Method for vaccine administration to salmonidae fish

Abstract

The present disclosure provides compositions and methods for salmonidae fish vaccination against PMCV and at least one other pathogen.

Description

Field of the Invention

[0001] The present invention generally belongs to the field of vaccination of salmonid fish. Background Art

[0002] Cardiomyopathy syndrome (CMS) is a disease that mainly affects large Atlantic salmonid fish in seawater approaching harvest in the second year, and thus the economic impact is significant. Affected fish may die suddenly without showing signs of the disease, or may show symptoms such as abnormal swimming behavior and anorexia. CMS is diagnosed based on histopathology, showing severe inflammation and degeneration of spongy myocardium in the atria and ventricles. Possible side effects of circulatory disorders are frequently observed multifocal hepatic necrosis.

[0003] As reported in Norwegian patent application no. 2008 2869, the infectious nature of CMS was ultimately demonstrated by isolating and culturing the virus causing CMS in fish. The virus was named piscine myocarditis virus (PMCV). It is characterized by the ability to cause one or more symptoms selected from the group consisting of: skin hemorrhage, raised scales, epicardial warts, ascites, fibrin tubes on the liver capsule, blood or blood clots filling the pericardial cavity, rupture of the atrial wall of the heart, dilation of the atrial heart, ventricular compression, spongy myocardium and inflammation of the epicardium and endocardium, liver damage (including multifocal anastomosing necrosis of hepatocytes and fibrin coating of the capsule), congestion of the spleen and / or gills.

[0004] The host cells for culturing PMCV disclosed in 2008 2869 have shown to result in low yields of the virus. There is still a need for additional knowledge in order to be able to develop effective means for controlling the disease and to be able to develop effective vaccines, such as recombinant vaccines. To this end, WO2011 / 131600 discloses nucleic acid sequences from PMCV.

[0005] DNA vaccines have become an attractive method for generating antigen-specific immune responses due to their stability and simplicity of delivery. DNA vaccines can be easily prepared in large quantities with high purity, administered repeatedly, and are highly stable relative to proteins and other biopolymers.

[0006] During the life cycle of salmonid fish, there are only a limited number of times fish can be vaccinated before their death and overall health are affected, preferably before the fish are transferred to seawater. At the same time, as more and more pathogens affecting the health of salmonid fish are discovered, it is desirable to vaccinate fish against as many pathogens as possible. Fish vaccination is a relatively labor-intensive operation - for practical reasons, it is advantageous to vaccinate against as many antigens as possible at one time. Therefore, there is a need in the art for methods of co-administering multiple vaccines to salmonid fish. Summary of the Invention

[0007] The present disclosure provides a DNA vaccine comprising a nucleic acid sequence encoding SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence that is at least 90% identical to SEQ ID NO:3 or SEQ ID NO:4, for protecting salmonid fish against PMCV and at least one other pathogen, wherein the DNA vaccine is administered intramuscularly; a second vaccine is administered intraperitoneally substantially simultaneously with the DNA vaccine; the second vaccine comprises an antigen that elicits a protective response against the at least one other pathogen; and the second vaccine is formulated as a W / O emulsion.

[0008] In certain embodiments, the nucleic acid sequence in the DNA vaccine disclosed herein encodes SEQ ID NO:5 or an amino acid sequence that is at least 90% identical thereto. In a more preferred embodiment, the nucleic acid sequence encodes SEQ ID NO:5 or an amino acid sequence that is at least 95% identical thereto, and at least 50% of the amino acids that are different from SEQ ID NO:5 are conservative substitutions.

[0009] The DNA vaccine according to any of the above embodiments may further comprise a nucleic acid sequence encoding a molecular adjuvant. In certain embodiments, the molecular adjuvant is IFNβ comprising SEQ ID NO:7 or IFNβ1 comprising SEQ ID NO:9, or a sequence that is at least 95% identical to SEQ ID NO:7 or SEQ ID NO:9. Preferably, the amino acids that are different from SEQ ID NO:7 or SEQ ID NO:9 are conservative substitutions.

[0010] The use of the DNA vaccine according to any of the above embodiments comprises two or three intramuscular injections, which are administered substantially simultaneously. Preferably, the total dose of the molecular adjuvant in the DNA vaccine administered in two or three injections is not greater than the dose of the molecular adjuvant in the DNA vaccine administered in a single injection. More preferably, the total dose of the antigen in the DNA vaccine administered in two or three injections is not greater than the dose of the antigen in the DNA vaccine administered in a single injection.

[0011] The DNA vaccine according to any of the above embodiments is administered together with a second vaccine, which is a water-in-oil emulsion and is administered intraperitoneally. In different embodiments, the second vaccine comprises one or more of the following antigens: Moritella viscosa, Piscirickettsias sp., Aeromonas sp., Vibrio sp, Aliivibrio sp., Listonella sp., Tenacibaculum sp., Pasteurella sp., Photobacterium sp., Flavobacterium sp., Yersinia sp., Renibacterium sp., Streptococcus sp., Lactococcus sp., Leuconostoc sp., Bifidobacterium sp., Pediococcus sp., Brevibacterium sp., Edwarsiella sp., Francisella sp., Pseudomonas sp., Cytophaga sp., Nocardia sp., Mycobacterium sp., viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmon pancreas disease virus (SPDV), iridovirus, nodavirus, piscine myocarditis virus (PMCV), and piscine orthoreovirus (PRV).

[0012] In more specific embodiments, the second vaccine comprises one or more (including all) of the following antigens: IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio salmonicida (Listonella anguillarum), Yersinia ruckeri O1, and Moritella viscosa. In other embodiments, the second vaccine comprises one or more (including all) of the following antigens: IPNV, Moritella viscosa, Aeromonas salmonicida, Vibrio anguillarum serotype 1 and O2, and Vibrio salmonicida (Listonella anguillarum).

[0013] In certain embodiments, the vaccines as described in any of the above embodiments and the vaccines administered as described in any of the above embodiments are administered to salmonids selected from the group consisting of: Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), and chinook salmon (Oncorhynchus tshawytscha); and most preferably Atlantic salmon.

[0014] The DNA vaccine as used in any of the above embodiments reduces the frequency, intensity, or duration of at least one clinical sign selected from the group consisting of death, lack of weight gain, skin ulcers, and heart defects.

[0015] In certain embodiments, the above DNA vaccine is administered to salmonids that have not been subjected to a smoltification signal within two weeks (preferably within six weeks) of injection of the DNA vaccine. In certain embodiments, the smoltification signal is light, feed, or water temperature. Detailed Description

[0016] The term "about" as applied to a reference numeral means that reference numeral plus or minus 10% of that value.

[0017] The term "different injection sites" refers to injection points that are positioned far enough apart from each other so as to effect different transdermal injections at these injection points. For example, the injection sites can be at least 0.5 mm apart. In different embodiments, the injection sites are 0.5 to about 100 mm apart or 0.5 to about 50 mm apart from each other, or about 1 to about 40 mm apart, or about 3 to about 30 mm apart or about 5 to about 25 mm apart, or about 5 to about 20 mm apart, or about 10 to about 20 mm apart, or about 10 mm to about 30 mm apart, or about 10 mm to about 40 mm apart, or about 10 mm to about 50 mm apart, or about 10 mm to about 60 mm apart, or about 10 mm to about 70 mm apart, or about 10 mm to about 80 mm apart, or about 10 mm to about 90 mm apart, or about 20 mm to about 30 mm apart, or about 20 mm to about 40 mm apart, or about 20 mm to about 50 mm apart, or about 20 mm to about 70 mm apart, or about 30 mm to about 40 mm apart.

[0018] The term "effective dose" refers to the amount of antigen that, if administered as a single injection in a given formulation, will provide the desired level of protection against the intended pathogen.

[0019] The term "lack of weight gain" means that infected, unvaccinated fish do not gain weight as well as uninfected fish. More specifically, it refers to a weight difference of at least 5%, and more preferably 10%, or 15%, or 20% between uninfected fish and infected, unvaccinated fish, where the weight difference of at least 5% (compared to uninfected fish) persists for at least four weeks or longer, such as at least five weeks or at least six weeks.

[0020] The term "not greater than" means equal to or less than a reference value, allowing for measurement error in the injection system.

[0021] The terms "protect", "protection", etc. mean that the vaccines disclosed herein are capable of reducing or eliminating the duration or severity of at least one clinical sign of the pathogen against which the salmonid fish are vaccinated. The clinical signs vary depending on the pathogen and include, but are not limited to, cardiac defects, skeletal muscle defects, skin defects, lack of weight gain, and death. Clinical signs also include a reduced viral count in the relevant organs and a decreased ability to infect other salmonid fish.

[0022] The term "substantially simultaneous" refers to the timing of multiple injections. If all of these multiple injections are administered within 5 minutes (from the first injection to the last injection), preferably within 4 minutes, or within three minutes, or within 2 minutes, or within 1 minute or within 30 seconds or within 15 seconds, then the injections are administered substantially simultaneously.

[0023] In a broad aspect, the present invention provides a DNA vaccine comprising a nucleic acid sequence encoding a PMCV protein or a fragment thereof, the nucleic acid sequence being capable of eliciting a protective immune response, for use in a method of protecting salmonid fish against PMCV and at least one other pathogen, wherein: the DNA vaccine is administered intramuscularly, and a second vaccine is administered intraperitoneally substantially simultaneously with the DNA vaccine, the second vaccine comprising an antigen that elicits a protective response against the at least one other pathogen; and the second vaccine is formulated as a W / O emulsion.

[0024] PMCV DNA vaccine

[0025] The PMCV DNA vaccine disclosed herein comprises a nucleic acid sequence encoding a PMCV protein or a fragment thereof that is capable of eliciting a protective immune response. In certain embodiments, the PMCV protein is encoded by PMCV ORF-1 and comprises the protein according to SEQ ID NO:3 or a sequence that is at least 90% identical to SEQ ID NO:3 (e.g., at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 97%, or at least 98%, or at least 99% or 100% identical to SEQ ID NO:3). If the PMCV protein has an identity to SEQ ID NO:3 of less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:3 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80% or at least 90% or 100%) of the different amino acids are conservative substitutions.

[0026] In other embodiments, the PMCV protein comprises an internal deletion such that the resulting protein is at least 95% identical to SEQ ID NO:4 (e.g., at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 97%, or at least 98%, or at least 99% or 100% identical to SEQ ID NO:4). If the PMCV protein has an identity to SEQ ID NO:4 of less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:4 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80% or at least 90% or 100%) of the different amino acids are conservative substitutions.

[0027] In other embodiments, the antigen in the PMCV DNA vaccine is provided in the form of a fusion protein, comprising, from 5' to 3':

[0028] a) an N-terminal secretion signal sequence of a secreted protein or a first membrane-bound protein;

[0029] b) a PMCV protein or a fragment thereof, as described in any of the above embodiments;

[0030] c) The transmembrane domain of the second membrane-binding protein.

[0031] In certain embodiments, the first membrane protein and the second membrane protein are the same. Preferably, the first (and second) protein is the viral hemorrhagic septicemia virus G-protein (VHSV-G).

[0032] Thus, in some embodiments, the N-terminal secretory signal sequence is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO:1 or SEQ ID NO:11. If the identity of the N-terminal secretory signal sequence to SEQ ID NO:1 or SEQ ID NO:11 is less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:1 or SEQ ID NO:11 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80%, or at least 90% or 100%) of the different amino acids in the N-terminal secretory signal are conservative substitutions.

[0033] In certain embodiments, the transmembrane domain is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO:2 or SEQ ID NO:12. If the identity of the transmembrane sequence to SEQ ID NO:2 or SEQ ID NO:12 is less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:2 or SEQ ID NO:12 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80%, or at least 90% or 100%) of the different amino acids in the transmembrane are conservative substitutions.

[0034] In one preferred embodiment, the fusion protein comprises SEQ ID NO:5 or an amino acid sequence that is at least 90% identical thereto. In various embodiments, the amino acid sequence is at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO:5. If the fusion protein has an identity to SEQ ID NO:5 of less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:5 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80%, or at least 90% or 100%) of the different amino acids in the fusion protein are conservative substitutions.

[0035] Molecular adjuvant

[0036] The DNA vaccine according to any of the embodiments described herein may further comprise a nucleic acid sequence encoding a molecular adjuvant. In certain embodiments, the molecular adjuvant is interferon. In a particularly preferred embodiment, the interferon is IFNβ or IFNβ1.

[0037] The interferon IFNβ protein comprises SEQ ID NO:7 or an amino acid sequence that is at least 90% identical thereto. In various embodiments, the amino acid sequence is at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO:7. If IFNβ has an identity to SEQ ID NO:7 of less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:7 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80%, or at least 90% or 100%) of the different amino acids in the IFNβ protein are conservative substitutions.

[0038] The interferon IFNβ1 comprises SEQ ID NO:9 or an amino acid sequence that is at least 90% identical thereto. In various embodiments, the amino acid sequence is at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO:9. If IFNβ1 has an identity to SEQ ID NO:9 of less than 100%, then preferably, the mutations that result in differences from SEQ ID NO:9 are substitutions. In certain advantageous embodiments, at least half (or at least 60% or at least 70% or at least 80%, or at least 90% or 100%) of the different amino acids in the IFNβ1 protein are conservative substitutions.

[0039] Those skilled in the art will further recognize that changes in nucleic acid sequences that result in modifications to the amino acid sequences of the encoded proteins may have little (if any) effect on the three-dimensional structure of the resulting proteins. For example, the codon for the amino acid alanine, a hydrophobic amino acid, can be replaced with a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue such as valine, leucine, or isoleucine. Similarly, changes that result in the replacement of one residue with a negatively charged residue, such as aspartic acid replacing glutamic acid; or one positively charged residue replacing another residue, such as lysine replacing arginine, are also expected to produce proteins with substantially the same functional activity.

[0040] The following six groups each contain amino acids that typically undergo conservative substitutions with each other: [1] alanine (A), serine (S), threonine (T); [2] aspartic acid (D), glutamic acid (E); [3] asparagine (N), glutamine (Q); [4] arginine (R), lysine (K), histidine (H); [5] isoleucine (I), leucine (L), methionine (M), valine (V); and [6] phenylalanine (F), tyrosine (Y), tryptophan (W), (see, for example, U.S. Patent Publication 20100291549).

[0041] In certain embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% or all 100% of the amino acids that differ from the reference sequence are conservative substitutions.

[0042] The protein and / or nucleic acid sequence identity according to any of the embodiments described herein can be evaluated using any of a variety of sequence comparison algorithms and programs known in the art. For sequence comparison, typically one sequence serves as a reference sequence (e.g., a sequence disclosed herein) against which the test sequence is compared. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0043] The percent identity between two amino acid or two nucleic acid sequences can be determined, for example, by using the computer program GAP, which is the Genetics Computer Group (GCG; Madison, WI) Wisconsin Package Version 10.0 program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12:387-95). When calculating the percent identity, the sequences to be compared are generally aligned in a manner that maximizes the matches between the sequences. Preferred default parameters of the GAP program include: (1) the GCG implementation of a unary comparison matrix for nucleotides (having a value of 1 for identity and 0 for non-identity), and the weighted amino acid comparison matrix of Gribskov and Burgess ((1986) Nucleic Acids Res. 14:6745), as described in Atlas of Polypeptide Sequence and Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pages 353-358 (1979) or other comparable comparison matrices; (2) for amino acid sequences, a penalty of 8 points for each gap and an additional penalty of 2 points for each symbol in each gap; or for nucleotide sequences, a penalty of 50 points for each gap and an additional penalty of 3 points for each symbol in each gap; (3) no penalty for terminal gaps; and (4) no maximum penalty for long gaps.

[0044] Sequence identity and / or similarity can also be determined by using the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444; computerized implementations of these algorithms (BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0045] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pair-wise alignment. A tree showing the clustering relationships used to create the alignment can also be drawn. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, 1987, J. Mol. Evol. 35:351-360; this method is similar to the method described in Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0046] Another example of a useful algorithm is the BLAST algorithm described in the following: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses a number of search parameters, most of which are set to default values. The adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself based on the composition of the specific sequence and the composition of the specific database being searched for the sequence of interest; however, these values can be adjusted to increase sensitivity.

[0047] Another useful algorithm is gapped BLAST, as reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses a BLOSUM-62 substitution score; the threshold T parameter is set to 9; a two-hit method that triggers gapless extension, charging a gap length of k, with a cost of 10 + k; u X is set to 16, and g X is set to 40 (for the database search phase) and 67 (for the output phase of the algorithm). Alignment of gaps is triggered by a score corresponding to approximately 22 bits.

[0048] Expression cassettes and vectors

[0049] One of ordinary skill in the art can design a nucleic acid sequence encoding the antigen of the present invention based on the amino acid sequence of the above antigen. In certain embodiments, the nucleic acid sequence encoding the PCMV antigen comprises SEQ ID NO:6.

[0050] The nucleic acid sequence encoding the antigen is subcloned into an expression cassette and is operably controlled by a first promoter. The first promoter can be selected from exemplary promoters such as: simian virus 40 early promoter (SV40), cytomegalovirus immediate early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1α promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken β-actin promoter coupled with CMV early enhancer (CAGG). Generally, the expression cassette further comprises a polyadenylation signal that terminates the transcription of the nucleic acid sequence encoding the antigen.

[0051] Optionally, the expression cassette may further comprise a nucleic acid sequence encoding a molecular adjuvant that is operably controlled by a second promoter. In certain preferred embodiments, the nucleic acid encoding SEQ ID NO:7 comprises SEQ ID NO:8, and the nucleic acid encoding SEQ ID NO:9 comprises SEQ ID NO:10.

[0052] Generally, the second promoter should be capable of initiating transcription in the host organism. In embodiments where the host is a salmonid fish (such as Salmo salar), suitable promoters include, but are not limited to, simian virus 40 early promoter (SV40), cytomegalovirus immediate early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1α promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken β-actin promoter coupled with CMV early enhancer (CAGG). Generally, the expression cassette of the present invention further comprises a polyadenylation signal that terminates the transcription of the nucleic acid sequence of the present invention.

[0053] The expression cassette according to any of the above embodiments can be subcloned into a vector. Different vectors suitable for the present invention are known in the art, including both plasmid vectors and viral vectors. Suitable plasmids include, but are not limited to, pUC-based vectors, pVAX vectors, pcDNA vectors, and NTC vectors. In a set of preferred embodiments, the vector is NTC9385R (Nature Technology Corporation) or a variant thereof, as described in the examples.

[0054] In other embodiments, a relatively new "doggybone" or dbDNA TM plasmid can be used as a vector. dbDNA TMPlasmids and methods for preparing these plasmids have been described at least in WO2018033730, WO2016034849, WO2019193361, WO2012017210, and WO2021161051. The advantage of this method is that the vector can be synthesized in a cell-free process, thereby improving production efficiency. The cell-free process preferably involves amplifying the template via strand displacement replication. This synthesis releases single-stranded DNA, which can in turn be replicated into double-stranded DNA using a polymerase. Alternatively, strand displacement can be achieved by providing a DNA polymerase and a separate helicase. The replicative helicase can unwind double-stranded DNA and facilitate the progression of the leading strand polymerase. The resulting double-stranded DNA concatemers are enzymatically cleaved and ligated to form a DNA construct in a doggybone shape.

[0055] Suitable viral vectors include, but are not limited to: alphaviruses, such as salmonid alphavirus (SAV), also known as salmon pancreas disease virus (SPDV) SAV; rhabdoviruses, such as viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV); paramyxoviruses, such as Atlantic salmon paramyxovirus (ASPV); adenoviruses; poxviruses, such as salmon gill poxvirus, etc. These viruses can be genetically modified to remove the part of the viral genome responsible for replication. Thus, the resulting viruses are infectious to fish cells and suitable for antigen production, but are not pathogenic.

[0056] Preferably, the same vector carries both the antigen and (if required) the molecular adjuvant. However, two different vector molecules can be provided, where one molecule carries the antigen and the other molecule carries the molecular adjuvant. Then, if required, these vector molecules can be combined in the desired ratio, either at the manufacturing stage or before use.

[0057] Those of ordinary skill in the art should understand that the differences between the described amino acid sequences and the reference amino acid sequences (whether in the context of a fusion protein or a molecular adjuvant) can be in the form of insertions, deletions, or substitutions. Preferably, the mutations are substitutions, and more preferably, at least some of these substitutions are conservative substitutions.

[0058] One of ordinary skill in the art will appreciate that there are a variety of methods for preparing the amino acid sequences, nucleic acid sequences, expression cassettes, and vectors disclosed herein. For example, nucleic acid sequences can be designed using software tools (such as CLC Main Workbench) and synthesized manually or generated using targeted mutagenesis. These sequences can be subcloned into expression cassettes and vectors by genetic engineering techniques widely available to those skilled in the art. See, for example, Molecular cloning: a laboratory manual (Sambrook & Russell: 2000, Cold Spring Harbor Laboratory Press; ISBN: 0879695773), and: Current protocols in molecular biology (Ausubel et al., 1988+ updates, Greene Publishing Assoc., New York; ISBN: 0471625949).

[0059] Multiple-dose vaccine administration

[0060] It has previously been found (see, for example, U.S. Provisional Application 63 / 375,285, entitled "Method of administration of aquaculture vaccines", filed on September 12, 2022) that PMCV and PD DNA vaccines administered in several doses are more effective than the same amount of the same DNA vaccine administered in a single dose. Thus, in certain embodiments, the DNA vaccine according to the invention is divided into two or three portions and delivered to different injection sites by two or three injections, the two or three injections being administered substantially simultaneously. Preferably, the last injection of the DNA vaccine is administered no later than 4 minutes, no later than 3 minutes, no later than 2 minutes, no later than 1 minute, no later than 45 seconds, no later than 30 seconds, no later than 15 seconds, and no later than 5 seconds, and no later than 1 second after the first injection.

[0061] The volume of the DNA vaccine per injection (whether the vaccine is delivered as a single injection or as two or three injections) can be determined by one of ordinary skill in the art, but typically the volume can independently be selected from 0.01 ml to about 0.25 ml and can include about 0.02 ml, about 0.025 ml, about 0.05 ml, about 0.075 ml, about 0.1 ml, about 0.15 ml, about 0.2 ml, or 0.25 ml. Currently preferred is that the multiple injections should contain substantially the same amount of antigen.

[0062] There are various methods for administering injections substantially simultaneously. In some embodiments, commercially available fish vaccination devices can be equipped with multi-needle injection heads, where the needles are configured to be at a desired distance from each other. Suitable vaccination machines include the NFT series products (Pharmaq), and specific models include NFT 20, NFT 25, and NFT 30. NFT 20 and NFT 25 administer the vaccine intraperitoneally, but can also be reconfigured for intramuscular injection. NFT 30 has a special DNA module that is capable of delivering DNA vaccine intramuscularly to fish fillets.

[0063] These machines process fish sized 120 mm to 250 mm (20 - 150 grams). Once completed, it sorts the vaccinated fish into three different sizes. In addition, it has channels for mispositioned, undersized, or discarded fish.

[0064] On the other hand, the vaccine can be manually injected via a syringe containing one or several needles. See, for example, the syringe sold by Pharmaq. This product is available in both single-size and double-size specifications. The single syringe is available in two sizes: 0.05 ml per dose and 0.1 ml per dose. In addition, if needed, the 0.05 ml syringe can be provided with an interchangeable 0.025 ml piston. The double syringe is available in three different dose size combinations: 0.05 ml + 0.05 ml per dose; 0.05 ml + 0.1 ml per dose; and 0.1 ml + 0.1 ml per dose. If needed, the 0.05 ml syringe can be provided with an interchangeable 0.025 ml piston. These syringes can be used for both water-based and oil-based vaccine formulations. The dose size can be easily adjusted by + / - 10%. The dose accuracy is recorded as a deviation of less than 3%.

[0065] The pressure for delivering the injection is not critical and can be derived from a hydraulic source, a pneumatic source, an electrical source, or a mechanical source.

[0066] The DNA vaccine according to any of the above embodiments is administered intramuscularly to salmonid fish. Muscles suitable for administering the DNA vaccine disclosed herein include, but are not limited to, skeletal muscle (whole fish fillets) and the epaxial muscle (muscles of the upper half of the fish).

[0067] Second vaccine

[0068] The DNA vaccine according to any of the above embodiments is administered intramuscularly substantially simultaneously with a second vaccine, which is formulated as a water-in-oil (W / O) injectable and is administered intraperitoneally.

[0069] A variety of antigens suitable for the second vaccine are known and are already commercially available. In addition, representative isolates of relevant fish pathogens are also available from a variety of sources.

[0070] In a specific embodiment of the present invention, the antigen from a bacterial source is selected from the group consisting of inactivated bacteria: Moraxella viscosa, Piscirickettsia species, Aeromonas species, Vibrio species, Allivibrio species, Listonella species, Tenacibaculum species, Pasteurella species, Photobacterium species, Flavobacterium species, Yersinia species, Renibacterium species, Streptococcus species, Lactococcus species, Leuconostoc species, Bifidobacterium species, Pediococcus species, Brevibacterium species, Edwardsiella species, Francisella species, Pseudomonas species, Cytophaga species, Nocardia species, Mycobacterium species, parts or subunits of these bacteria, and any combination thereof.

[0071] Isolates of such bacteria can be obtained, for example, from the LGC Promochem / ATCC (American Type Culture Collection) Storage and Distribution Center (ATCC), including strains of Aeromonas salmonicida (ATCC 33658), Vibrio salmonicida (ATCC 43839), Vibrio anguillarum serotype O1 (ATCC 43305) and O2 (ATCC 19264). In addition, cultures of Piscirickettsia salmonis have been deposited on June 9, 2006, at the European Collection of Cell Cultures (ECACC), Porton Down, Salisbury, Wiltshire, SP4 0JG UK, with the following accession numbers: 06050901, 06050902, 06050903 and 07032110.

[0072] Other specific embodiments relate to a vaccine, wherein the antigenic material obtained from a viral source other than a fish virus as defined above is from a virus selected from the group consisting of viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmon pancreas disease virus (SPDV), iridovirus, nodavirus, piscine myocarditis virus (PMCV), and piscine orthoreovirus that causes heart and skeletal muscle inflammation (this virus is sometimes referred to as the HSMIV or HSMI virus). These antigens can include modified live or inactivated organisms, parts or subunits of any of these viruses, DNA vaccines, and / or combinations thereof. Representative species of such viruses are available to those skilled in the art, for example, from the following deposits: infectious pancreatic necrosis virus (IPNV, ATCC VR-1318, country of origin: unknown), viral hemorrhagic septicemia virus (VHSV, ATCC VR_1389, country of origin: Denmark); infectious hematopoietic necrosis virus (IHNV, ATCC VR-1392, country of origin: United States)); infectious pancreatic necrosis virus; infectious salmon anemia (ISA) virus (ATCC VR-1554, country of origin: Canada). The applicant has previously made patent deposits of the following virus species: PRV (patent deposit number ECACC 04050401, country of origin: Norway).

[0073] In a more specific embodiment, the antigenic material is a subunit that can be selected from the group consisting of the glycoprotein of viral hemorrhagic septicemia virus (VHSV), the nucleoprotein of viral hemorrhagic septicemia virus (VHSV), the glycoprotein of infectious hematopoietic necrosis virus (IHNV), the nucleoprotein structural protein of infectious pancreatic necrosis virus (IPNV), antigenic fragments of any of these proteins, and combinations thereof.

[0074] In other embodiments, the antigenic material from an additional parasite source is from a source selected from the following: Lepeophtheirus Sp., Caligus Sp., and Ichthyophthirius Sp., parts of any of these parasites, and combinations thereof. In still other embodiments, the antigenic material is from a fungal source, the fungal source being selected from the group consisting of Saprolegnia Sp., Branchiomyces sanguinis, Branchiomyces demigrans, and Icthyophonus hoferi.

[0075] In certain embodiments, the antigen to be included in the second vaccine is selected from the group consisting of: IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio salmonicida (Listonella anguillarum), Yersinia ruckeri O1, and Moritella viscosa.

[0076] In other embodiments, the antigen in the second vaccine is selected from the group consisting of: IPNV, Moritella viscosa, Aeromonas salmonicida, Vibrio anguillarum serotype 1 and O2, and Vibrio salmonicida (Listonella anguillarum).

[0077] Suitable vaccines are commercially available and include, but are not limited to, ALPHA micro 6, ALPHA 7ILA / ISA, ALPHA JECT 1PD, ALPHA ERM SALAR, Micro4-2, and ALPHA 5-1.

[0078] The vaccines of the present invention (both the DNA vaccine and the second vaccine) may further comprise a suitable pharmaceutical carrier and / or adjuvant. The pharmaceutical carrier may be a sterile liquid, such as water or a buffered solution, such as physiological saline, as well as aqueous solutions of glucose and glycerol. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition may also contain a small amount of wetting agent or emulsifying agent or pH buffering agent. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should be suitable for the mode of administration.

[0079] Preferably, the second vaccine is administered in microdoses by injection such that the volume of one dose is less than 500 μl, or less than 400 μl, or less than 300 μl, or less than 200 μl, or about 100 μl, or less than 100 μl, or about 50 μl, or about 25 μl.

[0080] In certain embodiments, the uses described herein do not include the substantially simultaneous administration of a DNA vaccine encoding a salmon pancreas disease virus antigen, whether as a separate vaccine or in the same vector as the PMCV DNA vaccine described herein.

[0081] In other embodiments, the uses described herein do not include administering a DNA vaccine encoding a pancreatic disease viral antigen within a few days of each other, and co-administering substantially simultaneously a DNA vaccine encoding a PMCV antigen and a second vaccine as described above. Thus, the DNA vaccine encoding a PMCV antigen and the DNA vaccine encoding a pancreatic disease viral antigen are not administered within about 1 day, about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days, about 20 days, or about 21 days of each other. In other words, the DNA vaccine encoding a PMCV antigen and the DNA vaccine encoding a pancreatic disease viral antigen can be administered at least about 1 day apart, or about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days, about 20 days, or about 21 days apart.

[0082] In still other embodiments, the uses described herein do not include administering a DNA vaccine encoding a pancreatic disease viral antigen at the same site as the administration of a DNA vaccine containing a PMCV antigen according to the embodiments described herein. In this group of embodiments, the injection sites of the DNA vaccine encoding a PD antigen and the DNA vaccine encoding a PMCV antigen should be at least 3 mm apart from each other, or about 5 mm apart, or about 7 mm apart, or about 9 mm apart, or about 10 mm apart, or about 12 mm apart, or about 14 mm apart, or about 16 mm apart, or about 18 mm apart, or about 20 mm apart, or even greater distances.

[0083] The vaccines disclosed herein can be used to protect a variety of salmonid fish species from infection. Suitable salmonid fish include, but are not limited to, Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), chinook salmon (Oncorhynchus tshawytscha), and other species.

[0084] Salmonid fish of different ages (or weights) can be vaccinated according to the present invention. In certain embodiments, the weight of the salmonid fish at the time of vaccination is between about 15 and about 200 grams. Thus, the weight of the salmonid fish at the time of vaccination can be between about 25 and about 150 grams, or between about 40 and about 110 grams, or between about 50 and about 100 grams.

[0085] The inventors have surprisingly found that if salmonids treated with the vaccines described herein are not subjected to a smoltification signal within one week (or longer) after vaccination, the immune response against PMCV is further improved. Thus, in certain embodiments, after vaccination according to the methods disclosed herein, the fish are not subjected to smoltification for at least 1 week, or at least 10 days, or at least 14 days, or at least 18 days, or at least 21 days, or at least 28 days, or at least 30 days, or at least 35 days, or at least 42 days or even longer after vaccination. Thus, in more specific embodiments, salmonids treated with a vaccine for use according to any of the embodiments described herein are not subjected to a smoltification signal within six weeks after vaccination with the vaccine disclosed herein.

[0086] A variety of smoltification signals are known in the art. The smoltification signal can be selected without limitation from the group consisting of light, feed, water temperature, and any combination thereof. See, for example, EP 3197290A1.

[0087] The present invention will now be described in the following illustrative examples.

[0088] Examples

[0089] Example 1

[0090] Atlantic salmon (n = 240) with an average weight of 24 g were reared in four 150 L tanks containing fresh water (12°C) (n = 60 per tank). The 60 fish in each tank were starved for one day, then they were anesthetized using MS222 (Tricain, PHARMAQ AS), and then marked and vaccinated with one of four different vaccine regimens (Table 1). The vaccine regimens included vaccinating the fish with the CMSDNA vaccine alone or in combination with the multivalent commercial vaccine ALPHA micro 6 (PHARMAQ AS) (15 fish per group per tank). ALPHA micro 6 (AJm6) is a water-in-oil emulsion that contains the following inactivated antigens: Aeromonas salmonicida subsp., Listonella anguillarum (Vibrio anguillarum) serotypes O1 and O2, Vibrio salmonicida, Moritella viscosa, and infectious pancreatic necrosis virus.

[0091] The CMSDNA vaccine has an NTC9385R NANOPLASMID TM backbone, in which a nucleic acid sequence encoding a PMCV antigen having the amino acid sequence of SEQ ID NO:3 and a nucleic acid sequence encoding IFNβ having the amino acid sequence of SEQ ID NO:7 or IFNβ1 having the amino acid sequence of SEQ ID NO:9 are subcloned, each under the operable control of a CMV promoter.

[0092] Administration of the DNA vaccine was accomplished by intramuscularly giving two injections of 0.05 ml (one on each side) into skeletal muscle, with 10 μg of plasmid per injection; while ALPHA micro 6 was administered intraperitoneally as a single injection of 0.05 ml. The groups receiving both the DNA vaccine and ALPHA micro 6 were vaccinated during the same anesthesia period.

[0093] Two of the tanks were silvered by subjecting the fish to a 24:0 light:dark lighting regime for 6 weeks after vaccine administration. After these 6 weeks, the fish in these two tanks were transferred to seawater and a 12:12 light:dark regime. The remaining two tanks were not silvered and were kept in a 12:12 light:dark regime and freshwater throughout the study period. The experimental setup is summarized in Table 1.

[0094] After a 48-day immunization period, all groups were anesthetized and challenged with PMCV by intraperitoneal injection (0.1 ml) of a tissue homogenate containing infectious PMCV (isolate ID AL V1289), which was derived from a Norwegian CMS field outbreak. Two of the tanks (one freshwater and one seawater) were terminated and sampled 3 weeks after challenge, while the remaining two tanks were sampled 7 weeks after challenge. For the first sampling point, samples of the heart and kidney were taken for subsequent RNA extraction and quantification of viral RNA using a commercially available PMCV-specific real-time PCR assay (PHARMAQ Analytiq AS). The process was repeated for the last sampling point, but with the addition of heart sampling in formalin for histological analysis and semi-quantitative scoring of heart lesions according to severity, where 0 indicates no pathological findings and 3 indicates severe pathology (services provided by PHARMAQ Analytiq in Norway).

[0095] Table 1

[0096]

[0097]

[0098] AF – adipose fin; LM – left maxilla, RM – right maxilla

[0099] At both sampling points, blood was sampled on heparin-coated vacutainers from the groups that had received ALPHA micro 6. Plasma was subsequently used in ELISA assays to quantify anti-Aeromonas salmonicida (ALPHA The amount of IgM (one of the components in micro 6). These fish were also evaluated for local reactions caused by ALPHA using the modified Spielberg scale. induced by micro 6.

[0100] Table 2. Real-time PCR results from the hearts of fish challenged with PMCV 3 weeks ago. By default, negative fish gave a Ct value of 40.

[0101]

[0102] Table 3. Histological analysis of the atrium. Histological lesions were scored from 0 (normal) to 3 (severe pathology). The number of fish for each score in each group was indicated.

[0103]

[0104]

[0105] Table 4. Local reactions (adhesion, modified Spielberg score) and IgM titers against Aeromonas salmonicida 14 weeks after vaccination.

[0106]

[0107] The results showed that the co-administration of the multivalent vaccine ALPHA JECT micro 6 did not weaken the protection provided by the CMS vaccine under the silvering protocol. In the co-administration groups, the local reactions induced by ALPHA micro 6 were the same or lower (p < 0.05 in saline, p > 0.05 in fresh water, using the Mann-Whitney test), and after co-administration, only a slight and statistically non-significant decrease in the IgM titer against Aeromonas salmonicida was observed (p > 0.05, using the Mann-Whitney test).

[0108] This indicates that there is only a limited interaction between the DNA vaccine against CMS and the multivalent core vaccine, which is unlikely to affect the performance of any vaccine. Surprisingly, the co-administration of the DNA vaccine and ALPHA micro 6 slightly increased the virus count (i.e., reduced viral load) in both the freshwater and seawater groups (see Table 2, comparison between Group 1 and Group 3 in the freshwater and seawater tanks).

[0109] All publications, patent publications and non-patent publications cited in this specification indicate the state of the art of those skilled in the art to which the present invention pertains. All such publications are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0110] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments merely illustrate the principles and applications of the invention. Accordingly, it should be understood that many modifications can be made to the illustrative embodiments and other arrangements can be designed without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. A DNA vaccine comprising a nucleic acid sequence encoding SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence that is at least 90% identical to SEQ ID NO:3 or SEQ ID NO:4, for use in a method of protecting salmonid fish against PMCV and at least one other pathogen, wherein a) the DNA vaccine is administered intramuscularly; b) a second vaccine is administered intraperitoneally substantially simultaneously with the DNA vaccine; c) the second vaccine comprises an antigen that elicits a protective response against the at least one other pathogen; and d) the second vaccine is formulated as a W / O emulsion.

2. The DNA vaccine according to claim 1, wherein the nucleic acid sequence encodes SEQ ID NO:5 or an amino acid sequence that is at least 90% identical thereto.

3. The DNA vaccine according to claim 2, wherein the nucleic acid sequence encodes SEQ ID NO:5 or an amino acid sequence that is at least 95% identical thereto, and wherein further at least 50% of the amino acids that are different from SEQ ID NO:5 are conservative substitutions.

4. The DNA vaccine according to any one of claims 1 to 3, wherein the nucleic acid sequence encodes SEQ ID NO:

5.

5. The DNA vaccine according to any one of claims 1 to 4, further comprising a molecular adjuvant.

6. The DNA vaccine according to claim 5, wherein the molecular adjuvant is IFNβ1.

7. The DNA vaccine according to claim 5, wherein the molecular adjuvant is at least 95% identical to SEQ ID NO:7 or to SEQ ID NO:

9.

8. The DNA vaccine according to claim 7, wherein the molecular adjuvant is a conservative substitution variant of SEQ ID NO:7 or SEQ ID NO:

9.

9. The DNA vaccine according to claim 7, wherein the molecular adjuvant comprises SEQ ID NO:7 or SEQ ID NO:

9.

10. The DNA vaccine according to claim 9, wherein the molecular adjuvant is encoded by a nucleic acid sequence identical to SEQ ID NO:8 or SEQ ID NO:

10.

11. The DNA vaccine according to any one of claims 5 to 10, wherein the DNA vaccine is administered in two or three intramuscular injections, the intramuscular injections being administered substantially simultaneously, and wherein the total dose of the molecular adjuvant in the DNA vaccine administered in two or three injections is not greater than the dose of the molecular adjuvant in the DNA vaccine administered in a single injection.

12. The DNA vaccine according to any one of claims 4 to 11, wherein the DNA vaccine is administered in two or three intramuscular injections, the intramuscular injections being administered substantially simultaneously, and wherein the total dose of the antigen in the DNA vaccine administered in two or three injections is not greater than the dose of the antigen in the DNA vaccine administered in a single injection.

13. The DNA vaccine according to any one of claims 1 to 12, wherein the second vaccine comprises one or more of the following: Moritella viscosa, Piscirickettsia sp., Aeromonas sp., Vibrio sp., Aliivibrio sp., Listonella sp., Tenacibaculum sp., Pasteurella sp., Photobacterium sp, Flavobacterium sp., Yersinia sp., Renibacterium sp., Streptococcus sp., Lactococcus sp., Leuconostoc sp., Bifidobacterium sp., Pediococcus sp., Brevibacterium sp., Edwarsiella sp., Francisella sp., Pseudomonas sp., Cytophaga sp., Nocardia sp., Mycobacterium sp., viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmon pancreas disease virus (SPDV), iridovirus, nodavirus, piscine myocarditis virus (PMCV), and piscine orthoreovirus (PRV).

14. The DNA vaccine according to any one of claims 1 to 12, wherein the second vaccine comprises one or more of the following: IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, Vibrio anguillarum O2, Vibrio salmonicida (Aliivibrio), Yersinia ruckeri O1, and Moritella viscosa.

15. The DNA vaccine according to claim 14, wherein the second vaccine comprises IPNV, Moraxella viscosa, Aeromonas salmonicida, Vibrio anguillarum serotypes 1 and O2, and Vibrio salmonicida (Listonella anguillarum).

16. The DNA vaccine according to any one of claims 1 to 15, wherein the salmonid fish is Atlantic salmon (Salmo salar).

17. The DNA vaccine according to any one of claims 1 to 16, wherein the weight of the salmonid fish is about 40 to about 110 grams.

18. The DNA vaccine according to any one of claims 1 to 17, wherein the vaccine reduces the frequency, intensity, or duration of at least one clinical sign selected from the group consisting of death, lack of weight gain, skin ulcers, and heart defects.

19. The vaccine according to any one of claims 1 to 18, wherein the salmonid fish is not subjected to a silvering signal for at least one week after injection of the DNA vaccine.

20. The vaccine according to any one of claims 1 to 18, wherein the salmonid fish is not subjected to a silvering signal within six weeks after injection of the DNA vaccine.

21. The vaccine according to any one of claims 19 or 20, wherein the silvering signal is light, feed, water temperature, or any combination thereof.

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