EP4587053A1 - Vaccines against infectious salmon anemia and uses thereof - Google Patents

Abstract

Compositions, nucleic acids and polypeptides for treating and preventing infectious salmon anaemia (ISA) in finfish are provided. In one embodiment, a vaccine is provided comprising one or more nucleic acid molecules, optionally mRNA, encoding one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof and a nucleic acid carrier.

Description

VACCINES AGAINST INFECTIOUS SALMON ANEMIA AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This disclosure claims the benefit of United States Patent Appl. No. 63/407,434, filed September 16, 2022, the contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] The disclosure relates to vaccines for treating and preventing infectious salmon anemia (ISA) in finfish. More particularly, the disclosure relates to mRNA vaccines against ISA.

INTRODUCTION

[0003] Infectious salmon anemia (ISA) is a finfish disease caused by the ISA virus, a member of a family of viruses called Orthomyxoviridae. It is primarily a disease of farmed Atlantic salmon. ISA can occur as a single case or as more commonly as a localized epidemic. Clinical signs of ISA include anemia, hemorrhaging (eyes and skin), pale gills, ascites, discoloured liver and swollen spleen. There is currently no treatment for ISA once fish are infected with the ISA virus. Cumulative mortality can reach 90% or more. As the disease can occur at any time in the growing cycle, and the only current means to prevent spread of the disease is to immediately harvest the entire sea farm, financial losses to salmon farmers from most often premature harvests can be very significant.

[0004] Despite the impact of ISA on salmon farming, there has been little success in developing an effective and durable vaccine against ISA. Past vaccine versions were observed to quickly lose their effectiveness in commercial farms as the virus mutated, nullifying protection. Given the characteristically high rate of mutation of ISA virus and subsequent increased diversity of strains across all global salmon growing regions, new vaccine technologies are needed to rapidly and specifically control locally dominant ISA type strains to properly meet the global ISA disease challenge.

SUMMARY

[0005] In this context, the inventors aimed to generate nucleic acid vaccines useful for preventing, treating and otherwise inducing an immune response against infectious salmon anemia (ISA). Accordingly, provided herein are compositions, nucleic acids, polypeptides and methods and uses thereof. [0006] An aspect of the present disclosure includes a 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 a carrier, optionally a nucleic acid carrier.

[0007] In one embodiment, the one or more infectious salmon anemia virus (ISAv) antigenic polypeptides is acetylcholinesterase (P3) or fusion (F) protein (P3/F), hemagglutinin-esterase (HE) protein or polymerase basic protein 2 (PB2).

[0008] In another embodiment, the one or more nucleic acid molecules comprise or consist of (a) a sequence selected from SEQ ID Nos: 1-8, (b) an RNA molecule transcribed from (a) or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to (a) or (b).

[0009] In another embodiment, the ISAv antigenic polypeptide is P3/F, optionally long P3/F or short P3/F.

[0010] In another embodiment, the nucleic acid is RNA, optionally mRNA.

[0011] In another embodiment, the mRNA has a poly-A tail.

[0012] In another embodiment, the mRNA is capped.

[0013] In another embodiment, the mRNA is uncapped.

[0014] In another embodiment, the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle.

[0015] In another embodiment, the ISAv is selected from HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type.

[0016] In another embodiment, the ISAv is HPR 4 type.

[0017] In another embodiment, 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. Optionally, 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. [0018] 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.

[0019] In one embodiment, 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).

[0020] In another embodiment, 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.

[0021] In another embodiment, the ISAv antigenic polypeptide is P3/F, optionally long P3/F or short P3/F.

[0022] In another embodiment, the ISAv is selected from HPR 4 type, HPR 7a type,

HPR 7b type or HPR 14 type.

[0023] In another embodiment, the ISAv is HPR 4 type.

[0024] In another embodiment, the composition comprises a plurality of infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof.

[0025] In another embodiment, 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.

[0026] Another aspect of the present disclosure includes a composition as described herein for use in inducing an immune response against ISAv in fish.

[0027] A further aspect of the present disclosure includes a composition as described herein for use in treating or preventing ISA in fish.

[0028] In one embodiment, the fish is salmon or trout. Optionally, the fish is Salmo salar (Atlantic salmon), Salmo trutta (brown trout) or Oncorhynchus mykiss (rainbow trout).

[0029] 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.

[0030] In one embodiment, the immune response comprises induction of mx1 gene expression.

[0031] In another embodiment, the fish is administered a single dose of the vaccine, optionally followed by a second dose.

[0032] In another embodiment, the composition is administered to the subject by intramuscular injection, intraperitoneal injection or oral delivery.

[0033] In another embodiment, the method treats or prevents ISAv infection.

[0034] Another aspect of the present disclosure includes a method of making a nucleic acid vaccine against infectious salmon anemia virus (ISAv) in fish, comprising 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.

[0035] In one embodiment, the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle.

[0036] Another aspect of the present disclosure includes nucleic acid molecule comprising or consisting of the sequence set out in any one SEQ ID Nos: 1-4 or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to in any one SEQ ID Nos: 1-4.

[0037] 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.

[0038] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

DRAWINGS [0039] Embodiments are described below in relation to the drawings in which:

[0040] Figure 1 (A and B) 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.

[0041] 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, with Lane 7 containing 0.5- 3 kb DNA ladder.

[0042] 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, with Lane 7 containing 0.5-3 kb DNA ladder.

[0043] Figure 4 shows cytotoxicity of sense inserts compared to negative and positive controls. Cytotoxicity was determined using the neutral red cytotoxicity test (Sigma), comparing OD absorbance of treated wells in comparison to untreated controls. PBS was used as a negative control, Lipofectamine high (H=undiluted) and lipofectamine low (L=diluted; 1 :10) are compared here against PB-2 and the fusion P3/F genes, capped/uncapped (C/UC). ISAv 1x10⁴ TCID 50 was used as a positive control.

[0044] 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). Infectious salmon anemia virus (ISAv) was administered at 1x10⁴ TCID50 cells per well, L = low dose, 1/10 dilution of final solution; H = high dose, undiluted final solution; C = Capped mRNA; UC = Uncapped mRNA.

[0045] 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). Infectious salmon anemia virus (ISAv) was administered at 1x10⁴ TCID50 cells per well, L = low dose, 1/10 dilution of final solution; H = high dose, undiluted final solution; C = Capped mRNA; UC = Uncapped mRNA. [0046] Figure 7 shows infectious salmon anemia mortality curve in Atlantic salmon smolts at 10 and 20°C.

[0047] Figure 8 (A-C) 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.

[0048] Figure 9 (A and B) shows daily mortality of commercial (Micro Forte V2; A) and sham (PBS; B) vaccinated Atlantic salmon smolts (total of all three replicates).

[0049] Figure 10 shows total % survival of the PB-2 vaccines, (c = capped; uc= uncapped; 1-1 , 1-2 = undiluted; 2-1= diluted).

[0050] Figure 11 shows total % survival of the P3/F vaccines, (c = capped; uc= uncapped; 1-x = undiluted; 2-x = diluted).

DESCRIPTION OF VARIOUS EMBODIMENTS

[0051] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0052] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

General Definitions

[0053] Terms of degree such as "about", "substantially", and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other.

[0054] The terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide”, “primer” as used herein 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. The term "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.

[0055] The term “polypeptide” (or “protein”) as used herein 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.

[0056] The term “primer” as used herein 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.

[0057] With reference to nucleic acids, the terms “anneal” and “hybridize” as used herein refer to the ability of a nucleic acid to non-covalently interact with another nucleic acid through base-pairing. The terms “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.

[0058] The term “gene” and its derivatives, as used herein, refer to a DNA sequence that comprises a coding sequence associated with the production of a polypeptide or polynucleotide product (e.g., mRNA, rRNA, tRNA). Depending on the context, 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.

[0059] The term "sequence identity" as used herein 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = [number of identical overlapping positions] I [total number of positions] X 100%). In one embodiment, 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. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. T o obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). 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. When utilizing the ALIGN program for comparing amino acid sequences, 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.

[0060] The term “immune response” as used herein 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.

[0061] The phrase "inducing an immune response" as used herein refers to a method whereby an immune response is activated. The phrase "enhancing an immune response" refers to augmenting an existing immune response.

[0062] As used herein, the term “effective amount” or “therapeutically 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. For example, in the context the present disclosure, 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.

[0063] The term "vaccine" as used herein refers to a composition that induces an immune response upon administration to a subject. In one embodiment, the induced immune response provides protective immunity.

Compositions

[0064] The disclosure provides compositions that are useful for inducing an immune response against infectious salmon anemia (ISA).

[0065] In one embodiment, provided is a 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.

[0066] In another embodiment, provided is a composition comprising one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof and optionally a carrier.

[0067] Infectious salmon anemia virus (ISAv) belongs to the genus Isavirus, family Orthomyxoviridae. Various strains and subtypes of ISAv are known, some of which have differing levels of virulence. As used herein, 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. In one embodiment, the ISAv is HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type. In another embodiment, the ISAv is HPR 4 type. Also contemplated herein is any other existing or emergent pathogenic strain or subtype of ISAv.

[0068] As used herein, 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. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The skilled artisan will also understand that 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. Furthermore, one skilled in the art will understand that an antigen need not be encoded by a full-length nucleotide sequence of a gene or a full-length protein. As used herein, the term “infectious salmon anemia virus (ISAv) antigenic polypeptide” refers to an antigenic polypeptide from ISAv.

[0069] 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); and segment 4, the RNA polymerase (PA). 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. Without being bound by theory, 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.

[0070] Thus, in one embodiment, the antigenic polypeptide is an ISAv protein. In another embodiment, 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. In another embodiment, the antigenic polypeptide is selected from acetylcholinesterase (P3) or fusion (F) protein (P3/F), hemagglutinin-esterase (HE) protein and polymerase basic protein 2 (PB2).

[0071] Sequences of the various ISAv proteins, and the nucleic acids that encode the proteins are known in the art. For example, in one embodiment, 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. In another embodiment, 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. As used herein, 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.

[0072] The following table provides information on each of the 8 RNA segments:

Table 1. Replicon information

[0073] In one embodiment, 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.

[0074] In another embodiment, the one or more nucleic acid molecules are RNA molecules corresponding to 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. As used herein, the expression “RNA molecules corresponding” to a particular DNA sequence refers to RNA transcribed from the DNA sequence. [0075] Thus, in one embodiment, the composition comprises at least one RNA molecule encoding one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof.

[0076] In one embodiment, the RNA is mRNA. The mRNA optionally has a poly-A tail and can be capped or uncapped.

[0077] As used herein, the term “immunogenic variant or fragment thereof” refers to a variant or fragment of an antigenic polypeptide that induces an immune response to ISAv.

[0078] In one embodiment, 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.

[0079] In one embodiment, 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:

[0080] In one embodiment, 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. In a further embodiment, 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.

[0081] In one embodiment, 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. For example, 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.

[0082] The nucleic acid molecules of the composition are optionally formulated, or encapsulated, in a nucleic acid carrier. As used herein, the term “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. In one embodiment, the nucleic acid carrier is a lipid nanoparticle. In another embodiment, the nucleic acid carrier is a phospholipid, sterol, PEG-lipid or cationic phytoglycogen nanoparticle.

[0083] In another embodiment, the nucleic acid carrier is an expression vector. Examples of 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).

[0084] In another embodiment, the composition comprises more than one infectious salmon anemia virus (ISAv) antigenic polypeptide or an immunogenic variant or fragment thereof. For example, 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. [0085] The antigenic polypeptides or immunogenic variants or fragments thereof are optionally formulated, or encapsulated, in a protein carrier. As used herein, the term “protein carrier” refers to compounds that facilitate transfer of the polypeptide into an organism or its cells. In one embodiment, the polypeptides described herein are optionally modified for cell permeability, improved stability, and/or better bioavailability.

[0086] The 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. The expression “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.

[0087] In one embodiment, the polypeptides are isolated from ISAv using methods known in the art.

[0088] The 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).

[0089] The polypeptides may also be modified with an enhancer moiety. In one embodiment, the polypeptide is conjugated directly or indirectly to the enhancer moiety. As used herein, an enhancer moiety can increase or enhance the activity of the polypeptide. For example, the enhancer may be a permeability enhancer, a stability enhancer or a bioavailability enhancer. In another embodiment, the enhancer moiety is a PEG moiety. [0090] The polypeptides may also be modified with a cell-penetrating moiety. As used herein, the term “cell-penetrating moiety” refers to a moiety that promotes cellular uptake of the peptide upon delivery to a target cell.

[0091] The polypeptides can also be conjugated to a carrier protein, thereby forming a fusion protein.

[0092] The composition also optionally comprises a pharmaceutically acceptable carrier. As used herein, the term “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.

[0093] In one embodiment, the pharmaceutically acceptable carrier is a carrier acceptable for administration to fish.

[0094] In one embodiment, a composition described herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include intramuscular injection, intraperitoneal injection or oral delivery.

[0095] In one embodiment, the composition is formulated in a dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the fish to be treated.

[0096] In one embodiment, the 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.

Nucleic acids, vectors and recombinant cells

[0097] The disclosure also provides novel nucleic acids that when administered to a fish can induce an immune response against ISAv. [0098] In particular, the present disclosure provides a nucleic acid corresponding to SEQ ID NO: 1 which was identified as a sequence encoding ISAv protein PB2, SEQ ID NOs: 2 and 3, which were identified as sequences encoding ISAv protein P3/F and SEQ ID NO: 4, which was identified as a sequence encoding ISAv protein HE.

[0099] Thus, in one embodiment, the disclosure provides a nucleic acid comprising or consisting of any one of SEQ ID Nos: 1-4.

[00100] In another embodiment 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.

[00101] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6 (Log 10 (Na+)+0.41 (% (G+C)-600/l), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule 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. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5xDenhardt's solution/1.0% SDS at Tm (based on the above equation) -5° C., followed by a wash of 0.2xSSC/0.1 % SDS at 60° C. 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.

[00102] In another embodiment, 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. As used herein, the expression “RNA molecules corresponding” to a particular DNA sequence refers to RNA transcribed from the DNA sequence.

[00103] In one embodiment, the RNA is mRNA. The mRNA optionally has a poly-A tail and can be capped or uncapped.

[00104] 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. In an embodiment, the vector is a viral vector such as a retroviral, lentiviral, adenoviral oradeno- associated viral vector.

[00105] 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.

[00106] Also provided in another aspect is a recombinant cell expressing a nucleic acid or vector described herein. In an embodiment, the cell is a bacterial cell, yeast cell, a mammalian cell, or a plant cell.

Methods and Uses

[00107] The disclosure also provides uses and methods relating to the compositions, nucleic acids and polypeptides described herein. [00108] In particular, the 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.

[00109] Accordingly, provided is 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.

[00110] 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.

[00111] As used herein, the term “preventing” or “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.

[00112] The term “treating” or “treatment” as used herein and as is well understood in the art, 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.

[00113] In one embodiment, the fish is a finfish. In another embodiment, the fish is a salmon or a trout, optionally Salmo sa/ar (Atlantic salmon), Salmo trutta (brown trout) or Oncorhynchus mykiss (rainbow trout).

[00114] In one embodiment, 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.

[00115] In another embodiment, 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.

[00116] In another embodiment, the 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.

[00117] In one embodiment, the fish is administered a single dose of the composition, nucleic acid or polypeptide. In another embodiment, 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. In one embodiment, 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.

[00118] In one embodiment, 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. In one embodiment, the dose is 0.001 pg/g to 3 pg/g body weight. In another embodiment, 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. In another embodiment, 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.

Method of production

[00119] Also provided is a method of making a vaccine against infectious salmon anemia virus (ISAv). In one embodiment, 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. In another embodiment, the method comprises formulating one or more infectious salmon anemia virus antigenic polypeptides or immunogenic variants or fragments thereof in a protein carrier.

[00120] In one embodiment, 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.

[00121] In another embodiment, 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.

[00122] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00123] The following non-limiting examples are illustrative of the present disclosure:

EXAMPLES

Example 1.

Materials and Methods

Construction of ISAv HE, F and PB2 expressing mRNAs by Rt-PCR

[00124] Primers (Table 2) were designed to target the ISAv HE, P3/F and PB2 segments and amplified using Superscript™ IV One-Step RT-PCR System (Invitrogen) and RNA isolated from ASK cells exposed to ISAv. Amplicons (Figure 1) were resolved by electrophoresis on 1 % agarose gels and purified with the QIAquick® Gel Extraction Kit (QIAGEN) following the manufacturer’s instructions. Amplicons were eluted with 30 pl Buffer EB (QIAGEN) and stored at -20°C.

Table 2. Primers

[00125] Following gel purification, 10 pl of the gene fragments were A-tailed with 1 .25 U of Taq polymerase (New England BioLabs Inc.) in the presence of 1 .5 mM MgCI2 (NEB), 1 X PCR Buffer (NEB) and 2mM dATP (NEB) in a 25 pl reaction. Following incubation for approximately 2 hr at 72°C, the A-tailed inserts were purified with the QIAquick® PCR Purification kit (QIAGEN) following the manufacturer’s instructions and eluted with 20 pl Buffer EB (QIAGEN) (Figure 2).

[00126] The purified A-tailed inserts were quantified on a NanoDrop™ 1000 Spectrophotometer (Thermo Scientific) and ligated together in a 10 pl reaction with the pCR®2.1-TOPO® vector at a 1 :3 vector to insert molar ratio using 10 ng of vector and 1 pl salt solution following the manufacturer’s instructions (Invitrogen). In addition, a control ligation reaction of vector only (i.e. no insert present) was performed to evaluate background. Following incubation at room temperature (RT) for approximately 30 min, the ligation reactions were placed on ice prior to transformation. One Shot® Top10 Chemically competent E. coli cells (Invitrogen) were thawed on ice and gently mixed with 2 pl of the ligation reaction followed by 5 min incubation on ice. Cells were heat-shocked for 30 s at 42°C and immediately transferred to ice. 250 pl of room temperature S.O.C. medium was immediately added to each of the transformation mixtures and transferred to 16 X 125 mm borosilicate culture tubes and incubated for 1 hr at 37°C and 225 rpm shaking. Following incubation 75 pl of the 2X diluted cells were spread on pre-warmed (37°C) LB agar plates containing 40 pg/ml kanamycin (Teknova) and 160 pg X-gal (BioShop). Plates were incubated overnight at 37°C.

[00127] Recombinant bacteria were identified by blue/white screening where positive colonies were inoculated into 3 ml LB broth containing 40 pg/ml kanamycin and grown for approximately 16 h at 37°C and 185 rpm. Isolation of plasmid DNA was performed on 1.5 ml of culture using the PureLink™ Quick Plasmid Miniprep kit (Invitrogen) following the manufacturer’s instructions and eluted with 75 pl TE Buffer (Invitrogen). Plasmids were screened for the presence of inserts by digestion of 2 pl of the miniprep with FastDigest® (Fermentas) restriction enzymes Bam HI and Xhol in 20 pl reactions followed by a 15 min incubation at 37°C. Restriction digests were resolved by electrophoresis on 1 % agarose gels (Figure 3), and those plasmids carrying inserts of the predicted size were quantified on a NanoDrop™ 1000 Spectrophotometer (Thermo Scientific). Mutation-free (i.e. error-free) target sequences were confirmed by Sanger sequencing in both directions with the universal primers M13F(-20) and M13R(-20) at The Centre for Applied Genomics (TCAG). mRNA Synthesis workflow [00128] Error-free clones, positive for HE, F and PB2 were selected for mRNA synthesis following the manufacturer’s instructions (Invitrogen/ThermoFisher Scientific). Briefly, 1.5 pg of the insert positive plasmids were linearized in a 20 pl reaction with the FastDigest® (Fermentas) restriction enzyme BamHI for 15 min at 37°C, followed by inactivation at 80°C for 5 min. For each clone, approximately 0.8pg of the linearized template was prepared twice for RNA transcription in individual reactions containing 10X reaction buffer, 100mM each of ATP, CTP, GTP and UTP, 2 pl of IVT Enzyme mix (AOF) and nuclease-free water to 20 pl total volume. 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.

[00129] The denatured 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. 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.

Preparation for in vivo work

[00130] Preparation of the mRNA vaccines for injections in salmon was performed following the Invivofectamine® 3.0 Reagent Complexation Protocol (Invitrogen, ThermoFisher Scientific), which recommended a dose of 1 mg/kg as a starting point for experiments. Briefly, 50 pl of the polyA-tailed transcripts at 2.4 mg/ml for each of the capped and uncapped segments was mixed with an equal volume of Complexation Buffer in a 1 :1 ratio. The diluted transcript was then immediately added to 100 pl of Invivofectamine® 3.0 Reagent and mixed by vortexing to ensure complexation, followed by incubation of the duplex mixture for 30 min at 50°C. The complex was diluted 6-fold with the addition of 1 ml of Phosphate Buffered Saline (PBS, pH 7.4) to give a final concentration of 0.1 mg/ml of which 200 pl was removed and further diluted 10-fold with the addition of 2 ml of PBS, pH 7. The complexes were loaded into 1 ml luer lock syringes with 21 gauge needles and stored short term for no more than seven days at 4°C prior to in vivo delivery.

Fish maintenance and husbandry

[00131 ] Atlantic salmon smolts (20g; n = 750) were donated by Cooke Aquaculture Inc. and transferred from Oak Bay Hatchery, New Brunswick to the Aquatic Biological Containment Level II Facility at the Atlantic Veterinary College, Charlottetown, PEI. Fish were maintained in 1500L tanks on flow-through freshwater (FW) at 11 °C for several weeks and PIT (passive integrated transponder)-tagged during this period. At approximately 30g, fish were injected intramuscular (i.m.) in the dorsal muscle (just lateral to the midline of the dorsal fin) with a 200ul dose of either high or low mRNA vaccine prep, 200 ul i.m. PBS as a negative control or 100 ul intraperitoneal dose of Micro Forte V2 vaccine, based on their randomized vaccine grouping. Each group was equally represented in each of three experimental tanks. Fish recirculation biofiltration was seeded with bacterial biofilter culture (1500 L water volume; 10.5 ± 1 °C, 15-17 ppt salinity). The fish were acclimated to increasing salt, through the addition of Instant Ocean®, over a period of two weeks until salinity reached 33 ± 3 ppt. Fish were kept under a 14h: 10h light-dark photoperiod and hand fed commercial pellets twice daily while on the FW system and transitioned to a commercial marine diet for the recirculation seawater portion of the study.

ISAv viral cultures and infection

[00132] Viral preparations of infectious salmon anemia virus (ISAv), were obtained from the Department of Fisheries and Oceans, in Moncton, New Brunswick, initially provided by the Research and Productivity Council (RPC) in Fredericton, New Brunswick. The prepared high-virulence ISAv isolate (ISAV-HPR4; RPC/NB 04-085-1 ) was suspended in L-15 culture media following isolation from Atlantic salmon head kidney tissue by Ritchie et al. [2009], The ISAv isolate (TCID50 of 1x10⁵/ml) virulence was confirmed on an Atlantic salmon kidney (ASK) cell-line monolayer in vitro using the Spearmean-Karber method.

[00133] Viral preparations were thawed on ice and aliquoted into 1 ml syringes. Atlantic salmon housed in a spare tank in each module (n = 48 fish) were transported to a quarantined module for viral inoculation at 9 dpi. Each fish was lightly anesthetized using tricaine methanesulphonate (MS-222; Syndel, Nanaimo, BC, Canada) and were IP- injected with 0.1 ml of the ISAv isolate. Fish were allowed to recover from anesthesia in an aerated auxiliary tank, identified by reading pit tags and placed into their respective tanks. Viral donor fish remained in the quarantined module for 6 days following injection. Donor fish (ca. 45 per tank) were added to each experimental cohabitation tank to obtain a 5:1 ratio of cohabitant to donor fish. Fish were monitored and checked for mortalities 4 times daily. In the case of a mortality, fish were necropsied, externally/internally examined for clinical signs of disease and posterior kidney samples were taken and stored in RNAIater™ RNA Stabilization Solution for 24 hr at 4°C (ThermoFisher Scientific, Missasauga, ON, Canada) prior to long term storage at -80°C. Fish care and husbandry practices were in accordance with the guidelines of the Canadian Council of Animal Care protocol approved by the University of Prince Edward Island and Atlantic Veterinary College Animal Care Committee (21-029).

Transfection

[00134] Preparation of the mRNA vaccines for transfection in Atlantic salmon kidney (ASK) cells (ATCC CRL 2747) was performed following the procedure provided with the use of Lipofectamine™ MessengerMAX mRNA Transfection Reagent (Invitrogen, ThermoFisher Scientific). Briefly, ASK cells were grown to > 80% confluence in T-75 (BD Falcon, VWR) cell culture flasks at 20°C in 1X Leibovitz’s L- 15 medium with L-glutamine (Multicell, Wisent Inc.), supplemented with 10% heat inactivated fetal bovine serum (FBS) (Multicell, Wisent Inc.) and 1 % antibiotic-antimycotic (Gibco, ThermoFisher Scientific). After confluency was reached, the media was removed, and the cell layer was rinsed three times with 1X PBS, pH 7.4 (Growcells.com, VWR). The PBS was removed, and the cell layer was covered with pre-warmed (20°C) complete ASK media and the cells were dislodged from the flask substrate with a 1 .8 cm width cell scraper (Greiner Bio-One, VWR). The detached cells were mixed by gentle trituration and the cell suspension was transferred to a 50 ml Falcon tube (VWR) for centrifugation at 100 x g for 5 min. The supernatant was carefully discarded by aspiration and the cell pellet was resuspended in fresh complete ASK media, where the volume of resuspension was dependent on the number of cells harvested (i.e. approximately 15 ml complete ASK media for every two, T75 cell culture flasks). To determine the number of viable cells per ml, a sample of the resuspended cells was diluted 1 :2 with 0.4% T rypan Blue and mounted on a cell counting slide (EVE™, NanoEnTek, MBI Lab Equipment) to count on an image based automated cell counter (EVE™-MC, NanoEnTek, MBI Lab Equipment). [00135] Cells were diluted with complete ASK media to a concentration of 1 .5 X 10⁵ cells/ml and used to seed six well tissue culture plates (BD Falcon, VWR) at a cell density of 0.3 X 10⁶ cells/2 ml media and maintained in an incubator at 20°C. After24 hr passaging in the 6 well tissue culture plates and reaching about 80% confluence, cells were transfected with the mRNA transcripts and Lipofectamine MessengerMAX™ Reagent according to the manufacturer’s instructions (Invitrogen, ThermoFisher Scientific). The mRNA transcripts used for transfection included the capped and uncapped versions of PB2 and P3/F and the following protocol are the components for a single well of a 6 well tissue culture plate where reaction volumes were scaled to adjust for triplicate wells per transcript. Briefly, 3.75 pl of the Lipofectamine™ Messenger MAX™ Reagent was diluted in 125 pl of Opti-MEM™ Medium (Gibco, ThermoFisher Scientific). While the diluted Messenger MAX™ Reagent was incubating at room temperature for 10 min the mRNA master mix was prepared by adding 2.5 pg of the mRNA transcript to 125 pl Opti-MEM™ Medium (Gibco, ThermoFisher Scientific). The diluted mRNA was then added to the diluted Messenger MAX™ Reagent in a 1 :1 ratio and following 5 min of incubation at room temperature, the mixture (250 pl) was added to the ASK cells in a single well containing 2 ml of complete ASK media. The procedure was repeated for each version of the transcripts, but with two times (7.5 pl) the amount of Lipofectamine™ Messenger MAX™ Reagent to start.

[00136] Control plates that contained wells with only ASK cells and no mRNA transcripts and wells that contained ASK cells that received only the diluted Messenger MAX™ Reagent were maintained along with an additional set of plates that had ASK cells exposed to ISAv. All plates were maintained at 20°C and after 24 and 48 hours of transfection, the cells were harvested with TRIreagent for RNA isolation.

RNA extraction, cDNA synthesis and Q-RT-PCR of Immune Gene Expression

[00137] Tri-reagent and the phenol-chloroform method was used for RNA isolation following the Chomsky method. The quantity and quality of RNA was determined using a NanoDrop 2000 (260/280 1.9-2.0; 260/230 between 2.0 and 2.3) Spectrophotometer (ThermoFisher Scientific). The integrity of RNA samples was further assessed with a 3% agarose gel (I Bl Scientific, Peosta, IA, USA). RNA was DNase treated using the DNA- free™ kit (Ambion, Austin, TX, US). cDNA was synthesized from 2000ng of DNase treated total RNA using iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.). Negative reverse transcriptase (noRT) samples were generated by pooling up to 15 individual RNA samples. In the case of genomic DNA contamination in a noRT pool, all RNA samples included were retreated with DNase and tested again to ensure no impact on downstream quantification. Total RNA and cDNA were stored at -80°C and -20°C, respectively.

[00138] Reverse transcription quantitative PCR (RT-qPCR) was performed with the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories Inc.) in triplicate using clear 96-well Multiplate® PCR Plates™ (Bio-Rad Laboratories Inc.) on CFX Connect™ Real-Time qPCR System (Bio-Rad Laboratories Inc.) for standard curves and CFX384 (Bio-Rad Laboratories Inc.) when running samples. Primers were diluted to a concentration of 10 mM and 2 pl of template (100 ng) was used, resulting in a total reaction volume of 11 pl (5 pl Supermix, 1 pl of F and 1 pl R primer and 2 pl nuclease free water). Ten-fold and five-fold serial dilution standard curves were used to determine the amplification efficiency of the primer pairs for reference genes and genes of interest, respectively. Negative RT and NTCs were run in triplicate in conjunction with the standard curves to check for any contamination. An IPC was used as a calibrator to link the plates of each gene. Ribosomal protein S20 (rps20), and eukaryotic translation elongation factor 1 -alpha (efl ab) were tested as reference genes and using geNorm software, were found to be stable for inclusion in the analyses (M=0.997; CV= 0.349).

[00139] Genes of interest were selected (n=15) based on representation of interferon/anti-viral and cytokine signalling, inflammation. The PCR amplification profile used in analysis was based on the optimized efficiencies of primers tested. The PCR profile consisted of 1 cycle of an initial denaturation step at 95°C for 30s, followed by 40 cycles of annealing at 95°C for 15s and extension at 65°C for 15s, with fluorescence detections following each 60°C step. Following this protocol, melt curve analyses were performed at 0.5°C intervals from 65°C to 95°C for 5s.

Statistical analysis

[00140] Statistical analysis of gene expression data was performed using Maestro (BioRad) software with geNorm implementation to fully normalize and evaluate quality control of the data set. Reference genes selected for analysis were rps20 and ef1A based on stability, and a calibrated normalized relative expression determined using a geometric mean of the two reference genes. Data were Log2 transformed and tested for normality before an analysis of variance (two-way, p < 0.05) followed by a post-hoc Tukey’s honest significant difference (HSD) test was used to determine significance against control. Kaplan-Meier survival curves, logistic regression and gene expression analyses were performed in RStudio 1.1.456 (RStudio, Inc., Boston, MA, USA).

Results and Discussion

[00141] The HE (segment 6), F (segment 5) and PB (segment 1 ) genes for ISAv were cloned into two expression vectors. The first was a modified pBluescript vector system developed at AVC and the second a modified TOPO cloning vector from Invitrogen. An additional 5’ UTR in the F protein was included in the cloning process to see if this has any impact on transcription/translation and processing downstream in the host. The PCRs for each gene, and a-tailed purified amplicons are shown in FigurelA (TOPO system) and 1 B (pBLU system). The confirmed expression of this gene in the plasmid transformed into the different cell populations are shown in Figure 2. Sequencing confirmed the appropriate insert/gene and orientation in each case and single representatives for each gene were followed up with transcribing both sense and antisense versions of the gene for exposure to host/ASK cells.

[00142] Sequences of four clones are set out below in Table 3. There is some variability in the sequences due to error rates in the proofreading capacity of DNA polymerase during sequencing. R = G or A; K= G or T; M = A or C; S = G or C; Y =T or C; W= A or T.

Table 3. Clone sequences.

Target Animal Safety

[00143] Both sense and anti-sense versions of each gene were capped/or not and encapsulated within the lipofectamine lipid nanoparticle (LNP) system and exposed to ASK cells for 48 hr, or within the invivofectamine LNP and administered to Atlantic salmon parr. Cytotoxicity of Lipofectamine encapsulated ISAv transcripts ranged from 7-23%, which in some cases was 2x higher than PBS. Neither sense nor anti-sense ISAv gene transcripts tested induced further cytotoxicity in the ASK cell line than that induced by the lipofectamine vehicle (Figure 4). ISAv positive control induced 100% cytotoxicity by 48 hr.

[00144] Individually PIT tagged fish were weighed and vaccinated based on random distribution of fish to different treatment groups. Following 48 hrs post vaccination, a subset of fish (n=5) from each of the vaccine and control groups were euthanized with an overdose of MS-222, blood and head kidney removed for later analysis. Injection sites were also examined for external/internal discolouration/melanization and hemorrhage. Further, over the course of the trial, approximately 85% of all mortalities and sampled fish had injection sites examined in a similar fashion during necropsy. Initial external melanization at the injection site that was observed in some i.m. fish (PBS and vaccinates) within 24 h, had disappeared by 48. Some hemorrhage remaining within the dermis and muscle, however by final sampling of fish at mortality or end of trial sampling, injection site hemorrhage only remaining in a few fish including the PBS control, and therefore was considered no different in vaccinates than negative controls. There was also no mortality in salmon prior to addition of ISAv donor fish, and it was therefore concluded that all mRNA vaccines were well tolerated at these encapsulation concentrations, volumes and salmon size.

In vitro response to vaccines

[00145] mRNA vaccine delivery to ASK cells was also used to assess anti-viral responses, through RT-qPCR of common biomarkers of anti-viral responses in vertebrates. Mx1 , ISG-15, and IL-12 have all been shown to be important interferon/cytokine signals during early responses to viral infection, and commonly in Atlantic salmon infected with ISAv (Carvalho et al., 2018; Groves et al., 2022). In particular, mx1 gene expression is often the first indicator of ISAv infection, and as can be seen in vitro, expression of this gene increased significantly (10-fold) from 24 to 48 hr post addition of the virus in ASK cells (Figures 5,6). PBS and lipofectamine by itself had no impact on the expression of mx1 at 24 or 48 hr, however, mRNA encapsulated vaccines did induce differential expression based on timing, dose and capping. Variability was quite high between replicates over this time frame, suggesting that cellular uptake of the message may be variable with 24-48 hr, with some cells initiating strong anti-viral mx1 response within 24 hr and other within 48 hr. At 24 hr the only treatments that induced significant increases in mx1 expression were in the P3/F, fusion proteins. Here the capped and diluted vaccine (CL) and uncapped, undiluted vaccine showed strong induction of gene expression (Figure 5). Expression of mx1 was much greater by 48 hr in the capped fusion protein (P3/F) vaccinates regardless of dose (diluted and undiluted), compared to all groups at 24 hr and all but the capped vaccinates at 48 hr (Figure 6). At 48 hr, the capped PB-2 undiluted vaccine also showed variable but strong induction of Mx1 gene in ASKs. There was no other induction of expression in other mRNA vaccinates. The fusion induces responses >10-fold higher and sooner than the virus itself. The capped vaccines appeared to induce a greater response than uncapped with respect to mx1. The P3/F capped vaccine showed consistent results, with induction at 24 hr and increased induction at low and high doses, whereas the uncapped P3/F high showed induction at 24 hr. Without being bound by theory, the uncapped vaccines are possibly less stable, they may have a shorted time course for induction, which may be missed by assessing only at 24 and 48 hr.

[00146] Induction of other genes was less consistent across all ASK replicates compared to mx1. The relative expression of isg15 and il-12 was generally low, and in consideration of the variation in expression of mx1 within 48 hr, suggests that in ASKs consistent expression of these genes might require more time (i.e. 72 hr) post stimulation. Interleukin-12 however inconsistent, showed greatest induction in P3/F and PB-2 uncapped and undiluted vaccines at 48 hr (data not shown). Without being bound by theory, this also suggests that the timing of the induction by uncapped vaccines may be slightly quicker than the capped vaccines or may induce different targets not assessed here.

Vaccine efficacy

[00147] Based on prior work, donor fish injected i.p. with 1x10⁴ TCID50, were introduced to the cohabitation study 1 week after exposure. This was expected to initiate mortality within cohabitation fish at 3 weeks post introduction (Figure 7). Multiple donor mortalities began within 10 days post introduction and peaked between 13-16 dpi depending on replicate tank (Figure 8ABC). Mortality of donors within all replicate tanks ended in approximately 3 weeks, and just preceded mortality of cohabitant fish. In one of the tank replicates (tank 3) a low oxygen event was associated with the high level of mortality in donor fish. While it is unknown whether the oxygen event preceded the majority of mortalities, it was almost exclusively donor fish, and was an acute event of a short period (< 5 mg/L oxygen for less than 1 hour). Low oxygen has been anecdotally associated with ISAv mortality in the field in Atlantic Canada, as well, and since it did not change the trajectory of the mortality curve across the study was considered to have little effect on the outcome of the study.

[00148] Commercial and sham vaccinate cohabitants showed a similar mortality trajectory, and had no significant difference in total mortality over the course of the study (Figure 9). Total mortality of these two groups was >90%. Furthermore, total donor mortality was 87%, which is in line with prior work (Carvalho et al., 2020; Groves et al., 2022).

[00149] Four of the P3/F vaccines showed significant delays in mortality and overall mortality over the course of the study (Figure 10). The two best performing vaccines were the P3/F (2-6) uncapped/diluted vaccine with 75% survival and the P3/F (2-9) capped/diluted vaccine at 50%. Other versions of the P3/F capped/uncapped vaccines both diluted and undiluted also showed some delays in mortality. Based on in vitro evidence the Fusion protein genes stimulated the quickest and most significant anti-viral response and may be in part responsible for the higher survival. The anti-viral responses observed were mostly in the capped vaccines, but both capped and uncapped versions increased protection.

Conclusion and Future Work

[00150] The current work confirmed prior work showing lack of protection in current commercially available ISAv vaccines. Further, it is shown here that mRNA vaccines can be constructed for multiple ISAv targets and administered to Atlantic salmon smolts, without target animal safety concerns. In vitro assessment of these vaccines was able to identify the ability of these vaccines to stimulate anti-viral responses and this anti-viral in vitro data are in agreement with the ability of these targets to protect in vivo. Without being bound by theory, as low dose/diluted vaccines had the two highest % survivals it is suggested that dosing will not have to rely on significant amounts of mRNA to produce effective host responses.

Example 2

[00151 ] Both long (L) and short (S) versions of segment 5 (P3/fusion protein) of ISAv showed protective responses in salmon whether capped or uncapped, and at different concentrations. [00152] Examination of the P3/F vaccinates, capped and uncapped, diluted and undiluted, are examined both for protection through assessing exposure to ISAv donor fish and in terms of their ability to induce viral neutralization and induce cellular/antibody mediated immunity.

[00153] 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.

[00154] Upregulated anti-viral responses such as overexpression of genes encoding for Mx1 , ISG15, and IL-12 were observed in the P3/Fusion protein in vitro. Upregulated anti-viral responses such as overexpression of genes encoding for Mx1 , ISG15, and IL- 12 are assessed in vivo in animals post vaccination and compared against PBS-controls in addition to other adaptive immune and anti-viral responses to assess peak immune response.

Example 3

In vivo assessment of Immune response and protection

[00155] Multiple vaccine delivery formulations (Invivofectamine LNP [iLNP], other LNP [oLNP] source, and oral boost delivery [ob]) and doses (3 doses: 1 pg/g bw, 0.1 pg/g bw and 0.01 pg/g bw) are examined with single vs. prime/boost. Following a minimum 2 week acclimation to facilities (12°C), fish are individually PIT tagged and given a further 2 weeks post PIT tagging to allow for randomization by weight into study groups and dispersion to replicate study tanks. Following the above acclimation period, the following groups (n=25 fish each) are administered (100 pl)) vaccine/sham injections: (1 ) negative PBS- control group i.m., (2, 3) 0.01 ug L-P3 iLNP [capped and uncapped], (4,5) 0.1 ug L- P3 iLNP [capped and uncapped], (6, 7) 1.0 ug L-P3 iLNP [capped and uncapped], (8, 9) 0.01 ug L-P3 oLNP [capped and uncapped], (10, 11) 0.1 ug L-P3 oLNP [capped and uncapped], (12, 13) 1.0 ug L-P3 oLNP [capped and uncapped], (14, 15) 0.01 ug S-P3 iLNP [capped and uncapped], (16, 17) 0.1 ug S-P3 iLNP [capped and uncapped], (18, 19) 1.0 ug S-P3 iLNP [capped and uncapped], (20, 21) 0.01 ug S-P3 oLNP [capped and uncapped], (22, 23) 0.1 ug S-P3 oLNP [capped and uncapped], (24, 25) 1.0 ug S-P3 oLNP [capped and uncapped]. [00156] These 25 groups constitute the single dosage at 20 g. Another 25 groups receive the same initial dose and a follow up dose at 100g [im-boost], PBS controls receive only PBS injections either single or 2x. 5 fish per group are sampled for each vaccinate group within 3 days of onset of mortality to assess immune response to each vaccine. A lower ratio of donor fish with HPR4 i.p. injected compared to cohabitant vaccinates are used in this study compared to earlier work (i.e. 15% rather than 20%).

Table 4. Sequences

References

Chomczynski P, Sacchi N. 1987. “Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction.” Analytical Biochemistry. 162(1): 156-59.

Ritchie RJ, McDonald JT, Glebe B, Young-Lai W, Johnsen E, Gagne N. 2009. “Comparative Virulence of Infectious Salmon Anaemia Virus Isolates in Atlantic Salmon, (Salmo salar L.).” Journal of Fish Diseases. 32(2): 157-171 .

Groves, S.K. Whyte, S.L. Purcell, A.F. Garber, M. D. Fast. Climate change and infectious salmon anemia (ISA): How will the Atlantic salmon (Salmo salar) immune system defend against the ISA virus with increasing ocean temperatures? NACIW, June 6-9, 2022. Banff, Alberta, Canada.

Carvalho LA, Whyte SK, Braden LM, Purcell SL, Taylor RG, Rise ML, Gagne N, Fast MD. Functional feeds impact molecular responses of Atlantic salmon (Salmo salar) to Co- infection with Lepeophtheirus salmonis and infectious salmon anemia virus. Sea lice international conference. Patagonia, Chile, November 4-8, 2018.

Claims

CLAIMS:

1. A 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 a carrier, optionally a nucleic acid carrier.

2. The composition of claim 1 , wherein the one or more infectious salmon anemia virus (ISAv) antigenic polypeptides is acetylcholinesterase (P3) or fusion (F) protein (P3/F), hemagglutinin-esterase (HE) protein or polymerase basic protein 2 (PB2).

3. The composition of claim 1 or 2, wherein the one or more nucleic acid molecules comprise or consist of (a) a sequence selected from SEQ ID Nos: 1-8, (b) an RNA molecule transcribed from (a) or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to (a) or (b).

4. The composition of any one of claims 1-3, wherein the ISAv antigenic polypeptide is P3/F, optionally long P3/F or short P3/F.

5. The composition of any one of claims 1-4, wherein the nucleic acid is RNA.

6. The composition of claim 5, wherein the RNA is mRNA.

7. The composition of claim 6, wherein the mRNA has a poly-A tail.

8. The composition of claim 6 or 7, wherein the mRNA is capped.

9. The composition of claim 6 or 7, wherein the mRNA is uncapped.

10. The composition of any one of claims 1-9, wherein the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle.

11 . The composition of any one of claims 1-10, wherein the ISAv is selected from HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type.

12. The composition of claim 11 , wherein the ISAv is HPR 4 type.

13. The composition of any one of claims 1-12, comprising a plurality of nucleic acid molecules encoding one or more infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof.

14. The composition of claim 13, wherein 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.

15. 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.

16. The composition of claim 15, wherein 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).

17. The composition of claim 15 or 16, wherein 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.

18. The composition of any one of claims 15-17, wherein the ISAv antigenic polypeptide is P3/F, optionally long P3/F or short P3/F.

19. The composition of any one of claims 15-18, wherein the ISAv is selected from HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type.

20. The composition of claim 19, wherein the ISAv is HPR 4 type.

21. The composition of any one of claims 15-20, comprising a plurality of infectious salmon anemia virus (ISAv) antigenic polypeptides or immunogenic variants or fragments thereof.

22. The composition of claim 21 , wherein 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.

23. The composition of any one of claims 1 -22, for use in inducing an immune response against ISAv in fish.

24. The composition of any one of claims 1-22, for use in treating or preventing ISA in fish.

25. The composition for use of claim 23 or 24, wherein the fish is salmon or trout.

26. The composition for use of claim 25, wherein the fish is Salmo salar (Atlantic salmon), Salmo trutta (brown trout) or Oncorhynchus mykiss (rainbow trout).

27. The composition for use of any one of claims 23-26, wherein the ISAv is selected from HPR 4 type, HPR 7a type, HPR 7b type or HPR 14 type.

28. The composition for use of claim 27, wherein the ISAv is HPR 4 type.

29. A method of inducing an immune response against ISAv in a fish, the method comprising administering to the fish the composition of any one of claims 1-22 in an amount effective to produce an immune response in the fish.

30. The method of claim 29, wherein the immune response comprises induction of mx1 gene expression.

31 . The method of claim 29 or 30, wherein the fish is administered a single dose of the vaccine, optionally followed by a second dose.

32. The method of any one of claims 29-31 , wherein the composition is administered to the subject by intramuscular injection, intraperitoneal injection or oral delivery.

33. The method of any one of claims 29-32, wherein the fish is salmon or trout.

34. The method of any one of claims 29-33, wherein the method treats or prevents ISAv infection.

35. A method of making a nucleic acid vaccine against infectious salmon anemia virus (ISAv) in fish, comprising 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.

36. The method of claim 35, wherein the nucleic acid carrier is a nanoparticle, optionally a lipid nanoparticle or a cationic phytoglycogen nanoparticle.

37. A nucleic acid molecule comprising or consisting of the sequence set out in any one SEQ ID Nos: 1-4 or a sequence with at least 75, 80, 85, 90, 95 or 99% sequence identity to in any one SEQ ID Nos: 1-4.

38. A use of the nucleic acid molecule of claim 37 or RNA transcribed from the nucleic acid molecule of claim 37 for inducing an immune response against ISAv in fish.