NO346254B1 - Isolated salmonid alphavirus and its use - Google Patents

Description

Technical field of the invention

The present invention relates to veterinary immunology and in particular to isolated salmonid alphavirus of subtpe SAV3 as well as ways to prevent or control infections of salmonid alphavirus and outbreaks of fish pancreatic disease (fish pancreatic disease) or sleeping sickness by using an inactivated salmonid alphavirus of subtpe SAV3.

The background of the invention

Pancreas disease has become a serious disease in salmon farming in several geographical areas. The disease was first reported from Scotland and has subsequently been reported from Ireland, Norway and North America. Since 2007, pancreatic disease has become endemic in most farms in Ireland and isolated outbreaks of pancreatic disease have been reported in North America. Another diagnosis, sleeping sickness, is endemic in parts of France and has also been reported in Italy and Spain (McLoughlin & Graham, 2007). Recently, there have been unofficial reports that pancreatic disease has also been observed in Chile.

In Norway, the disease has spread over the past 10 years, with 58 confirmed infected areas in 2006 and 98 confirmed infected areas in 2007. In 2006, the disease also spread to Møre og Romsdal, which is north of the previously defined geographical area that was affected by salmonid pancreatic disease. Pancreatic disease is now the biggest health problem for the aquaculture industry in the western part of Norway. Pancreatic disease (PD) and the related sleeping sickness are both caused by alphaviruses and cannot be treated with any therapeutics.

The first alphavirus was isolated from fish in 1995 (Nelson et al. 1995). Today, alphaviruses have been isolated from both Atlantic salmon suffering from pancreatic disease and rainbow trout suffering from sleeping sickness (McLoughlin and Graham 2007).

The Salmonis alphaviruses (SAV) are spherical (approximately 65 nm in diameter) enveloped RNA viruses. They contain a single-stranded positive-sense RNA genome of 11–12 kB (McLoughlin and Graham 2007). In European patent EP 712 926 it is described to be sensitive to chloroform, quickly inactivated at pH=3 and at 50 <o>C and with a liquid density of 1.20 g/ml. Nucleotide sequencing studies assign salmonid alphaviruses to three genetically distinct subtypes (Powers et al. 2001, Hodneland et al. 2005, Weston et al. 2005). Salmonid alphavirus 2 (SAV2) causes sleeping sickness in rainbow trout, salmonid alphavirus 1 (SAV1) causes pancreatic disease in Atlantic salmon in Ireland and Scotland, while salmonid alphavirus 3 (SAV3) causes pancreatic disease in Atlantic salmon and sea-farmed rainbow trout in Norway. This is further supported by Karlsen et al. (2006) who found that the SAV3 subtypes that cause pancreatic disease in Norway are genetically homogeneous, but different from SAV1 and SAV2. Recently, SAV4, SAV5 and SAV6 have been identified as subtypes related to SAV1. Andersen et al. (2007) have further identified that the pseudobranches and the heart are the most suitable tissues for RT-PCR diagnostics in exposure experiments with SAV1 and SAV3.

In Norway, all farmed Atlantic salmon are vaccinated against the most common bacterial diseases and very often against pancreatic necrosis virus (IPNV) before they are transferred to the sea. Although comprehensive vaccination programs are certainly desired to avoid economic losses and further spread of infectious diseases, such programs are also associated with technical challenges. Administering vaccines requires extensive handling of the fish, which causes stress and mortality because the fish must be pumped into holding tanks, transferred to an anesthetized tank, injected with vaccine, and pumped back into holding tanks. Due to the large scale of modern aquaculture, such vaccination programs are expensive and labor intensive and cause stress to the fish. For these reasons, vaccines against the various infectious diseases are preferably administered in combined, polyvalent vaccines containing several antigens.

Theoretically, combining vaccines should be uncomplicated. In practice, however, combining several vaccine antigens often leads to a reduced effect of each individual antigen. Such problems associated with polyvalent vaccines are discussed, for example, in André, E.F. (1999), which provides an overview of strategies for human pediatric vaccination programs. Similar problems raise concerns in animal vaccination programmes, for example in the case of vaccines against foot-and-mouth disease in sheep, as reviewed by O'Meara et al. (1993).

Despite the fact that salmonid alphavirus was isolated and characterized more than a decade ago, vaccination against pancreatic disease is still a challenge. In European patent EP 712 926, example 11 presents data showing that a composition intended for vaccination purposes based on pancreatic disease virus SAV1, inactivated by the addition of beta-propiolactone and NaOH, is ineffective for protection against subsequent infection. In the same experiment, a composition based on PDV treated with 0.1% formalin (35–38%) appears to elicit a protective immune response in an experimental setting. However, since treatment with 0.1% formalin is insufficient to inactivate PDV, one must conclude that the latter composition is one with live virus. Both compositions contained PD virus of the SAV1 subtype with a titer of 10<7.5>TCID50ml<-1>.

As of February 2008, only one PD vaccine, sold under the name Norvac compact PD, was commercially available. The vaccine is based on inactivated PD virus of the SAV1 subtype: A first-generation product contained antigen in amounts of 10<7.2>TCID50/dose with a dosing volume of 100 μl. A second generation product has been developed that contains 10<7.5>TCID50/dose. Rodger & Mitchell (2005) investigated the effect of this vaccine: in 2003 they found that vaccination did not reduce the risk of outbreaks of pancreatic disease; in 2004 it seemed that vaccinated fish had a slightly lower risk of infection, but the result was not statistically significant. Furthermore, in mixed populations with vaccinated and non-vaccinated fish, there was a tendency for lower mortality in vaccinated fish.

In the summary of product characteristics, Norvac compact PD is described as incompatible with other vaccines and immunological products. Consequently, this monovalent PD vaccine is only recommended for injection at least 210 degree days before injection of other multivalent vaccines that protect against common bacterial diseases and IPNV in farmed fish (degree days are calculated as the product of days and temperature: 10 days with a temperature of 10 ºC is the same like 100 degree days). Under normal conditions, Norvac compact PD must be administered 2-3 weeks before the administration of a polyvalent vaccine.

According to the Summary of Product Characteristics, no information is available on the safety and efficacy of the simultaneous use of this vaccine with any other immunological products. However, the stated lack of compatibility with other vaccines is consistent with previous reports of multivalent PD vaccines failing to induce good protection against pancreatic disease (Christie et al. 1999 A, Intervet newsletter 2002, Christie et al.

1999B). Christie reported the induction of neutralizing antibodies in 60% of subjects vaccinated with a monovalent PD vaccine. The modest efficacy was greatly compromised when the vaccine was combined with other immunological products since the resulting polyvalent vaccine induced neutralizing antibodies in only 20% of vaccinated individuals.

In EP 1 075 523 the inventors are also concerned about the problem of PD vaccines being incompatible with other vaccines. In paragraph [0004] it is stated: “A disadvantage in the production of inactivated vaccines from the PD virus described in EP-A-712 926 is the slow growth of the virus, especially in cell cultures, which makes the production of the vaccines relatively inefficient process. A further disadvantage of the inactivated vaccines is the instability of the inactivated virus in the presence of other inactivated pathogens which leads to loss of potency. Fish vaccines are generally produced as multivalent vaccines, and significantly higher amounts of inactivated virus are required in the multivalent vaccines than would be required in a monovalent vaccine to compensate for the loss of potency." The inventors refer to recombinant protein vaccines as a solution, but such recombinant vaccines have never been implemented in the treatment of pancreatic disease. In WO2007031572 A1, the inventors identified an epitope for SAV E2 capable of inducing a virus-neutralizing immune response for use in vaccine development and diagnostics.

In conclusion, existing vaccination programs against fish pancreatic disease are still ineffective and have not been able to stop the spread of the disease. In addition, according to the general opinion in the field, PD vaccines based on inactivated viruses are only available at high production costs and they are incompatible with vaccines against other infectious diseases prevalent in farmed fish.

There is therefore still an urgent need for effective measures that can control fish pancreatic disease, both for ethical and economic reasons.

Summary of the invention

An object of the present invention is to provide i) an isolated alpha virus of subtype SAV 3; as well as ways to prevent or control salmonid alphavirus infections and outbreaks of fish pancreatic disease (fish pancreatic disease) or sleeping sickness (sleeping disease) using an inactivated salmonid alphavirus of subtpe SAV3.

In particular, one aspect of the invention relates to an isolated salmonid alphavirus of subtype SAV3 which comprises a nucleic acid sequence that is at least 90% identical to the sequence indicated in SEQ ID NO: 1 and is further characterized by

a. a visible cytopathogenic effect during early passage in cell culture, and

b. the ability to induce mortality of at least 30% in a laboratory infection model, the model includes smoltification of Atlantic salmon according to standard procedures and post-smolt challenge by intraperitoneal injection with a SAV3 dose of at least 10<8>TCID50 per fish, such as at least 10<9>TCID50, at least 10<10>TCID50 or preferably at least 3.5 x 10<8>TCID50 per fish within one day of transfer to seawater at 12 <o>C, and

c. the ability to grow in vitro to a titer of at least 1x10<8 >TCID50/ml in the supernatant/growth medium cultures of CHH-1 cells or CHSE-214 cells.

The invention also provides isolated virus as defined above, wherein the virus has a detectable cytopathogenic effect during the first, second, third or fourth passage on a culture of CHSE cells.

The invention also provides isolated virus as defined above, wherein said titer of at least 1x10<8 >TCID50/ml is achieved when:

i) Cells are cultured using host cells that have been seeded at a density of 0.1-1x10<5 >cells cm<-2>;

ii) The host cells are cultured for 4-6 days prior to virus infection;

iii) The host cells are cultured to a density of 0.1-1.0 x10<6 >cells cm<-2 >at the time of infection with said virus isolate;

iv) The host cells are cultured in a growth medium comprising EMEM (EBSS) + 10% fetal bovine serum (FBS) 2 mM L-glutamine 1% non-essential amino acids (NEAA) 0.1% gentamicin and

v) The infected cells are cultured at a temperature of 15 ºC for a period of 10-14 days.

The invention also provides isolated virus as defined above, wherein the virus comprises a nucleic acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 4.

The invention also provides isolated virus as defined above, wherein the virus comprises a nucleic acid sequence that is at least 98% identical to the sequence set forth in SEQ ID NO: 1.

The invention also provides isolated virus as defined above, which virus is positive in a SAV reverse transcriptase quantitative PCR identity test using a set of primers such as the primers set forth in SEQ ID NO: 7 and 8 and the conditions 50 <o>C, 30 min - 95 <o>C, 10 min –(95 <o>C, 30 sec - 57 <o>C, 60 sec -72 <o>C, 30 sec)*40.

The invention also provides isolated virus as defined above, wherein the virus is a genetic variant of a single of the virus strains deposited under the Budapest Convention at the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 0JG UK 12 December 2007 under deposit numbers 07121201 and 07121202.

Another aspect of the invention provides a use of an inactivated salmonid alpha virus of subtype SAV3, wherein the virus comprises a nucleic acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 1 and is further characterized by

a. a visible cytopathogenic effect during early passage in cell culture, and b. the ability to induce mortality of at least 30% in a laboratory infection model, the model includes smoltification of Atlantic salmon according to standard procedures and post-smolt challenge by intraperitoneal injection with a dose of SAV3 of at least 10<8>TCID50 per fish, such as at least 10<9>TCID50, at least 10<10>TCID50 or preferably at least 3.5 x 10<8>TCID50 per fish within one day of transfer to seawater at 12 <o>C, and c. the ability to grow in vitro to a titer of at least 1x10<8 >TCID50/ml in the supernatant/growth medium cultures of CHH-1 cells or CHSE- 214 cells; in the manufacture of a vaccine to prevent or reduce the incidence of infection with salmonid alphavirus.

The invention also provides a use as defined above, in which the salmonid alphavirus is inactivated by the addition of formaldehyde.

The invention also provides a use as defined above, in which the salmonid alphavirus is inactivated using a procedure comprising the addition of 1.5-2.5 g/kg, formaldehyde and subsequent incubation for 48-96 hours at a temperature of 14-17 <º>C.

Brief description of the figures

Figure 1 shows SAV3 titers in crude supernatants from CHH-1 cell cultures inoculated with different SAV3 isolates or passages of isolates.

Figure 2 is a representation of SAV3 titers in crude supernatants from SAV3-infected CHSE-214 cell cultures.

Figure 3 A and B shows % accumulated mortality after challenge of Atlantic salmon smolt with salmonid alphavirus type 3 (SAV3). The figure further shows the effect of vaccinating fish in the mating stage against salmonid pancreatic disease using a polyvalent vaccine according to the invention. Two parallel experiments were conducted in separate challenge tanks (A and B).

The present invention will be described in more detail below.

Detailed description of the invention

The present invention is based on the observation that, despite previous reports to the contrary, the development of polyvalent vaccines combining inactivated salmonid alpha virus with vaccines against other fish pathogens is actually the most effective way to protect farmed salmon against pancreatic disease.

In particular, the inventors made the following observations:

i) Despite testing different vaccine formulations, it was not possible to establish any adverse effect of other vaccine components on the stability of the pancreatic disease antigen. In other words: PD vaccines based on inactivated viruses are highly compatible with vaccines against currently widespread fixed pathogens;

ii) In vaccines based on inactivated virus, a minimum antigen titer in the range of 3.75 x 10<7 >TCID50/dose seems desirable to effectively induce protective immunity, indicating that previous vaccination programs have been based on vaccines containing insufficient amounts antigen;

iii) Titers of salmonid alpha virus sufficient for the large-scale production of vaccines with good efficacy can certainly be obtained using conventional virus propagation technology and at a reasonable cost.

Microbiological material

In connection with the present invention, isolates of salmonid alphavirus have been deposited under the Budapest Convention at the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 0JG UK on 12 December 2007 under the following accession numbers: 07121201 , which corresponds to isolate ALV 405 in the examples; 07121202, which corresponds to isolate ALV 407 in the examples; and 07121203 which corresponds to isolate ALV 409 in the examples.

Polyvalent vaccines containing antigens from typical fish pathogens except salmonid alphaviruses are well known in the art and are already commercially available. The identification and isolation of suitable antigens to be used in such vaccines is described in the literature and thus enables the person skilled in the art to generate and prepare such polyvalent vaccines. In addition, representative isolates of relevant fish pathogens are available without restriction from different sources. For example, the following bacterial species are available from the LGCPromochem/American Type Culture Collection ATCC Storage and Distribution Center (ATCC): A. salmonicida (ATCC 33658<TM >country of origin: not stated), Aeromonas hydrophila, V. salmonicida (ATCC 43839<TM>, country of origin: Norway), V. anguillarum serotype O1(ATCC 43305<TM>, country of origin: Denmark) and O2(ATCC 19264)<TM>, country of origin: not stated) and Moritella viscosa (ATCC BAA-105<TM>, country of origin: Norway). Flavobacterium columnaris (deposited as Cytophaga columnaris) is available under ATCC number 23463™ (Country of origin: USA).

Regarding Piscirickettsia salmonis, the present applicant has deposited several useful isolates under the Budapest Treaty with the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 0JG UK: three isolates were deposited on 9 June 2006 under accession numbers 06050901, 06050902 and 06050903 (isolate AL10005, AL10007 and AL10008). A single isolate was deposited on 21 March 2007 under accession number 07032110 (country of origin: Chile). Similarly, representative isolates of relevant virus species are available including infectious pancreatic necrosis virus (IPNV, ATCC VR-1318, country of origin: not stated), Viral Hemorrhagic Septicemia Virus (VHSV, ATCC VR_1389, country of origin: Denmark); Infectious Hematopoietic Nerosis Virus (IHNV, ATCC VR-1392, Country of Origin: USA)); Pancreatic Necrosis Virus; Spring Viremia of Carp (SVC, ATCC VR-1390, country of origin: Denmark); Channel Catfish Virus (CCV) (ATCC VR-665, Country of Origin: USA); Infectious Salmon Anemia (ISA) virus (ATCC VR-1554, country of origin:

Canada).

Patent filings have previously been made by the current applicant for the following virus species: Heart and Skeletal Muscle Infection Virus (HSMIV, patent filing no. ECACC 04050401, country of origin: Norway); Cardiomyopathic syndrome virus (CMSV, patent deposit no. ECACC 07032902, country of origin: Norway).

Definitions

Before the present invention is discussed in further detail, the following terms and conventions will be defined:

"An antigenic and/or immunogenic material" in the present context refers to a material containing one or more protective epitopes. An epitope is protective if it can induce an immune response capable of effectively interfering with the extent or progression of infection with salmonid alphaviruses, including SAV1, SAV2 and especially SAV3. The term includes polypeptides, including polypeptides produced using recombinant techniques and parts of such polypeptides.

The term "genotypic characteristics" broadly refers to the composition of one or more parts of an individual's genome that contribute to determining a specific characteristic.

With regard to the virus according to the invention, the expression "related genotypic characteristics" is used in relation to viruses that contain nucleic acid sequences with a high degree of sequence identity compared to known nucleic acid sequences from previously isolated salmonid alphaviruses. Comparison can be made with the nucleic acid sequences encoding a single one of the nsP1, nsP2, nsP3 and nsP4 nonstructural proteins. Comparison can further be made with the nucleic acid sequences that code for capsid, glycoprotein E2, glycoprotein E3 or 6K and gpE1 proteins. The term is used in particular to define viruses that have nucleic acid sequences with a high degree of sequence identity compared to all such known amino acid sequences of deposited isolates ALV 405, ALV 407 and/or ALV 409. With the aim of defining the viral isolates used in connection with the present invention reference is made in particular to nucleic acid sequences of glycoprotein E2 and the non-structural protein nsP3 as provided herein and identified by SEQ ID NOs: 1-6. It should therefore be understood that the virus according to the invention contains nucleic acid sequences that are at least 80% identical, such as at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% identical to the nucleic acid sequences of glycoprotein E2 and the non-structural protein nsP3 as provided herein and identified by SEQ ID NOs: 1-6.

The term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to provide the best possible match, allowing the insertion of gaps or alternatively truncation at the ends of the polypeptide sequences or

the nucleotide sequences. The sequence identity can be calculated as where Ndif is the total number of non-identical residues in the two sequences when aligned, and where Nref is the number of residues in one of the sequences. Therefore, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref=8). A gap is considered non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8).

With respect to all embodiments of the invention that relate to nucleotide sequences, the percentage of sequence identity between one or more sequences can also be based on alignment using clustalW software (http:/www.ebi.ac.uk/clustalW/index.html) with default settings. For nucleotide sequence alignments, these settings are: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB).

Alternatively, nucleotide sequences can be analyzed using the program DNASIS Max, and the comparison of the sequences can be done at http://www.paralign.org/. This service is based on the two comparison algorithms called Smith-Waterman (SW) and ParAlign. The first algorithm was published by Smith and Waterman (1981) and is a well-established method that finds the optimal local fit for two sequences. The second algorithm, ParAlign, is a heuristic method of sequence alignment; details of the method are published in Rognes (2001). Default settings for score matrix and Gap handicap as well as E-values were used.

The term "phenotypic characteristics" refers just as generally to one or more observable characteristics of an organism that are produced by the interaction of the inherited genotype of the individual with transmitted epigenetic factors and/or non-inherited environmental variation. In the context of the present invention, the term "related phenotypic characteristics" includes any of the following characteristics: size, shape, pH stability, temperature stability, chloroform sensitivity and hemagglutination.

"Phenotypic characteristics" further includes the ability to elicit any of the clinical signs of pancreatic disease and sleeping sickness, including gross pathological signs and histopathological signs as described in the present application. "Phenotypic characteristics" also includes the ability of the virus to introduce such clinical signs in a laboratory challenge experiment as described herein. Furthermore, the expression refers to the behavior of the virus when it grows on cell cultures, including growth kinetics and the ability to induce a cytopathogenic effect.

The terms SAV1, SAV2 and SAV3 define subtypes of salmonid alphaviruses: the SAV1 subtype is defined and characterized in Nelson et al. Diseases of Aquatic Organisms, 22, 25-32, 1995 and a representative isolate has been deposited at ECACC under accession number V94090731. The SAV2 subtype, on the other hand, is the trigger for "sleeping sickness" and was first isolated and characterized by Castric et al. Bulletin of the European Association of Fish Pathologists 17, 27-30, 1997. The SAV3 subtype was first isolated and characterized by Hodneland et al. as described in Dis Aquat Organ. 2005 Sep 5; 66(2):113-20 (Erratum in: Dis Aquat Organ. 2005 Nov 9; 67(1-2):181). The terms SAV4, SAV5 and SAV6 define new subtypes of salmonid alphavirus found near Scotland and Ireland.

As those skilled in the art will understand, "cytopathogenic effect" refers to visible morphological changes in cells infected with virus. In particular, it may include shutdown of cellular RNA and protein synthesis, cell fusion, release of lysosomal enzymes, changes in cell membrane permeability, diffuse changes in intracellular structures, presence of viral inclusion bodies and chromosomal aberrations.

The expression "component or part of said virus" refers to a component or part of the nucleic acid core of the virus or the surrounding protein coat.

"Cell culture" refers to cultures of cells such as transformed cells established in vitro. In particular, as used herein, "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single stem cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be completely identical to the stem cells or cultures, and the cell line referred to includes such variants. The term "cell lines" also includes immortalized cells.

The term "adjuvant" is used in its normal sense and defines an agent that can stimulate the immune system and increase the response to a vaccine without having any specific antigenic effect in itself.

Similarly, the term "emulsifier" is usually used to define a substance that stabilizes an emulsion, often a surfactant.

The term "detergent" defines another surfactant class that will interact chemically with both oil and water and thus stabilize the interface between oil or water droplets in a suspension.

Emulsions comprising water and oil are generally referred to either as water-in-oil emulsions, oil-in-water emulsions or water-in-oil-in-water emulsions. Whether an emulsion becomes a water-in-oil emulsion or an oil-in-water emulsion depends on the volume fraction of both phases and on the type of emulsifier. In general, emulsifiers and emulsifying particles tend to promote dispersion of the phase in which they do not dissolve very well. Proteins are dissolved, e.g. better in water than in oil and thus tend to form oil-in-water emulsions (ie, they promote the spreading of oil droplets through a continuous water phase).

In the present context, a "surfactant", also known as "a surfactant", is a wetting agent that lowers the surface tension in a liquid, allows easier spreading and lowers the interfacial tension between two liquids.

Finally, in the context of the present invention, a "polyvalent vaccine" (also known as multivalent vaccine) is used to define a combination of several antigens in one vaccine. Therefore, a polyvalent vaccine can protect against more than one disease. In contrast to a polyvalent vaccine, a "monovalent vaccine" is a vaccine that contains vaccine components directed against only one pathogen. In particular, a monovalent vaccine may contain only one antigen that protects against one particular disease.

According to a first aspect, the present invention provides an isolated salmonid alphavirus of subtype SAV3 which comprises a nucleic acid sequence which is at least 90% identical to the sequence indicated in SEQ ID NO: 1 and is further characterized by

a. a visible cytopathogenic effect during early passage in cell culture, and

b. the ability to induce mortality of at least 30% in a laboratory infection model, the model includes smoltification of Atlantic salmon according to standard procedures and post-smolt challenge by intraperitoneal injection with a SAV3 dose of at least 10<8>TCID50 per fish, such as at least 10<9>TCID50, at least 10<10>TCID50 or preferably at least 3.5 x 10<8>TCID50 per fish within one day of transfer to seawater at 12 <o>C, and

c. the ability to grow in vitro to a titer of at least 1x10<8 >TCID50/ml in the supernatant/growth medium cultures of CHH-1 cells or CHSE-214 cells.

Also described is a composition comprising a salmonid alphavirus or an antigenic and/or immunogenic material derived therefrom, combined with one or more components selected from the group consisting of:

a. a live, weakened, killed or inactivated bacterium,

b. a virus that is not salmonid alphavirus, said virus is preferably weakened or inactivated,

c. a mushroom,

d. a parasite and

e. an antigenic and/or immunogenic material derived from said bacterium in a), from said virus in b), from said fungus in c) or from said parasite in d).

Although compositions based on subunits of the virus, for example compositions comprising recombinant antigens, may be contemplated, it is preferred that the composition be prepared on the basis of cultures of the virus. For that purpose, it is desirable that the composition contains salmonid alphavirus at a titer of at least 7.5x10<8>TCID50/ml.

Although naturally occurring non-virulent strains of salmonid alphavirus and strains of the virus that have been attenuated in the laboratory may be contemplated, the composition preferably contains a salmonid alphavirus that has been either killed or inactivated.

In a similar way, the bacterial, fungal and parasite components of the vaccine are preferably killed or otherwise rendered incapable of infecting/attacking the fish.

Inactivation of the virus can be achieved by chemical or physical methods.

Chemical inactivation can be carried out by treating the viruses with, for example, but not limited to, treatment with enzymes, with formaldehyde, βpropiolactone or ethyleneimine or a derivative thereof, with organic solvent (e.g. halogenated hydrocarbon) and/or detergent, f .ex. Triton<® >or Tween<®>. Physiological inactivation can advantageously be carried out by subjecting the viruses to high-energy radiation such as UV light, gamma irradiation or X-rays. If necessary, the inactivating agent can be neutralized with thiosulphate. If necessary, the pH is then returned to about pH 7.

While formaldehyde inactivation has previously been considered suboptimal as a method of salmonid alphavirus inactivation, the present inventors have learned that formaldehyde inactivation is indeed a useful approach. In a preferred embodiment, the virus is inactivated by the addition of formaldehyde. In a preferred embodiment, the virus is inactivated by the addition of 1.5-2.5 g/kg formaldehyde and subsequent incubation for 12-96 hours, such as for 48-84 hours at a temperature from 13-17 ºC, such as from 14- 16 ºC.

The virus is incubated with formaldehyde for a period of 12 - 96 hours, such as from 24 - 96 hours, from 36 - 96 hours, from 36 - 72 hours, from 36 - 60 hours or such as from 48 - 96 hours, from 48 - 72 hours or something like from 48 - 60 hours. The inventors have found that when 2.0 g/kg of formaldehyde is used, a 48-hour incubation is usually sufficient to ensure satisfactory inactivation of the virus. However, to satisfy specific regulatory requirements, longer incubation periods may be preferred, such as an incubation period of at least 72 hours. The virus is preferably incubated with formaldehyde at a temperature of 13-17 <º>C, such as a temperature of 14-17 <º>C, 15-17 <º>C, 16-17 <º>C, 13-16 < º>C, 13-15 <º>C, 14-16 <º>C or such as a temperature of 14-15 <º>C.

When it comes to the composition's bacterial components, it should likewise be understood that several applicable methods of inactivation are available to the person skilled in the art. The bacteria can be inactivated by using a procedure that includes the addition of 3.5 - 4.5 g/kg formaldehyde and subsequent incubation for 1-2 hours at a temperature of 20 <º>C.

Although each of the viral, bacterial, fungal and parasitic components of the vaccine can be killed or inactivated before being combined with the other components, they can also be inactivated or killed after they have been added to the vaccine.

Said live weakened, killed or inactivated bacterium may be of a species which is a recognized fish pathogen. Accordingly, the bacterium is preferably selected from the group consisting of bacteria of the species Piscirickettsias spp., Aeromonas spp., Vibrio spp., Listonella spp., Moritella viscosa, Photobacterium damsela, Flavobacterium spp., Yersinia spp., Renibacterium spp., Streptococcus spp., Lactococcus spp., Leuconostoc spp., Bifidobacterium spp., Pediococcus spp., Brevibacterium spp., Edwarsiella spp., Francisella spp., Pseudomonas spp., Cytophaga spp., Nocardia spp., Haphnia spp. and Mycobacerium spp.

The bacteria can further be selected from the group consisting of Aeromonas spp., Vibrio spp., Flavobacterium spp., Haphnia spp., Piscirickettsia spp. and Moritella viscosa. Even more specifically, the bacterium is selected from the group consisting of Aeromonas hydrophila, Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum, Vibrio ordali, Flavobacterium columnaris, Haphnia sp., Piscirickettsia salmonis and Moritella viscosa.

The composition may further comprise killed or inactivated bacteria of one or more species selected from the group consisting of A. salmonicida, V. salmonicida, V. anguillarum and M. viscosa.

The composition can further comprise said bacteria of the species Vibrio ordali and/or Piscirickettsia salmonis.

The composition may also comprise bacteria of one or more species selected from the group consisting of Aeromonas hydrophila, Flavobacterium columnaris and Haphnia sp.

In certain cases, the composition is intended primarily for use in connection with fish farming of Atlantic salmon, especially in the North Atlantic. Bacteria of the species Aeromonas sp. especially A. salmonicida; Vibrio sp., especially V. salmonicida and V. anguillarum serotype O1 and O2; and Moritella viscosa present a challenge for fish farming in Norwegian waters, and therefore it may be preferred that the live weakened, killed or inactivated bacteria in the composition is selected from this special group of bacterial species.

In other cases, the composition is intended for use in South America, including Chile, and in these cases the bacterium is selected from the group of bacterial species consisting of Vibrio sp, especially Vibrio ordali and Piscirickettsia sp., especially Piscirickettsia salmonis.

In still other cases mainly intended for use in Southeast Asia, the bacterium is selected from the group consisting of Aeromonas sp., especially Aeromonas hydrophila, Flavobacterium sp., especially Flavobacterium columnaris and Haphnia sp.

In a similar way, said non-salmonid alpha virus is a recognized fish pathogen. Accordingly, it is preferably selected from the group consisting of: infectious pancreatic necrosis virus (IPNV), Viral Hemorrhagic Septicemia Virus (VHSV); Viral Hemorrhagic Septicemia Virus (VHSV); Infectious Hematopoietic Necrosis virus (IHNV); Pancreatic Necrosis Virus; Spring Viremia of Carp (SVC); Channel Catfish Virus (CCV); Infectious Salmon Anemia (ISA) virus; nodavirus; iridovirus, koi herpes virus; Heart and Skeletal Muscle Inflammation Virus (HSMV) and Cardiomyopathy Syndrome Virus.

Of these viruses, infectious pancreatic necrosis virus (IPNV) is particularly relevant in connection with Norwegian salmon farming. It is therefore preferred in certain cases that said virus, apart from salmonid alpha virus, is infectious pancreatic necrosis virus (IPNV).

The fungus is preferably selected from the group consisting of: Saprolegnia Sp., Branchiomyces sanguinis, Branchiomyces demigrans and Icthyophonus hoferi.

Furthermore, the parasite can be particularly selected from the group consisting of: Lepeophtheirus Sp., Caligus Sp. and Ichthyophthirius Sp.

For reasons of convenience, it is often preferred that approaches to control infections in fish populations target all or at least most of the potentially relevant pathogens. It may therefore be preferred that the composition comprises two or more viral, bacterial, fungal and/or parasitic components as defined above, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more components as defined above. In particular, compositions may comprise 2, 3, 4, 5, 6, 7, 8 or 10 viral, bacterial, fungal and/or parasitic components selected from the above.

It will be understood that the composition should contain antigens in amounts sufficient to elicit an immune response, preferably a protective response, upon injection of the composition into a fish. In general, it is preferred that the composition has a relatively high content of salmonid alphavirus since this is believed to improve the protective immune response when the composition is used for vaccination purposes. The present inventors have observed that it is generally possible to grow salmonid alphavirus to a satisfactory titer in cell culture and that the cost of production does not hinder the development of multivalent vaccines. Furthermore, the availability of isolates with a particular SAV3 subtype that exhibit excellent growth kinetics when grown in cell culture has allowed the present inventors to develop an extremely cost-effective viral vaccine component for pancreatic disease.

The vaccine may in particular comprise an amount of salmonid alpha virus antigen, such as an amount of SAV3 antigen, which corresponds to 1.5x10<8>-1x10<11 >TCID50/ml vaccine, such as from 1.5x10<8>-5x10<10> , from 1.5x10<8>-1x10<10>, from 1.5x10<8>-5x10<9>, from 1.5x10<8>-2x10<9>, from 1.5x10<8>-9x10< 8>, from 1.5x10<8>-8x10<8>, from 1.5x10<8>-7x10<8>, 1.5x10<8>-6x10<8>, 2.5x10<8>-1x10< 11>, 5x10<8>-1x10<11>, 7.5x10<9>-1x10<11>, 1x10<10>-1x10<11>, 2.5x10<10>-1x10<11>, 5x10<10 >-1x10<11>, 7.5x10<10>-1x10<11>, 2.5x10<8>-7.5x10<10>, 5x10<8>-5x10<10>, 5x10<8>-7x10< 9>, 5x10<8>-6x10<9>, 5x10<8>-5x10<9>, 5x10<8>-4x10<9>, 5x10<8>-3x10<9>, 5x10<8>-2x10< 9>, from 5x10<8>-1x10<9>, 6x10<8>-7x10<9>, 6x10<8>-6x10<9>, 6x10<8>-5x10<9>, 6x10<8>-4x10 <9>, 6x10<8>-3x10<9>, 6x10<8>-2x10<9>, 6x10<8>-1x10<9>, 7x10<8>-7x10<9>, 7x10<8>-6x10 <9>, 7x10<8>-5x10<9>, 7x10<8>4x10<9>, 7x10<8>-3x10<9>, 7x10<8>-2x10<9>, 7x10<8>-1x10< 9>, 8x10<8>-7x10<9>, 7.5x10<8>-1x10<10>, 8x10<8>-6x10<9>, 8x10<8>-5x10<9>, 8x10<8>- 4x10<9>, 8x10<8>-3x10<9>, 8x10<8>-2x1 0<9>, 8x10<8>-1x10<9>, 9x10<8>-7x10<9>, 9x10<8>-6x10<9>, 9x10<8>-5x10<9>, 9x10<8>- 4x10<9>, 9x10<8>-3x10<9>, or from 9x10<8>-2x10<9>, 1x10<9>-7.5x10<10>, 7.5x10<8>-5x10<10> , 1x10<9>-2.5x10<10>, 1x10<9>-1x10<10>, 1x10<9>-8x10<9>, 1x10<9>-5x10<9>, 1x10<9>-2, 5x10<9>, 2.5x10<9>-1x10<10 >or from 5x10<9>-7.5x10<9 >TCID50/ml vaccine.

As those skilled in the art will know, several methods and modes of administration of vaccines may be applicable in aquaculture. It is therefore to be understood that the vaccine may be formulated for administration by a route selected from the group consisting of: intraperitoneal injection, bath, immersion, intramuscular injection and oral administration or combinations thereof.

When vaccines are to be administered by injection, a relatively small dosage volume is generally required. Thus, it is desirable to formulate the composition so that each dose has a volume of 25-200 μl. More preferably, each dose has a volume of 40-120 μl, such as a volume of 90-110 μl or 45-55 μl. Currently, a dosage volume of 50 μl is preferred.

When the composition is formulated for injection, it may particularly be formulated for intraperitoneal injection into a teleostei including but not limited to salmonids, sea bass, bream, cod, snapper, flounder, catfish, yellowtail and tilapias. Preferably, the vaccine is formulated for injection into a salmonid fish.

When the composition is intended for injection, it may be formulated such that an aliquot of 25-200 μl, preferably an aliquot of 40-120 μl, such as an aliquot of 90-110 μl or 45-55 μl, and most preferably an aliquot of 50 μl, contains salmonid alphavirus in amounts corresponding to 0.75x10<7>-0.5x10<10 >TCID50, such as from 0.75x10<7>-2.5x10<9>, from 0.75x10<7>-0 .5x10<9>, from 0.75x10<7>-2.5x10<8>, from 0.75x10<7>-1x10<8>, from 0.75x10<7>-4.5x10<7>, from 0.75x10<7>-4x10<7>, from 0.75x10<7>-3.5x10<7>, 0.75x10<7>-3x10<7>, 1.25x10<7>-0.5x10< 10>, 2.5x10<7>-0.5x10<10>, 3.75x10<8>-0.5x10<10>, 0.5x10<9>-0.5x10<10>, 1.25x10<9 >-0.5x10<10>, 2.5x10<9>-0.5x10<10>, 3.75x10<9>-0.5x10<10>, 1.25x10<7>-3.75x10<9> , 2.5x10<7>-2.5x10<19>, 2.5x10<7>-3.75x10<8>, 2.5x10<7>-3x10<8>, 2.5x10<7>-2, 5x10<8>, 2.5x10<7>-2x10<8>, 2.5x10<7>-1.5x10<8>, 2.5x10<7>-1x10<8>, from 2.5x10<7> -0.5x10<8>, 3x10<7>-3.5x10<8>, 3x10<7>-3x10<8>, 3x10<7>-2.5x10<8>, 3x10<7>-2x10<8 >, 3x10<7>-1.5x10<8>, 3x10<7>-1x10<8>, 3x10<7>-0.5x10<8>, 3.5x10<7 >-3.5x10<8>, 3.5x10<7>-3x10<8>, 3.5x10<7>-2.5x10<8>, 3.5x10<7>-2x10<8>, 3.5x10 <7>-1.5x10<8>, 3.5x10<7>-1x10<8>, 3.5x10<7>-0.5x10<8>, 4x10<7>-3.5x10<8>, 3 ,75x10<7>-0.5x10<9>, 4x10<7>-3x10<8>, 4x10<7>-2.5x10<8>, 4x10<7>-2x10<8>, 4x10<7>- 1.5x10<8>, 4x10<7>-1x10<8>, 4x10<7>-0.5x10<8>, 4.5x10<7>-3.5x10<8>, 4.5x10<7>- 3x10<8>, 4.5x10<7>-2.5x10<8>, 4.5x10<7>-2x10<8>, 4.5x10<7>-1.5x10<8>, or from 4.5x10 <7>-1x10<8>, 0.5x10<8>-3.75x10<9>, 3.73x10<7>-2.5x10<9>, 0.5x10<8>-1.25x10<9> , 0.5x10<8>-0.5x10<9>, 0.5x10<8>-4x10<8>, 0.5x10<8>-2.5x10<8>, 0.5x10<8>-1, 25x10<9>,1.25x10<8>-0.5x10<9 >or to 2.5x10<8>-3.75x10<8 >TCID50.

As mentioned above, it is desirable that the composition contains salmonid alpha virus at a titer of at least 7.5x10<8>TCID50/ml. This preferably gives an amount of salmonid alphavirus of at least 3.75x10<7>TCID50/dose.

It should be understood that for the purposes of the present invention, the specified amounts of salmonid alphavirus are determined by titration of CHH cells as illustrated in the examples. An alternative approach is the titration of CHSE cells. As established in Example 3, CHH cells are approximately twice as sensitive to salmonid alphavirus as CHSE cells. Therefore, when determining virus titers for CHH cells, the values obtained are approximately twice as high as when determined by titrating CHSE cells.

In European patent EP 712,926 CHSE cells are used for titration. It is therefore reasonable to assume that the virus titers in the PD vaccine, which is commercially available from the holder of EP 712,926, are also determined by titration of CHSE cells. The stated antigen content of 10<7.2>TCID50/dose in the first generation monovalent vaccine from the patentee would therefore correspond to 3.2x10<7>TCID50/dose if determined using CHH cells for titration. Likewise, the stated antigen content of 10<7.5>TCID50/dose in the second generation vaccine would correspond to 6.4x10<7>TCID50/dose when titrated with CHH cells.

The composition preferably comprises inactivated salmonid alpha virus (SAV) at a titer of 5x10<8 >- 5x10<10 >TCID50/ml (which gives an antigen content of 2.5x10<7 >- 2.5x10<9 >TCID50/dose at a dosage volume of 50 μl).

Furthermore, it is preferred that said living weakened or killed bacteria are present in amounts corresponding to 0.2 x10<8>-2.5x10<9 >cells/ml (0.1 x10<7>-1.25x10<8 >cells /dose), such as 0.5 x10<8>-2.5x10<9 >cells/ml (0.25 x10<7>-1.25x10<8>cells/dose), 0.5 x10<8> -1x10<9 >cells/ml (0.25x10<7>-0.5x10<8>cells/dose), 0.5 x10<8>-0.5x10<9 >cells/ml (0.25 x10< 7>-0.25x10<8>cells/dose), 1x10<8>-5x10<9>cells/ml (0.5x10<7>-0.25x10<8>cells/dose), 2.5x10<8> -5x10<9 >cells/ml (1.25x10<7>-2.5x10<8 >cells/dose), 5x10<8>-5x10<9 >cells/ml (2.5x10<7>-2.5x10 <8>cells/dose), 7.5x10<8>-5x10<9>cells/ml (3.75x10<7>-2.5x10<8>cells/dose), 1-5x10<9>cells/ml (0.5-2.5x10<8>cells/ml), 2-5x10<9>cells/ml (1-2.5x10<8>cells/dose), 3-5x10<9>cells/ml (1 ,5-2.5x10<8>cells/dose) or 4-5x10<9>cells/ml (2-2.5x10<8>cells/dose). It is further preferred that the bacterial antigen is added in amounts that elicit protection resulting in a relative survival rate (RPS) greater than 70 for the relevant disease using a challenge model for the disease using intraperitoneal injection as a challenge.

The bacterium is usually present in amounts corresponding to 1x10<8>-8x10<9 >cells/ml. At a dosing volume of 50 μl, this corresponds to 5x10<6 >– 4x10<8 >cells/dose. Preferably, the bacterium is present in amounts corresponding to 0.9 - 5 x10<9 >cells/ml, corresponding to 0.45 - 2.5x10<8 >cells/dose at a dosage volume of 50 μl.

In cases mainly aimed at the Norwegian market, the live weakened or killed bacterium is present in quantities corresponding to ≥2.9x10<8 >cells/ml (≥1.45x10<7 >cells/dose) Aeromonas salmonicida, ≥7.4x10 <7 >cells/ml (≥3.7x10<6>cells/dose) Vibrio salmonicida, ≥ 4.3x10<8 >cells/ml (≥ 1.65x10<7>cells/dose) V. anguillarum serovar O2 α, ≥ 3.2x10<8 >cells/ml (≥1.6x10<7 >cells/dose) V. anguillarum serovar O1 and ≥ 2.7x10<7 >cells/ml (≥ 1.35x10<6 >cells/dose) Moritella viscosa . In further cases intended for the same purpose, the composition includes inactivated Aeromonas salmonicida, inactivated Vibrio.

Salmonicida, inactivated Vibrio anguillarum and inactivated Moritella viscosa, and each bacterial antigen is present in an amount corresponding to 0.9 -5x10<9 >cells/ml.

In cases currently intended for the Chilean market, the composition comprises: inactivated Vibrio ordali and inactivated Piscirickettsia salmonis, both in an amount corresponding to 0.9 -5x10<9 >cells/ml.

In cases currently intended for East Asia, the composition comprises inactivated Aeromonas hydrophila, inactivated Flavobacterium columnaris and inactivated Haphnia sp., each in an amount corresponding to 0.9 -5x10<9 >cells/ml.

The virus, except salmonid alpha virus, may preferably be present in amounts corresponding to ≥5x10<8 >PFU/ml. These amounts are particularly relevant when the virus is infectious pancreatic necrosis virus (IPNV). In specific cases, the virus is present in amounts corresponding to 1.0 - 5x10<9 >PFU/ml or 2-8 antigen units (AU)/ml in cases of infectious pancreatic necrosis virus (IPNV). For dosage sizes of 50 μl, this corresponds respectively to 0.5 – 2.5x10<8 >PFU/dose and 0.2-0.8 antigen units/dose.

As those skilled in the art will appreciate, it is possible to include one or more recombinantly produced antigens in the vaccines. In particular, the antigenic and/or immunogenic material derived from the salmonid alphavirus may be a material produced using recombinant techniques. In addition, the antigenic and/or immunogenic material derived from said bacteria in point a), from said virus in point b), from said fungus in point c) or from said parasite in point d) as defined above can be produced using recombinant techniques.

Such techniques, including techniques for cloning, expression, synthesis and purification of peptides and polypeptides, are of course available and well known to those skilled in the art.

Suitable recombinant antigens can be polypeptides cloned from fish pathogens or immunogenic parts of such polypeptides. An immunogenic portion of a polypeptide may comprise a T-cell and/or a B-cell epitope. T-cell epitopes, which are linear, can be identified by examining the effect of deletion mutations systematically introduced into the polypeptide sequence. B cell epitopes can be identified by analyzing the B cell responses to overlapping peptides covering the polypeptide sequence of interest. Those skilled in the art will be aware that immunogenic amino acid sequences will generally have a minimum length of 6 amino acids in a subsequent sequence. Longer amino acid sequences may of course be preferred, including amino acid sequences of at least 7, 8, 9, 10, 12, 15, 20, 30, 50, 100, 150 or 200 amino acids in a subsequent sequence.

When intended for the purpose of eliciting a protective immune response, the skilled person will recognize that the composition may further comprise an organic adjuvant and/or an inorganic adjuvant.

The organic adjuvant is preferably selected from the group consisting of: mineral oil, squalene 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane), virosomes, Montanide and CpG oligodeoxynucleotides . Other examples of frequently used adjuvants in fish and shellfish farming are muramyl dipeptides, lipopolysaccharides, several glucans and glycans, mineral oil and Carbopol<®>. Adjuvants such as interleukin and glycoproteins can also be used. A comprehensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review article by Jan Raa (1996), the contents of which are incorporated herein by reference in their entirety.

Useful inorganic adjuvants will be known to the skilled practitioner and are described by JC Aguilar and EG Rodriguez, 2007, Vaccine 25, 3752-3762, the contents of which are incorporated herein by reference in their entirety.

In particular, the inorganic adjuvant which is optionally included in the composition is selected from the group consisting of Al(OH)3 (aluminium hydroxide), Ca3(PO4)2 (calcium phosphate) and water-insoluble salts of aluminium, calcium, iron or zirconium.

The composition may further comprise a suitable pharmaceutical carrier. The vaccine may be formulated as a water-in-oil emulsion. The vaccine can also include a so-called "vehicle". A vehicle is an agent to which the antigen attaches without being covalently bound to it. Such vehicles are i.a. biodegradable nano/microparticles or capsules of PLGA (polylactide-co-glycolic acid), alginate or chitosan, liposomes, niosomes, micelles, multiple emulsions and macrosols, all known in the art. A special form of such a vehicle where the antigen is partially encapsulated in the vehicle is the so-called ISCOM (European patents EP 109,942, EP 180,564 and EP 242,380, the contents of which are incorporated herein by reference in their entirety).

In addition, the composition may comprise one or more suitable surface-active compounds, detergents and/or emulsifiers.

In particular, the detergent is selected from the group consisting of non-ionic detergents, cationic detergents and anionic detergents.

Those skilled in the art will recognize that esters of non-PEG-ylated or PEG-ylated sorbitan with fatty acids are examples of useful emulsifiers. As appears in the examples, the emulsifier can in particular be polysorbate or sorbitan oleate. More particularly, said detergent and/or emulsifier can be selected from the group consisting of polyoxyethylene (20) sorbitan monooleate (Tween<®>80), sorbitan monooleate (Span<®>80), cremophor, Tween® and Span®.

In certain cases, the composition is selected from the group consisting of a water-in-oil emulsion, a water-in-oil-in-water emulsion and an oil-in-water emulsion. Currently, the preferred formulation is a water-in-oil emulsion.

In specific cases, the composition comprises a mineral oil.

Certain cases relate to a vaccine comprising an oil phase and a water phase, each phase constituting from 20-80% (vol/vol) of the total volume of the vaccine.

In further cases, the vaccine comprises an oil phase which constitutes from 50-80% (vol/vol) of the total volume of the vaccine such as 55-75% (vol/vol), from 45-62% (vol/vol), from 45-60 % (vol/vol), from 50-70% (vol/vol), from 55-70% (vol/vol), from 50-65% (vol/vol), from 50-60% (vol/vol) from 55-60% (vol/vol) or from 57-60% (vol/vol) of the total volume of the vaccine.

In still further cases, the vaccine comprises an aqueous phase which constitutes from 20-55% (vol/vol) of the total volume of the vaccine such as 20-50% (vol/vol), from 20-45% (vol/vol), from 20- 40% (vol/vol), from 30-55% (vol/vol), from 30-52% (vol/vol), from 30-50% (vol/vol), from 37-55% (vol/vol ) from 37-50% (vol/vol) or from 37-45% (vol/vol) of the total volume of the vaccine.

In even further cases, the composition comprises an oil phase and a water phase, each phase constituting from 30-70% (vol/vol) of the total volume of the vaccine.

In still further cases, the composition comprises an oil phase comprising from 45-70% (vol/vol) of the total volume of the vaccine such as from 45-65% (vol/vol), from 45-62% (vol/vol), from 45 -60% (vol/vol), from 50-70% (vol/vol), from 55-70% (vol/vol), from 50-65% (vol/vol), from 50-60% (vol/ vol) from 55-60% (vol/vol) or from 57-60% (vol/vol) of the total volume of the vaccine.

In still further cases, the composition comprises an aqueous phase comprising from 30-55% (vol/vol) of the total volume of the vaccine such as from 30-50% (vol/vol), from 30-45% (vol/vol), from 30 -40% (vol/vol), from 35-55% (vol/vol), from 35-52% (vol/vol), from 35-50% (vol/vol), from 37-55% (vol/ vol) from 37-50% (vol/vol) or from 37-45% (vol/vol) of the total volume of the vaccine.

In further cases, the composition comprises an oil phase which constitutes from 50-80% (vol/vol) of the total volume of the vaccine such as from 55-75% (vol/vol), from 45-62% (vol/vol), from 45- 60% (vol/vol), from 50-70% (vol/vol), from 55-70% (vol/vol), from 50-65% (vol/vol), from 50-60% (vol/vol ) from 55-60% (vol/vol) or from 57-60% (vol/vol) of the total volume of the vaccine.

In still further cases, the composition comprises an aqueous phase comprising from 20-55% (vol/vol) of the total volume of the vaccine such as from 20-50% (vol/vol), from 30-45% (vol/vol), from 20 -40% (vol/vol), from 30-55% (vol/vol), from 30-52% (vol/vol), from 30-50% (vol/vol), from 37-55% (vol/ vol) from 37-50% (vol/vol) or from 37-45% (vol/vol) of the total volume of the vaccine.

It should be understood that the salmonid alphavirus in the composition may be a virus of any of the currently known SAV subtypes, SAV1, SAV 2, SAV 3, SAV4, SAV5 and SAV6 as defined above. The present inventors have observed that previously described strains of salmonid alphavirus are effective when used as an antigenic component in polyvalent vaccines and provide good protection against subsequent infection.

Representative isolates of SAV1 and SAV 2 subtypes are described in the subject: A patent deposit of SAV1 has previously been made under the Budapest Convention at ECACC under deposit number V94090731. The deposited SAV1 isolate corresponds to the isolate described in Nelson et al. 1995 and the isolate provided in EP 712,926.

Preferably, the salmonid alphavirus in the composition is a salmonid alphavirus subtype 3 (SAV3) virus. It is most preferred that the salmonid alphavirus in the composition is selected from the group consisting of the virus strains deposited under the Budapest Convention at the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 0JG UK on 12 December 2007 under deposit numbers 07121201, 07121202 and 07121203.

Related genotypic characteristics:

In further embodiments, the virus according to the first aspect is a strain or isolate with genotypic characteristics related to or similar to those in the deposited strains indicated above, in particular any of the deposited SAV3 strains.

Although the genome organizations of all characterized SAVs are similar, the nucleotide sequence similarity between SAV3 and SAV1 is only 91.6%, and between SAV3 and SAV2 92.9% (Hodneland et al. 2005). If the comparison is limited to specific genes, for example the non-structural protein nsP3, the similarity between subtypes is as low as 81.5% (Weston et al. 2005).

In particular embodiments, the salmonid alphavirus used in connection with the present invention comprises a nucleic acid sequence that is at least 90%, such as 95%, 97% or 99% identical to a single one of the sequences indicated in SEQ ID NO: 1.

SEQ ID NO: 1

aagaagtgca ccagattttc caccaccccg aagaagtccg ccccctacct cgttgacgtg tacgacgctc tgccgatttc tgtagagatt agcaccgttg taacatgcaa cgacaatcag tgcacagtga aggtgccacc cggtaccaca gtgaaattcg ataagaagtg caagagcgct gcccaagcga ccgttacctt taccagcgac tcccagacgt ttacgtgtga ggagccggtt ctgacggccg ccagtatcac ccagggcaag ccgcacctta gatcatctat gttgcccagc ggaggcaagg aagtgaaggc gaggatccca ttcccgttcc cgccagagac cgcgacc;

SEQ ID NO: 2:

aagaaatgta ccagattttc cactactcca aaaaagtccg cactatacct cgttgatgtg tacgacgctc tgccgatttc cgtagagatt agcaccgtcg taacatgcaa cgatagccag tgcacagtga gggtgccacc cggcaccaca gtgaaattcg acaaaaaatg caagagcgct gcctcggcga ccgtcacttt caccagcgac tcccagacgt ttacgtgcga ggagccggtc ctaacggctg ccagtatcac ccagggcaag ccacacctca gatcggcaat gttgcctagt ggaggcaagg aagtgaaagc aaggatcccg ttcccgttcc cgccggaaac tgcaact;

SEQ ID NO: 3

aagaaatgca ccaggttttc caccaccccg aagaagtccg cgctctatct cgttgatgtg tatgatgctc tgccgatttc tgtagagatc agcaccgtgg tgacatgcaa cgaaagacag tgcacagtga gggtgccacc cggtaccaca gtgaaattcg ataagaagtg caagaacgtt gccaaagaga ccgtcacctt caccagcgac tcccagacgt ttacgtgcga ggagccggtc ctaacggccg ccagcatcac ccagggcaag ccgcacctta gatcgtcaat gttgcccagc ggaggcaaag aggtgaaagc gaggattcca ttcccgttcc cgccagagac tgcgact.

In further embodiments, the salmonid alphavirus used in connection with the present invention comprises a nucleic acid sequence that is at least 80%, such as 85%, 90%, 95%, 98% or 99% identical to the sequence set forth in a single of SEQ ID NO: 4.

SEQ ID NO: 4

gttacgccag cggcatcatt ggcgggcagc gtccacggcc atagtgtacg cagcgcccct gccatgaggg ccgccagcac aggtgccaga agcgtgcgca gtgtccagtc cggttcagcc gggcatagaa ctgacgtcgc cagcgtcgcc ggctcagcgg ggctgcctag agggctaaca cgggaccagt tcggcgccgt gagagctagg gcccgcaggg acttagagct ggagggatca gagcatggca gtcagactag cttccgttcc ggctcgctga tggtggagag caccgctagt ggctacagcc aacgttctga cgatcaggac acgggctcc

SEQ ID NO: 5:

gtcgcgccag cggcatcatt ggcgggcagc gtccacagcc atagtgtgcg cagcgcccct gccattctga gggccgccag cacaggagcc agaagcgtgc gcagtgtcca gtccggctta accgggcaca gagatgatgc cgttagcgtc gccggttcgg tgagacagcc cagtgggccg cccagcagcg tgagcacgcc cgccgcgcct agagggctaa cacgggaaca gttcggcgcc gtaagagcta gggcccgcag ggacctagag ttggagggac cggagcatgg cagccaggcc agcttccgtt ccggctcgct ggtggtgggg agcaccgcca gtagctacag ccaacgtcct gacgaccagg acacgggctc t;

SEQ ID NO: 6:

gttgtgccgg cgacatcgtc gacgggcagc gtccacagcc gcagtagcgc ccctgctatt ttgagggccg ccagcgcggg tgccagaagc gtgcgcagcg cccaacccgg ccccgccggg catagagcgg gcgccttcag cgtcgctggc tcggtgagac agcccagcgg gccgcctagt agcgtgagca cgcccgccgc gaccagaggg ctgacacggg accagtttga cgtcgtgaga gctagggccc gtaggaactt ggaaccggag gggtcggagc atggcagcca agccagcttc cgctccggct cgctgacggt ggggagctct gctagtagct acagccaacg ttccgacgat caggacacgg gcacc

In preferred embodiments of the present invention which relate to viruses of the SAV3 variant, it shall be understood that the virus comprises a nucleic acid sequence which is at least 98%, 99% or 99.5% identical to the sequence indicated in SEQ ID NO: 1.

For the purposes of the present invention, a suitable test to determine whether a virus isolate has genotypic characteristics related to or similar to the deposited strains above is given in Example 2: one skilled in the art will appreciate that in preferred embodiments, the salmonid alphavirus of the invention is a virus, which is positive in the SAV reverse transcriptase quantitative PCR (RT-QPCR) identity test as described in Example 2, particularly when using a set of primers designed to amplify part of a salmonid alphavirus glycoprotein E2 nucleotide sequence, such as the primers indicated in SEQ ID NO: 7 and 8.

Related phenotypic characteristics:

In still further cases, the virus is a strain or isolate with phenotypic characteristics related to or similar to those of the deposited strains, particularly any of the deposited SAV3 strains.

Phenotypic differences exist between isolates within the three SAV groups, as well as between groups (Christie et al. Dis. Aquat. Org. 75: 13-22, 2007, Hodneland et al. 2005, Taksdal et al.): I a study by Taksdal et al. all Atlantic salmon and 76% of rainbow trout infected with viruses of the SAV3 subtype had severe pancreatic lesions in the late/regenerative phase. This was in contrast to reports from Irish and Scottish cases where fish infected with SAV1 usually had normal, probably healed, exocrine pancreatic tissue. A further characteristic feature of pancreatic disease caused by the SAV3 subtype is the presence of numerous cells in the kidney containing cytoplasmic eosinophilic granules (EG). In Taksdal's studies, EG-containing cells were found in 40% of rainbow trout and in 64% of Atlantic trout. In addition, it is characteristic that rainbow trout and Atlantic salmon are equally sensitive to infection with SAV3, although rainbow trout appear to be slightly resistant to infection with SAV1. Finally, the heart appears to recover earlier after infections with SAV3 compared to infections with SAV1.

According to further embodiments, the virus included in the composition according to the invention is capable of inducing mortality of at least such as at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or preferably at least 60% in a laboratory challenge model, the model includes smoltification of Atlantic salmon according to standard procedures and challenge of post-smolt by intraperitoneal injection with a SAV3 dose of at least 10<8>TCID50 per fish, such as at least 10<9>TCID50, at least 10<10>TCID50 or preferably at least 3.5 x 10<8>TCID50 per fish within one day of transfer to seawater (25 ‰) at 12 <o>C.

The new variant of salmonid alphavirus subtype SAV3 according to the invention is characterized by the ability to grow to high titers in vitro in cultures of host cells. Thus, according to the invention, this virus may be able to grow to a titer in the supernatant/growth medium of at least 1x10<8>, 5x10<8>, 1x10<9>, 5x10<9>, 1x10<10 > and 5x10<10 > or such as at least 1x10<11 >TCID50/ml when cultured using host cells selected from the group consisting of CHH-1 cells or CHSE-214 cells.

In particular, such high virus titres according to the invention can be achieved when:

i) Cells are cultured using host cells that have been seeded at a density of 0.1-1x10<5 >cells cm<-2>;

ii) The host cells are cultured for 4-6 days prior to virus infection;

iii) The host cells are cultured to a density of 0.1-1.0 x10<6 >cells cm<-2 >at the time of infection with said virus isolate;

iv) The host cells are grown in a growth medium comprising EMEM (EBSS) + 10% fetal bovine serum (FBS) 2 mM L-glutamine 1% non-essential amino acids (NEAA) 0.1% gentamicin and v) The infected cells are grown at a temperature of 15 ºC for a period of 10-14 days.

It will further be understood that the SAV1, SAV3, SAV4, SAV5 or SAV6 virus included in the composition is a virus which, if not attenuated or inactivated, is capable of causing the symptoms associated with fish pancreatic disease. General symptoms of fish pancreatic disease have been reviewed in McLoughlin and Graham, Journal of Fish Disease 2007, 30, 511-531, the contents of which are incorporated herein in their entirety. The symptoms include:

i) Death/mortality

ii) Lack of appetite

iii) Impaired swimming skills

iv) Lethargy

v) Pancreatic necrosis

vi) Cardiomyopathy and skeletal myopathy, including esophageal muscle lesions

vii) Renal lesions, affected kidneys containing numerous interstitial cells filled with eosinophilic material

viii) Focal gliosis in the brain.

In general, the course of the disease as a result of infections with fish pancreatic disease virus can be characterized as comprising an acute phase and a late/chronic phase, where each phase is associated with different histopathological symptoms. In particular, the virus provided may be capable of causing at least one of the following histopathological symptoms in the acute phase:

i) destruction of the main part of pancreatic acinar tissue and a variable inflammatory response ranging from no inflammation to moderate inflammation;

ii) fibrosis of periacinar tissue.

The virus provided may also be capable of causing at least one of the following histopathological symptoms in the late/chronic phase.

i) significant loss of pancreatic acinar tissue with or without fibroplasia of periacinar tissue;

ii) multifocal cardiomyocytic necrosis, affected cells have a shrunken, deeply eosinophilic cytoplasm and pyknotic nuclei.

In contrast to infections with SAV1 and -3, infections with SAV2 in rainbow trout have been reported to cause lower mortality, but mortality of up to 22% has been reported (Boucher and Baudin 1994). However, sleeping sickness can cause the fish to lie on their side at the bottom of tanks or water channels. If disturbed, the fish swims a little and then returns to the bottom. This sign is primarily due to extensive necrosis in red skeletal muscle.

Macroscopic lesions do not occur, but occasionally secondary ulcers on the skin or petechiae on the pyloric organ. Necrosis of red skeletal muscle can be found, and histopathological examination can reveal inflammation in the exocrine pancreas and heart. In the individual fish, these lesions can be present with or without any behavioral changes.

It will be understood that the SAV2 virus, which may be included in the composition, is a virus which, if not attenuated or inactivated, is capable of causing the symptoms associated with sleeping sickness in fish.

It should further be understood that the virus according to the invention can have a visible cytopathogenic effect during early passage in cell culture, such as during the first, second, third or fourth passage on a culture with CHSE cells. As mentioned, the present inventors have identified a new variant of salmonid alphavirus subtype SAV3. This variant of the virus showed a visible cytopathogenic effect during the first passage in cell culture. While previous isolates of salmonid alphavirus have not had a visible cytopathogenic effect until they are exposed to the in vitro culture conditions, the ability to cause a cytopathogenic effect without having been passed on a cell line can therefore be used as a further characteristic of the virus according to the present invention.

It should also be understood that the deposited viruses can mutate or otherwise change their characteristics, for example when they are passed on host cells in vitro (JD Watson et al., 1987). Those skilled in the art will appreciate that replication of RNA virus genomes is accompanied by very high mutation rates (Watson et al., 1987). This is due to a lack of proofreading activity in RNA virus polymerases, which leads to a constant generation of new genetic variants, for example during virus propagation (Elena and Sanjuán, 2005). Domingo and Holland (1997) also conclude that different constellations of mutations can be associated with a similar biological behavior. Salmonid alphavirus used for the purpose of the present invention can therefore be a strain that can be obtained by introducing one or more substitutions, omissions and/or additions of nucleotides in the genome of a single one of the deposited strains mentioned above. In other words, the salmonid alphavirus used for the purposes of the present invention may be a genetic variant of any of the above-mentioned deposited strains.

During the screening of new SAV 3 isolates from outbreaks in Norwegian salmon farms, the inventors have discovered a variant of the virus with characteristics that differ significantly from those previously described. The finding that this variant of the SAV3 subtype showed visible cytopathogenic effect during its first passage in cell culture, and produced lethality when injected into Atlantic salmon, was therefore highly surprising. In addition, it is very encouraging that vaccines based on antigenic material from this variant of the virus provide excellent protection against subsequent infection with salmonid alphavirus, also when the vaccine is given as a polyvalent vaccine comprising additional antigens. In addition, this newly discovered variant of the virus has good growth characteristics when grown in vitro on cultures of host cells.

It will be understood that the cultures of salmonid alpha virus used are essentially free of other viral or microbial material. In particular, contamination of virus isolates with IPNV is a source of concern since fish infected with salmonid alphavirus are often also infected with IPNV. During the isolation of the virus, it may therefore be necessary to add an antibody directed against IPNV to eliminate the IPNV virus from isolates or cultures of the virus. In cultures of virus on host cells, this antibody will inhibit infection of the host cells with IPNV.

In a specific and currently preferred case, which is currently intended for use in Northern Europe, including Norway, the composition includes:

i) inactivated Salmonid alphavirus, such as subtype SAV3 in an amount corresponding to 7.5x10<8 >- 5x10<9 >TCID50/ml (to preferably give 3.75x10<7 >– 2.5x10<8 >TCID50/dose at a dosing volume of 50 μl), preferably 1x10<9 >- 5x10<9 >TCID50/ml (to preferably give 0.5x10<8 >– 2.5x10<8 >TCID50/dose at a dosing volume of 50 μl),

ii) inactivated Aeromonas salmonicida in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose),

iii) inactivated Vibrio. salmonicida in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosage volume of 50 μl),

iv) inactivated Vibrio anguillarum in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosage volume of 50 μl),

v) inactivated Moritella viscosa in an amount corresponding to 0.5 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosing volume of 50 μl),

vi) inactivated infectious pancreatic necrosis virus (IPNV) in an amount corresponding to 1.0-5.0x10<9 >PFU/ml live virus (to preferably provide 0.5 – 2.5x10<8 >PFU/dose at a dosage volume of 50 μl) or 2-8 antigen units (AU)/ml (to preferably give 0.2-0.8 antigen units/dose at a dosing volume of 50 μl),

vii) an adjuvant, preferably mineral oil; and

viii) a detergent and/or emulsifier.

In alternative cases currently intended for use in South America, including Chile, the composition includes:

i) inactivated salmonid alphavirus, such as subtype SAV3 in an amount corresponding to 7.5x10<8 >- 5x10<9 >TCID50/ml (to preferably give 3.75x10<7 >– 2.5x10<8 >TCID50/dose at a dosing volume of 50 μl), preferably 1x10<9 >- 5x10<9 >TCID50/ml (to preferably give 0.5x10<8 >– 2.5x10<8 >TCID50/dose at a dosing volume of 50 μl),

ii) inactivated Vibrio ordali in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosage volume of 50 μl),

iii) inactivated Piscirickettsia salmonis in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosing volume of 50 μl),

iv) inactivated infectious pancreatic necrosis virus (IPNV) in an amount corresponding to 1.0-5.0x10<9 >PFU/ml live virus (to preferably provide 0.5 – 2.5x10<8 >PFU/dose at a dosage volume of 50 μl) or 2-8 antigen units (AU)/ml (to preferably give 0.2-0.8 antigen units/dose at a dosing volume of 50 μl),

v) an adjuvant, preferably mineral oil; and

vi) a detergent and/or emulsifier.

In other alternative cases currently intended for use in Southeast Asia, the composition includes:

i) inactivated salmonid alphavirus, such as subtype SAV3 in an amount corresponding to 7.5x10<8 >- 5x10<9 >TCID50/ml (to preferably give 2.5x10<7 >– 2.5x10<8 >TCID50/dose at a dosing volume of 50 μl), preferably 1x10<9 >- 5x10<9 >TCID50/ml (to preferably give 0.5x10<8 >– 2.5x10<8 >TCID50/dose at a dosing volume of 50 μl),

ii) inactivated Aeromonas hydrophila in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosing volume of 50 μl),

iii) inactivated Flavobacterium columnaris in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosage volume of 50 μl),

iv) inactivated Haphnia sp. in an amount corresponding to 0.9 -5x10<9 >cells/ml (to preferably give 0.45 – 2.5x10<8 >cells/dose at a dosage volume of 50 μl),

v) inactivated infectious pancreatic necrosis virus (IPNV) in an amount corresponding to 1.0-5.0x10<9 >PFU/ml (to preferably give 0.5 – 2.5x10<8 >PFU/dose at a dosage volume of 50 μl) or 2-8 antigen units (AU)/ml (to preferably give 0.2-0.8 antigen units/dose at a dosing volume of 50 μl),

vi) an adjuvant, preferably mineral oil; and

vii) a detergent and/or emulsifier.

Dosage form

A dosage form of a composition is also described.

Although other dosage forms may be contemplated, including solid dosage forms based on fish feed, the dosage form is preferably a liquid dosage form, more preferably a liquid dosage form for injection such as intraperitoneal injection. When the dosage form is intended for injection, it preferably has a volume of 25-200 μl, more preferably a volume of 40-120 μl, such as a volume of 90-110 μl or 45-55 μl. Currently, a dosing volume of 50 μl is most preferred for practical reasons.

In connection with the dosage form, the same preferences apply with regard to dosage volume and antigen content as described in connection with the composition.

In particular, the dosage form preferably comprises salmonid alphavirus in amounts corresponding to 0.75 x 10<7>-0.5 x 10<10 >TCID50/dose, such as from 0.75 x 10<7>-2.5 x 10<9> , from 0.75 x 10<7>-0.5 x 10<9>, from 0.75 x 10<7>-2.5 x 10<8>, from 0.75 x 10<7>-1 x 10<8>, from 0.75 x 10<7>-4.5 x 10<7>, from 0.75 x 10<7>-4 x 10<7>, from 0.75 x 10<7 >-3.5 x 10<7>, 0.75 x 10<7>-3 x 10<7>, 1.25 x 10<7>-0.5 x 10<10>, 2.5 x 10 <7>-0.5 x 10<10>, 3.75 x 10<8>-0.5 x 10<10>, 0.5 x 10<9>-0.5 x 10<10>, 1 .25 x 10<9>-0.5 x 10<10>, 2.5 x 10<9>-0.5 x 10<10>, 3.75 x 10<9>-0.5 x 10< 10>, 1.25 x 10<7>-3.75 x 10<9>, 2.5 x 10<7>-2.5 x 10<19>, 2.5 x 10<7>-3, 75 x 10<8>, 2.5 x 10<7>-3 x 10<8>, 2.5 x 10<7>-2.5 x 10<8>, 2.5 x 10<7>- 2 x 10<8>, 2.5 x 10<7>-1.5 x 10<8>, 2.5 x 10<7>-1 x 10<8>, from 2.5 x 10<7> -0.5 x 10<8>, 3 x 10<7>-3.5 x 10<8>, 3 x 10<7>-3 x 10<8>, 3 x 10<7>-2.5 x 10<8>, 3 x 10<7>-2 x 10<8>, 3 x 10<7>-1.5 x 10<8>, 3 x 10<7>-1 x 10<8>, 3 x 10<7>-0.5 x 10<8>, 3.5 x 10<7>-3.5 x 10<8>, 3.5 x 10<7>-3 x 10<8>, 3.5 x 10<7>-2.5 x 10<8>, 3.5 x 10<7>-2 x 10<8>, 3.5 x 10<7>-1.5 x 10<8>, 3.5 x 10<7>-1 x 10<8>, 3.5 x 10<7>-0.5 x 10<8>, 4 x 10<7>-3.5 x 10<8>, 3.75 x 10<7>-0, 5 x 10<9>, 4 x 10<7>-3 x 10<8>, 4 x 10<7>-2.5 x 10<8>, 4 x 10<7>-2 x 10<8> , 4 x 10<7>-1.5 x 10<8>, 4 x 10<7>-1 x 10<8>, 4 x 10<7>-0.5 x 10<8>, 4.5 x 10<7>-3.5 x 10<8>, 4.5 x 10<7>-3 x 10<8>, 4.5 x 10<7>-2.5 x 10<8>, 4 .5 x 10<7>-2 x 10<8>, 4.5 x 10<7>-1.5 x 10<8>, or from 4.5 x 10<7>-1 x 10<8> , 0.5 x 10<8>-3.75 x 10<9>, 3.73 x 10<7>-2.5 x 10<9>, 0.5 x 10<8>-0.5 x 10<9>, 0.5 x 10<8>-4 x 10<8>, 0.5 x 10<8>-2.5 x 10<8>, 0.5 x 10<8>-1, 25 x 10<9>, 1.25 x 10<8>-0.5 x 10<9 >or from 2.5 x 10<8>-3.75 x 10<8 >TCID50/dose.

Vaccine

It is further described a vaccine comprising a composition or a dosage form as defined above. It should be understood that all features described above will also apply to the vaccine.

As those skilled in the art will know, several methods and modes of administration of vaccines may be applicable in aquaculture. It is therefore to be understood that the vaccine may be formulated for administration by a route selected from the group consisting of: intraperitoneal injection, bath, immersion, intramuscular injection and oral administration or combinations thereof.

For reasons of convenience, it is preferred that the vaccine is administered to pairs of salmon, preferably pairs of 10-60 grams. As those skilled in the art will appreciate, however, the vaccine can also be administered to salmon in other stages, including all saltwater stages.

As shown in the examples, the compositions provide extremely effective protection against salmonid alphavirus infection when tested in laboratory challenge experiments where the vaccinated fish are challenged with high titers of highly virulent SAV3 isolates. In the area where the fish are exposed to much lower titers of the virus, it is expected that the protective effect of the vaccines provided is even higher than that shown in the present examples.

Medical application/procedure for prophylaxis

A use of inactivated salmonid alpha virus in the preparation of a drug has also been described, e.g. as an immunological composition such as a vaccine, and/or dosage form for administration simultaneously or in combination with one or more antigenic components selected from the group consisting of:

a) a killed bacterium,

(b) an inactivated virus other than a salmonid alphavirus;

c) a mushroom,

d) a parasite and

e) an antigenic and/or immunogenic material derived from said bacterium in a), from said virus in b), from said fungus in c) or from said parasite in d).

It should therefore be understood that the aforementioned dosage form preferably contains inactivated salmonid alphaviruses in an amount corresponding to at least 3.75 x 10<7 >TCID50/dose. Likewise, it is preferred that said vaccine / immunological composition contains inactivated salmonid alphavirus in an amount corresponding to at least 7.5 x 10<8 >TCID50/ml.

It is also described a use of an inactivated salmonid alphavirus in the preparation of a vaccine or an immunological composition compatible with other immunological products, in which the vaccine comprises inactivated salmonid alphavirus in an amount corresponding to at least 7.5 x 10<8 >TCID50/ ml (equivalent to 3.75 x 10<7 >TCID50/dose with a dosing volume of 50 μl).

The composition as described above is also for use in medicine. In particular, the composition may be for use in preventing or reducing the incidence of fish pancreatic disease or, in other words, for use in preventing or reducing the incidence of salmonid alphavirus infection. Also disclosed is a vaccine according to any of the features described above for use in preventing or reducing the incidence of fish pancreatic disease.

Other aspects of the invention provide a vaccine as described above for use in preventing or reducing the incidence of infection with salmonid alphavirus. It is being considered whether a polyvalent vaccine based on a composition comprising a combination of viral, bacterial, fungal and/or parasitic antigens as described above may provide a successful approach to control infections with any of the currently known groups of salmonid alphavirus, SAV1, SAV2, SAV3, SAV4, SAV5 and SAV6.

Further descriptions relate to the use of a composition as described above for the preparation of a drug/vaccine to prevent or reduce the occurrence of infection with salmonid alphavirus.

Even further descriptions relate to the use of a composition as described above for the production of a drug/vaccine to prevent or reduce the occurrence of fish pancreatic disease and/or sleeping sickness. In yet another description, a method is provided for reducing the occurrence of and/or treating or preventing infection with salmonid alpha virus in a fish population, where the method comprises administering a composition, vaccine or dosage form as defined above to the fish.

Procedure for making a vaccine

A method for producing a composition and/or vaccine and/or dosage form as defined above is also described. The method comprises combining a preferably attenuated or inactivated salmonid alphavirus or an antigenic and/or immunogenic material derived therefrom with one or more components selected from the group consisting of:

a. a live, weakened, killed or inactivated bacterium,

b. a virus that is not salmonid alphavirus, said virus is preferably weakened or inactivated,

c. a mushroom,

d. a parasite and

e. an antigenic and/or immunogenic material derived from said bacterium in a), from said virus in b), from said fungus in c) or from said parasite in d).

Preferably, the amount of salmonid alphavirus is adjusted to provide a composition or vaccine comprising at least 7.5 x 10<8 >TCID50/ml of inactivated salmonid alphavirus or a dosage form comprising at least 2.5 x 10<7 >TCID50/dose.

In this context, the viral, bacterial, fungal and parasitic antigen components as specified above are preferably dependent on the fish pathogens that are widespread in the area where the vaccine is to be used.

Since the salmonid alphavirus must be grown on cultures of host cells, the method according to the description can further include

a. establishment of a culture with host cells;

b. infecting the host cells with an isolate of a virus of salmonid alphavirus and c. culturing infected cells under conditions which allow said virus to reach a titer in the supernatant/growth medium of at least 7.5 x 10<8 >TCID50/ml, such as at least 1 x 10<9>, 5 x 10<9>, 1 x 10<10 >or at least 5 x 10<10 >TCID50/ml.

In the method, the host cells are grown either on a solid medium or as suspension cultures.

To provide optimal growth conditions, the host cells are preferably grown at a pH between 6.5 and 8.5, such as between 7.0 and 8.0, between 7.2 and 8.0, between 7 and 7.5 or between 7, 2 and 7.5.

The process has been developed for the specific purpose of producing the salmonid alphavirus vaccine component in large quantities. Therefore, the process in the currently preferred features is adapted to produce salmonid alphavirus material in batches of 25-2000 liters, such as 25-1000 liters, 50-2000 liters, 50-1000 liters or 50-500 liters.

It is a further feature of the method that in certain cases the host cells are seeded at a density of 0.1-1.5 x 10<5 >cells cm<-2>, 0.2-1.5 x 10<5 >cells cm <-2>, 0.2-1.25 x 10<5 >cells cm<-2>, 0.2-1 x 10<5 >cells cm<-2 >or 0.2-1 x 10<5 >cells cm<-2 >when grown on a solid support.

According to further description, the host cells are cultured for 3-8 days prior to virus infection, such as from 4-8 days, 4-7 days or from 4-6 days.

In even further descriptions, said host cells are grown to a density of 8 x 10<5 >cells cm<-2 >at the time of infection with said virus isolate.

In particular, the host cells can be cultured to a density of from 5 x 10<5 >cells cm<-2 >to 5 x 10<6 >cells cm<-2 >at the time of infection with said virus isolate.

As regards the growth medium, it may be preferred that the host cells are cultured in a growth medium selected from the group consisting of EMEM (EBSS) + 10% fetal bovine serum (FBS) 2 mM L-glutamine 1% non-essential amino acids (NEAA) 0.1% gentamicin .

After infecting the cells, the infected cells are preferably cultured at a temperature of 12-16 ºC, such as at a temperature of 13-16 ºC, 13-15 ºC, 14-16º C or 14-15 ºC, and for a period of 6 -20 days, such as a period of 8-20 days, 8-18 days, 8-16 days, 9-20 days, 9-18 days, 9-16 days, 10-20 days, 10-18 days, 10 -16 days or 10-14 days.

Furthermore, it may be preferred that the infected cells are cultured until 30% of the host cells are lysed or detached, such as until 40%, 50%, 60%, 70%, 80% or 90% of the host cells are lysed or have a cytopathogenic effect consisting of rounded cells.

As explained above, it may be preferred that said salmonid alphavirus is a virus of the SAV3 subtype.

As regards the host cells, these are preferably selected from the group consisting of cells derived from heart tissue from young chum salmon (Onchorhynchus keta), cells derived from Chinook salmon (Oncorhynchus tshawytscha) embryos.

More particularly, it is preferred to use host cells selected from the group consisting of CHH-1 cells and CHSE-214 cells, CHH-1 cells are available from ATCC under accession number CRL-1680, CHSE-214 cells are available from ATCC , deposit number CRL 1681 and from ECACC under deposit number 91041114.

Bacterial antigens for use in polyvalent vaccines can be prepared using conventional techniques, e.g. by growing the bacteria in tryptic soy broth with 2% NaCl at 12-15 <o>C, or in brain/heart infusion broth with 3% NaCl at 12-15 <o>C (Atlas, RM,2004, Handbook of Microbiological Media , third edition, CRC press, pages 1820 and 246).

As for infectious pancreatic necrosis virus (IPNV), this virus can be propagated for the purpose of producing a vaccine in CHSE-214 cells according to Dobos P and Roberts TE, 1982, Canadian Journal of Microbiology, 29, 377 – 384.

The cultivation of these representative pathogens is also described at http://www.lgcpromochem-atcc.com/.

A method for improving a polyvalent vaccine is also described. The method comprises including (an immunogenic amount of) an attenuated or inactivated salmonid alphavirus in the polyvalent vaccine. In one description, the polyvalent vaccine comprises one or more components selected from the group consisting of:

a. a live, weakened, inactivated or killed bacterium,

b. a virus that is not salmonid alphavirus, said virus is preferably weakened or inactivated,

c. a mushroom,

d. a parasite and

e. an antigenic and/or immunogenic material derived from said bacterium in a), from said virus in b), from said fungus in c) or from said parasite in d).

In connection with this method, the same preferences apply in relation to dosage volume and antigen content as described in connection with the composition.

An example of such a polyvalent vaccine that could be advantageously improved by including an antigenic preparation of salmonid alphavirus is AJ 6-2 already marketed by the present applicant.

It should be noted that embodiments and features described in connection with one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application are hereby incorporated by reference in their entirety.

The invention will now be described in further detail in the following non-limiting examples.ju

Examples

Example 1: Isolation of PD virus from fish with clinical PD infection (ALV-405, ALV 406 and ALV-407)

Materials

Organ material

Hearts were taken from fish with clinical symptoms of pancreatic disease in commercial fish farms. The hearts were kept cold at 4<º>C.

Cell cultures

● CHH-1, 145th passage

● CHSE-214, 36th passage

● GF-1, 4th passage

Cells were seeded two to three days before use.

Growth media

● CHH-1 growth medium: EMEM (Sigma M7278), 10% FBS (Sigma F-3885), 1% L-glutamine (Sigma G-7513), 1% NEAA (Sigma M-7145), 0.1% gentamicin (Sigma G1397).

● CHSE-214 growth medium: EMEM (Sigma M7278), 10% FBS (Sigma F-3885), 1% L-glutamine (Sigma G-7513), 0.1% gentamicin (Sigma G1397). ● GF-1 growth medium: L-15 (Sigma L-5520 lot), 10% FBS (Sigma F-3885), 1% L-glutamine (Sigma G-7513), 0.1% gentamicin (Sigma G1397).

Filter:

Minisart plus (Sartorius #17829)

Anti-IPNV antibodies: Rabbit anti-IPNV antiserum (CP54/1996)

Hearts from Atlantic salmon were homogenized using a porcelain mortar and quartz sand with some added medium (about 8-20 ml). The homogenate was centrifuged at 2000 x g for 10 min at 4<o>C before filtering through a 0.45 μm syringe filter. The homogenates were distributed in cryobottles and frozen at –80 <o>C.

Samples of some of the organ homogenates were mixed 1:100 with anti-IPNV antiserum and incubated for two hours at 15°C. Then 50-200 μl homogenate with or without added anti-IPNV antiserum was inoculated into the different cell cultures (50-90% confluent) in 75/175 cm<2 >Nunc cell flasks with 15/50 ml of appropriate medium according to table 1.

When passing the virus in new cell cultures, 1 ml of supernatant was transferred to new cell culture bottles.

Table 1

,++,+++ denote the degrees of cytopathogenic effect observed in the cells

X X dpi; x x days after infection when cytopathogenic effect was observed in the cells X : X x 10 <x >TCID50 Titration result of supernatant from the specific bottle.

Conclusions

● SAV3 was isolated from hearts collected from Atlantic salmon with pancreatic disease ● Cytopathogenic effect was visible in the cell cultures from passage 1

● The passage 1 SAV3 isolates had a titer of up to 1.3 x 10<8 >TCID50 ● SAV3 can be isolated in passage 1 on several different cell lines

Example 2: Identity test for SAV3, and test for the absence of IPNV in SAV3 isolates

To confirm the identity of different isolates of SAV3 and to test them for the absence of infectious pancreatic necrosis virus (IPNV), a reverse transcription quantitative PCR (RTQPCR) procedure using primers specific for SAV and IPNV was applied.

Materials

Virus isolates: ALV-405 p5 and p6, ALV 407 p1, ALV 408 p1, ALV 4091pA and 1pB (two separate isolates). Positive control: IPNV ALV 103.

RNA Isolation: QIAamp Viral RNA Mini Spin Protocol

Reverse transcription and QPCR: Brilliant® II QPCR and QRT-PCR reagents from Stratagene.

Table 2: Primers

RT-PCR conditions:

IPNV test: 50 <o>C, 30 min – 95 <o>C, 10 min –(95 <o>C, 30 sec - 57 <o>C, 60 sec - 72 <o>C, 30 sec) *40

SAV3 test: 50 <o>C, 30 min - 95 <o>C, 10 min –(95 <o>C, 30 sec - 57 <o>C, 60 sec -72 <o>C, 30 sec) *40

Results: All SAV3 isolates were positive in SAV RT-QPCR and negative in IPNV RT-QPCR. These tests show that all the SAV3 isolates are indeed SAV and not contaminated with IPNV.

Example 3: Titration of salmon pancreas disease virus using different cell lines.

SAV3 was titrated on two different cell lines; CHH and CHSE as described in Examples 4 and 5 respectively. The TCID50 values obtained with the two cell lines are shown in Table 3. The CHH cell line is approx. twice as sensitive to the virus as the CHSE cell line.

Table 3:

Example 4: Cultivation of SAV3 in CHH-1 cell culture flasks at high cell density.

Intention

The aim was to evaluate potential PD yields for different virus isolates in CHH-1 cell culture flasks at high cell density.

Materials and methods

Cell cultures and growth media

3 x 175 cm<2 >flasks, CHH-1, 160th passage with 100 x 10<6 >cells per bottle (5.7 x 10<5 >C/cm<2>)

CHH-1 growth medium: EMEM (Sigma M7278), 10% FBS (Invitrogen), 1% L-glutamine (Sigma G-7513), 1% NEAA (Sigma M-7145), 0.1% gentamicin (Sigma G1397) .

Virus/infectious material

ALV-405, p6, titer: 5.01 x 10<8 >TCID50/ml

ALV-407, p1, titer: 3.41 x 10<7 >TCID50/ml

ALV-407, p4, titer: 4.14 x 10<7 >TCID50/ml

Table 4. Cell cultures and experimental data.

Just before infection, the growth medium in the bottles was replaced with 50 ml of fresh growth medium, and then the respective infection materials were added (Table 1). All bottles were incubated at 15 °C with tightly closed caps.

Sampling, titration and microscopic observations

Microscopic observations and titration of samples were carried out on 7, 10, 13 and

18 days pi on CHH-1.

Results/discussion

Microscopic observations

Loosening and rounding of cells appeared approx. a week pi in all bottles. At the end of the observation period (18 days pi), flask 2 had the most prominent CPE with 80% of cells detached while flasks 1 and 3 had 20% and 40% detached cells, respectively.

Titration results

The three isolates/passages all had titres of 10<9 >TCID50/ml or higher.

Surprisingly, yields were highest in the bottle infected with the 1st passage of ALV-407 (Figure 1, ALV-407 p2), but this bottle also had the most prominent CPE. The results confirm that CHH-1 cells produce high SAV3 yields also from virus strains with few passages. Results are shown in Figure 1.

Conclusions

● The highest yields were 1.78 x 10<9 >TCID50/ml in a sample obtained 13 days after inoculation with ALV-407 p1 at MOI 0.2.

● Production of SAV3 with few passages in CHH-1 cells gives a good yield of SAV3.

Example 5: Cultivation of SAV3 in CHSE cells.

Intention

The aim was to evaluate the potential SAV3 yields of different virus strains in CHSE-214 cell cultures.

Materials and methods

Cell culture and growth medium

● CHSE-214: 19th passage with approx. 80% confluence.

● CHSE-214 growth medium: EMEM (Sigma M7278), 10% FBS (Invitrogen), 1% L-glutamine (Sigma G-7513), 0.1% gentamicin (Sigma G1397).

Virus strain

● ALV-404, 8p.

Table 5. Cell cultures and experimental data. All bottles were 75 cm<2>, had 20 ml of growth medium and were inoculated with 100 µl of the virus strain.

The bottles were cooled to 15 °C before being infected without changing the medium. All bottles were incubated at 15 °C with tightly closed caps.

Samples of the virus inoculums were titrated in 96-well plates.

Microscopic observation and titration of samples from all bottles were performed 6, 12, 15, 20 and 27 days pi.

Results/discussion

Microscopic observations

The infection (lysis and detachment of cells) progressed rapidly in the CHSE cultures, with more than 90% lysis at 12 days pi. Samples from the two bottles were pooled and titrated in 96-well plates on days 6, 12, 15, 20 and 27 days post-infection.

The average values from the two parallel bottles are shown in Figure 2.

This experiment shows that CHSE-214 is a suitable cell line for the production of SAV3 since high virus titers are produced from low-passage strains of SAV3.

Example 6: Vaccination of juvenile salmon (parr) using a polyvalent vaccine including SAV3

The present example presents a multivalent vaccine against pancreatic disease based on salmonid alphavirus subtype 3 antigenic material and shows excellent protection in an experimental challenge model with high mortality in the unvaccinated control group.

Table 6: Main features of the experiment

Atlantic juvenile salmon (parr) with an average weight of 25.7 grams were vaccinated with 0.1 ml of a polyvalent water-in-oil vaccine. The vaccine contained inactivated antigen from the following pathogens:

Table 7: Vaccine components

All components were inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15 <º>C.

Table 8: Labeling and vaccination

After smoltification, the fish were divided into two tanks containing seawater (25 ‰) and challenged by injection of 2nd passage, recently produced ALV-407. The challenge dose was 5.36 x 10<8 >TCID50 per fish. In the challenge tanks, mortality reached 86% in the control group in tank 1 and 66% in tank 2, while no mortality was recorded in the groups vaccinated with the polyvalent vaccine that protects against salmonid pancreatic disease. Results are shown in Figure 3. This experiment shows that a polyvalent vaccine can indeed protect against salmonid pancreatic disease.

Example 7: Dose/response – vaccination with monovalent and polyvalent SAV3 vaccine.

The present example presents a dose-response experiment with monovalent and multivalent vaccine against pancreatic disease.

Table 9: Main features of the experiment

Atlantic juvenile salmon (parr) with an average weight of 25.7 grams were vaccinated with 0.1 ml of a polyvalent water-in-oil vaccine. The vaccine contained inactivated antigen from the following pathogens:

Table 10: Vaccine components, multivalent vaccines.

Table 11: Vaccine components, monovalent vaccines.

All components were inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15 <º>C.

Labeling and vaccination

For each group, 40 fish were tagged by clipping the fin and vaccinated with 0.05 ml vaccine or 0.05 ml PBS and left for immunization and smoltification. After smoltification, the fish were divided into two tanks containing seawater (25 ‰) and challenged by injection of 2nd passage, recently produced ALV-407. The challenge dose was 6.5 x 10<8 >TCID50 per fish.

Results:

Table 12:

In the challenge, the polyvalent vaccine provided better protection than the monovalent vaccine at the lowest antigen doses. This experiment shows that a polyvalent vaccine with a PD antigen content of 1 x 10<9 >TCID50 /ml can effectively protect against salmonid pancreatic disease when given at doses of 0.5 x 10<8 >TCID50 /dose. Since the vaccine provides some protection against subsequent infection, even when given at doses of 10<6 >TCID50, it is reasonable to conclude that a satisfactory degree of protection can also be obtained when the vaccine is administered at doses containing somewhat less than 0.5 x 10<8 >TCID50 /dose.

Example 8: Vaccination of juvenile salmon (parr) using a polyvalent vaccine comprising SAV1

The present example presents a multivalent vaccine against pancreatic disease containing SAV1 antigenic material. The vaccine shows good protection in an experimental challenge model with high mortality in the unvaccinated control group.

An isolate of salmonid alphavirus subtype 1 is isolated from an outbreak of pancreatic disease in Atlantic salmon in Scotland. SAV1 antigenic material is prepared from a culture of this virus grown on CHSE cells according to a similar procedure as described in previous examples.

A vaccine containing the following components is prepared:

Table 13: Vaccine components

The SAV1 virus was inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15<º>C.

The vaccine is administered to juvenile Atlantic salmon (parr). The fish are vaccinated and marked, and vaccinated fish and control fish are then smoltified as described in the previous example.

After smoltification, the fish are distributed in tanks containing seawater (25 ‰) and challenged by injection of the 2nd passage, recently produced SAV3. The challenge dose is 3-6 x 10<8 >TCID50 per fish. This experiment shows that polyvalent vaccine comprising salmonid alphavirus type 1 antigenic material can indeed protect against salmonid pancreatic disease SAV3.

Example 9: Vaccination of juvenile salmon (parr) using a vaccine comprising SAV1

The present example presents a multivalent vaccine against pancreatic disease containing SAV1 antigenic material. The vaccine shows good protection in an experimental challenge model with high mortality in the unvaccinated control group.

An isolate of salmonid alphavirus subtype 1 is isolated from an outbreak of pancreatic disease in Atlantic salmon in Scotland. SAV1 antigenic material was prepared from a culture of this virus according to a similar procedure as described in the previous examples.

A vaccine containing the following components was prepared:

Table 14: Vaccine components

Vaccine A:

Vaccine B and C:

The SAV1 virus was inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15<º>C. The vaccines were formulated as water-in-oil emulsions using standard methods.

60 fish were marked by fin clipping and vaccinated with 0.05 ml vaccine or 0.05 ml PBS and left for immunization and smoltification as described in the previous example.

After smoltification, the fish were distributed in a tank containing seawater (25 ‰) and challenged by injection of the 2nd passage, recently produced SAV3. The challenge dose was 3.01 x 10<8 >TCID50 per fish.

Table 15: Results.

Mortality in the control group reached 70%, while mortality in the group vaccinated with a polyvalent vaccine comprising 2 x 10<9 >TCID50 /ml inactivated SAV1 reached 3.3%, giving a relative survival rate of 95.2. This experiment shows that a polyvalent vaccine comprising salmonid alphavirus type 1 antigenic material can indeed protect against salmonid pancreatic disease. In this experiment, the SAV1 antigen dose is doubled compared to the monovalent SAV1 vaccine C, and there is an increase in observed RPS. This suggests that the same antigen dose can be used in polyvalent PD vaccines that elicit the same protection as monovalent PD vaccines. This is in contrast to conventional knowledge in the field of fish vaccinology.

Example 10: Vaccination of juvenile salmon (parr) using a polyvalent vaccine including SAV2

The present example presents a multivalent vaccine against pancreatic disease containing SAV2 antigenic material. The vaccine shows good protection in an experimental challenge model with high mortality in the unvaccinated control group.

An isolate of salmonid alphavirus subtype 2 is isolated from an outbreak of sleeping sickness in rainbow trout reared in freshwater in France. SAV2 antigenic material is prepared from a culture of this virus grown on CHSE cells according to a similar procedure as described in previous examples.

A vaccine containing the following components is prepared:

Table 16: Vaccine components

The SAV2 virus is inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15 <º>C.

The vaccine is administered to juvenile Atlantic salmon (parr). The fish are vaccinated and marked, and vaccinated fish and control fish are then smoltified as described in the previous example.

After smoltification, the fish are distributed in tanks containing seawater (25 ‰) and challenged by injection of the 2nd passage, recently produced SAV3. The challenge dose is 3-6 x 10<8 >TCID50 per fish. This experiment shows that a polyvalent vaccine comprising salmonid alphavirus type 2 antigenic material can indeed protect against subsequent infection with salmonid alphavirus SAV3.

Example 11: Vaccination of juvenile salmon (parr) using a polyvalent vaccine including SAV3

The present example presents a multivalent vaccine against pancreatic disease containing SAV3 antigenic material. The vaccine shows good protection in an experimental challenge model with high mortality in the unvaccinated control group.

An isolate of salmonid alphavirus subtype 3 is isolated from an outbreak of pancreatic disease in Atlantic salmon in Scotland. SAV3 antigenic material is prepared from a culture of this virus grown on CHSE cells according to a similar procedure as described in previous examples.

A vaccine containing the following components is prepared:

Table 17: Vaccine components

The SAV3 virus was inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15<º>C. Bacterial antigens were inactivated by the addition of 4 g/kg formaldehyde and subsequent incubation for 1.5 hours at a temperature of 20 <º>C.

A water-in-oil emulsion is made according to standard methods.

The vaccine is administered to juvenile Atlantic salmon (parr). The fish are vaccinated and marked, and vaccinated fish and control fish are then smoltified as described in the previous example.

After smoltification, the fish are distributed in tanks containing seawater (25 ‰) and challenged by injection of the 2nd passage, recently produced SAV3. The challenge dose is 3-6 x 10<8 >TCID50 per fish. This experiment shows that a polyvalent vaccine comprising salmonid alphavirus antigenic material and several bacterial antigens can indeed protect against salmonid pancreatic disease.

Example 12: Vaccination of juvenile salmon (parr) using a polyvalent vaccine including SAV3

The present example presents a multivalent vaccine against pancreatic disease containing SAV3 antigenic material. The vaccine shows good protection in an experimental challenge model with high mortality in the unvaccinated control group.

An isolate of salmonid alphavirus subtype 3 is isolated from an outbreak of pancreatic disease in Atlantic salmon in Scotland. SAV3 antigenic material is prepared from a culture of this virus grown on CHSE cells according to a similar procedure as described in previous examples.

A vaccine containing the following components is produced:

Table 18: Vaccine components

SAV3 virus was inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15<º>C. Bacterial antigens were inactivated by the addition of 4 g/kg formaldehyde and subsequent incubation for 1.5 hours at a temperature of 20 <º>C.

A water in oil emulsion is prepared according to standard methods.

The vaccine is administered to juvenile Atlantic salmon (parr). The fish are vaccinated and marked, and vaccinated fish and control fish are then smoltified as described in the previous example.

After smoltification, the fish are distributed in tanks containing seawater (25 ‰) and challenged by injection of the 2nd passage, recently produced SAV3. The challenge dose is 3-6 x 10<8 >TCID50 per fish. This experiment shows that a polyvalent vaccine comprising salmonid alphavirus antigenic material and several bacterial antigens can indeed protect against salmonid pancreatic disease.

Example 13: Vaccination of juvenile salmon (parr) using a polyvalent vaccine including SAV3

The present example presents a multivalent vaccine against pancreatic disease containing SAV3 antigenic material. The vaccine shows good protection in an experimental challenge model with high mortality in the unvaccinated control group.

An isolate of salmonid alphavirus subtype 3 is isolated from an outbreak of pancreatic disease in Atlantic salmon in Scotland. SAV3 antigenic material is prepared from a culture of this virus grown on CHSE cells according to a similar procedure as described in previous examples.

A vaccine containing the following components is prepared:

Table 19: Vaccine components

The SAV3 virus is inactivated by the addition of 2.0 g/kg formaldehyde and subsequent incubation for 72 hours at a temperature of 15 <º>C. Bacterial antigens were inactivated by the addition of 4 g/kg formaldehyde and subsequent incubation for 1.5 hours at a temperature of 20 <º>C.

A water-in-oil emulsion is made according to standard methods.

The vaccine is administered to juvenile Atlantic salmon (parr). The fish are vaccinated and marked, and vaccinated fish and control fish are then smoltified as described in the previous example.

After smoltification, the fish are distributed in tanks containing seawater (25 ‰) and challenged by injection of the 2nd passage, recently produced SAV3. The challenge dose is 3-6 x 10<8 >TCID50 per fish. This experiment shows that a polyvalent vaccine comprising salmonid alphavirus antigenic material and Montanid ISA 736 B can indeed protect against salmonid pancreatic disease.

Receipts for deposits of microorganisms:

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Virus Salmon Alphavirus 3 ALV-407 Deposit Reference 07121202

has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on

12 December 2007

Health Protection Agency, Porton Down and

European Collection of Ceil Cultures

This document certifies that

Virus Salmon alphavirus 3 ALV-409 Deposit Reference 07121203

has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on

12 December 2007

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Virus HEART & SKELETAL MUSCLE INFLAMMATION VIRUS (HSMI) Deposit Reference 04050401 has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977, with the European Collection of Cell Cultures on

04 May 2004

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Virus Cardiomyopathy syndrome virus (CMS) Deposit Reference 07032902

has been accepted as a patent deposit, in accordance with

The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on

29 March 2007

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Bacteria AL10005

Deposit Reference 06050901

has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on 09 May 2006

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Bacteria AL10007

Deposit Reference 06050902

has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on 09 May 2006

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Bacteria AL 10008

Deposit Reference 06050903

has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on 09 May 2006

Health Protection Agency, Porton Down and

European Collection of Cell Cultures

This document certifies that

Bacteria Piscirickettsia salmonis Deposit Reference 07032110

has been accepted as a patent deposit, in accordance with The Budapest Treaty of 1977,

with the European Collection of Cell Cultures on 21 March 2007

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WO2007031572 A1

Claims (10)

New patent requirements 1. An isolated salmonid alphavirus of subtype SAV3 comprising a nucleic acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 1 and is further characterized by a. a visible cytopathogenic effect during early passage in cell culture, and b. the ability to induce mortality of at least 30% in a laboratory infection model, the model includes smoltification of Atlantic salmon according to standard procedures and post-smolt challenge by intraperitoneal injection with a SAV3 dose of at least 10<8>TCID50 per fish, such as at least 10<9>TCID50, at least 10<10>TCID50 or preferably at least 3.5 x 10<8>TCID50 per fish within one day of transfer to seawater at 12 <o>C, and c. the ability to grow in vitro to a titer of at least 1x10<8 >TCID50/ml in the supernatant/growth medium cultures of CHH-1 cells or CHSE-214 cells. 2. Isolated virus according to claim 1, wherein the virus has a visible cytopathogenic effect during the first, second, third or fourth passage on a culture of CHSE cells. 3. Isolated virus according to claim 1 or 2, where said titer of at least 1x10<8 >TCID50/ml is achieved when: i) Cells are cultured using host cells that have been seeded at a density of 0.1-1x10<5 >cells cm<-2>; ii) The host cells are cultured for 4-6 days prior to virus infection; iii) The host cells are cultured to a density of 0.1-1.0 x10<6 >cells cm<-2 >at the time of infection with said virus isolate; iv) The host cells are cultured in a growth medium comprising EMEM (EBSS) + 10% fetal bovine serum (FBS) 2 mM L-glutamine 1% non-essential amino acids (NEAA) 0.1% gentamicin and v) The infected cells are cultured at a temperature of 15 ºC for a period of 10-14 days. 4. Isolated virus according to any one of claims 1-3, wherein the virus comprises a nucleic acid sequence which is at least 80% identical to the sequence indicated in SEQ ID NO: 4. 5. Isolated virus according to any one of claims 1-4, wherein the virus comprises a nucleic acid sequence which is at least 98% identical to the sequence indicated in SEQ ID NO: 1. 6. Isolated virus according to any one of claims 1-5, wherein viruses are positive in a SAV reverse transcriptase quantitative PCR identity test using a set of primers such as the primers set forth in SEQ ID NO: 7 and 8 and the conditions 50 <o >C, 30 min - 95 <o>C, 10 min –(95 <o>C, 30 sec - 57 <o>C, 60 sec -72 <o>C, 30 sec)*40. 7. Isolated virus according to any one of claims 1-6, wherein the virus is a genetic variant of a single of the virus strains deposited under the Budapest Convention at the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire ( UK), SP4 0JG UK 12 December 2007 under deposit numbers 07121201 and 07121202. 8. Use of an inactivated salmonid alpha virus of subtype SAV3, where the virus comprises a nucleic acid sequence that is at least 90% identical to the sequence indicated in SEQ ID NO: 1 and is further characterized by a. a visible cytopathogenic effect during early passage in cell culture, and b. the ability to induce mortality of at least 30% in a laboratory infection model, the model includes smoltification of Atlantic salmon according to standard procedures and post-smolt challenge by intraperitoneal injection with a dose of SAV3 of at least 10<8>TCID50 per fish, such as at least 10<9>TCID50, at least 10<10>TCID50 or preferably at least 3.5 x 10<8>TCID50 per fish within one day of transfer to seawater at 12 <o>C, and c. the ability to grow in vitro to a titer of at least 1x10<8 >TCID50/ml in the supernatant/growth medium cultures of CHH-1 cells or CHSE-214 cells; in the manufacture of a vaccine which is for the prevention or reduction of the incidence of infection by salmonid alphavirus. 9. Use according to claim 8, in which the salmonid alpha virus is inactivated by the addition of formaldehyde. 10. Use according to claim 9, in which the salmonid alphavirus is inactivated using a procedure comprising the addition of 1.5-2.5 g/kg, formaldehyde and subsequent incubation for 48-96 hours at a temperature of 14-17 <º >C.