Method for administration of vaccines for aquaculture

Abstract

FIELD: biotechnology.SUBSTANCE: use of a vaccine containing a nucleic acid with a sequence encoding an antigen obtained from a pathogen infecting fish in a method for actively protecting fish from the pathogen is described. In this case, the said method includes the administration of the said vaccine to the said fish intramuscularly in the form of multiple injections.EFFECT: administration of an injection from multiple injections provides a synergistic protective immune response, which is higher than the protective immune response caused by the same dose of the same vaccine, administered as a single effective dose, while both groups of fish are kept under the same conditions.18 cl, 8 tbl, 2 ex

Description

FIELD OF INVENTION

[0001] This invention generally relates to the field of DNA vaccines for aquaculture.BACKGROUND OF THE INVENTION

[0002] Aquaculture has seen a dramatic increase in productivity, reaching >527% from 1990 to 2018. In 2018, aquaculture accounted for approximately 46% of global total aquatic production (179 million tonnes) and 52% of seafood for human consumption (fish, crustaceans, mollusks, and other aquatic animals excluding aquatic mammals, reptiles, seaweeds, and other aquatic plants).

[0003] Commercial aquaculture is susceptible to infectious diseases caused primarily by bacteria, viruses, parasites, and, to a lesser extent, fungi. Bacterial diseases can cause significant biological and, therefore, economic losses. Although they can generally be controlled with antibiotics, the indiscriminate use of these pharmaceuticals ultimately poses a threat to human health due to the development and transmission of resistance mechanisms among bacterial species, some of which are also human pathogens. Furthermore, some traditional vaccines based on inactivated viruses can cause unacceptable side effects, and / or some pathogens are difficult to culture.

[0004] Pancreatic disease (PD) is an economically important viral disease affecting seawater-farmed Atlantic salmon (Salmo salar) in Ireland, the United Kingdom, and Norway. The causative agent is officially named salmon pancreatic disease virus (SPDV), but is commonly referred to as salmonid alphavirus (SAV), partly because the virus also infects other salmonid species. Oil-adjuvanted vaccines containing inactivated whole SPDV virus particles have been used for over a decade to reduce the impact of PD in the salmonid aquaculture industry. However, in some cases, the use of these vaccines has resulted in significant side effects such as spinal column deformity and fibrosis of the surrounding musculature, leading to downgrades at slaughter, reduced growth, and animal welfare issues. See Bseverfjord et al, NOFIMAreport 35V2021; and Holm et al, Aquaculture, 526, 2020.

[0005] Cardiomyopathy syndrome (CMS) is an inflammatory heart disease that primarily affects farmed Atlantic salmon (Salmo salar L.). The disease was first identified in Norway in 1985 and has since been diagnosed in both farmed and wild Atlantic salmon. The causative agent of CMS has been identified as fish myocarditis virus (PMCV), and the isolation and characterization of the virus are described in publication WO 2011 / 131600. Methods for virus control are urgently needed, but despite intensive research, it has not been possible to effectively cultivate PMCV and, consequently, produce vaccines based on attenuated or inactivated virus.

[0006] DNA vaccines have become an attractive approach for generating antigen-specific immune responses due to their stability and ease of delivery. DNA vaccines can be easily produced on a large scale with high purity, administered repeatedly, and are highly stable with respect to proteins and other biological polymers.

[0007] Among the many forms of nucleic acid-based vaccines that can be created, circular DNA plasmids are the simplest. DNA vaccination involves immunization with a circular DNA plasmid, which contains the gene (or genes) encoding the antigen. Indeed, the administration of free DNA (naked DNA) stimulates an effective and long-lasting immune response to the protein (antigen) encoded by the gene vaccine. When plasmid DNA is administered to a person, the plasmid is taken up by cells, and its genetic information is translated into an immunizing protein. This allows the host's immune system to respond to the antigen.

[0008] However, DNA vaccines are relatively new, and there is a need to improve the efficacy of these vaccines. SUMMARY OF THE INVENTION

[0009] In one aspect of the present invention, there is provided a vaccine comprising a nucleic acid sequence encoding an antigen obtained from a pathogen that infects fish, for use in a method for actively protecting fish against the pathogen, wherein said method comprises administering said vaccine to said fish intramuscularly in the form of a plurality of injections, wherein a. the injections of the plurality of injections are performed at different sites; b. each of said plurality of injections contains a dose of said vaccine that is less than an effective dose; and c. the total dose of said vaccine does not exceed the effective dose; and d. all injections of the plurality of injections are performed to said fish substantially simultaneously.

[0010] Also provided are vaccines as described above in which multiple injections act synergistically.

[0011] A vaccine characterized in that a total dose provides a more rapid formation of immunity than an effective dose given as a single injection, when administered to fish kept under the same conditions.

[0012] In some aspects of vaccination carried out in accordance with the methods described herein, the total dose of said vaccine is less than the effective dose.

[0013] In another aspect, the present invention provides a vaccine comprising a nucleic acid sequence encoding an antigen obtained from a pathogen that infects fish for use in a method for actively protecting fish against the pathogen, said method comprising administering said vaccine to said fish intramuscularly in the form of a plurality of injections, and wherein a. the injections of the plurality of injections are performed at different sites; b. all injections of the plurality of injections are performed to said fish substantially simultaneously, and wherein the injections of the plurality of injections act synergistically, and the total dose administered in the form of a plurality of injections provides a more rapid formation of immunity than the same dose administered in the form of a single injection of the same composition, when administered to fish kept under the same conditions.Preferably, the injections from multiple injections act synergistically and the total dose administered as multiple injections provides a more rapid development of immunity than the same dose administered as a single injection of the same composition, when administered to fish kept under the same conditions.

[0014] Preferably, the plurality of injections consists of 2, 3, or 4 injections. The plurality of injections can be administered within 60 seconds, or more preferably within 30 seconds, or even more preferably within 15 seconds.

[0015] Also provided is a vaccine as described above, wherein the fish is a salmon, and the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISA) (ISAV), fish myocarditis virus (PMCV), and fish orthoreovirus (PRV).

[0016] In certain embodiments of the vaccine described above, the pathogen is a salmonid alphavirus and the antigen comprises SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments of the present invention, the pathogen is PMCV and the antigen comprises SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments of the present invention, said vaccine comprises antigens against both a salmonid alphavirus and PMCV.

[0017] Also provided is a vaccine as described above, wherein the fish is tilapia and the pathogen is selected from the group consisting of nerve necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and Lake Tilapia virus.

[0018] Also provided are vaccines as described above, in which the nucleic acid sequence encoding said antigen is delivered by a heterologous vector, such as a plasmid or viral vector.

[0019] Also provided are vaccines as described above, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator. In certain embodiments of the present invention, the molecular immunomodulator is interferon.

[0020] Vaccines as described above, additionally containing an adjuvant, are also offered.

[0021] Also provided are vaccines as described above, wherein said vaccine comprises means for transporting said nucleic acid-based vaccine across a cell membrane. In certain embodiments, said means comprise a lipid coating.

[0022] In another aspect of the present invention, there is provided a finished article comprising: a) a reservoir containing a vaccine according to claims, wherein said reservoir is operatively connected to a plurality of needles, wherein said plurality of needles are adapted to deliver an injection from a plurality to different injection sites; and b) means for applying pressure to said reservoir, which allows the vaccine to be pushed through the plurality of needles.

[0023] In another aspect of the present invention, there is provided a finished article comprising:a. a plurality of reservoirs, each of which contains one of a plurality of injections of a vaccine according to claims, wherein each reservoir is operatively connected to a needle, and wherein needles connected to different reservoirs are adapted to administer injections from the plurality to different injection sites;b. one or more means for applying pressure to said reservoir, which allows pushing the vaccine through the plurality of needles.

[0024] In both this and the previous aspect of the present invention, the means may be obtained from a variety of sources, including, without limitation, pneumatic, hydraulic, electrical, or mechanical sources.

[0025] In another aspect of the present invention, there is provided a method of administering an effective dose of a nucleic acid vaccine to a fish, wherein said method comprises delivering a plurality of portions of said effective dose to different injection sites of said fish, and wherein said plurality of portions are administered substantially simultaneously.

[0026] In certain embodiments of the method described herein, the plurality of injections consists of 2, 3, or 4 injections and can be delivered within 60 seconds, 30 seconds, or 15 seconds. In certain embodiments of the present invention, the plurality of injections consists of 2 injections and is delivered in 15 seconds or less. In certain embodiments of the present invention, the plurality of injections consists of three injections and is delivered in 15 seconds or less.

[0027] In certain embodiments of the method described in this aspect, the fish is a salmonid and the pathogen is selected from the group consisting of salmonid alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmonid anemia virus (ISA) (ISAV), fish myocarditis virus (PMCV) and fish orthoreovirus (PRV).

[0028] In certain embodiments of the method of this aspect of the present invention, the pathogen is a salmonid alphavirus and the antigen comprises SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments of the present invention, the pathogen is PMCV and the antigen comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[0029] Also provided is a method according to this aspect of the present invention, wherein the fish is tilapia and the pathogen is selected from the group consisting of nerve necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV) and Tilapia Lake virus.

[0030] In certain embodiments of the method according to this aspect of the present invention, said nucleic acid sequence encoding said antigen is delivered via a heterologous vector. In certain embodiments, said heterologous vector is a plasmid vector. In other embodiments, said heterologous vector is a viral vector.

[0031] A method according to this aspect of the present invention is also provided, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator. In certain embodiments of the present invention, the molecular immunomodulator is interferon.

[0032] Also provided is a method according to this aspect of the present invention, wherein the vaccines further comprise an adjuvant.

[0033] Also provided is a method according to this aspect of the present invention, wherein said vaccine comprises means for transporting said nucleic acid-based vaccine across a cell membrane. In certain embodiments, said means comprise a lipid coating.

[0034] Also provided is a method according to this aspect of the present invention, wherein the injections from multiple portions act synergistically.

[0035] Also provided is a method according to this aspect of the present invention, wherein the total dose provides a more rapid formation of immunity than the effective dose administered as a single injection.DETAILED DESCRIPTION

[0036] For a better understanding of this invention, the following definitions are provided

[0037] The term "about" when applied to the reference number refers to the reference number plus or minus 10 percent of the specified value.

[0038] The term "different injection sites" refers to injection points that are spaced sufficiently apart so that injections at those points result in different penetrations of the skin. For example, the injection sites may be spaced at least 0.5 mm apart. In various embodiments of the present invention, the injection sites are spaced from 0.5 to about 100 mm apart, or from 0.5 to about 50 mm from one another, or from about 1 to about 40 mm, or from about 3 to about 30 mm, or from about 5 to about 25 mm, or from about 5 to about 20 mm, or from about 10 to about 20 mm, or from about 10 mm to about 20 mm, or from about 10 mm to about 20 mm, or from about 10 mm to about 30 mm, or from about 10 mm to about 40 mm, or from about 10 mm to about 50 mm, or from about 10 mm to about 60 mm, or from about 10 mm to about 70 mm, or from about 10 mm to about 80 mm, or from about 10 mm to about 90 mm, or from about 20 mm to about 30 mm, or from about 20 mm to about 40 mm, or from about 20 mm to about 50 mm, or at a distance from 20 mm to 70 mm from one another, or from 30 mm to 40 mm from one another.

[0039] The term “effective dose” refers to the amount of antigen that, when administered as a single injection in a given formulation, will provide the desired level of protection against the intended pathogen.

[0040] The term "synergistically acting plural injections" refers to a) the ability of a total dose to elicit a stronger protective immune response than the same formulation containing an effective dose, if the total dose is substantially equal to the effective dose; or b) the ability of a total dose to elicit the same protective immune response than the same formulation containing an effective dose, if the total dose is less than the effective dose.

[0041] The term "nucleic acid-based vaccine" refers to a vaccine in which the antigen is encoded by a nucleic acid sequence. The nucleic acid sequence encoding the antigen must enter the cell, replicate, and be expressed as the antigen. Nucleic acid-based vaccines include, but are not limited to, DNA vaccines and mRNA vaccines. DNA vaccines can be vectored by a plasmid or a heterologous viral vector.

[0042] The term "substantially simultaneously" refers to the time interval between injections of the portions of the effective dose of the vaccine described herein. The portions are considered to be administered substantially simultaneously if the last portion of the vaccine is administered no later than 5 minutes after the first portion, preferably 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, no later than 5 seconds, and no later than 1 second.

[0043] The term "substantially the same amount of antigen" in injections from a plurality of injections refers to the amount of antigen in each of a plurality of N injections, which varies between 1 / 2N and 1.5 / N of the total dose of antigen. Thus, if there are two injections from a plurality of injections, each injection from the plurality preferably contains from 1 / 4 of the total dose to 3 / 4 of the total dose. If there are three injections from a plurality, then each injection from the plurality preferably contains from 1 / 6 to 1 / 2 of the total dose. In the most preferred embodiment of the present invention, each injection from a plurality of N injections should contain approximately 1 / Nth part of the total dose of antigen. Thus, if N=2 (two injections from a plurality of injections), then each injection from the plurality should contain about 1 / 2 of the total dose. If N=3 (three injections from a plurality of injections), then each injection from the plurality should contain about 1 / 3 of the total dose.

[0044] The term "protect against a pathogen" refers to both the absence (or reduced level) of infection and the absence (or reduced level) of symptoms associated with the infection. If the protective titer against a specific pathogen is known, then protection against the pathogen also means that a statistically significant percentage of fish achieved the protective titer after vaccination, as described in this document.

[0045] The term "total dose" refers to the sum of the antigen doses administered with each injection of multiple injections. For example, and without limitation, if a vaccine is administered in three injections and each injection contains 100 ng of vector containing the nucleotide sequence encoding the antigen, the total dose is 300 ng.

[0046] The phrase "total dose substantially equal to the effective dose" refers to a total dose that is at least 90% and no more than 100% of the effective dose. Conversely, the phrase "total dose less than the effective dose" refers to a total dose that is less than 90% of the effective dose.

[0047] The term "the same protective immune response" refers to a response that is at least 95% and no more than 110% of the reference value, which is the immune response elicited by a single injection containing an antigen dose equal to the total dose. Conversely, the term "a stronger protective immune response" refers to a response that is greater than 110% of the reference value, unless tissue damage is being measured; in this case, the term "a stronger protective immune response" refers to a response that is less than 95% and greater than 110% of the reference value.

[0048] It is believed that administering the same amount of vaccine in multiple injections is likely to improve the uptake of antigen by host cells, leading to increased antigen expression and allowing the antigen dose to be spared to achieve essentially the same protective immune response as a larger dose of antigen administered in a single injection (i.e., the effective dose), and / or to improve the protective immune response if the total dose of vaccine administered in multiple injections is the same as the effective dose.

[0049] In a first broad aspect, the present invention provides a vaccine comprising a nucleotide sequence encoding an antigen obtained from a pathogen for use in a method for protecting fish from infection by said pathogen, wherein said method comprises administering said vaccine to fish intramuscularly in the form of a plurality of injections, and wherein each of said plurality of injections contains a dose of said vaccine that is less than an effective dose; the total dose of said vaccine does not exceed the effective dose; and all injections of the plurality of injections are administered to said fish substantially simultaneously.

[0050] In a set of preferred embodiments of the present invention, said administration of a plurality of injections provides a synergistic protective immune response that is higher than the protective immune response induced by the same dose of the same vaccine administered as a single effective dose, while both groups of fish (the group treated with a single effective dose and the group treated in accordance with the present invention) are kept under the same conditions. This may be important because fish are poikilothermic animals, and the water temperature in the aquarium may be important. The temperature itself is not as important as keeping the two groups of fish under the same conditions.

[0051] Also provided is a vaccine administered in the manner described above, wherein the total dose of said vaccine is less than the effective dose.

[0052] The present invention also provides a vaccine comprising a nucleic acid sequence encoding an antigen obtained from a pathogen that infects fish for use in a method for actively protecting fish against the pathogen, wherein said method comprises administering said vaccine to said fish intramuscularly in the form of a plurality of injections, and wherein the injections of the plurality of injections are performed at different sites; all injections of the plurality of injections are performed to said fish substantially simultaneously, and wherein the injections of the plurality of injections act synergistically or wherein the total dose administered in the form of a plurality of injections provides a more rapid formation of immunity than the same dose administered in the form of a single injection of the same composition, when administered to fish kept under the same conditions.Preferably, the injections from multiple injections act synergistically and the total dose administered as multiple injections provides a more rapid development of immunity than the same dose administered as a single injection of the same composition, when administered to fish kept under the same conditions.

[0053] The immune response can be measured in several ways, depending on the pathogen and technical knowledge. For example, the titer of antibodies specific to the pathogen can be determined. In other embodiments of the present invention, the protective immune response can be measured by the percentage of seroconverted animals. This assessment criterion is particularly suitable for pathogens for which a protective titer has been established.

[0054] In other embodiments of the present invention, mortality or survival may be a suitable endpoint for determining a synergistic effect. The number of viruses may also be a suitable endpoint for determining a synergistic effect. Alternatively, a suitable endpoint may be the prevalence of infection (i.e., the ratio of the number of virus-positive fish to the total number of fish) in at least one organ at one or more time points. The immune response may also be measured by organ or tissue damage (or, rather, the absence of organ or tissue damage) in response to virus infection. Synergy in at least one of these endpoints indicates synergism of the protective immune response.

[0055] Vaccines administered according to the methods described can provide earlier immunity than the same vaccines administered as a single injection when administered to fish maintained under the same conditions. Immunity can be measured by the same methods as the immune response, including, but not limited to, virus counts, tissue damage, or the percentage of fish free of virus infection. An earlier immune response after virus challenge in the group receiving multiple injections compared to the group receiving a single injection indicates earlier immunity, even though the immune response elicited by single and multiple injections may be less pronounced at a later stage.

[0056] Vaccines suitable for use in accordance with the method described herein are vaccines in which a nucleic acid molecule encoding an antigen enters host cells and expresses the antigen. Such vaccines can be DNA vaccines or mRNA vaccines. Both types of such vaccines are known in the art.

[0057] mRNA-based vaccines against COVID-19 have been approved for use in humans. Thus, in certain embodiments of the present invention, the vaccine comprises an mRNA sequence of a protein derived from a pathogen that causes disease in fish.

[0058] DNA vaccines are also known. At least one DNA vaccine, CLYNAV®, has been approved in Europe for the protection of salmon against salmonid pancreatic disease caused by salmonid alphavirus. CLYNAV® consists of a DNA plasmid (pUK-SPDV-poly2#l) dissolved in phosphate-buffered saline. This vaccine does not contain adjuvants or preservatives. The quantitative and qualitative composition has been properly defined as "pUK-SPDV-poly2#l DNA plasmid encoding salmon pancreatic disease virus (SPDV) proteins", dose 5.1-9.4 μg / 0.05 ml (101.6-188.4 μg / ml).

[0059] Thus, in DNA vaccines according to various embodiments of the present invention, the nucleic acid sequence encoding the antigen is contained in a heterologous vector. Various heterologous vectors known in the art are suitable for implementing the present invention. Vectors include, but are not limited to, viral vectors, plasmid vectors, and circular DNA vectors, also known as "dogbone" vectors.

[0060] Suitable heterologous viral vectors include, but are not limited to, alphaviruses such as SAV, rhabdoviruses such as VHSV and IHNV, paramyxoviruses such as ASP, adenoviruses, poxviruses such as Salmon gill poxvirus, and the like. These viruses can be genetically modified to remove portions of the viral genome responsible for replication. The resulting viruses will thus be infectious to fish cells and suitable for antigen production, but will not be pathogenic.

[0061] Suitable plasmids include, but are not limited to, pUC-based vectors, pVAX vectors, pcDNA vectors, and NTC vectors. In certain preferred embodiments of the present invention, the vector is NTC9385R (Nature Technology Corporation) or a variant thereof.

[0062] In other embodiments of the present invention, a relatively new dog bone plasmid or DBDNA™ may be used as a vector. DBDNA™ plasmids, as well as the process for creating these plasmids, are described in at least WO2018033730, WO2016034849, WO2019193361, WO2012017210, and WO2021161051.

[0063] The advantage of this approach is that the vector can be synthesized cell-free, increasing production efficiency. The cell-free process preferably involves amplification of the template by strand-displacement replication. This synthesis releases single-stranded DNA, which can in turn be copied into double-stranded DNA by a polymerase. Alternatively, strand displacement can be achieved by feeding a DNA polymerase and a separate helicase. Replicative helicases can open the DNA duplex and promote the advancement of the leading-strand polymerase. The resulting double-stranded DNA concatemer is enzymatically cut and ligated, thus forming a dog-bone-shaped DNA construct. Dog-bone vectors consist of a telomerase recognition site, the desired construct (including the antigen-coding sequence, promoter, and polyA site), and a sequence complementary to it.Thus, the complementary parts of the vector hybridize with each other and form a helix.

[0064] Various pathogens are suitable for use as an antigen source in the nucleic acid-based vaccines described herein. In certain embodiments of the present invention, the pathogen is a virus. Suitable viruses can be selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISA) (ISAV), fish myocarditis virus (PMCV), fish orthoreovirus (PRV), lumpfish virus, viral nerve necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and Lake Tilapia virus. In a specific embodiment of the present invention, the virus is salmon alphavirus. In another embodiment of the present invention, the virus is PMCV.

[0065] In other embodiments of the present invention, the pathogen is a bacterium. Suitable bacteria include, but are not limited to, Piscirickettsia sp., Aeromonas sp., Vibrio sp., Listonella sp., Moritella viscosa, Photobacterium damselae, 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., and Mycobacerium sp.

[0066] Particularly preferred are antigens of intracellular bacterial pathogens, including, but not limited to, Piscirickettsia sp., Edwardsiella sp., Yersinia sp., Francisella sp., Photobacterium sp., Mycobacterium sp., and Renibacterium sp.

[0067] Surface proteins of viruses and bacteria may be suitable candidates for the nucleic acid-based vaccines described in this document.

[0068] In other embodiments of the present invention, the pathogen is a parasite, such as, for example, a sea louse. In certain embodiments of the present invention, the sea louse is selected from the genus Lepeophtheirus or Caligus, and the antigen can be a midgut protein or an immunogenic fragment thereof. See, for example, U.S. Patent 11,167,017.

[0069] Certain vaccines, particularly suitable for administration to salmonids such as Salmo salar, contain nucleic acid sequences encoding immunogens that elicit a protective immune response against salmonid alphavirus. Suitable, non-limiting examples of such antigens include, but are not limited to, SEQ ID NO: 1 or SEQ ID NO: 2.

[0070] Other vaccines also suitable for administration to salmonids, such as Salmo salar, contain nucleic acid sequences encoding immunogens that elicit a protective immune response. A suitable, non-limiting example of such an antigen is the protein encoded by ORF-1 from PMCV (e.g., SEQ ID NO: 3), or a fragment thereof, including, but not limited to, SEQ ID NO: 4.

[0071] Other vaccines suitable for administration to species such as Salmo salar include nucleic acid sequences encoding immunogens that elicit a protective immune response against fish myocarditis virus (PMCV) and immunogens that elicit a protective immune response against salmonid alphavirus, as described above.

[0072] In other embodiments of the present invention, the pathogen is a parasite, such as a sea louse, particularly of the genus Lepeophtheirus, and more particularly of the species Lepeophtheirus salmonis. Sea louse peptides have been proposed as potential vaccine antigens. Some suitable candidates are described in U.S. Patent 11,167,017.

[0073] The nucleic acid sequence in the DNA vaccine is typically under the control of a suitable promoter. Promoters suitable for the vaccine of the present invention should be capable of initiating transcription in the host organism. In embodiments of the present invention where the host is salmon, such as Salmo salar, suitable promoters include, but are not limited to, the early promoter of simian virus 40 (SV40), the slow-early promoter of cytomegalovirus (CMV), the human ubiquitin C (UBC) promoter, the human elongation factor 1a (EF1A) promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-actin promoter in combination with the CMV early enhancer (CAGG).

[0074] The vector may also contain a nucleic acid sequence encoding a molecular immunomodulator. Suitable molecular immunomodulators include interferons. Nucleic acid sequences encoding salmon interferons, delivered in a DNA vaccine, have previously been shown to enhance antigen-specific immune responses. Accordingly, in some embodiments, the molecular immunomodulator is an interferon selected from the group consisting of salmon IFNa, IFNb, IFNb1, IFNa2, and IFNc.

[0075] The vaccine may also contain an adjuvant. Suitable adjuvants include, but are not limited to, saponins (e.g., Quil A), alum, CpG oligonucleotides, oligoribonucleotides, cytokines, glycolipids such as VAU™ 1005, quaternary amines such as dimethyldioctadecylammonium bromide (hereinafter "DDA"). Complexes containing a saponin, a sterol (e.g., cholesterol), and, optionally, a phospholipid are described in the art. Combinations of CpG oligonucleotides and saponin, CpG and cholesterol, and CpG and alum have been reported to cause synergistic effects.

[0076] In some embodiments of the present invention, the vaccine may include a liposomal adjuvant and / or carrier to facilitate vector transport across the cell membrane and thereby result in enhanced expression of the antigen and / or molecular immunomodulator. A suitable, non-limiting example of such a liposomal adjuvant / carrier system is described, for example, in U.S. Patent 10,456,459.

[0077] The vaccine according to the present invention may further contain auxiliary substances such as preservatives, stabilizers, buffers, etc.

[0078] Various fish species are suitable for vaccination according to the method described in this document. Suitable species include salmonids (including species of the genus Salmo and the genus Oncorhynchus), grouper (Dicentrarchus labrax), and warm-water fish including tilapia (Oreochromis niloticus) and pangasius (Pangasius hypophthalmus). Other species include beluga whale, Arctic char, mandarin grouper, and largemouth bass. Fish can be vaccinated according to the methods described in this document if they weigh between 15 and 200 grams, and more preferably between 40 and 110 grams.

[0079] As mentioned above, the vaccine according to the present invention is administered in multiple portions by a plurality of injections performed substantially simultaneously (the first portion and the last portion are administered within five minutes. It is preferable that the last portion 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, no later than 5 seconds, and no later than 1 second after the administration of the first portion of the vaccine.

[0080] The exact number of injections of the plurality of injections is usually 2, 3, 4 or 5, more preferably 2 or 3.

[0081] The injection volumes of the plurality of injections can be determined by a person skilled in the art, but in general, the volumes can be independently 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, it is preferable that the injections of the plurality of injections contain substantially the same amount of antigen.

[0082] There are many methods for administering injections simultaneously. In certain embodiments of the present invention, commercially available fish vaccination equipment may be equipped with a multi-needle injection tip in which the needle tips are spaced at a desired distance from each other. Suitable vaccination machines include the NFTT (Pharmaq) product line, and specific models are the NFT 20, NFT 25, and NFT 30. The NFT 20 and NFT 25 deliver vaccines intraperitoneally, but can also be converted for intramuscular injections. The NFT 30 is equipped with a specialized DNA module capable of injecting a DNA vaccine intramuscularly into fish fillets.

[0083] The machine processes fish ranging in size from 120 mm to 250 mm (20-150 grams). The machine then sorts the vaccinated fish into three different sizes. It also has a channel for non-compliant, undersized, or rejected fish.

[0084] Alternatively, the vaccine can be administered manually using a syringe containing one or more needles. See, for example, the MICRO-MATIC® syringe sold by Pharmaq. This product is available in single and double sizes. The single syringe is available in two sizes: 0.05 ml per dose and 0.1 ml per dose. In addition, the 0.05 ml syringe can be supplied with a replaceable 0.025 ml plunger, if required. 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. The 0.05 ml syringe can be supplied with a replaceable 0.025 ml plunger, if required. These syringes can be used for both water-based and oil-based vaccine formulations. The dose size can be easily adjusted within + / - 10%. The dose accuracy deviation has been documented to be less than 3%.

[0085] The pressure for injection is not important and can be obtained from hydraulic, pneumatic, electrical or mechanical sources.

[0086] Generally, the exact number of injections (i.e., the number of injections from a set of injections) can be determined experimentally. Preferably, this number should be between two and five, even more preferably between two and four, and even more preferably between two and three.

[0087] In one embodiment of the present invention, there are three injections of a plurality of injections, and the antigen comprises a PMCV antigen, such as, for example, SEQ ID NO: 3 or SEQ ID NO: 4. In these embodiments of the present invention, the vaccine may also contain an immunomodulator, such as, for example, interferon. Preferably, the interferon is IFNb. In another embodiment of the invention, there are two injections of a plurality of injections, and the antigen comprises a PMCV antigen, such as, for example, SEQ ID NO: 3 or SEQ ID NO: 4. In these embodiments of the present invention, the vaccine may also contain an immunomodulator, such as, for example, interferon. Preferably, the interferon is IFNb.

[0088] In another embodiment of the invention, there are two injections of a plurality of injections, and the antigen comprises an SAV antigen, such as, for example, SEQ ID NO: 1 or SEQ ID NO 2.

[0089] The following examples are presented as illustrative embodiments, but should not be construed as limiting the scope of the invention. Many changes, variations, modifications and other uses and applications of the present invention will be apparent to those skilled in the art. EXAMPLES Example 1. Multiple injections of SAV DNA vaccine result in lower viral load and lower clinical scores than a single injection. Materials and methods: Juvenile Atlantic salmon with an average weight of 26 g were vaccinated in fresh water by intramuscular administration of a standard dose of PD DNA vaccine. One group received the entire 0.05 ml dose in one injection. The other group received the 0.05 ml dose in two consecutive injections of 0.025 ml each. Fish injected with phosphate-buffered saline (PBS) served as a negative control. The fish were kept in the same tank throughout the study.Beginning the day after vaccination, fish were continuously exposed to light to induce smoltification, inducing the physiological changes necessary to prepare them for transfer to seawater. Following a 6-week immunization period (42 days, 500 degree days), fish were transferred to seawater and exposed to SAV3 virus in the same tank as control fish. SAV3 infection was achieved by intraperitoneal injection of naive fish with SAV3 infectious material, which was introduced into the same tank to expose vaccinated and control fish to SAV3 under conditions simulating a natural outbreak. Each vaccinated group was infected with 29-30 fish, while the negative control group was infected with 20 fish. All surviving fish were collected after 5 weeks (36 days) of co-infection in the same tank.Fish were weighed, and heart and pancreas samples were placed in formalin and RNALATER® (both organs are important targets for PD). Since no deaths were recorded in the two vaccinated groups during the observation period, and only one death (5%) was recorded in the negative control group, efficacy was assessed primarily based on weight, the severity of heart and pancreatic tissue damage, and the number of SPDV in the heart and pancreas.

[0090] Statistical analysis of viral load in the heart and pancreas in the vaccinated groups compared to the negative control group was performed using the Mann-Whitney test with GRAPHPAD PRISM® v.8.1.1 software. Statistical analysis of the weight of individuals from the vaccinated groups at the end of the infection period compared to the negative control group was performed using the unpaired t-test with Welch's correction using GRAPHPAD PRISM® v.8.1.1 software.

[0091] Quantification of SPDV in heart and pancreatic tissue samples was performed using a probe-based quantitative reverse transcriptase-PCR (RT-qPCR). This assay targets the nonstructural protein 1 (nsPl) of SPDV and is capable of detecting all known salmonid alphaviruses (Hodneland & Endresen, 2006). Analyses were performed in an officially accredited diagnostic laboratory (PHARMAQ Analytiq), authorized to officially confirm SPDV infection in salmonids in Norway.

[0092] The severity of tissue lesions in surviving fish at the end of 5 weeks post-infection (wpc) was analyzed histopathologically on a scale of 0-3 (pancreas) and 0-4 (heart) using the scoring system described in Graham et al., Journal of fish diseases, vol 34, issue 4, 237-286. 2011. This scoring system is presented in Table 1.

[0093] The results are shown in Tables 2 (number of viruses), 3 (severity of tissue damage) and 4 (weight).

[0094] The PCR results showed that both single and double injections resulted in lower infection prevalence and viral load (higher levels), but prevalence was lower in the double-injection group. Viral counts in the heart were significantly different from the control group in both the single-injection and double-injection groups. Viral counts in the pancreas were significantly different from the control group in the double-injection group. These results suggest that double injection of the 2 x 0.025 ml vaccine is more effective than a single injection of the 0.05 ml vaccine.

[0095] The pancreatic and cardiac tissue lesions of fish in the single-injection group and the double-injection group were different from those of the control group. Notably, the double-injection group tended to have smaller lesions in both the pancreas and heart than the single-injection group (0.13 versus 0.41 in the heart and 0.17 versus 0.31 in the pancreas).

[0096] The weights of fish from vaccinated groups 5 weeks after the start of co-inoculation with the virus in the same tank show that both vaccinated groups were highly protected from the reduced weight gain associated with PD, with significantly higher weight and growth rate compared to the negative control group. No statistically significant differences were observed between the single-injection and double-injection groups.

[0097] Increasing the number of injections from one to two resulted in a reduction in viral load and viral prevalence in both the heart and pancreas five weeks after infection. Prevalence in both the heart and pancreas was more than halved in the double-injection group compared to the single-injection group, even though the total amount of vaccine administered was the same in both groups. Example 2: Multiple injections of PMCV DNA vaccine result in lower viral load and lower histopathological scores than a single injection.

[0098] Atlantic salmon (n=150) with an average weight of 29 g were maintained in a 500 L freshwater tank (12C) under a 12:12 light:dark cycle. The fish were fasted for one day, then anesthetized with MS222 (Tricaine, PHARMAQ AS), divided into five groups (n=30 per group) by shortening the adipose fin or upper jaw, and vaccinated intramuscularly with a DNA plasmid expressing PMCV ORF1 partial antigen and salmon IFNb as a molecular adjuvant. One of the five groups was administered a negative control vaccine (PBS), while the other four groups were administered vaccines containing the PMCV ORF1 coding sequence 1, 2, 3, or 4 times during the same anesthesia period. The plasmid concentration was adjusted for each group so that the total plasmid amount was 10 μg per fish, regardless of the number of injections. Table 5 shows the data by vaccination group.

[0099] After 7 weeks of immunization, all fish were anesthetized again and injected intraperitoneally with 0.1 ml of kidney homogenate containing infectious PMCV particles. Then, at 3 and 7 weeks post-infection, hearts were collected from 15 fish from each group at each time point. Hearts were preserved in RNALATER® and sent to the PHARMAQ Analytiq diagnostic laboratory (Bergen, Norway) for PMCV RNA detection by real-time PCR. Heart samples collected at 7 weeks post-infection were also examined for cardiac pathology (PHARMAQ Analytiq, Bergen, Norway). Virus amounts at 3 and 7 weeks post-infection are presented in Tables 6 and 7, respectively. Histological analysis of the cardiac atria is presented in Table 8.

[00100] Increasing the number of injections from 1 to 3 resulted in both an increase in the number of viruses (a decrease in the viral load) and an increase in the percentage of fish that tested negative (from two in the one-injection group to eight in the three-injection group) three weeks after challenge. Seven weeks after challenge, the number of negative fish in the one-, two-, and four-injection group was 5 out of 15, and in the three-injection group, the number of negative fish was 7 out of 15. These results indicate that multiple administration of the vaccine promotes more rapid development of immunity. No significant differences in weight were found between the one-, two-, three-, and four-injection groups.

[00101] All publications cited in the description, both patent and non-patent publications, indicate the level of skill of specialists in the field of technology to which this invention pertains.All of these publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[00102] Although the present invention has been described with reference to specific embodiments of the invention, it should be understood that these embodiments of the invention merely illustrate the principles and direction of practice of the present invention. 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Claims

1. The use of a vaccine containing a nucleic acid with a sequence encoding an antigen obtained from a pathogen that infects fish in a method for actively protecting fish from the pathogen, wherein said method comprises administering said vaccine to said fish intramuscularly in the form of a plurality of injections, wherein said fish is a salmon, and wherein a. injections from multiple injections are performed in different places; b. each of said plurality of injections contains a dose of said vaccine that is less than the effective dose; c. the total dose of the said vaccine does not exceed the effective dose; and d. all injections of the plurality of injections are administered to the specified fish substantially simultaneously, and where (1) the specified set consists of two or three injections and the pathogen is PMCV (fish myocarditis virus), or (2) The given set consists of two injections and the pathogen is SAV (salmon alphavirus).

2. The use according to paragraph 1, characterized in that the injections from the plurality act synergistically.

3. Use according to paragraph 1 or 2, characterized in that the total dose provides a more rapid formation of immunity than the effective dose administered as a single injection, when administered to fish kept under the same conditions.

4. The use according to any of the preceding paragraphs, characterized in that the total dose of said vaccine is less than the effective dose.

5. The use according to any of the preceding paragraphs, characterized in that the administration of said plurality of injections is carried out within 1 minute.

6. The use according to any of the preceding paragraphs, characterized in that the administration of said plurality of injections is carried out within 30 seconds.

7. The use according to any of the preceding paragraphs, characterized in that the administration of said plurality of injections is carried out within 15 seconds.

8. The use according to any of the preceding claims, characterized in that each injection of said plurality contains essentially the same amount of antigen.

9. The use according to paragraph 8, characterized in that a. the set consists of two injections and each injection of said set contains approximately 1 / 2 of the total dose of antigen; or b. The set consists of three injections and each injection from the set contains approximately 1 / 3 of the total dose of antigen.

10. The use according to any of the preceding paragraphs, characterized in that: a) the set consists of three injections and the pathogen is PMCV, or b) the set consists of two injections and the pathogen is SAV.

11. The use according to any of the preceding claims, characterized in that said nucleic acid with a sequence encoding said antigen is delivered by means of a heterologous vector.

12. The use according to claim 11, characterized in that the heterologous vector is a plasmid vector or a viral vector.

13. The use according to paragraph 11 or 12, characterized in that the heterologous vector additionally contains a nucleic acid with a sequence encoding a molecular immunomodulator.

14. The use according to paragraph 13, characterized in that the molecular immunomodulator is interferon.

15. The use according to any of the preceding paragraphs, characterized in that said vaccine additionally contains an adjuvant.

16. The use according to claim 15, characterized in that the adjuvant is selected from the group consisting of MPLA, CpG-containing oligodeoxyribonucleotides, oligoribonucleotides, saponins, sterols and cationic lipids.

17. The use according to any of the preceding claims, characterized in that said vaccine contains means for transporting said nucleic acid-based vaccine through a cell membrane.

18. The use according to paragraph 17, characterized in that said means contain a lipid coating.

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