WO2023285976A1

WO2023285976A1 - Composition and uses thereof

Info

Publication number: WO2023285976A1
Application number: PCT/IB2022/056440
Authority: WO
Inventors: Ian Thompson
Original assignee: Stonehaven Incubate AG
Priority date: 2021-07-13
Filing date: 2022-07-13
Publication date: 2023-01-19
Also published as: CA3226725A1; EP4370151A1

Abstract

The present disclosure relates generally to polynucleotide compositions encoding a salmon alphavirus-like particle and uses thereof, wherein the composition comprises (i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein and (ii) a second nucleic acid sequence encoding one or more structural proteins, wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

Description

COMPOSITION AND USES THEREOF

FIELD

[0001] The present invention relates generally to a polynucleotide composition encoding an alphavirus-like particle capable of inducing an immune response against alphaviruses, and uses thereof.

BACKGROUND

[0002] Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive-sense RNA virus with a ~12 kb genome, belonging to the family Togaviridae, genus Alphavirus. First described in Scotland in 1976, SAV is known to be a serious pathogen for Atlantic salmon ( Salmo salar), rainbow trout ( Oncorhynchus mykiss) and potentially other salmonid species, where it is responsible for conditions such as pancreas disease (PD). A similar disease, termed “sleeping disease” (SD) and affecting rainbow trout has been described and widely reported in Europe. Comparative histopathological and genomic studies suggested both diseases are caused by very similar viruses (Boucher et al, (1996) J Fish Dis 19, 303- 310; Weston et al. (2002) J Virol 76, 6155-6163). Recently, SAV has been detected in wild- caught non-salmonid marine fish species, namely flatfish spp. in Europe.

[0003] There is some evidence to suggest that infected farmed salmonids are a reservoir and source of contamination of SAV. Both PD and SD are becoming more prevalent, with rising economic importance for aquaculture industries worldwide. Serious quarantine and biosecurity measures have been implemented, with SAV-free countries declining to import salmon products or livestock from regions that have not been declared SAV-free to contain transmission of the pathogen.

[0004] Early studies suggested that pre-exposed fish develop some resistance to re infection. Subsequent studies have demonstrated that inactivated virus vaccines, sub-unit protein vaccines and (most recently) DNA vaccines may provide protection against PD.

[0005] Vaccines against SAV are available, some of which have been reviewed by Deperasmska et al. (J Vet Res. 2018; 62(1): 1-6). However, many SAV vaccines have yet to be proven highly effective at reducing mortality and have generally failed to prevent pathological lesions in the pancreas and heart, or growth retardation. Indeed, despite the introduction of these vaccines and increased biosecurity measures, the number of recorded SAV outbreaks in the most affected country (Norway) have remained fairly constant (http://trination.org/wp-content/uploads/2019/06/Hilde-Sindre.pdf).

[0006] Therefore, there remains an urgent need for an effective vaccine for inducing protective immunity against SAV infection.

SUMMARY

[0007] In an aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises (i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein under the control of a first promoter and (ii) a second nucleic acid sequence encoding one or more structural proteins under the control of a second promoter, wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

[0008] The present disclosure also extends to a host cell comprising the polynucleotide composition described herein.

[0009] In another aspect disclosed herein, there is provided a method of producing a salmon alphavirus-like particle, the method comprising (a) culturing the host cell, as herein described, under conditions and for a period of time sufficient to allow expression of the salmon alphavirus-like particle by the host cell, and (b) optionally isolating the salmon alphavirus-like particle from the culture.

[0010] The present disclosure also extends to a salmon alphavirus-like particle encoded by the polynucleotide composition, as herein described.

[0011] Also disclosed herein is a salmon alphavirus-like particle produced by the methods described herein.

[0012] In another aspect disclosed herein, there is provided a method for treating or protecting against salmon alphavirus infection in a subject, the method comprising administering to a subject in need thereof the polynucleotide composition, the host cell, or the salmon alphavirus-like particle, as herein described.

[0013] The present disclosure also extends to a vaccine composition for use in treating or protecting against salmon alphavirus infection in a subject, the vaccine composition comprising the polynucleotide composition, the host cell, or the salmon alphavirus-like particle described in the preceding paragraphs.

[0014] The present disclosure also extends to use of the composition as described herein in the manufacture of a vaccine composition for treating or protecting against salmon alphavirus infection in a subject.

[0015] In another aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(ii) a second nucleic acid sequence encoding one or more structural proteins; and

(iii) at least one promoter; wherein at least one promoter regulates the independent expression of the first nucleic acid sequence and the second nucleic acid sequence, and wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

[0016] In a further aspect disclosed herein is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(iii) a bidirectional promoter; wherein the bidirectional promoter is situated between the first nucleic acid sequence and the second nucleic acid sequence; the first nucleic acid sequence and the second nucleic acid sequence are located on complementary strands of the polynucleotide; the first nucleic acid sequence and the second nucleic acid sequence are under the control of the bidirectional promoter; and the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

[0017] In another aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(ii) a second nucleic acid sequence encoding one or more structural proteins;

(iii) at least one promoter; and

(iv) a spacer sequence between the first nucleic acid sequence and the second nucleic acid sequence, wherein the spacer is capable of disrupting expression of a contiguous polypeptide comprising the capsid protein and the one or more structural proteins; wherein the at least one promoter regulates the expression of the first nucleic acid sequence and the second nucleic acid sequence, and wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

[0018] Further aspects and illustrative embodiments of the invention are also described in the detailed description below.

BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 shows the amino acid sequence of 28 different isolates of salmon alphavirus isotype 3 (SAV3). The amino acid sequences for the SAV subtype-3 structural polyprotein from 28 isolates were aligned using MUSCLE (Multiple Sequence Alignment tool from EMBL-EBI, using consensus threshold of >50%). The sequences are arranged according to earliest to most recent isolate disclosure. Sequence #4 in the alignment corresponds to SEQ ID NO:7 disclosed herein. The amino acid differences are highlighted in yellow. From this alignment, four unique sequences were generated (Variants 1-4; SEQ ID NOs: 8-11, respectively; see also Table 1). [0020] Figure 2 is an identity matrix showing the homology of two variant sequences (Variants 2 and 4; SEQ ID NOs:9 and 11, respectively) with the non-redundant data set of known SAV3 subtype sequences. Sequence #1 in the matrix corresponds to SEQ ID NO:7 disclosed herein.

[0021] Figure 3 shows the structural elements of the 3-db SAV3 nucleic acid construct corresponding to SEQ ID NO: 50.

[0022] Figure 4 shows the structural elements of the 6-CMV-CP-EFlα-ENV nucleic acid construct relative to SEQ ID NO:51.

[0023] Figure 5 shows the structural elements of the 7-CMV-ENV-EFlα-CP nucleic acid construct relative to SEQ ID NO:52.

[0024] Figure 6 shows the structural elements of the 8-CMV-CP-CMV-ENV nucleic acid construct relative to SEQ ID NO: 53.

[0025] Figure 7 shows the structural elements of the 9-CMV-CP-T2A-ENV nucleic acid construct relative to SEQ ID NO: 54.

[0026] Figure 8 shows the cumulative number of virus positive samples recorded during the study in which fish were administered saline (as negative controls) or doggybone DNA (dbDNA) vaccine Construct #3 (db-SAV3, under the control of a single CMV promoter; SEQ ID NO:50) or Construct #7 (db-CMV-ENV-EFlα-CP, under the control of a single CMV promoter; SEQ ID NO:52) at 2.5pg/fish. The data, represented as the cumulative number of virus positive samples over time, show that vaccination with a dual-promotor contruct (Construct #7) provided greater protection against virus challenge when compared to vaccination with a single promoter construct (Construct #3).

[0027] Figure 9 shows the level of viraemia represented as SAV RNA copies (logio) on Day 21 post-virus challenge in fish that had been treated with saline (as negative controls) or dbDNA vaccine. Constructs #3, #7 or #9 at 2.5pg/fish (Construct #9 is a single promoter CMV-CP-T2A-ENV construct; SEQ ID NO: 54). The data show that vaccination with a dual- promotor construct (Construct #7) reduced the level of viraemia post-virus challenge when compared to vaccination with either of single promoter Construct #3 or single promoter Construct #9. [0028] Figure 10 shows the level of viraetnia in heart tissue from fish administered saline (as negative controls) or dbDNA vaccine Construct #3 or Construct #7 at l.Opg/fish (A) and at 2.5pg/fish (B). The data are represented as SAV RNA copies (logio). Figure 10A shows that vaccination at l.Opg/fish with a dual-promotor conduct (Construct #7) provided a greater reduction in virus load in heart tissue post-virus challenge (~ 100-fold reduction compared to saline controls) when compared to vaccination with single promoter Construct #3 (~10-fold compared to saline controls). Figure 10B shows that vaccination at 2.5pg/fish with either single promoter Construct #3 or dual promotor Construct #7 reduced virus load in heart tissue post-virus challenge.

[0029] Figure 11 shows the level of viraemia (A; SAV RNA copies (logio)) and heart viral load (B; SAV RNA copies (logio)) in fish administered saline (as negative controls) or dbDNA vaccine Construct #6 or Construct #7 at 2.5pg/fish. Construct #6 with the arrangement db-CMV-CP-EFlα-ENV showed no efficacy in reducing viraemia (A) or heart viral load (B), whereas Construct #7 with the reverse arrangement db-CMV-ENV-EFlla-CP was effective at reducing viraemia and heart viral load.

DETAILED DESCRIPTION

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice the present invention. Practitioners may refer to Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, N.Y., and Ausubel et al. (1999) Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York, Murphy et al. (1995) Virus Taxonomy Springer Verlag:79-87, for definitions and terms of the art and other methods known to the person skilled in the art.

[0031] As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 10% ( e.g , by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) to a reference quantity, level, value, dimension, size, or amount. [0032] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0033] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of'. Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0034] As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an organism" includes one organism, as well as two or more organism; and so forth.

[0035] Nucleotide and amino acid sequences are referred to by sequence identifier numbers (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc. A summary of sequence identifiers is provided herein.

[0036] All sequence reference numbers (e.g. , GenBank ID, EMBL-Bank ID, DNA Data Bank of Japan (DDBK) ID, etc.) provided herein were current as at the filing date.

[0037] As used herein, the terms "encode," "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and / or translated, typically in a host cell, to produce the polypeptide or if it can be processed into a form that can be transcribed and / or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode," "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0038] The present disclosure is predicated, at least in part, on the inventors' surprising finding that a polynucleotide composition encoding a salmon alphavirus-like particle unexpectedly provides improved protection against salmon alphavirus infection, where the polynucleotide employs (i) one or more promoters to independently regulate expression of the salmon alphavirus capsid protein and expression of the salmon alphavirus structural proteins or (ii) where the polynucleotide comprises a spacer sequence (such as T2A) between the capsid and structural protein coding regions that is capable of disrupting the translation of the capsid and structural proteins as a single polypeptide sequence.

[0039] Thus, in an aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein under the control of a first promoter and (ii) a second nucleic acid sequence encoding one or more structural proteins under the control of a second promoter, wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

[0040] In another aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(iii) at least one promoter; wherein the at least one promoter regulates independent expression of the first nucleic acid sequence and the second nucleic acid sequence, and wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein. [0041] In another aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(ii) a second nucleic acid sequence encoding one or more structural proteins;

(iii) at least one promoter; and

Salmon alphavirus

[0042] As noted herein, salmonid alphavirus (SAV) is an enveloped, single-stranded, positive-sense RNA virus with a ~12 kb genome, belonging to the family Togaviridae, genus Alphavirus. The genomes of the reference strains of the viruses thought to cause PD and SD have been sequenced and compared demonstrating that these strains are subtypes of the same virus, as suggested by an earlier comparative histopathology study. Hereafter, the terms SAV and SPDV are used interchangeably, and understood to mean salmonid alphaviruses that underlie SPD and SD in salmonid species. Based on genomic analyses, at least six subtypes of SAV have been described that are the causative agents of significant diseases of farmed Atlantic salmon, rainbow trout and other salmonids (SAV1-6). In Europe, SAV1 causes PD in farmed Atlantic salmon in Ireland. SAV2 or SDV causes sleeping disease in England, France, Germany, Italy, Scotland and Spain. SAV3 or Norwegian salmon alphavirus is responsible for PD in Norway, exclusively. SAV4 consists of Atlantic salmon strains from Ireland, SAV5 consists of Scottish strains and SAV6 contains one virus, isolated from Atlantic salmon in Ireland. SAV2 has been divided into two subgroups named freshwater variant (SAV2 FW) and marine variant (SAV2 MW). Infections with SAV2 FW cause sleeping disease in freshwater-reared rainbow trout in England, Scotland, and mainland European countries. The marine variant of SAV2 (SAV2 MW) is responsible for pancreas disease in seawater-reared Atlantic salmon. The third subtype of salmonid alphavirus (SAV3) has been isolated from farmed Atlantic salmon in Norway, hence it is also named Norwegian salmonid alphavirus (NSAV). SAV3 causes pancreas disease in Atlantic salmon and sea-reared rainbow trout. It has been shown that SAV3 has genomic organisation identical to that of SAV1 and SAV2. Until 2010, SAV3 was the only subtype recognised in Norway but now SAV2 is also well established. Subtypes 4—6 of SAV (SAV4— 6) have been detected in Scotland and Ireland in connection with PD outbreaks and detected there along with SAV1.

[0043] The transmission vector for SAV has not been identified, but direct horizontal transmission has been demonstrated. It is not known whether aquatic invertebrates play a significant role in the epizootiology of the SAV-associated diseases. The main transmission route of salmonid alphavirus appears to be horizontal and via water contact, while there is suggestion that vertical transfer (parent to offspring) may be possible as well. All life stages are considered susceptible to infection with SAV. Transport of fish across borders or different waterways will likely increase the risk of spreading SAV (reviewed by Jansen et al. (2017) Journal of Fish Diseases 40(1): 141).

[0044] Clinical signs associated with PD include sudden inappetence, lethargy and an increased number of faecal casts in the cages, increased mortality and ill-thrift. The histopathological changes in fish affected by PD and SD primarily occur in the pancreas, heart, and skeletal muscles. From the six different SAV subtypes, SAV1 and SAV3 are particularly associated with pathology in the cardiovascular and muscular tissue of cultured salmon. Changes in the dermal bacterial microflora composition (characterised most prominently by the loss of Proteobacteria ) of Atlantic salmon has also been detected in response to infection with SAV3 which may render the fish more susceptible to secondary infections by opportunistic bacterial pathogens present in the environment or within the host indigenous microbial reservoir.

[0045] Sleeping disease (SD) is an infectious disease similar to pancreas disease; however, it affects rainbow trout reared in fresh water. SD is an increasing problem throughout Europe, causing high mortality and growth retardation of fish. The characteristic sign of sleeping disease is the unusual behaviour of affected fish, manifested in their laying on their side on the bottom of the tank, hence the name “sleeping” disease. Extensive necrosis of skeletal red muscles is considered to be the cause of this behaviour. This is followed by the characteristic development of histological lesions of the pancreas and heart.

[0046] Based on the field observations showing that fish surviving SAV infections were less susceptible to reinfection, it has been suggested that vaccination may be applied for control of PD and SD. A study has indicated that a vaccine containing a single subtype strain may protect against PD caused by different strains of SAV, independent of their subtype grouping (Graham et al. (2014) J Fish Dis 37: 683-691), although efficacious vaccines remain elusive.

Salmon alphavirus-like particle

[0047] The present disclosure provides a polynucleotide composition encoding a salmon alphavirus-like particle that can be used for treating, or for inducing protection against, SAV infection.

[0048] The terms "virus-like particle", "sub-viral particle", "VLP" and the like are used interchangeably herein to refer to anon-replicating (i.e., non-virulent) viral shell. Thus, VLP engender a protective immune response in the host without harming the host or host cell. In some embodiments, the VLP is derived entirely or partially from the viral proteins of an alphavirus. The VLP is generally composed of one or more viral proteins. Suitable viral proteins would be known to persons skilled in the art, illustrative examples of which include those referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from such proteins, including structural proteins as described herein. Suitable alphavirus capsid and other structural proteins would be known to persons skilled in the art.

[0049] By "particle-forming polypeptide" derived from a particular viral protein is meant a full-length or near full-length viral protein, as well as a fragment thereof, or a viral protein with internal deletions, insertions or substitutions, which retains the ability to form VLPs under conditions that favour VLP formation. The polypeptide may comprise the full- length sequence, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. The term “particle-forming polypeptide" therefore extends to deletions, insertions and substitutions to the polypeptide sequence, although it would be understood that such modified polypeptides suitably retain the ability to form a VLP. The term also extends to natural variations of the specified polypeptides, since variations in coat proteins often occur between viral isolates. The term also extends to deletions, additions and / or substitutions of the polypeptide sequence that do not naturally occur in the reference protein (i.e., in nature), as long as the modified polypeptide retains the ability to form a VLP. Suitable substitutions include those which are conservative in nature; that is, those that take place within a family of amino acids that are related in their side chains. For example, amino acids are generally divided into four families: (1) acidic- aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine, phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.

[0050] A salmon alphavirus-like particle (SAVLP) will typically comprise one or a plurality of virus structural proteins (or fragments or portions thereof) assembled into a molecule which has a quaternary structure that mimics or resembles the overall structure of a corresponding wild-type, native and / or authentic salmon alphavirus particle. Therefore, it will be appreciated that an SAVLP is morphologically similar to a corresponding authentic, wild-type and / or native salmon alphavirus particle since the SAVLP will typically have a similar conformation to native viral structural proteins. An SAVLP may be engineered or a product of recombinant technology and, more particularly, recombinant DNA technology, although without limitation thereto.

[0051] An SAVLP can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing SAVLP will be familiar to persons skilled in the art, illustrative examples of which are discussed herein.

[0052] As used herein, the term "recombinant" is understood to mean artificial nucleic acid structures (i.e., non-replicating cDNA or RNA; or replicons, self-replicating cDNA or RNA) which can be transcribed and / or translated in a host cell. Recombinant nucleic acid molecules may initially be inserted into a vector. Non-viral vectors such as plasmid expression vectors or viral vectors may be used. Suitable vectors would be known to persons skilled in the art, an illustrative example of which includes an alphavirus vector. [0053] The one or more virus structural proteins which form an SAVLP includes any structural protein amino acid sequence of a virus which can form part of a virus particle structure, illustrative examples of which include an envelope protein as herein described (e.g., E1 , E2, E3 and 6K). Without being bound by theory or by a particular mode of application, it is generally understood that the more structural proteins are included in the SAVLP, the greater the immune response is likely to be, insofar as there are more B cell and T cell epitopes to which an immune response can be raised, and additionally, the conformational veracity of the VLP is likely to be maximised. However, an effective immune response may nevertheless be generated where the SAVLP comprises only a single structural envelope protein.

[0054] In an embodiment disclosed herein, the SAVLP comprises a salmon alphavirus capsid protein, and one or more structural proteins selected from the group consisting of salmon alphavirus E1 , E2, E3 and 6K envelope proteins. In an embodiment disclosed herein, the SAVLP comprises a salmon alphavirus capsid protein, and at least two structural proteins selected from the group consisting of salmon alphavirus E1 , E2, E3 and 6K envelope proteins. In an embodiment disclosed herein, the SAVLP comprises a salmon alphavirus capsid protein, and at least three structural proteins selected from the group consisting of salmon alphavirus E1 , E2, E3 and 6K envelope proteins. In an embodiment disclosed herein, the SAVLP comprises a salmon alphavirus capsid protein, and envelope proteins E1 , E2, E3 and 6K. In an embodiment disclosed herein, the SAVLP comprises a salmon alphavirus capsid protein, and envelope proteins E1 , E2 and E3.

[0055] In an embodiment disclosed herein, the second nucleic acid sequence encodes at least two structural proteins selected from the group consisting of salmon alphavirus E1 , E2, E3 and 6K envelope proteins. In an embodiment disclosed herein, the second nucleic acid sequence encodes at least three structural proteins selected from the group consisting of salmon alphavirus E1 , E2, E3 and 6K envelope proteins. In an embodiment disclosed herein, the second nucleic acid sequence encodes envelope proteins E1 , E2, E3 and 6K. In an embodiment disclosed herein, the second nucleic acid sequence encodes envelope proteins E1 , E2 and E3.

[0056] The SAVLP may include other individual structural proteins, such as protein monomers, or dimers, or protein complexes spontaneously formed upon purification of recombinant structural proteins, such as self-assembling or intact SAVLP. The SAVLP may also be in the form of a capsid monomer, protein or peptide fragment of SAVLP or capsid monomer, or mixtures thereof. It is further contemplated that the SAVLP may further comprise a lipid envelope and / or an isolated genetic material such as DNA and / or RNA. As noted herein, the present disclosure also extends to SAVLP produced using structural protein fragments or mutated forms thereof, such as structural proteins that have been modified by the insertion, substitution or deletion of one or more amino acids, although without limitation thereto.

[0057] In an embodiment, the capsid and / or structural protein has at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to any one of the corresponding amino acid sequences of the capsid and / or structural protein shown in any one of SEQ ID NOs: 1-49, for example, after optimal alignment or best fit analysis.

[0058] Reference to "at least 80%" includes 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the recited sequences, including SEQ ID NOs: 1-49, after optimal alignment or best fit analysis.

[0059] Optimal alignment of sequences within a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment ( i.e resulting in the highest percentage homology over the comparison window) generated by any suitable method known to persons skilled in the art. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (1997) Nucl. Acids. Res. 25:3389. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al. (1994-1998) In: Current Protocols in Molecular Biology, John Wiley & Sons Inc.

[0060] The term "sequence identity", as used herein, refers to the extent that sequences are identical or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by- amino acid basis over a window of comparison. Two or more peptide sequences may be compared by determining their "percent identity". The percent identity of two sequences may be described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalised by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). Suitable methods and computer programs for performing an alignment of two or more amino acid sequences and determining their sequence identity or homology are well known to persons skilled in the art. For example, the percentage of identity or similarity of two amino acid sequences can be readily calculated using algorithms, for example, BLAST, FASTA, or the Smith-Waterman algorithm. A "percentage of sequence identity" may therefore be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lle, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" is the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0061] The term "sequence identity", as used herein, includes exact identity between compared sequences at the nucleotide or amino acid level. Sequence identity, as herein described, typically relates to the percentage of amino acid residues in the candidate sequence that are identical with the residues of the corresponding peptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions, nor insertions shall be construed as reducing sequence identity or homology.

[0062] The present disclosure also extends to non-exact identity (i.e., similarity) of sequences at the nucleotide or amino acid level where any difference(s) between sequences are in relation to amino acids (or in the context of nucleotides, amino acids encoded by said nucleotides) that are nevertheless related to each other at the structural, functional, biochemical and / or conformational levels. For example, where there is non-identity (similarity) at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and / or conformational levels. In an embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. For example, leucine may be substituted for an isoleucine or valine residue. This may be referred to as a conservative substitution. In an embodiment, the amino acid sequences may be modified by way of conservative substitution of any of the amino acid residues contained therein, such that the modification has no or negligible effect on the functional activity of the modified polypeptide when compared to the unmodified polypeptide.

[0063] By "corresponding amino acid sequence" means the amino acid sequence of the same region of the salmon alphavirus polypeptide of any one of SEQ ID NOs:l-ll. For example, an amino acid sequence that corresponds to a salmon alphavirus capsid protein includes amino acid residues 1 to 282 of any one of SEQ ID NOs:l, 2 and 4-6, or or amino acid residues 1 to 283 of SEQ ID NO:3, or amino acid residues 1 to 281 of any one of SEQ ED NOs:7-ll. Similarly, an amino acid sequence that corresponds to salmon alphavirus envelope protein E1 or E1 glycoprotein includes amino acid residues 860 to 1320 of any one of SEQ ID NOs:l, 2 and 5, or amino acid residues 861 to 1322 of SEQ ID NO:3, or amino acid residues 847 to 1307 of SEQ ID NO:4, or amino acid residues 860 to 1310 of SEQ ID NO:6, or amino acid residues 859 to 1319 of any one of SEQ ID NO:7-ll. In another example, an amino acid sequence that corresponds to a salmon alphavirus salmon alphavirus envelope protein E2, or E2 glycoprotein includes amino acid residues 354 to 791 of any one of SEQ ID NOs: 1, 2, 5 and 6, or amino acid residues 355 to 792 of SEQ ID NO:3, or amino acid residues 353 to 790 of any one of SEQ ID NOs:7-l 1. In yet another example, an amino acid sequence that corresponds to a salmon alphavirus salmon alphavirus envelope protein E3, or E3 glycoprotein includes amino acid residues 283 to 353 of any one of SEQ ID NOs: 1, 2, 5 and 6, or amino acid residues 284 to 354 of SEQ ID NO:3, or amino acid residues 282 to 352 of any one of SEQ ID NOs:7-l 1. Similarly, an amino acid sequence that corresponds to salmon alphavirus 6K protein glycoprotein includes amino acid residues 792 to 859 of any one of SEQ ID NOs: 1, 2, 5 and 6, or amino acid residues 793 to 860 of SEQ ID NO:3, or amino acid residues 779-846 of SEQ ID NO:4, or amino acid residues 791 to 858 of any one of SEQ ID NOs:7-l 1. In another example, an amino acid sequence that corresponds to salmon alphavirus envelope proteins or glycoproteins E2 and E3 (i.e., combined) includes amino acid residues 283 to 778 of SEQ ID NO:4.

[0064] In an embodiment disclosed herein, the salmon alphavirus capsid protein comprises, consists or consists essentially of an amino acid sequence corresponding to amino acid residues 1 to 282 of SEQ ID NO: 1, or to amino acid residues 1 to 282 of SEQ ID NO:2, or to amino acid residues 1 to 283 of SEQ ID NO:3, or to amino acid residues 1 to 282 of any one of SEQ ID NOs:4-6, or to amino acid residues 1 to 281 of any one of SEQ ID NOs: 7- 11, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

[0065] In an embodiment, the salmon E1 envelope protein comprises, consists or consists essentially of an amino acid sequence corresponding to amino acid residues 860 to 1320 of SEQ ID NO: 1 or to amino acid residues 860 to 1320 of SEQ ID NO:2, or to amino acid residues 861 to 1322 of SEQ ID NO:3, or to amino acid residues 847 to 1307 of SEQ ED NO:4, or to amino acid residues 860 to 1320 of SEQ ID NO: 5, or to amino acid residues 860 to 1310 of SEQ ID NO:6, or to amino acid residues 859 to 1319 of any one of SEQ ID NO:7-ll, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

[0066] In an embodiment, the salmon E2 envelope protein comprises, consists or consists essentially of an amino acid sequence corresponding to amino acid residues 354 to 791 of SEQ ID NO: 1 or to amino acid residues 354-791 of SEQ ID NO:2, or to amino acid residues 355 to 792 of SEQ ID NO:3, or to amino acid residues 354 to 791 of SEQ ID NO:5 or SEQ ID NO:6, or to amino acid residues 353 to 790 of any one of SEQ ID NOs:7-l 1, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

[0067] In an embodiment, the salmon E3 envelope protein comprises, consists or consists essentially of an amino acid sequence corresponding to amino acid residues 283 to 353 of SEQ ID NO:l, or to amino acid residues 283 to 353 of SEQ ID NO:2, or to amino acid residues 284 to 354 of SEQ ID NO:3, or to amino acid residues 283 to 353 of SEQ ID NO:5 or SEQ ID NO:6, or to amino acid residues 282 to 352 of any one of SEQ ID NOs:7-

11, or to amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0068] In an embodiment, the salmon E2 and E3 envelope proteins comprise, consist or consist essentially of an amino acid sequence corresponding to amino acid residues 283 to 778 of SEQ ID NO:4, or an amino acid sequence having at least 80% sequence identity thereto. In an embodiment, the combined E2 and E3 envelope proteins comprises, consists or consists essentially of an amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 80% sequence identity thereto.

[0069] In an embodiment, the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of an amino acid sequence corresponding to amino acid residues 792 to 859 of SEQ ID NO: 1 or SEQ ID NO:2, or to amino acid residues 793 to 860 of SEQ ID NO:3, or to amino acid residues 779-846 of SEQ ID NO:4, or to amino acid residues 792 to 859 of SEQ ID NO:5 or SEQ ID NO:6, or to amino acid residues 791 to 858 of any one of SEQ ID NOs:7-l 1, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

[0070] hi an embodiment, the salmon alphavirus capsid protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ED NOs:12 and 21-26, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0071] In an embodiment, the salmon alphavirus E3 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:13 and 27-31, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0072] In an embodiment, the salmon alphavirus E2 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:14-18 and 32-36, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0073] In an embodiment, the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:19 and 37-42, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0074] In an embodiment, the salmon alphavirus E1 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:20 and 43-48, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0075] In an embodiment, the second nucleic acid sequence encodes envelope proteins E1, E2 and E3. In another embodiment, the second nucleic acid sequence encodes envelope proteins E1, E2, E3 and 6K.

[0076] In an embodiment, the first and second nucleic acid sequences encode a salmon alphavirus polypeptide comprising, consisting or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0077] In an embodiment, the salmon alphavirus capsid protein comprises, consists or consists essentially of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thgereto.

[0078] In an embodiment, the salmon alphavirus E3 envelope protein comprises, consists or consists essentially of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity thereto.

[0079] In an embodiment, the salmon alphavirus E2 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-18, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0080] In an embodiment, the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto.

[0081] In an embodiment, the salmon alphavirus E1 envelope protein comprises, consists or consists essentially of an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto.

[0082] In an embodiment, the salmon alphavirus capsid protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:21-26, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0083] In an embodiment, the salmon alphavirus E3 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:27-31, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0084] In an embodiment, the salmon alphavirus E2 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:32-36, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0085] In an embodiment, the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:37-42, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0086] In an embodiment, the salmon alphavirus E1 envelope protein comprises, consists or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:43-48, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

[0087] In an embodiment, the polynucleotide composition comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 52-54, and nucleic acid sequences having at least 70% sequence identity to any of the foregoing. In an embodiment, the polynucleotide composition comprises a nucleic acid sequence of SEQ ID NQ:52, or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:52. In an embodiment, the polynucleotide composition comprises a nucleic acid sequence of SEQ ID NO:53, or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:53. In an embodiment, the polynucleotide composition comprises a nucleic acid sequence of SEQ ID NO: 54, or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:54, Reference to " at least 70%" includes 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the recited sequence(s), including to any one of SEQ ID NOs:52-54, after optimal alignment or best fit analysis. In an embodiment, polynucleotide composition comprises a nucleic acid sequence of SEQ ID NO:52. In an embodiment, polynucleotide composition comprises a nucleic acid sequence of SEQ ID NO: 53. In an embodiment, polynucleotide composition comprises a nucleic acid sequence of SEQ ID NO:54.

[0088] The present disclosure extends to polynucleotide compositions comprising first and second nucleic acid sequences that encode any combination of the capsid proteins and the one or more structural proteins described herein. Suitable combinations may comprise capsid and structural proteins of the same salmon alphavirus subtype (e.g., subtypes 1-6), or they may comprise capsid and structural proteins of two or more different salmon alphavirus subtypes. In an embodiment, the combination comprises a capsid protein of salmon alphavirus subtype 1 and one or more structural proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 2-6, illustrative examples of which are described herein. In an embodiment, the combination comprises a capsid protein of salmon alphavirus subtype 2 and one or more structural proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1 and 3-6, illustrative examples of which are described herein. In an embodiment, the combination comprises a capsid protein of salmon alphavirus subtype 3 and one or more structural proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1, 2 and 4-6, illustrative examples of which are described herein. In an embodiment, the combination comprises a capsid protein of salmon alphavirus subtype 4 and one or more structural proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-3, 5 and 6, illustrative examples of which are described herein. In an embodiment, the combination comprises a capsid protein of salmon alphavirus subtype 5 and one or more structural proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-4 and 6, illustrative examples of which are described herein. In an embodiment, the combination comprises a capsid protein of salmon alphavirus subtype 6 and one or more structural proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-5, illustrative examples of which are described herein. [0089] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes one or more structrural proteins selected from the group consisting of:

(i) an E3 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity thereto;

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity thereto;

(iii) a 6K protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto; and

(iv) an E1 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto.

[0090] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes one or more structural proteins selected from the group consisting of:

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto; (iii) a 6K protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto; and

[0091] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes one or more structural proteins selected from the group consisting of:

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 80% sequence identity thereto;

[0092] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes one or more structural proteins selected from the group consisting of: (i) an E3 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity thereto;

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto;

[0093] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes one or more structural proteins selected from the group consisting of:

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereto;

(iv) an E1 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto. [0094] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes two or more structrural proteins selected from the group consisting of:

[0095] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes two or more structural proteins selected from the group consisting of:

[0096] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes two or more structural proteins selected from the group consisting of:

[0097] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes two or more structural proteins selected from the group consisting of: (i) an E3 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity thereto;

[0098] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes two or more structural proteins selected from the group consisting of:

(iv) an E1 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto. [0099] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes three or more structrural proteins selected from the group consisting of:

[0100] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes three or more structural proteins selected from the group consisting of:

[0101] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes three or more structural proteins selected from the group consisting of:

[0102] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes three or more structural proteins selected from the group consisting of: (i) an E3 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity thereto;

[0103] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes three or more structural proteins selected from the group consisting of:

(iv) an E1 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto. [0104] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes:

[0105] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of

SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes:

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto;

(iii) a 6K protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto; and (iv) an E1 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto.

[0106] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes:

[0107] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of

(ii) an E2 protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto; (iii) a 6K protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto; and

[0108] In an embodiment, the first nucleic acid sequence encodes a salmon alphavirus capsid protein comprising, consisting or consisting essentially of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto, and the second nucleic acid sequence encodes:

Promoters

[0109] As described herein, the present disclosure is predicated, at least in part, on the inventors' surprising finding that a polynucleotide composition encoding a salmon alphavirus-like particle unexpectedly provides improved protection against salmon alphavirus infection, where the polynucleotide employs (i) one or more promoters to independently regulate expression of the salmon alphavirus capsid protein and expression of the salmon alphavirus structural proteins or (ii) where the polynucleotide employs a spacer sequence (such as T2A) between the capsid and structural protein coding regions, wherein the spacer is capable of disrupting expression of a contiguous polypeptide comprising the capsid protein and the one or more structural proteins.

[0110] For example, a first promoter may be used to drive expression of the salmon alphavirus capsid protein and a second promoter may be used to drive expression of the salmon alphavirus structural envelope proteins. In another example, a single bidirectional promoter may be used to drive expression of the salmon alphavirus capsid protein and the salmon alphavirus structural envelope proteins.

[0111] A promoter is understood to mean a region of DNA that is the site of initiation of transcription of a particular gene. They are typically located near the transcription start sides of genes, upstream of the open reading frame.

[0112] Suitable promoters will be known to persons skilled in the art, illustrative examples of which include CMV (Human Cytomegalovirus), EFlα (E1ongation Factor- 1 alpha), EFlα - HTLV (EFlα - Human T cell Leukaemia Virus composite), RSV (Rous Sarcoma Virus), CAG (chimeric CMV/chicken beta actin/Rabbit beta globulin composite) and CBAG (chimeric CMV/salmon beta actin/Rabbit beta globulin). Other illustrative examples of suitable promoters are discussed in Toro-Ascuy et al. (Appl Environ Microbiol. 2015; 81(4): 1210-1224) and in Hølvold et al. (Vet Res. 2014; 45(1): 21), the entire contents of which are incorporated herein by reference. In some embodiments, the promoter is a fish promoter (i.e. derived from a fish genome). Exemplary fish promoters are known in the art (e.g. interferon regulatory factor 1A (IRF1A; Alonso et al. Vaccine. 2003; 21(15):1591- 600)) and can be selected for use in accordance with the present disclosure.

[0113] In an embodiment, the first promoter is selected from the group consisting of a CMV promoter and an EFlα promoter. In an embodiment, the first promoter is a CMV promoter.

[0114] In an embodiment, the second promoter is selected from the group consisting of a CMV promoter and an EFlα promoter. In an embodiment, the second promoter is a CMV promoter.

[0115] In an embodiment, the first promoter is different to the second promoter. For example, the first promoter is a CMV promoter and the second promoter can be selected from the group consisting of EFlα (E1ongation Factor-1 alpha), EFlα - HTLV (EFlα - Human T cell Leukaemia Virus composite), RSV (Rous Sarcoma Virus), CAG (chimeric CMV/chicken beta actin/Rabbit beta globulin composite) and CBAG (chimeric CMV/salmon beta actin/Rabbit beta globulin). In another example, the first promoter is an EFlα promoter and the second promoter can be selected from the group consisting of CMV promoter, EFlα - HTLV (EFlα - Human T cell Leukaemia Virus composite), RSV (Rous Sarcoma Virus), CAG (chimeric CMV/chicken beta actin/Rabbit beta globulin composite) and CBAG (chimeric CMV/salmon beta actin/Rabbit beta globulin). In an embodiment, the first promoter is a CMV promoter and the second promoter is an EFlα promoter. In another embodiment, the first promoter is an EFlα promoter and the second promoter is a CMV promoter. In an embodiment, the first promoter is an EFlα promoter and the second promoter is a separate EFlα promoter. In another embodiment, the first promoter is a CMV promoter and the second promoter is a separate CMV promoter.

[0116] In an embodiment, the first promoter is a strong promoter and the second promoter is a weak promoter. In an embodiment, the first promoter is a weak promoter and the second promoter is a strong promoter. If is to be understood that, in the context of promoters, the terms "strong" and "weak" are typically used to describe promoters according to their effects on transcription rates and thereby on gene expression, wherein a strong promoter provides a faster rate of transcription as compared to a "weak" promoter. Suitable strong and weak promoters will be familiar to persons skilled in the art, illustrative examples of which include CMV promoter as the "strong" promoter and EFlα promoter as the comparative "weak" promoter in that pairing.

[0117] In another embodiment, the first promoter is the same as the second promoter, such that the first nucleic acid sequence is under the control of the same promoter as the second nucleic acid sequence, wherein the first nucleic acid sequence is linked to the second nucleic acid sequence by a spacer, and wherein the spacer comprises a nucleic acid sequence that disrupts expression of a contiguous polypeptide comprising the capsid protein and the one or more structural proteins. It is to be understood that, in this context, "same" means that the first promoter and the second promoter are the same promoter, such that the polynucleotide may suitably be referred to as a single promoter construct. [0118] In another aspect disclosed herein, there is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(ii) a second nucleic acid sequence encoding one or more structural proteins;

(iii) at least one promoter; and

[0119] Suitable spacers will be familiar to persons skilled in the art, illustrative examples of which include an internal ribosome entry site (IRES) and a nucleic acid sequence encoding a self-cleaving peptide (e.g., a T2A sequence), as described, for example, in Hobemik and Bros (Int JMol Sci. 2018 Nov; 19(11): 3605), the entire contents of which is incorporated herein by reference. The term "internal ribosomal entry site" or "IRES" typically refers to a viral, cellular, or synthetic (e.g., a recombinant) nucleic acid sequence which allows for initiation of translation of an mRNA at a site internal to a coding region within the same mRNA or at a site 3' of the 5' end of the mRNA, to provide for translation of an operably linked coding region located downstream of (i.e., 3' of) the internal ribosomal entry site. This makes translation independent of the 5' cap structure, and independent of the 5' end of the mRNA. An IRES sequence typically provides necessary cis-acting sequences required for initiation of translation of an operably linked coding region. Suitable nucleic acid sequences encoding a self-cleaving peptide will be known to persons skilled in the art, illustrative examples of which include 2A self-cleaving peptides (e.g., T2A) that share a core sequence motif of DX1EX2NPGP and can induce ribosomal skipping during translation of a protein in a cell. Cleavage is understood to be triggered by ribosomal skipping of the peptide bond between the proline (P) and glycine (G) residues in C-terminal end of the 2A peptide, resulting in the peptide upstream of the 2A peptide having extra amino acids on its C- terminal end, while the peptide downstream of the 2A peptide will have an extra proline on its N-terminal end. The molecular mechanism of 2A-peptide-mediated cleavage is understood to involve ribosomal skipping of the glycyl-prolyl peptide bond formation.

[0120] In an embodiment disclosed herein, the polynucleotide composition described herein comprises a nucleic acid sequence encoding the salmon alphavirus capsid protein and the one or more structural envelope proteins, wherein the nucleic acid sequence encoding the salmon alphavirus capsid protein is separated from the nucleic acid sequence encoding the salmon alphavirus capsid protein and the one or more structural envelope proteins by a spacer (e.g. , capsid-spacer-E3-E2-6K-E 1 ). Without being bound by theory or by a particular mode of application, this illustrative orientation would allow the use of a single promoter (e.g., a CMV promoter). This illustrative orientation also advantageously provides the option of an independent heterologous promotor/independent cassette for a bivalent vaccine expressing antigens from (example) cardiomyopathy syndrome virus (CMSV) or heart and skeletal muscle inflammation virus (HSMIV). In an embodiment, the spacer is an internal ribosome entry site (IRES). In another embodiment, the spacer is a nucleic acid sequence encoding a 2 A self-cleaving peptide. In another embodiment, the 2 A self-cleaving peptide is a T2A self-cleaving peptide.

[0121] In an embodiment, the promoter is a unidirectional promoter. In another embodiment, the promoter is bidirectional. Bidirectional promoters are regulatory DNA sequences that sit between two bidirectional gene pairs that drives the coordinated transcription of the bidirectional genes in opposite directions. A bidirectional gene pair refers to two adjacent genes located on complementary strands of DNA with their 5' ends orientated in a head-to-head orientation. Thus, in an embodiment disclosed herein, the polynucleotide composition described herein comprises a first nucleic acid sequence encoding the salmon alphavirus capsid protein and a second nucleic acid sequence encoding one or more structural envelope proteins of a salmon alphavirus, wherein the first nucleic acid is separated from the second nucleic acid sequence by a bidirectional promoter capable of independently driving the expression of the salmon alphavirus capsid protein and one or more structural envelope proteins of a salmon alphavirus. [0122] In a further aspect disclosed herein is provided a polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(iii) a bidirectional promoter; wherein the bidirectional promoter is situated between the first nucleic acid sequence and the second nucleic acid sequence; wherein the first nucleic acid sequence and the second nucleic acid sequence are located on complementary strands of the polynucleotide; wherein the first nucleic acid sequence and the second nucleic acid sequence are under the control of the bidirectional promoter; and wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

[0123] In an embodiment, the polynucleotide further comprises one or more regulatory elements selected from the group consisting of enhancers, origin of replication, selectable markers, multiple cloning sites, ribosomal binding sites, start codons, termination codons, transcription termination sequences, Shine-Dalgamo sequences and Kozak consensus sequences. Suitable regulatory elements will be known to persons skilled in the art, illustrative examples of which include polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES) and nucleic acid sequences that modulate mRNA stability.

[0124] The present disclosure also extends to plasmids comprising the polynucleotide composition described herein. Suitable plasmids will be familiar to persons skilled in the art, illustrative examples of which are described in Garver et al. ( Marine Biotechnology, 2005; 7(5):540-553), Xing et al. ( Frontiers in Immunology, 2019; 10:499) and Hølvold et al. (Veterinary Research; 2014; 45: 21), the entire contents of which are incorporated herein by reference.

[0125] The delivery of the polynucleotide composition described herein to a subject in need thereof (e.g., a salmonid) can be achieved by any means known to persons skilled in the art. Illustrative examples of suitable polynucleotide delivery systems include DNA- ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA (e.g. naked DNA consisting of the polynucleotide), calcium phosphate / DNA precipitation, gene gun techniques, electroporation, and colloidal dispersion systems (e.g., macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes). In an embodiment, the delivery system / vehicle is a liposome. DNA (and optionally intact SAVLP) can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see, for example, Fraley et al. Trends Biochem. Sci., 6:77, 1981, the entire contents of which is incorporated herein by reference). The liposome may comprise a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids (e.g., cholesterol). The physical characteristics of liposomes may depend on pH, ionic strength, and the presence of divalent cations. Illustrative examples of suitable lipids useful in liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Other illustrative examples include diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, preferably from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidyl-choline. In other embodiments, the polynucleotide composition is delivered as naked DNA.

[0126] In an embodiment dsiclsored herein, the polynucleotide composition comprises a nucleic acid sequence of SEQ ID NO: 52, or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 52. In another embodiment, the polynucleotide composition comprises the nucleic acid sequence of SEQ ID NO: 52.

Vectors

[0127] Nucleic acid molecules corresponding to and / or derived from and / or encoding salmon alphavirus proteins and / or one or more antigens (and / or immunogens) thereof may also be contained within a suitable vector (e.g., a recombinant vector) such as one or more non-viral and / or viral vectors. "Non-viral" vectors may include, for instance, plasmid vectors (e.g., compatible with bacterial, insect, yeast and / or mammalian host cells). Suitable vectors will be known to persons skilled in the art, illustrative examples of which include retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others. [0128] In an embodiment, a vector is employed to deliver the polynucleotide composition (e.g., to a cell in vitro or in vivo). Where such vectors are used to induce and / or enhance an immune response, the vector may also encode other proteins (e.g., co stimulatory molecules, cytokines or chemokines) and / or be combined with other factors (e.g., exogenous cytokines). Other strategies may also be utilised to improve the efficiency of gene expression and delivery, including, for example, the use of self-replicating viral replicons, in vivo electroporation, incorporation of stimulatory motifs such as CpG, sequences for targeting of the endocytic or ubiquitin-processing pathways, prime-boost regimens, proteasome-sensitive cleavage sites, and the mucosal delivery systems.

[0129] Methods for preparing and using such non-viral vectors, viral vectors, and variations thereof are available in the art, illustrative examples of which can be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), and PCR Protocols: A Guide to Methods and Applications (Innis et al, 1990. Academic Press, San Diego, CA), for instance.

Expression Cassettes

[0130] In an embodiment, the polynucleotide compositions, as herein described, are incorporated into a nucleic acid cassette, also referred to herein as an expression cassette. A nucleic acid cassette or expression cassette is intended to mean a nucleic acid sequence designed to introduce a nucleic acid sequence, typically a heterologous nucleic acid sequence (e.g., the nucleic acid construct as described herein) into a vector. The expression cassette may include a terminal restriction enzyme linker (i.e., Restriction Enzyme recognition nucleotides) at each end of the sequence of the cassette to facilitate insertion of the nucleic acid sequence or sequences of interest. The terminal restriction enzyme linkers at each end may be the same or different terminal restriction enzyme linkers. In some embodiments, the terminal restriction enzyme linkers may include rare restriction enzyme recognition/cleavage sequences, such that unintended digestion of the nucleic acid or the alphavirus genome into which the cassette is to be introduced does not occur. Suitable terminal restriction enzyme linkers would be known to persons skilled in the art. In an embodiment, the Restriction Enzyme recognition nucleotides for Pac I (TTAATTAA) is added to the 5’ end of each expression cassette and the Restriction Enzyme recognition nucleotides for Sbf I (CCTGCAGG) is added to the 3’ end of each expression cassette.

[0131] In an embodiment, the transcriptional and translational regulatory control sequences include a promoter sequence, a 5’ non-coding region, a cis-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream open reading frame, ribosomal-binding sequences, transcriptional start site, translational start site, and / or nucleotide sequence which encodes a leader sequence, termination codon, translational stop site and a 3’ non-translated region.

[0132] In an embodiment, the transcriptional control sequence includes a promoter. Suitable promoters would be known to persons skilled in the art, an illustrative example of which is a vaccinia virus early/late promoter, as shown in GenBank Accession No. X55811. In one embodiment, the promoter is a bidirectional promoter.

[0133] In an embodiment, an expression cassette can initially be created to express the capsid and envelope (structural) proteins, as herein described, by linking the first and second nucleic acid sequences together by using an Internal Ribosome Entry Site (IRES) nucleic acid sequence from encephalomyocarditis virus (EMCV) in the configuration capsid protein- IRES-envelope, so that the IRES preferred start codon is also the start codon for the capsid protein. In this configuration, transcription from a single promoter will lead to the translation of two open reading frames. The term "internal ribosomal entry site" or "IRES" typically refers to a viral, cellular, or synthetic (e.g., a recombinant) nucleic acid sequence which allows for initiation of translation of an mRNA at a site internal to a coding region within the same mRNA or at a site 3' of the 5' end of the mRNA, to provide for translation of an operably linked coding region located downstream of {i.e., 3' of) the internal ribosomal entry site. This makes translation independent of the 5' cap structure, and independent of the 5' end of the mRNA. An IRES sequence typically provides necessary cis-acting sequences required for initiation of translation of an operably linked coding region.

[0134] In some embodiments, the expression cassette can be cloned into a Homologous Recombination Plasmid. The expression cassettes as herein described may be cloned into the restriction enzyme sites {e.g., Pad and Sbfl) of the Homologous Recombination plasmid so that it is located between the FI repeat and F2 arm. [0135] Expression cassettes contemplated herein may also comprise one or more selectable marker sequences suitable for use in the identification of host cells which have or have not been infected, transformed or transfected with the expression cassette. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., b-galactosidase, luciferase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., various fluorescent proteins such as green fluorescent protein, GFP).

[0136] In an embodiment, the expression cassette comprises, consists or consists essentially of a nucleic acid sequence of SEQ ID NO: 50, or a nucleic acid sequence having at least 70% sequence identity thereto.

[0137] In an embodiment, the expression cassette comprises, consists or consists essentially of a nucleic acid sequence of SEQ ID NO:51, or a nucleic acid sequence having at least 70% sequence identity thereto.

[0138] In an embodiment, the expression cassette comprises, consists or consists essentially of a nucleic acid sequence of SEQ ID NO:53, or a nucleic acid sequence having at least 70% sequence identity thereto.

[0139] In an embodiment, the expression cassette comprises, consists or consists essentially of a nucleic acid sequence of SEQ ID NO: 54, or a nucleic acid sequence having at least 70% sequence identity thereto.

Host cells

[0140] The present disclosure also extends to a host cell comprising the polynucleotide composition described herein.

[0141] As used herein, a host cell is understood to mean a cell comprising the polynucleotide composition described herein. The host cell can be a bacterial cell, a yeast cell, insect or a mammalian cell line. In a preferred embodiment, the host cell is an internal cell of the subject to which the polynucleotide composition described herein will be administered. [0142] The host cell may be transfected and / or infected by a vector or progeny thereof such that it may express the polynucleotide composition described herein and produce the salmon alphavirus-like particles, as herein described.

[0143] Suitable host cell lines are known to those of skill in the art and are commercially available, for example, through established cell culture collections. Such cells may then be used to produce viral particles, or for other uses as may be required. An exemplary method may comprise culturing a cell comprising the polynucleotide composition (e.g., optionally under the control of an expression sequence) under conditions that allow for the production of the SAVLP. The SAVLP may then be isolated from the cell or the cell culture medium using standard techniques known to persons skilled in the art.

[0144] Suitable host cells would be known to persons skilled in the art as any cell that is capable of being infected with the alphavirus, as herein described, and forming a VLP upon infection. In an embodiment, the cell is a continuous cell line, although it is not imperative that the host cell is a cell line able to divide continuously. For example, a suitable mammalian or higher eukaryotic cell may be used as a host cell in accordance with the present disclosure. Illustrative examples of suitable host cells include RK18, BHK, VERO, HBOC-143B, HaCat, HepG2, HeLa, HT1080, HEK-293, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, myeloma cells (e.g., SB20 cells) and CEMX174 are available, for example, from the ATCC. In an embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell. In other embodiments, the host cell is a fish cell. Fish (including salmonid) cell lines are well known in the art and include, for example, CHSE/F (ATCC CRL 1681; formerly known as CHSE-214), CHH-1 (ATCC CRL 1680), and others described in Lannin et al., (In Vitro, 1984, 20:671).

Methods of producing virus-like particles

[0145] Also disclosed herein is a method of producing a salmon alphavirus-like particle comprising culturing a host cell comprising the polynucleotide, as herein described, under conditions and for a period of time sufficient to allow expression of the salmon alphavirus- like particle by the host cell.

[0146] In an embodiment, the salmon alphavirus-like particle (known interchangeably herein as VLP or SAVLP) is a product of an assembly reaction in which one or more virus structural proteins are recombinantly expressed and assembled into a VLP under conditions that promote self-assembly of the virus structural protein into a VLP.

[0147] In an embodiment, a VLP may be produced by introduction into a host cell, tissue or organ, of the alphavirus vector, as herein described.

[0148] VLPs can form spontaneously upon recombinant expression of the polynucleotide in an appropriate expression system. Methods for producing particular VLPs are known to persons skilled in the art, illustrative examples of which are discussed herein.

[0149] The presence of SAVLPs following recombinant expression of the polynucleotide composition described herein can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterisation, and the like (see, e.g., Baker etal.,Biophys. J. (1991) 60:1445-1456; andHagensee etal.,J. Virol. (1994) 68:4503-4505). For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the SAVLP preparation in question, and images recorded under appropriate exposure conditions.

[0150] The salmon alphavirus-like particle, as described herein, encoding the proteins that can form a VLP, provide an efficient means for the production of VLPs using a variety of different cell types, suitable examples of which would be known to persons skilled in the art (e.g., bacterial, insect, fungal (yeast) and animal (e.g. human) cells), illustrative examples of which are described herein.

[0151] Transcription of polynucleotide composition within the host cell nucleus and translation of the encoded product takes place in the transfected host cell and is sufficient for generation of VLPs in the modified host cell cytoplasm.

[0152] In an embodiment, the nucleic acid sequences encoding of the peptide sequences disclosed herein are codon optimised for expression in the host cell. Suitable methods of codon optimisation will be familiar to persons skilled in the art, illustrative examples of which are described in Mauro and Chappell (2014; Trends Mol. Med., 20(11):604-613) and in Mauro (2018; BioDrugs, 32:69-81), the contents of which are incorporated herein by reference. Host cells expressing one or more of the sequences described herein can readily be generated given the disclosure provided herein by stably integrating the one or more sequences (e.g., in the form of one or more expression vectors or expression cassettes) encoding the necessary structural proteins to allow for the formation of a VLP. The promoter regulating expression of the stably integrated nucleic acid sequences(s) may be constitutive or inducible. Thus, a host cell can be generated in which one or more viral structural proteins are stably integrated, so that non-replicating VLPs are formed.

[0153] When the polynucleotide composition encoding the salmon alphavirus-like particles are introduced into a host cell and subsequently expressed at the necessary level, the VLP assembles and can then be released from the cell into the culture media, where it can be further processed (e.g., purified), if necessary, having regard to their intended use.

[0154] Depending on the expression system and host cell selected, the VLPs are typically produced by growing the host cell comprising the expression vector or cassette comprising the polynucleotide compositions described herein under conditions whereby the VLP-forming polypeptides are expressed and VLPs can be formed. The selection of the appropriate growth conditions is within the skill of the art. If the VLPs accumulate intracellularly, the cells can be disrupted (e.g., by using chemical, physical or mechanical means), which lyse the cells, yet keep the VLPs substantially intact. Such methods are known to persons skilled in the art, illustrative examples of which are described in, e.g., Protein Purification Applications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds., 1990). Alternatively, VLPs may be secreted and harvested from the surrounding culture media.

[0155] The VLP can then be isolated (or substantially purified) using methods that preserve the integrity thereof, such as, by density gradient centrifugation, e.g., sucrose gradients, PEG-precipitation, pelleting, and the like (see, e.g., Kimbauer et al. J. Virol. (1993) 67:6929-6936), as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography. For example, a composition or preparation comprising an isolated VLP prepared according to the method of the present disclosure may comprise at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100% of an isolated VLP, as measured by methods known to persons skilled in the art.

[0156] In certain embodiments, preparations and / or compositions comprising the polynucleotides described herein are also provided. For example, a preparation or composition may comprise, for example, a salmon alphavirus nucleic acid, as a partially purified (e.g., about any of 50%, 60%, 75%, 90%, 95% purity (e.g, w/w)) or purified (e.g., about 98-100% (w/w)) preparation or composition. Typically, such preparations include a buffer such as phosphate- or tris-buffered saline (PBS or TBS, respectively). The preparations may also be formulated to contain excipients, like stabilisers, for example. The nucleic acids according to the invention may also be combined with one or more pharmaceutically acceptable carriers prior to use (e.g., administration to a host). A pharmaceutically acceptable carrier may be a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a cell and / or subject, without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimise any degradation of the active ingredient and to minimise any adverse side effects in the subject, as would d be well known to one of skill in the art.

Vaccine compositions and uses thereof

[0157] The present disclosure also extends to methods of producing a vaccine composition for targeting multiple antigens associated with salmon alphavirus infection. In an embodiment disclosed herein, the salmon alphavirus-like particle encoded by the polynucleotide composition or produced by the methods disclosed herein, when administered, induces an immune response directed against the antigens associated with PD or SD. In another embodiment disclosed herein, the salmon alphavirus-like particle produced by the methods disclosed herein is designed such that, when administered, induces an antibody response directed against more than one antigen associated with PD or SD (e.g., 2, 3, 4 or more antigens associated with PD or SD).

[0158] Suitable pharmaceutical carriers and their formulations that may be suitable are available to those of ordinary skill in the art as described in, for example, Remington's: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5. Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing polypeptides or fragments thereof. Matrices may be in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, surface active agents, adjuvants, immunostimulants, in addition to the binding agent and / or nucleic acid. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents and anaesthetics. Adjuvants may also be included in the immunuostimulatory compositions to stimulate or enhance the immune response. Non-limiting examples of suitable classes of adjuvants include those of the gel-type (e.g., aluminum hydroxide/phosphate ("alum adjuvants"), calcium phosphate, microbial origin (muramyl dipeptide (MDP)), bacterial exotoxins (cholera toxin (CT), native cholera toxin subunit B (CTB), E. coli labile toxin (LT), pertussis toxin (PT), CpG oligonucleotides, BCG sequences, tetanus toxoid, monophosphoryl lipid A (MPL) of, for example, Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella exseri), particulate adjuvants (biodegradable, polymer microspheres), immunostimulatory complexes (ISCOMs)), oil-emulsion and surfactant-based adjuvants (Freund's incomplete adjuvant (FIA), microfluidised emulsions (MF59, SAF), saponins (QS-21)), synthetic (muramyl peptide derivatives (murabutide, threony-MDP), non-ionic block copolymers (L121), polyphosphazene (PCCP), synthetic polynucleotides (poly A :U, poly I :C), thalidomide derivatives (CC-4407/ACTIMID), RH3-ligand, or polylactide glycolide (PLGA) microspheres, among others. Metallic salt adjuvants such as alum adjuvants are well-known in the art as providing a safe excipient with adjuvant activity. The mechanism of action of these adjuvants are thought to include the formation of an antigen depot such that antigen may stay at the site of injection for up to 3 weeks after administration, and also the formation of antigen/metallic salt complexes which are more easily taken up by antigen presenting cells. In addition to aluminium, other metallic salts have been used to adsorb antigens, including salts of zinc, calcium, cerium, chromium, iron, and berilium. The hydroxide and phosphate salts of aluminium are the most common. Formulations or compositions containing aluminium salts, antigen, and an additional immunostimulant are known in the art. An example of an immunostimulant is 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Other homologs and / or derivatives of any of these toxins may also suitable, provided that they retain adjuvant activity.

[0159] The compositions comprising a salmon alphavirus or VLP, as described herein, may include a pharmaceutically acceptable excipient or diluent, suitable examples of which would be known to persons skilled in the art. To the extent that the compositions are intended for inducing an immune response in a subject in need thereof against infection by SAV, the composition can be referred to interchangeably herein as a vaccine composition, an immunogenic composition or an immunomodulating composition. The compositions of the present disclosure are typically used for prophylactic purposes, as discussed herein, but can also be used for ameliorative, palliative, or therapeutic purposes, such as for inducing an immune response in a subject who has already been infected by SAV.

[0160] In some embodiments, the composition can comprise a pharmaceutically acceptable excipient, suitable examples of which would be known to persons skilled in the art. Illustrative examples of pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3.sup.rd ed. Amer. Pharmaceutical Assoc.

[0161] Vaccine composition may be in a form suitable for administration by injection, or in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection, or for dispersal into waterways / culture medium or environment of the fish.

[0162] Vaccine compositions of the present disclosure may also be provided in a kit. The kit may comprise additional components to assist in performing the methods as herein described, such as administration device(s), excipients(s), and / or diluent(s). The kits may include containers for housing the various components and instructions for using the kit components in such methods.

[0163] The polynucleotide compositions described herein, including vectors, expression cassettes, plasmids and host cells comprising said polynucleotide composition, as well as the SAVLP encoded by the polynucleotide compositions, may be used, for example, to stimulate an immune response against salmon alphavirus described herein in a host. In some embodiments, immunogenic compositions and vaccines comprising the polynucleotide compositions described herein, including vectors, expression cassettes, plasmids and host cells comprising said polynucleotide composition, as well as the SAVLP encoded by the polynucleotide compositions, may be used to treat diseases caused by or associated with the presence of salmon alphavirus in salmon. An immunological composition is typically one that, upon administration to a host such as salmon, induces or enhances an immune response directed against the antigen or immunogen (e.g., SAV polypeptide(s)) contained within the composition. This response may include the generation of antibodies (e.g, through the stimulation of B cells) or a T cell-based response (e.g., a cytolytic response). These responses may or may not be protective or neutralising. A protective or neutralising immune response is one that may be detrimental to the cell containing or expressing the antigen (e.g., from which the antigen was derived) and beneficial to the host (e.g., by reducing or preventing tumour growth). As used herein, protective or neutralising antibodies and / or cellular responses may be reactive to SAV polypeptide(s) and / or an antigen thereof. An immunological composition that, upon administration to a host, results in a protective or neutralising immune response may be considered a vaccine. Immunological compositions comprising at least one polynucleotide compositions described herein, including vectors, expression cassettes, plasmids and host cells comprising said polynucleotide composition, as well as the SAVLP encoded by the polynucleotide compositions, may also include one or more additional antigens.

[0164] The present disclosure also extends to methods for treating disease caused by or associated with salmon alphavirus in a host by administering to the host at least one or more effective doses of one or more of the polynucleotide compositions described herein, including vectors, expression cassettes, plasmids and host cells comprising said polynucleotide composition, as well as the SAVLP encoded by the polynucleotide compositions.

[0165] Thus, in an aspect disclosed herein, there is provided a method for treating or protecting against salmon alphavirus infection in a subject, the method comprising administering to a subject in need thereof the polynucleotide composition, the host cell, or the salmon alphavirus-like particle as herein described. [0166] In an embodiment, the polynucleotide composition, the host cell or the salmon alphavirus-like particle is administered to the subject by intramuscular injection.

[0167] In an embodiment, the subject has, or is at risk of developing, salmonoid pancreatic disease.

[0168] The present disclosure also extends to a vaccine composition for use in treating or protecting against salmon alphavirus infection in a subject, the vaccine composition comprising the polynucleotide composition, the host cell, and / or the salmon alphavirus- like particle, as herein described.

[0169] The present disclosure also extends to use of the polynucleotide composition, the host cell, and / or the salmon alphavirus-like particle, as herein described, in the manufacture of a vaccine composition for treating or protecting against salmon alphavirus infection in a subject.

[0170] Suitable methods of treating or preventing salmon alphavirus infection in a subject employing the polynucleotide compositions, host cells and / or SAVLP, as herein described, will be familiar to persons skilled in the art, an illustrative example of which is described in WO 2014/041189, the entire contents of which is incorporated herein by reference.

[0171] In some embodiments, the SAVLP described herein are administered to a subject in a suitable dose ( e.g ., about 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ viral particles) and dosing schedule (e.g., once, twice, or three times a day / week / month), as may be determined by one of ordinary skill in the art. The polynucleotide compositions described herein, including vectors, expression cassettes, plasmids and host cells comprising said polynucleotide composition, may also be administered to a subject in a suitable dose and dosing schedule (e.g., once, twice, or three times a day / week / month), as may be determined by one of ordinary skill in the art. In some embodiments, these reagents may be administered via any route (e.g., bath immersion, intraperitoneally, intradermally, intravenously, orally, or intramuscularly) at one or more times. The dose may be administered intramuscularly. When multiple doses are administered, the doses may comprise about the same or different types and or amounts of reagent (e.g., in a prime-boost format). The doses may also be separated in time from one another by the same or different intervals. For instance, the doses may be separated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, one week, 1.5 weeks, two weeks, 2.5 weeks, three weeks, 3.5 weeks, one month, 1.5 months, two months, 2.5 months, three months, 3.5 months, four months, 4.5 months, five months, 5.5 months, six months, 6.5 months, seven months, 7.5 months, eight months, 8.5 months, nine months, 9.5 months, 10 months, 10.5 months, 11 months, 1 1.5 months, 12 months, 1.5 years, 2 years, , or any time period before, after, and / or between any of these time periods. In an embodiment, the reagents are administered in a single administration. In another embodiment, where the subject is a salmonid, the administration is once or twice, given at a young age, for example when the fish weigh 10-30g.

[0172] In an embodiment, the polynucleotide composition, the host cell and / or the SAVLP, as described herein, is administered to the subject in an effective amount of from about 20 nanograms to about 20 micrograms. In an embodiment, the polynucleotide composition, as described herein, is administered to the subject in an effective amount of from about 0.5 microgram to about 5 micrograms. In an embodiment, the polynucleotide composition, as described herein, is administered to the subject in an effective amount of from about 1.0 microgram to about 3 micrograms.

[0173] The composition comprising nucleic acid sequences encoding the capsid and structural proteins of salmon alphavirus is capable of inducing an immune response in a host, upon infection, against homologous strains salmon alphaviruses, as well as cross-protective immunity against a heterologous strain of salmon alphaviruses; that is, against a strain of salmon alphavirus that is different from the strain of salmon alphavirus from which the salmon alphavirus capsid and structural proteins have been derived. Persons skilled in the art would understand that the expression of capsid or structural proteins from different SAV subtypes, will provide a composition that is capable of inducing an immune response against homologous strains of SAV, thus providing greater protection from subsequent infection with SAV. In addition, and without being bound by theory or by a particular mode of action, it is expected that a polynucleotide construct encoding a salmon alphavirus-like particle, comprising nucleic acid sequences encoding two or more SAV proteins, is more likely to induce a cross-protective immune response against infection by a heterologous strain of SAV. [0174] It is to be understood that the polynucleotide compositions, host cells and / or SAVLP will be administered, or formulated for administration, to a subject in need thereof in an effective amount. By "effective amount" is meant an amount that is effective to facilitate protection of the host against infection, or symptoms associated with infection, by a salmon alphavirus, e.g., to reduce a symptom associated with an infection, and / or to reduce the number of infectious agents in the individual. An effective amount will typically reduce a symptom associated with infection and / or reduces the number of infectious agents in an individual by at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, preferably at least about 80%, or more preferably at least about 90%, or more, when compared to the symptom or number of infectious agents in an individual not so treated.

[0175] Persons skilled in the art will understand that the effective amount is likely to depend on the health and physical condition of the subject to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through methods known to persons skilled in the art.

[0176] An effective amount (number) of salmon alphavirus-like particles may generally be in a range of from about 10² to about 10⁷, from about 10³ to about 10⁶, or from about 10⁴ to about 10⁵ plaque forming units (PFU). An optimal amount for a particular modulatory effect, such as an immunomodulating composition against S AV infection, can be ascertained by standard studies involving observation of antibody titers and types, levels of immune responses including immunosuppressive cells and other responses in a subject. The level of immunity provided by the composition can be monitored to determine the need, if any, for boosters. For instance, following an assessment of antibody titers in the serum, optional booster immunisations may be desired. The immune response to the homologous and / or heterologous strain of salmon alphavirus is likely to be enhanced using an adjuvant and / or an immunostimulant, as described herein.

[0177] The polynucleotide compositions, host cells and / or SAVLP, as described herein, may be administered to a subject in need thereof in isolation or in combination with other additional therapeutic agent(s). In embodiments where a pharmaceutical composition is administered with therapeutic agent(s), the administration may be simultaneous or sequential (i.e., pharmaceutical composition administration followed by administration of the agent(s) or vice versa). Thus, here two or more entities are administered to a subject "in conjunction", they may be administered in a single composition at the same time, or in separate compositions at the same time, or in separate compositions separated in time.

[0178] It will be apparent to persons skilled in the art that the optimal quantity and spacing of individual dosages, if required to induce the desired immune response, can be determined, for example, by the form, route and site of administration, and the nature of the particular subject to be treated, as is described herein. Optimum conditions can be determined using conventional techniques known to persons skilled in the art.

[0179] In some instances (e.g., preventative applications), it may be desirable to have several or multiple administrations of the polynucleotide compositions, host cells and / or SAVLP, as herein described. For example, the polynucleotide compositions, host cells and / or SAVLP may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations may be from about one to about twelveweek intervals, and in certain embodiments from about one to about four week intervals. Periodic re-administration may be desirable in the case of recurrent exposure or risk of exposure to salmon alphavirus. It will also be apparent to persons skilled in the art that the optimal course of administration can be ascertained using conventional course of treatment or efficacy or immune status determination tests.

[0180] Methods for measuring an immune response to the polynucleotide compositions, host cells and / or SAVLP, following administration, will be known to persons skilled in the art, illustrative examples of which include plaque-reduction neutralisation assay, micro neutralisation assay, solid-phase heterogeneous assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., electrochemiluminescence assay), amplified luminescent proximity homogeneous assays, flow cytometry, intracellular cytokine staining, functional T-cell assays including suppressor T-cell assays, functional B-cell assays, functional monocyte-macrophage assays, dendritic and reticular endothelial cell assays, measurement of NK cell responses, oxidative burst assays, cytotoxic-specific cell lysis assays, pentamer binding assays, and phagocytosis and apoptosis evaluation. [0181] It will be understood that "inducing" an immune response or cross-protective immunity, as contemplated herein, includes eliciting or stimulating an immune response and / or enhancing a previously existing immune response to obtaining a desired physiologic effect; namely, protection from an otherwise potentially lethal infection by salmon alphavirus, whether the alphavirus is of the same species as the capsid and / or envelope proteins that are incorporated into the SAVLP or encoded by the polynucleotide compositions and host cells, as described herein. The effect is prophylactic in terms of completely or partially preventing a disease or symptom associated with SAV infection, including those discussed herein.

[0182] The terms “immune response”, "immunological response" will be understood as meaning the development in a subject of a humoral and / or a cellular immune response to salmon alphavirus. A "humoral immune response" typically refers to an immune response mediated by antibody molecules, while a "cellular immune response" is typically mediated by T-lymphocytes and / or other white blood cells.

[0183] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entireties.

[0184] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

EXAMPLES

Materialas and Methods

[0185] A list of representative peptide sequences for the structural polyproteins for each of the 6 SAV subtypes were retrieved from publically available repositories (i.e. NCBI GenBank), and are listed in Table 1.

[0186] The amino acid sequences for the SAV subtype-3 structural polyprotein from 28 isolates were aligned using MUSCLE (Multiple Sequence Alignment tool from EMBL-EBI, using consensus threshold of >50%), and edited. The alignment is shown in Figure 1, where the sequences are arranged according to earliest to most recent isolate discovery. Sequence #4 in the alignment of Figure 1 corresponds to SEQ ID NO:7. The amino acid differences are highlighted in yellow. The amino acid variations in the Variant sequences occur predominantly in the E2 envelope protein region.

[0187] From this alignment, four unique variant sequences were generated (SEQ ID NOs:8-ll). An identity matrix that shows the homology of two of the variant sequences (Variant 2 (SEQ ID NO:9) and Variant 4 (SEQ ID NO:l 1)) with the non-redundant data set of known SAV3 subtype sequences is shown in Figure 2. A list of representative nucleic acid conducts encoding SAV3 structural polyprotein for in vivo testing comprising both single and dual promoters are provided in Table 4. The structural elements of each of the nucleic acid constructs is shown in Figures 3-7. In vivo testing was conducted using the single and dual promoter nucleic acid constructs at two doses alongside relevant controls, as shown in Tables 5 and 6.

Table 1. Illustrative examples of Salmon alphavirus amino acid sequences.

Table 2. Illustrative examples of the amino acid sequences of the polyprotein subunits of Salmon alphavirus subtype 3.

Table 3. Illustrative examples of the amino acid sequences of the polyprotein subunits of Salmon alphavirus subtypes 1, 2, 4, 5 and 6.

Table 4. Illustrative examples of single and dual promoter nucleic acid contructs encoding the polyprotein subunits of Salmon alphavirus subtype 3

Example 1: Fish and rearing conditions

1.1 Experimental fish

1.2 Husbandry management

[0188] The fish were produced at VESO Vikan hatchery and acclimatised at the test facility for seven days prior to the vaccination. The fish were identified by Visible Implant E1astomer (VIE) tags, vaccinated at parr stage, then kept in freshwater throughout the trial. Any dead fish were collected daily. Environmental parameters were also recorded daily. Observations in relation to abnormal or unexpected behaviour, loss of appetite or any unexpected increase in mortality were recorded.

Example 2: Study Design

2.1 Doggybone DNA vaccine constructs

[0189] Closed linear Doggybone DNA constructs comprising one of SEQ ID NOs:50- 54 were generated (see Table 5) for this study. [0190] Doggybone DNA (dbDNA™) is a proprietary synthetic closed linear double- stranded DNA construct. Closed linear DNA is generally understood to be double-stranded DNA covalently closed at each end. The double stranded section of the DNA is therefore complementary. When denatured, closed linear DNA may form a single stranded circle. The DNA may be closed at each end by any suitable structure, including a cruciform, a hairpin or a hairpin loop, depending on preference. The end of the closed linear DNA may be composed of a non-complementary sequence, thus forcing the DNA into a single stranded configuration at the cruciform, hairpin or hairpin loop. Alternatively, the sequence can be complementary. It may be preferred that the end is formed by a portion of a target sequence for a protelomerase enzyme. A protelomerase target sequence is any DNA sequence whose presence in a DNA template allows for the enzymatic activity of protelomerase, which cuts a double stranded section of DNA and re-ligates them, leaving covalently closed ends. In general, a protelomerase target sequence comprises any perfect palindromic sequence; that is, any double-stranded DNA sequence having two-fold rotational symmetry, or a perfect inverted repeat. The closed linear DNA may have a portion of a protelomerase target sequence at one or both ends. The protelomerase target sequence can have the same cognate protelomerase at each end, or require a different protelomerase for each end. Closed linear DNA constructed via the action of various protelomerase enzymes have been previously disclosed in WO2010/086626, W02012/017210 and WO2016/132129, the entire contents of which are incorporated herein by reference. Closed linear DNA constructed using in vitro DNA amplification followed by cleavage with a protelomerase enzyme has the advantage that the closed linear DNA is produced in an in vitro, cell-free environment, and can be scaled up for commercial production.

2.2 Study design

[0191] The aim of this study was to evaluate protection in freshwater salmon following vaccination against SAV subtype 3 at 500-degree days after vaccination. A positive control DNA vaccine (dbDNA-SAV3; 3-db SAV3; SEQ ID NO:50), eight test DNA vaccine groups (comprising four DNA vaccines - SEQ ID NOs:51-54 - provided at two different doses - 1.0 pg/fish or 2.5 pg/fish; intramuscular administration (IM)) and a negative control group (saline) were included in this study (Tables 5 and 6). The positive control was a SAV3 DNA vaccine construct under the control of a single CMV promoter. Table 5: Single and dual promoter nucleic acid conducts encoding the polyprotein subunits of Salmon alphavirus subtype 3 that were employed in this study

Table 6: Administration regimen; Vaccination was spread over two days, with six random groups vaccinated each day

NA: not applicable.

*R: Red, O: Orange; G: Green. Pos 1: base of anal fin; Pos 6: ventrolateral tissue of right mandibulae; Pos 7: ventrolateral tissue of left mandibulae, Pos 8: Top of head ** About 10% extra in case of unexpected mortality *** 0.05mL per dose

[0192] The two doses (1.0 pg/fish and 2.5 pg/fish; IM) were selected on results from a prior study in the same model and were intended to bridge that part of the dose response where vaccine efficacy is expected to drop from a high level to lower or negligible level. Testing at a level around the anticipated minimum protective dose was aimed at determining which of the DNA vaccines provides superior or inferior efficacy versus the positive control.

[0193] Vaccination was performed by intramuscular (i.m.) injection (0.05 mL/fish). Duration of immunisation was 500-degree days. During the immunisation period, vaccinated fish were kept in one tank. Shedders (SAV negative fish which are experimentally infected with the SAV challenge virus and mixed with the test groups at an appropriate ratio in order to provide an authentic source of infectious virus) were kept in another tank to minimise handling at challenge.

[0194] After the immunisation, the fish were challenged with SAV3 using a cohabitation challenge model. An illustrative example of suitable cohabitation challenge models is described in Graham et al (2011, J Fish Dis 34: 273-286), the contents of which are incorporated herein by reference in their entirety. Shedders were anesthetised, injected intraperitoneally (i.p.) with 0.1 ml thawed and dose-adjusted SAV3 solution. The i.p. injected shedders were then introduced to the tank with vaccinated fish. Duration of the challenge was 35 days, and sampling was carried out at 0, 4, 14, 18, 21, 25, 28 and 35 days post challenge. Details of sampling is described in Example 3, below.

2.3 Day 0 definition

[0195] The first day of vaccination is day 0.

2.4 Marking

[0196] The fish were marked at the time of vaccination by VIE tags according to VESO Standard Operating Procedures (SOPs). Shedders were marked by adipose fin clipping at challenge according to VESO SOPs.

2.5 Vaccination

[0197] The fish were acclimatised for seven days and starved for minimum 48 hours prior to vaccination. Fish were randomly selected, anesthetised, marked and i.m. injected with the vaccines or control substances according to Table 3 and VESO SOPs. Vaccination was spread over two days, with six random groups vaccinated each day according to table 3. Random.org software was used to generate random numbers.

2.6 Challenge procedure

2.6.1. Challenge isolate

[0198] Norwegian isolate from a field outbreak in 2007 grown in CHSE-214 at Norwegian Veterinary Institute. Original concentration 10^5.5TCID₅₀/mL . Challenge inoculum was diluted with PBS in 1:5 ratio. Published: Taksdal T et al., J Fish Dis 2015 (12): 1047-61.

2.6.2. Challenge

[0199] The fish were challenged with a cohabitant model according to VESO SOPs, 6 weeks post after vaccination. The shedders were starved minimum 24 hours prior to the challenge. At the time of challenge, 204 shedders were anesthetised and i.p. injected with 0.1 ml of SAV3 inoculum according to VESO SOPs and then introduced to the tank with vaccinated fish. 2.7 Termination

[0200] The challenge trial was terminated after an observation period of 35 days post challenge (dpc).

2.8 Endpoint

[0201] Fish at the terminal stage were taken out of the study and killed by an overdose of anaesthetic and / or by a lethal blow to the head. Culled fish were equated with dead fish in subsequent efficacy calculations and reporting. Only fish that were not able to self-propel or maintain their position in the water, and/ or fish lying on the floor of the trial tank or were not able to return to an upright position when prodded or turned on their side were culled.

2.9 Test validity and total mortality

[0202] To verify presence of SAV genome, heart tissues were sampled from randomly selected five shedders at the termination of the challenge for qPCR analyses.

Example 3: Sampling 3.1 Sampling

[0203] Blood: At each sampling time point, randomly selected 10 fish from each group were sampled (Table 7). Sampling of blood was performed using heparinised vacutainer tubes and needles. Blood was transferred to empty tubes from Patogen AS and shipped chilled, but not frozen, on the same day to the analytical laboratory.

[0204] Heart: For each fish sampled, two pieces of heart apex were sampled. Samples from incidental mortalities were stored overnight at 4C in RNA later®. The following day, these samples were transferred to -80C for long term storage. These samples were shipped to the analytical laboratory on the day of termination.

[0205] Samples from the single scheduled time point (35 dpc) were shipped to the analytical laboratory on the same day. These samples were shipped chilled, but not frozen, in RNA later®. Table 7: Sampling scheme

Table 8: Number of fish sampled at termination

3.2 Quantitative analysis of SAV RNA in blood and heart post-virus challenge

[0206] A quantitative polymerase chain reaction (qPCR) assay was used to quantify SAV RNA levels in whole blood and heart tissue post-virus challenge in the fish of this study. The qPCR analysis of the blood and heart samples was carried out using a validated and ISO17025 accredited method by Patogen AS (Patogen AS, Alesund, Norway), as previously described by Hodneland, K and Endresen, C (2006); J. Virol. Methods 131 (2) 184-192), the entire contents of which is incorporated herein by reference. The cut off Ct- value was set to 37. Ct-values were converted to SAV RNA copy number by Patogen.

RESULTS

[0207] As shown in the cumulative data in Figure 8, vaccinating fish with a dual promoter construct (Construct #7; db-CMV-ENV-EFlα-CP; SEQ ID NO:52) provided greater protection against virus challenge, as evidenced by a lower cumulative number of virus positive samples over the course of the study, when compared to vaccination with the single promoter Construct #3 (db-SAV3, under the control of a single CMV promoter; SEQ ID NO:50).

[0208] This was reflected in a reduction in the level of viraemia (SAV RNA copies), including on Day 21 post-virus challenge (see Figure 9). These data show that vaccination with a dual promotor construct reduced the level of viraemia post-virus challenge when compared to vaccination with a single promoter construct. The data also showed that vaccination with Construct #9 also reduced the level of viraemia post- virus challenge when compared to vaccination with a single promoter construct at Day 21. Construct #9 (SEQ ID NO:54) is a single promoter dbDNA construct comprising a polynucleotide sequence encoding a ribosomal skipping sequence sequence (T2A) between the capsid and envelope proteins, allowing the subsequent translation of two distinct proteins from a single polycistronic molecule.

[0209] Following initial infection, dissemination of virus through the blood to sites of secondary replication is a critical stage in the progression of pancreas disease. Consistent with the demonstration of a marked reduction in viraemia, vaccination with a dual promoter construct also reduced viral load in the heart tissue of these fish (Figure 10). Heart samples from fish vaccinated with dual-promotor Construct #7 at l.Opg dose had a mean viral load of only 380 SAV RNA copies compared with corresponding mean values of 61,660 SAV RNA copies in the negative control (saline) group and 12,023 SAV RNA copies in the positive control (single promotor) Construct #3.

[0210] Reference to Figure 11 reveals that the benefits of the dual promotor system are not universal. The selection and assignment of genes to specific promotors and the order in which those promoters are cloned within the vaccine construct are in fact critical in terms of clinical outcomes. In the specific example described in Figure 11, Construct #6 with the arrangement db-CMV-CP-EFlα-ENV showed no efficacy. The level of viraemia and heart viral load was extremely similar to the negative control (saline) group. In contrast, Construct #7 with the reverse arrangement db-CMV-ENV-EFlα-CP was more effective than the positive control (single promotor) Construct #3 at reducing levels of virus in the heart at the highly disciminating 1.0 microgram dose.

[0211] Great advances have been made in the understanding of alphavirus replication (reviewed by Joyce et al Future Microbiol. 2009 September; 4: 837-856). However, there are still very significant knowledge gaps, and these are particularly apparent in the specific case of SAV, which has been the subject of less research than those alphaviruses of significance in human health.

[0212] The present inventors have demonstrated that DNA vaccines employing either a dual promotor system or a single promoter system incorporating a disruptive spacer sequence such as T2A between the Cap and Env coding regions, provide unexpectedly superior protective immunity against SAV challenge when compared to a single promotor approach relying on native translation mechanisms. Without being bound by theory or by a particular mode of application, it is hypothesised that the present invention facilitates the expression of Cap and structural Env proteins at optimal ratios with respect to the efficient formation of VLPs and, in turn, provides improved protective immunity against the virus.

Claims

1. A polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises (i) a first nucleic acid sequence encoding a salmon alpfaavirus capsid protein under the control of a first promoter and (ii) a second nucleic acid sequence encoding one or more structural proteins under die control of a second promoter, wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

2. The composition of claim 1, wherein the second nucleic acid sequence encodes envelope proteins E1, E2 and E3,

3. The composition of claim 1, wherein the second nucleic acid sequence encodes envelope proteins E1, E2, E3 and 6K.

4. The composition of any one of claims 1 to 3, wherein the salmon alphavirus capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:12 and 21-26 and amino acid sequences having at least 80% sequence identity to any of the foregoing.

5. The composition of any one of claims 1 to 3, wherein the salmon alphavirus capsid protein comprises an amino acid sequence corresponding to amino acid residues 1 to 282 of any one of SEQ ID NOs:l, 2 and 4-6, or to amino acid residues 1 to 283 of SEQ ID NO:3, or to amino acid residues 1 to 281 of any one of SEQ ID NOs:7-ll, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

6. The composition of any one of claims 1 to 5, wherein the salmon alphavirus E1 envelope protein comprises an amino acid sequence selected from the group consisting of SEQ ID NG:20 and 43-48, and an amino acid sequence having at least 80% sequence identity to any of the foregoing.

7. The composition of any one of claims 1 to 5, wherein the salmon E1 envelope protein comprises an amino acid sequence corresponding to amino acid residues 860 to 1320 of any one of SEQ ID NOs:l, 2 and 5, or to amino acid residues 861 to 1322 of SEQ ID NO:3, or to amino acid residues 847 to 1307 of SEQ ID NO:4, or to amino acid residues 860 to 1310 of SEQ ID NO:6, or to amino acid residues 859 to 1319 of any one of SEQ ID NO:7-l 1, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

8. The composition of any one of claims 1 to 7, wherein the salmon alphavims E2 envelope protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 14-18 and 32-36, and amino a.cid sequences having a.t least 80% sequence identity to any of the foregoing.

9. The composition of any one of claims 1 to 7, wherein the salmon E2 envelope protein comprises an amino acid sequence corresponding to amino acid residues 354 to 791 of any one of SEQ ID NOs: 1, 2, 5 and 6, or to amino acid residues 355 to 792 of SEQ ID NO:3, or to amino acid residues 353 to 790 of any one of SEQ ID NOs:7-ll, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

10. The composition of any one of claims 1 to 9, wherein the salmon alphavims E3 envelope protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 27-31, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

11. The composition of any one of claims 1 to 9, wherein the salmon E3 envelope protein comprises an amino acid sequence corresponding to amino acid residues 283 to 353 of any one of SEQ ID NOs: 1, 2, 5 and 6, or to amino acid residues 284 to 354 of SEQ ID NO:3, or to amino acid residues 282 to 352 of any one of SEQ ID NOs:7-ll, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

12. The composition of any one of claims 1 to 11, wherein the salmon alphavims 6K envelope protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 37-42, and amino acid sequences having at least 80% sequence identity to any of the foregoing.

13. The composition of any one of claims 1 to 11, wherein the salmon alphavims 6K envelope protein comprises an amino acid sequence corresponding to amino acid residues 792 to 859 of any one of SEQ ID NOs: 1 , 2, 5 and 6, or to amino acid residues 793 to 860 of SEQ ID NO:3, or to amino acid residues 779-846 of SEQ ID NO:4, or to amino acid residues 791 to 858 of any one of SEQ ID NOs:7-l 1, or an amino acid sequence having at least 80% sequence identity to any of the foregoing.

14. The composition of any one of claims 1 to 13, wherein the salmon E2 and E3 envelope proteins comprise an amino acid sequence corresponding to amino acid residues 283 to 778 of SEQ ID NO:4, or an amino acid sequence having at least 80% sequence identity thereto.

15. The composition of any one of claims 1 to 14, wherein the first promoter is selected from the group consisting of a CMV promoter and an EFlα promoter.

16. The composition of claim 15, wherein the first promoter is a CMV promoter.

17. The composition of any one of claims 1 to 16, wherein the second promoter is selected from the group consisting of a CMV promoter and an EFlα promoter.

18. The composition of claim 17, wherein the second promoter is an EFlα promoter.

19. The composition of any one of claims 1 to 18, wherein the first promoter is different to the second promoter.

20. The composition of any one of claims 1 to 19, wherein the first promoter is the same as the second promoter, such that the first nucleic acid sequence is under the control of the same promoter as the second nucleic acid sequence, wherein the first nucleic acid sequence is linked to the second nucleic acid sequence by spacer, and wherein the spacer comprises a nucleic acid sequence that disrupts expression of a contiguous polypeptide comprising the capsid protein and the one or more structural proteins.

21. The composition of claim 1 , comprising a nucleic acid sequence of SEQ ID NO: 52, or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:52.

22. The composition of claim 21, comprising the nucleic acid sequence of SEQ ID NO:52.

23. A host cell comprising the polynucleotide composition of any one of claims 1 to 22.

24. A method of producing a salmon alphavirus-like particle, the method comprising (a) culturing the host cell of claim 23 under conditions and for a period of time sufficient to allow expression of the salmon alphavirus-like particle by the host cell and, (b) optionally , isolating the salmon alphavirus-like particle from the culture.

25. A salmon alphavirus-like particle encoded by the polynucleotide composition of any one of claims 1 to 22.

26. A salmon alphavirus-like particle produced by the method of claim 24.

27. A method for treating or protecting against salmon alphavirus infection in a subject, the method comprising administering to a subject in need thereof the polynucleotide composition of any one of claims 1 to 22, the host cell of claim 23, or the salmon alphavirus- like particle of claim 25 or claim 26,

28. The method of claim 27, wherein the polynucleotide composition, the host cell or the salmon alphavirus-like particle is administered to the subject by intramuscular injection.

29. The method of claim 27 or claim 28, wherein from about 20 nanograms to about 20 micrograms of the polynucleotide is administered to the subject.

30. The method of any one of claims 26 to 28, wherein the subject has, or is at risk of developing, salmonoid pancreatic disease.

31. A vaccine composition for use in treating or protecting against salmon a!phavirus infection in a subject, the vaccine composition comprising the polynucleotide composition of any one of claims 1 to 22, the host cell of claim 23, or the salmon alphavirus-like particle of claim 25 or claim 26,

32. Use of the polynucleotide composition of any one of claims 1 to 22, the host cell of claim 23, or the salmon alphavirus-like particle of claim 25 or claim 26, in the manufacture of a vaccine composition for treating or protecting against salmon alphavirus infection in a subject.

33. A polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(iii) at least one promoter; wherein the at least one promoter regulates independent expression of the first nucleic acid sequence and the second nucleic acid sequence, and wherein the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

34. A polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises: (i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(iii) a bidirectional promoter; wherein the bidirectional promoter is situated between the first nucleic acid sequence and the second nucleic acid sequence; the first nucleic acid sequence and the second nucleic acid sequence are located on opposite ends of the polynucleotide; the first nucleic acid sequence and the second nucleic acid sequence are under the control of the bidirectional promoter; and the one or more structural proteins is selected from the group consisting of salmon alphavirus E1 envelope protein, E2 envelope protein, E3 envelope protein and 6K envelope protein.

35. A polynucleotide composition encoding a salmon alphavirus-like particle, wherein the composition comprises:

(i) a first nucleic acid sequence encoding a salmon alphavirus capsid protein;

(ii) a second nucleic acid sequence encoding one or more structural proteins;

(iii) at least one promoter; and