WO2020078941A1

WO2020078941A1 - Emulsion vaccine for fish

Info

Publication number: WO2020078941A1
Application number: PCT/EP2019/077851
Authority: WO
Inventors: Theodorus Jansen; Petter Frost; Shou Wang
Original assignee: Intervet International B.V.; Intervet Inc.
Priority date: 2018-10-16
Filing date: 2019-10-15
Publication date: 2020-04-23
Also published as: CL2021000894A1; CA3114485A1; EP3866843A1

Abstract

The present invention discloses the new and advantageous properties of an emulsion of water and oil that can be used to prepare an emulsion vaccine for fish which has improved safety properties. The emulsion employs a specific class of polymeric emulsifiers instead of prior art emulsifiers. When used in fish, vaccines based on this adapted emulsion induced a smaller drop in appetite after vaccination, and the vaccinated fish showed a faster recovery to normal apetite. This while providing equal or better immune-protection as compared to current emulsion vaccines. The polymeric emulsifier 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. Preferred emulsifier is a PEG-30-di-(polyhydroxystearate).

Description

Emulsion vaccine for fish

The present invention relates to the field of veterinary vaccinology, more specifically the invention relates to an emulsion vaccine for fish. In particular 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.

In the quest to gather animal protein, wild-catch fishing has for ages been a convenient source of food. However in modern times the high demand has led to overfishing of many fish species, and alternate means have been found: for several fish species a system of fish farming (pisciculture) has been developed whereby fish are raised in containment. Worldwide there has been a steep rise in the tonnage of fish produced from these operations since the 1980’s, and today about half of all fish consumed were produced via fish farming. This covers fish from salt-, brackish, and freshwater species, and from all temperature zones. For example: coldwater species such as salmon, trout, cod, turbot and halibut; temperate water species such as trout, eel, and carp; and warm water-species such as grouper, sea bass, bream, catfish, barramundi, amberjack, tilapia and pangasius.

Because of the large variety in biological characteristics of the various fish species being farmed, different systems of breeding and rearing are applied. Most efficient is when the fish can be raised in containment during their whole life cycle, from fertilized egg to adult form. The breeding can be done indoors in tanks, or outside in ponds, or in closed nets in rivers, lakes or in sheltered areas of sea water. For example Atlantic salmon (Salmo salar) can be fertilised and raised indoors in fresh water tanks for 12 - 20 months to the so-called smolt stadium, after which they need to be transferred to salt water environment to mature for another 1 - 2 years.

While the fish farming of carp and tilapia are numbers 1 and 2 in total numbers of fish produced, the aquaculture of salmonids, especially of Atlantic salmon, has the highest value and is the most industrialised type of fish farming. The largest producers of farmed Atlantic salmon are in: Norway, Chili, Canada, Scotland, Iceland, and the Faroe Islands.

Because they are living together in large groups, and often are kept outside in a natural environment, fish in fish farms are susceptible to a number of infectious diseases. Several aquatic viral, bacterial, fungal, and parasitic agents are known, causing serious effects on health and well-being, as well as considerable economic losses. While initially large amounts of antibiotics were used in fish farming, however that was not sustainable in the long term. Currently the major method of treatment and prevention of diseases in fish is by the active immunisation of the fish, using a growing number of vaccines for various pathogens. Most used vaccines are adjuvated inactivated vaccines, comprising killed virus and/or killed bacteria. Some reviews on fish vaccines are: Sommerset et al. (2005, Expert Rev. Vaccines, vol. 4, p. 89 - 101 ); and Assefa & Abunna (2018, Vet. Med. Int., vol. 2018, article ID: 5432497). The excellent efficacy of these vaccines was of paramount importance for the success and fast expansion of worldwide fish farming. The method of administration of vaccine to fish varies depending on the type of farming and the vaccine; if possible mass vaccination is applied such as by oral vaccination with the feed, alternatively the immersion of fish in a vaccine solution is an option. However, when optimal control and efficacy are required, fish can also be individually injected, typically by intraperitoneal or intramuscular route. This requires temporary anesthetizing the fish.

Vaccines comprising non-replicative antigens often require an immune stimulant for optimal efficacy: 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.

For ease of administration, and for enhanced adjuvant effect, 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. In such emulsion 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. The choice for one or the other type of emulsion can be based on the type of immune-response that is desired.

To generate and maintain such an emulsion requires the input of both mechanical as well as chemical energy: the separate liquids are mixed in an appropriate device using certain levels of shear-force, pressure, and temperature to disperse one phase into another. The chemical energy is provided by the use of an emulsifier (also: surfactant) which stabilises the dispersed phase by taking position at the interphase of water and oil. A vaccine emulsion can be made up of one or more adjuvants, with one or more emulsifiers.

A large number of emulsifiers for use in emulsion vaccines is available, and more are constantly being developed. A short review of this field was made by Ascarateil & Dupuis (2006, Vaccine vol. 24S2, p. S2/83 - S2/85).

Examples of combinations of adjuvants and emulsifiers as used in commercial veterinary vaccines are: Amphigen® (Zoetis), containing a light mineral oil with lecithin as emulsifier; Xsolve® (previously called: Microsol-Diluvac Forte®, MSD Animal health), which contains a combination of the adjuvants light mineral oil and vitamin E-acetate, with the emulsifier Tween® 80 (Polysorbate 80, or polyoxyethylene sorbitan mono-oleate); and MetaStim® (Zoetis), comprising squalane, Pluronic® (a nonionic tri-block copolymer of blocks of polyoxyethylene and polyoxypropylene), and Tween 80.

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). Next to the paramount requirements for emulsion vaccines to be efficacious and stable, there are some special requirements for vaccines for use in fish. These refer to aspects of ease of use, and especially to costs. This because fish farming is very much a high volume - low margin enterprise. For these reasons, 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

administration. Examples of such multivalent vaccines are: 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.

In the use of adjuvated vaccines, a balance between safety and efficacy is an important factor, especially for oil-adjuvated vaccines. While these vaccines are highly effective at relatively low cost, however these are known to cause some vaccination side-effects. See for reviews: Midtlyng, 1997 (Dev. Biol. Stand., ol. 90, p. 371-379); and Berg et al., 2006 (Dis. of Aq. Organ., vol. 69, p. 239-248). While undesirable, such side effects are generally considered acceptable when taking into account the inevitable serious disease or mortality in case the fish would not be vaccinated. For example in salmon, 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.

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

PharmSciTech, vol. 13, p. 247-253).

NB: These emulsifiers are not the same as the non-ionic block copolymeric emulsifiers that are known generally as Poloxamer™ (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.

Through the years several attempts have been made to analyse, overcome or prevent the vaccination side-effects of oil-adjuvants used in fish vaccination: change of the conditions of the vaccination, in timing, the size of the fish, or the temperature of the water (Berg et al., supra),

use of other oils: non-mineral oil instead of mineral oil, see Thim et al. (2014, vaccines, vol. 2, p. 228-251 ),

use of other adjuvants: Toll like receptor agonists, or particulate structures, see Brudeseth (2013, Fish & Shellf. Imm., vol. 35, p. 1759-1768), and Villumsen et al. (2017, Sci. Reports, vol. 7, doi: 10.1038/s41598-017-06324-7), and

use of non-adjuvanted types of vaccines such as DNA based vaccines, e.g. Apex®-IHN or Clynav® (Novartis/Elanco), etc.

However for large scale application in commercial vaccines, these adaptations were not considered satisfactory in terms of the vaccine efficacy and/or the duration of the immune response. Also, such other adjuvants are generally more expensive, and the use of a DNA- or recombinant protein-based vaccine may not be appropriate or possible for each type of pathogen. So although the side-effects of oil- adjuvated vaccines have been a concern for a long time, this is still the major type of vaccine being used in fish vaccination, for lack of an alternative that is equally effective, affordable, and broadly applicable.

Consequently, there is a need in the field to further improve oil-adjuvanted vaccines for fish, while keeping them effective and affordable.

It is therefore an object of the present invention to overcome a disadvantage in the prior art, and to accommodate to this need in the field by providing an emulsion of water and oil with an antigen of a fish pathogen, that can be used in an improved vaccine for fish.

When using a current W/O emulsion vaccine, vaccination side-effects in vaccinated fish have been described. For example for oil-adjuvated vaccines using Polysorbate 80 and sorbitan mono-oleate as emulsifiers. Next to mild side-effects in the abdominal region, a reduction of feed intake in the two weeks post vaccination could be observed.

Because such multivalent emulsion vaccines are such complex mixtures, there was no direct indication which factor(s) was responsible; other workers had replaced the mineral oil by other adjuvants, however that reduced vaccine efficacy and duration, as well as increase the cost-price of the vaccine. Consequently, there was no clear indication in the art how the benefits of classic mineral oil-emulsions vaccines could be maintained while reducing their side-effects.

Surprisingly it was found that the object can be met, and consequently one or more disadvantages of the prior art can be overcome, by using as the emulsifier 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. In addition 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.

It is not known exactly how this class of polymeric emulsifiers reduces the vaccination side-effects.

Although the inventors do not want to be bound by any theory or model that might explain these findings, they speculate that the prior art use of sorbitan-based emulsifiers, in particular of Polysorbate, somehow played a key role in causing the drop in apetite in the vaccinated fish. Consequently it is speculated that the replacement of Polysorbate and sorbitan mono-oleate by a polymeric emulsifier as described for the invention, provides a safer emulsion vaccine for fish, even when using mineral oil as an adjuvant.

This was not at all obvious from any disclosure in the prior art, as there was no indication that a sorbitan- based emulsifier was the cause of these vaccination side-effects in fish; many changes have been made to fish vaccines in the past but no studies varied the emulsifier to improve safety. Also, even with a plan to reduce or remove Polysorbate, the inventors had no indication on which compound would be suitable as replacement, while being safe and effective in an emulsion vaccine for fish.

Therefore in a one aspect 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.

Typically the droplets of the dispersed phase are very small, in the range of micrometers.

Procedures and equipment for the preparation of an emulsion at any scale are well-known in the art, and are for instance described in handbooks such as:“Remington: the science and practice of pharmacy” (2000, Lippincot, USA, ISBN: 683306472), and:“Veterinary vaccinology” (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681 ).

The term“comprising” (as well as variations such as“comprise”,“comprises”, and“comprised”) as used herein, refer(s) to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and does not refer to the exclusion of any of such element(s) or combinations. Consequently, 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. Depending on the type of the emulsion, the oil-phase is the continuous phase (as in a W/O emulsion), or is the dispersed phase (as in an O/W emulsion). When used for the formulation of a vaccine, 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.

For the invention,“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. Preferably 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.

The term“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. For example 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. All members share the newly discovered and advantageous property that when used as the emulsifier of oil-emulsion vaccines for fish, the vaccinated fish showed less reduction of appetite post vaccination, faster return to normal feed intake, and therefore a much lower dip in their growth curve, as compared to prior art use of emulsifiers. Consequently, the skilled person is perfectly capable of selecting a particular type of the polymeric emulsifier for the invention to achieve a certain emulsifying effect.

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).

Also, one or more further emulsifiers can be added, to provide the combination of emulsifiers with certain desired properties. A well-known way to characterise the properties of (mixtures of) emulsifiers is the HLB number (hydrophile-lipophile balance; Griffin 1949, J. Soc. Cosm. Chem., vol. 1 , p. 311-326). Typically 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.

Details of embodiments and of further aspects of the invention will be described below. It is preferred that 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.

Therefore in an embodiment 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. Another is the type of adjuvant. To provide good‘duration of immunity’, i.e. provide immune-stimulation over a long period of time such as many weeks or months, 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.

Therefore in an embodiment of the emulsion according to the invention, the emulsion is a water-in-oil (W/O) emulsion.

For the preparation and stabilisation of a W/O emulsion according to the invention, a polymeric emulsifier for the invention is selected with the right properties, preferably having an HLB number of 10 or less.

Therefore, in an embodiment of the W/O emulsion according to the invention, components A each have a molecular weight of at least 500 g/mol.

In an embodiment of the W/O emulsion according to the invention, component B has a molecular weight of at least 500 g/mol.

In a preferred embodiment of the W/O emulsion according to the invention, 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. 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.

Preferably the hydroxystearic acid is a 12-hydroxystearic acid.

In an embodiment of the W/O emulsion according to the invention, component B is a polymer of an ethylene glycol.

In a preferred embodiment of the W/O emulsion according to the invention, component A is a

polyhydroxystearic acid, and component B is a polyethylene glycol.

Preferably the polyhydroxystearic acid is a poly(12-hydroxystearic acid).

Polyethylene glycol (PEG) is also known as polyethylene oxide (PEO) or polyoxyethylene (POE).

In an embodiment of the W/O emulsion according to the invention, 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 A has 2 - 50 units of hydroxystearic acid, and component B has 8 - 60 units of ethylene glycol. For the invention, cited ranges also include the end points.

Especially favourable results in the formulation of safe vaccines for fish based on W/O emulsions according to the invention, were obtained using as the emulsifier a PEG-30-di-(polyhydroxystearate).

Therefore, in an embodiment of the W/O emulsion according to the invention, the polymeric emulsifier is a PEG-30-di-(polyhydroxystearate).

The term“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.

NB: 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. As described in the Examples hereinafter, a stable multivalent W/0 emulsion could be prepared by using Cithrol DPHS instead of the two prior art emulsifiers: Polysorbate 80 (Tween® 80) and Sorbitan mono- oleate (Span® 80). When used to prepare a vaccine, 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.

Therefore in an embodiment, 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.

Preferably 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.

In a preferred embodiment, 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.

For the invention“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.

In an embodiment, 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.

Preferably 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.

In an embodiment of the emulsion according to the invention, the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites.

In an embodiment, 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.

Preferably, the bacterial pathogen is one or more selected from Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum, and Moritella viscosa. In an embodiment, 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 one or more selected from Salmon pancreas disease virus (SPDV) and Infectious pancreatic necrosis virus (IPNV).

In an embodiment, the fungal pathogen is selected from: Saprolegnia, Achyla, Aphanomyces, lchthyophonus, Branchiomyces and Dermocystidium.

In an embodiment, the algal pathogen is selected from: Chlorochytrium and Scenedesmus.

In an embodiment, the endo- or ecto-parasitic pathogen is selected from: Amoebae, Flagellates, Ciliates, Microsporidia, Myxosporeans, Monogeneans, Cestodes, and Crustaceans.

In a preferred embodiment, the crustacean parasite is an ecto-parasite, preferably from the taxonomic family Caligidae; more preferably from the genera Lepeophtheirus or Caligus.

The names indicated above refer to the taxonomic groups to which these pathogens are currently assigned. However that is a taxonomic classification that could change in time as new insights can lead to reclassification into a new or different taxonomic group. However, as this does not change the pathogen itself, or its antigenic repertoire, but only its scientific name or classification, such re-classified (micro-) organisms remain within the scope of the invention.

The reference to the various taxonomic groups includes any pathogen that is a species, subtype, variant, biotype, serotype or genotype within that group.

In a preferred embodiment, the fish pathogen is a pathogen that is infectious to salmonid fish.

Such 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.

As described, 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.

In an embodiment 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.

Preferably the two or more fish pathogens are selected from bacteria and viruses.

More preferably, 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.

As described, 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. However, the use of 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.

Therefore in an embodiment of the emulsion according to the invention, 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. Also 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. Depending on the characteristics of the emulsion according to the invention, 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.

For the invention the non-live antigen can be of any degree of purity.

Therefore in an embodiment of the emulsion according to the invention, 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. What constitutes 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). For the invention 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.

Methods and materials to prepare such an inactivated culture or to prepare such a part thereof are generally known and available at any scale. For example: 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.

As is most often applied for antigens from bacterial pathogens for use in fish vaccines, the antigen comprises inactivated bacterial cells, also known as a bacterin.

Therefore, in an embodiment of the emulsion according to the invention, 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.

In a preferred embodiment 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.

With the proven performance of mineral oils in emulsion vaccines for fish, and their affordable price, the only disadvantage of their use so-far were the vaccination side-effects. However, this is overcome to a large degree by the present invention.

Therefore, in an embodiment of the emulsion according to the invention, the oil phase comprises a mineral oil.

Preferably 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®

(Sonneborn).

In an embodiment the emulsion according to the invention comprises one or more of the features selected from the group consisting of:

the emulsion is a water-in-oil (W/O) emulsion.

in the 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;

in the W/O emulsion components A and component B all have a molecular weight of at least 500 g/mol;

in the W/O emulsion, component A is a polymer of a hydroxystearic acid; preferably the hydroxystearic acid is a 12-hydroxystearic acid;

in the W/O emulsion, component B is a polymer of an ethylene glycol;

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). 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

A has 2 - 50 units of hydroxystearic acid, and component B has 8 - 60 units of ethylene glycol; in the W/O emulsion according to the invention, the polymeric emulsifier is a PEG-30-di-

(polyhydroxystearate);

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,

Francisella, Flexibacter, Cytophaga, Corynebacterium, Renibacterium, Flavobacterium,

Fusarium, Bacillus, Yersinia, Mycobacterium, Neorickettsia, Piscirickettsia, Streptococcus, Pseudomonas, Photo bacterium, Clostridium, Tenacibaculum, Lactococcus, Leucothrix, and Nocardia; preferably 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,

Microsporidia, Myxosporeans, Monogeneans, Cestodes, and Crustaceans; in a preferred embodiment, 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. Preferably the two or more fish pathogens are selected from bacteria and viruses. More preferably, 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 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. In an embodiment of the emulsion according to the invention, 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. salmonicida, Vibrio salmonicida, Vibrio anguillarum serotypes 01 and 02a, and Moritella viscosa; and 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.

Similarly, when preparing an O/W emulsion, the polymeric emulsifier for the invention 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.

For making an O/W type emulsion, where the antigen is initially not present in the aqueous phase, the use of high intensity emulsification of water and oil is a further option, for example using microfluidisation. However, as the skilled person will be well aware, when preparing a W/O emulsion, where the non-live bacterial antigen is comprised in the aqueous phase to be emulsified, the type and the intensity of the emulsification process applied, needs to be compatible with the fragile nature of the antigen to keep its immunological properties intact.

Therefore in a further aspect 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:

a. admixing the aqueous phase and the polymeric emulsifier,

b. emulsifying the mixture of step a. with the oil phase, and

c. admixing the emulsion of step b. with the antigen from a fish pathogen.

Each of the aqueous phase, the polymeric emulsifier, the oil phase, and the antigen from a fish pathogen, are as defined hereinabove. In a similar aspect, the invention relates to a method for the manufacture of the W/O emulsion according to the invention, the method comprising the steps of:

a. admixing the oil phase and the polymeric emulsifier, and

b. emulsifying the mixture of step a. with the aqueous phase, whereby the aqueous phase

comprises the antigen from a fish pathogen.

Again, each of the oil phase, the polymeric emulsifier, the aqueous phase, and the antigen from a fish pathogen, are as defined hereinabove.

Preferably 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. Commonly this regards the use of ingredients that are pharmaceutically acceptable, and complying with quality regulations such as good manufacturing practice standards. All these are well known to a skilled person, and are prescribed in Governmental regulations such as the Pharmacopoeia, and in handbooks such as: Remington and Pastoret (both supra). Typically such manufacture is done aseptically.

As described, the emulsion according to the invention is particularly advantageous when applied as a constituent of a vaccine for fish.

Therefore, in a further aspect 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.

In a further aspect 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.

In an embodiment 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.

In an embodiment of the vaccine according to the invention, the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites.

As the skilled person will be well aware, the emulsion according to the invention can be applied“for use in a vaccine” in different ways. For example, the emulsion itself can be applied as a vaccine. Alternatively 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. Also, 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. Depending on the type of the emulsion, 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. Also, or consequentially, 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’.

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. For example the antigen dose can be expressed as a virus titre or a number of bacterial cells. Alternatively the antigen can be quantified by a serologic- or bio-chemical test such as an ELISA or an AlphaLisa™, 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. Alternatively, 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. Such 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.

Therefore, in an embodiment, 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. Alternatively 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.

In a preferred embodiment, the target fish are salmonid fish.

As described, 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.

Therefore in a preferred embodiment of the vaccine 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.

More preferably 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.

For ease of handling, and to facilitate the use of the vaccine according to the invention, and specifically of its administration by injection, the emulsion vaccine should not have a viscosity that is too high. In addition, by choosing a certain viscosity, the occurrence of sedimentation or creaming of the dispersed phase in the emulsion vaccine can be reduced or prevented.

Therefore in an embodiment the vaccine according to the invention has a viscosity below 500 mPa.s.

Preferably 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.

Therefore in an embodiment of the vaccine according to the invention, the mean droplet size (diameter) of the dispersed phase is less than 25 pm.

Methods and equipment to measure particle sizes are well-known, and can for example employ laser-light scattering measurements.

In a preferred embodiment of the vaccine according to the invention, 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;

In a preferred embodiment 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.

It is well within reach of the skilled person to further optimise a vaccine according to the invention.

Generally 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.

Further aspects of the present invention relate to novel and inventive uses and combinations of the elements of the invention such as the polymeric emulsifier, the antigen from a fish pathogen, or the oil phase, all as described for the invention.

Therefore, in a further aspect 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.

In a further aspect 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.

In a further aspect 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. In a further aspect 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.

In a preferred embodiment 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.

Therefore, in a further aspect 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. Typically 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. Depending on whether the vaccine according to the invention is based on an O/W or on a W/O emulsion, different techniques of administration can be applied. For example as an O/W emulsion vaccine 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.

“Parenteral” refers to administration through the skin, for example by intramuscular,

intraperitoneal, intradermal, submucosal, or subcutaneous route.

It goes without saying that the optimal route of administration of a vaccine according to the invention will depend on the specifics of the vaccine that is used, and on the particular characteristics of the fish. A skilled person is perfectly capable of selecting and optimising such route and method of administration.

The volume of a dose of the vaccine according to the invention, e.g. when administered by parenteral route, is a volume that is acceptable for the fish, and can for instance be between about 0.001 and about 5 ml. Preferably 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

administered in a simultaneous, concurrent or sequential fashion, in a manner compatible with their registered use. The invention will now be further described by the following, non-limiting, examples.

Examples

1. Example 1 : Safety and efficacy trials

1.1. Introduction

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.

1.2. Materials and methods

1.2.1. Experimental design

For assessing the appetite loss and the serological response 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

intraperitoneally vaccinated with vaccines Hepta-P (heptavalent antigen containing emulsion, with polysorbate and sorbitan-oleate as emulsifiers), or Hepta-C (similar vaccine, comprising Cithrol as emulsifier), as test groups. A control group was also included that was injected with saline. The three groups, each consisting of 50 fish, were kept in separate tanks at 17 °C during the nine weeks immunization period. The fish were observed daily.

For a period of 17 days post vaccination (pv), feed intake was determined daily, by subtracting collected remaining uneaten feed the amount of from feed given. At 9 weeks 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.

The assessment of the early effects of vaccination was performed in separate fish. One week before vaccination Atlantic salmon parr (approximately 35 grams) were acclimatized to water at 12 °C. Groups of twenty fish were injected in the same way as in the 9 week safety and efficacy experiment, and kept together in one tank, differentiated by group specific labelling. The fish were sampled 3 days pv to evaluate early reactions to vaccination such as adhesions and melanisation in the abdominal cavity.

1.2.2. Test vaccines

Both test vaccines Hepta-P and Hepta-C contained identitical quantities of the following inactivated antigens per dose (0,1 ml) of vaccine:

Salmon pancreas disease virus (SPDV), strain F93-125, > 75 % RPP (1 )

Infectious pancreatic necrosis virus (IPNV), serotype Sp, > 1.5 ELISA units (2)

Aeromonas salmonicida subsp. salmonicida, > 10.7 log2 ELISA units (3)

Vibrio salmonicida, > 90 % RPS (4)

Vibrio anguillarum serotype 01 , > 75 % RPS

Vibrio anguillarum serotype 02a, > 75 % RPS

Moritella viscosa, > 5.8 log2 ELISA units (3)

(1 ) RPP: relative percentage protection in a laboratory test in Atlantic salmon

(2) antigen amount measured in finished product

(3) serological response in Atlantic salmon

(4) RPS: relative percent survival in a laboratory test in Atlantic salmon

The 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.

1.2.3. Test animals and husbandry

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. In addition, the batch of experimental fish used, tested negative for IPNV, SPDV and ISAV by PCR.

The vaccinated salmon for assessment of early reaction (n=20 per group) were individually marked by maxillae clipping or adipose fin clipping; the salmon injected with the control substance remained unlabelled.

Test animals were kept in fresh water tanks, at 17 °C ± 2 °C with at least 85% oxygen, or at 12 °C ± 2 °C with at least 75 % oxygen, pH = 6.8 - 7.2. 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.

1.2.4. Vaccination

Prior to vaccination the experimental fish were starved for 36 hrs and anaesthetized. The 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.

1.2.5. Monitoring of results

Early reactions to vaccination were monitored in the fish kept at 12 °C. These were starved for 24 hrs, and at 3 d. pv the fish were killed by an overdose of anaesthetic, bled, and autopsy was performed, checking for adhesions and melanisation in the abdominal cavity.

The scoring of adhesions (Speilberg score) and melanisation was done according to standard practices in this field.

Further safety and serology data were collected from the fish kept in separate tanks at 17 °C for 9 weeks: appetite loss was measured by weighing the food given to each tank and by collection and weighing of the food remaining. This was done daily for 17 days after vaccination. Analysis of feed intake over time was done using a two way ANOVA MIXED model with vaccine and time as fixed effects for each of the periods 1-9 d. pv. and 10-17 d. pv., and difference between groups were estimated based on the differences of Least Square Means (a=0.05; using the program SAS®).

Late local reaction scores were monitored at 9 weeks pv in 35 fish from each group, by checking for adhesions and melanisation in the abdominal cavity upon autopsy. The scores were statistically evaluated using the Kruskal-Wallis test, followed by the Dunn test as post-hoc test for pairwise group comparisons (a=0.05; SAS)

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).

For serology determination, blood was collected at 9 w. pv. from 35 fish from each test group kept at 17 °C, and of 12 fish from the control group. After overnight clotting, the sera were mixed 1 :1 with 86 % glycerol and stored at -20°C until analysis by ELISA.

The 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. In short, 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 CBA™ 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.

1.3. Results and Discussion

1.3.1. Results of tests for early local reactions at 3 d py and 12 °C

1.3.1.1. Early adhesions scores

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.

1.3.1.2. Melanisation at 3 d pv

No signs of melanin were found on organs at 3 d pv in any of the groups.

1.3.2. Results of tests for feed intake for 17 days, and for safety and serology at 9 w py and 17 °C

1.3.2.1. Evaluation of loss of appetite post immunization

The change in feed intake was recorded (in grams of feed ingested) for each tank (treatment group) over a period of 17 days following vaccination. The results clearly showed that appetite was much less reduced after vaccination with the Hepta-C vaccine, as resulting from the vaccination with the Hepta-P vacine (p=0.045).

Also, the fish vaccinated with Hepta-C were back to the same level of daily feed intake as the saline group already after 9 d pv, while for the fish receiving the Hepta-P vaccine, this took 15-16 days. Results are presented in Figure 1. 1.3.2.2. Adhesion scores at 9 w pv

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).

1.3.2.3. Melanisation at 9 w pv

No significant differences were found in level of melanisation of organs or of abdominal wall between the two vaccine groups. Only the melanisation of organs was significantly higher for both vaccines as compared to saline group.

1.3.2.4. Antibody response against A. salmonicida at 9 w pv

Specific antibodies against M. viscosa and A. salmonicida were measured in the same sera.

Both vaccine groups induced significantly increased antibody titres as compared to the control group. The antibody titres against A. salmonicida induced by the Hepta-C vaccine were significantly higher than those induced by the Hepta-P vaccine (ANOVA, p< 0.0001 ), while the antigens used and their amounts were the same. Both vaccines induced titres that were above the potency requirement (10.7 Log2).

As is also evident from the smaller standard deviation, the antibody titres induced by the Hepta-C vaccine also showed less spread between fish than for the Hepta-P vaccinates.

The ELISA titres in the saline group were below the detection limit (6.6). Results are presented in

Table 1.

Table 1 : Serology results (ELISA) against A. salmonicida at 9 w pv, and at 17 °C

1.3.2.5. Antibody response against M. viscosa at 9 w pv

For the induction of seroresponse against M. viscosa, a similar picture emerged as for A. salmonicida: the titres induced by the Hepta-C vaccine were significantly higher (ANOVA, p< 0.0001 ) and with less spread, than the titres induced by the Hepta-P vaccine, even though both contained the same antigens and at the same amounts. Results are presented in Table 2. Both vaccines induced titres that were above the potency requirement (5.8 Log2).

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.

Table 2: Serology results (ELISA) against M. viscosa at 9 w pv, and at 17 °C

1.4. Conclusions

The safety and efficacy profile of the 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:

Significantly less loss of appetite after vaccination, and a faster return to normal appetite, Similar scores for intra-abdominal adhesions and melanisation, and

Significantly better immune-response against A. salmonicida and against M. viscosa; both with higher antibody titres and with smaller spread.

2. Example 2: Efficacy against SPDV

2.1. Introduction

In this experiment the inventors expanded on the efficacy results as described in Example 1 above. Using the exact same vaccine formulations the protective capacity was tested against a challenge infection with salmon pancreas disease virus (SPDV). The side-by-side comparison was made between a prior art formulation of a heptavalent vaccine with Tween 80 + Span 80 emulsifiers, against a heptavalent vaccine based on the novel emulsion with Cithrol DPHS as emulsifier. Vaccinations used only a half dose per animal, conform the registered potency test for release of efficacious batches of SPDV vaccines. 2.2. Materials and methods

2.2.1. Experimental design

In short: acclimatized Atlantic salmon parr was i.p. vaccinated with half of a full dose of each vaccine. Sterile saline was used as mock vaccine.

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.

2.2.2. Test vaccines

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.

2.2.3. Test animals:

Atlantic salmon, 38 per group, strain: Salmobreed, of mixed sex, and mean weight at vaccination was 28 grams. The different groups were kept in the same tank, separated by different markings.

2.2.4. Challenge infection

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.

2.2.5. Post challenge blood sampling

Ten days post SPDV challenge, individual blood samples for PCR testing were collected from the caudal vein of anesthetized fish. After o/n clotting serum was collected and kept frozen until use.

2.2.6. SPDV Real Time PCR

RNA was extracted from individual sera, that had been spiked with inactivated Equine Influenza Virus, EIV. 35 samples per group were analysed with the SPDV gene nsP1 , in a real-time PCR assay, to detect SPDV viremia prevalence as measure of protection against challenge. A real time PCR assay specific for EIV HA gene was also performed to detect the EIV spike added to the serum, as a positive control on the quality of the RNA extraction.

Relative (test vs control) prevalence of SPDV infection (by PCR detection of SPDV in sera) were used to calculate the potency of the tested vaccines, by the prevalence of fish positive for SPDV after challenge. The level of protection was expressed as absolute (+ or -) and as the relative difference in infection prevalence between vaccinated groups and the control group, as a: relative percentage protection (RPP), which is calculated as follows: RPP= [1- (% PCR positive fish in vaccinated group / % PCR positive fish in control group)] x 100.

Statistical analysis of the proportion of infected fish in the groups with PCR detected SPDV in serum was performed by the Fisher’s exact test, comparing the vaccinated groups to the saline group. In addition the prevalence of positive fish in groups receiving the same dose was also compared pairwise to each other using the Fisher exact test. The level of significance (a) was set to 0.05, and the test was two-sided. Statistical calculations were executed using the SAS program.

2.3. Results and Discussion

All samples were PCR positive for the EIV spike gene, which served as internal control for the RNA purification. Therefore all samples were valid for analysis.

An overview of the SPDV PCR results on sera sampled at 10 d post challenge is presented in Table 3, indicating the prevalence and calculated RPP for each group, versus prevalence in the saline group.

Table 3: Prevalence and RPP values based on PCR detection of SPDV in serum at 10 d. p.c.

The PCR results for the saline group showed that the SPDV i.m. challenge was successful since the number of positive fish in this group was 97 % (34 out of 35).

The prevalence of SPDV positive fish in all vaccinated groups was significantly lower than that in the control group, as demonstrated by a p value in Fisher’s exact test of 0.0001.

Importantly, even though all vaccinated animals only received a half dose of vaccine, the two groups (Hepta-P and Hepta-C) were not significantly different from each other in the prevalence of SPDV infection after challenge; the p value in the two sided Fisher test was 0.133. 2.4. Conclusion

The results show that 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.

3. Example 3: Optimisation of vaccine composition

3.1. Introduction

After 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.

Some level of sedimentation is common for water-in-oil emulsion vaccines, and most product leaflets will recommend a brief shaking of the emulsions before administration. Nevertheless such sedimentation could make a commercial product less attractive visually. Therefore the inventors developed some variants of the formulations tested, to optimise also this aspect of the new emulsion and vaccine.

3.2. Variations tested:

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.

To prevent, or at least to considerably reduce sedimentation of the aqueous phase, 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.

In a smal experiment, similar to the setup in Example 1 , the variants of the emulsion vaccines were also tested for their capacity to induce protective levels of antibodies against A. salmonicida, and M. viscosa. Table 5 shows the results of the ELISA titrations; protective Ab titres are levels above 10.7 or 5.8 Log2 respectively.

3.3. Results

Combined results of viscosity and sedimentation are presented in Table 4. Serology results are presented in Table 5.

Table 4: Effect of variations in composition, on viscosity of water-in-oil formulations with Cithrol as

emulsifier

Table 5: Log2 ELISA titres induced by vaccination with the variants of the emulsion vaccines

3.4. Conclusions

Several observations could be made:

the increase in water content in the emulsion had more effect on viscosity than the increase of Cithrol content

(almost) complete prevention of sedimentation (after 24 hrs at 4 °C) could be achieved by increasing the water content in the emulsion to a 70:30 wateroil ratio, and/or by increasing the Cithrol content to 1 % w/w.

all vaccine compositions induced protective levels of antibodies against A. salmonicida and M. viscosa.

Legend to the figures

Figure 1 :

Daily feed intake in grams/group for the different test groups, over a period of 17 days post-vaccination

Claims

1. 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.

2. The emulsion according to claim 1 , characterised in that the emulsion is a water-in-oil (W/O)

emulsion.

3. The W/O emulsion according to claim 2, characterised in that components A and component B all have a molecular weight of at least 500 g/mol.

4. The W/O emulsion according to claims 2 or 3, characterised in that component A is a

polyhydroxystearic acid, and component B is a polyethylene glycol.

5. The W/O emulsion according to any one of claims 2 - 4, characterised in that the polymeric emulsifier is a PEG-30-di-(polyhydroxystearate).

6. The emulsion according to any one of claims 1 - 5, characterised in that the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites.

7. The emulsion according to any one of claims 1 - 6, characterised in that the antigen of a fish

pathogen comprises inactivated bacterial cells.

8. The emulsion according to any one of claims 1 - 7, characterised in that the oil phase comprises a mineral oil.

9. Method for the manufacture of the W/O emulsion according to any one of claims 2 - 8, the method comprising the steps of:

a. admixing the oil phase and the polymeric emulsifier, and

comprises the antigen from a fish pathogen.

10. The emulsion according to any one of claims 1 - 8, for use in the vaccination of fish against infection or disease caused by a fish pathogen.

1 1. 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 any one of claims 1 - 8.

12. Vaccine according to claim 11 , characterised in that the fish pathogen is selected from: bacteria, viruses, and endo- or ecto-parasites.

13. 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 in any one of claims 1 and 3 - 5.

14. Use of the polymeric emulsifier as defined in any one of claims 1 and 3 - 5, 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.

15. 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 in any one of claims 1 and 3 - 5.

16. 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 in any one of claims 1 and 3 - 5.

17. 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 claims 1 1 or 12.