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EP4587053A1 - Vaccins contre l'anémie infectieuse du saumon et leurs utilisations - Google Patents

Vaccins contre l'anémie infectieuse du saumon et leurs utilisations

Info

Publication number
EP4587053A1
EP4587053A1 EP23864220.1A EP23864220A EP4587053A1 EP 4587053 A1 EP4587053 A1 EP 4587053A1 EP 23864220 A EP23864220 A EP 23864220A EP 4587053 A1 EP4587053 A1 EP 4587053A1
Authority
EP
European Patent Office
Prior art keywords
composition
isav
nucleic acid
fish
hpr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23864220.1A
Other languages
German (de)
English (en)
Inventor
Mark David FAST
Sara PURCELL
Shona WHYTE
Andrew Keith Swanson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gmg Fish Services Inc
Original Assignee
Gmg Fish Services Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gmg Fish Services Inc filed Critical Gmg Fish Services Inc
Publication of EP4587053A1 publication Critical patent/EP4587053A1/fr
Pending legal-status Critical Current

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    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07048—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01007—Acetylcholinesterase (3.1.1.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01053—Sialate O-acetylesterase (3.1.1.53)
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle.
  • the ISAv is selected from HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type.
  • the composition comprises a plurality of nucleic acid molecules encoding one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof.
  • the plurality of nucleic acid molecules encode one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof from more than one HPR strain type.
  • An aspect of the present disclosure includes a composition comprising one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof and a carrier, optionally a protein carrier.
  • the one or more ISAv antigenic polypeptides is acetylcholinesterase (P3) or fusion (F) protein (P3/F), hemagglutinin-esterase (HE) protein or polymerase basic protein 2 (PB2).
  • P3 acetylcholinesterase
  • F fusion protein
  • HE hemagglutinin-esterase
  • PB2 polymerase basic protein 2
  • the one or more ISAv antigenic polypeptides are encoded by nucleic acid molecules that comprise or consist of a sequence selected from SEQ ID Nos: 1-8 or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID Nos: 1-8.
  • the ISAv antigenic polypeptide is P3/F, optionally long P3/F or short P3/F.
  • the ISAv is selected from HPR 4 type, HPR 7a type,
  • HPR 7b type or HPR 14 type are HPR 7b type or HPR 14 type.
  • Another aspect of the present disclosure includes a composition as described herein for use in inducing an immune response against ISAv in fish.
  • the fish is salmon or trout.
  • the fish is Salmo salar (Atlantic salmon), Salmo trutta (brown trout) or Oncorhynchus mykiss (rainbow trout).
  • Another aspect of the present disclosure includes inducing an immune response against ISAv in a fish, the method comprising administering to the fish a composition as described herein in an amount effective to produce an immune response in the fish.
  • the fish is administered a single dose of the vaccine, optionally followed by a second dose.
  • the method treats or prevents ISAv infection.
  • the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle.
  • a further aspect of the present disclosure includes a use of a nucleic acid as described herein or RNA transcribed as described herein for inducing an immune response against ISAv in fish.
  • Figure 1 shows PCR product results for each different ISAv segment and linearized plasmid.
  • S Segment, 1 , 6, 5 (segment 5-1 , 5-2 for the two different reading frames from the Fusion protein gene).
  • pBLU pBLU plasmid. DNA ladder 0.5 - 3.0 kb included in each gel.
  • Figure 2 shows PCR inserts cloned into pBLU expression vector for proper orientation and sequence confirmation.
  • Higher Kb band is linearized plasmid
  • Lanes 1-6 are Segment 5-1 inserts
  • Lanes 8-13 are segment 5-2 inserts
  • Lane 7 containing 0.5- 3 kb DNA ladder.
  • Figure 3 shows PCR inserts cloned into pCR2.1 vector expression for proper orientation and sequence confirmation.
  • Higher Kb band is linearized plasmid
  • Lanes 1-6 are Segment 1 inserts
  • Lanes 9-13 are segment 6 inserts
  • Lane 7 containing 0.5-3 kb DNA ladder.
  • Figure 5 shows mean ( ⁇ SEM) calibrated normalized relative quantities (CNRQ) of Mx1 gene in Atlantic salmon kidney (ASK) cells, 24 hours after exposure to individual vaccinates, + control (ISAv) or - control (PBS).
  • Figure 6 shows mean ( ⁇ SEM) calibrated normalized relative quantities (CNRQ) of Mx1 gene in Atlantic salmon kidney (ASK) cells, 48 hours after exposure to individual vaccinates, + control (ISAv) or - control (PBS).
  • Figure 7 shows infectious salmon anemia mortality curve in Atlantic salmon smolts at 10 and 20°C.
  • Figure 8 shows donor daily mortality and survival across tanks.
  • A shows daily mortality with the O2 event in tank 3, whereas B shows the daily mortality without this event.
  • C shows daily survival across individual replicate tanks with the O2 event included.
  • Figure 9 shows daily mortality of commercial (Micro Forte V2; A) and sham (PBS; B) vaccinated Atlantic salmon smolts (total of all three replicates).
  • nucleic acid means two or more covalently linked nucleotides. Unless the context clearly indicates otherwise, the term generally includes, but is not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which may be single-stranded (ss) or double stranded (ds).
  • the nucleic acids can be any length depending upon the application, for example from 30 bp to 8kb or longer, optionally up to 200 base pairs in length or for example up to 8kb or longer, and may be single-stranded or double-stranded.
  • nucleic acid and its derivatives, as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA and includes isolated nucleic acids.
  • polypeptide refers to two or more amino acids linked by a peptide bon, and includes synthetic and natural polypeptides as well as polypeptides that are modified. Various lengths of polypeptides are contemplated herein.
  • primer generally refers to single-stranded DNA for example from about 30 to up to 200 base pairs in length that can be used to produce a transcription product.
  • anneal and hybridize refer to the ability of a nucleic acid to non-covalently interact with another nucleic acid through base-pairing.
  • complementary or complementary nucleic acid refer to a nucleic acid or a portion of a nucleic acid that is able to anneal with a nucleic acid of a given sequence. In some cases this is referred to as the “reverse complement” or “anti-sense” of a given sequence.
  • gene refers to a DNA sequence that comprises a coding sequence associated with the production of a polypeptide or polynucleotide product (e.g., mRNA, rRNA, tRNA).
  • a polypeptide or polynucleotide product e.g., mRNA, rRNA, tRNA.
  • the term “gene” may be used refer to a genomic DNA sequence comprising for example introns, exons, promoters, and other regulatory sequences, or may be used to refer to the coding sequence, or open reading frame, which encodes the polypeptide or polynucleotide product.
  • sequence identity refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules.
  • the default parameters of the respective programs e.g. of XBLAST and NBLAST
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • immune response can refer to activation of either or both the adaptive and innate immune system cells such that they shift from a dormant resting state to a state in which they are able to elaborate molecules typical of an active immune response.
  • inducing an immune response refers to a method whereby an immune response is activated.
  • enhancing an immune response refers to augmenting an existing immune response.
  • an effective amount means an amount of a composition or nucleic acid of the application that is effective, at dosages and for periods of time necessary to achieve the desired result.
  • an effective amount is an amount that, for example, induces inducing an immune response against ISAv a fish or treats or prevents ISA in fish. Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the fish. The amount of a given composition, nucleic acid or polypeptide that will correspond to such an amount will vary depending upon various factors, such as the pharmaceutical formulation, the route of administration and the like, but can nevertheless be routinely determined by one skilled in the art.
  • vaccine refers to a composition that induces an immune response upon administration to a subject.
  • the induced immune response provides protective immunity.
  • compositions that are useful for inducing an immune response against infectious salmon anemia (ISA).
  • composition comprising one or more nucleic acid molecules encoding one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof and optionally a carrier.
  • ISAv infectious salmon anemia virus
  • composition comprising one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof and optionally a carrier.
  • ISAv infectious salmon anemia virus
  • Infectious salmon anemia virus belongs to the genus Isavirus, family Orthomyxoviridae. Various strains and subtypes of ISAv are known, some of which have differing levels of virulence.
  • the term “ISAv” refers to ISAv of any strain or subtype, including for example the North American subtype and the European subtype.
  • the HPR classification system is commonly used for North American and European ISAv strains.
  • the ISAv is HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type.
  • the ISAv is HPR 4 type.
  • Also contemplated herein is any other existing or emergent pathogenic strain or subtype of ISAv.
  • the term “antigen” or “antigenic” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • any DNA or RNA which comprises a nucleotide sequence or a partial nucleotide sequence encoding a polypeptide that elicits an adaptive immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded by a full-length nucleotide sequence of a gene or a full-length protein.
  • infectious salmon anemia virus (ISAv) antigenic polypeptide refers to an antigenic polypeptide from ISAv.
  • the ISAv genome comprises eight negative-sense single-stranded RNA segments that encode at least 10 proteins.
  • Segment 1 encodes polymerase basic protein 2 (PB2), a component of the virion RNA polymerase;
  • segment 2 encodes polymerase basic protein 1 (PB1 );
  • segment 3 the nucleocapsid protein (NP);
  • segment 4 the RNA polymerase (PA).
  • PB2 polymerase basic protein 2
  • PB1 polymerase basic protein 1
  • segment 3 the nucleocapsid protein
  • PA RNA polymerase
  • Unlike influenza A viruses where fusion and hemagglutinin activity are present on the same polypeptide chain in ISAv these correspond to two separate gene segments: segment s, acetylcholinesterase (P3) or fusion (F) protein (P3/F) and segment 6, hemagglutinin-esterase (HE).
  • Segment 7 encodes proteins P4 and P5; and segment 8, proteins P6 and P7.
  • proteins P4 and P5 may be the ISAv counterparts to the membrane proteins M1 and M2 of influenza A virus and proteins P6 and P7 may be related to the nonstructural proteins NS1 and NEP of influenza A virus.
  • the antigenic polypeptide is an ISAv protein.
  • the antigenic polypeptide is an ISAv protein selected from polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1 ), nucleoprotein (NP), polymerase acidic (PA) protein, acetylcholinesterase (P3) or fusion (F) protein (P3/F), hemagglutinin-esterase (HE) protein, P4, P5, P6 and P7.
  • the antigenic polypeptide is selected from acetylcholinesterase (P3) or fusion (F) protein (P3/F), hemagglutinin-esterase (HE) protein and polymerase basic protein 2 (PB2).
  • PB2 protein is encoded by to SEQ ID NO: 5 (from SEGMENT 1) and HE protein (from SEGMENT 6) is encoded by SEQ ID NO: 8.
  • P3/F protein is encoded by SEQ ID NO: 6 (from SEGMENT 5-1) or SEQ ID No: 7 (from SEGMENT 5-2). The sequences encoding P3/F are both on segment 5 but correspond to different lengths of the same P3/F gene based on the potential different open reading frames.
  • the version of P3/fusion protein encoded by SEGMENT 5-1 is referred as the “short” (S) version of P3/fusion protein and the version of P3/fusion protein encoded by SEGMENT 5-2 is referred to as the “long” (L) version of P3/fusion protein.
  • the one or more nucleic acid molecules comprise or consist of a sequence selected from SEQ ID Nos: 1-8 or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to any one of SEQ ID Nos: 1-8.
  • the RNA is mRNA.
  • the mRNA optionally has a poly-A tail and can be capped or uncapped.
  • immunogenic variant or fragment thereof refers to a variant or fragment of an antigenic polypeptide that induces an immune response to ISAv.
  • the variant has at least 60, 65, 70, 75, 80, 85, 90, 95 or 99% sequence identity to the full length of the salmon anemia virus (ISAv) antigenic polypeptide or a fragment thereof.
  • ISAv salmon anemia virus
  • a “variant” polypeptide is a “conservatively substituted variant”.
  • a “conservatively substituted variant” refers to a variant with at least one conservative amino acid substitution.
  • a “conservative amino acid substitution” as used herein, refers to the substitution of an amino acid with similar hydrophobicity, polarity, and R-chain length for one another. In a conservative amino acid substitution, one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Without the intention of being limited thereby, in one embodiment, the substitutions of amino acids are made that preserve the structure responsible for the ability of the peptide to increase glucose uptake or decrease hepatic glucose production as disclosed herein. Examples of conservative amino acid substitutions include:
  • the fragment is at least 5, 10 or 20 amino acids in length. In another embodiment, the fragment is 100% identical to the full-length antigenic polypeptide except missing at least one amino acid from the N and/or C terminal, in each case with or without signal peptides and/or a methionine at position 1.
  • the fragment comprises 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91 % or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the full length antigenic polypeptide and optionally additionally comprises an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.
  • the composition comprises a nucleic acid molecule or a plurality of nucleic acid molecules that encode more than one infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof.
  • ISAv infectious salmon anemia virus
  • the composition can comprise nucleic acid molecules that encode different ISAv antigenic polypeptides, different immunogenie fragments and/or variants of the same ISAv antigenic polypeptides, or immunogenic variants or fragments thereof of different strain types or any combination thereof.
  • nucleic acid molecules of the composition are optionally formulated, or encapsulated, in a nucleic acid carrier.
  • nucleic acid carrier refers to compounds that facilitate transfer of the nucleic acid into cells, such as, for example, nanoparticles, polylysine compounds, liposomes, and the like.
  • the nucleic acid carrier is a lipid nanoparticle.
  • the nucleic acid carrier is a phospholipid, sterol, PEG-lipid or cationic phytoglycogen nanoparticle.
  • the nucleic acid carrier is an expression vector.
  • expression vectors include, but are not limited to, plasmids and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the nucleic acid molecule(s).
  • the composition comprises more than one infectious salmon anemia virus (ISAv) antigenic polypeptide or an immunogenic variant or fragment thereof.
  • the composition can comprise different ISAv antigenic polypeptides, different immunogenic fragments and/or variants of the same ISAv antigenic polypeptides, or immunogenic variants or fragments thereof of different strain types or any combination thereof.
  • the antigenic polypeptides or immunogenic variants or fragments thereof are optionally formulated, or encapsulated, in a protein carrier.
  • protein carrier refers to compounds that facilitate transfer of the polypeptide into an organism or its cells.
  • the polypeptides described herein are optionally modified for cell permeability, improved stability, and/or better bioavailability.
  • polypeptides described herein may be prepared using recombinant DNA methods. These polypeptides may be purified and/or isolated to various degrees using techniques known in the art. Accordingly, nucleic acid molecules having a sequence which encodes a polypeptide of the disclosure may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the polypeptide. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.
  • vectors suitable for transformation of a host cell means that the expression vectors contain a nucleic acid molecule encoding a polypeptide of the disclosure and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. “Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
  • polypeptides are isolated from ISAv using methods known in the art.
  • polypeptides may be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
  • the polypeptides may also be modified with an enhancer moiety.
  • the polypeptide is conjugated directly or indirectly to the enhancer moiety.
  • an enhancer moiety can increase or enhance the activity of the polypeptide.
  • the enhancer may be a permeability enhancer, a stability enhancer or a bioavailability enhancer.
  • the enhancer moiety is a PEG moiety.
  • the polypeptides may also be modified with a cell-penetrating moiety.
  • the term “cell-penetrating moiety” refers to a moiety that promotes cellular uptake of the peptide upon delivery to a target cell.
  • polypeptides can also be conjugated to a carrier protein, thereby forming a fusion protein.
  • composition also optionally comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Optional examples of such carriers include, but are not limited to, water, saline, ringer’s solution and dextrose solution.
  • the pharmaceutically acceptable carrier is a carrier acceptable for administration to fish.
  • a composition described herein is formulated to be compatible with its intended route of administration.
  • routes of administration include intramuscular injection, intraperitoneal injection or oral delivery.
  • the composition is formulated in a dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the fish to be treated.
  • compositions described herein further comprise an agent that enhances its function.
  • the composition can also contain other active ingredients as necessary or beneficial for the particular indication being treated, optionally those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the disclosure provides a nucleic acid comprising or consisting of any one of SEQ ID Nos: 1-4.
  • the disclosure provides a nucleic acid having at least 50, 60, 70, 80, 90, 95 or 99% sequence identity with any one of SEQ ID NO: 1 to 4 or a nucleic acid that hybridizes to a nucleic acid comprising or consisting of any one of SEQ ID NO: 1 to 4 under at least moderately stringent hybridization or stringent hybridization conditions.
  • the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature.
  • a 1 % mismatch may be assumed to result in about a 1 ° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C.
  • stringent hybridization conditions are selected.
  • Moderately stringent hybridization conditions include a washing step in 3xSSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in Ausubel, 1989 and in Sambrook et al., 1989.
  • the one or more nucleic acid molecules are RNA molecules corresponding to SEQ ID Nos: 1-4, a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to SEQ ID Nos: 1-4 or a nucleic acid that hybridizes to a nucleic acid comprising or consisting of any one of SEQ ID NO: 1 to 4 under at least moderately stringent hybridization or stringent hybridization conditions.
  • RNA molecules corresponding to a particular DNA sequence refers to RNA transcribed from the DNA sequence.
  • the RNA is mRNA.
  • the mRNA optionally has a poly-A tail and can be capped or uncapped.
  • the disclosure further contemplates a vector comprising a nucleic acid described herein, optionally a recombinant expression vector containing a nucleic acid molecule that encodes a peptide of the disclosure and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence.
  • the vector is a viral vector such as a retroviral, lentiviral, adenoviral oradeno- associated viral vector.
  • Recombinant expression vectors can be introduced into host cells to produce a transformed host cell.
  • the term “transformed host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the disclosure.
  • the terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • a recombinant cell expressing a nucleic acid or vector described herein.
  • the cell is a bacterial cell, yeast cell, a mammalian cell, or a plant cell.
  • compositions, nucleic acids and polypeptides described herein are useful for inducing an immune response against ISAv in fish, and for treating and preventing ISA in fish.
  • a method of inducing an immune response against ISAv in a fish comprising administering a therapeutically effective amount of a composition, nucleic acid or polypeptide as described herein to a fish. Also provided is a use of a composition, nucleic acid or polypeptide as described herein for inducing an immune response against ISAv in a fish. Also provided is a use of a composition, nucleic acid or polypeptide as described herein for preparation of a medicament for inducing an immune response against ISAv in a fish. Further provided is a composition, nucleic acid or polypeptide as described herein for use in inducing an immune response against ISAv in a fish.
  • Also provided is a method of preventing or treating ISA in a fish comprising administering a therapeutically effective amount of composition, nucleic acid or polypeptide as described herein to a fish. Also provided is a use of a composition, nucleic acid or polypeptide as described herein for preventing or treating ISA in a fish. Also provided is a use of a composition, nucleic acid or polypeptide as described herein for preparation of a medicament for preventing or treating ISA in a fish. Further provided is a composition, nucleic acid or polypeptide as described herein for use in preventing or treating ISA in a fish.
  • prevention refers partially or completely preventing or delaying the onset of one or more symptoms or features of a disease (i.e. , ISA). Prevention is causing the symptoms of the disease to not develop i.e., inhibiting the onset of disease in a fish who may be exposed to or predisposed to a disease state, but does not yet experience or display symptoms of the disease. Prevention may be administered to a subject who does not exhibit signs of the diseases. Prevention includes prophylactic treatment.
  • treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • the fish is a finfish.
  • the fish is a salmon or a trout, optionally Salmo sa/ar (Atlantic salmon), Salmo trutta (brown trout) or Oncorhynchus mykiss (rainbow trout).
  • the induction of immunity by a composition, nucleic acid or polypeptide described herein can be detected by expression of the mx1 gene in the fish.
  • mx1 gene expression is often the first indicator of ISAv infection in fish. Accordingly, an increase of mx1 gene expression in a fish that has been administered a composition or nucleic acid(s) described herein compared to a control fish (for example a fish that has been administered no composition/ nucleic acid or a sham composition/nucleic acid) can be used to detect induction of immunity.
  • the induction of immunity by the composition, nucleic acid(s) or polypeptide(s) can be detected by observing in vivo or in vitro the response of all or any part of the immune system in the fish against the ISAv antigen.
  • the induction of immunity by the composition or nucleic acid(s) can be also confirmed by observing the induction of antibody production against the ISAv antigen. For example, when antibodies against an antigen are induced in a laboratory fish immunized with the composition encoding the antigen, and when antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.
  • composition, nucleic acid or polypeptide is administered or for use by intramuscular injection, intraperitoneal injection or orally. In one embodiment, the composition, nucleic acid or polypeptide is for administration in a feed.
  • the fish is administered a single dose of the composition, nucleic acid or polypeptide.
  • the fish is administered a first dose of the composition, nucleic acid or polypeptide followed by a second (booster) dose of the composition, nucleic acid or polypeptide.
  • the second dose is optionally provided prior to sea water entry of the fish.
  • the first dose when the fish weighs 10 to 30g, about 20g or 20g and the second dose is administered when the fish weighs at least a 50 to 250g, or 100 to 200g.
  • the dose is 0.01 ng/g to 3 p/g body weight, optionally 0.01 ng/g to 1 ng/g body weight, 0.01 ng/g to 0.5 ng/g body weight, 0.01 ng/g to 0.1 ng/g body weight or 0.01 ng/g to 0.05 ng/g body weight.
  • the dose is 0.001 pg/g to 3 pg/g body weight.
  • the dose is 0.005 pg/g to 0.15 pg/g body weight, 0.05 pg/g to 0.15 pg/g body weight or 0.5 pg/g to 3 pg/g body weight.
  • the dose is about 0.01 pg/g, 0.1 pg/g or 1 pg/g body weight or 0.01 pg/g, 0.1 pg/g or 1 pg/g body weight. In another embodiment, the dose is 0.5 to 2 pg/g body weight, about 1 pg/g body weight or 1 pg/g body weight. Where the fish is administered more than one dose, each dose may be the same or different.
  • ISAv infectious salmon anemia virus
  • the method comprises formulating one or more nucleic acid molecules encoding one or more infectious salmon anemia virus antigenic polypeptides or immunogenic variants or fragments thereof in a nucleic acid carrier.
  • the method comprises formulating one or more infectious salmon anemia virus antigenic polypeptides or immunogenic variants or fragments thereof in a protein carrier.
  • the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle and one or more nucleic acid molecules are encapsulated in the nanoparticle.
  • the method comprises isolating one or more nucleic acid molecules, optionally RNA molecules, encoding one or more infectious salmon anemia virus antigenic polypeptides or immunogenic variants or fragments thereof and mixing the isolated nucleic acid molecules with a lipid nanoparticle.
  • transcript reactions Following a 2 h incubation at 37°C one of the transcript reactions was held at 4°C (uncapped transcript), while its second reaction was diluted two times with nuclease-free water and denatured with heating for 10 min at 70°C and then placed on ice for 5 min in preparation for capping and 2’-O-Methylation.
  • RNA solution was mixed with 10X capping buffer, 10mM GTP, 2.5 mM SAM, 4 pl Vaccinia mRNA capping enzyme, 4 pl Vaccinia mRNA Cap 2’- O-Methyltransferase to make an 80 pl reaction volume and left to incubate for 60 min at 37°C.
  • 10X capping buffer 10mM GTP, 2.5 mM SAM, 4 pl Vaccinia mRNA capping enzyme, 4 pl Vaccinia mRNA Cap 2’- O-Methyltransferase to make an 80 pl reaction volume and left to incubate for 60 min at 37°C.
  • 4 pl Vaccinia mRNA Cap 2’- O-Methyltransferase to make an 80 pl reaction volume and left to incubate for 60 min at 37°C.
  • To remove any remaining DNA template both the capped and uncapped RNA were incubated for 15 min at 37°C with 1 pl DNasel.
  • Poly(A) tailing was then performed at 37°C for 60 min with the addition 5X E-PAP reaction buffer, 25 mM MnCI2, 10 mM ATP, 8 units PolyA Polymerase (E-PAP) and 36 pl nuclease-free water to the DNase-treated capped and uncapped RNA. Transcripts were store at -80°C until required for future in vivo and in vitro work.
  • the two P3/F segments (SEGMENT 5-1 and SEGMENT 5-2), capped and uncapped are studied at 3 concentrations (1 pg/g bw, 0.1 pg/g bw and 0.01 pg/g bw) using the following dosing schedule: single vs booster with Invivofectamine LNP; single vs booster with other LNP; Single plus booster post SW using Glysantis formulation).
  • Initial immunization occurs at 20 g or just prior to salt water (SW) transition and ISAv exposure.

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Abstract

L'invention concerne des compositions, des acides nucléiques et des polypeptides pour le traitement et la prévention de l'anémie infectieuse du saumon (AIS) chez les poissons à nageoires. Dans un mode de réalisation, l'invention concerne un vaccin comprenant une ou plusieurs molécules d'acide nucléique, éventuellement de l'ARNm, codant pour un ou plusieurs polypeptides antigéniques du virus de l'anémie infectieuse du saumon (vAIS) ou des variants ou des fragments immunogènes de ceux-ci et un support d'acide nucléique.
EP23864220.1A 2022-09-16 2023-09-15 Vaccins contre l'anémie infectieuse du saumon et leurs utilisations Pending EP4587053A1 (fr)

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US202263407434P 2022-09-16 2022-09-16
PCT/CA2023/051229 WO2024055123A1 (fr) 2022-09-16 2023-09-15 Vaccins contre l'anémie infectieuse du saumon et leurs utilisations

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WO2002079231A2 (fr) * 2001-03-30 2002-10-10 Microtek International (1998) Ltd. Acides nucleiques codant pour des polypeptides du virus de l'anemie infectieuse du saumon
US7501128B2 (en) * 2002-09-17 2009-03-10 Novartis Animal Health Us, Inc. IHNV G protein for immune stimulation
CL2014003146A1 (es) * 2014-11-20 2015-06-12 Univ Santiago Chile Método para producir virus arn monocatenario negativo; plásmido recombinante funcional en células animales, que consta de un esqueleto pss-urg; método de obtención de partículas virales; y uso para expresar arn, proteínas autógenas o exógenas al virus.

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