xsolve 2.0 (Eumulgin B1 / Marcol 52) appears homogeneous after 1 week at 37° C. In contrast, disruption is observed with the reference adjuvant composition (Polysorbate 80 / Mineral Oil).

[0364] Mastersizer results in μm after 1 week at 37°C: [Table 5]

[0365] Conclusion: Polysorbate 80 can be replaced with Eumulgin B1.

[0366] · The use of Eumulgin B1 results in stable emulsions compared to emulsions using Polysorbate 80, which destroys them.

[0367] [Example 8] Efficacy, safety and stability of SVEA-E / FMD vaccine composition Vaccination-challenge experiment An animal study in cattle was performed to compare an FMD vaccine composition based on SVEA-E adjuvant and containing FMD virus-like particles as antigen (referred to as SVEA-E / FMD vaccine) with a classical FMD vaccine formulated with Montanide ISA206. Both vaccines contained a suboptimal but identical dose (i.e., 5 μg / dose) of FMDV Asia1 / Shamir / ISR / 89 VLP antigen so that differences between the groups could be seen.

[0368] Cows were vaccinated intramuscularly (IM) with 2 ml of vaccine on the left side of the neck. Three weeks after vaccination, all animals were challenged with FMDV, Asia1 / Shamir / ISR / 89 strain, by intradermal lingual (IDL) inoculation. Blood samples were taken at several time points after vaccination and challenge to measure serological responses during the study. After challenge, animals were examined under anesthesia for FMD-specific lesions.

[0369] Using the SVEA-E / FMD vaccine composition, virus neutralization titers were found at the same level as those induced by the classical FMD vaccine (Figure 2A). The SVEA-E / FMD vaccine provided complete protection against challenge, whereas 80% protection was observed for the classical FMD vaccine (Figure 2B). In contrast to control animals and the classical ISA206 group, no FMDV-associated viremia was observed after challenge in the SVEA-E / FMD group.

[0370] No local reactions were observed after vaccination in both vaccinated groups.

[0371] Duration of immunity (DOI) in cattle, pigs and goats Animals were immunized with a bivalent FMD vaccine that contained VLPs of two FMD antigens: O / TUR / 5 / 2009 and Asia1 / Shamir / ISR / 89. Only virus neutralization titers against the O / TUR / 5 / 2009 component were determined.

[0372] Duration of immunity in cattle (Figure 3), pigs (Figure 4) and goats (Figure 5) using the SVEA-E adjuvanted FMD vaccine composition was comparable to the DOI observed from vaccination with the corresponding classical Montanide ISA206 adjuvanted vaccine composition.

[0373] Stability of SVEA-E with high antigen loading against Montanide ISA206 [Table 6]

[0374] From these data it can be concluded that vaccine compositions with high antigen loads are stable when based on SVEA-E emulsions but not when based on Montanide ISA206 emulsions.

[0375] NOTE: The O / TUR / 05 / 2009 and Asia1 / Shamir / ISR / 89Shm VLP capsid antigens used in this experiment were of pre-development quality and therefore relatively crude compared to the future FMD VLP antigens of fully developed and optimized commercial product quality, which only makes the stability results even more impressive.

[0376] Antigen mass ELISA can be used to quantitate the VLPs in the vaccine composition without disrupting the emulsion, which is not possible with Montanide ISA206-based formulations. The VLPs in the vaccine are stable. Vaccine composition based on quantification of inactivated FMDV O / TUR / 5 / 2009 VLPs by VHH-ELISA Vaccines formulated with the SVEA-E adjuvant composition A strong immune response was induced in cattle after prime (0 dpv) and booster vaccination 42 days later (see results in table below) After vaccine formulation: Same quantification as before formulation (VHH ELISA) No need to destroy emulsion vaccines based on SVEA-E Vaccine stability monitored: stable for >9 months at 4°C (see Figure 6)

[0377] [Table 7]

[0378] Antigen information: Asia1 / Shamir / ISR / 89 Asia1 / Shamir / ISR / 89 VLPs (containing capsid-stabilizing mutations in the VP2 protein: S093C and K190N) were produced by baculovirus expression system. Tnao38 insect cells were infected with the corresponding recombinant baculovirus at MOI 0.1 in a 2-liter bioreactor. Cell culture supernatants were harvested at 5 dpi by centrifugation and treated with binary ethylenimine (BEI) to inactivate the recombinant baculovirus, followed by concentration by filtration. The vaccine was formulated with SVEA-E adjuvant composition. The concentration of intact VLPs was determined by ELISA using M332F antibody (Harmsen et al., 2017, Front. Immunol., vol. 8, p. 960). Serially diluted samples were incubated at 37°C for 1 h on microtiter plates coated with M332F antibody overnight at 4°C. After removing the samples and washing three times with PBS-Tween, a fixed amount of biotinylated M332F was added to the plate and incubated for 1 h at 37° C. After removing the biotinylated antibody and washing the plate three times with PBS-Tween, peroxidase-conjugated streptavidin was added to the plate for chromophore detection.

[0379] O / TUR / 5 / 2009 Virus-like particles Similarly, O / TUR / 5 / 2009 VLPs (containing a capsid-stabilizing mutation in the VP2 protein: S093C) were produced in a 2-liter bioreactor containing Tnao38 insect cells infected at moi of 1. Cell culture supernatants were harvested at 5 dpi by centrifugation at 200xg. The concentration of intact VLPs was determined by ELISA using VHH C1 (Wang et al., 2015, BMC Veterinary Research 11:120, DOI 10.1186 / s12917-015-0437-2). For this, serially diluted samples were incubated for 1 h at 37°C on microtiter plates coated with C1 overnight at 4°C. After removing the samples and washing three times with PBS-Tween, a fixed amount of biotinylated C1 was added to the plates and incubated for 1 h at 37°C. After removing the biotinylated antibody and washing the plates three times with PBS-Tween, peroxidase-conjugated streptavidin was added to the plates for chromophore detection.

[0380] conclusion Particularly surprising from these results were the following findings: 1. The O / W emulsion type vaccine composition can be used as an FMD vaccine. 2. The efficacy of the vaccine against FMDV challenge infection was even better than that obtained using a similar dose of antigen but formulated in the format of the classical ISO206 W / O / W emulsion vaccine, which is the gold standard type of FMDV vaccine. 3. These vaccines are cross-species effective, and 4. Besides being more effective than current standard type FMD vaccines, the vaccine composition according to the invention has several important further advantages, in particular in terms of emulsion stability but also in handling and processing.

Claims

1. An adjuvant composition comprising an emulsion of water, tocopherol or a pharmaceutically acceptable ester thereof, and polyethoxyethylene cetostearyl ether.

2. 2. The adjuvant composition of claim 1, wherein the polyethoxyethylene cetostearyl ether is selected from polyethoxyethylene 12 cetostearyl ether, polyethoxyethylene 20 cetostearyl ether, and polyethoxyethylene 30 cetostearyl ether.

3. 2. The adjuvant composition of claim 1, wherein the polyethoxyethylene cetostearyl ether is polyethoxyethylene 12 cetostearyl ether.

4. 2. The adjuvant composition of claim 1, wherein the pharmaceutically acceptable ester of tocopherol is α-tocopheryl acetate.

5. The adjuvant composition of claim 1 further comprising squalane.

6. 10. The adjuvant composition of claim 1, which is free of esters of fatty acids.

7. 2. The adjuvant composition of claim 1, further comprising a non-mineral oil, preferably a synthetic oil.

8. 2. The adjuvant composition of claim 1, wherein the adjuvant composition is an oil-in-water emulsion.

9. 2. The adjuvant composition of claim 1, wherein the polyethoxyethylene cetostearyl ether is present in an amount of about 1 to about 9% (by weight) based on the volume of the adjuvant composition.

10. 2. The adjuvant composition of claim 1, wherein the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 1 to about 20% (by weight) based on the volume of the adjuvant composition.

11. 6. The adjuvant composition of claim 5, wherein squalane is present in an amount of about 5 to about 20% (by weight) based on the volume of the adjuvant composition.

12. 7. The adjuvant composition of claim 6, wherein the mineral oil is present in an amount of about 5 to about 40% (by weight) based on the volume of the adjuvant composition.

13. 7. The adjuvant composition of claim 6, wherein the non-mineral oil is present in an amount of about 5 to about 40% (by weight) based on the volume of the adjuvant composition.

14. 10. The adjuvant composition of claim 1, further comprising silica.

15. 2. The adjuvant composition of claim 1, further comprising emulsion particles having a particle size D50 of about 80 to about 200 nm, preferably about 40 to about 100 nm.

16. 10. Use of the adjuvant composition of claim 1 for formulating an emulsion vaccine, preferably an oil-in-water emulsion vaccine.

17. A vaccine composition comprising the adjuvant composition of claim 1 and an antigen.

18. 18. The vaccine composition of claim 17, wherein the antigen comprises a crude antigen.

19. 19. The vaccine composition of claim 17 or 18, wherein the antigen comprises an esterase, preferably a lipase.

20. 18. The vaccine composition of claim 17, wherein the vaccine composition comprises one or more antigens selected from non-live bacterial, parasitic and viral antigens.

21. 21. The vaccine composition of claim 20, wherein the non-live antigen is derived from Porcine Circovirus Type 2, Mycoplasma hyopneumoniae and / or Foot and Mouth Disease Virus.

22. 18. The vaccine composition of claim 17, wherein the vaccine composition is an emulsion vaccine.

23. 23. The vaccine composition of claim 22, wherein the emulsion vaccine is an oil-in-water emulsion vaccine.

24. 18. A method for producing the vaccine composition of claim 17, said method comprising: - preparing an aqueous phase containing the antigen; - mixing said aqueous phase with the adjuvant composition of claim 1; A method comprising:

25. 18. The vaccine composition of claim 17 for use in a method for protecting a human or animal subject from infection and / or disease caused by a pathogen.

26. 26. The vaccine composition for use according to claim 25, wherein the animal subject is a pig or a ruminant.

27. 27. A vaccine composition for use according to claim 25 or 26, wherein the pathogen is selected from bacteria, parasites and viruses.

28. 26. The vaccine composition for use according to claim 25, wherein the pathogen is selected from one or more of Porcine Circovirus Type 2, Mycoplasma hyopneumoniae and Foot and Mouth Disease Virus.

29. 26. The vaccine composition for use according to claim 25, wherein said vaccine composition is administered by intradermal or intramuscular route.

30. 18. A vaccine composition according to claim 17 for use in vaccinating a human or animal subject against infection and / or disease caused by a pathogen.

31. 10. Use of the adjuvant composition of claim 1 for the manufacture of a vaccine composition for protecting a human or animal subject from infection and / or disease caused by a pathogen.

32. 20. A method for vaccination of a human or animal subject against infection and / or disease caused by a pathogen, said method comprising administering to said subject a vaccine composition according to claim 17.

33. A kit of parts comprising at least two containers, one container containing the adjuvant composition of claim 1 and one container containing an antigen.

34. 34. The kit of parts of claim 33, wherein the antigen is lyophilized live PRRSV or a lyophilized preparation of non-live Lawsonia intracellularis.

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