NO335784B1 - Liposome vaccine comprising antigenic material from fin-fish pathogens. - Google Patents

Abstract

Described are liposome vaccine formulations for fin fish which comprise antigenic material derived from one or more fin fish pathogens in association with liposomes and which optionally include other therapeutic compounds. The vaccines can be used for immunization and / or for therapeutic treatment of fin fish against infectious diseases. The vaccines can result in reduced side effects such as less adhesion and pigmentation compared to oil-based vaccine formulations. Methods for preparing and storing the liposome vaccines and methods for administering the vaccine formulation to fin fish are further described.

Description

FIELD OF THE INVENTION

The present invention relates to fish vaccines and in particular vaccines for immunogenic and therapeutic use in finfish.

BACKGROUND OF THE INVENTION

Disease, caused by a number of pathogens, has been responsible for large economic losses in the aquaculture industry. With the advances in technology and increased knowledge of fish pathogens, aquaculture health products have been developed to help fish farmers manage disease. Traditionally, aquaculture vaccines have been oil-based formulations. Especially in salmon farming, oil adjuvant vaccines are standard within the industry for long-term protection against furunculosis (Aeromonas salmonicida). Furthermore, because of the difficulty in generating long-term protection against A. salmonicida, the standard formulation for A. salmonicida typically dictates the formulation for other vaccine components. As a result, all vaccine components are generally formulated with oil adjuvants.

While these oil-based formulations are effective against certain fish pathogens, they are associated with serious side effects at the injection site such as adhesions and pigment formations that result in a deterioration of the quality of the fish at harvest. A further side effect can be growth retardation. These side effects potentially affect earnings. In addition, the time and costs associated with injecting fish can increase significantly due to the difficulties associated with administering a viscous oil adjuvant formulation to large quantities of fish.

It has been difficult to generate long-term protection against certain fish pathogens. So far, injection is the only route of administration that has provided reasonable immunity to fish diseases. Despite a strong commercial need and extensive research, an effective vaccine against fish-related pathogenic organisms that does not require administration by injection has eluded scientists since Duff's first oral vaccine trials (Duff, 1942). Experiments have shown that injection of a vaccine preparation provides the best result with the longest duration of protection and immunization, while diving and oral administration provide less effective protection against the infections in question.

Inactivated viral or bacterial vaccines have been administered by oral methods where the vaccine is added directly to the water or incorporated into the fish feed. Oral vaccines have historically been shown to be inconsistent and with relatively low levels of protection, suggesting that they may be most useful as a revaccination method.

Gene expression systems leading to in situ production of antigens have been used as a method to introduce antigens directly into animals, typically by using live viral vectors containing special sequences from an adenovirus, an adeno-associated virus or a retrovirus genome. In viral sequences, it allows the appropriate processing and packaging of a gene into a virion, which can then be introduced into animals via invasive or non-invasive infection. Viral vectors have several shortcomings. As, for example, viral vectors are living pathogens, they still carry a risk of unintended infection. Furthermore, the protein from viral vector sequences includes unwanted inflammatory or other immune responses that may prevent the use of the same vector for subsequent vaccine or boost. Viral vectors also limit the size of the target gene that can be expressed due to viral packaging limitations.

"Naked DNA", i.e. DNA not incorporated into a vector, transfects relatively efficiently when injected into skeletal muscle, but poorly or not at all when injected into other tissues (Wolff et al., Science 247: 1465-1468 (1990) , which is considered part of the description). Plasmid DNA coated on the surface of small gold particles and introduced into the skin by a helium-powered particle accelerator or "gene gun" can be used to directly transfect cells in the epidermis or dermis (Pecorino and Lo, Current Biol., 2: 30-32 (1992) ).

Other immunization methods described include a method of immunizing fish against disease by spraying with vaccine (U.S. Patent No. 4,223,014) and a hyperosmotic immersion method for treating aquatic animals by immersing the animals in a hyperosmotic solution followed by immersion in a vaccine containing solution (U.S. Patent No. 4,009,259). U.S. patent no. 6,180,614 describes methods for immunization of aquaculture species by introducing DNA expression systems into the aquaculture species by various methods of administration, including the use of liposomes as agents for transfection.

Liposomes are vesicles comprising phospholipids and other components that spontaneously form into concentric bilayers when they come into contact with aqueous solutions. Particles contained in solution are trapped between or attached to the objects which, being biodegradable, slowly release their contents as they break down in a biological system. Liposomes can therefore act as a vaccine adjuvant due to the slow release of antigen. Phosphatidylcholine is itself a poor antigen (Alving, In: Sela, M.

(ed.) The Antigens, Vol 4, Academic Press. New York, 1-15, 1977), but has been used successfully as an immunoadjuvant (van Rooijen & van Nieuwmegan, Immunol. Commun. 9: 747-757, 1980). Thus, the liposomes act not only as a carrier, but, possibly more importantly, as an adjuvant.

Liposomes have primarily been used in the human health area to deliver various compounds to target organs and to reduce the toxicity of certain chemotherapeutic agents. In mammalian studies, liposomes are considered to be suitable carriers for a number of drugs and vaccines because they can a) serve as a depot system for sustained release of a compound; b) alter the biodistribution of biologically active substances (Profitt et al, 1983; Lui and Huang, 1992); c) protect the encapsulated materials from inactivation due to host defense mechanisms (Ahmad et al. 1993; Vaage et al. 1993); and d) reduce side effects (Graner et al. 1985; Philips et al. 1991). Furthermore, liposomes have been used as drug delivery carriers because they can be safely administered without serious side effects and because of their biodegradability and non-toxic nature.

Power et al., J. Fish Disease, 13: 329-323, 1990, examine tissue distribution of liposomes after injection into rainbow trout, but have no data on safety. Furthermore, it is most important that Power's formulations do not contain antigen, and thus the survey does not address the issue of effectiveness. Indeed, Power concludes that "more work is needed to determine the effectiveness of liposomes in delivering pharmaceuticals to fish"; see the end of the paragraph on page 331.

Uptake and tissue distribution of empty liposomes after parenteral administration to rainbow trout has been studied (Power et al., J. Fish Disease, 13: 329-323, 1990) in the same way as uptake and biodistribution of a radiolabeled form of liposomally incorporated lipopolysaccharide from Aeromonas salmonicida in rainbow trout (Nakhla et al., J. of Liposome Research 4(2): 1029-1048,1994). However, the latter study does not show how the use of liposomes can be linked to vaccine effectiveness expressed in terms of mortality. In another study, bacterial lipopolysaccharide from A. salmonicida was incorporated into liposomes and the effect on the antibody responses in rainbow trout was analyzed (Nakhla et al., Fish and Shellfish Immunology, 7: 387-401, 1997), however, no correlation between the antibody response was demonstrated and the protective immunity. Furthermore, it was found that more than a single priming injection of liposomal LPS was required before challenge with free LPS to generate a prolonged anti-LPS response.

Nahkla et al, 1997, show that the humoral immune response to lipopolysaccharides from Æ salmonicida is prolonged, when lipopolysaccharides are incorporated into unilamellar or multilamellar liposomes. Nahkla et al. do not demonstrate that the liposome:lipopolysaccharide formulations result in protective immunity. In addition, Nahkla et al do not combine antigenically the material from several different pathogens, and Nahkla does not assess the severity of any of the side effects. In discussing the results, Nahkla suggests that "liposome-incorporated LPS can be safely administered to fish..."; see the end of the first paragraph on page 398. In contrast, there is no indication that liposome will be safer and have fewer side effects than conventional oil-based vaccine formulations: Allegedly, oil-based vaccine formulations were considered "safe" when Nahkla et al. released its publication in 1997.

The relationship between antibody response and protective immunity is not clear. There are reports that there is a poor correlation between agglutinating antibodies against A. salmonicida and protective immunity (McCarthy et al, 1983; Olivier et al, 1985). Other studies show that there is a correlation between antibody responses against A-layer protein and protection against furunculosis after intraperitoneal injection of a number of different vaccines with oil as adjuvant (Midtlyng et al. Fish & Sellfish Immunol. 6: 335-350, 1996).

The use of liposomes as vaccine adjuvants has also been studied in rainbow trout. In Rodgers (Dis. Aquat. Org. 8: 69-72, 1990) the use of liposomes with fish vaccines in which about 30% of the components were contained in liposomal vesicles was studied. However, the efficacy results obtained in this study were suboptimal for use in the field, suggesting that these are not effective vaccines.

Rodgers (Dis. Aquat. Org. 8: 69-72, 1990) thus compares the effectiveness of a three-component vaccine against A. salmonicida comprising whole cells, toxoid and lipopolysaccharides, and phosphatidycholine liposomes with a similar three-component vaccine without liposomes. The vaccine comprising liposomes resulted in a slightly lower overall mortality compared to the vaccine without any liposomes. Although the vaccine in Rodgers is a multi-component vaccine, all antigenic material (whole cell, inactive toxin and LPS) is from the same pathogenic species: Aeromonas salmonicida. Therefore, Rodgers does not show that the vaccine is effective against several different finfish pathogens. Unvaccinated fish served as the control group. Therefore, Rodgers fails to show whether the safety and effectiveness of his vaccine can be compared to conventional oil-based vaccines. Rodgers therefore does not concern a multivalent vaccine in the same sense as the present liposome vaccine formulation, where antigenic material from two or more specific finfish pathogens must be included and therefore cannot be compared.

Fernandez-Alonzo et al. (Vaccine, vol. 19, no. 23-24, 2001, pp. 3067-3075) examines the possibility of using liposomes as a carrier in the vaccination of rainbow trout against viral haemorrhagic septicemia virus (VHSV). The authors conclude that DNA vaccination by electroporation can be effective, while immersion in the formulation containing DOTAP liposomes (DOTAP:cholesterol:phosphatidylcholine) or formulations containing commercial liposomes, fugene-6, is not effective. The case manager may have interpreted that Fernandez-Alonzo's document shows electroporation of liposome formulations, but as far as the Applicant can see, immersion in liposome formulations is an ineffective alternative to electroporation. Thus, Fernandez-Alonzo's conclusion is that liposomes are not effective as carriers in DNA vaccinations against VHSV.

Finally, WO 2000/037100 provides formulations for immune contraceptives in fish comprising teleost homologues of the zona pellucida, optionally with an adjuvant. WO 2000/037100 does not seem relevant in the context of vaccination against finfish pathogens and the safety and effectiveness issue associated therewith.

Furthermore, repeated injections of a vaccine are not cost-effective. From an economic perspective, one of the problems faced in developing fin-fish vaccines that incorporate strategies such as DNA vaccines, or repeated injection of the vaccine to boost the immune response, is that these are not sufficiently cost-effective strategies to be commercial viable.

There is therefore a need for a finfish vaccine with an acceptable minimum effectiveness for immunizing finfish against disease.

This background information is provided to illustrate the technology believed to be the closest.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a liposome vaccine formulation for finfish. According to one aspect of the invention, a Liposome vaccine formulation for finfish is provided, characterized in that it comprises:

a) liposomes, and

b) antigenic material derived from two or more different finfish pathogens selected

from the group: Vibrio anguillarum, Aeromonas salmonicida, Vibrio salmonicida, infectious

pancreatic necrosis virus (IPNV) or Photobacterium damselae subsp. piscicida, where at least one part of the antigenic material is liposome-associated.

According to another aspect of the invention, a Liposome vaccine formulation is provided, characterized in that it comprises antigenic material selected from the group consisting of proteins, peptides, nucleic acids, whole-cell living or weakened fin-fish pathogens, cell fragments, cell secretions, cells, cell organelles, cell membranes, cell membrane fragments , tissue, tissue fragments, and tissue extracts, where at least one part of the antigenic material is liposome-associated.

According to a further aspect of the invention, a method for producing a liposome vaccine formulation is provided, characterized in that it comprises:

(a) preparing a liposome formulation, and

(b) associating antigenic material derived from two or more fish pathogens with the liposome formulation to form the liposome vaccine formulation.

DESCRIPTION OF THE FIGURES

Figure 1 shows an electron micrograph of multilamellar liposomes (ML) and antigenic material. Liposomes were prepared by mechanical dispersion. Micrograph suggests that whole-cell bacterin (WC) is encapsulated (E) in liposomes or adhered to (A) clusters of liposomes. The formulation also contains free (F) antigenic material. Figure 2 shows the effect of varying concentrations of dextran on the present association of liposomes and antigenic material incorporated into lyophilized LAMB vaccines reconstituted with water or saline. Figure 3 shows the effect of varying concentrations of dextran on the herein described association of lyophilized empty liposomes reconstituted with antigenic material to form LAMB vaccines. Figure 4 shows the effect of long-term storage on the percentage association of liposomes and antigenic material incorporated into a LAMB vaccine formulation. Figure 5 shows the effectiveness of LAMB vaccine formulations incorporating liposomes, whole bacterin and hexaflumuron against re-emergent sea lice infestation.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and definitions

Below are the abbreviations used here: cholesterol (CH); dipalmitoylphosphatidylcholine (DPPC); dipalmitoylphosphatidylglycerol (DPPG); diphytanylphosphatidylcholine (D(PHY)PC); phosphatidylcholine, purified from eggs (eggPC); liposome-associated antigenic material-based (LAMB); non-liposomal mineral oil adjuvant (NLMA); phosphatidylcholine (PC); Relative Percent Survival (RPS); stearylamine (SA).

The fish pathogen Vibrio anguillarum has recently been given the name Listonella anguillarum. Both names are used in the technique and refer to the same organism. References here to Vibrio anguillarum or V. anguillarum must therefore be considered a reference to the bacterium now known as Listonella anguillarum.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as is usually used by professionals in the field to which the invention relates.

The term "vaccine" as used herein refers to a material capable of producing an immune response. A vaccine according to the invention will produce immunity against disease in finfish or can be used to treat disease in finfish.

As used herein, the term "treating", "treated" or "treatment", as used in connection with an infectious finfish disease or finfish pathogen, means a treatment which increases the resistance of an individual to infection by a pathogen (ie, the likelihood before the individual will be infected with the pathogen) as well as a treatment after the individual is infected to combat an infection (eg reduce, eliminate, alleviate or stabilize an infection).

The term "disease" as used here refers to a finfish disease which is caused by infection of a species of finfish by contact with one or more infectious organisms such as species of bacteria, viruses, parasites or fungi or with infectious or toxic agents formed by these or other organisms. The term also incorporates diseases caused by other infectious agents such as prions.

The term LAMB vaccine is used to indicate the liposome-associated antigenic material-based (LAMB) vaccine formulations described according to the invention.

The term "liposome" as used herein refers to a preparation of artificial membrane vesicles that are useful as a delivery vector in vivo or in vitro. Single-lamellar or multi-lamellar vesicles are formed by dispersing lipid molecules in aqueous solutions. Liposomes can include positively charged, neutral or negatively charged lipids, or combinations of these, and can optionally also include one or more amphiphilics, for example dipalmitoylphosphatidylcholine, cholesterol, stearylamine, phosphatidylcholine, phosphatidylglycerol, diphytanylphosphatidylcholine or dipalmitoylphosphatidylglycerol.

The terms "antigen" and "antigenic material" are used interchangeably here to refer to a molecule, molecule ring, part or parts of a molecule, or a combination of molecules, up to and including whole cells and tissues, which are capable of to induce an immune response in a finfish. The antigenic material may comprise a single epitope or antigenic determinant or it may comprise a number of epitopes or antigenic determinants.

The term "immune response" as used herein refers to a change in the reactivity of the immune system of a finfish in response to an antigen or an antigenic material and may involve antibody production, induction of cell-mediated immunity, complement activation and/or development of immunological tolerance.

The term "liposome-associated" as used herein, in reference to antigenic material, refers to antigenic material that is encapsulated by, adhered to, embedded in, trapped between, mixed with, or otherwise combined with one or more liposomes. The term also includes antigenic material to which one or more liposomes are attached.

The term "encapsulated by" as used herein, in reference to a liposome-associated antigen, refers to an antigen that is completely encapsulated or contained within the interior of a liposome.

The term "adhered to" as used herein in reference to a liposome-associated antigen refers to an antigen that is attached to or trapped between liposomes, or clusters of liposomes, but is not necessarily enclosed in the interior of a portion of a liposome .

The term "empty liposome" as used herein refers to a liposome that is not associated with any antigenic material.

The expression "reduced side effects" means that the formulations result in a significantly reduced assessment on the Speilberg scale (see Table 16) and the Pigmentation scale in relation to an oil adjuvant vaccine.

The term "incorporation" or equivalent as used herein is the procedure of combining components such as antigenic material and one or more liposomes, and possibly other bioactive agents such as immunostimulants, drugs and antibiotics, or other components including polymers, adjuvants, excipients, carriers and similar, to form the LAMB vaccine formulation.

The terms "immunization" and "vaccination" are used interchangeably herein to refer to the administration of a LAMB vaccine to a finfish. According to the invention, the immunization can have a prophylactic effect, a therapeutic effect or a combination thereof. In one embodiment of the invention, immunization with the LAMB vaccine induces an immune response in finfish. Immunization can be accomplished using methods including, but not limited to, intraperitoneal injection (i.p.), intravenous injection (i.v.), intramuscular injection (i.m.), oral administration, spray administration, or immersion.

The term "bacterin" as used herein refers to inactivated or weakened whole-cell bacteria, or components or fragments of bacteria.

Liposome vaccine formulations

The present invention provides LAMB vaccines comprising antigenic material and liposomes. The LAMB vaccines may optionally additionally include other bioactive agents such as prophylactic compounds or therapeutic compounds, and/or other components such as polymers, adjuvants, excipients, carriers and the like. The LAMB vaccines can be used for immunization and/or treatment of finfish against infection of viral, bacterial, fungal, parasitic or other pathogenic origin and/or their associated components or secretions.

According to the invention, the LAMB vaccines show a minimum efficiency. Where the LAMB vaccine is a multivalent vaccine formulation capable of inducing an immune response against more than one pathogen, each vaccine type in the formulation exhibits a minimum efficacy. In one embodiment of the invention, "minimum efficacy" or the like is defined as being an efficacy comparable to minimum acceptable standards for authorized fish vaccines. These standards, where available, are defined in various pharmacopoeias and appropriate government regulations and are typically measured as the relative percentage survival (RPS) at a predetermined percentage control mortality. For example, the minimum acceptable European pharmacopoeia standards, where the control mortality is 60%, are established as: 1) for inactivated, oil-adjuvanted furunculosis (A. salmonicida) vaccines: the RPS is not less than 80% for injectable vaccines; 2) for inactivated cold-water vibrosis (V. salmonicida) vaccines: RPS is not less than 60% for immersion vaccines and not less than 90% for injectable vaccines, and 3) for inactivated vibrosis (V. anguillarum, V. ordalli) vaccines : RPS is not less than 60% for immersion vaccines and not less than 75% for injectable vaccines.

However, those skilled in the art will recognize that currently available standards generally relate to oil-adjuvanted and water-based vaccines and are not set for liposomal vaccines.

Those skilled in the art will recognize that RPS may be dependent on the length of time post-immunization to challenge with the pathogen. According to the present invention, the RPS is determined at least around 6 weeks post-immunization. In one embodiment, the RPS is determined at least about 12 weeks post-immunization. In yet another embodiment, the RPS is determined at least about 16 weeks post-immunization.

The liposomal adjuvant in the LAMB vaccines is more benign than other known adjuvants such as alum or mineral oil, as the induction of local or inflammatory responses due to liposomal adjuvants is significantly less than that of other adjuvants. In particular, liposomal adjuvants result in fewer adhesions and less pigmentation. In addition and because fish are less stressed when administering liposome-based vaccines, characteristically less weight loss is observed than when the oil is used as an adjuvant. In one embodiment of the invention, the LAMB vaccines therefore show reduced side effects such as fewer adhesions and/or less pigmentation compared to oil-based vaccine formulations. In another embodiment, the use of LAMB vaccines results in less weight loss in vaccinated finfish than the use of oil-based vaccine formulations.

According to the invention, the LAMB vaccines are administered to finfish and the antigenic component of the vaccine induces an immune response that provides protection against natural infection from a pathogen or supports overcoming an ongoing and possibly chronic infection. Thus, the immunization procedure may be prophylactic to prevent infection from occurring or may be therapeutic to treat pre-existing infections.

Finfish that can be treated with LAMB vaccines include vertebrate fish that can be either bony or cartilaginous. Examples of these fin fish include, but are not limited to, salmonids (such as Atlantic salmon, Pacific salmon, Coho salmon), rainbow trout, brown trout, brown trout, Atlantic char, carp, catfish, yellowtail, bream, perch, catfish, grayling, whitefish , turbot, cod, halibut, perch, tropical fish, sturgeon or sea bass.

Types of pathogens and finfish diseases

The LAMB vaccines and methods according to the invention are useful for immunizing finfish species against a number of pathogens and diseases. Such pathogens and diseases include, infectious pancreatic necrosis virus (WNV)^- ieromonas salmonicida causing furunculosis; Vibrio salmonicida causing Vibrio salmonicida septicemia (VSS) or Hitra disease; Photobacterium damsela; Vibrio anguillarum.

Antigenic material

The LAMB vaccines comprise one or more liposome-associated antigens or liposome-associated antigenic material. The antigen or the antigenic material can be a small molecule or a whole cell or a fragment or an organelle thereof. Examples of antigenic material that can be used in the LAMB vaccines include, but are not limited to, proteins, peptides, saccharides, polysaccharides, lipopolysaccharides, nucleic acids and derivatives or analogs thereof. Other examples include lipids; complete, large parts or fragments of bacterial, viral, parasitic or fungal material, including whole-cell live or attenuated fin-fish pathogens; cell extracts; cell secretion; toxins; glycolipids; whimpers; cell organelles; cell membranes or fragments thereof; tissues and fragments or extracts of tissues from multicellular systems. Different antigens or antigenic material can be combined in the LAMB vaccines. When more than one molecule is included in the vaccine, the molecules may be covalently combined or they may be included in the vaccine as individual elements. The present invention also aims at the use of one or more pharmaceutical compounds known for use in aquatic-related industries to prevent diseases in finfish in the LAMB vaccines.

Examples of suitable antigenic material derived from known finfish pathogens include, but are not limited to, an iron-regulated outer membrane protein (IROMP) of Aeromonas salmonicida, an outer membrane protein (OMP) of Aeromonas salmonicida, an A-layer protein of Aeromonas salmonicida , an OMP of Vibro anguillarum or Vibro ordalli, a fiagellary protein of V. anguillarum or V. ordalli and combinations thereof. Examples of whole cell antigenic material obtained from a bacterial source include, but are not limited to, live, attenuated or inactivated A. salmonicida, V. salmonicida, V. anguillarum Ol, V. anguillarum 02, Moritellaviscosa, and combinations thereof.

Examples of antigenic material obtained from a viral source include VP1 of infectious pancreatic necrosis virus (IPNV); VP2 of infectious pancreatic necrosis virus (IPNV); VP3 of infectious pancreatic necrosis virus (IPNV); N structural protein of infectious pancreatic necrosis virus (IPNV);.

The antigenic material may comprise combinations of antigens, for example one or more different antigens from the same pathogen, two or more antigens, each from a different pathogen or a number of antigens comprising two or more antigens from different finfish pathogens. One or more parts of such antigenic material can induce an immune response against one or more pathogens and can therefore be used to form multivalent LAMB vaccines. Multivalent LAMB vaccines may contain multiple antigens against one disease or against different diseases, or multiple antigens against multiple diseases. Portions of antigenic material may optionally be antigenic, for example portions that are not antigenic to a particular finfish disease may serve to enhance an immune response to another, unrecognized pathogen. The present invention further aims at adding protein helper epitopes, cytokines, carrier polypeptides, cholera toxin subunits, and/or other immunostimulants to the antigenic material to support improvement of an immune response.

Isolation and production of antigenic material

Antigenic material for use in the LAMB vaccines can be isolated, purified or synthesized using a number of techniques well known to those skilled in the art.

Pathogenic organisms that serve as a natural source of antigenic material can be obtained and purified directly from infected finfish or alternatively can be cultured in vitro using suitable culture techniques well known to those skilled in the art. If whole cells or fragments thereof are to be used, cells from the pathogenic organism can be easily isolated, washed, fractionated or purified as required. For example, antigenic material isolated and purified directly from finfish pathogenic organisms can be washed by centrifugation and resuspension to remove extracellular material containing enzymes, media, metabolites and salt. Alternatively, unwashed, antigenic material can be used in the LAMB vaccines. Various methods of combining methods which are well known in the art can be used to isolate, purify, weaken or wash antigenic material to achieve this.

When the antigenic material is to be isolated from the cells, alternatively, once sufficient amounts of cells have been harvested, the antigenic material can be extracted, isolated and purified as needed by well-known techniques such as centrifugation, column chromatography and the like.

When the antigenic material is a peptide or polypeptide, it can also be synthesized on

per se known methods, for example by the well-known solid phase method, see for example Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963), Houghten et al., Int. J. Pept. Pro. Res. 16: 311-320 (1980) and Parker and Hodges, J. Prot. Chem. 3: 465-478

(1985), for a discussion of these techniques. Peptide synthesizers suitable for performing solid-phase polypeptide syntheses are commercially available (eg, Beckman Model 990B Peptide Synthesizer, Beckman Instruments Co., Berkeley, Calif, U.S.A.).

According to the invention, peptides useful for preparing synthetically derived antigenic material for a LAMB vaccine formulation are those comprising an amino acid sequence of a known peptide or protein antigenic determinant or epitope of a finfish pathogen.

In addition to known, antigenic amino acid sequences, potential epitopes that may be usable for polypeptide forms of antigenic material can be selected using different physicochemical principles that support prediction as to which parts of the polypeptide are most likely to be surface-oriented and therefore immunogenic. These include the hydrophilicity plots of Hopp and Woods (Proe. Nat. Acad. Sei. 78 3824-3828,1981), and similar work by Kyte g Doolittle (J. Mol. Biol. 157, 105-132, 1982). Furthermore, the empirical prediction of protein conformation (Chou and Fasman, Ann. Rev. Biochem., 47, 251-276, 1978) is a useful guide in predicting which parts of a polypeptide are likely to be immunogenic.

A number of antigenic materials useful for finfish immunization are well known in the art and commercially available. Such commercially available, antigenic materials can then be used in the LAMB vaccines.

Preparation of liposomes

Liposomes are systems consisting of hydrated mono- or multi-lamellar bilayers of amphipathic compounds that are dispersed in an aqueous medium and are generally derived from phospholipids and/or other lipid substances, as is well known in the art. A variety of non-toxic, physiologically acceptable lipids are capable of forming liposomes and can be used in the LAMB vaccines. Liposomes can be prepared from extracts containing lipids from natural sources and/or synthetic lipids. In addition, lipids used to prepare liposomes can be "ether" lipids or stable analogues of biological lipids. Lipids that are used to form the liposomes can also be derivatized, functionalized or modified using methods known per se. The present invention also aims at the use of cochelates which are stable, non-toxic phospholipid-calcium precipitates consisting of phosphatidylserine, cholesterol and calcium, in the liposome formulation.

Examples of lipids that can be used alone or in combination to prepare the liposomes include, but are not limited to, lipids and phospholipids such as soy lecithin, partially refined lecithin, hydrogenated phospholipids, lysophosphate, cholesterol (CH), phosphatidylcholine (PC), phosphatidylcholine purified from egg (eggPC), phosphatidylethanolamine (PE), diphytanylphosphatidylcholine (D(PHY)PC), phosphatidylserine, phosphatidylinositol, cardiolipin, sphingolipids, gangliosides, cerebrosides, ceramides, other ester analogues of phosphatidylcholine (such as PAF and lysoPAF); synthetic phospholipids (such as L-α-lecithin, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dilinoloylphosphatidylcholine, distearoylphosphatidylcholine, diarchidoylphosphatidylcholine); PE derivatives (such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine, 1 -acyl-2-acyl-sn-glycero-3-phosphoethanolamine, dinitrophenyl- and dinitrophenylamino-caproylphosphatidylethanolamine, 1,2-diacyl-sn- glycero-3-phosphoethanolamine-N-polyethylene glycol (PEG-PE), N-biotinyl-PE, N-caproylamine PE, N-dodecylamine-PE, N-MPB-PE, N-PDD-PE, N-succinyl-PE, N-glutaryl-PE); phosphatidyl glycerols (such as dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol; phosphatidic acids, 1,2-diacyl-sn-glycero-3-phosphate salt, l-acyl-2-acyl-sn-glycero-3-phosphate sodium salt); phosphatidylserines (such as 1,2-diacyl-sn-glycero-3-[phospho-L-serine] sodium salt, 1-acyl-2-acyl-sn-glycero-3-[phospho-L-serine] sodium salt, lysophosphatidic acid); cationic lipids (such as 1,2-diacyl-3-trimethylammonium propane (TAP), 1,2-diacyl-3-dimethylammonium propane (DAP), N-[l-(2,3-dioleoyloxy)propyl]-N,N', N"-trimethylammonium chloride (DOTMA)); phospholipids with multidifferent head groups (such as phosphatidylethanol, phosphatidylpropanol and phosphatidylbutanol, phosphatidylethanolamine-N-mono-methyl, 1,2-distearoyl(dibromo)-sn-glycero-3-phosphocholine); polymerizable lipids ( such as diyne PC and diyne PE, for example, 1,2-bis(10,12-tricosadinoyl-sn-glycero-3-phosphocholine) and phospholipids with partially or fully fluorinated fatty acid chains.

The LAMB vaccines may comprise antigenic material associated with liposome formulations that are positively charged, neutral or negatively charged. As is well known in the art, the selection of the type of lipid (natural or synthetic), the total charge of the lipid (positive, negative or neutral), the size of the lipid (large or small) and the number and ratio of lipids will determine the charge, size and lamellarity of the final liposomes and will depend on the type of angiogenic material to be associated with the liposome. Such a selection is considered to be within the specialist's area of knowledge.

The ratio of the liposome components may vary according to the type of organ or tissue to be targeted and the amount of liposome-associated antigenic material required in a LAMB vaccine for a particular disease state. For bacteria, for example, higher degrees of association of antigenic material are often observed when using positively charged liposome formulations than when using neutral formulations, and the lowest degrees of association are usually observed when using negatively charged liposome formulations. Examples of suitable positively charged liposome formulations for the LAMB vaccines include, but are not limited to, PC:CH:SA and DPPC:CH:SA; examples of neutral liposome formulations include, but are not limited to, PC:CH and DPPC:CH, and examples of negatively charged liposome formulations include, but are not limited to, PC:CH:PG and DPPC:CH:DPPG.

The liposome formulations for use in LAMB vaccines may comprise one or more lipids selected from a single class, or they may comprise combinations of lipids from different classes. When lipids from a single class are used, the lipids can be of a single type or of different types. For example, a molecular species of phosphatidylcholine can be used, so that the liposome comprises a completely homogeneous population of molecules. Alternatively, the liposome can comprise a number of distinct, molecular species of, for example, phosphatidylcholine, if egg phosphatidylcholine is used as lipid.

In one embodiment of the invention, the liposome formulations for use in the LAMB vaccines comprise lipids selected from two classes of lipids where one or more molecular species of each class are selected. Representative examples of this embodiment would be PC:CH and DPPC:CH.

In another embodiment, the liposome formulations for use in the LAMB vaccines comprise lipids selected from three classes of lipids, wherein one or more molecular species are selected from each class. Thus, for example, the lipids can be mixed together to form liposomes with the formulas X: Y:Z, where X means a phospholipid or sphingolipid; Y means a neutral lipid, and Z means a charged amphipathic molecule or amphiphile. The phospholipid or sphingolipid (X) may be one or more than one from a group of natural and/or synthetic compounds including, but not limited to, PC, PE, phosphatidylglycerol (PG) and sphingomyelin. Examples of suitable neutral lipids Y include, but are not limited to, cholesterol and its precursors and derivatives, diglycerides or triglycerides. Examples of suitable charged amphiphiles (Z) include, but are not limited to, amphiphilic compounds such as stearylamine, fatty acids and other phospholipids having overall positive, neutral or negative charges.

Ratios between the lipid components used to form the liposomes will depend on the desired formulation of liposomes and can for example range from around 6:3:3 to around 1:0:0 including ratios between these ranges, for example 4:3:3, 6:3:1 and 2:1:0.

As is well known in the art, particular combinations of lipids can be chosen to form liposome formulations according to the invention which, for example, provide insight into organs in finfish with antigenic material or pharmaceutical compounds, increased loading capacity and/or carrying capacity for a particular antigenic material or pharmaceutical compound, increased or decreased uptake of antigenic material or pharmaceutical compounds at the target sites, increased association of antigenic material or pharmaceutical compounds with certain lipids of liposome formulation, or more benign formulations, expressed by reduced side effects or increased potency or adjuvantity. The lipid components can be varied depending on the type of antigenic material or drug one wishes to associate with the liposome formulation. Furthermore, the lipids used to form the liposomes as described here can be modified using methods known per se to make these lipids more or less hydrophobic or hydrophilic, or more resistant to degradation by chemical or biochemical processes, depending on the desired vaccine formulation.

Any additives for LAMB vaccines

Other constituents or components may possibly be included in the LAMB vaccines. For example, the LAMB vaccines may comprise one or more bioactive agents such as therapeutic compounds (including, for example, antibiotics and anti-apoptotic agents), prophylactic compounds (including, for example, purified protein antigens), immunostimulants (such as glucan, mucin, dextran, cytokines, chemokines and saponins) and other pharmaceutical compounds such as vitamins, antioxidant compounds or growth-regulating compounds. These bioactive substances can be liposome-associated or they can remain unassociated with the liposomes in the vaccine. The LAMB vaccines may also include other auxiliary vaccine ingredients such as biopolymers, carriers, buffers, stabilizers, preservatives, excipients and/or solubilizers. Examples include, but are not limited to, chitin, chitosan, mineral oil, vegetable oil, and other compounds or components well known to those skilled in the art.

The LAMB vaccines can be formulated in combination with polymers. Biodegradable microspheres consisting of polymers such as polyester poly(lactide-coglyceride) have also been used to microencapsulate antigenic material isolated from various pathogens. Other polymers can also be used, for example chitin, chitosan, dextran, alginate or glucans.

In addition, LAMB vaccines may contain "empty" liposomes, i.e., liposomes that are not associated with any antigenic material, bioactive agent, or any other component of the vaccine formulation. These empty liposomes can serve as adjuvants for the LAMB vaccines.

Other adjuvants for immunization are well known in the art and can be combined by those skilled in the art with the LAMB vaccines as described herein to provide a pharmaceutical preparation. Oil adjuvants are less desirable because they give undesirable side effects such as visceral adhesions, (which can be associated with limited growth) and melanized granuloma formations (which can reduce the quality of fish on the market) and because they cannot give a homogeneous mixture with any vaccine preparation. However, the use of liposomes in combination with an oil adjuvant can help to protect against some of the negative effects described above.

One or more of these additional components may be added to the LAMB vaccines before, during or after the LAMB vaccine manufacturing process. The present invention also aims at administering one or more of these components to a finfish before, during or after immunization of the finfish with the LAMB vaccine.

Production of LAMB vaccines

The LAMB vaccines comprise one or more types of antigenic material associated with a liposome formulation. The LAMB vaccines may be monovalent, comprising a liposome formulation with a single source of antigenic material, or they may be multivalent, comprising a liposome formulation with two or more antigens or sources of antigenic material from the same or from different pathogens. The LAMB vaccines may also include one or more bioactive agents or other additives.

Different types of liposome formulations can be combined with one or more types of antigenic material to form the LAMB vaccines. Typically, the hydrophobic components of the LAMB vaccine will adhere to, or be captured by, the membrane (lipid) portion of the liposome and hydrophilic components will be encapsulated in the inner portion of a liposome. However, the person skilled in the art will recognize that this will not always be the case and that the means for associating a compound with a liposome is dependent on the particular compound to be included in the vaccine and the liposome formulation used.

Incorporation of antigenic material into one or more liposome formulations to prepare the LAMB vaccines can be accomplished by a number of methods well known in the art. Examples include, but are not limited to, hand mixing, grinding, extrusion, French pressing through a sieve, emulsifying or homogenizing lipids together with antigenic material. After the incorporation procedure, the vaccine formulations can be sonicated to increase the association of antigenic material with the liposomal formulation if this is desired. Alternatively to, or after sonication, the mixture of lipids and antigenic material can be subjected to gentle heating and cooling or freeze-thaw cycles to further enhance the association of antigenic material with the liposomes. The LAMB vaccines can be prepared using sterile components under an inert atmosphere to provide sterile vaccines if desired, or the preparation can be completed under non-sterile conditions.

The suitable ratio of antigenic material to lipid in the LAMB vaccine will depend, for example, on the loading capacity required for the liposome formulation, the amount of antigenic material or pharmaceutical agent required and the total charge for the liposome. A representative example of a suitable range for the ratio between antigenic material and lipid in a LAMB vaccine is from around 1 mg of antigenic material per 5 mg of total lipid to around 1 mg of antigenic material per 120 mg total lipid. Those skilled in the art will recognize that the amount of antigenic material associated with a liposome can affect the efficacy of the finished vaccine formulation and modify its overall "efficacy" and will be able to determine the amount of antigenic material to be added to provide a suitable, effective vaccine.

The amount of antigenic material required for incorporation into LAMB vaccines will depend on the type of disease the vaccine is to immunize against, the state of the course of the disease as well as the size of the animal to be immunized, among other factors, and can be easily determined by the person skilled in the field of fish vaccine production.

Tests used to measure the amount of antigenic material incorporated into the LAMB vaccine formulation and the degree of liposome association will depend on the type of antigenic material used. If, for example, the antigenic material is a whole-cell preparation, electron microscopy can be used. Where the antigenic material is a polypeptide or lipopolysaccharide, protein or carbohydrate, suitable measurement assays are well known to those skilled in the art. Combinations of analyzes may be necessary when the antigenic material is a combination of whole cells, polypeptides, proteins, lipopolysaccharides and/or other components. A representative example of a method for assessing the degree of liposome association is given in Example 1.

The LAMB vaccines may be combined with a suitable carrier to form a unit dosage form. The amount of LAMB vaccine required to provide a single dose form will vary depending on the individual being treated and the particular mode of administration. The specific dosage and treatment regimen for any particular fish will depend on a number of factors, including the particular liposome formulation used, the stability and activity of the particular vaccine formulation used, age, body weight, general health, fish species and disease state being treated, the nature of the disease who are immunized or treated. The amount of vaccine may also depend on whether other therapeutic or prophylactic agents, including additional pathogenic proteins, whole inactivated pathogens, or other adjuvants, or associated compounds, are to be co-administered, and whether other components are to be administered with, before or after the LAMB vaccine.

Test of the LAMB vaccines

After manufacturing the LAMB vaccines according to the invention, it may be desirable to test the effectiveness of the vaccines with a view to given immunogenicity for immunized animals and/or with a view to side effects.

A number of tests which are well known to those skilled in the art can be used for this purpose. Below are examples of methods for testing LAMB vaccines for side effects and for effectiveness in preventing or protecting against diseases commonly found in finfish.

A method that can be used to assess the degree of side effects is the Speilberg scale and involves the evaluation of intra-abdominal lesions after autopsy. Each test fish is scored and given a score from 0 to 6 that reflects the severity of the lesions according to the visual appearance of the body cavity (Midtlyng et al, Fish & Shellfish Immunol. 6: 335-350, 1996) (see, for example, Table 12).

Another method that can be used to assess the degree of side effects uses a pigmentation scale and involves a visual assessment of the degree of pigmentation on the fish's exterior. Each fish is assessed and given a score from 0 to 3 which reflects the degree of pigmentation where 0 = no pigmentation; 1 = some pigmentation; 2 = medium pigmentation; and 3 = high pigmentation.

One method that can be used to determine the potential effectiveness of a LAMB vaccine is the measurement of the distribution of the vaccine formulation in tissues and/or organs of immunized animals. After immunization and at various time intervals, tissue and/or organ samples are taken from the animals and assessed for vaccine content using standard methods. One method for measuring the incorporation of the vaccine formulations into tissues or organs of animals is to label either the antigenic material or the liposome part of the vaccine using a tracer or a marker, for example a radioactive, chemiluminescent or fluorescent marker. LAMB vaccines comprising these tracers or markers can then be administered to the animal and monitored accordingly. By taking samples from tissue at different time intervals and measuring the amount of marker present in the tissue, or the rate of uptake of the marker, one can determine the level of vaccine incorporation and thereby the given immunogenicity likely to be given to the immunized animal.

Another method of measuring the potential effectiveness of a LAMB vaccine is to measure the levels of circulating antibodies. Once serum or tissue samples are collected, the level of antibodies can be measured by standard techniques such as enzyme-linked immunoassay (ELISA).

An additional method that can be used to determine the potential efficacy of a LAMB vaccine is the measurement of the relative percent survival (RPS) of immunized and non-immunized animals challenged with pathogenic materials. One such method is described in detail below. Other methods well known to those skilled in the art to measure the level of given immunogenicity for a vaccine formulation according to the invention can be used in combination with the above strategies.

Testing the LAMB vaccines for minimum effectiveness

According to the invention, the LAMB vaccines show a minimum effectiveness. As described above, the minimum efficiency is defined by the relative percent survival (RPS). The "minimum effectiveness" can be defined as an effectiveness comparable to the minimum acceptable standards for authorized fish vaccines or can be defined as an effectiveness of at least 50% RPS, where RPS is defined as:

RPS - [ 1-( mortality for vaccinated fish/ mortality for non-vaccinated fish)] x 100

A number of standard tests well known to those skilled in the art can be used to test the vaccine formulations of the invention for minimum efficacy. An example is a protocol provided by the "European Pharmacopoeia Commission" (see Example 13).

RPS can be determined by routine procedure as is well known in the art. In general, a separate test is carried out for each species and each serovar included in the LAMB vaccine. Randomly selected fish are divided into test and control groups and marked for identification. The LAMB vaccine is administered to the test group by a selected mode. The control group is subjected to dummy vaccination. If necessary, additional control groups to which commercially available vaccines are administered may be included in the test for comparison. The fish are kept as much as possible in the same tank or an equal number of checks and vaccinations are mixed in each tank if more than one tank is used. At a fixed time interval after vaccination, a challenge is carried out by injection using cultures of the selected pathogen whose virulence has been verified. The fish are observed daily until a predetermined percent mortality is reached in the control group. A curve for mortality versus time from challenge is plotted for both vaccinees and controls, and the time corresponding to the previously selected percent mortality in the controls is determined by interpolation. The mortality (M) at the time corresponding to the previously selected percent mortality in the controls is read from the curve for the vaccines. The relative percent survival (RPS) is calculated as from the expression:

(1-M/preselected % mortality in controls) x 100

Preservation and storage of LAMB vaccines

There are a number of processes aimed at extending the time of the vaccine, which can be used for the preservation and storage of LAMB vaccines. Examples include lyophilization and/or freezing of prepared LAMB vaccines or their components. Effective preservation of the liposomes and the antigenic material incorporated into the liposome formulations will enable their effective use at a later time. LAMB vaccines comprising antigenic material and liposome formulations can thus be lyophilized and/or frozen and stored. Alternatively, antigenic material and liposome formulations can be frozen separately and thawed and rehydrated at a later time for combining to form a LAMB vaccine formulation.

LAMB vaccines can be frozen by freeze drying or spray drying or by other well known processes. Before freezing, the LAMB vaccines can be dehydrated or lyophilized. One or more cryoprotective compounds may be added if necessary to support preservation of the LAMB vaccines in the deep-frozen state.

Methods of vaccine delivery

The present LAMB vaccines are generally administered to finfish in an amount sufficient to produce an immune response or an immune system enhancement. The amount necessary to induce the immune system enhancement will vary on an individual basis and will be based, at least in part, on considerations of the size of the fish, the severity of the symptoms, the virulence of the pathogen or toxin, and the intended results. Determining the correct dosage for a particular situation is within the expert's area of knowledge. LAMB vaccine formulations may, if desired, also contain other compatible bioactive agents.

A therapeutically effective dose of the LAMB vaccine can be administered to the fish in a manner known per se. For example, the LAMB vaccine can be introduced into the fish orally, which is the least stressful method of immunization. LAMB vaccines for oral use can be formulated with biodegradable polymers as described previously. The LAMB vaccines can be coated on or ground into lining in the form of a paste or a liquid suspension or incorporated into gelatin capsules and introduced into finfish environments. Formulations of LAMB vaccines for oral use may include lactose and corn starch and other carbohydrates well known to those skilled in the art. The LAMB vaccine formulation can be used with or without polymers or products that improve entry of the vaccine into the cells of the intestinal epithelium or deeper cells.

Alternatively, the LAMB vaccines can be administered to finfish by injection with a needle, a needle-free beam injection system, or another injection method well known to those skilled in the art. Injection routes for finfish include, but are not limited to, intraperitoneal, intramuscular, or subcutaneous modes. An example of an injectable dosage unit for a finfish under 100 g would be around 0.05 - 0.5 ml of a solution containing the LAMB vaccine.

The LAMB vaccines can also be administered to fish by spraying techniques. Typically, a fish is exposed to a spray for at least 2 seconds. The fish can pass through a mist of vaccine formulation solution, created by passing the vaccine through high-pressure dispersing nozzles. Pressures up to 90 psi are generally satisfactory for this purpose. LAMB vaccines can be used with or without products known to improve the entry of vaccines into and through skin cells.

An alternative method that can be used to immunize a large number of fish at the same time is by immersion in a solution containing one or more LAMB vaccines. Immersion in vaccine solution typically takes place for at least a few seconds, after which the fish are returned to the holding tanks. Immunization by immersion can also be achieved by placing the finfish in tanks containing relatively small volumes of water. One or more concentrated LAMB vaccines are then added to the tank and the fish are allowed to remain there for a suitable period of time (up to several hours) before the tank is refilled with water to restore the normal aquatic environment. This immersion method is particularly suitable for small fish that cannot be immunized by direct injection.

Immersion techniques allow the LAMB vaccines to penetrate the cells of the skin epithelium, gills or intestinal wall. With injection, the LAMB vaccines can penetrate muscle cells or other cells in the muscle tissue (for example fibroblasts, immune cells) or cells in the viscera in the intraperitoneal cavity. Finfish exposed to antigenic material using the techniques described above, or other techniques well known to those skilled in the art, may experience induction of appropriate immune responses in regional or systemic lymphoid tissue.

Set

The present invention additionally provides kits containing the LAMB vaccines. The kit may contain the LAMB vaccine formulation or may contain individual components that allow the user to prepare the LAMB vaccine. Individual components of the kit will be packaged in separate containers and, in connection with small containers, there may be written guidelines in a form determined by the authorities regulating the manufacture, use or sale of pharmaceuticals or biological products, where the guidelines reflect approval at the time of manufacture, use or sale for animal management.

When the components of the kit are provided in one or more liquid solutions, the liquid solution may be an aqueous solution, for example a sterile aqueous solution. In this case, the container means can be, for example, a suitable syringe, a pipette or another apparatus of this type, from which the preparation can be administered to an individual.

The components in the set can also be provided in frozen form or they can be provided in dried or lyophilized form and the set can also contain a suitable solvent for reconstituting the lyophilized components. Regardless of the number or type of containers, the set according to the invention can also include an instrument to support the administration of the vaccine. Such an instrument may be a syringe, pipette or other delivery carrier.

To provide a better understanding of the invention, reference is made to the following examples. These must be illustrative and not in any way limiting the invention.

EXAMPLES

EXAMPLE 1: LAMB vaccine formulation comprising whole cell bacterins and

freaks out

The antigenic material in this example comprises weakened or inactivated whole-cell bacteria (bacterin) of various fish pathogens and infectious pancreatic necrosis virus (IPNV). In particular, 5 types of antigenic material were used, alone or in combination, in the vaccine formulation. The antigenic material consisted of whole cell preparations from A. salmonicida, V. salmonicida, V. anguillarum Ol, V. anguillarum 02 bacterin and a virus (IPNV). The liposome formulations used in this example were: 1) eggPC:CH:SA; 2) DPPC:CH; 3) DPPC:CH:DPPG; 4) DPPC:CH:SA.

In this example, the bacterin was washed twice in 2% NaCl solution with centrifugation at 4500 rpm for 24 minutes between each wash.

The procedure described above is an example of a method that can be used to prepare the liposome component of the LAMB vaccine. Other methods that are well known to those skilled in the art can also be used. Briefly, suitable ratios of lipids were dissolved, at known concentrations, in chloroform/methanol in a volume ratio of 2:1 in an autoclaved Erlenmeyer flask, round flask or a glass beaker. The solvents were then evaporated under a constant stream of N 2 gas or under reduced pressure using a Brinkmann rotary evaporator. After evaporation of the solvents, the lipid films were placed overnight under vacuum in a desiccator. The dried films were hydrated with the appropriate volume of solution containing antigenic material (called antigenic material medium) and placed on an orbital shaker for 1 hour at room temperature in a shaking water bath if higher temperatures were required. The resulting liposome preparations were then subjected to 3x1 minute vortex sessions of mechanical mixing with a one minute rest period between vortex sessions. Alternatively, the dried films can be hydrated with water or saline to form empty liposomes where an appropriate volume of antigenic material is added to the empty liposomes followed by incubation on an orbital shaker for 1 hour at room temperature or in a shaking water bath if higher temperatures are required.

Figure 1 shows an electron micrograph of multilamellar liposomes (ML) and antigenic material prepared as above. Micrographs suggest that whole-cell bacteria (WC) are encapsulated (E) in liposomes or adhered (A) to clusters of liposomes. The formulation also contains free (F), antigenic material. The scale bar represents 1 uM.

The amount of antigenic material associated with the liposomes can be assessed by gradient centrifugation as described below. Other methods which are well known to the person skilled in the art can also be used for this purpose. In short, part of the vaccine formulation can be layered on top of a 5% and 10% metrizamide step density gradient. The material is centrifuged for 2 hours at 40,000 rpm using a Beckman SW 50.1 rotor. The resulting pellicle was carefully and gently removed and resuspended with 0.9% saline. The suspension was further centrifuged at 45,000 rpm in a 60 Ti rotor for 1 hour to yield a pellet. Similarly, the remaining part of the gradient (minus the pellicle) was resuspended with 0.9% saline and centrifuged at 45,000 rpm in a Beckman 60 Ti rotor for 1 hour to give a second pellet. Both pellet samples were dialyzed overnight and then analyzed for lipid and protein content. In this example, the degree of association is expressed as the amount of protein material that is incorporated into the pellicle as a proportion of the total, original protein material (Table 1).

Data suggest that the LAMB vaccines comprise encapsulated, adhered (trapped) and free, antigenic material (Figure 1). In the example shown in Table 1, the percentage of antigenic material associated with the liposome formulations appears to be dependent on the total charge of the liposome. Higher levels of association are evident with positively charged liposomes (eggPC:CH:SA, DPPC:CH:SA).

EXAMPLE 2: Efficacy and side effects of multivalent LAMB vaccines comprising whole-cell bacterins, outer membrane fragments (OMF), A-layer protein, or infectious pancreatic necrosis virus (IPNV)

The liposomes and antigenic material incorporated into the LAMB vaccines according to this example were prepared as described in Example 1. In this example, 5 types of antigenic material were used, alone or in combination, in the vaccine. The antigenic material consisted of the following: 1) a whole cell preparation containing 4 types of bacterin, including: A. salmonicida, V. anguillarum Ol, V. anguillarum 02 and V. salmonicida; 2) an outer cell membrane fraction (OMF) of A. salmonicida; 3) a purified A-layer protein from the outer membrane of A. salmonicida; 5) IPNV. The liposome formulations used in this example were: 1) eggPC:CH:SA; 2) eggPC:CH:PG; 3) DPPC:CH; 4) DPPC:CH:DPPG; 5) DPPC:CH:SA. The LAMB vaccine formulations are shown in Table 2.

About. 2000 Atlantic salmon {Salmo salar L.) with an average weight of 34 grams were used to determine the effectiveness and side effects of different vaccines against A. salmonicida (furunculosis). The fish were randomly selected from a general population, allocated to the appropriate experimental groups, marked (freeze marking) and placed in freshwater tanks kept at 12°C. In this example, the fish were vaccinated intraperitoneally with 0.1 ml of different LAMB vaccines, 0.2 ml of a non-liposomal mineral oil adjuvant (NLMA) vaccine (ALPHA JECT 6002) and 0.2 ml control (PBS) vaccine. The fish were anesthetized with benzocaine before vaccination. 6 and 12 weeks after vaccination, 10 fish from each group were sampled for blood, weight and side effects. The level of side effects at the injection site was analyzed using a standard adhesion scale (0 - 6) and a pigmentation scale (0 - 3). 6 and 16 weeks after vaccination, respectively, the fish were challenged with an effective dose of A. salmonicida. The challenge was carried out according to standard procedures well known to those skilled in the art. In short, fish from the suitable tanks and experimental groups injected intraperitoneally with an effective dose of A. salmonicida. The fish were monitored for 21 days after challenge and percent mortality for the challenged fish and for the control fish was measured daily. A rear teriological assessment was carried out on all dead fish before and during the challenge period to determine the presence of A. salmonicida bacteria.

Data show that the vaccination of Atlantic salmon with LAMB vaccine formulations provides protection against infectious challenge with A. salmonicida (Table 2). The protective effect appears 6 and 16 weeks after vaccination, with an increase in protection between 6 and 16 weeks for the main part of the LAMB vaccine. The level of protection obtained for some of the LAMB vaccines incorporating inactivated whole-cell bacterins and IPNV is comparable to NLMA vaccine containing the same bacterins and IPNV. In this example, LAMB vaccines containing whole-cell bacterins plus IPNV provided somewhat better protection against A. salmonicida than LAMB vaccines containing OMF or A-layer protein. The LAMB vaccine provided equivalent protection using half the standard dose of the NLMA vaccine (0.1 ml vs 0.2 ml). Furthermore, the adhesion and pigment ratings for the LAMB vaccine are lower than for an NLMA vaccine, suggesting that the level of side effects is reduced (Table 3). The obtained data also show that the LAMB vaccines containing different types of antigenic material are able to induce an immune response against A. salmonicida and/or IPNV in Atlantic salmon (Table 3).

EXAMPLE 3: Efficacy of multivalent LAMB vaccines against Vibrio

salmonicida and Vibrio anguillarum (Serotypes Ol and 02)

About. 500 Atlantic salmon (Salmo salar L.) with an average weight of 15.7 grams were used to demonstrate the effectiveness of different vaccines against V salmonicida and V anguillarum (Ol and 02). The liposomes and antigenic material incorporated into the LAMB vaccines were prepared and produced as described in Example 1. The antigenic material used in this example consisted of whole-cell bacterin preparations of A. salmonicida, V samonicida, V. anguillarum (Ol), V. anguillarum ( 02) and a virus (IPNV). The liposome formulation used in this example was

DPPC:CH:DPPG.

The fish were randomly selected from the general population, allocated to the correct ones

experimental groups, marked (freeze marking) and placed in fresh water tanks that were kept at 12°C. In this example, the fish were vaccinated with 0.2 ml of a LAMB vaccine, 0.2 ml of an NLMA vaccine (ALPHA JECT 5000) and 0.2 ml of control (PBS) vaccine. The fish were anesthetized with benzocaine before vaccination. 12 weeks, respectively 33 or 36 weeks after vaccination, the fish were challenged with an infectious dose of V. salmonicida, V. anguillarum Ol or V. anguillarum 02. The challenge was carried out according to standard procedures in the technique. For the group of fish that were infected with V. salmonicida, V. anguillarum Ol or V. anguillarum 02X2 weeks after vaccination, this occurred by co-habitation challenge. Briefly, unvaccinated fish were injected intraperitoneally with an infective dose of the pathogen in question. The infected fish (cohabitants) were placed in the relevant tanks containing control fish and fish that had been vaccinated against V. salmonicida, V. anguillarum Ol or V. anguillarum 02. As the disease developed in the cohabitant fish, the bacteria were released to the water and transferred to vaccinated and control fish. The fish were monitored for 21 days after the challenge and percent mortality for vaccinated and control fish measured daily. For the group of fish that were challenged 33 and 36 weeks after vaccination, fish from the respective tanks and experimental groups were injected intraperitoneally with an infectious dose of V. salmonicida, V. anguillarum Ol or V. anguillarum 02. The fish were monitored for 21 days after the challenge and percent mortality for vaccinated and control fish measured daily.

Obtained data show that vaccination of Atlantic salmon with a multivalent LAMB vaccine formulation provides protection against an infectious challenge with V. salmonicida, V. anguillarum Ol or V. anguillarum 02 (Table 4). The protective effect is shown 12, respectively 33 or 36 weeks after vaccination. In this example, the level of protection provided by the LAMB vaccine formulations with whole cell bacterins and IPNV is comparable to an NLMA vaccine containing the same bacterins and IPNV.

EXAMPLE 4: Efficacy and side effects of monovalent and multivalent LAMB vaccines comprising washed and unwashed whole cell A.

salmonicida and V. salmonicida bacteria

About. 3400 Atlantic salmon (Salmo salar) with an average weight of 24.5 grams were used to demonstrate the effectiveness and side effects of different LAMB vaccines against A. salmonicida (furunculosis) and V. salmonicida (Hitra disease). The liposomes and antigenic material incorporated into the LAMB vaccines were prepared as described in Example 1. The antigenic material used in this example was washed and unwashed whole cell bacterin preparations and A. salmonicida and V. salmonicida used alone or in combination. The liposome formulations used in this example were: 1) egg PC:CH:SA; 2) DPPC:CH; 4) DPPC:CH:DPPG; 5) DPPC:CH:SA. The LAMB vaccine formulations are shown in Tables 5 and 6.

The fish were randomly selected from the general population, allocated to the appropriate experimental group, marked (freeze tagging and adipose fin/jaw cutting) and placed in freshwater tanks maintained at 12°C. In this example, the fish were vaccinated with 0.2 ml of various LAMB vaccines, 0.2 ml of an NLMA vaccine (Apoject-2-Fural) and 0.2 ml of a control (PBS) vaccine. The fish were anesthetized with benzocaine before vaccination. 16 weeks after vaccination, 10 fish from each of the groups were tested for blood, weight and side effects. The level of side effects at the injection site was analyzed according to a standard adhesion scale (0 - 6) and a pigmentation scale (0 - 3). 6 or 16 weeks after vaccination, the fish were infected with V. salmonicida (intraperitoneal challenge) or A. salmonicida (co-habitant challenge). The challenge is conducted according to standard procedures well known in the art. For challenge with V. salmonicida, fish from the respective groups were injected intraperitoneally with an infectious dose of the pathogen. For the cohabitation challenge with A. salmonicida, unvaccinated fish were injected intraperitoneally with an infective dose of the pathogen. The infected fish (co-habitant) were placed in the relevant tanks containing control fish and fish vaccinated against A. salmonicida. As funiculosis developed in the co-habitant fish, bacteria were released into the water and transferred to vaccinated and control fish. The fish used in each challenge method were monitored for 17-21 days after infection where percent mortality was measured daily. A bacteriological assessment was carried out on 50% of the dead co-habitants, 30% of the fish in the control groups and all dead fish in the vaccinated groups to determine the presence of V. salmonicida or A. salmonicida bacteria.

The obtained data show that vaccination of Atlantic salmon with LAMB vaccines provides protection against infectious challenge with V. salmonicida (Table 5). The level of protection achieved by the monovalent and multivalent LAMB vaccines incorporating whole-cell washed and unwashed bacterin is comparable to an NLMA vaccine containing V. salmonicida and A. salmonicida bacterin, 6 and 16 weeks after vaccination, respectively. The data obtained also show that monovalent and multivalent LAMB vaccines are able to induce an immune response against V. salmonicida.

The data obtained further show that vaccination of Atlantic salmon with LAMB vaccines provides protection against infectious challenge with A. salmonicida (Table 6). The level of protection achieved by some of the monovalent formulations incorporating whole-cell washed and unwashed A. salmonicida bacterin is comparable to an NLMA vaccine containing V. salmonicida and A. salmonicida bacterin, 6 weeks after vaccination. Data also show that adhesion and pigmentation ratings for the LAMB vaccines are lower than an NLMA vaccine, indicating that the level of side effects is reduced.

EXAMPLE 5: Efficacy and side effects for monovalent and multivalent LAMB vaccines of varying concentration and antigenic material comprising whole cell V. salmonicida and A. salmonicida bacterin.

About. 3350 Atlantic salmon (Salmo salar) with an average weight of 18.6 g were used in the challenge test to show the effectiveness of different vaccines against A. salmonicida (furunculosis) and V. salmonicida (Hitra disease). The liposomes and antigenic material incorporated into the LAMB vaccines were prepared as described in Example 1. The antigenic material used in this example includes unwashed whole cell bacterin preparations of V. salmonicida and A. salmonicida, used alone or in combination. The liposome formulations used in this example were 30 mg/ml, 60 mg/ml and 90 mg/ml preparations of 1) DPPC:CH; 4) DPPC:CH:DPPG; 5) DPPC:CH:SA. The LAMB vaccine formulations are shown in Tables 7 and 9.

The fish were randomly selected from the general population, allocated to the relevant experimental groups, marked (freeze marking) and placed in freshwater tanks kept at 12°C. In this example, the fish were vaccinated intraperitoneally with 0.2 ml of various LAMB vaccines, 0.2 ml of an NLMA vaccine (ALPHA JECT 4000) and 0.2 ml of a control (PBS) vaccine. The fish were anesthetized with benzocaine before vaccination. 6 and 16 weeks after vaccination, 10 fish from each group were taken for testing of blood, weight and side effects. The level of adverse reaction at the injection site was analyzed according to a standard adhesion scale (0 - 6) and a pigmentation scale (0 - 3). 6 and 16 weeks after vaccination, respectively, the fish were challenged with an infectious dose of V. salmonicida or A. salmonicida. The challenge was conducted according to standard procedures well known in the art. Briefly, fish from the relevant tanks and experimental groups were injected intraperitoneally with an infectious dose of V. salmonicida or A. salmonicida. The farts were monitored for 21 days after the challenge and percent mortality for the challenged fish and control fish was measured daily. A bacteriological assessment was carried out on all dead fish and 30% of the control fish during the challenge period to determine the presence of V. salmonicida or A. salmonicida bacteria.

Data show that the vaccination of Atlantic salmon with monovalent and multivalent LAMB vaccine formulations at 30 or 60 mg/ml lipids and unwashed bacterin provides protection against challenge with V. salmonicida (Table 7). The protective effect appears 6 and 16 weeks after vaccination. The level of protection provided by the vaccines is comparable to a multivalent NLMA vaccine containing V. salmonicida and A. salmonicida whole cell bacterin. Adhesion and pigment scores for the majority of the LAMB vaccines are lower than the NLMA vaccine, suggesting that the level of side effects is reduced (Table 8). The data obtained also show that LAMB vaccines are able to induce an immune response against V. salmonicida with antibody titers increasing between weeks 6 and 16 (Table 8). There is also a good correlation between RPS and antibody response to V. salmonicida. Adherence ratings for the monovalent and multivalent LAMB vaccines are lower than the NLMA vaccine, indicating that the level of side effects is reduced.

Data also show that vaccination of Atlantic salmon with monovalent and multivalent formulations with 30, 60 or 90 mg/ml lipids and unwashed bacterin provides protection against challenge with A. salmonicida (Table 9). The protective effect is evident 6 weeks after vaccination. The protective effect provided by some of the LAMB vaccines is comparable to a multivalent NLMA vaccine containing V. salmonicida and A. salmonicida whole cell bacteria. Adhesion and pigment scores for the monovalent and multivalent LAMB vaccines are lower than an NLMA vaccine, suggesting that the level of side effects is reduced (Table 9).

EXAMPLE 6: Lyophilized whole-cell multivalent LAMB vaccines

bacterin

LAMB vaccines incorporating liposomes and antigenic material including whole cell bacterin were prepared using a method involving a lyophilization (freeze drying) process. In this example, a cryoprotectant was incorporated into the vaccine formulation. The antigenic material used in this example consisted of a whole cell bacterin preparation containing A. salmonicida, V. salmonicida, V. viscosis, V. anguillarum 01 and V. anguillarum 02. The liposome formulation used in this example was PC:CH:SA (30 mg lipid/ml). Some of the LAMB vaccines contained varying amounts of dextran (molecular weight 42,000) in the range 10-90 mg dextran per 30 mg lipid.

Briefly, suitable ratios of lipids were dissolved, at known concentrations, in chloroform:methanol in a volume ratio of 2:1, in an autoclaved Erlenmeyer flask or glass beaker. The solvents were then evaporated under a constant stream of N 2 gas or under reduced pressure using a Brinkmann rotary evaporator. After evaporation of the solvents, the lipid films were placed overnight in a vacuum desiccator. The dried films were hydrated with the appropriate volume of antigenic material medium and placed in an orbital shaker for 1 hour at room temperature or in a shaking water bath at higher temperatures if necessary. The resulting liposome preparations were then subjected to three 1 minute vortex sessions with mechanical mixing, with a one minute rest between vortex sessions.

The LAMB vaccines were, in a suitable container, reduced to a low temperature to produce an ice shell inside the container. The frozen formulation was then placed in a freeze dryer until the aqueous component was completely removed. After lyophilization, the vaccine formulation was reconstituted with saline or water and placed on an orbital shaker for 1 hour at room temperature or in a shaking water bath if higher temperatures were required. The formulation was then adjusted to 3 x 1 minute vortex sessions with mechanical mixing with a 1 minute rest period between vortex sessions.

Alternatively, the dried lipid films were hydrated with saline or water to form empty liposomes. The empty liposomes were reduced to a low temperature to form an ice shell, freeze-dried and reconstituted with antigenic material. The cryoprotectant, dextran, was added either to the antigenic material or to the empty liposome before freeze-drying. The amount of antigenic material associated with the liposomes comprising the vaccine formulation can be assessed using gradient centrifugation as outlined in Example 1.

Data show that dextran provides a general effect of maintaining and in some cases also increasing the percentage association of liposomes and antigenic material in lyophilized LAMB vaccines reconstituted with saline or water (Figure 2). Dextran, in the range 10-25 per 30 mg of lipid, increased the percentage association of liposomes and antigenic material compared to lyophilized LAMB vaccines without cryoprotectant. Furthermore, percent association is significantly higher in the presence of dextran, at concentrations of 15 and 30 mg/30 mg lipid, when the lyophilized LAMB vaccines are reconstituted with water versus saline.

Data obtained also show that a high level of association of liposomes and antigenic material is achieved after reconstitution of lyophilized empty liposomes with antigenic material to form the vaccine formulation (Figure 3). In this example, dextran, in the range of 15-45 mg per 30 mg lipid, not increasing association after reconstitution of lyophilized empty liposomes with antigenic material to form the vaccine formulation. Percent association of liposomes and antigenic material in the presence of dextran is lower than that shown by LAMB vaccines prepared without cryoprotectant.

NOTE: in both Figures 2 and 3, the asterisk (<*>) indicates a significant difference at p<0.05 for comparing the formation of empty liposomes using water or saline at any given concentration of dextran (t-test). EXAMPLE 7: The effect of different long-term storage conditions for LAMB vaccines Liposomes and antigenic material incorporated into the LAMB vaccines were prepared as described in Example 1. The LAMB vaccines were stored at 4°C and -20°C in a 12 months period. The amount of antigenic material associated with the liposomes comprising the vaccine formulation was assessed at defined time points (0, 3, 6 and 12 months respectively) using gradient centrifugation as outlined in Example 1. The antigenic material used in this example consisted of a whole cell preparation containing A. salmonicida, V. salmonicida, V. anguillarum Ol, V. anguillarum 02 and V. viscosis bacterins. The liposome formulation used in this example was DPPC:CH:SA. The data obtained show that the association levels did not decrease significantly during a 12-month period in any of the chosen storage conditions (Figure 4). This suggests that the LAMB vaccines remained stable over a 12 month period because the antigenic material did not appear to leak or dissociate from the liposomes. Asterisk (<*>) in Figure 4 indicates a significant difference p<0.05 for comparison of percentage association of the formulations stored at 4°C and

-20°C at any time period (t-test).

EXAMPLE 8: LAMB vaccine containing immunostimulants and

polymers

Around 1000 Atlantic salmon (Salmo salar L.) with an average weight of 30 g were used to demonstrate the effectiveness of different vaccines against A. salmonicida. The liposomes, antigenic material, one or more polymers and one or more immunostimulants that were incorporated into the LAMB vaccines were prepared as described in Example 1. The antigenic material used in this example was A-layer protein and single-cell bacterin preparations of A. salmonicida. The polymers used in this example were mucin and chitosan. The immunostimulant used in this example was glucan. The liposome formulations used in this example were DPPC:CH,

DPPC:CH:SA, DPPC:CH:DPPG, DPPC:CH:SA and D(PHY)PC:CH:SA.

The fish were randomly selected from the general population, allocated to the relevant experimental group, marked (tattooed) and placed in freshwater tanks maintained at 12°C. In this example, the fish were vaccinated with 0.2 ml of a LAMB vaccine, 0.2 ml of an NLMA vaccine (ALPHA JECT 4000) and 0.2 ml of a control (PBS) vaccine. The fish were anesthetized with benzocaine before vaccination. 6 and 19 weeks after vaccination, 10 fish from each group were taken for testing of blood, weight and side effects. The level of adverse reactions at the injection site was analyzed according to a standard adhesion scale (0-6) and pigmentation scale (0-3). 16 weeks after vaccination, the fish were infected with A. salmonicida using a co-habitant challenge model. The challenge was conducted according to standard procedures well known in the art. Briefly, vaccinated fish were not injected intraperitoneally with an infective dose of pathogens. The infected fish (co-habitants) were placed in tanks containing the control fish and fish vaccinated against A. salmonicida. 34 days after the co-habitant challenge, low mortality was observed in the control fish, which indicates a low challenge pressure. The fish were injected intraperitoneally with an infective dose of A. salmonicida. The fish were monitored for 21 days after challenge with percentage mortality for experimental and control fish measured daily.

Data show that vaccination of Atlantic salmon with LAMB vaccine formulations provides protection against infectious challenge against A. salmonicida (Table 10B). The level of protection provided by the LAMB vaccines with whole-cell bacterin and A-layer protein, whole-cell bacterin and charged polymers, whole-cell bacterin and an immunostimulant is comparable to an NLMA vaccine containing whole-cell A. salmonicida. Adhesion and pigmentation ratings for the LAMB vaccines are lower than an NLMA vaccine, suggesting that the level of side effects is reduced (Table 10B). These data also show that the LAMB vaccines containing antigenic material are capable of inducing an immune response against A. salmonicida (Table 10B).

EXAMPLE 9: LAMB vaccines containing hexaflumuron.

About. 600 Atlantic salmon (Salmo salar) with an average weight of 100 g were used in this trial to determine the effectiveness of different LAMB vaccines against sea lice infestation. The fish were divided into 4 groups, each containing 150 fish. In this example, the fish were injected intraperitoneally with 0.2 ml of different LAMB vaccines containing hexaflumuron and 0.2 ml of a LAMB vaccine without hexaflumuron (negative control). 5 weeks after vaccination, the fish were transferred from fresh water to sea water (mini cage). After transfer to seawater, the fish were exposed to a natural salmon lice infestation. 10 fish from each group were sampled every 3 weeks to assess the number of salmon lice and to determine whether the number of salmon lice varied between groups. The water temperature varied between 3 and 8°C during the test. The test ended approx. 18 weeks after the transfer to seawater.

The liposomes and antigenic material incorporated into the LAMB vaccines were prepared as described in Example 1. The antigenic material used in this example was whole cell bacterin preparations of A. salmonicida, V. anguillarum, V. salmonicida and V. viscosis as well as a drug , hexaflumuron. The liposome formulations used in this example were preparations of eggPC:CH:SA and DPPC:CH:SA.

In this example, liposome formulations incorporating whole cell bacteria were mixed with liposomal formulations with hexaflumuron. The liposomal formulations containing hexaflumuron were prepared by hydration of the dried lipid film with a standard suspension of hexaflumuron. Alternatively, the hexaflumuron powder can be mixed with empty liposomes. The LAMB vaccines with liposomes, whole cell bacterin and hexaflumuron are shown in Table 11.

Figure 5 shows the average number of salmon lice in each group during the sample. Tl and T2 are not shown in the figure as salmon lice were not detected at these times. The figure shows that fish treated with LAMB-hexaflumuron have a significantly lower number of salmon lice after 9 and 12 weeks after the transfer to seawater (T3 and T4). After completion of the salmon lice registration after 12 weeks after the transfer to the seawater, the fish in all groups were de-liced using the bath treatment preparation ALPHA MAX (deltamethrin). Only the fish in the control group needed de-lice, but as all groups were kept in the same cages, all groups were exposed to the bath treatment. The treatment was successful and counts 15 weeks after lice removal showed that salmon lice were not found in any of the groups.

These data show that LAMB vaccines incorporating liposomes, whole-cell bacterin and hexaflumuron protect fish against re-infestation for at least 12 weeks after transfer to seawater (18 weeks after injection). It is possible that the protection will last longer, but the trials were terminated after 18 weeks because there was no new salmon lice infestation.

EXAMPLE 10: The effect of LAMB vaccines on weight and

adhesion assessments.

About. 200 Atlantic salmon with an average weight of around 75 g were used to compare side effects and weight after vaccination with NLMA and LAMB vaccines. The liposomes and the antigenic material incorporated into the LAMB vaccine were prepared as described in Example 1. the antigenic material used in this example was whole cell bacterin preparations of A. salmonicida, V. salmonicida, V. anguillarum Ol, V. anguillarum 02 and V. viscosis. The liposome formulation used in this example was eggPC:CH:SA.

The fish were randomly selected from the general population, allocated to the appropriate experimental group, coded and placed in freshwater tanks maintained at 12°C. In this example, the fish were vaccinated with 0.2 ml of an NLMA and 0.2 ml of a LAMB vaccine. The fish were anesthetized with benzocaine before vaccination. Around 2 months after vaccination, the fish were transferred to seawater. The average temperature of the sea water was 9.2°C. 10 fish from each group were sampled after around 4.6 and 9 months respectively after vaccination. At each time point, weight and adhesion ratings were noted. The Spielberg scale was used to analyze the adhesion ratings. The tests were terminated after 10 months and the body weight was noted in the remaining fish from each experimental group.

Data show that after 4, 6, 9 and 10 months after vaccination, the mean weight of fish that were vaccinated with the LAMB vaccines is higher than fish that were vaccinated with the NLMA vaccine (Table 12). Furthermore, the adhesion ratings for the LAMB vaccines according to the invention are lower than for an NMLA vaccine (Table 13). The increased weight and lower adhesion observed with the LAMB vaccines indicates that the level of side effects is reduced compared to a standard oil-adjuvanted vaccine.

EXAMPLE 11: Efficacy of monovalent LAMB vaccines containing inactivated infectious pancreatic necrosis virus (IPNV).

About. 400 Atlantic salmon (Salmo salar) with an average weight of 33 grams were used to demonstrate the effectiveness of different LAMB vaccines against IPNV. The liposomes and the antigenic material incorporated into the LAMB vaccines were prepared as described in Example 1. The antigenic material used in this example were inactivated preparations of IPNV. The liposome formulations used in this example were: 1) egg PC:CH; 2) eggPC:CH:SA. The LAMB vaccine formulations are shown in Table 14.

Before vaccination, a random sample of 10 fish was analyzed for the presence of IPNV to ensure that they were free of the viral pathogen. The fish were randomly selected from the general population, allocated to the appropriate experimental group, marked (Panjet dye mark) and placed in freshwater tanks maintained at 12°C. In this example, the fish were vaccinated with 0.1 ml of different LAMB vaccines, 0.1 ml of different NLMA vaccines and 0.1 ml of a control (PBS) vaccine. The fish were anesthetized with benzocaine before vaccination. After a winter photoperiod, the fish were brought on a summer photoperiod for smoltification and transfer to seawater. The level of smoltification was examined by measuring plasma chloride. 86 days after vaccination, the fish were challenged by introducing virulent IPNV into the tanks. The challenge was conducted according to standard procedures well known in the art. The fish were monitored for 47 days after infection with percent mortality versus daily. Fish mortality was diagnosed using a test kit (co-agglutination test with IPNV-specific antibodies) and the presence of IPNV was further verified by a head kidney culture of 10 dead fish per tank.

These data show that dosing Atlantic salmon with the LAMB formulation provides protection against infectious challenge with IPNV (Table 14). The level of protection provided by the monovalent LAMB vaccines incorporating inactivated IPNV is comparable to NLMA vaccines 86 days after vaccination.

EXAMPLE 12: Effectiveness of LAMB vaccine containing whole cell

inactivated Photobacterium damsela bacterin.

About. 225 sea bass (Dicentrarchus labrax) with an average weight of 8.1 grams were used to demonstrate the effectiveness of LAMB vaccines against P. damsela subspecies piscicida. The liposomes and the antigenic material incorporated into the LAMB vaccine were prepared as described in Example 1. The antigenic material used in the LAMB vaccine in this example were unwashed whole-cell bacterin preparations of P. damsela and V. anguillarum Ol. The liposome formulations used in this example were: 1) egg PC:CH:SA, 2) eggPC:CH; 3) eggPC:CH:PG. The LAMB vaccine formulations are shown in Table 15.

The fish were randomly selected from the general population, allocated to the appropriate experimental group and placed in freshwater tanks maintained at 20°C. In this example, the fish were vaccinated with 0.1 ml of the LAMB vaccine, 0.1 ml of a water-based, non-liposome vaccine (Alpha Dip 2000) and 0.1 ml of a control (0.9% NaCl) vaccine. The fish were anesthetized with benzocaine before vaccination. 16 weeks after vaccination, the fish were infected with P. damsela subspecies piscidia. The challenge was conducted according to standard procedures well known in the art. The fish were injected intraperitoneally with an infective dose of the pathogen and were monitored 21 days after infection while percent mortality was measured daily. A bacteriological assessment was carried out on 30% of the dead fish in the control group and on all dead fish in the vaccinated groups to determine the presence of P. damsela subspecies piscidia.

Data show that the vaccination of sea bass with LAMB formulations provides protection against infectious challenge with P. damsela subspecies piscidia (Table 15). The level of protection provided by LAMB vaccines incorporating unwashed whole-cell bacterin is comparable to a water-based non-liposomal vaccine containing P. damsela subspecies piscidia and V. anguillarum Ol 16 weeks after vaccination.

EXAMPLE 13: Test protocol set up by the "European Pharmacopoeia Commission" for vibriosis vaccine (inactivated) for Salmonids

DEFINITIONS

Vibriosis vaccine (inactivated) for salmonids is prepared from cultures of one or more suitable strains or serovars of Vibrio anguillarum; the vaccine may also include Vibrio ordalii.

PRODUCTION

The strains of Vibrio anguillarum and Vibrio ordalii are grown separately. The suspensions are inactivated in a suitable manner. They can be purified and concentrated. Whole or disrupted cells can be used and the vaccine can contain extracellular products of the bacteria released into the growth medium.

CHOICE OF VACCINE COMPOSITION

The strains of Vibrio anguillarum and Vibrio ordalii that were used have been shown to be suitable for the production of antigens of presumed immunological importance. The vaccine has been shown to be satisfactory in terms of safety and immunogenicity in the species of fish for which they are intended. The following tests can be used to demonstrate safety and immunogenicity.

Safety. The safety is tested in three different batches using test A or B, depending on the recommendations for use.

A. Vaccines intended for administration by injection. A test is carried out for each fish species for which the vaccine is intended. The fish used are from a population which does not have specific antibodies against the relevant serovars of Vibrio anguillarum, or where applicable, Vibrio ordalii and which has not been vaccinated or exposed to vibriosis. The test is carried out under the conditions recommended for use of the vaccine with a water temperature of no less than 12°C. A quantity of the vaccine corresponding to 2 times the recommended dose per mass unit of fish of the minimum body mass recommended for vaccination is administered intraperitoneally to each of not fewer than 50 fish of the minimum recommended body mass. The fish are observed for 21 days. No abnormal local or systemic reactions occur. The test is invalid if more than 6% of the fish die due to causes not due to the vaccine.

B. Vaccines intended for administration by immersion. A test is carried out for each fish species for which the vaccine is intended. The fish used are from a population which does not have specific antibodies against the relevant serovars of Vibrio anguillarum, or where applicable, Vibrio ordalii and which has not been vaccinated or exposed to vibriosis. The test is carried out under the conditions recommended for use of the vaccine with a water temperature of no less than 12°C. A dipping bath is prepared at twice the recommended concentration. No fewer than 50 fish with the minimum body mass recommended for vaccination are used. The fish is bathed 2 times the recommended time. The fish are observed for 21 days. No abnormal local or systemic reactions occur. The test is invalid if more than 6% of the fish die due to causes not due to the vaccine. C. Safety is also demonstrated in field trials by administering the intended dose to a sufficient number of fish distributed in more than one set of habitats. No abnormal reactions occur.

Immunogenicity: The test described under "Potency" below is suitable for demonstrating the immunogenicity of the vaccine.

BATCH TESTING

Batch potency test. For routine tests of vaccine batches, the test described below under the section "Potency" can be carried out using groups of not fewer than 30 fish of one of the species for which the vaccine is intended; alternatively, a validated test based on antibody response can be carried out where the criteria are set up with reference to a vaccine batch that has given satisfactory results in the test as described under "Potency". The following test can be used.

Fish are used from a population that does not have specific antibodies against the relevant serovars of Vibrio anguillarum and, where applicable, Vibrio ordalii, and that lie within the specified limits for body mass. The test is carried out at a defined temperature. In each of no fewer than 25 fish, a dose of the vaccine is injected intraperitoneally according to the instructions for use. Dummy vaccinations are carried out on a control group of no fewer than 10 fish. Blood samples are collected at defined intervals after vaccination. For each sample, the level of specific antibodies against the different serovars of Vibrio anguillarum and Vibrio ordalii included in the vaccines is determined using a suitable immunochemical method. The vaccines are approved with the test if the mean levels of the antibodies are not significantly lower than that found for a batch that gave satisfactory results in the test described under "Potency". The test is not valid if the control group shows antibodies against the relevant serovars of Vibrio anguillarum and Vibrio ordalii.

IDENTIFICATION

When injected into healthy fish that do not have specific antibodies against Vibrio anguillarum and, where applicable, Vibrio ordalii, the vaccine stimulates the production of such antibodies.

TESTS

Safety. No fewer than 10 fish of one of the species for which the vaccine is intended are used, each with no less than the minimum body mass recommended for vaccination; if fish of the minimum body mass are not available, fish of not more than twice this mass are used, and, where the vaccine is administered by injection, the dose is increased proportionately. Fish are used from a population that does not have specific antibodies against Vibrio anguillarum, and possibly Vibrio ordalii and that has not been vaccinated or exposed to vibriosis. The test is carried out under the conditions recommended when using the vaccine with a water temperature of no less than 12°C. For vaccines administered by injection or immersion, an amount of vaccine equivalent to twice the recommended dose is injected intraperitoneally into each fish. For vaccines that are only administered by immersion, a bath with twice the recommended concentration is used and the fish is bathed for twice the recommended immersion time. The animals are observed for 21 days. No abnormal local or systemic reactions occurred that could be due to the vaccine. The test is invalid if more than 10% of the fish die for reasons not due to the vaccine.

Sterility. The vaccine satisfies the test for sterility as prescribed in the monograph "Vaccines for veterinary use ( 62").

POTENCY

A separate test is carried out for each species and each serovar incorporated into the vaccine. The test is carried out according to a protocol that defines limits for the fish's body mass, the water source, the water flow and the temperature limits, and preparations for a standardized challenge. No fewer than 100 fish are vaccinated in a recommended manner according to the instructions for use. Dummy vaccinations are carried out on a control group with a similar composition to the vaccinated group; vaccinated and control fish are marked for identification. All fish are kept in the same tank or an equal number of controls and vaccines are mixed in each tank if more than one tank is used. The challenge is carried out by injection a fixed period of time after vaccination, defined according to what is stated with a view to the development of protective immunity. For challenge, cultures of Vibrio anguillarum or Vibrio ordalii are used if virulence has been verified. The fish are observed daily until at least 60% specific mortality is reached in the control group. The results for both vaccinees and controls are plotted on a curve with mortality against the time from challenge and by interpolation the time corresponding to 60% mortality in controls is determined. The test is invalid if the specific mortality is less than 60% in the control group 21 days after the first fish death. From the curve for the vaccinations, one can read the mortality (M) at the time which corresponds to 60% mortality in the controls. The calculation of the relative percentage survival (RPS) takes place from the equation:

The vaccine is approved with this test if the RPS is not less than 60% for vaccines administered by immersion and 75% for vaccines administered by injection.

The invention is described in detail, but it is clear that much can be varied in many ways. Such variations are intended to be within the scope of the invention as they are within the scope of the skilled person's knowledge.

Claims (42)

patent claim 1. Liposome vaccine formulation for finfish, characterized in that it comprises: a) liposomes, and b) antigenic material derived from two or more different finfish pathogens selected from the group: Vibrio anguillarum, Aeromonas salmonicida, Vibrio salmonicida, infectious pancreatic necrosis virus (IPNV) or Photobacterium damselae subsp. piscica, where at least one part of the antigenic material is liposome-associated. 2. Liposome vaccine formulation according to claim 1, characterized in that the liposome vaccine formulation is formulated so that a dose unit has a volume of 0.05 - 0.5 ml. 3. Liposome vaccine formulation according to claim 1 or 2, characterized in that it is able to provide a minimum efficiency in finfish of at least 50% relative percentage survival. 4. Liposome vaccine formulation according to any one of claims 1 to 3, characterized in that it comprises antigenic material selected from the group consisting of proteins, peptides, nucleic acids, whole-cell live or weakened fin-fish pathogens, cell fragments, cell secretions, viruses, cell organelles, cell membranes, cell membrane fragments, tissue, tissue fragments and tissue extracts, where at least one part of the antigenic material is liposome-associated. 5. Liposome vaccine formulation according to any one of claims 1 to 4, characterized in that it further comprises one or more bioactive agents. 6. Liposome vaccine formulation according to any one of claims 1 to 4, characterized in that the bioactive agent is a drug, an antibiotic or an immunostimulant. 7. Liposome vaccine formulation according to any one of claims 1 to 4, characterized in that the antigenic material, which is derived from two or more different finfish pathogens, comprises a number of antigenic determinants derived from the same finfish pathogen. 8. Liposome vaccine formulation according to any one of claims 1 to 7, characterized in that it further comprises one or more adjuvants. 9. Liposome vaccine formulation according to any one of claims 1 to 8, characterized in that it further comprises a pharmaceutically acceptable carrier. 10. Liposome vaccine formulation according to any one of claims 1 to 9, characterized in that the antigenic material comprises one or more proteins, peptides, saccharides, polysaccharides, lipopolysaccharides, nucleic acid, lipids, glycolipids or derivatives or analogues thereof. 11. Liposome vaccine formulation according to any one of claims 3 to 10, characterized in that said minimum efficacy is determined by challenging a finfish to which the liposome vaccine formulation has been administered with one or more finfish pathogens at least 6 weeks after administration of said liposome vaccine formulation. 12. Liposome vaccine formulation according to one of claims 3 to 11, characterized in that the relative percent survival is determined when the mortality in non-vaccinated control fin fish is 60%. 13. Liposome vaccine formulation according to any one of claims 1 to 12, characterized in that the liposome vaccine formulation is capable of reducing side effects relative to a corresponding oil-based vaccine formulation. 14. Liposome vaccine formulation according to any one of claims 1 to 13, characterized in that the liposome vaccine formulation is able to minimize the side effect to a Speilberg scale rating of less than 2. 15. Liposome vaccine formulation according to any one of claims 1 to 14, characterized in that the antigenic material is derived from one or more bacterial sources. 16. Liposome vaccine formulation according to claim 15, characterized in that said antigenic material comprises one or more components selected from the group: weakened whole cells, inactivated whole cells, cell fragments, cell organelles, cell organelle fragments, cell extracts, cell secretions, toxins, membranes and membrane fragments. 17. Liposome vaccine formulation according to claim 16, characterized in that the antigenic material comprises one or more components selected from the group consisting of: a jem-regulated outer membrane protein (IROMP) from Aeromonas salmonicida, an outer membrane protein (OMP) of Aeromonas salmonicida, an A-layer protein of Aeromonas salmonicida, an OMP of Vibro anguillarum, a flagellar protein of V. anguillarum, live, attenuated or inactivated A. salmonicida, V. salmonicida, V. anguillarum 01 or V. anguillarum 02. 18. Liposome vaccine formulation according to any one of claims 1 to 14, characterized in that the antigenic material is derived from one or more viral sources. 19. Liposome vaccine formulation according to claim 18, characterized in that the antigenic material comprises one or more components selected from the group consisting of: weakened whole viruses, inactivated whole viruses and fragments of viruses. 20. Liposome vaccine formulation according to claim 18, characterized in that said antigenic material comprises one or more components selected from the group: ; VP1 of infectious pancreatic necrosis virus (IPNV); VP2 of infectious pancreatic necrosis virus (IPNV); VP3 of infectious pancreatic necrosis virus (IPNV); and N structural protein of infectious pancreatic necrosis virus (IPNV). 21. Liposome vaccine formulation according to any one of claims 1 to 20, characterized in that the fin fish is selected from the group: Atlantic salmon, Pacific salmon, Coho salmon, rainbow trout, brown trout, brook trout, char, carp, catfish, yellowtail, bream, perch , catfish, grayling, whitefish, turbot, cod, halibut, , , sturgeon or sea bass. 22. Liposome vaccine formulation according to any one of claims 2 to 4, characterized by the fin fish being rainbow trout. 23. Liposome vaccine formulation according to any one of claims 1 to 14, characterized in that one or more of the fish pathogens are selected from the group: infectious pancreatic necrosis virus (IPNV); Aeromonas salmonicida causing furunculosis; Vibrio salmonicida causing Vibrio salmonicida septicemia (VSS) or Hitra disease; Photobacterium damselae subspecies piscicida and Vibrio anguillarum. 24. Method for the preparation of a liposome vaccine formulation according to any one of claims 1 to 23, characterized in that it comprises: (a) preparation of a liposome formulation, and (b) associating antigenic material derived from two or more fish pathogens with the liposome formulation to form the liposome vaccine formulation. 25. Method according to claim 24, characterized in that it comprises formulation of liposome as the formulation and the liposome-associated antigenic material such that a dose unit has a volume of 0.05 - 0.5 ml. 26. Method according to claim 24 or 25, characterized in that the liposome formulation comprises one or more classes of lipids selected from the group consisting of phospholipids, neutral lipids, glycolipids and amphiphiles. 27. Method according to any one of claims 24 to 26, characterized in that it further comprises combining one or more bioactive agents with said liposome vaccine formulation. 28. Method according to any one of claims 24 to 27, characterized in that it further comprises combining one or more adjuvants with said liposome vaccine formulation. 29. Method according to claim 27, characterized in that said bioactive agent is a drug, an antibiotic or an immunostimulant. 30. Method according to any one of claims 24 to 29, characterized in that it further comprises combining a pharmaceutically acceptable carrier with said liposome vaccine formulation. 31. The liposome vaccine formulation according to any one of claims 1 to 23 for use as a medicament. 32. Liposome vaccine formulation for use according to claim 31, characterized in that the use is by inducing an immune response in a finfish. 33. Liposome vaccine formulation for use according to claim 31 or 32, characterized in that the use is for the treatment of a fin fish that is infected with one or more fish pathogens. 34. Liposome vaccine formulation for use according to any one of claims 31 to 33, characterized in that the liposome vaccine formulation is administered by injection. 35. Liposome vaccine formulation for use according to any one of claims 31 to 34, characterized in that the liposome formulation is administered by intraperitoneal, intramuscular or subcutaneous injection. 36. Liposome vaccine formulation for use according to claim 34 or 35, characterized in that the liposome vaccine formulation is administered in a volume of 0.05 to 0.5 ml. 37. Liposome vaccine formulation for use according to any one of claims 31 to 36, characterized in that the liposome vaccine formulation comprises two or more lipids. 38. Liposome vaccine formulation for use according to any one of claims 31 to 36, characterized in that the use reduces side effects compared to a corresponding, oil-based vaccine formulation. 39. Liposome vaccine formulation for use according to any one of claims 31 to 38, characterized in that the use minimizes side effects to a Speilberg scale rating of less than 2. 40. Liposome vaccine formulation for use according to any one of claims 31 to 39, characterized in that the use is prophylactic to prevent infection from occurring. 41. Liposome vaccine formulation for use according to any one of claims 31 to 40, characterized in that the use is therapeutic to treat pre-existing infections. 42. Liposome vaccine formulation for use according to any one of claims 31 to 41, characterized in that the use is for inducing an immune response that supports the overcoming of an ongoing and possibly chronic infection.