EP3866843A1 - Vaccin de type émulsion destiné aux poissons - Google Patents

Vaccin de type émulsion destiné aux poissons

Info

Publication number
EP3866843A1
EP3866843A1 EP19786574.4A EP19786574A EP3866843A1 EP 3866843 A1 EP3866843 A1 EP 3866843A1 EP 19786574 A EP19786574 A EP 19786574A EP 3866843 A1 EP3866843 A1 EP 3866843A1
Authority
EP
European Patent Office
Prior art keywords
fish
emulsion
vaccine
oil
pathogen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19786574.4A
Other languages
German (de)
English (en)
Inventor
Theodorus Jansen
Petter Frost
Shou Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Intervet International BV
Original Assignee
Intervet International BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Intervet International BV filed Critical Intervet International BV
Publication of EP3866843A1 publication Critical patent/EP3866843A1/fr
Pending legal-status Critical Current

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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
    • A61K39/02—Bacterial antigens
    • 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
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/107—Vibrio
    • 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
    • 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
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Definitions

  • the present invention relates to the field of veterinary vaccinology, more specifically the invention relates to an emulsion vaccine for fish.
  • the invention relates to an emulsion comprising an oil phase, an aqueous phase, an emulsifier and an antigen from a fish pathogen; to the emulsion as a water-in-oil emulsion; to a method for the manufacture, and to uses of the emulsion; to a vaccine for fish comprising the emulsion; and to medical uses of the components of the emulsion.
  • coldwater species such as salmon, trout, cod, turbot and halibut
  • temperate water species such as trout, eel, and carp
  • warm water-species such as grouper, sea bass, bream, catfish, barramundi, amberjack, tilapia and pangasius.
  • Vaccines comprising non-replicative antigens often require an immune stimulant for optimal efficacy: an adjuvant.
  • an adjuvant As an excipient, such an adjuvant needs to be pharmaceutically acceptable, and cost effective.
  • Well known adjuvants used in fish vaccines are: aluminium salts, liposomes, glucans, alginate, and in particular: oils.
  • an oil adjuvant can be emulsified with an antigen in an aqueous phase to form an emulsion that 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
  • the choice for one or the other type of emulsion can be based on the type of immune-response that is desired.
  • 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
  • Pluronic® a nonionic tri-block copolymer of blocks of polyoxyethylene and polyoxypropylene
  • 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 increase of phase separation. Maintaining the stability of the emulsion is thus important for the use and efficacy of an emulsion vaccine: a 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).
  • fish vaccines will often be directed at several diseases or pathogens at once, by containing several different antigens in a single vaccine formulation. This is favourable to reduce stress for a target animal from prevention of the need for repeated treatments, as well as to reduce labour costs for the
  • multivalent vaccines examples include: Forte® VII (Aqua Health, Novartis), ALPHA JECT® micro 6 (Pharmaq), Aquavac® PD7 Vet (MSD Animal health), and the multivalent inactivated vaccines from Centrovet (Virbac); these vaccines comprise combinations of several bacterins and viral antigens with a mineral oil adjuvant.
  • a bacterin is an antigenic preparation of inactivated bacterial cells.
  • the intra-peritoneal administration of an oil-adjuvanted vaccine can give rise to pigmentation (melanisation), intra-abdominal adhesions, and a temporary drop in feed-intake in the period after vaccination. These can have consequences on the fish’s well-being and on the economy of the operation.
  • Non-ionic A-B-A block copolymeric emulsifiers of polyalkylene glycol and monocarboxylic acids were first described in EP 0000424 for use in dispersion of water in fuels, and in WO 96/07689 for dispersion of pigments or toners in organic medium. The different uses depend from a difference in molecular weight of the component B.
  • EP 0000424 A practical use for the emulsifiers of EP 0000424 was also found in the cosmetics industry as emulsifiers for skin creams (Jang et al., 2015, Toxicol. Res., vol. 31 , p. 105-136). Further, a described pharmaceutical use is for enhancing the skin-penetration of drugs (Casiraghi et al., 2012, AAPS
  • emulsifiers are not the same as the non-ionic block copolymeric emulsifiers that are known generally as PoloxamerTM (BASF).
  • the Poloxamers do not contain a fatty acid component A, but are co-polymers of blocks of polyoxyethylene and polyoxypropylene.
  • WO 2002/067899 describes the use of A-B-A block copolymeric emulsifiers of polyalkylene glycol and monocarboxylic acids in oil emulsion vaccines.
  • the specific disclosure is of the emulsifier Arlacel® P135, and its use in low-viscosity W/O or W/O/W (water-in-oil-in-water) emulsion vaccines with antigens from two inactivated avian viruses.
  • Non-adjuvanted types of vaccines such as DNA based vaccines, e.g. Apex®-IHN or Clynav® (Novartis/Elanco), etc.
  • a polymeric emulsifier of a specific class namely: a tri-block copolymer of polyalkylene glycol and fatty acids.
  • Vaccines based on emulsions made with this emulsifier when administered to fish, showed significantly less reduction of appetite post vaccination, and better feed intake, and therefore a lesser dip in their growth curve, as compared to prior art vaccines. All this while maintaining excellent vaccine efficacy and no increase of the cost-price.
  • These results are an important improvement to the health and well-being of the vaccinated fish.
  • they allow the continued employment of the classic oil-based emulsion vaccines with all their beneficial effects on the economy of fish farming operations, but then using safer emulsions.
  • the invention relates to an emulsion comprising an oil phase, an aqueous phase, an emulsifier and an antigen from a fish pathogen, characterised in that the emulsifier is a polymeric emulsifier which is a block copolymer having a general formula A-B-A in which component B is the divalent residue of a water-soluble polyalkylene glycol and component A is the residue of an oil- soluble complex monocarboxylic acid.
  • An“emulsion” is a mixture of at least two immiscible liquids, whereby one is dispersed in another.
  • the droplets of the dispersed phase are very small, in the range of micrometers.
  • 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.
  • An“oil phase” is a liquid based on an oil.
  • An‘oil’ is used here in its common meaning and refers to a nonpolar chemical substance that is hydrophobic and lipophylic, 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 nonmineral oils are metabolisable.
  • the oil phase may contain excipients such as an emulsifier.
  • the oil-phase is the continuous phase (as in a W/O emulsion), or is the dispersed phase (as in an O/W emulsion).
  • the oil-phase can serve as adjuvant.
  • Much used mineral oil adjuvant in veterinary vaccines is a light (or white) 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).
  • An“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 aqueous phase may contain the antigen from a fish pathogen for the invention, depending on the type of the emulsion according to the invention.
  • An“antigen” is a substance that is capable of inducing an immunological reaction in a target, 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 micro-organism (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.
  • “fish” refers to fin fish, both cartilaginous and bony fin fish, from any climate area: cold-, temperate- or tropical waters, and living in any type of water: sweet-, brackish, or salt water.
  • the fish may be grown in captivity as farmed fish, breeding fish or ornamental fish.
  • a fish is selected from: bass, grouper, snapper, Tilapia, yellowtail, amberjack, flounder, Pangasius, carp, bream, sturgeon, catfish, eel, trout, salmon, whitefish, halibut, cod, Koi, and goldfish.
  • A’’pathogen is an organism or micro-organism that can cause signs of disease, typically with negative consequences to health and well-being of the target that it affects.
  • Typical pathogens are bacteria, viruses, protozoa, fungi, algae and endo- or ecto-parasites.
  • the pathogen can be a primary or a secondary (opportunistic) pathogen.
  • A“fish pathogen” is then a pathogen that can affect the health and/or well-being of a fish.
  • the fish pathogen may be known to be a fish pathogen or not.
  • the fish pathogen can e.g. be from: bacteria, viruses, protozoa, fungi, algae and endo- or ecto-parasites.
  • An“antigen from a fish pathogen” is an antigen that is based on, or derived or obtained from a fish pathogen.
  • the antigen can be the pathogen (replicative or not), or can be a part thereof, such as a molecule from such a fish pathogen.
  • An“emulsifier” 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.
  • a compound is“polymeric” when it consists of repeated (molecular) units. As is common with polymeric compounds, the number of subunit repetitions may not be exactly known, but is statistically distributed around an average value lying within a certain range.
  • the molar ratio between the components A and B may vary from 125:1 to 2:1.
  • the weight proportion of the component B in the polymeric emulsifier for the invention may be up to 80 % w/w.
  • the monocarboxylic acid may have up to 25 carbon atoms.
  • the polymeric emulsifier for the invention is an amphiphilic molecule, in that the components A are“oil- soluble”, i.e. have a hydrophobic nature, and the component B is“water-soluble”, i.e. is hydrophilic.
  • the components A and component B each consist of subunits as defined herein which subunits are connected to each other by ether bonds.
  • the components A and B themselves are connected by a COO- ester bond, making the detailed general structure of the polymeric emulsifier for the invention: A- COO-B-OOC-A.
  • complex indicates that the polymer of the monocarboxylic acids, component A, incorporates different monocarboxylic acid moieties, to determine chain-length.
  • the specific composition of the polymeric emulsifier for the invention can be selected depending on the required emulsifying properties, while considering also: the type of emulsion desired, the type of oil used, and the characteristics of the (multiple) antigen(s) incorporated.
  • variations may include the size and composition of components A and B, their molar ratio, and their weight percentage of the complete emulsifier molecule. The properties of those molecules are known, and they are available commercially.
  • Polymeric emulsifiers for use in the invention are generally available commercially, and in different qualities and purities, from a number of suppliers of fine chemicals. Examples are the families of polymers known as: Atlox® and Hypermer® (Uniqema); Termul® (Huntsman); Kolliphor®, Dehymuls®, and Solutol® (BASF); and Cithrol® (Croda).
  • one or more further emulsifiers can be added, to provide the combination of emulsifiers with certain desired properties.
  • HLB number hydrophile-lipophile balance; Griffin 1949, J. Soc. Cosm. Chem., vol. 1 , p. 311-326.
  • HLB number hydrophile-lipophile balance
  • an emulsifier or emulsifier mixture with HLB number below 10 favours W/O emulsions
  • an emulsifier (mixture) with HLB number of 10-16 will favour O/W emulsions.
  • the emulsion according to the invention induces as little as possible vaccination side- effects over and above those caused by the oil adjuvant, or the vaccination process itself.
  • the emulsion according to the invention does not contain (i.e. is free from) a sorbate-based emulsifier; or even: does not contain a Polysorbate, more preferably: does not contain a Polysorbate and a Sorbitan mono-oleate.
  • Immunity in fish may take a long time to develop.
  • One relevant factor is the environmental temperature.
  • fish vaccines can be developed as water-in-oil (W/O) emulsions: the continuous oil phase provides a depot function at the site of immunisation that provides persistent presentation of the antigen to the fish’s immune system.
  • W/O water-in-oil
  • the emulsion is a water-in-oil (W/O) emulsion.
  • a polymeric emulsifier for the invention is selected with the right properties, preferably having an HLB number of 10 or less.
  • components A each have a molecular weight of at least 500 g/mol.
  • component B has a molecular weight of at least 500 g/mol.
  • the components A and component B all have a molecular weight of at least 500 g/mol.
  • the W/O emulsion according to the invention may itself be used for the formulation of a further emulsion, such as a W/O/W emulsion.
  • a further emulsion such as a W/O/W emulsion.
  • This may require the use of an additional emulsifier, either a variant of the polymeric emulsifier for the invention, or another emulsifier. Selection and optimisation of such conditions are within the capabilities of the skilled person.
  • Preferred components of the polymeric emulsifier for the invention are polyethylene glycol, and polyhydroxystearic acid. Emulsifiers with these building blocks were shown to have favourable properties in regard to the safety of fish vaccines prepared from these emulsions. In addition they provided goof vaccine efficacy, and excellent stability even when the antigens for the invention where (relatively) impure. Also they only require the use of a relatively low weight percentage of the emulsifier, and are effective even at a relatively high amount of water phase dispersed in the oil. This leaves much room for including antigen in aqueous phase into the W/O emulsion according to the invention. Therefore, in an embodiment of the W/O emulsion according to the invention, component A is a polymer of a hydroxystearic acid.
  • the hydroxystearic acid is a 12-hydroxystearic acid.
  • component B is a polymer of an ethylene glycol.
  • component A is a
  • component B is a polyethylene glycol
  • the polyhydroxystearic acid is a poly(12-hydroxystearic acid).
  • Polyethylene glycol is also known as polyethylene oxide (PEO) or polyoxyethylene (POE).
  • component A is a polyhydroxystearic acid (molecular weight 300 g/mol)
  • component B is a polyethylene glycol (molecular weight 62 g/mol) whereby each component A has 2 - 50 units of hydroxystearic acid, and component B has 8 - 60 units of ethylene glycol.
  • cited ranges also include the end points.
  • the polymeric emulsifier is a PEG-30-di-(polyhydroxystearate).
  • PEG-30 indicates that the average number of moles of ethylene oxide reacted per mole of substance is: 30.
  • PEG-30-di-(polyhydroxystearate) has CAS nr. 70142-34-6, and has HLB nr. 5.5.
  • Another name for PEG is Macrogol; Macrogol 30 dipolyhydroxystearate is described in the European Pharmacopoiea under monograph no. 07/201 1 :2584.
  • PEG-30-di-(polyhydroxystearate) is commercially available, for example as: Cithrol DPHS, Atlox 4912 (Uniqema), Termul 2510, Sabowax PIS, and Dehymuls LE.
  • Cithrol DPHS (Croda), has an average molecular weight of about 5000 g/mol, and has 5 - 15 units of 12-hydroxystearic acid per component A, and 15 - 35 units of ethylene glycol per component B.
  • Previously Cithrol DPHS was known as Arlacel® P135.
  • Arlacel P135 is not to be confused with Arlacel A, the emulsifier that is used in Freund’s complete adjuvants, which is a mixture of a mineral oil and bacteria.
  • Arlacel A is not a block copolymer, but a mono-oleate ester of a mannitol sugar, and has CAS nr. 25339-93-9.
  • Cithrol DPHS Polysorbate 80 (Tween® 80) and Sorbitan mono- oleate (Span® 80).
  • this W/O emulsion vaccine comprising several bacterins and inactivated viruses, was found to show significantly less vaccination side-effects, specifically a smaller drop in appetite after vaccination, while providing equal or better immune-protection.
  • the amount of the polymeric emulsifier for the invention in the emulsion according to the invention is determined based on differences in the desired properties of the resulting emulsion, and of the intended use of the emulsion as a vaccine.
  • the lower limit is determined by the limit of efficacy of the specific polymeric emulsifier used; the upper limit is determined by practical considerations of usability, as some variants of the polymeric emulsifier for use in the invention have a wax-like constitution.
  • the emulsion according to the invention comprises an amount of the polymeric emulsifier for the invention that is 0.01 - 15 % w/w, expressed in weight percent of the vaccine prepared from the emulsion.
  • the emulsion according to the invention comprises an amount of the polymeric emulsifier for the invention that is 0.05 - 10; 0.1 - 5; 0.2 - 3; 0.3 - 2; 0.4 - 1.5; or even 0.5 - 1 % w/w of the weight of the vaccine prepared from the emulsion, in this order of preference.
  • the emulsion according to the invention comprises an amount of the polymeric emulsifier for the invention of about 0.5 % w/w, by weight of the vaccine prepared from the emulsion.
  • “about” indicates that a number can 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, in that order of preference.
  • the emulsion according to the invention comprises an amount of oil of 10 - 90 % w/w; the percentage of oil is expressed by weight of the vaccine prepared from the emulsion.
  • the emulsion according to the invention comprises an amount of oil of 20 - 80; 25 - 70; or even 30 - 60 % w/w, by weight of the vaccine prepared from the emulsion, in this order of preference.
  • the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites.
  • the bacterial pathogen is from a bacterial family of: Aeromonas, Vibrio, Moritella, Edwardsiella, Francisella, Flexibacter, Cytophaga, Corynebacterium, Renibacterium, Flavobacterium, Fusarium, Bacillus, Yersinia, Mycobacterium, Neorickettsia, Piscirickettsia, Streptococcus, Pseudomonas, Photobacterium, Clostridium, Tenacibaculum, Lactococcus, Leucothrix, and Nocardia.
  • the bacterial pathogen is one or more selected from Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum, and Moritella viscosa.
  • the viral pathogen is selected from: infectious haematopoietic necrosis virus, salmon pancreas disease virus, spring viremia of carp virus, viral haemorrhagic septicemia virus, cyprinid herpesvirus, piscine myocarditis virus, gill pox virus, Koi herpesvirus, piscine orthoreovirus, yellowtail ascites virus, viral nervous necrosis virus, infectious salmon anemia virus, Hirame rhabdovirus, epizootic hematopoietic necrosis virus, striped jack nervous necrosis virus, red-spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, barfin flounder nervous necrosis virus, channel catfish virus, grass carp hemorrhage disease virus, infectious pancreatic
  • the viral pathogen is one or more selected from Salmon pancreas disease virus (SPDV) and Infectious pancreatic necrosis virus (IPNV).
  • SPDV Salmon pancreas disease virus
  • IPNV Infectious pancreatic necrosis virus
  • the fungal pathogen is selected from: Saprolegnia, Achyla, Aphanomyces, lchthyophonus, Branchiomyces and Dermocystidium.
  • the algal pathogen is selected from: Chlorochytrium and Scenedesmus.
  • the endo- or ecto-parasitic pathogen is selected from: Amoebae, Flagellates, Ciliates, Microsporidia, Myxosporeans, Monogeneans, Cestodes, and Crustaceans.
  • the crustacean parasite is an ecto-parasite, preferably from the taxonomic family Caligidae; more preferably from the genera Lepeophtheirus or Caligus.
  • the reference to the various taxonomic groups includes any pathogen that is a species, subtype, variant, biotype, serotype or genotype within that group.
  • the fish pathogen is a pathogen that is infectious to salmonid fish.
  • pathogens for the preparation of an antigen for the invention can be obtained from a variety of sources, e.g. as field isolate from an aquatic organism in the wild or in a fish farm, or from various laboratories, (depository) institutions, or (veterinary) universities.
  • fish vaccines preferably comprise more than one antigen for convenience, and economy of operation. Therefore in an embodiment the emulsion according to the invention comprises at least one further antigen from a fish pathogen.
  • the emulsion according to the invention comprises antigens from two or more fish pathogens, the fish pathogens being selected from bacteria, viruses, and endo- or ecto-parasites.
  • the two or more fish pathogens are selected from bacteria and viruses.
  • the bacterium is selected from Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum, and Moritella viscosa, and the virus is selected from Salmon pancreas disease virus and Infectious pancreatic necrosis virus.
  • the antigen of a fish pathogen for the invention can be in any form: a live (attenuated) pathogen, a killed pathogen, or a part of a pathogen.
  • live antigens in fish vaccination is in many countries strictly regulated, because of the danger of reversion to virulence and the easy spread into nature. Consequently most fish vaccines are prepared from killed pathogens.
  • the antigen from a fish pathogen is a non-live antigen.
  • A“non-live antigen” is any antigen that is not a live (i.e. a replicative) antigen. Often this will be an antigen preparation based on inactivated (killed) virus or -bacteria.
  • a preparation of inactivated bacteria is also called: a bacterin.
  • Such an inactivated preparation can contain inactivated cells, e.g. the (infected) eukaryotic cells used to grow the virus, or the bacterial cells. The cells can be more or less damaged or ruptured from the inactivation.
  • a non-live antigen can also be a subunit, i.e. a part of a viral or bacterial inactivated preparation, such as e.g. an extract, fraction, homogenate, or sonicate.
  • a non-live antigen can be a synthetic or a recombinant product, such as an expression vector or an expressed protein. All these are well-known in the art.
  • the non-live bacterial antigen for the invention is typically contained in a liquid, such as a watery buffer.
  • a liquid such as a watery buffer.
  • the non-live bacterial antigen for the invention will either be contained in the internal aqueous phase (in case the emulsion according to the invention is a W/O emulsion), or will be added to the aqueous phase after the emulsification (in case the emulsion according to the invention is an O/W emulsion), as will be outlined below.
  • non-live antigen can be of any degree of purity.
  • the non-live antigen is an inactivated viral or -bacterial culture, or is a part thereof.
  • the part of the inactivated culture is selected from: a pellet, supernatant, concentrate, dialysate, extract, sonicate, lysate and fraction of such a culture.
  • a“viral or -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 viral or bacterial culture is used either as a whole, i.e. as the full volume of the inactivated 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, 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, aceton, 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 from a whole that is purified from the rest, for example by filtration or precipitation, whereby the fraction is the retentate.
  • the antigen comprises inactivated bacterial cells, also known as a bacterin.
  • the antigen of a fish pathogen comprises inactivated bacterial cells.
  • 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 be without the culture medium, for example resulting from sedimentation, centrifugation, or concentration.
  • the inactivated bacterial cells are from Aeromonas salmonicida and/or from Moritella viscosa.
  • Suitable oil phases for use in the emulsion according to the present invention are mineral- or non-mineral oils, and mixtures of mineral- and non-mineral oils.
  • Mineral oils for the invention include but are not limited to paraffin oils.
  • Non-mineral oils for the invention include but are not limited to vegetable oils, animal oils, natural hydrocarbons, metabolisable synthetic or semi-synthetic oils (such as Miglyol® and Cetiol®), fatty acid esters of propylene glycol and C6 to C24 fatty acids such as oleyl oleates, diesters of capric- or caprylic acids and the like.
  • Suitable vegetable oils for the invention are peanut oil, soybean oil, sunflower oil, and derivatives such as tocopherol.
  • Suitable animal oils for the invention are squalane and squalene and the like. All are widely available commercially.
  • the oil phase comprises a mineral oil.
  • the mineral oil is a light liquid paraffin oil.
  • Such light liquid paraffin oil is generally available, examples are: Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile), and Klearol®
  • the emulsion is a water-in-oil (W/O) emulsion.
  • components A each have a molecular weight of at least 500 g/mol; or component B has a molecular weight of at least 500 g/mol;
  • components A and component B all have a molecular weight of at least 500 g/mol;
  • component A is a polymer of a hydroxystearic acid; preferably the hydroxystearic acid is a 12-hydroxystearic acid;
  • component B is a polymer of an ethylene glycol
  • component A in the W/O emulsion, component A is a polyhydroxystearic acid, and component B is a polyethylene glycol; preferably the polyhydroxystearic acid is a poly(12-hydroxystearic acid).
  • component A in the W/O emulsion, component A is a polyhydroxystearic acid (molecular weight 300 g/mol), and component B is a polyethylene glycol (molecular weight 62 g/mol), whereby each component
  • the polymeric emulsifier is a PEG-30-di-
  • the emulsion comprises an amount of the polymeric emulsifier for the invention that is 0.01 - 15 % w/w, expressed in weight percent of the vaccine prepared from the emulsion; preferably the emulsion comprises an amount of the polymeric emulsifier for the invention that is 0.05 - 10; 0.1 - 5; 0.2 - 3; 0.3 - 2; 0.4 - 1.5; or even 0.5 - 1 % w/w of the weight of the vaccine prepared from the emulsion, in this order of preference;
  • the emulsion comprises an amount of the polymeric emulsifier for the invention of about 0.5 % w/w, by weight of the vaccine prepared from the emulsion;
  • the emulsion comprises an amount of oil of 10 - 90 % w/w, the percentage of oil is expressed by weight of the vaccine prepared from the emulsion; preferably the emulsion comprises an amount of oil of 20 - 80; 25 - 70; or even 30 - 60 % w/w, by weight of the vaccine prepared from the emulsion, in this order of preference;
  • the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites;
  • the bacterial pathogen is from a bacterial family of: Aeromonas, Vibrio, Moritella, Edwardsiella,
  • the bacterial pathogen is selected from Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum, and Moritella viscosa;
  • the viral pathogen is selected from: infectious haematopoietic necrosis virus, salmon pancreas disease virus, spring viremia of carp virus, viral haemorrhagic septicemia virus, cyprinid herpesvirus, piscine myocarditis virus, gill pox virus, Koi herpesvirus, piscine orthoreovirus, yellowtail ascites virus, viral nervous necrosis virus, infectious salmon anemia virus, Hirame rhabdovirus, epizootic hematopoietic necrosis virus, striped jack nervous necrosis virus, red- spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, barfin flounder nervous necrosis virus, channel catfish virus, grass carp hemorrhage disease virus, infectious pancreatic necrosis virus, Tilapia lake virus and red sea bream iridovirus; preferably the viral pathogen is selected from Salmon pancreas disease virus and Infectious pancreatic necrosis virus;
  • the fungal pathogen is selected from: Saprolegnia, Achyla, Aphanomyces, lchthyophonus, Branchiomyces and Dermocystidium;
  • the algal pathogen is selected from: Chlorochytrium and Scenedesmus;
  • the endo- or ecto-parasitic pathogen is selected from: Amoebae, Flagellates, Ciliates,
  • the crustacean parasite is an ecto-parasite, preferably from the taxonomic family
  • Caligidae more preferably from the genera Lepeophtheirus or Caligus;
  • the fish pathogen is a pathogen that is infectious to salmonid fish
  • the emulsion according to the invention comprises at least one further antigen from a fish pathogen
  • the emulsion according to the invention comprises antigens from two or more fish pathogens, the fish pathogens being selected from bacteria, viruses, and endo- or ecto-parasites.
  • the two or more fish pathogens are selected from bacteria and viruses.
  • the bacterium is selected from Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum, and Moritella viscosa
  • the virus is selected from Salmon pancreas disease virus and Infectious pancreatic necrosis virus;
  • the antigen from a fish pathogen is a non-live antigen
  • the non-live antigen is an inactivated viral or -bacterial culture, or is a part thereof; preferably the part of the inactivated culture is selected from: a pellet, supernatant, concentrate, dialysate, extract, sonicate, lysate and fraction of such a culture;
  • the antigen of a fish pathogen comprises inactivated bacterial cells; preferably the inactivated bacterial cells are from Aeromonas salmonicida and/or from Moritella viscosa; and
  • the oil phase comprises a mineral oil; preferably the mineral oil is a light liquid paraffin oil.
  • the antigen of a fish pathogen comprises antigens from two or more fish pathogens, the fish pathogens are selected from at least two bacteria and at least two viruses; the bacteria are selected from: Aeromonas salmonicida subsp.
  • the at least two viruses are infectious pancreatic necrosis virus and salmon anemia virus;
  • the antigen of a fish pathogen comprises inactivated bacterial cells;
  • the oil phase comprises a mineral oil;
  • the mineral oil is a light liquid paraffin oil;
  • the emulsion is a water-in-oil (W/O) emulsion;
  • the polymeric emulsifier is a PEG-30-di- (polyhydroxystearate);
  • the emulsion comprises an amount of the polymeric emulsifier that is 0.5 - 1 % w/w by weight of the vaccine prepared from the emulsion; and the emulsion comprises an amount of oil of 30 - 60 % w/w, by weight of the vaccine prepared from the emulsion.
  • the emulsion according to the invention can be prepared using well-known methods and materials. The details of these procedures will be dependent on the characteristics of the polymeric emulsifier for the invention used, and the type of the emulsion to be prepared. For example, when the emulsion according to the invention is of the O/W type, an emulsion of oil and aqueous phase can be prepared separately, and subsequently the antigen from a fish pathogen for the invention is added. However this is usually not applied for an emulsion of the W/O type, where the aqueous phase commonly contains the antigen from the start as it will become the internal phase.
  • the polymeric emulsifier for the invention when preparing an O/W emulsion, is dissolved in the aqueous phase. However when preparing a W/O emulsion, the polymeric emulsifier for the invention is dissolved into the oil phase. Occasionally it may be required to apply some heating of the solvent, for example to 50-60 °C, to get the emulsifier completely dissolved. When required, further emulsifiers can be comprised in the oil and/or in the aqueous phase. For both types of emulsions, the aqueous phase and the oil phase can be emulsified using suitable equipment such as by ultrasonic, or rotor-stator type mixing.
  • the invention relates to a method for the manufacture of an oil-in-water (O/W) emulsion according to the invention, the method comprises the steps of:
  • step b emulsifying the mixture of step a. with the oil phase
  • step b. admixing the emulsion of step b. with the antigen from a fish pathogen.
  • the invention relates to a method for the manufacture of the W/O emulsion according to the invention, the method comprising the steps of:
  • step b emulsifying the mixture of step a. with the aqueous phase, whereby the aqueous phase
  • each of the oil phase, the polymeric emulsifier, the aqueous phase, and the antigen from a fish pathogen are as defined hereinabove.
  • the method for the manufacture according to the invention is performed in a way that allows a medical use of the emulsion produced, such as in a vaccine.
  • a medical use of the emulsion produced such as in a vaccine.
  • manufacture is done aseptically.
  • the emulsion according to the invention is particularly advantageous when applied as a constituent of a vaccine for fish.
  • the invention relates to the emulsion according to the invention for use in the vaccination of fish against infection or disease caused by a fish pathogen.
  • the invention relates to a vaccine for use in the protection of a fish against infection or disease caused by a fish pathogen, characterised in that the vaccine comprises the emulsion according to the invention.
  • the fish is a salmonid fish; preferably the salmonid fish is selected from Atlantic-, steelhead-, Chinook-, coho-, pink-, chum-, and sockeye salmon, rainbow-, brook-, lake-, and brown trout, and char.
  • the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites.
  • the emulsion according to the invention can be applied“for use in a vaccine” in different ways.
  • the emulsion itself can be applied as a vaccine.
  • the emulsion can be used as ingredient in further processing for example into a W/O/W emulsion, which can then be applied as a vaccine.
  • the use as a vaccine may require 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, gelatin, amino acids, or buffers.
  • the further ingredients may be added during- or after the manufacture of the emulsion according to the invention.
  • A“vaccine” is a well-known composition with a medical effect, and comprises an immunologically active component, and a pharmaceutically acceptable carrier.
  • As‘carrier’ for the invention functions the aqueous phase, or the emulsion itself.
  • The‘immunologically active component’ for the invention is the antigen from a fish pathogen.
  • the vaccine stimulates the immune system of a fish, and induces a protective immunological response.
  • the response may originate from the fish’s 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 or disease” by reducing in a vaccinated fish the severity of a subsequent infection or infestation, for example by reducing the number of pathogens, or shortening the duration of the pathogen’s replication in or on the fish, and reducing the number, the intensity, or the severity of lesions caused by an infection or infestation.
  • a vaccine is effective in reducing or ameliorating the (clinical) symptoms of disease that may be caused by such infection, infestation or replication, or by the target’s response to that infection, infestation or replication.
  • a reference for such diseases and clinical signs is: "The Merck veterinary manual” (10th ed., 2010, C.M. Kahn edt., ISBN: 091 191093X.
  • Such a vaccine is colloquially referred to as a: vaccine ‘against’ the particular pathogen, or as a‘viral, bacterial, etc. 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 challenge infection, e.g. by monitoring the fish’s signs of disease, clinical scores, or by re-isolation of the pathogen, and comparing these results to a vaccination-challenge response seen in mock-vaccinated fish.
  • the amount of the antigen from a fish pathogen for the invention can be expressed in different ways, depending on the type of the antigen employed.
  • the antigen dose can be expressed as a virus titre or a number of bacterial cells.
  • 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 according to the invention can be used as a prophylactic-, metaphylactic-, or therapeutic treatment.
  • the vaccine 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 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 according to the invention may itself be added to a vaccine.
  • the vaccine according to the invention can advantageously be combined with one or more further antigens, e.g. derived from a micro-organism pathogenic to the intended human or animal target.
  • a further antigen may itself be an infectious micro-organism, or be inactivated, or a subunit.
  • the further antigen may consist of a biologic or synthetic molecule such as a protein, a carbohydrate, a
  • lipopolysacharide a lipid, or a nucleic acid molecule.
  • the vaccine according to the invention comprises at least one additional antigen.
  • the targets for the vaccine according to the invention are fish in need of a vaccination against infection or disease caused by the particular pathogen from which an antigen in the vaccine is obtained or derived. While size and age of the fish to be vaccinated can be relevant parameter, generally it is favourable to vaccinate healthy, uninfected fish, and to vaccinate as early as possible.
  • the selection of the species or type of fish as the target for the vaccination is mainly determined by the host range of the pathogen involved.
  • the pathogen can be pathogenic to humans but not (significantly) to a fish carrying that pathogen. In that case it may still make sense to vaccinate fish against that pathogen, in order to prevent zoonotic infection and food-borne illness of humans that would otherwise consume an infected product prepared from such fish.
  • the target fish are salmonid fish.
  • the emulsion according to the invention allows for the use of a relatively large volume of water as compared to the oil. This is favourable for including a relatively large mass of aqueous phase containing antigen in the vaccine that is prepared from the emulsion according to the invention.
  • the ratio of wateroil in the vaccine is 40:60 % w/w or is higher with respect to the relative amount of the water.
  • the % w/w is expressed by weight of the vaccine.
  • the wateroil ratio in the vaccine according to the invention is 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, or even 90:10 % w/w expressed by weight of the vaccine, in this order of preference.
  • the emulsion vaccine should not have a viscosity that is too high.
  • the occurrence of sedimentation or creaming of the dispersed phase in the emulsion vaccine can be reduced or prevented.
  • the vaccine according to the invention has a viscosity below 500 mPa.s.
  • the vaccine has a viscosity of less than 400 mPa.s., less than 300 mPa.s., or even between 100 and 300 mPa.s., in this order of preference.
  • Such viscosity is to be determined at about 20 °C, using a Brookfield DV-I+ viscometer, utilising spindle type No. 62 for 30 sec. at 60 r.p.m.
  • Methods and materials to influence the viscosity of an emulsion (vaccine) of an oil phase and an aqueous phase are well-known to a skilled person. For example by varying the amount of water in the emulsion, or the size of the droplets of the dispersed phase.
  • the mean droplet size (diameter) of the dispersed phase is less than 25 pm.
  • the mean droplet size (diameter) of the dispersed phase is less than 20 pm; less than 15 pm, less than 10 pm, between 10 and 0.1 pm; or even between 5 and 0.5 pm, in this order of preference;
  • the vaccine according to the invention is based on a W/O emulsion; comprises a mineral oil as the oil-phase; comprises 0.5 - 1 % w/w expressed by weight of the vaccine PEG-30-di- (polyhydroxystearate) as emulsifier; has a wateroil ratio of 60:40 - 70:30 % w/w expressed by weight of the vaccine; has a viscosity below 400 mPas; and has a mean droplet size (diameter) of the dispersed phase of 20 pm or less.
  • this involves the fine-tuning of the efficacy of the vaccine to further improve its provided immune-protection. This can be done by adapting the dose, volume, adjuvant or antigen content of the vaccine, or by application via a different route, method, or regime. All these are within the scope of the invention.
  • the invention relates to the use of an emulsifier in an emulsion, the emulsion further comprising an oil phase, an aqueous phase, and an antigen from a fish pathogen, characterised in that the emulsifier is the polymeric emulsifier as defined hereinabove.
  • the invention relates to a use of the polymeric emulsifier as defined hereinabove, for the manufacture of an emulsion vaccine for fish, the emulsion vaccine further comprising an oil phase, an aqueous phase, and an antigen from a fish pathogen.
  • the invention relates to a use of an antigen from a fish pathogen for the manufacture of an emulsion vaccine for fish, the emulsion vaccine further comprising an oil phase, an aqueous phase, and the polymeric emulsifier as defined hereinabove.
  • the invention relates to a use of an oil phase for the manufacture of an emulsion vaccine for fish, the emulsion vaccine further comprising an aqueous phase, an antigen from a fish pathogen, and the polymeric emulsifier as defined hereinabove.
  • the oil-phase comprises a mineral oil, more preferably a light liquid paraffin oil.
  • the vaccine according to the invention needs to be administered to a fish, in order to achieve its beneficial immunogenic effect.
  • the invention relates to a method for the vaccination of a fish against infection or disease caused by a fish pathogen, the method comprising the administration to said fish of the vaccine according to the invention.
  • The“administration” of the vaccine according to the invention to a fish can be performed using any feasible method and route.
  • the optimal way of administration will be determined by the type of the vaccine applied, and the characteristics of the fish and of the disease caused by the pathogen that it is intended to protect against.
  • different techniques of administration can be applied.
  • the vaccine according to the invention can be administered e.g. by an enteral or mucosal route, i.e. by immersion.
  • Other possibility is via a method of mass administration, such as via the feed.
  • 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 is intraperitoneal, intradermal, submucosal, or subcutaneous route.
  • the volume of a dose of the vaccine according to the invention is a volume that is acceptable for the fish, and can for instance be between about 0.001 and about 5 ml.
  • one dose is a volume between 0.005 and 3 ml, between 0.01 and 1 ml, or even between 0.025 and 0.5 ml, in this order of preference.
  • the method, timing, and volume of the administration of an emulsion vaccine according to the invention is preferably integrated into existing vaccination schedules of other vaccines that the target fish may require, in order to reduce stress to the fish and to reduce labour costs.
  • these other vaccines can be
  • the following experiment was performed to test the effect of the replacement of prior art emulsifiers Tween 80 + Span 80 as commonly used in oil-emulsion fish vaccines, by Cithrol DPHS as emulsifier, on the safety and the efficacy of the vaccine.
  • the experimental vaccines tested comprised several antigens, mineral oil as adjuvant, and were formulated as water-in-oil emulsions. Special attention was given to the loss of appetite after vaccination.
  • Atlantic salmon parr (of approximately 35 grams) were treated one week before vaccination with an increase of their water temperature from 12 °C to 17 °C, by adjusting +2 °C every second day. Next the fish were
  • Hepta-P heptavalent antigen containing emulsion, with polysorbate and sorbitan-oleate as emulsifiers
  • Hepta-C similar vaccine, comprising Cithrol as emulsifier
  • feed intake was determined daily, by subtracting collected remaining uneaten feed the amount of from feed given.
  • pv blood was sampled from 35 fish from each group and vaccination reactions were scored for fish in the different groups. Immune response against Aeromonas salmonicida and Moritella viscosa were evaluated by performing ELISA’s on individual serum samples.
  • Atlantic salmon parr (approximately 35 grams) were acclimatized to water at 12 °C.
  • test vaccines Hepta-P and Hepta-C contained identitical quantities of the following inactivated antigens per dose (0,1 ml) of vaccine:
  • SPDV Salmon pancreas disease virus
  • IPNV Infectious pancreatic necrosis virus
  • Aeromonas salmonicida subsp. salmonicida > 10.7 log2 ELISA units (3)
  • Vibrio anguillarum serotype 01 > 75 % RPS
  • test vaccines were formulated as water-in-oil emulsions, with light liquid paraffin oil as adjuvant, and contained as emulsifier either Polysorbate 80 and sorbitan mono-oleate, or contained 0.5% w/w Cithrol DPHS as emulsifier. Wateroil weight ratio of the vaccine was 45:55. Mock vaccine was sterile saline (0.9 % NaCI).
  • the vaccine bottles were incubated overnight at ambient temperature (15 °C) and hand-shaken prior to use.
  • Atlantic salmon, strain: Stofnfiskur, Iceland, of mixed sex, and mean weight at vaccination was 33.5 grams.
  • Test animals were given 7 days of acclimatisation at experimental conditions.
  • Routine disease monitoring was performed on the experimental population by a veterinarian responsible for fish health.
  • the batch of experimental fish used tested negative for IPNV, SPDV and ISAV by PCR.
  • the vaccinated salmon for assessment of early reaction were individually marked by maxillae clipping or adipose fin clipping; the salmon injected with the control substance remained unlabelled.
  • Feed was commercial fish feed, available to appetite. Feeding and environmental controls were carried out daily. After vaccination, the fish were observed until they had properly recovered from anaesthesia.
  • test- and the control groups were given the vaccine or control substance by i.p. injection, at 0.1 ml/dose, using single use syringes of 0.5 x 4 mm.
  • Adhesion scores were determined macroscopically according to the Speilberg scale (Midtlyng et al., 1996, Fish & Shellfish Imm., vol. 6, p. 335-350). This scale runs from 0 (no visible lesions) to 6 (very severe lesions, adhesions, granulomas, major carcass damage). Melanisation scores were graded between 0 (no melanisation) and 3 (heavy pigmentation on several organs, fillet unremovable from abdominal wall, fish will be downgraded).
  • Elisa methods applied are the standard tests for these antigens, and are well-known to be indicative of in vivo efficacy.
  • Antibodies against M. viscosa in serum were measured using a direct ELISA.
  • ELISA plates were coated with inactivated M. viscosa and test and control sera were added in serial two-fold dilutions to the plate.
  • Bound antibodies were detected using rabbit anti-salmon IgM, followed by HRP-conjugated mouse anti-rabbit IgG.
  • a colour reaction reflecting bound salmon antibodies was developed by adding a TMB substrate, and the colour measured using an ELISA reader.
  • the antibody titre was expressed as Log2 value of the maximum dilution of the sample that gave an OD-value equal to 3 times the mean found for a negative control serum measured on each plate.
  • Antibodies against A. salmonicida in serum were measured using a similar direct ELISA as described for M. viscosa, except that the ELISA plates were coated with inactivated A. salmonicida.
  • the antibody titre was expressed as Log2 value of the maximum dilution of the sample that gave an OD-value equal to 5 times the mean found for a negative control serum measured on each plate.
  • the antibody titres were calculated using the CBATM program (Abend Vertical) and the titres were expressed in Log2 values as the maximum dilution giving 5 times the mean background. Validity was based on the scores of test- and control samples being within certain value ranges.
  • the test for early adhesion induced by either of the test vaccines showed only very mild local reaction scores at 3 d pv and 12 °C.
  • the average Speilberg scores were: Hepta-P: 0.4, Hepta-C: 0.2, and Saline: 0.0. With standard deviations of 0.5 and 0.4 respectively for the two vaccine groups, these were not found to be statistically different from the control group.
  • the adhesion scores for the different groups at 9 w pv. showed no significant difference between the vaccine groups (average Speilberg scores were 1.2 and 1.3), and both were significantly higher than those in the Saline group (0.0).
  • the antibody titres induced by the Hepta-C vaccine also showed less spread between fish than for the Hepta-P vaccinates.
  • Both vaccine groups induced significantly increased antibody titres as compared to the control group. Again, both vaccine groups induced significantly increased antibody titres as compared to the control group. Whereby the Hepta-C vaccine performed even better than the Hepta-P vaccine in regard to efficacy against M. viscosa.
  • Cithrol-based heptavalent vaccine formulation is better than that of the similar vaccine emulsified with Tween 80 and Span 80, because the Hepta-C vaccinates showed:
  • the treatment groups were reared in freshwater at 12 °C for 6 weeks whereby intramuscular challenge infection was performed with SPDV. Potency against SPDV was measured as relative percentage protection (RPP) of the vaccinated group compared to the control group, by means of detection of infection with SPDV via PCR of serum.
  • RPP relative percentage protection
  • Vaccines used were the same as described in Example 1 : Hepta-P and Hepta-C.
  • the control group received sterile Saline (0.9 % NaCI).
  • the vaccine given was a half dose: 0.05 ml, delivered intra- peritoneally by injection.
  • the different groups were kept in the same tank, separated by different markings.
  • SPDV challenge was performed at 6 weeks after vaccination. Prior to challenge, all fish were transferred to a challenge facility and kept in one tank for the remaining 10 days of the experiment.
  • Challenge material was SPDV SAV3 strain PD03-13p2, at 4.75 Log 10 TCID50/ml. Challenge was administered to individual fish by intramuscular injection of 0.05 ml, at the lateral line anterior to the dorsal fin.
  • Relative (test vs control) prevalence of SPDV infection were used to calculate the potency of the tested vaccines, by the prevalence of fish positive for SPDV after challenge.
  • Cithrol based emulsion vaccine formulations protect fish effectively against a challenge infection with SPDV, and to a level of protection (from a half dose) that was not significantly less than that of current commercial vaccines, while providing the improved safety profile.
  • Example 3 Optimisation of vaccine composition
  • the new vaccine formulation using a polymeric emulsifier according to the invention was demonstrated to be useful as a safe and efficacious vaccine for fish, other aspects could be optimised. Specifically the viscosity of the new formulation, such as tested in the Hepta-C vaccine described in the above examples, was rather low. Although this clearly did not affect safety or efficacy, it was observed that the new vaccine showed so-called‘sedimentation’ upon storage. This means that upon storage, the dispersed aqueous phase tended to move downwards under gravity. This is not the same as breaking of the emulsion, i.e. losing dispersion and showing phase separation. Also, other than breaking, sedimentation is fully reversible, and the phases can be rapidly redistributed by simple shaking by hand prior to administration.
  • the formulation of the Hepta-C vaccine as tested had a water to oil ratio of 45:55 % w/w, and comprised 0.5 % w/w Cithrol DPHS; both percentages are expressed by weight of the vaccine. This resulted in a formulation with a viscosity of about 70 mPa.s. The viscosity was measured as described herein.
  • both the water content and the Cithrol content were varied to increase viscosity. Variations tested were: wateroil ratios of 50:50, 60:40, and 70:30 % w/w. Also, Cithrol content was increased to 1.0 % w/w for some of the samples.
  • the composition of the vaccine-variants tested was essentially the same as that of Hepta-C, apart from the test variables. To assess the effect of the different compositions on sedimentation, the different vaccine compositions were filled into 500 ml bottles, all to the same volume, and these were stored static for 24 hrs at 4 °C. After this period the vertical height of a sedimentation line (if visible) was measured in millimetres, and this was divided by the vertical height of the total volume. Any result of this height ratio below 1 indicated that some level of sedimentation had occurred.

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Abstract

La présente invention concerne les propriétés nouvelles et avantageuses d'une émulsion d'eau et d'huile, qui peut être utilisée pour préparer un vaccin de type émulsion destiné aux poissons qui présente des propriétés de sécurité d'emploi améliorées. L'émulsion utilise une classe spécifique d'émulsifiants polymères au lieu des émulsifiants classiquement utilisés jusqu'à présent. Lorsqu'ils sont utilisés chez les poissons, des vaccins basés sur cette émulsion adaptée induisent une moindre perte de l'appétit après la vaccination, et les poissons vaccinés présentent une récupération plus rapide d'un appétit normal. Ceci tout en assurant une protection immunitaire égale ou supérieure par rapport aux vaccins de type émulsion actuels. L'émulsifiant polymère est un copolymère à blocs de formule générale A-B-A dans laquelle le composant B est le résidu divalent d'un polyalkylène glycol hydrosoluble et le composant A est le résidu d'un acide monocarboxylique complexe liposoluble. L'émulsifiant préféré est un PEG-30-di-(polyhydroxystéarate).
EP19786574.4A 2018-10-16 2019-10-15 Vaccin de type émulsion destiné aux poissons Pending EP3866843A1 (fr)

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EP18200708 2018-10-16
PCT/EP2019/077851 WO2020078941A1 (fr) 2018-10-16 2019-10-15 Vaccin de type émulsion destiné aux poissons

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DE2862369D1 (en) 1977-07-12 1984-03-08 Ici Plc Linear or branched ester-ether block copolymers and their use as surfactants either alone or in blends with conventional surfactants
US5646212A (en) 1994-09-02 1997-07-08 Ici Americas Inc. Polyalkylene glycol anhydroxy carboxylic acid dispersant
AU2002253085B2 (en) 2001-02-28 2006-03-09 Intervet International B.V. Injectable water-in-oil emulsions

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