WO2024069549A1 - Construction de vaccin et ses utilisations - Google Patents

Construction de vaccin et ses utilisations Download PDF

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Publication number
WO2024069549A1
WO2024069549A1 PCT/IB2023/059738 IB2023059738W WO2024069549A1 WO 2024069549 A1 WO2024069549 A1 WO 2024069549A1 IB 2023059738 W IB2023059738 W IB 2023059738W WO 2024069549 A1 WO2024069549 A1 WO 2024069549A1
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nucleic acid
acid sequence
seq
alphavirus
sequence encoding
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PCT/IB2023/059738
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English (en)
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Ian Thompson
Sung Won Kim
Lisa Jayne Caproni
Samantha Busfield
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Touchlight Aquaculture Limited
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Priority claimed from AU2022902819A external-priority patent/AU2022902819A0/en
Application filed by Touchlight Aquaculture Limited filed Critical Touchlight Aquaculture Limited
Publication of WO2024069549A1 publication Critical patent/WO2024069549A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • C12N7/045—Pseudoviral particles; Non infectious pseudovirions, e.g. genetically engineered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates generally to a vaccine construct comprising a polynucleotide encoding an alphavirus-like particle and uses thereof.
  • Salmonid alphavirus is an enveloped, single-stranded, positive-sense RNA virus with a ⁇ 12 kb genome, belonging to the family Togaviridae, genus Alphavirus.
  • SAV Salmonid alphavirus
  • 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).
  • PD pancreas disease
  • SD pancreas disease
  • Vaccines against SAV are available, some of which have been reviewed by Deperasinska et al. (J Vet Res. 2018; 62(1): 1-6). Whilst some SAV vaccines have proven to be modestly effective at reducing mortality, most have generally failed to prevent pathological lesions in the pancreas and heart, or growth retardation. However, despite the introduction of these vaccines and increased biosecurity measures, SAV continues to be a major health and welfare issue in salmonid aquaculture, with 100 new cases registered in the most affected country (Norway) in 2021, and 98 new cases in 2022 (Norwegian Veterinary Institute, Norwegian Fish Health Report 2022 Norwegian ...Fish..Health.. Report.,2022 (vetinst.no).
  • vaccine construct comprising a polynucleotide encoding a salmon alphavirus-like particle, wherein the polynucleotide comprises a nucleic acid sequence encoding a salmon alphavirus capsid protein and at least one nucleic acid sequence encoding a salmon alphavirus envelope protein selected from the group consisting of an E3, E2, 6K and E1 envelope protein, wherein the nucleic acid sequence encoding the salmon alphavirus capsid protein and/or the at least one nucleic acid sequence encoding the salmon alphavirus envelope protein is codon-optimised to resemble codon usage in a fish cell, and wherein the polynucleotide provides an enhanced immune response against salmon alphavirus when compared to a polynucleotide encoding a salmon alphavirus-like particle in which the nucleic acid sequences are not codon-optimised.
  • a vaccine construct comprising polynucleotide encoding a salmon alphavirus-like particle, wherein the polynucleotide comprises:
  • nucleic acid sequence encoding a salmon alphavirus capsid protein comprises the nucleic acid sequence set forth in SEQ ID NO: 5 or a nucleic acid sequence having at least 76.5% sequence identity thereto;
  • the nucleic acid sequence encoding the E3 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:7 or a nucleic acid sequence having at least 80.5% sequence identity thereto;
  • the nucleic acid sequence encoding the E2 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:9 or a nucleic acid sequence having at least 74.5% sequence identity thereto;
  • the nucleic acid sequence encoding the 6K envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 11 or a nucleic acid sequence having at least 73% sequence identity thereto;
  • the nucleic acid sequence encoding the E1 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 13 or a nucleic acid sequence having at least 76.5% sequence identity thereto.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein comprises the nucleic acid sequence set forth in SEQ ID NO:5;
  • the nucleic acid sequence encoding the E3 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:7;
  • the nucleic acid sequence encoding the E2 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:9;
  • the nucleic acid sequence encoding the 6K envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 11;
  • the nucleic acid sequence encoding the E1 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 13.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein consists of the nucleic acid sequence set forth in SEQ ID NO: 5;
  • the nucleic acid sequence encoding the E3 envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO:7;
  • the nucleic acid sequence encoding the E2 envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO:9;
  • the nucleic acid sequence encoding the 6K envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO: 11;
  • the nucleic acid sequence encoding the E1 envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO: 13.
  • the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:3 or a nucleic acid sequence having at least 76% sequence identity thereto.
  • the polynucleotide consists of the nucleic acid sequence set forth in SEQ ID NO:3.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein is operatively linked to a promoter.
  • the promoter is a CMV promoter or an EF1 ⁇ promoter.
  • the at least one nucleic acid sequence encoding the salmon alphavirus envelope protein is operatively linked to a promoter.
  • the promoter is a CMV promoter or an EFla promoter.
  • the polynucleotide further comprises a SV40 poly(A) sequence.
  • the polynucleotide further comprising a telomeric sequence.
  • telomeric sequences flank both ends of the polynucleotide construct.
  • the polynucleotide is a closed linear polynucleotide.
  • the present disclosure also extends to a cell comprising the polynucleotide described herein.
  • the present disclosure also extends to a salmon alphavirus-like particle encoded by the polynucleotide described herein.
  • the present disclosure also extends to a vaccine composition
  • a vaccine composition comprising the vaccine construct, the cell or the salmon alphavirus-like particle described herein.
  • the present disclosure also extends to a method for treating or protecting against salmon alphavirus infection in a subject, the method comprising administering to a subject in need thereof the vaccine construct, the salmon alphavirus-like particle or the composition described herein.
  • the vaccine construct, the salmon alphavirus-like particle or the composition described herein is administered to the subject by intramuscular injection.
  • the method described herein comprises administering to the subject the vaccine construct in an amount of from about 0.5 micrograms to about 10 micrograms.
  • the subject has, or is at risk of developing, salmonoid pancreatic disease or sleeping disease.
  • the present disclosure also extends to a use of the vaccine construct, the cell or the salmon alphavirus-like particle described herein, in the manufacture of a vaccine composition for treating or protecting against salmon alphavirus infection in a subject.
  • the present disclosure also extends to the vaccine construct described herein for use in treating or preventing against salmon alphavirus infection in a subject.
  • the present disclosure also extends to a method of inducing an immune response against salmonid alphavirus in a subject, the method comprising administering to a subject in need thereof the vaccine construct, the salmon alphavirus-like particle or the composition described herein.
  • the salmonid alphavirus is a SAV2 subtype.
  • the salmonid alphavirus is a SAV3 subtype.
  • the immune response comprises an antibody response, wherein the antibodies are capable of binding specifically to and neutralising the salmon alphavirus.
  • the antibodies are capable of binding specifically to and neutralising SAV2 and SAV3 subtypes.
  • the polynucleotide, the salmon alphavirus-like particle or the composition described herein is administered to the subject by intramuscular injection.
  • Figure 1 shows a schematic of the cohabitation challenge model trial design.
  • DPC days post challenge.
  • Figure 2 shows a schematic of Doggybone closed linear constructs (dbDNA) comprising a polynucleotide construct comprising capsid, E3, E2, 6K and E1 nucleic acid sequences encoding for a salmon alphavirus-like particle (A).
  • Figure 3 shows a schematic of Doggybone closed linear constructs (dbDNA) comprising a polynucleotide construct comprising codon-optimised modified capsid, E3, E2, 6K and E1 nucleic acid sequences encoding for a salmon alphavirus-like particle (A).
  • Figure 4 shows SAV viraemia post-challenge expressed as SAV genome copy number (log 10 ; mean + S.E.M.) in fish receiving negative control (-ve Control; dbDNA eGFP 7.5 ⁇ g), 3-dbDNA SAV3 (7.5 ⁇ g); or ClyNav® at the recommended dose of 6.0-9.4 ⁇ g plasmid DNA per 0.05 ml.
  • Figure 5 shows SAV viraemia post-challenge expressed as SAV genome copy number (logio; mean + S.E.M.) in fish receiving negative control dbDNA eGFP (-ve Control), very low dose of 3-dbDNA SAV3 (1.0 ⁇ g); or very low dose of 5-dbDNA SAV3 CO (1.0 ⁇ g).
  • Figure 6 shows SAV3 neutralising antibody titres in fish receiving a reference dose (7.5 ⁇ g) of either 3-dbDNA SAV3 or 5-dbDNA SAV3 CO, at 500 degree days post- immunisation, with individual data points around the median.
  • Figure 7 shows SAV3 neutralising antibody titres in fish receiving a reference dose of 5-dbDNA SAV3 CO (7.5 ⁇ g) or recommended dose of ClyNav®, at 500 degree days post- immunisation, with individual data points around the median.
  • Figure 8 shows SAV2 and SAV3 neutralising antibody titres in fish receiving a reference dose of 5-dbDNA SAV3 CO (7.5 ⁇ g) or recommended dose of ClyNav®, at 500 degree days post-immunisation, with individual data points around the median.
  • Figure 9 shows the nucleotide and polypeptide sequences disclosed herein.
  • sequence identifier numbers 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.
  • All sequence reference numbers e.g., GenBank ID, EMBL-Bank ID, DNA Data Bank of Japan (DDBK) ID, etc.
  • the present disclosure is predicated, at least in part, on the inventors' surprising finding that a vaccine construct encoding a salmon alphavirus-like particle, wherein the polynucleotide comprises nucleic acid sequences that have been codon-optimised to resemble codon usage in a fish cell, unexpectedly provides improved protection against salmon alphavirus infection when compared to a polynucleotide encoding a salmon alphavirus-like particle in which the nucleic acid sequences are not codon-optimised.
  • This finding is entirely surprising, as one would have expected salmon alpha virus to have naturally evolved towards optimal codon usage in fish cells.
  • active agent and “therapeutic agent” are used interchangeably herein and refer to agents that prevent, reduce or ameliorate at least one symptom of a disease or disorder.
  • administering concurrently or “co- administering” and the like refer to the administration of a single composition containing two or more agents, or the administration of each agent as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such agents are administered as a single composition.
  • simultaneous is meant that the agents are administered at substantially the same time, and desirably together in the same formulation.
  • temporary it is meant that the agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful.
  • the agents when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject.
  • the term “same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
  • the term “separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The agents may be administered in either order.
  • the term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the agents may be administered in a regular repeating cycle.
  • corresponding as used herein in reference to a particular gene is intended to mean an analogous or equivalent or comparable gene.
  • a corresponding endogenous gene it is intended to mean the analogous, equivalent or comparable naturally-occurring gene.
  • a corresponding exogenous gene it is intended to mean an analogous, equivalent or comparable exogenous gene.
  • the corresponding gene has analogous or equivalent function or having sequence similarity.
  • the corresponding gene may be identical in function and/or sequence.
  • the corresponding gene may have about the same function or activity.
  • the corresponding gene may have reduced function or activity.
  • the phrase “corresponds to” or “corresponding to” is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence.
  • the nucleic acid sequence will display at least about 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 even up to 100% sequence identity to the reference nucleic acid sequence.
  • encode refers to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide.
  • 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.
  • 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.
  • a processed RNA product e.g., mRNA
  • an effective amount in the context of treating a disease or condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition.
  • the effective amount will vary depending upon the age, health and physical condition of the individual to be treated and whether symptoms of disease are apparent, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject.
  • Optimum dosages may vary depending on the relative potency in an individual subject, and can generally be estimated based on EC50 values found to be effective in in vitro and in vivo animal models. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • expression typically refers to any step involved in the production of an RNA molecule or a polypeptide, such as by transcription, post- transcriptional modification, translation, post-translational modification, and secretion.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • nucleic acid refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA, or a combination thereof.
  • the term typically refers to polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single-, double- or triple- stranded forms of DNA and RNA.
  • nucleic acids of the present disclosure can be in isolated or purified form, and made, isolated and /or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology.
  • the nucleic acids can also be synthesised in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.
  • peptide “polypeptide” and “protein” are to be understood as referring to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain.
  • Amino acids are typically represented by their one-letter or three-letters code, according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (IIe); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai)
  • a “promoter” refers to one or more a nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter may include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter may optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • “Promoter” includes a minimal promoter that is a short nucleic acid sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which control elements (e.g., cis-acting elements) are added for control of expression.
  • Promoter also refers to a nucleotide sequence that includes a minimal promoter plus control elements (e.g., cis-acting elements) that are capable of controlling the expression of a coding sequence or functional RNA.
  • This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an “enhancer” is a nucleic acid sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter.
  • promoters bind sequence-specific nucleic acid-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic nucleic acid segments. A promoter may also contain nucleic acid sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as “minimal or core promoters.” In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison (e.g., over 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200 or more nucleotides or amino acids residues).
  • a “percentage of sequence identity” is 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) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu,IIe, 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.
  • the identical nucleic acid base e.g., A, T, C, G
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu,IIe, Phe, Tyr, Trp, Lys, Arg
  • sequence identity will be understood to mean the “match percentage” calculated by an appropriate method.
  • sequence identity analysis may be carried out using 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.
  • Sequences may be aligned using a global alignment algorithms (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)).
  • a global alignment algorithms e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970
  • a local alignment algorithm e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005).
  • Alignment for the purposes of determining percent amino acid sequence identity can be achieved by any means available to persons skilled in the art, illustrative examples of which include publicly available computer software, such as is available at http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Persons skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein, % sequence identity typically refers to values generated using pair wise sequence alignment that creates an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm).
  • sequence identity 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.
  • 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.
  • identity i.e., similarity
  • nucleotide and sequence comparisons are made at the level of identity rather than similarity.
  • leucine may be substituted for an isoleucine or valine residue. This may be referred to as a conservative substitution.
  • 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.
  • salmonid alphavirus 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.
  • SAV and SPDV are used interchangeably, and understood to mean salmonid alphaviruses that underlie PD and SD in salmonid species.
  • SAV1- 6 the causative agents of significant diseases of farmed Atlantic salmon, rainbow trout and other salmonids.
  • 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 the original subtype identified in Norway.
  • SAV4 consists of Atlantic salmon strains from Ireland
  • SAV5 consists of Scottish strains
  • 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).
  • SAV2 FW 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.
  • SAV3 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. There are currently two PD epidemics ongoing in Norway. SAV3 was the only subtype recognised in Norway until 2010, but since that time, SAV2 has become 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.
  • 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.
  • SAV1 and SAV3 are associated with cardiovascular and muscular tissue pathologies (Graham et al. (2011) J Fish Dis 34:273).
  • SD Sleeping disease
  • 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.
  • vaccination may be applied for control of PD and SD, although efficacious vaccines remain elusive.
  • the present disclosure provides a vaccine construct comprising polynucleotide encoding a salmon alphavirus-like particle, wherein the polynucleotide comprises a nucleic acid sequence encoding a salmon alphavirus capsid protein and at least one nucleic acid sequence encoding a salmon alphavirus envelope protein selected from the group consisting of an E3, E2, 6K and E1 envelope protein, wherein the nucleic acid sequence encoding the salmon alphavirus capsid protein and/or the at least one nucleic acid sequence encoding the salmon alphavirus envelope protein is codon-optimised to resemble codon usage in a fish cell, and wherein the polynucleotide provides an enhanced immune response against salmon alphavirus when compared to a polynucleotide encoding a salmon alphavirus-like particle in which the nucleic acid sequences are not codon-optimised.
  • the present disclosure also provides a vaccine construct comprising a polynucleotide encoding a salmon alphavirus-like particle, wherein the polynucleotide comprises:
  • nucleic acid sequence encoding a salmon alphavirus capsid protein comprises the nucleic acid sequence set forth in SEQ ID NO: 5 or a nucleic acid sequence having at least 76.5% sequence identity thereto;
  • the nucleic acid sequence encoding the E3 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:7 or a nucleic acid sequence having at least 80.5% sequence identity thereto;
  • the nucleic acid sequence encoding the E2 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:9 or a nucleic acid sequence having at least 74.5% sequence identity thereto;
  • the nucleic acid sequence encoding the 6K envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 11 or a nucleic acid sequence having at least 73% sequence identity thereto; and (iv) the nucleic acid sequence encoding the E1 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 13 or a nucleic acid sequence having at least 76.5% sequence identity thereto.
  • VLP Virus-like particles
  • capsid and I or envelope proteins also referred to herein as viral structural proteins
  • VLP may also lack or possess dysfunctional copies of certain genes of the native virus, and this may result in the virus-like-particle being incapable of some function that is otherwise characteristic of the native virus, such as replication and I or cell-cell movement.
  • VLP possess biologically desirable traits that are attributed, at least in part, to the particulate viral structure. Of particular interest is their efficient recognition, cellular uptake, and processing by host immune systems.
  • VLP are also amenable to a broad range of modifications including encapsulation, chemical conjugation, and genetic manipulation (see, e.g., Roldao et al. Expert Rev Vaccines 2010; 9(10): 1149-76), allowing VLP to be employed as suitable delivery agents for immunotherapy, illustrative examples of which include the prophylactic VLP vaccines Gardasil®, Cervarix®, Hecolin®, and Porcilis PCV®.
  • VLP also overcome some of the drawbacks associated with traditional vaccine production; namely, the infectious nature associated with live and inactivated vaccines and lengthy production time.
  • self-assembly typically refers to a process in which a system of pre- existing components, under specific conditions, adopts a more organised structure through interactions between the components themselves.
  • self-assembly refers to the intrinsic capacity of the encoded structural proteins (the capsid and the one or more envelope proteins), once expressed, to self-assemble into VLP, including in the absence of other viral proteins.
  • Self-assembly does not preclude the possibility that cellular proteins such as chaperones participate in the process of intracellular VLP assembly.
  • the self- assembly process may be influenced by factors such as choice of expression host, choice of expression conditions, and conditions for maturing the VLP.
  • virus capsid and / or envelope proteins may be able to form VLP on their own, or in combination with several virus capsid and / or envelope proteins, these optionally all being identical or related essential components of the virus structure.
  • virus-like particle and VLP are therefore used interchangeably herein to refer to one or several recombinantly expressed viral structural (capsid and envelope) proteins, which spontaneously assemble into macromolecular particulate structures mimicking the morphology of a virus coat, but lacking infectious genetic material.
  • the polynucleotide encodes a salmon alphavirus and at least one (e.g., 1, 2, 3 or 4) salmon alphavirus envelope proteins, preferably at least three or more preferably at least four salmon alphavirus envelope proteins.
  • the polynucleotide encodes a salmon alphavirus VLP comprising, consisting or consisting essentially of a salmon alphavirus capsid protein and one salmon alphavirus envelope protein.
  • the polynucleotide encodes a salmon alphavirus VLP comprising, consisting or consisting essentially of a salmon alphavirus capsid protein and two salmon alphavirus envelope proteins.
  • the polynucleotide encodes a salmon alphavirus VLP comprising, consisting or consisting essentially of a salmon alphavirus capsid protein and three salmon alphavirus envelope proteins. In an embodiment, the polynucleotide encodes a salmon alphavirus VLP comprising, consisting or consisting essentially of a salmon alphavirus capsid protein and four salmon alphavirus envelope proteins. It is to be understood that the VLP may comprise any number of two or more viral structural proteins, including any combination of viral capsid and I or envelope proteins, as long as the two or more viral structural proteins, once liberated following host cell expression, are capable of self-assembly to form a VLP.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein comprises, consists or consists essentially of the nucleic acid sequence of SEQ ID NO:5. In another embodiment, the nucleic acid sequence encoding the salmon alphavirus capsid protein comprises, consists or consists essentially of the nucleic acid sequence having at least 76.5% sequence identity to SEQ ID NO:5.
  • sequence identity By “at least 76.5% sequence identity” is meant 76.5%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, for example, after optimal alignment or best fit analysis.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein comprises, consists or consists essentially of a nucleic acid sequence having at least 76.5%, at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, 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% or more preferably at least 99% sequence identity to SEQ ID NO:5, for example, after optimal alignment or best fit analysis.
  • a salmon alphavirus capsid protein encoded by a nucleic acid sequence having at least 76.5% sequence identity to SEQ ID NO:5, as herein described will suitably be a “functional variant” of the reference sequence (i.e., of SEQ ID NO:5).
  • a “functional variant”, as used herein means a nucleotide sequence that differs from the reference sequence to which it is being compared, which may include a natural (i.e., native) sequence or a synthetic variant thereof, yet encodes a salmon alphavirus capsid protein capable of self-assembly (with other viral structural proteins) to form a VLP, as described herein.
  • the nucleic acid sequence encoding the salmon alphavirus E3 envelope protein comprises, consists or consists essentially of the nucleic acid sequence of SEQ ID NO:7.
  • the nucleic acid sequence encoding the salmon alphavirus E3 envelope protein comprises, consists or consists essentially of the nucleic acid sequence having at least 80.5% sequence identity to SEQ ID NO:7.
  • sequence identity 80.5%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:7, for example, after optimal alignment or best fit analysis.
  • the nucleic acid sequence encoding the salmon alphavirus E3 envelope protein comprises, consists or consists essentially of a nucleic acid sequence having at least 80.5%, having at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, 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% or more preferably at least 99% sequence identity to SEQ ID NO:7, for example, after optimal alignment or best fit analysis.
  • a salmon alphavirus E3 envelope protein encoded by a nucleic acid sequence having at least 80.5% sequence identity to SEQ ID NO:7, as herein described will suitably be a “functional variant” of the reference sequence (i.e., of SEQ ID NO:7).
  • a “functional variant”, as used herein means a nucleotide sequence that differs from the reference sequence to which it is being compared, which may include a natural (i.e., native) sequence or a synthetic variant thereof, yet encodes a salmon alphavirus E3 envelope protein capable of self-assembly (with other viral structural proteins) to form a VLP, as described herein.
  • the nucleic acid sequence encoding the salmon alphavirus E2 envelope protein comprises, consists or consists essentially of the nucleic acid sequence of SEQ ID NO:9. In another embodiment, the nucleic acid sequence encoding the salmon alpha virus E2 envelope protein comprises, consists or consists essentially of the nucleic acid sequence having at least 74.5% sequence identity to SEQ ID NO:9.
  • sequence identity By “at least 74.5% sequence identity” is meant 74.5%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9, for example, after optimal alignment or best fit analysis.
  • the nucleic acid sequence encoding the salmon alphavirus E2 envelope protein comprises, consists or consists essentially of a nucleic acid sequence having at least 74.5%, having at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, 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% or more preferably at least 99% sequence identity to SEQ ID NO:9, for example, after optimal alignment or best fit analysis.
  • a salmon alphavirus E2 envelope protein encoded by a nucleic acid sequence having at least 74.5% sequence identity to SEQ ID NO:9, as herein described will suitably be a “functional variant” of the reference sequence (i.e., of SEQ ID NO:9).
  • a “functional variant”, as used herein means a nucleotide sequence that differs from the reference sequence to which it is being compared, which may include a natural (i.e., native) sequence or a synthetic variant thereof, yet encodes a salmon alphavirus E2 envelope protein capable of self-assembly (with other viral structural proteins) to form a VLP, as described herein.
  • the nucleic acid sequence encoding the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of the nucleic acid sequence of SEQ ID NO: 11. In another embodiment, the nucleic acid sequence encoding the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of the nucleic acid sequence having at least 73% sequence identity to SEQ ID NO:11.
  • sequence identity 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% or 99% sequence identity to SEQ ID NO: 11, for example, after optimal alignment or best fit analysis.
  • the nucleic acid sequence encoding the salmon alphavirus 6K envelope protein comprises, consists or consists essentially of a nucleic acid sequence having at least 73%, having at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, 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% or more preferably at least 99% sequence identity to SEQ ID NO: 11, for example, after optimal alignment or best fit analysis.
  • a salmon alphavirus 6K envelope protein encoded by a nucleic acid sequence having at least 73% sequence identity to SEQ ID NO: 11, as herein described will suitably be a “functional variant” of the reference sequence (i.e., of SEQ ID NO:11).
  • a “functional variant”, as used herein means a nucleotide sequence that differs from the reference sequence to which it is being compared, which may include a natural (i.e., native) sequence or a synthetic variant thereof, yet encodes a salmon alphavirus 6K envelope protein capable of self-assembly (with other viral structural proteins) to form a VLP, as described herein.
  • the nucleic acid sequence encoding the salmon alphavirus E1 envelope protein comprises, consists or consists essentially of the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, the nucleic acid sequence encoding the salmon alphavirus E1 envelope protein comprises, consists or consists essentially of the nucleic acid sequence having at least 76.5% sequence identity to SEQ ID NO: 13.
  • sequence identity By “at least 76.5% sequence identity” is meant 76.5%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13, for example, after optimal alignment or best fit analysis.
  • the nucleic acid sequence encoding the salmon alphavirus E1 envelope protein comprises, consists or consists essentially of a nucleic acid sequence having at least 76.5%, 77%, 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, 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% or more preferably at least 99% sequence identity to SEQ ID NO: 13, for example, after optimal alignment or best fit analysis.
  • a salmon alphavirus E1 envelope protein encoded by a nucleic acid sequence having at least 76.5% sequence identity to SEQ ID NO: 13, as herein described will suitably be a “functional variant” of the reference sequence (i.e., of SEQ ID NO: 13).
  • a “functional variant”, as used herein means a nucleotide sequence that differs from the reference sequence to which it is being compared, which may include a natural (i.e., native) sequence or a synthetic variant thereof, yet encodes a salmon alphavirus E1 envelope protein capable of self-assembly (with other viral structural proteins) to form a VLP, as described herein.
  • nucleotide and protein are sequences provided in Figure 9.
  • polynucleotides comprising, consisting, or consisting essentially of nucleic acid sequences encoding fragments (e.g., truncated and partial sequences) of any one or more of the capsid and I or envelope proteins encoded by the polynucleotide described herein.
  • fragments e.g., truncated and partial sequences
  • Such fragments will suitably retain the ability to self- assemble into VLP under conditions that favour VLP formation.
  • the present disclosure also extends to natural variations of the specified capsid and I or envelope proteins encoded by the polynucleotide sequences described herein.
  • Such variants may comprise 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 variant retains the ability to self-assemble 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.
  • amino acids can be generally classed as: (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, and (5) aromatic— phenylalanine, tryptophan, and tyrosine.
  • the VLP will have a quaternary structure that mimics or substantially resembles the overall structure of a corresponding wild-type, native and/or authentic salmon alphavirus particle.
  • a salmon alphavirus VLP will be morphologically similar to a corresponding authentic, wild-type and/or native salmon alphavirus particle, since the VLP will typically have a similar conformation to native viral structural proteins.
  • the VLP may be engineered or a product of recombinant technology and, more particularly, recombinant DNA technology, although without limitation thereto.
  • the VLP may form spontaneously upon recombinant expression of the capsid and the at least one envelope proteins in an appropriate expression system. Methods for producing VLP will be familiar to persons skilled in the art, illustrative examples of which are discussed elsewhere herein.
  • recombinant is to be understood as meaning 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.
  • the more structural proteins are included in the VLP 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.
  • the VLP comprises only the capsid protein and the at least one envelope protein, as described herein.
  • the VLP 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 VLP.
  • the VLP may also be in the form of a capsid monomer, protein or peptide fragment of VLP or capsid monomer, or mixtures thereof. It is further contemplated that the VLP may further comprise a lipid envelope and/or an isolated genetic material such as DNA and/or RNA.
  • the present disclosure also extends to VLP produced using structural protein fragments or variant forms thereof, such as structural proteins that have been modified by the addition, substitution or deletion of one or more amino acids, although without limitation thereto.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein comprises the nucleic acid sequence set forth in SEQ ID NO:5;
  • the nucleic acid sequence encoding the E3 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:7;
  • the nucleic acid sequence encoding the E2 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO:9;
  • the nucleic acid sequence encoding the 6K envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 11;
  • the nucleic acid sequence encoding the E1 envelope protein comprises the nucleic acid sequence set forth in SEQ ID NO: 13.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein consists of the nucleic acid sequence set forth in SEQ ID NO: 5;
  • the nucleic acid sequence encoding the E3 envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO:7;
  • the nucleic acid sequence encoding the E2 envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO:9:
  • the nucleic acid sequence encoding the 6K envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO: 11;
  • the nucleic acid sequence encoding the E1 envelope protein consists of the nucleic acid sequence set forth in SEQ ID NO: 13.
  • the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:3 or a nucleic acid sequence having 76% sequence identity thereto.
  • at least 76% sequence identity is meant 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3, for example, after optimal alignment or best fit analysis.
  • the polynucleotide comprises the nucleic acid sequence having at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, 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% or more preferably at least 99% sequence identity to SEQ ID NO: 3, for example, after optimal alignment or best fit analysis.
  • nucleic acid sequence having at least 76% sequence identity to SEQ ID NO: 3, as herein described will suitably be a “functional variant” of the reference sequence (i.e., of SEQ ID NO: 3).
  • a “functional variant”, as used herein means a nucleotide sequence that differs from the reference sequence to which it is being compared, which may include a natural (i.e., native) sequence or a synthetic variant thereof, yet encodes the salmon alphavirus capsid and the at least one envelope proteins capable of self-assembly to form a VLP, as described herein.
  • the polynucleotide comprises, consists or consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 3. In an embodiment, the polynucleotide consists of the nucleic acid sequence set forth in SEQ ID NO:3.
  • the nucleic acid sequence encoding the salmon alphavirus capsid protein is operatively linked to a promoter.
  • Suitable promoters will be familiar to persons skilled in the art, illustrative examples of which include a CMV promoter, an EFla promoter, IRF1A promoter or the beta actin promoter.
  • the promoter is an IRF1A promoter.
  • the promoter is a beta actin promoter.
  • the promoter is a CMV promoter or an EFla promoter.
  • the promoter is a CMV promoter.
  • the promoter is an EFla promoter.
  • the at least one nucleic acid sequence encoding the salmon alphavirus envelope protein is operatively linked to a promoter.
  • the polynucleotide further comprises a poly(A) sequence. Suitable poly(A) sequences will be known to persons skilled in the art, illustrative examples of which are described in Proudfoot NJ (Genes Dev. 2011; 25(17): 1770-82).
  • the polynucleotide further comprises a SV40 poly(A) sequence.
  • the polynucleotide further comprises telomeric sequences.
  • the telomeric sequences flank both ends of the polynucleotide.
  • flank typically means that the telomeric sequence is next to or adjacent the end of the polynucleotide.
  • the polynucleotide is a closed linear polynucleotide.
  • the closed linear polynucleotide is a linear DNA covalently closed at both ends by hairpin loops.
  • the term ’’codon optimisation refers to the replacement of certain codons with synonymous codons which allows improved expression of the resultant polypeptide or protein, while keeping the amino acid sequence of a translated protein unchanged. This is based on the discovery that although there are 64 different codons (61 codons encoding for amino acids and 3 stop codons) but only 20 different translated amino acids, that is many amino acids can be encoded for by more than one codon.
  • Codon usage bias for a variety of organisms is known, such that a particular nucleotide sequence can be codon- optimised for expression in a host cell.
  • Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.
  • Codon-optimised coding regions can be designed by various different methods. This optimisation may be performed using methods which are available on-line, published methods, or a company which provides codon optimising services. One codon optimising method is described, e.g., in International Patent Publication No.
  • the nucleic acid sequence encoding the product is modified with synonymous codon sequences.
  • the entire length of the open reading frame (ORF) for the product can be modified.
  • only a fragment of the ORF may be altered.
  • only a few codons in the open reading frame are altered.
  • only one codon in the open reading frame is altered.
  • polynucleotides described herein may suitably be incorporated in a vector (e.g., a recombinant vector).
  • a vector e.g., a recombinant vector.
  • Suitable vectors will be familiar to persons skilled in the art, illustrative examples of which include non-viral and I or viral vectors.
  • Non-viral vectors may include, for instance, plasmid vectors (e.g., compatible with bacterial, insect, yeast and I or mammalian host cells).
  • Suitable polynucleotide constructs or 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.
  • a vector is employed to deliver the polynucleotide (e.g., to a cell in vitro or in vivo).
  • the polynucleotide or 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).
  • RNA replicons may also be utilised to improve the efficiency of gene expression and delivery, including, for example, the use of self-replicating viral replicons, RNA 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 mucosal delivery systems.
  • the polynucleotide or vector is a mini-chromosome.
  • mini-chromosomes include PAC, BAC, YACs and artificial chromosomes).
  • the polynucleotide or vector is a closed linear polynucleotide / DNA vector.
  • the closed linear polynucleotide / vector is a ministring DNA.
  • the closed linear polynucleotide / vector is a minigene.
  • the closed linear polynucleotide / vector is a close-ended linear duplex DNA (CELiD or ceDNA).
  • the closed linear polynucleotide / vector is a doggybone (dbDNATM) DNA.
  • dbDNATM 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.
  • 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.
  • 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.
  • a protelomerase target sequence is any DNA sequence whose presence in double stranded DNA template allows for its conversion into a telomeric end by the enzymatic activity of protelomerase.
  • protelomerase target sequences are, in general, palindromic sequences; that is, 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. This portion remains after the protelomerase has cleaved and re-ligated its cognate sequence.
  • the portion of the protelomerase target sequence at each end can be the same or different; for instance, the portion may be generated from protelomerase target sequences that have different cognate enzymes, or the same cognate enzyme.
  • 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.
  • the closed linear polynucleotide comprises covalently closed ends also described as hairpin loops, where base-pairing between complementary DNA strands is not present.
  • the hairpin loops join the ends of complementary DNA strands.
  • the closed linear polynucleotide construct comprises telomeric sequences.
  • the telomeric sequences are situated such that they cap the the closed linear polynucleotide.
  • the telomeric sequences flank both ends of the promoter-linked polynucleotide.
  • the polynucleotide construct comprises 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.
  • 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.
  • 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. [0104] The present disclosure also extends to a cell comprising the polynucleotide described herein.
  • Suitable host cells permissive for viruses and for the formation of VLP will be familiar to persons skilled in the art, illustrative examples of which are described in Sarkar et al. ⁇ Korean J. Microbiol. 2019;55(4):327-343), Santi et al. ⁇ Methods. 2006; 40(1): 66- 76), Roldao et al. ⁇ Expert Rev. Vaccines', 2010; 9(10): 1149-1176) and Ghislain Masavuli et al. ⁇ Front Microbiol. 2017; 8: 2413), and include bacteria, yeast, fungi, plant, insect, mammalian and avian cells.
  • the cell is a bacterium.
  • Suitable virus-permissive bacteria will be familiar to persons skilled in the art, an illustrative example of which includes Escherichia coli (Huang et al., NPJ Vaccines. 2017; 2:3).
  • the cell is a yeast cell.
  • Suitable virus permissive yeast cells will be familiar to persons skilled in the art, illustrative examples of which are described in Kumar and Kumar ⁇ FEMS Yeast Research, 2019; 19(2): foz007) and include Pichia Pastoris (Kim and Kim, Letters Appl. Micro., 2017; 64(2): 111-123), Saccharomyces cerevisiae (Zhao et al. Appl Microbiol Biotechnol. 2013; 97(24): 10445-52) and Hansenula polymorpha (Wetzel et al. J Biotechnol. 2019; 20;306:203-212).
  • the cell is a plant cell.
  • Suitable virus permissive plant cells will be familiar to persons skilled in the art, illustrative examples of which are described in Makarkov et al. ⁇ npj Vaccines', 2019; 4(17)) and Santi et al. ⁇ Methods. 2006; 40(1): 66-76), and include Nicotiana tabacum and Arabidopsis thaliana (Greco et al. Vaccine. 2007; 28;25(49):8228-40), Samsun NN and Solanum tuberosum cv. Solara.
  • the cell is an insect cell.
  • Suitable virus permissive insect cells will be familiar to persons skilled in the art, illustrative examples of which include Spodoptera frugiperda (sf9; Wagner et al. PLoS One. 2014; 9(4):e94401), Trichoplusia ni (BTI-TN5B 1- 4; Krammer et al., Mol Biotechnol. 2010; 45(3):226-234) and Drosophila Schneider 2 (S2; Park et al., J Virol Methods. 2018; 261:156-159).
  • the cell is a fish cell.
  • the cell is a salmon cell.
  • Fish cells suitably allow expression of the peptides encoded by the polynucleotide described herein, including because the polynucleotides described herein are codon-optimised for expression in fish.
  • the present disclosure also extends to a composition comprising the polynucleotide, the cell or the salmon alphavirus-like particle described herein.
  • the present disclosure also extends to a salmon alphavirus-like particle (VLP) encoded by the polynucleotide described herein.
  • VLP salmon alphavirus-like particle
  • VLP salmon alphavirus-like particle
  • Suitable culture methods and conditions I times suitable for producing VLP will be familiar to persons skilled in the art, understanding that the method steps and conditions I times will likely vary depending, for example, on the type of host cell employed. Illustrative examples of suitable methods of production are set out elsewhere herein and also described in Cheng and Mukhopadhyay (Virology, 2011; 413(2): 153-160), Charlton Hume et al. (ibid), Ghislain Masavuli et al. (ibid) and Vacher et al. (Mol. Pharmaceutics 2013; 10: 1596-1609).
  • the recombinant structural virus proteins used to prepare the VLP described herein can be readily prepared by standard genetic engineering techniques by the skilled person provided with the sequence of the wild-type protein. Methods of genetically engineering proteins are well known in the art, illustrative examples of which are described, for example, in Ausubel et al. (1994 and updates) Current Protocols in Molecular Biology, John Wiley & Sons, New York). Isolation and cloning of the nucleic acid sequence encoding the structural proteins can be achieved using standard techniques (see, e.g., Ausubel et al., ibid.).
  • the nucleic acid sequence can be obtained directly from the virus by extracting RNA by standard techniques and then synthesising cDNA from the RNA template (e.g., by RT-PCR).
  • the nucleic acid sequence encoding the structural proteins is then inserted directly or after one or more subcloning steps into a suitable expression vector.
  • suitable vectors include plasmids, phagemids, cosmids, bacteriophage, baculoviruses, retroviruses or DNA viruses.
  • the structural protein(s) can then be expressed and purified as described in more detail below.
  • the nucleic acid sequence encoding the structural protein(s) can be further engineered to introduce one or more mutations, such as those described above, by standard in vitro site-directed mutagenesis techniques known to persons skilled in the art.
  • the nucleic acid sequence encoding the structural proteins may also be rationally designed and synthesised. Mutations can be introduced by deletion, insertion, substitution, inversion, or a combination thereof, of one or more of the appropriate nucleotides making up the coding sequence. This can also be achieved, for example, by PCR based techniques for which primers are designed that incorporate one or more nucleotide mismatches, insertions or deletions.
  • the presence of the mutation can be verified by a number of standard techniques, for example by restriction analysis or by DNA sequencing.
  • Virus proteins may also be engineered to produce fusion proteins comprising one or more immunogens fused to virus coat protein. Methods for making fusion proteins are well known to those skilled in the art. DNA sequences encoding a fusion protein can be inserted into a suitable expression vector as noted above. Persons skilled in the art will appreciate that the DNA encoding the coat protein or fusion protein can be altered in various ways without affecting the activity of the encoded protein. For example, variations in DNA sequence may be used to optimise for codon preference in a host cell used to express the protein, or may contain other sequence changes that facilitate expression.
  • the expression vector may further include regulatory elements, such as transcriptional elements, required for efficient transcription of the DNA sequence encoding the coat or fusion protein.
  • regulatory elements such as transcriptional elements
  • suitable regulatory elements include promoters, enhancers, terminators, and polyadenylation signals.
  • the present disclosure therefore also provides vectors comprising a regulatory element operatively linked to one or more nucleic acid sequences encoding the VLP described herein.
  • suitable regulatory elements may be dependent on the host cell chosen for expression of the VLP and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, plant, mammalian or insect genes.
  • the expression vector may additionally contain heterologous nucleic acid sequences that facilitate the purification of the expressed VLP.
  • suitable heterologous nucleic acid sequences include affinity tags such as metal-affinity tags, histidine tags, avidin/streptavidin encoding sequences, glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences.
  • GST glutathione-S-transferase
  • the amino acids corresponding to expression of the nucleic acids can be removed from the expressed VLP prior to use, including clinical use, as described elsewhere herein. Alternatively, the amino acids corresponding to expression of heterologous nucleic acid sequences can be retained on the VLP if they do not interfere with subsequent use.
  • the expression vector can be introduced into a suitable host cell by one of a variety of methods known in the art, illustrative examples of which include stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors.
  • suitable host cells include bacterial, yeast, insect, plant and mammalian cells.
  • the VLP will suitably assemble 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 the intended use.
  • the VLP are typically produced by growing the host cell transformed by the expression vector under conditions whereby the viral proteins encoded by the polypeptide I vector are expressed and VLP can be formed.
  • the selection of the appropriate growth conditions will be familiar to persons skilled in the art.
  • the VLP can 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.
  • density gradient centrifugation e.g., sucrose gradients, PEG- precipitation, pelleting, and the like
  • standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • 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.
  • the presence of the VLP can be detected using conventional techniques known in the art, such as by electron microscopy, atomic force microscopy, biophysical characterisation, and the like (see, e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; and Hagensee et al., J. Virol.
  • the VLP can be isolated by density gradient centrifugation and / or identified by characteristic density banding.
  • cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.
  • the present disclosure also extends to a VLP produced by the methods described herein and a vaccine composition comprising the vaccine constructs and / or VLP, as described herein.
  • the present disclosure also extends to 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, the salmon alphavirus-like particle or the composition described herein.
  • the polynucleotide, the salmon alphavirus-like particle or the composition described herein is administered to the subject by intramuscular injection.
  • the method described herein comprises administering to the subject the polynucleotide construct in an amount of from about 0.1 microgram to about 20 micrograms. In an embodiment, the method described herein comprises administering to the subject the polynucleotide construct in an amount of from about 0.2 microgram to about 18 micrograms. In an embodiment, the method described herein comprises administering to the subject the polynucleotide construct in an amount of from about 0.3 microgram to about 15 micrograms. In a preferred embodiment, the method described herein comprises administering to the subject the polynucleotide construct in an amount of from about 0.5 microgram to about 10 micrograms.
  • the method described herein comprises administering to the subject the polynucleotide construct in an amount of from about 0.5 microgram to about 5 micrograms. In another preferred embodiment, the method described herein comprises administering to the subject the polynucleotide construct in an amount of from about 1 microgram to about 5 micrograms. [0126] In an embodiment, the subject has, or is at risk of developing, salmonoid pancreatic disease.
  • the present disclosure also extends to a use of the polynucleotide, the cell or the salmon alphavirus-like particle described herein, in the manufacture of a vaccine composition for treating or protecting against salmon alphavirus infection in a subject.
  • the present disclosure also extends to the polynucleotide described herein for use in treating or preventing against salmon alphavirus infection in a subject.
  • the present disclosure also extends to a method of inducing an immune response against a salmonid alphavirus in a subject, the method comprising administering to a subject in need thereof the polynucleotide, the salmon alphavirus-like particle or the composition described herein.
  • the salmonid alphavirus is a salmon alphavirus that results in salmon pancreas disease or sleeping disease in salmonid fish.
  • the salmonid alphavirus is a subtype selected from the group consisting of SAV1, 2, 3, 4, 5 and 6.
  • the salmonid alphavirus is a SAV1 subtype.
  • the salmonid alphavirus is a SAV4 subtype.
  • the salmonid alphavirus is a SAV5 subtype.
  • the salmonid alphavirus is a SAV6 subtype.
  • the salmonid alphavirus is a SAV2 subtype.
  • the salmonid alphavirus is a SAV3 subtype.
  • the present disclosure also extends to polynucleotides comprising a nucleic acid sequence encoding combinations of salmon alphavirus capsid proteins and one or more salmon alphavirus envelope proteins, including where the capsid and the one or more salmon alphavirus envelope proteins are from different salmon alphavirus subtypes, as described herein.
  • Suitable combinations may comprise capsid and envelope proteins of the same salmon alphavirus subtype (e.g., subtypes 1-6), or they may comprise capsid and envelope proteins of two or more different salmon alphavirus subtypes.
  • the combination comprises a capsid protein of salmon alphavirus subtype 1 and one or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 2-6, illustrative examples of which are described herein.
  • the combination comprises a capsid protein of salmon alphavirus subtype 2 and one or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1 and 3-6, illustrative examples of which are described herein.
  • the combination comprises a capsid protein of salmon alphavirus subtype 3 and one or more envelope 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.
  • the combination comprises a capsid protein of salmon alphavirus subtype 4 and one or more envelope 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.
  • the combination comprises a capsid protein of salmon alphavirus subtype 5 and one or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-4 and 6, illustrative examples of which are described herein.
  • the combination comprises a capsid protein of salmon alphavirus subtype 6 and one or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-5, illustrative examples of which are described herein.
  • a polynucleotide comprising a nucleic acid sequence encoding a combination of two or more salmon alphavirus envelope proteins from different salmon alphavirus subtypes, as described herein.
  • the combination comprises two or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 2-6, illustrative examples of which are described herein.
  • the combination comprises two or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1 and 3-6, illustrative examples of which are described herein.
  • the combination comprises two or more envelope 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.
  • the combination comprises two or more envelope 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.
  • the combination comprises two or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-4 and 6, illustrative examples of which are described herein.
  • the combination comprises two or more envelope proteins of a salmon alphavirus subtype selected from the group consisting of subtypes 1-5, illustrative examples of which are described herein.
  • the immune response comprises an antibody response.
  • the antibody response is the induction of antibodies capable of binding specifically to and neutralising the salmon alphavirus.
  • the antibodies are capable of binding specifically to and neutralising SAV2 and SAV3 subtypes.
  • the vaccine construct, VLP and I or compositions described herein can be formulated for administration by a variety of routes.
  • the vaccine construct, VLP and I or compositions described herein can be formulated for oral, topical, rectal or parenteral administration or for administration by inhalation, intranasally or spray.
  • parenteral includes subcutaneous injections, intradermal, intravenous, intramuscular, intrathecal, intrastemal injection and infusion techniques.
  • the vaccine construct, the VLP or the composition described herein is administered to the subject by intramuscular injection.
  • the vaccine construct, the VLP or the composition described herein is administered to the subject by intraperitoneal injection.
  • the vaccine construct, the VLP or the composition described herein is administered to the subject through immersion (i.e., via uptake through the gills), by introducing the vaccine construct, the VLP or the composition described herein into the water in which the fish inhabits.
  • the vaccine construct, VLP and I or compositions described herein will suitably comprise a therapeutically effective amount of the vaccine construct and I or VLP.
  • therapeutically effective amount typically means an amount of the vaccine construct and I or VLP, as described herein, necessary to attain the desired response, for example, the inducement of an immune response to the target (i.e., to the salmon alphavirus).
  • the appropriate dosage of the vaccine construct and / or VLP, as described herein may depend on a variety of factors including, but not limited to, a subject’s physical characteristics (e.g., age, weight, sex), whether the vaccine construct and / or VLP, as described herein, is being used as single agent or as part of adjuvant therapy, the progression (i.e., pathological state) of any underlying virus infection or disease, and other factors that may be recognised by persons skilled in the art.
  • an appropriate dosage of the vaccine composition see, e.g., in Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; and Gilman et al., (Eds), (1990), “Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press). 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.
  • Illustrative examples of a suitable therapeutically effective amount of vaccine construct and I or VLP for administration to a subject include from about 0.1 microgram to about 20 micrograms, from about 0.2 microgram to about 18 micrograms, from about 0.3 microgram to about 15 micrograms, from about 0.5 microgram to about 10 micrograms, from about 0.5 microgram to about 5 micrograms, or from about 1 microgram to about 5 micrograms.
  • Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • a therapeutically effective amount of the vaccine construct and I or VLP, as described herein, as defined herein, is effective to induce an immune response to the target, irrespective of genotype.
  • immune response typically refers to the development in a subject of a humoral and/or a cellular immune response to the target.
  • 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.
  • the vaccine construct and I or VLP, as described herein, when administered to a subject induces an immune response selected from one or more of a neutralising antibody response, a cytotoxic T lymphocyte (CTL) response, a natural killer T cell response and / or a helper T lymphocyte (e.g., CD4 + T cell) response and innate immune response to the target antigen.
  • CTL cytotoxic T lymphocyte
  • helper T lymphocyte e.g., CD4 + T cell
  • Methods for measuring an immune response 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), Western immunoblot, 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 or NKT cell responses, oxidative burst assays, cytotoxic- specific cell lysis assays, pentamer binding assays, and phagocytosis and apoptosis evaluation.
  • solid-phase heterogeneous assays e.g., enzyme
  • the vaccine construct and I or VLP can be administered to a subject in need thereof by any suitable route of administration, including administration of the composition orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, intradermally, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients.
  • suitable routes of administration including administration of the composition orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, intradermally, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients.
  • suitable routes of administration including administration of the composition orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, intradermally, in tablet,
  • administration is accomplished by intramuscular injection of the vaccine construct and I or VLP.
  • the vaccine construct and I or VLP, as described herein can be administered in a manner compatible with the route of administration and physical characteristics of the recipient (including health status) and in such a way that it elicits the desired effect(s) (e.g., the induction of a protective immune response against the target).
  • the vaccine construct and I or VLP may be administered to a recipient in isolation or in combination with other additional therapeutic agent(s).
  • a pharmaceutical composition comprising the vaccine construct and I or VLP, as described herein, is formulated for administration with additional therapeutic agent(s)
  • the administration may be simultaneous or sequential (i.e., administration of the vaccine construct and I or VLP is followed by administration of the additional agent(s) or vice versa).
  • 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.
  • the vaccine construct and / or VLP may be administered in conjunction with another antiviral agent.
  • An "antiviral agent” typically means an agent which, when administered to a subject, is capable of significantly reducing the virus titre in the blood or serum either directly (e.g., by inhibiting a viral enzyme activity) or indirectly ⁇ e.g., via modulation of the antiviral responses of a host cell), either transiently or in a sustained way.
  • the vaccine construct and I or VLP, as described herein may suitably be given in an appropriate single dosage in order to elicit an immune response.
  • the initial dose may be followed by boosting dose.
  • the boosting dose may comprise the same vaccine construct and I or VLP as the initial (priming) dose, whether at an equivalent dose (e.g., the same or similar dose), a lower dose or a higher dose as compared to the initial dose.
  • the administration regime need not differ from any other generally accepted vaccination programs. For instance, a single administration in an amount sufficient to elicit an effective immune response may be used. Alternatively, as noted above, other regimes of initial administration of the complex followed by boosting, including as described above. Boosting may occur at times that take place well after the initial administration if the immune response (as measured, e.g., by antibody titres) falls below acceptable levels.
  • the vaccine construct and I or VLP can be used in combination an additional immunopotentiator or adjuvant to enhance an immune response in the subject.
  • the present disclosure therefore extends to compositions further comprising an immunopotentiator or adjuvant.
  • the immunopotentiator or adjuvant is administered concomitantly with the polynucleotide and I or VLP, as described herein.
  • the immunopotentiator or adjuvant can be administered prior or subsequently to the polynucleotide and I or VLP, as described herein, depending on the need as can be suitably determined by persons skilled in the art.
  • immunopotentiator is intended to mean a substance that, when mixed with an immunogen, elicits a greater immune response than the immunogen alone.
  • an immunopotentiator can enhance immunogenicity and provide a superior immune response.
  • an adjuvant or other active ingredient optionally may be included in the compositions.
  • the polynucleotide and I or VLP, or compositions thereof as described herein are capable of efficiently potentiating an immune response in the absence of an additional adjuvant.
  • Suitable immunopotentiators or adjuvants will be familiar to persons skilled in the art, illustrative examples of which include mineral and non-mineral oil based emulsions, such as water in oil, oil in water, or water/oil/water (WOW) formulations.
  • mineral and non-mineral oil based emulsions such as water in oil, oil in water, or water/oil/water (WOW) formulations.
  • WOW water/oil/water
  • Another example of an emulsion based adjuvant includes squalene-based, oil-in-water nano-emulsion vaccine adjuvants (e.g. AddaVax (Invivogen)).
  • Immunopotentiating cytokines may also be used.
  • cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T- lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (see, e.g., US 5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL- 12, IL-15, IL-23, IL-7, IL-18, IFN-alpha. IFN-beta).
  • immunopotentiating cytokines can be included as polypeptide molecules in the compositions, or be encoded for by nucleic acids, which can be co-administered with the polynucleotide and I or VLP, as described herein, or be included in the polynucleotide as described herein.
  • CpG immunostimulatory oligonucleotides have also been reported to have immunopotentiating effects. Without being bound by theory or by a mode of application, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll- like receptors (TLR), mainly TLR9. CpG-triggered TLR9 activation enhances antigen- specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines.
  • TLR Toll- like receptors
  • Th1 cytotoxic T-lymphocyte
  • CTL cytotoxic T-lymphocyte
  • the Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a Th2 bias.
  • vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a Th2 bias.
  • CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak.
  • a CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany) which is a preferred component of the pharmaceutical composition of the present invention.
  • TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.
  • Suitable adjuvants include chemically modified CpGs (e.g., CpR, Idera), dsRNA analogues such as Poly(I:C) and derivates thereof (e.g.
  • AmpliGen RTM Hiltonol RTM, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, Bevacizumab.RTM., celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP- 547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system, which may act therapeutically and/or as an adjuvant.
  • the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan without undue experimentation.
  • the vaccine construct and I or VLP, or compositions thereof as described herein can further comprise one or more immunostimulatory or adjuvanting lipids.
  • Suitable immunostimulatory lipids will be familiar to persons skilled in the art, illustrative examples of which include glycolipids and phospholipids.
  • the immunostimulatory lipid is an immunostimulatory glycolipid or an immunostimulatory phospholipid.
  • the immunostimulatory lipid may be a naturally-occurring lipid or it may be synthetically derived (synthesised).
  • the immunostimulatory lipid is a synthetic lipid.
  • the synthetic lipid may resemble, in part or in whole, a naturally-occurring lipid.
  • immunostimulatory lipid analogues that is, synthetic immunostimulatory lipids that resemble, at least in part, naturally-occurring lipids and yet are still capable of acting as an adjuvant to potentiate a host's immune response to an immunogen.
  • Suitable immunostimulatory lipid analogues will be familiar to persons skilled in the art, illustrative examples of which are described in US patent publication nos. 20200009165 and 20190358318, Maeda et al. (Vaccine; 1989; 7(3):275-281); Jiang et al. (Carbohydr. Res., 2007; 342(6):784-796); Foster et al. (J. Med. Chem., 2018; 61(3):1045-1060), the contents of which are incorporated herein by reference in their entirety.
  • the immunostimulatory lipid may be a glycolipid. Suitable immunostimulatory glycolipids will be familiar to persons skilled in the art, illustrative examples of which are described in Kim et al. (Expert Rev Vaccines. 2008; 7(10): 1519-1532), Godfrey, etal. (Nat. Immunol 2015; 16: 1114-1123, and Cerundolo, et al. (Nat. Rev. Immunol. 2009; 9: 28-38).
  • the glycolipid is an alpha-anomeric glycolipid or a beta-anomeric glycolipid.
  • the glycolipid is an alpha-anomeric glycolipid.
  • the alpha-anomeric glycolipid is an alpha-anomeric glycosphingolipid.
  • the alpha-anomeric glycosphingolipid is ⁇ -galactosylceramide or ⁇ - glucosylceramide.
  • the alpha-anomeric glycosphingolipid is ⁇ - glucosylceramide.
  • the glycolipid is a beta-anomeric glycolipid.
  • the beta-anomeric glycolipid is a beta-anomeric glycosphingolipid.
  • the beta-anomeric glycolipid is selected from the group consisting of ⁇ -mannosylceramide, ⁇ -glucosylceramide and ⁇ -galactosylceramide.
  • the beta-anomeric glycolipid is ⁇ -mannosylceramide.
  • the immunostimulatory lipid may be a phospholipid. Suitable immunostimulatory phospholipids will be familiar to persons skilled in the art, illustrative examples of which are described in Godfrey, et al. (2015; ibid).
  • the phospholipid is lyso- phosphatidylcholine or lysophosphatidyl-ethanolamine.
  • Immunostimulatory lipids, as described herein, including immunostimulatory glycolipids and phospholipids, can vary in the length and saturation of their fatty acid chains (including the acyl and / or sphingosine chains).
  • ⁇ -GalCer C26 ⁇ -GalCer C24, ⁇ -GalCer C20:2 etc.
  • ⁇ -GalCer also known as KRN7000
  • KRN7000 has an 18C phytosphingosine and a 26C acyl chain
  • ⁇ -GalCer C20:2 has a C20 acyl chain and cis-diunsaturation at C11 and C14.
  • Suitable immunostimulatory lipids of varying length and saturation of their fatty acid chains will be familiar to persons skilled in the art, illustrative examples are described in Wun et al. (2011.
  • Immunity 34: 327-339 Such variation in the length and saturation of their fatty acid chains may impact on the immunostimulatory capacity of such lipids. Nevertheless, it is to be understood that the immunostimulatory lipids described herein, when administered to a host with an immunogen (e.g., by enhancing the update of the VLP I polynucleotides, as described herein, by antigen presenting cells), will be capable of potentiating the host's immune response to the immunogen, irrespective of the length and saturation of their fatty acid chains.
  • an immunogen e.g., by enhancing the update of the VLP I polynucleotides, as described herein, by antigen presenting cells
  • an immunostimulatory lipid is capable of potentiating the host's immune response to an immunogen according to the methods described herein, irrespective of the length and saturation of their fatty acid chains, will be known to persons skilled in the art. Where two or more immunostimulatory or adjuvanting lipids are contemplated, it is generally desirable to select a combination of two or immunostimulatory or adjuvanting lipids that further enhance or potentiate the host's immune response to the immunogen, irrespective of whether the enhancement or potentiation is additive or synergistic.
  • non-immunostimulatory lipids e.g., neutral, cationic and/or ionisable lipids, cholesterol and polyethylene glycol.
  • suitable lipids are also described in US 20190314291, the entire contents of which is incorporated herein by reference.
  • immunological preparation and “vaccination” are used interchangeably herein to refer to the administration of the vaccine construct and I or VLP, as described herein, to a subject for the purposes of raising an immune response and can have a prophylactic effect, a therapeutic effect, or a combination thereof.
  • the terms “treat,” “treated,” or “treating” when used with respect to a disease or pathogen refers to a treatment which increases the resistance of a subject to the disease or to infection with a pathogen (i.e. decreases the likelihood that the subject will contract the disease or become infected with the pathogen), as well as a treatment after the subject has contracted the disease or become infected with the pathogen in order to fight a disease or infection (e.g., to reduce, eliminate, ameliorate or otherwise stabilise a disease or infection).
  • the vaccine construct and I or VLP, as described herein are capable of providing protective immunity to a host.
  • protective immunity is intended to mean the ability of a host (e.g., salmon), to resist (delayed onset of symptoms, reduced severity of symptoms or lack of symptoms), as a result of its exposure to the vaccine construct and I or VLP, as described herein, disease or death that would otherwise follow exposure to a pathogen.
  • Protective immunity is typically achieved by one or more of mucosal, humoral, or cellular immunity.
  • subject or host refers to an animal in need of treatment.
  • the subject or host is a fish, more preferably salmon, including both fresh water and salt water species.
  • kits or packs comprising the vaccine construct and I or VLP and I or compositions, as described herein, including for use as a vaccine.
  • Individual components of the kit can be packaged in separate containers, associated with which, when applicable, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for administration.
  • the kits may optionally further comprise one or more other therapeutic agents for use in combination with the vaccine construct and I or VLP and I or compositions, as described herein.
  • the kit may optionally contain instructions or directions outlining the method of use or administration regimen.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the solution may be administered to a subject or applied to and mixed with the other components of the kit.
  • the components of the kit may also suitably be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components.
  • the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient.
  • Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or similar medically approved delivery vehicle.
  • Figure 1 is schematic describing the cohabitation challenge model trial design.
  • the fish in the trial were Atlantic salmon (Salmo salar), StofnFiskur Optimal strain.
  • the fish were 24.2 g average weight at vaccination.
  • the fish were maintained in freshwater, adjusted according to minimum 70% oxygen in effluent water, with the maximum stocking density of 40 kg/ms, at 12°C ⁇ 1° C.
  • the fish were fed 1-2% of biomass by automatic feeder. See table below for summary of hatchery conditions.
  • the fish were acclimatised at the test facility for a minimum of one week prior to vaccination, and were starved for a minimum of 48 hours prior to vaccination.
  • the fish were identified by visible implant elastomer (VIE) tags, unvaccinated at parr stage, and kept in freshwater throughout the trial.
  • Vaccination was performed by intramuscular (i.m.) injection (0.05mL/fish), using 1 mL disposable syringes supplied with 0.5 x 3 mm stainless steel needles positioned at 90° on the left epaxial muscle, central to the dorsal fin and above the midline. Duration of immunisation was 500-degree days.
  • 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
  • the SAV3 was a Norwegian isolate from a field outbreak in 2007, grown in CHSE- 2014 at Norwegian Veterinary Institute. Original concentration 10 5.5 TCID 50 /mL. Challenge inoculum was diluted with PBS in 1:5 ratio. Published: Taksdal T et al., J Fish Dis 2015 (12): 1047-61.
  • Duration the challenge was 28 days. The first day of vaccination is day 0. Samplings were carried out at 0, 14, 17, 21, 24 and 28 days post-challenge (dpc). At each sampling point, 10 randomly selected fish from each group were sampled.
  • Heart tissue was sampled from five randomly selected shedders at the termination of challenge for qPCR analysis to verify presence of SAV infection.
  • qPCR quantitative polymerase chain reaction
  • the assay was performed at Norwegian Veterinary Institute according to the general method of Graham et al, 2003 with some modifications.
  • plasma samples were titrated against SAV3 (Isolate 4 from Taksdal et al., 2015) or SAV2 (Isolate 1 from Taksdal et al., 2015, GenBank ref. HE863664) on CHSE-214 cells in 2 parallel wells (96 well plate) in a two-fold dilution series. After 7 days of incubation at 15° C, the cell layer was fixed with 80% acetone.
  • SAV-infected cells were then visualised in an indirect immuno- fluorescence test according to the procedure described by Falk, Namork & Dannevig, 1998, but with the use of monoclonal antibody 17H23 directed against the E2 glycoprotein of SAV (Merour et al. 2016) as the primary antibody and with biotin labelled goat anti-mouse Ig and FITC-labelled streptavidin included in the assay. Positive staining was scored in a fluorescence microscope. Antibody titres ⁇ 1:20 were recorded as positive results.
  • EXAMPLE 1 CLOSED LINEAR POLYNUCLEOTIDE IS EFFECTIVE AS A VEHICLE FOR IMMUNSING ATLANTIC SALMON AGAINST SAV
  • Plasmid DNA have been the predominant DNA-based immunisation vectors.
  • a closed linear polynucleotide construct comprising a polynucleotide sequence comprising SAV3 capsid, E3, E2, 6K and E1 nucleic acid sequences (SEQ ID NO:2) was generated (dbDNA SAV3 in Figure 2).
  • Doggybone DNA (dbDNATM) is a proprietary synthetic closed linear double-stranded DNA construct.
  • Construct 3-dbDNA SAV3 (SEQ ID NO: 14) was used to immunise fish, and its effects compared to plasmid DNA-based ClyNav® vaccine using a cohabitation challenge model trial design.
  • a dbDNA comprising polynucleotide GFP sequences (dbDNA eGFP) was used as a negative control.
  • dbDNATM is at least as capable as plasmid DNA-based vehicle as inducing a protective immune response. Further, this shows that dbDNATM construct comprising SAV3 nucleic acid sequences may be useful for immunising Atlantic salmon.
  • SEQ ID NO:2 is the wildtype polynucleotide sequence (GenBank: AY604238) that encodes for a SAV subtype-3 structural polyprotein having the amino acid sequence of SEQ ID NO: 1.
  • the polynucleotide sequence encoding for SEQ ID NO:1 was modified to comprise codons that were optimised for expression in fish cells.
  • SEQ ID NO: 3 is one such codon-optimised nucleotide sequence that was inserted into a closed linear proprietary synthetic closed linear double-stranded DNA construct to generate 5-dbDNA SAV3-CO construct (SEQ ID NO: 15), as shown in Figure 3.
  • Pairwise group non-parametric comparison of AUCLogGCN indicates the overall level of viraemia observed in fish vaccinated with 3-dbDNA SAV3 1.0 ⁇ g was not significantly lower than that seen in the negative control group (i.e. there was no meaningful impact of vaccination). In contrast, the overall level of viraemia seen for 5-db SAV3-CO 1.0 ⁇ g was significantly lower than that seen in the negative control group, indicating a positive impact of vaccination with the codon optimized form, even at very low dose (1.0 ⁇ g).
  • EXAMPLE 3 MODIFIED dbDNA SAV3 CONSTRUCT PROVIDES IMPROVED NEUTRALISING ANTIBODY RESPONSE
  • EXAMPLE 4 MODIFIED DBDNA SAV3 CONSTRUCT PROVIDES POTENT CROSS-NEUTRALISATION OF SAV2

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Abstract

La présente invention concerne de manière générale des constructions de vaccin comprenant un polynucléotide codant pour une particule de type alphavirus de saumon et leurs utilisations, y compris pour induire une réponse immunitaire contre un alphavirus de salmonidé.
PCT/IB2023/059738 2022-09-29 2023-09-29 Construction de vaccin et ses utilisations WO2024069549A1 (fr)

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