Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/125—Picornaviridae, e.g. calicivirus
  • A61K39/135—Foot- and mouth-disease virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
  • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to the field of vaccinology, more specifically of veterinary vaccinology.
• the invention relates to an adjuvant composition comprising an emulsion of water, tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxyethylene cetostearyl ether as an emulsifier.
• Said adjuvant composition can be used for formulating a vaccine, particularly an emulsion vaccine, including a bacterial, parasitic and/or viral antigen.
• the resulting vaccine composition can be used in a method of protecting a human or animal target against infection and/or disease caused by a pathogen, particularly caused by a bacterium, parasite or virus.
• the invention further relates to methods for the manufacture of such adjuvant compositions and for the manufacture of such vaccine composition.
• Bacterial, parasitic and viral vaccines both for humans and for animals, are well known for over a century, and are available in many forms.
• Such vaccines can be live, i.e. contain replicative (attenuated) bacteria, parasites or viruses, or non-live, i.e. comprise inactivated bacteria, parasites or viruses, or one or more of their components.
• Vaccines that comprise non-live (i.e. non-replicating) antigens often require an adjuvant to provide an immune-stimulation for the non-live antigen.
• an adjuvant for example: mineral oil e.g. BayolTM or MarkolTM, MontanideTM or paraffin oil; non-mineral oil such as squalene, squalane, or vegetable oils, e.g. ethyl oleate; aluminium salts, e.g.
• aluminium hydroxide or aluminium phosphate
• peptides such as dimethylglycine, or tuftsin
• bacterial cell-wall components such as lipid A and muramyldipeptide
• synthetic polymers such as pluronics, dextranes, carbomeres, pyran, or saponin
• cytokines and stimulators of toll-like receptors such as immunostimulatory oligodeoxynucleotides containing non-methylated CpG groups; etc.
• an oil adjuvant can be emulsified with an antigen that is in an aqueous phase, to form an emulsion which in turn can be used for the preparation of a vaccine.
• one liquid phase is dispersed in another, typically as a water-in-oil (W/O) or as an oil-in-water (O/W) type emulsion.
• W/O water-in-oil
• O/W oil-in-water
• a vaccine emulsion can be made up of one or more adjuvants, with one or more emulsifiers.
• Amphigen® Zoetis
• Xsolve® previously called: Microsol-Diluvac Forte®, MSD Animal health
• Tween® 80 Polysorbate 80 or polyoxyethylene sorbitan mono-oleate
• MetaStim® Zoetis
• squalane Pluronic® (a non-ionic tri -block copolymer of blocks of polyoxyethylene and polyoxypropylene), and Tween 80
• Fortasol® a.k.a.
• Diluvac Forte® comprises Vitamin-E acetate and Tween 80; SP-oilTM (PluronicTM, squalane, and Tween); and AS03TM (GSK), which contains 2.1 % w/v squalene and 2.4 % w/v vitamin E, with 1.0 % w/v Tween 80 as emulsifier.
• An emulsion for use as a vaccine should be stable and not ‘break’, meaning that the type, size, and number of the droplets of the dispersed phase should not change too much over time, which could eventually lead to reduction of dispersion and ultimately to a complete phase separation.
• Maintaining the stability of the emulsion is important to guarantee the use and efficacy of an emulsion vaccine during its registered shelf-life. Effects of gravity over time, such as sedimentation or creaming are harmless and can readily be reversed by shaking by hand. However actual breaking of an emulsion is irreversible, and the resulting sub-optimal distribution of the phases may lead to incorrect dosing, to safety issues, and can affect the immunological potency of the vaccine antigen(s).
• oil-adjuvanted emulsion vaccines with bacterial, parasitic and/or viral antigens are commercially available for a large number of pathogens, and for all the major animal target groups: swine, cattle, sheep, poultry, companion animals (e.g. cats, dogs, horses), and fish.
• Non-live bacterial, parasitic, or viral vaccines for use in agriculture will therefore typically comprise little purified antigens derived from, for example, an inactivated bacterial, parasitic or viral culture, or from extracts or fractions of such a culture.
• Such rather crude antigens can be based on inactivated bacteria or parasites or on inactivated viruses, perhaps washed or concentrated once; or antigen based on bacterial-, parasitic- or viral fractions, such as bacterial cells, parasites or viruses that were lysed or disrupted.
• these crude antigen preparations may contain undefined or unintended impurities which can have an effect on the safety, the efficacy, or the stability of a (combination) vaccine.
• vaccines as part of their development process, must undergo rigorous testing for safety, efficacy and stability, before they can receive a marketing authorisation from governmental- or regulatory authorities to place such vaccine on the market as a commercial product.
• the unspecified components of a non-live antigen preparation are not necessarily unwanted, as they may act as a further adjuvant and in this way provide an aspecific boost to the immune response. Also, potentially disturbing factors of a biological nature would be expected to become inactivated during the preparation of the non-live bacterial, parasitic or viral antigens. Nevertheless unwanted effects on safety, efficacy or stability of a vaccine can be observed during the development of an emulsion-based vaccine comprising a crude antigen preparation.
• Another obstacle to overcome in making adjuvanted vaccines is to prevent an interaction between the various vaccine components that would negatively influence the immune response or the vaccine’s safety or stability. Such interaction may for instance occur between the antigens themselves, e.g. because some are quite crude products. Also, the adjuvant may interfere with, or even damage a vaccine antigen. It is thus difficult to develop an adjuvanted vaccine which induces an effective immune-response against one or more antigens especially for complex combinations relating to antigens from multiple species of pathogens.
• the adjuvanted vaccine should be safe upon use in animals, i.e. not produce significant side reactions such as fever, local swelling, loss of appetite, etc. Also more practical properties are relevant: the emulsion vaccine should ideally be capable of economic production, be sufficiently stable during formulation and storage, and allow potency testing methods for each antigen, in the presence of the other antigens.
• Mycoplasma hyopneumoniae and Law sonia inlracellularis are caused by bacteria such as: Mycoplasma hyopneumoniae and Law sonia inlracellularis: and by viruses such as porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome virus (PRRSV).
• Mycoplasma hyopneumoniae (Mhyo) is the primary agent causing (porcine) enzootic pneumonia, a chronic respiratory disease in swine, occurring worldwide. Especially young piglets are vulnerable to this highly contagious disease.
• the bacterium is relatively small, lacks a cell wall, and belongs to the genus Mollicutes. These bacteria live a parasitic lifestyle on or inside host cells.
• Pulmonary disease from Mhyo is largely an immune-mediated pathology leading to consolidated pneumonia.
• the bacterium colonizes and damages the pulmonary ciliated epithelium, leading to loss of ciliary activity.
• the most problematic consequence of this disease is that it predisposes for different secondary infections of the porcine respiratory system, e.g. by other bacterial and viral pathogens. This gives rise to the so called: Porcine Respiratory Disease Complex (PRDC), displaying severe lung lesions.
• PRDC Porcine Respiratory Disease Complex
• Lawsonia intracellularis causes proliferative enteropathy, also known as ileitis, which is a common enteric disease of post-weaning pigs worldwide.
• the characteristic lesion is a proliferation of immature enterocytes in the ileal intestinal crypts, which cells contain the causative bacteria. Clearance of the bacteria from the enterocytes leads to resolution of the associated proliferative lesions. Histologic lesions can be confirmed as /.mi’.sr>///a-positive by visualization of 1.5 - 2.5 pm long, vibrioid shaped bacteria in the enterocytes, but also within intestinal macrophages.
• the bacteria can be detected via PCR in clinical or in subclinical cases. Clinical cases are usually present in the grower-finisher period.
• L. intracellularis bacteria are obligate intracellular, and non-motile gram-negative bacilli, from the Desulfovibrionaceae family.
• PCV2 Porcine circovirus type 2
• PMWS post-weaning multisystemic wasting syndrome
• PCV2 is a very small non-enveloped virus of the Circovirus genus. It contains a circular single stranded DNA genome with two major genes.
• the ORF2 gene encodes the viral capsid protein of about 233 amino acids.
• Recombinantly expressed PCV2 ORF2 proteins form virus-like particles which are highly effective as a subunit vaccine.
• PRRSV Porcine reproductive and respiratory syndrome virus
• WO 2018/115435 describes a combination vaccine for swine comprising non-live antigen from PCV2 and live PRRSV.
• the vaccine is an oil-in-water emulsion comprising squalane and vitamin E-acetate.
• WO 2021/048338 describes a combination vaccine for swine for protection against a pathogenic infection with PCV2 and Mhyo comprising non-live immunogen of PCV2 and non-live immunogen of Mhyo.
• the vaccine is an oil-in-water emulsion comprising squalane, vitamin E- acetate and silica.
• FMD which is a highly contagious and devastating disease of cloven-hoofed animals worldwide has had significant 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 inhibition of susceptible animal movement, slaughter of infected animals and vaccination.
• animals are immunized with inactivated whole virus vaccines to control the spread of foot-and-mouth disease virus (FMDV).
• FMDV foot-and-mouth disease virus
• the vaccine technology used comes with efficacy concerns, particularly with respect to cell-mediated immunity.
• alternative vaccines are currently developed such as recombinant protein and peptide vaccines, empty capsid vaccines and genetically engineered inactivated vaccines.
• the traditional FMD inactivated whole virus vaccines are often formulated in aqueous Al(0H)3 and saponins, or in oil-based adjuvants.
• Al(OH)3/saponin-based vaccines are not ideal for use in pigs as their protective efficacy is low in this species.
• W/O/W emulsion vaccines based on Montanide ISA-206 oil adjuvant are preferred for FMD prevention as they can be used to protect all susceptible species and are ideal for emergency vaccination.
• oil-adjuvanted vaccines generate higher and longer lasting immune responses than the Al(OH)3-adjuvanted vaccines.
• Newly developed mineral oil-based adjuvant Montanide ISA-201 is suggested as an alternative as it seems to induce earlier and higher neutralizing antibody responses, higher cellular immunity and protective efficacy in cattle, as compared to ISA-206.
• ISCOMs immunostimulating complexes
• saponin such as Quil-A
• cholesterol phospholipid
• antigen a group consisting of phospholipid and antigen.
• ISCOMs have been shown to elicit high-titred long-lasting antibodies and strong helper- and cytotoxic T lymphocyte responses.
• ISCOMs have been described as adjuvants for an FMD recombinant protein (C- terminal half of the VP1 protein) vaccine; it was found that a combination of recombinant protein in ISCOMs with Montanide ISA-206 could achieve early protective titres and longer lasting immunity in guinea pigs; it remains to be seen whether FMD vaccines formulated in ISCOMs will be as successful in target species. Also, this technology is rather expensive.
• CpG cytidine-phosphate-guanosine
• ODNs oligodeoxynucleotides
• FMD vaccine adjuvants suitable to induce the desired immunological enhancement such as strong and long-lasting humoral and cellular immunity, while complying with general requirements relating to safety, stability and ease of use, in order to overcome the drawbacks of the currently available adjuvants in FMDV emulsion vaccines.
• the present invention provides an adjuvant composition
• an adjuvant composition comprising an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxyethylene cetostearyl ether as an emulsifier.
• the adjuvant composition can advantageously be used for formulating an emulsion vaccine.
• the invention further provides a vaccine composition comprising said adjuvant composition and an antigen.
• the invention also provides a method for the manufacture of a vaccine composition comprising the claimed adjuvant composition and an antigen, comprising the steps of: a) preparing an aqueous phase comprising an antigen, and b) admixing said aqueous phase with the adjuvant composition as claimed.
• the invention provides the claimed vaccine composition for use in a method of protecting a human or animal target against infection and/or disease caused by a pathogen.
• an adjuvant composition comprising an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxyethylene cetostearyl ether. This was found to solve the occurrence of emulsioninstability, even in complex mixtures, and even when using relatively crude preparations of antigens.
• an adjuvant composition comprising particularly an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxyethylene cetostearyl ether as an emulsifier, provides stability to emulsions of oil and water that contain non-live antigens.
• the inventors do not want to be bound by any theory or model that might explain these findings, they speculate that the sorbitan-based emulsifiers used in the prior art, in particular Polysorbate, were being degraded over time, leading to a loss of emulsifying capacity, and the subsequent breaking of the emulsion.
• the factors degrading the prior art emulsifiers were enzymes that were present in the crude preparation of the non-live antigens.
• the esterases may have been the cause for degrading the emulsifiers, considering that an increase in free fatty acid levels could be observed to correlate with a reduction of the concentration of the prior art emulsifiers, in emulsions that showed breaking.
• esterase enzymes are produced by many of the known bacterial and parasitic genera and in vivo may serve as virulence factor, and/or to mobilise nutritional components. All major families of bacteria, both Gram-positive and Gram-negative, are involved, and both benign or pathogenic strains, such as: Bacillus, Staphylococcus, Pseudomonas, Salmonella, and many more. Consequently such enzymes can be present, in principle, in any preparation of non-live bacterial antigens. A similar consideration applies to crude preparations of non-live parasitic antigens.
• an emulsion vaccine is a complex formulation with a multitude of biological- and chemical compounds, for which it is very difficult to determine which factor is affected, and which component of the composition is the cause, upon the observation of instability of the emulsion.
• components of the culture medium used for growing the bacterial culture e.g. animal serum, broth or extract of animal tissues, peptones or hydrolysates of animal or vegetable origin, lipids, sugars, amino acids, vitamins and minerals, antifoam, antifungals, etc.
• inactivating agent e.g. thiomersal, benzalkonium chloride, formaldehyde, betapropiolactone, or detergent, etc.
• stabilisers dextrane, glycerol, gelatine, amino acids, buffers, etc.
• - preservatives thiomersal, phenoxyethanol, formaldehyde, antibiotics (e.g. gentamycin), components of the micro-organism: fragments of the cell wall, the internal cellular organelles, the nucleic acid; external bacterial organelles such as: pili, fimbriae, or flagella; bacterial proteins, lipids, and carbohydrates, such as: toxins, lipopolysaccharides, various enzymes, etc. one or more adjuvant(s), emulsifier(s), and their trace elements or impurities. Further the stability of the emulsion may be affected by one or more effects from physical factors in the preparation, purification, formulation, emulsification, filling, transport and storage of the vaccines.
• antibiotics e.g. gentamycin
• components of the micro-organism fragments of the cell wall, the internal cellular organelles, the nucleic acid; external bacterial organelles such as: pili, fimbriae, or flagella
• the invention relates to an adjuvant composition
• an adjuvant composition comprising an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxyethylene cetostearyl ether.
• the tocopherol or the pharmaceutically acceptable ester thereof acts as an oily adjuvant
• the polyethoxyethylene cetostearyl ether acts as an emulsifier
• the water used for the adjuvant composition according to the invention is preferably water of high purity, and is of pharmaceutical quality grade and suitable for parenteral injection; such quality water is typically sterile, and essentially free from pyrogens, and is for example: (multiply) distilled water, reverse osmosis water, or water for injection (WFI).
• quality water is typically sterile, and essentially free from pyrogens, and is for example: (multiply) distilled water, reverse osmosis water, or water for injection (WFI).
• An responsibleadjuvant“ is a substance that increases or modulates the immune response, e.g. to a vaccine.
• an adjuvant provides an immune-stimulation for an antigen, particularly a non-live antigen, which would otherwise not be sufficiently immunogenic. This will trigger different routes of the immune system, however the basic mechanisms of this process are not well understood.
• An “emulsion” is a mixture of at least two immiscible liquids, whereby one is dispersed in another. Typically the droplets of the dispersed phase are very small, with diameters of a few micrometres or less.
• An emulsifier for the invention is a molecule with amphiphilic properties, having both a hydrophobic- and a hydrophilic side. Many emulsifiers are known in the art with their various properties. Most are readily available commercially, and in different degrees of purity.
• the term “discy ethoxy ethylene cetostearyl ether“ refers to a class of hydrophilic, non-ionic emulsifiers that are used in the manufacturing of various O/W emulsions. This term is used interchangeably in this application with the term “polyoxyethylene cetostearyl ether”.
• Polyoxyethylene cetostearyl ether is an ether of cetostearyl alcohol. Cetostearyl alcohol, cetearyl alcohol or cetylstearyl alcohol is a mixture of fatty alcohols, consisting predominantly of cetyl (16 C) and stearyl alcohols (18 C) and is classified as a fatty alcohol.
• the term “polyethoxyethylene cetostearyl ether” has the same meaning as “a mixture comprising a poly ethoxy ethylene cetyl ether and a poly ethoxy ethylene stearyl ether”.
• Particular 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).
• Polyethoxyethylene 12 cetostearyl ether is commercially available under a variety of commercial names, e.g. as Eumulgin® Bl, Kolliphor® CS 12, Volpo® CS 12, Cremophor® A25, Simulsol tm , or Brij CS12®.
• Poly ethoxy ethylene 12 cetostearyl ether finds application in antiperspirants and deodorants, sun care (after-sun, self-tanning, sun protection), body-, colour- and face care products. It is also used in baby care and cleansing, face cleansing, hair colouring and conditioning formulations. It is further used as a mild, non-ionic emulsifier for pharmaceutical oil-in-water emulsions; delivering good sensory properties during product application.
• Polyethoxyethylene 12 cetostearyl ether is a component of the adjuvant known as AF03, which comprises: Squalene (NB: thus not squalane), Eumulgin® Bl (polyethoxyethylene 12 cetostearyl ether), MontaneTM 80 (sorbitan oleate), Mannitol, and phosphate buffered saline.
• Squalene NB: thus not squalane
• Eumulgin® Bl polyethoxyethylene 12 cetostearyl ether
• MontaneTM 80 sorbitan oleate
• Mannitol phosphate buffered saline
• Tocopherol refers to a class of organic chemical compounds having vitamin E activity.
• Tocopherol includes a-Tocopherol (CAS number: 10191-41-0), P-Tocopherol (CAS number: 148-03-8), y-Tocopherol (CAS number: 54-28-4), and 5-Tocopherol (CAS number: 119-13-1).
• esters of tocopherol particularly include alpha- tocopheryl-acetate which is also termed tocopheryl acetate or vitamin E-acetate having CAS number 58-95-7.
• Alpha-tocopheryl -acetate can be derived from vegetable materials such as seeds, nuts, fruits or leaves, or from fatty meats, but may also be produced synthetically.
• 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 different degrees of purity.
• alpha-tocopheryl -acetate for the adjuvant composition according to the invention is preferably DL-alpha-tocopherol -acetate, which is the racemate of the chemical with CAS number: 7695-91-2.
• any such text section, paragraph, claim, etc. can also relate to one or more embodiment s) wherein the term “comprises” (or its variations) is replaced by terms such as “consist of’, “consisting of’, or “consist essentially of’.
• the adjuvant composition may further comprise a pharmaceutically acceptable carrier.
• the adjuvant composition may be identical to the vaccine composition as described herein, but lacking an antigen.
• a “pharmaceutically acceptable carrier” for the invention is an aqueous liquid of a high grade of purity and preferably sterile, for example: water, a physiological salt solution, or a phosphate buffered saline solution.
• the carrier can comprise further additives, such as stabilisers or preservatives.
• the poly ethoxy ethylene cetostearyl ether is selected from poly ethoxy ethylene 12 cetostearyl ether, poly ethoxy ethylene 20 cetostearyl ether, and poly ethoxy ethylene 30 cetostearyl ether. Even more preferably, the polyethoxyethylene cetostearyl ether is polyethoxyethylene 12 cetostearyl ether.
• the pharmaceutically acceptable ester of tocopherol is an alpha-tocopheryl acetate.
• the adjuvant composition according to the invention comprises polyethoxyethylene 12 cetostearyl ether and alpha-tocopheryl acetate.
• the adjuvant composition according to the invention is free of ester surfactants, such as esters of fatty acids, particularly of esters formed from sugar alcohols or their derivatives and fatty acids such as sorbitan or mannide esters.
• ester surfactants such as esters of fatty acids, particularly of esters formed from sugar alcohols or their derivatives and fatty acids such as sorbitan or mannide esters.
• esters are commonly used as surfactants in oil-based adjuvant compositions.
• Sorbitan esters are also known as polysorbates, Tweens or Spans.
• Particular examples of esters include polyoxyethylene sorbitan mono-oleate (Polysorbate 80 or Tween® 80), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), sorbitan monolaurate (Span 20).
• Other examples include esters of octadecanoic acid and anhydromannitol (contained in Montanide ISA-206
• the vaccine composition according to the invention is free of esters of fatty acids.
• the adjuvant composition according to the invention further comprises squalane.
• “Squalane” is a well-known compound, and preferably refers to the chemical compound with CAS number 111-01-3. Some alternate names are: hydrogenated shark liver oil, hexamethyltetracosane, or perhydrosqualene. This is not to be confused with squalene (CAS nr. 111-02-4) which is a poly-unsaturated C30 oil and is metaboliseable as a compound of the cholesterol pathway. However, squalane is the fully hydrogenated form of squalene and is therefore not prone to oxidation. Thus, while squalane is a non-mineral oil, and is transported from the injection site, it is non-metaboliseable.
• squalane is commercially available in a variety of purities, for example: from vegetable source, from Worlee (Squalane, vegetable), or Croda (Pripure Squalane); or synthetic, e.g. from Kuraray (Squalane-PE).
• a high purity of the squalane is preferred: preferably over 75 % purity, more preferably over 80, 90, or even over 95 % purity, in that order of preference.
• the adjuvant composition according to the invention further comprises a mineral oil.
• mineral oil suitable for use in the adjuvant composition examples include, for example, BayolTM or MarkolTM, MontanideTM or a light (or white) liquid paraffin oil, such as Marcol® 52 (Exxon Mobile) or Drakeol® 6VR (Penreco) or Klearol® (Sonnebom).
• the adjuvant composition according to the invention further comprises a nonmineral oil.
• Non-mineral oils can be of synthetic, animal or vegetable origin.
• the non-mineral oils are preferably biodegradable (metabolisable) and biocompatible.
• synthetic oils suitable for use in the adjuvant composition include, for example, a Shell Ondina® oil, e.g. Shell Ondina X420.
• animal oils suitable for use in the adjuvant composition include, for example, fish oil.
• oils suitable for use in the adjuvant composition include oils derived from nuts, seeds and grains, such as peanut oil, soybean oil, coconut oil, and olive oil; jojoba oil; safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil; corn oil, or the oil of other cereal grains such as wheat, oats, rye, rice, teff, and triticale.
• the adjuvant composition according to the invention comprises polyethoxyethylene 12 cetostearyl ether, alpha-tocopheryl acetate, and squalane.
• the adjuvant composition according to the invention comprises polyethoxyethylene 12 cetostearyl ether, alpha-tocopheryl acetate, and a non-mineral oil.
• the non-mineral oil is a synthetic oil, such as a Shell Ondina X GTL- based medicinal white oil.
• the adjuvant composition according to the invention comprises polyethoxyethylene 12 cetostearyl ether, alpha-tocopheryl acetate, and a mineral oil.
• the mineral oil is a light liquid paraffin oil.
• the adjuvant composition according to the invention is an oil-in-water emulsion.
• oil-in-water emulsion is a well-known composition, comprising an outer aqueous phase, which contains an internal dispersed oily phase; for the invention, the dispersed phase is formed by the droplets of the oily adjuvant (tocopherol or a pharmaceutically acceptable ester thereof).
• oil phase is a liquid based on an oil.
• An “oil” is used herein in its common meaning and refers to a nonpolar chemical substance that is hydrophobic and lipophilic, with a high hydro-carbon content.
• An oil can be of mineral origin, or of non-mineral such as of synthetic, animal or vegetable origin. Some non-mineral oils are metabolisable.
• the oily phase may contain excipients such as an emulsifier.
• the oil-phase is the dispersed phase, as is typical for an O/W emulsion.
• the oilphase serves as adjuvant.
• Much used mineral oil adjuvant in veterinary vaccines is a 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).
• aqueous phase is a liquid based on water.
• the aqueous phase may contain e.g. a buffer or saline, and one or more excipients such as an emulsifier or a stabiliser.
• the size of the particles of the dispersed phase of the inventive adjuvant composition is preferably quite small.
• the average diameter of the particles of the dispersed phase is below about 1 micrometre, such emulsions are commonly called “submicron emulsions”.
• the emulsion is a submicron emulsion.
• particle size is expressed in nanometres (nm), and as an average particle size, also known as median diameter, expressed as the D50 of a cumulative particle size distribution.
• particle size is expressed in nm of D50, as determined using a MastersizerTM (Malvern Instruments). Particle size measurements can be made in the (concentrated) oily emulsion constituting the adjuvant composition or the vaccine composition; the particle refractive index of the oily phase for the invention is 1.48.
• the Malvern Mastersizer size analysis report presents D50 as D(0.50).
• Preferred process for high-energy emulsification for preparing the adjuvant composition according to the invention is the use of a high-pressure homogeniser, preferably a MicrofluidiserTM (Microfluidics). Typically 1 - 3 passages at a pressure of between 500 - 1500 bar (i.e. 7000 - 22000 psi) will be sufficient.
• Emulsions prepared in this way typically have dispersed phase particles with a D50 of 500 nm or less, and have a narrow size distribution. Such an emulsion is called a nano emulsion.
• emulsions with such very finely sized particles of the dispersed phase are prepared in several consecutive steps.
• an initial relatively coarse oily emulsion is prepared by low-energy mixing, which is followed by one or more subsequent high-energy treatments to achieve further reduction of particle size.
• the ‘microfluidised’ oily emulsion, comprising the adjuvant(s) and emulsifier in water is then combined with the aqueous phase with or without the antigens, to prepare the adjuvant composition, respectively the vaccine composition according to the invention.
• the oil-droplets i.e. (emulsion) particles
• the oil-droplets i.e. (emulsion) particles
• the emulsion particles have a D50 of 500 nm or less; preferably D50 is 250 nm or less. More preferred: D50 is 150 nm or less.
• the emulsion particles have a particle size D50 of about 80 to about 200 nm, more preferably of about 40 to about 100 nm.
• the size distribution of the oil-droplets in the submicron oil-in-water emulsion of the adjuvant composition or the vaccine composition according to the invention is preferably relatively narrow.
• An indicator of particle size distribution is the D90 of a cumulative particle size distribution. Therefore, in an embodiment of the submicron oil-in-water emulsion of the adjuvant composition or the vaccine composition according to the invention, the oil-droplets have a D90 below 900 nm, more preferred D90 is below 500 nm, 400 nm, or even below 300 nm, in that order of preference. Most preferred: D90 is equal to or smaller than about 250 nm.
• the adjuvant composition or the vaccine composition according to the invention comprises emulsion particles having a particle size D50 of about 80 to about 200 nm, preferably of about 40 to about 100 nm.
• the polyethoxy ethylene cetostearyl ether emulsifier is present in an amount of about 1 to about 9 % by weight based on the weight of the adjuvant composition (w/w).
• the polyethoxyethylene cetostearyl ether is present in an amount of about 2 to about 7 % w/w of the adjuvant composition.
• polyethoxy ethylene cetostearyl ether is present in an amount of about 6 to about 7 % w/w of the adjuvant composition.
• polyethoxy ethylene cetostearyl ether is present in an amount of about 2 to about 4% w/w of the adjuvant composition.
• “about” indicates that a number can vary between ⁇ 25 % around its indicated value.
• “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, in that order of preference.
• a tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 1 to about 20 % by weight based on the weight of the adjuvant composition.
• the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 6 to about 16 % w/w of the adjuvant composition.
• the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 14 to about 16 % w/w of the adjuvant composition. In another particularly preferred embodiment the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 2 to about 8 % w/w of the adjuvant composition. Most preferred: the tocopherol or a pharmaceutically acceptable ester thereof, particularly alpha- tocopheryl acetate, is present in an amount of about 15 % w/w of the adjuvant composition.
• squalane is present in an amount of about 5 to about 20 % by weight based on the weight of the adjuvant composition.
• squalane is present in an amount of about 6 to about 15 % w/w of the adjuvant composition.
• squalane is present in an amount of about 13 to about 14 % w/w of the adjuvant composition.
• the mineral oil is present in an amount of about 5 to about 40 % by weight based on the weight of the adjuvant composition. In a particularly preferred embodiment the mineral oil is present in an amount of about 6 to about 7 % w/w of the adjuvant composition.
• 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.
• 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. In 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.
• 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.
• the adjuvant composition according to the invention can be prepared as a concentrate.
• Said concentrate can be, for example, a 2x w/v concentrate which means that the concentrate represents 50 % of the final volume of the vaccine.
• the concentrate is, for example, a 4x w/w concentrate, this means that the concentrate represents 25 % of the weight of the final vaccine, and so forth.
• the adjuvant composition comprises about 5 to about 7 % w/w, preferably about 6 % w/w of poly ethoxy ethylene 12 cetostearyl ether, and about 14 to about 16 % w/w, preferably about 15 % w/w of alpha-tocopheryl acetate.
• this adjuvant composition is referred to as Fortasol-E.
• Fortasol-E is a 2x w/v concentrate of the final vaccine which means that the concentrate represents 50 % of the volume of the final vaccine.
• the Fortasol-E formulation is a nano emulsion.
• the adjuvant composition according to the invention comprises about 6 to about 7 % w/w of polyethoxyethylene 12 cetostearyl ether, about 15 to about 16 % w/w of alpha-tocopheryl acetate and about 13 to about 14 % w/w of squalane.
• this adjuvant composition is referred to as SVEA-E.
• SVEA-E is a 4x w/v concentrate, which is comprised as 25 % of the volume of the final vaccine.
• the SVEA-E adjuvant composition is a nano emulsion.
• the adjuvant composition according to the invention comprises about 3 to about 4 % w/w of poly ethoxy ethylene 12 cetostearyl ether, about 7 to about 8 % w/w of alpha-tocopheryl acetate and about 6 to about 7 % w/w of a non-mineral oil.
• this adjuvant composition is referred to as EVO420.
• EVO420 is a 2x w/v concentrate which is comprised as 50 % of the volume of the final vaccine.
• the EVO420 adjuvant composition is a nano emulsion.
• a vaccine composition according to the invention comprising the SVEA-E adjuvant composition and further comprising Orf2 antigen of PCV2 and crude bacterin antigen of Mhyo, was found to be stable even after 27 months at 4 °C.
• a similar vaccine composition with SVEA- E and comprising FMDV empty capsids as the antigen was found to be stable up to 8 months at 4 °C.
• the adjuvant composition according to the invention further comprises silica.
• 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, more preferably in an amount of about 0.4 % w/w of the adjuvant composition.
• Fumed silica refers to silicium dioxide with an average particle size of about 6 to about 13 nm. Fumed silica is commercially available under the tradename Aerosil 380 (average particle size of 7 nm). Pharmaceutical quality grade fumed silica is commercially available as Aerosil 300 (average particle size of 7 nm, which has a smaller surface area compared to Aerosil 380) or Aerosil 200 (average particle size of 12 nm).
• the adjuvant composition according to the invention comprises about 6 to about 7 % w/w of polyethoxyethylene 12 cetostearyl ether, about 15 to about 17 % w/w of alpha-tocopheryl acetate, about 13 to about 14 % w/w of squalane and further about 0.3 to 0.5 % w/w of fumed silica.
• this adjuvant composition is referred to as SVEA-E double plus.
• SVEA-E double plus is a 2x w/w concentrate, which is comprised as 50 % of the weight of the final vaccine.
• the SVEA-E double plus adjuvant composition is a nano emulsion.
• the adjuvant composition according to the invention comprises only one third of the amount of emulsifier of the SVEA-E composition, i.e. about 2 to about 2.3 % w/w of polyethoxyethylene 12 cetostearyl ether.
• this adjuvant composition is referred to as microSVEA-E.
• the microSVEA-E adjuvant composition has emulsion particles in the micrometre range and, therefore, represents a microemulsion.
• microSVEA-E double plus comprises only one third of the amount of emulsifier of the SVEA-E double plus composition, i.e. about 2 to about 2.3 % w/w of polyethoxyethylene 12 cetostearyl ether.
• this adjuvant composition is referred to as microSVEA-E double plus.
• the microSVEA-E double plus adjuvant composition has emulsion particles in the micrometre range and, therefore, represents a microemulsion.
• the adjuvant composition according to the invention comprises about 2 to about 3 % w/w polyethoxyethylene 12 cetostearyl ether, about 2 to about 3 % w/w alpha-tocopheryl acetate and about 30 to about 40 % w/v; preferably about 35 % of a non-mineral oil.
• this adjuvant composition is referred to as Xsolve2.0.
• the Xsolve2.0 adjuvant composition is a nano emulsion.
• the adjuvant composition according to the invention can be used for formulating an emulsion vaccine, e.g. a vaccine composition according to the invention.
• ‘formulation’ regards the preparation of the vaccine composition according to the invention, by the admixing of an aqueous phase comprising one or more antigens as described herein, and the adjuvant composition according to the invention.
• the adjuvant composition according to the invention can be used for formulating a non-live vaccine, e.g. a vaccine composition according to the invention comprising a non-live antigen as described herein.
• the adjuvant composition according to the invention does not contain (i.e. is free from) an emulsifier that is susceptible to degradation resulting from a crude bacterial- or parasitic antigen as defined for the invention; preferably susceptible to degradation by an esterase or lipase as defined herein. More preferably, the emulsion does not contain a sorbate- based emulsifier, or even: does not contain a Polysorbate; even more preferably: does not contain a Polysorbate and a Sorbitan mono-oleate. In a further aspect, the invention relates to a vaccine composition comprising the adjuvant composition according to the invention and an antigen.
• an “antigen” is a substance that is capable of inducing an immunological reaction in a target human or animal, possibly with the help of an immunostimulating compound such as an adjuvant.
• Antigens can be prepared synthetically or can be derived from a biological source, for example they can be a microorganism (replicative or not), or can be a part thereof, e.g. a protein, lipid, carbohydrate, or nucleic acid, or combinations thereof, e.g.: a peptidoglycan, a lipoglycan, a lipopeptide, or a lipopolysaccharide, etc.
• non-live (non-replicating) antigen relates to molecules such as proteins, carbohydrates, lipids or nucleic acids, or are complex combinations thereof, more or less pure.
• non-live antigen can refer to a killed (i.e. non-replicative) microorganism, or can be a part thereof such as an extract, fraction, homogenate, or sonicate.
• a non-live antigen can be a nucleic acid based, or recombinant product, such as an expression vector or an expressed protein, or the product of an in vitro expression system. All these are well-known in the art.
• Live antigen refers to live (i.e. replicative) bacteria, parasites, or viruses that are suitable for use as a vaccine component, i.e. having a reduced level of pathogenicity, also known as being attenuated, or modified live.
• Attenuated is defined as causing a lower level of lesions, and/or having a reduced rate of infection, or of replication. All, as compared to unmodified or ‘wildtype’ bacteria, parasites, or viruses.
• Attenuation of a micro-organism can be obtained in vitro, for instance by passageing through experimental animals or in cell-culture and selection, or via recombinant DNA technology, all well known in the art.
• live While it is biologically incorrect to refer to a virus as being “live”, that is the common way to refer to a virus that is replicative and not inactivated. Consequently, for the invention the term “live” as relating to a virus refers to a virus that is capable of replication under appropriate conditions, e.g. in suitable host cells or animals.
• the vaccine composition according to the invention comprises an antigen of a bacterium, a parasite, and/or a virus.
• the antigen is preferably a non-live antigen.
• the vaccine composition can comprise one or more further antigens, e.g. from a bacterium, parasite, virus, fungus, ectoparasite, etc.
• the further antigen can be live or non-live.
• the vaccine composition comprises one or more antigens selected from a non-live antigen of a bacterium, of a parasite, and of a virus.
• the vaccine composition contains multiple (two or more) antigens, more preferably two or more bacterial antigens.
• a “bacterial antigen” is an antigen that is derived, based on, or obtained from a bacterium.
• a bacterium is a prokaryotic microorganism that is currently classified in the taxonomic super kingdom of Bacteria.
• a “parasite antigen” is derived, based on, or obtained from a parasite.
• a parasite is a eucaryotic microorganism; for example as currently classified in the clade Apicomplexa, or the phylum Nematoda.
• Ectoparasites are e.g. flies, flees, mites, or ticks.
• Fungi are for example Aspergillus.
• a concentrated viral antigen is an antigen that is derived, based on, or obtained from a virus.
• the antigen included in the vaccine composition according to the invention is derived from a micro-organism that is pathogenic to the target species which is to be protected against infection and/or disease by the vaccine.
• micro-organisms pathogenic to porcine animals are:
• Viruses porcine circovirus type 2, porcine parvovirus, African swine fever virus, swine influenza virus, porcine respiratory coronavirus, porcine cytomegalovirus, porcine rotavirus, porcine poxvirus, porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus, classical swine fever virus, swine influenza virus, foot-and-mouth disease virus, porcine epidemic diarrhoea virus, transmissible gastro-enteritis virus, and vesicular stomatitis virus.
• porcine circovirus type 2 porcine parvovirus
• African swine fever virus African swine fever virus
• swine influenza virus porcine respiratory coronavirus
• porcine cytomegalovirus porcine rotavirus
• porcine poxvirus porcine reproductive and respiratory syndrome virus (PRRSV)
• PRRSV porcine reproductive and respiratory syndrome virus
• pseudorabies virus classical swine fever virus
• swine influenza virus foot
• Bacteria Mycoplasma, Lawsonia, Escherichia, Brachyspira, Streptococcus, Salmonella, Clostridium, Actinobacillus, Pasteurella, Haemophilus, Erysipelothrix, Leptospira, Burkholderia, Enterococcus, Mycobacterium, and Bordetella.
• Toxoplasma Toxoplasma, Isospora, and Trichinella.
• micro-organisms that are pathogenic to ruminants are: For cattle: Neospora, Dictyocaulus, Cryptosporidium, Ostertagia, bovine rotavirus, bovine viral diarrhoea virus, bovine coronavirus, infectious bovine rhinotracheitis virus (bovine herpes virus 1), bovine paramyxo virus, bovine parainfluenza virus, bovine respiratory syncytial virus, rabies virus, bluetongue virus, Escherichia, Salmonella, Staphylococcus, Streptococcus Mycobacterium, Brucella, Clostridium, Pasteurella, Yersinia Mannheimia, Haemophilus, Leptospira, Bacillus, Moraxella, Francisella, Histophilus, Trueperella, Fusobacterium. Actinomyces, Coxiella, Campylobacter, Erysipelothrix, Listeria, Burkholderia, Nocardia
• cervines Epizootic haemorrhagic disease virus, bluetongue virus, papilloma virus, Borrelia burgdorferi, Mycobacterium bovis, and Trueperella pyogenes.
• the vaccine composition according to the invention can comprise a crude preparation of a bacterial or parasitic antigen. Because of the emulsion-stabilizing effect provided by the components of the adjuvant composition according to the invention, no elaborate and expensive purification of bacterial and/or parasitic antigens is required.
• crude refers to an antigen from a bacterium, parasite or virus, as used herein, which was prepared without much (or any) further purification after they were harvested from an in vitro culture, and inactivated and/or lysed or disrupted. Such crude antigen preparations still contain undefined or unintended impurities which can have an effect on the stability of a vaccine based on an emulsion of oil and water.
• the antigen to be comprised in the vaccine composition according to the invention is typically contained in a liquid, such as an aqueous buffer.
• the advantageous effect of the present invention applies particularly to the use of such vaccine composition for the agricultural sector.
• the antigen is a nonlive bacterial antigen; preferably the non-live bacterial antigen is an inactivated whole bacterial culture (i.e. a bacterin), or is part of such culture.
• the part of the inactivated whole bacterial culture is selected from: a pellet, supernatant, concentrate, dialysate, extract, sonicate, lysate, and fraction of such a culture.
• a “bacterial culture” or “a part thereof’ is well-known to a skilled person, and is described in handbooks and manuals such as “Veterinary vaccinology” (supra).
• the inactivated bacterial culture is used either as a whole, i.e. as the complete content of a particular culture vessel, or as a part thereof.
• inactivation of bacteria can be performed using chemical or physical means; physical means are e.g. heating, irradiation, or very high pressure; chemical means are e.g. incubation with merthiolate, formalin, diethylamine, binary ethylenamine, beta propiolactone, benzalkonium chloride or glutaraldehyde.
• a supernatant or a pellet can be prepared by centrifugation.
• a concentrate or a dialysate can be prepared e.g. by a method of cross-flow filtration.
• An extract can be made for example by washing or incubation with a solvent or a detergent solution;
• the solvent can be a liquid or a gas, the liquid can e.g. be aqueous such as water or a buffer; an organic solvent such as an alcohol, acetone, or ether; or can be a supercritical liquid, etc.
• the extract is the part that is removed with the solvent, and is often retrieved from that solvent in a subsequent process.
• a sonicate can be prepared using a sonification device, for example a flow-through sonification cell.
• a lysate can be prepared by physical or (bio-)chemical means, e.g. using a French press, or using an enzymatic treatment.
• a fraction is a part of a whole that is purified from the rest, for example a filtrate or a precipitate, whereby the fraction is the retentate.
• bacterin Most used in bacterial vaccines for agricultural use, is a non-live bacterial antigen which comprises inactivated bacterial cells. Commonly such an antigen preparation of killed bacterial cells is called a bacterin.
• the inactivated bacterial cells can be in any form, and can be intact or can be damaged.
• the inactivated bacterial cells can be at any level of purity, for example can be with the bacterial culture medium in which they were fermented, or can be without the culture medium, for example resulting from sedimentation, centrifugation, or concentration. Therefore in an embodiment of the vaccine composition according to the invention, the non-live bacterial antigen comprises inactivated bacterial cells.
• the bacteria from which the non-live antigen is prepared can be any bacterium of human medical- and/or of veterinary relevance, for example any bacterium that is a (potential) pathogen, either as primary- or as secondary (opportunistic) pathogen.
• suitable bacterial antigens include Actinobacillus pleuropneumoniae. Mycoplasma hyopneumoniae (Mhyo), Lawsonia inlracellularis. Erysipelothrix rhusiopathiae , and Leptospira, or a part of any of those, alone or in any combination.
• combinations of non-live bacterial antigen from two or more bacteria can also be prepared and used. Also, combinations with antigens from viral- or parasitic pathogens are desired.
• 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.
• a viral antigen is preferably derived from an in vitro culture of virus and host cells; the virus may be obtained from the culture supernatant, from the pellet, or may be comprised in the host cells.
• pathogenic viruses are known to be relevant to veterinary medicine.
• Exemplary viral antigens include PCV2, PRRSV and FMDV, or a part of any of those, alone or in any combination.
• An antigen for the invention may also be comprised in the vaccine composition according to the invention by way of a recombinant vector; such a vector may be a nucleic acid or a replicon particle (RP); the nucleic acid is preferably a eukaryotic expression plasmid or an RNA molecule; the RP is preferably an Alphavirus RP.
• a recombinant vector may be a nucleic acid or a replicon particle (RP); the nucleic acid is preferably a eukaryotic expression plasmid or an RNA molecule; the RP is preferably an Alphavirus RP.
• the antigen comprises a viral antigen.
• the viral antigen comprises FMDV, preferably non-live antigen of FMDV, e.g. recombinantly expressed virus-like particles of FMDV.
• the FMDV antigen is particularly preferably a recombinantly expressed empty capsid of FMDV.
• the non-live antigen is selected from one or more of porcine circovirus type 2, Mycoplasma hyopneumoniae, and foot- and-mouth disease virus.
• one or more further non-live antigen may be comprised, selected from PRRSV, or L. intracellularis.
• a “vaccine” is a well-known composition with a medical effect, and comprises an immunologically active component, and a pharmaceutically acceptable carrier.
• carrier for the invention functions the aqueous phase, or the adjuvant composition according to the invention itself.
• the ‘immunologically active component’ for the invention is the antigen from a bacterium, parasite, or virus, or is a combination of antigens from one or more of those microorganisms.
• the vaccine composition according to the invention stimulates the immune system of a target human or animal, and induces a protective immunological response.
• the response may originate from the targets’ innate- and/or from the acquired immune system, and may be of the cellular- and/or of the humoral type.
• a vaccine provides protection “against infection and/or disease” by reducing in a vaccinated target the severity of an infection, for example by reducing the number of pathogens, or shortening the duration of the pathogen’s replication in the target, and reducing the number, the intensity, or the severity of lesions caused by infection with a pathogen. Also, or consequentially, a vaccine is effective in reducing or ameliorating the (clinical) symptoms of disease that may be caused by such infection or replication, or by the target’s response to that infection or replication.
• a reference for such diseases and clinical signs is: "The Merck veterinary manual" (supra).
• Such a vaccine is colloquially referred to as a: vaccine ‘against’ the particular pathogen, e.g. as an ‘Mhyo vaccine’, or as an ‘FMDV vaccine’ .
• a vaccine In order to be immunologically effective, a vaccine needs to contain a sufficient amount of the antigen. How much that is, is either already known from related vaccines, or can readily be determined e.g. by monitoring the immunological response following vaccination and (in the case of an animal target) a challenge infection, e.g. by monitoring the targets’ signs of disease, clinical scores, or by re-isolation of the pathogen, and comparing these results to a vaccinationchallenge response seen in mock-vaccinated animals.
• the amount of the bacterial-, parasitic-, or viral antigen for the vaccine composition according to the invention can be expressed in different ways, depending on the type of the antigen employed.
• the antigen dose can be expressed as a number of bacterial cells, parasites, or virions, counted before they were inactivated.
• the antigen can be quantified by a serologic- or bio-chemical test such as an ELISA or an AlphaLisaTM, and expressed in relative units, compared to an appropriate reference standard. All these are well known in the art.
• the vaccine composition according to the invention can be used as a prophylactic-, metaphylactic-, or therapeutic treatment.
• the vaccine composition according to the invention can serve as an effective priming vaccination, which can later be followed and amplified by a booster vaccination, with the same or with a different vaccine.
• the vaccine composition according to the invention can additionally comprise other compounds, such as an additional antigen or micro-organism, a cytokine, or an immunostimulatory nucleic acid comprising an unmethylated CpG, etc.
• the vaccine composition according to the invention may itself be added to a vaccine.
• the vaccine composition according to the invention is of particular relevance in the field of animal husbandry.
• the non-live bacterial antigen for the invention is derived from a bacterium a bacterial family such as:
• the bacterium is selected from one of: Pasteurella, Escherichia, Salmonella, Yersinia, Staphylococcus, Streptococcus, Mycobacterium, Moraxella, Bacillus, Brucella, Clostridia, Mannheimia, Haemophilus, Francisella, Fusobacterium, Histophilus, Fusobacterium, Trueperella (Arcanobacterium), Actinomyces, Clostridia, Coxiella, Campylobacter, Erysipelothrix, Leptospira, Listeria, Burkholderia, Nocardia, Mycoplasma, Bacteroides, and Chlamydia.
• the bacterium is selected from one of: Mycoplasma, Lawsonia, Escherichia, Brachyspira, Streptococcus, Salmonella, Clostridia, Actinobacillus, Pasteurella, Haemophilus, Erysipelothrix, Leptospira, Burkholderia, Enterococcus, Mycobacterium, and Bordetella.
• the non-live antigen is from porcine circovirus type 2, Mycoplasma hyopneumoniae, and/or foot- and-mouth disease virus.
• the emulsifier is present in an amount of about 1 to about 5 % by weight based on the volume of the vaccine composition (w/v).
• the emulsifier is present in an amount of about 1 to about 4 % w/v of the vaccine composition.
• 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.
• a tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 2 to about 9 % by weight based on the volume of the vaccine composition.
• the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 3 to about 8 % w/v of the vaccine composition.
• the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 2 to about 3 % w/v of the vaccine composition.
• the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 3 to about 4 % w/v of the vaccine composition.
• the tocopherol or a pharmaceutically acceptable ester thereof is present in an amount of about 7 to about 8 % w/v of the vaccine composition.
• squalane is present in an amount of about 3 to about 7 % by weight based on the volume of the vaccine composition.
• squalane is present in an amount of about 3 to about 4 % w/v of the vaccine composition.
• squalane is present in an amount of about 6 to about 7 % w/v of the vaccine composition.
• 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.
• 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.
• the noon-mineral oil is present in an amount of about 3 to about 4 % w/v of the vaccine composition.
• 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.
• 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 poly ethoxy ethylene 12 cetostearyl ether, and about 14 to about 16 % w/v, preferably about 15 % w/v of alpha-tocopheryl acetate.
• the density of a 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.
• the vaccine composition is based on the SVEA-E adjuvant composition and comprises about 1 to about 2 % w/v of polyethoxyethylene 12 cetostearyl ether, about 3 to about 4 % w/v of alpha-tocopheryl acetate and about 3 to about 4 % w/v of squalane.
• the density of a 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.
• a vaccine based on the SVEA-E double plus adjuvant composition is particularly suitable for intradermal administration, e.g. by means of the ID AL® device, particularly for intradermal administration to swine.
• the vaccine composition is based on the EVO420 adjuvant composition and comprises about 1 to about 2 % w/v of polyethoxyethylene 12 cetostearyl ether, about 3 to about 4 % w/v of alpha-tocopheryl acetate and about 3 to about 4 % w/v of squalane.
• the density of a 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.
• the vaccine composition is based on the SVEA-E double plus adjuvant composition and comprises about 3 to about 4 % w/v of polyethoxyethylene 12 cetostearyl ether, about 7 to about 8 % w/v of alpha-tocopheryl acetate, about 6 to about 7 % w/v of squalane, and about 0.1 to 0.3 % w/v of fumed silica.
• the density of a 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.
• the vaccine composition is based on the microSVEA-E adjuvant composition and comprises about 0.3 to about 0.7 % w/v of polyethoxyethylene 12 cetostearyl ether, about 3 to about 4 % w/v of alpha-tocopheryl acetate and about 3 to about 4 % w/v of squalane.
• the density of a 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.
• the vaccine composition is based on the microSVEA-E double plus adjuvant composition and comprises about 0.8 to about 1.2 % w/v of poly ethoxy ethylene 12 cetostearyl ether, about 7 to about 8 % w/v of alpha-tocopheryl acetate, about 6 to about 7 % w/v of squalane, and about 0.1 to 0.3 % w/v of fumed silica.
• the density of a 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.
• Vaccine compositions according to the invention based on microSVEA-E or on microSVEA-E double plus as the adjuvant composition according to the invention, are particularly suitable for intramuscular administration.
• vaccine compositions based on microSVEA-E or on microSVEA-E double plus are both microemulsions, these thus have dispersed particles in the emulsion in the micrometre range.
• these vaccine compositions did not exhibit any problems with stability such as breaking of the emulsion. Therefore, using an adjuvant composition that is a microSVEA-E or a microSVEA-E double microemulsion allows to obtain vaccine compositions having larger emulsion particles in the micrometre range, which nevertheless have good safety and no issues with stability. This can be used advantageously in relation to protection against certain pathogens and disease, as vaccines with a larger dispersed particle size can be expected to induce a different (i.e. less Thl-directed) type of immune response than do vaccines with nano emulsions
• the vaccine composition is based on the Xsolve2.0 adjuvant composition and comprises about 2 to about 3 % w/w polyethoxyethylene 12 cetostearyl ether, about 2 to about 3 % w/w alpha-tocopheryl acetate and about 30 to about 40 % w/v; preferably about 35 % of a non-mineral oil.
• the density of a vaccine composition based on the Xsolve2.0 adjuvant composition is about 0.9 to about 1.1 g/ml, preferably about 0.9 to about 1.0 g/ml.
• the vaccine composition has a high payload 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.
• the vaccine composition according to the invention is preferably an emulsion vaccine.
• the vaccine composition according to the invention was found to be very effective, safe and stable, when prepared as an oil-in-water emulsion. Therefore, in a preferred embodiment the vaccine composition according to the invention is an oil-in-water emulsion vaccine.
• the aqueous antigen can be combined with the adjuvant composition using low shear mixing techniques, as adjuvant composition itself has already been emulsified, potentially involving high shear mixing.
• the outer aqueous phase comprises the antigen in a pharmaceutically acceptable carrier; and the oily phase comprises alpha-tocopheryl acetate and optionally squalane as oily adjuvant(s).
• the O/W emulsion according to the invention may itself be used for the formulation of a further emulsion, such as a W/O/W or O/W/O emulsion. This may require the use of an additional emulsifier. Selection and optimisation of such conditions are within the capabilities of the skilled person.
• the adjuvant composition of the invention is suitable for formulating a vaccine comprising crude antigens, i.e. little purified antigens derived from, for example, an inactivated bacterial or -parasite culture, or from extracts or fractions of such a culture.
• the antigen comprises a crude antigen.
• the vaccine composition according to the invention is cost effective. Furthermore, reduced batch- to-batch variability was observed, along with suitability of the inventive composition for large- scale production.
• the adjuvant composition according to the invention is particularly useful for combination with antigens, particularly non-live bacterial- or -parasitic antigens, that contain an enzyme with esterase activity; such antigens can deteriorate the emulsifier used in prior art emulsions of an oily phase and an aqueous phase, causing the emulsion to break.
• antigens particularly non-live bacterial- or -parasitic antigens
• the antigen comprises an esterase, preferably a lipase.
• esterase refers to enzymes having esterase activity, specifically to esterases and lipases. Such enzymes are characterised in the IUBMB (International Union of Biochemistry and Molecular Biology) enzyme classification system under EC 3.1.1.1 or EC 3.1.1.3 respectively.
• Tests to determine whether an antigen “contains an esterase” for the invention refer to tests for esterase activity which exist in a great variety of forms and types of assays, and are available commercially.
• Any level of esterase activity in the antigen can be harmful to the stability of prior art emulsions, especially upon long-term storage. Therefore based on appropriate positive and negative reference standards, the presence of any detectable level of esterase activity qualifies an antigen as ‘contains an esterase’ according to the invention.
• the adjuvant composition according to the present invention and particularly a vaccine composition comprising said adjuvant composition in combination with an antigen, surprisingly exhibit several further benefits.
• the adjuvant composition and the vaccine composition are successfully used for porcines and for ruminants; preferably used for pigs, cattle, sheep and goats.
• the adjuvant composition and vaccine composition derived therefrom is comparatively easy to administer since its density is low as compared to prior art adjuvant compositions.
• the density of the adjuvant is from about 0.9 to about 1.1 g/ml, preferably from about 0.9 to about 1.0 g/ml. Additionally, the adjuvant composition and the vaccine composition derived therefrom is safe, as no serious adverse events were observed.
• the vaccine composition comprising high antigen amounts, such as in multivalent- or combination vaccines, is considerably more stable as compared to prior art compositions.
• the adjuvant composition and vaccine composition derived therefrom can tolerate higher temperatures as compared to prior art compositions, e.g. Montanide ISA206 which phasereverses above 32 °C.
• the vaccine composition according to the invention provides quick onset of immunity (OOI).
• the vaccine composition according to the invention provides for a longer duration of immunity (DOI).
• the vaccine composition according to the invention can be prepared using well-known methods and materials. The details of these procedures will be dependent on the type of the emulsion to be prepared. For example, as an O/W emulsion, the emulsification of the oily- and aqueous phase (without antigen) can be done separately (e.g. to constitute the adjuvant composition according to the invention), before the subsequent admixing of the antigen to prepare the emulsion vaccine according to the invention.
• emulsifiers can be comprised in the oil and/or in the aqueous phase.
• the aqueous- and the oily phases can be emulsified using suitable equipment such as by ultrasonic-, or rotor-stator type mixing.
• the inventors further discovered that the polyethoxy ethylene cetostearyl ether of the adjuvant composition according to the invention dissolved surprisingly well into the oily phase. This was unexpected for an emulsifier of high HLB number (e.g. Polyethoxyethylene 12 cetostearyl ether has an HLB of 14), especially as Polysorbate 80 having almost the same HLB number (15) does not dissolve well in the oil phase.
• an emulsifier of high HLB number e.g. Polyethoxyethylene 12 cetostearyl ether has an HLB of 14
• Polysorbate 80 having almost the same HLB number (15) does not dissolve well in the oil phase.
• the use of high intensity emulsification of water and oily phase is still a further option, for example by using microfluidisation.
• the invention relates to a method for the manufacture of a vaccine composition according to the invention, the method comprising the steps of: a) preparing an aqueous phase comprising an antigen, and b) admixing said aqueous phase with the adjuvant composition according to the invention.
• each of: the aqueous phase, the oily phase, the adjuvant composition, and the antigen are as defined herein.
• an aqueous phase may be prepared including an antigen and the emulsifier (i.e. a polyethoxyethylene cetostearyl ether).
• an oily phase including a tocopherol or a pharmaceutically acceptable ester thereof, and optionally squalane, a mineral oil, and/or a non-mineral oil, is prepared prior to admixing both phases in order to obtain the vaccine composition of the invention.
• the method for the manufacture according to the invention is performed in a way that allows a medical use of the vaccine composition.
• the vaccine composition according to the invention is particularly advantageous when applied as a vaccine against bacterial-, parasitic-, and/or viral disease, all as described herein. Therefore, in a further aspect the invention relates to the vaccine composition according to the invention for use in a method of protecting a human- or animal target against infection and/or disease caused by a pathogen.
• the selection of the target for the method of protecting according to the invention is mainly determined by the host range of the pathogen to be protected against: for humans, for animals, or for both.
• a pathogen can be pathogenic to humans but not to an animal carrying the pathogen. In that case it may still make sense to apply animal vaccination, in order to prevent zoonotic infection and food-borne illness of humans that could consume an infected animal product such as e.g. meat or milk.
• the age, weight, sex, immunological status, and other parameters of the target to be vaccinated are not critical, although it is clearly favourable to vaccinate healthy, uninfected targets, and to vaccinate as early as possible.
• an “animal” for the invention is any animal of veterinary relevance, e.g. bovine, porcine, caprine, ovine, cervine, canine, feline, equine, avian, fish, or shrimp.
• the animal target is a swine or a ruminant.
• swine refers to animals of the family Suidae, and preferably to animals of the genus Sus, which are also referred to as porcines. Examples are: a wild or a domestic pig, hog, wild boar, babirusa, or warthog. This also includes swine indicated by an arbitrary name, for example referring to their sex or age such as: sow, queen, boar, barrow, hog, gilt, weaner, or piglet. Further the term swine refers to porcine animals of any type such as of breeding- or fattening type, and to parental lines of any of these types.
• the term broughtruminant“ refers to large hoofed herbivorous grazing or browsing mammals that are able to acquire nutrients from plant-based food by fermenting it in a specialized stomach prior to digestion, principally through microbial actions. Ruminating mammals include cattle, all domesticated and wild bovines, goats, sheep, giraffes, deer, gazelles, and antelopes.
• the ruminant is selected from agricultural livestock, particularly cattle, bovines, sheep, and goats.
• the swine is a sow or a young swine.
• a ‘young swine’ for the invention is a swine of less than 6 months old, preferably less than 5, 4, 3, 2, or even less than 1 month old, in this order of preference.
• the pathogen is selected from a bacterium, a parasite, and a virus, all as described herein.
• the pathogen is selected from one or more of porcine circovirus type 2, Mycoplasma hyopneumoniae, and foot-and-mouth-disease virus.
• the vaccine composition for use in a method of protecting according to the invention, is administered by intradermal or intramuscular route.
• the invention relates to the vaccine composition according to the invention for use in the vaccination of a human or animal target against infection and/or disease caused by a pathogen.
• the animal target is a ruminant or a swine
• the pathogen is a bacterium, parasite, or virus, all as described herein.
• the vaccine composition according to the invention can be applied “for use in a method of protecting” or “for use in the vaccination” in different ways.
• the vaccine composition itself can be applied as a vaccine.
• the vaccine composition can be used as ingredient 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 may involve admixing or including certain further ingredients, for example stabilisers or preservatives.
• Preservatives are e.g. thiomersal, phenoxyethanol, formalin, antibiotics (e.g. gentamycin).
• Stabilisers are e.g. dextrane, glycerol, gelatine, amino acids, or buffers.
• the further ingredients may be added during or after the manufacture of the vaccine composition according to the invention.
• the invention relates to the use of the adjuvant composition according to the invention for the manufacture of a vaccine composition for protecting a human or animal target against infection and/or disease caused by a pathogen.
• the animal target is a ruminant or a swine
• the pathogen is a bacterium, parasite, or virus, all as described herein.
• the vaccine composition according to the invention needs to be administered to a human or animal target, in order to achieve its beneficial immunogenic effect.
• the invention relates to a method for the vaccination of a human or animal target against infection and/or disease caused by a pathogen, the method comprising the administration to said target of the vaccine composition according to the invention.
• the animal target is a ruminant or a swine
• the pathogen is a bacterium, parasite, or virus, all as described herein.
• the “administration” of the vaccine composition according to the invention to a human or animal target can be performed using any feasible method and route.
• the optimal route and method of administration will be determined by the type of the vaccine applied, and by the characteristics of the target and of the disease that it is intended to protect against.
• Different techniques of administration can be applied.
• the vaccine composition according to the invention is aqueous in character, and can therefore be administered by enteral or mucosal route, i.e. via eye drop, nose drop, oral, enteric, oro-nasal drop, spray.
• enteral or mucosal route i.e. via eye drop, nose drop, oral, enteric, oro-nasal drop, spray.
• Other possibility is via a method of mass administration, such as via drinking water, coarse spray, atomisation, on-feed, etc.
• a skilled person is perfectly capable of selecting and optimising such route- and method of administration.
• Preferred way of administration for a method of vaccination according to the invention is by parenteral route.
• Parenter refers to administration through the skin, for example by intramuscular, intraperitoneal, intradermal, submucosal, or subcutaneous route.
• the vaccine composition is administered by intradermal- or intramuscular route.
• the volume of a dose of the vaccine composition according to the invention is a volume that is acceptable for the target human or animal, and can for instance be between about 0.1 and about 10 ml.
• one dose is a volume between 0.1 and 5 ml, more preferably one dose is between 0.2 and 3 ml.
• the volume of one dose is preferably between about 0.5 and about 3 ml, more preferably between 1 and 2 ml.
• the volume of one dose is preferably between about 0.1 and about 0.5 ml, more preferably is about 0.2 ml.
• the method, timing, and volume of the administration of a vaccine composition according to the invention is preferably integrated into existing vaccination schedules of other vaccines that the target human or animal may require, in order to reduce stress to the target and to reduce labour costs.
• These other vaccines can be administered in a simultaneous, concurrent or sequential fashion, in a manner compatible with their registered use.
• the adjuvant composition according to the invention can be marketed as a stand-alone product. This allows it to be used by qualified persons for preparing an O/W emulsion vaccine with a preferred antigen shortly before administration. Such on-the-spot preparation is also called fieldside mixing, and allows flexibility in the selection and the combination of the antigens that are admixed. However a vaccine composition prepared in such a way does not have the guaranteed properties of quantity, quality, and sterility that are provided with a ready-mixed vaccine composition that was prepared in the pharmaceutical factory of a registered manufacturer.
• kits of parts An alternative is the provision of the adjuvant composition according to the invention in a kit of parts.
• a kit is typically a package comprising 2 or more containers, the content of which can be mixed to prepare the vaccine composition according to the invention shortly before administration.
• the kit of parts can comprise one container having the adjuvant composition according to the invention, and one or more further containers comprising one or more antigens.
• the one or more further antigens can be in liquid form.
• the one or more further antigens can be in a dried-, or in a freeze-dried form.
• the freeze-dried antigen can be a live- or a killed micro-organism, for example can be a freeze-dried preparation of live PRRSV, or can be a freeze-dried preparation of non-live L. intracellularis . Therefore in a further aspect the 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.
• the antigen is a freeze-dried live PRRSV, or is a freeze-dried preparation of non-live L. intracellularis .
• the kit of parts according to the invention may comprise a box with said two or more containers, and may comprise instructions for use. Said instructions may for example be written on the box that contains the constituents of the kit; may be present on a leaflet in- or on- or with that box; or may be viewable on, or downloadable from, an internet website from the manufacturer or the distributor of the kit, etc.
• Fortasol-E is a 2x w/v concentrate.
• a similar composition having Polysorbate 80 (PS80) instead of Eumulgin Bl as the emulsifier was prepared.
• the Mhyo antigen used was a crude antigen preparation of a bacterin of Mhyo grown in a culture using pig blood in the medium.
• Oily phase with Eumulgin B 1 was clear, the oily phase with Polysorbate 80 was turbid Both adjuvant compositions can be easy filtrated through a 0.2 pm Nylon filter
• the adjuvant composition with Eumulgin showed some slight clustering of the visible particles
• SVEA-E is a 4x w/v concentrate.
• the concentrate formed 25 % w/v of the final vaccine.
• the final vaccine contained the following components and strengths:
• the O/W emulsion was prepared as a 4x w/v concentrate. WFI was used as water phase. High pressure homogenization was used. 4x w/v concentrate:
• the SVEA-E 4x concentrate microscopically appeared as an O/W solubilisate with some droplets between 1 and 5 pm.
• SVEA-E Double Plus is a 2x w/w concentrate.
• Density concentrate 0.965 g/ml
• Density final vaccine 0.986 g/ml
• the final vaccine contained the following components and strengths:
• Vitamin E acetate (corrected for purity) 16.17 g Macrogol Cetostearyl Ether 12 NS 6.59 g
• Aerosil 300 pharma 0.41 g
• the SVEA-E double plus composition comprised in the final vaccine: 3.25 % w/v of polyethoxyethylene 12 cetostearyl ether; 7.97 % w/v of alpha-tocopheryl acetate; 6.77 % w/v of squalane; and 0.20 % w/v of fumed silica.
• the SVEA double plus reference composition comprised in the final vaccine: 3.24 % w/v of Polysorbate 80; 7.94 % w/v of alpha-tocopheryl acetate; 6.75 % w/v of squalane; and 0.20 % w/v of fumed silica.
• Emulsion vaccines comprising a crude Mhyo antigen preparation and comprising an adjuvant composition having Eumulgin Bl as emulsifier, are clearly more stable than the reference emulsion vaccines with the same antigen, but with Polysorbate 80 as the emulsifier.
• the reference breaks within 10 days at 37 °C, while the Eumulgin emulsions do not show any decline in emulsion quality even after 3 weeks at 37 °C.
• the EVO420 is a 2x w/v concentrate, comprising vitamin E-acetate, Shell Ondina X420, and Eumulgin Bl.
• the density is equal to SVEA.
• the final vaccine contained the following components and strengths:
• Density of final vaccine 0.9913 g/ml
• the O/W emulsion was prepared as a 2x concentrate. WFI was used as water phase. High pressure homogenization was used.
• composition of the 2x concentrate is composition of the 2x concentrate:
• Example 6 Microscopically the EVO420 2x w/v concentrate appears as an O/W nano emulsion with some droplets between 1 pm and 5 pm.
• Example 6 Xsolvel.O
• the concentrate is based on the Xsolve adjuvant except that the mineral oil was replaced in volume by a Shell Ondina X GTL-based medicinal white oil, and the Polysorbate 80 was replaced in weight by Eumulgin Bl
• a micro emulsion having oil droplets with 99 % ⁇ 1 pm should appear Microscopically the Microsol-E / Shell Ondina X GTL-based medicinal white oil adjuvant composition appears as a homogenous O/W emulsion with some droplets between 1 pm and 5 pm.
• Microsol Eumulgin B 1/ Shell Ondina X GTL-based medicinal white oil concentrate 22.31 g
• Example 7 Stability study using Xsolvel.O (Eumulgin Bl/Marcol 52)
• Eumulgin Bl to obtain Xsolve2.0 comprising: NB:
• these vaccine compositions contained crude Mhyo bacterin antigen, but in an amount that far exceeded the amount present in any comparable commercial Mhyo O/W vaccine; this in order to force the appearance of an effect of the crude antigen on the PS80 emulsifier and correspondingly on the stability of the emulsion.
• the above adjuvant composition was subjected to a stability study, wherein appearance (by light microscopic view) and particle sizes (by Mastersizer measurements) were determined.
• Polysorbate 80 can be replaced by Eumulgin Bl
• Example 8 Efficacy, safety and stability of a SVEA-E/FMD vaccine composition
• IM intramuscularly
• IDL intradermolingual
• DOI duration of immunity
• a bivalent FMD vaccine was used to immunize the animals, which vaccine contained two types of FMD antigens: VLPs of O/TUR/5/2009 type and of Asial/Shamir/ISR/89 type. Only the virus-neutralizing titres against the O/TUR/5/2009 component were determined.
• compositions are Compositions:
• Vaccine compositions with a mixture of 0/TUR/20172009 and Asial/Shamir/ISR/89Shm viruslike particles (1 : 1 v/v)
• Antigen solution 15 and 30 % (v/v) of the final vaccine 15 and 30 % (v/v) of the final vaccine.
• the density of the antigen solution is considered 1 g/ml, thus the antigen quantities are converted in weight.
• An antigenic mass ELISA can be used to quantify VLPs in the vaccine composition without breaking the emulsion, which is not possible with Montanide ISA206-based formulations. VLPs in vaccines are stable.
• Vaccine composition is based on quantification of inactivated FMDV O/TUR/5/2009 VLPs with VHH-ELISA
• the Asial/Shamir/ISR/89 VLPs (containing capsid-stabilizing mutations in the VP2 protein: S093C and K190N) were produced by way of a baculovirus expression system.
• Tnao38 insect cells were infected at MOI 0.1 with the corresponding recombinant baculovirus.
• Cell culture supernatant was harvested at 5 dpi by centrifugation, treated with binary ethylenimine (BEI) to inactivate the recombinant baculoviruses, and subsequently concentrated by filtration.
• Vaccine was formulated with SVEA-E adjuvant composition.
• the concentration of intact VLPs was determined by ELISA using the M332F antibody (Harmsen et al., 2017, Front. Immunol., vol. 8, p. 960). Serially diluted samples were incubated for 1 h at 37 °C on microtitre plates coated overnight at 4 °C with M332F antibody. After removing the samples and three washes with PBS-Tween, a fixed amount of a biotinylated M332F was added to plates and incubated for Ih at 37 °C.
• the O/TUR/5/2009 VLPs (containing a capsid-stabilizing mutation in the VP2 protein: S093C) were produced in 2-litre bioreactors containing Tnao38 insect cells that were infected at an m.o.i. of 1. Cell culture supernatant was harvested at 5 dpi by centrifugation at 200 xg. The concentration of intact VLPs was determined by ELISA using VHH Cl (Wang et al., 2015, BMC Veterinary Research 11 : 120, DOI 10.1186/sl2917-015-0437-2).
• an O/W emulsion type vaccine composition can be used as an FMD vaccine
• the vaccine compositions according to the invention have several important further advantages, most notably for the stability of the emulsion, but also in handling and processing.

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