Aquaculture vaccine application method

Abstract

A method of delivering a fish DNA vaccine is provided, wherein the vaccine is intramuscularly delivered in multiple injections.

Description

Technical Field

[0001] The present invention is generally in the field of DNA vaccines for use in aquaculture. Background Art

[0002] Aquaculture has experienced a huge growth in productivity, reaching >527% in the time frame between 1990 and 2018. In 2018, aquaculture contributed approximately 46% of total global production of aquatic organisms (179M tonnes) and 52% of seafood (fish, crustaceans, molluscs and other aquatic animals, excluding aquatic mammals, reptiles, seaweed and other aquatic plants) for human consumption.

[0003] Commercial aquaculture is affected by infectious diseases caused mainly by bacteria, viruses, parasites and (to a lesser extent) fungi. Bacterial diseases can cause significant biological losses and therefore economic losses. Although these can usually be controlled using antibiotics, the indiscriminate use of these drugs will eventually pose a threat to human health because of the development and transfer of resistance mechanisms between bacterial species (some of which are also human pathogens). In addition, some traditional vaccines based on inactivated viruses may cause unacceptable side effects and / or some pathogens are difficult to grow in culture.

[0004] Pancreatic disease (PD) represents an economically important viral disease affecting marine farmed Atlantic salmon (Salmosalar) in Ireland, the United Kingdom and Norway. The aetiological agent is formally named salmonid pancreatic disease virus (SPDV), but is often denoted salmonid alphavirus (SAV), partly because the virus also infects other salmonid species. Oil-adjuvanted vaccines containing inactivated SPDV whole virions 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 considerable side effects in the form of spinal deformities and fibrosis of the surrounding muscle tissue, leading to downgrading at slaughter, reduced growth and animal welfare issues. See also et al., NOFIMA report 35\2021; and Holm et al., Aquaculture, 526, 2020.

[0005] Cardiomyopathy syndrome (CMS) is an inflammatory heart disease that primarily affects farmed Atlantic salmon (Salmosalar L.). The disease was first detected in Norway in 1985 and has been diagnosed in farmed and wild Atlantic salmon since then. The causative agent of CMS has been identified as fish myocarditis virus (PMCV) and the isolation and characterization of the virus are described in WO 2011 / 131600. Methods for controlling the virus are urgently needed, but despite extensive research, PMCV has not yet been effectively cultivated, or vaccines have therefore been produced based on attenuated or inactivated viruses.

[0006] DNA vaccines are an attractive approach for generating antigen-specific immune responses due to their stability and simplicity of delivery. DNA vaccines can be easily prepared on a large scale with high purity, administered repeatedly, and are highly stable relative to proteins and other biopolymers.

[0007] Among the various forms of constructible nucleic acid vaccines, circular DNA plasmids are the simplest. DNA vaccination involves immunization with circular DNA plasmids containing genes (or multiple genes) encoding antigens. In fact, injection of free DNA (naked DNA) stimulates an effective and long-term immune response to proteins (antigens) encoded by gene vaccines. When plasmid DNA is injected into an individual, the plasmid is absorbed by the cells and its genetic information is translated into immunization proteins. This enables the host immune system to react to the antigen.

[0008] However, DNA vaccines are relatively new, and there is a need to improve the effectiveness of these vaccines. Summary of the invention

[0009] In one aspect, the present disclosure provides a vaccine comprising a nucleic acid sequence encoding an antigen derived from a pathogen affecting fish, the vaccine being used in a method of actively protecting fish from said pathogen, the method comprising administering said vaccine intramuscularly to said fish as multiple injections, wherein

[0010] a. delivering multiple members to different injection sites;

[0011] b. each member of said plurality of times contains less than an effective dose of said vaccine; and

[0012] c. the total dose of the vaccine is no greater than the effective dose; and

[0013] d. All members of the plurality of times are delivered to the fish substantially simultaneously.

[0014] Also disclosed is a vaccine as described above, wherein the members of the plurality act synergistically.

[0015] The vaccine of claim 1 or claim 2, wherein the total dose provides a faster onset of immunity than the effective dose administered as a single injection when administered to fish maintained under the same conditions.

[0016] In certain aspects of the vaccines practiced according to the methods described herein, the total dose of the vaccine is less than the effective dose.

[0017] In another aspect, the present disclosure provides a vaccine comprising a nucleic acid sequence encoding an antigen derived from a pathogen affecting fish, the vaccine being used in a method of actively protecting fish from the pathogen, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein

[0018] a. delivering multiple members to different injection sites;

[0019] b. All members of the multiple times are delivered to the fish substantially simultaneously, and wherein the members of the multiple times act synergistically, or wherein when administered to fish maintained under the same conditions, the total dose administered in the multiple injections provides a faster onset of immunity than the same dose administered in a single injection of the same formulation. Preferably, the members of the multiple times act synergistically, or wherein when administered to fish maintained under the same conditions, the total dose administered in the multiple injections provides a faster onset of immunity than the same dose administered in a single injection of the same formulation.

[0020] Preferably, the plurality has 2, 3 or 4 members. The plurality may be delivered within 60 seconds, or more preferably within 30 seconds, or even more preferably within 15 seconds.

[0021] Also disclosed is a vaccine as described above, wherein 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 salmon anaemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV).

[0022] In certain embodiments of the vaccines described above, the pathogen is a salmonid alphavirus and the antigen comprises SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the pathogen is PMCV and the antigen comprises SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the vaccine comprises antigens against salmonid alphavirus and against PMCV.

[0023] Also disclosed is the vaccine as described above, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral neurological necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV) and tilapia lake virus.

[0024] Also disclosed is a vaccine as described above, wherein the nucleic acid sequence encoding the antigen is delivered by a heterologous vector, such as a plasmid vector or a viral vector.

[0025] Also disclosed is a vaccine as described above, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator. In certain embodiments, the molecular immunomodulator is interferon.

[0026] Also disclosed is a vaccine as described above, further comprising an adjuvant.

[0027] Also disclosed is a vaccine as described above, wherein the vaccine comprises a device for transporting the nucleic acid-based vaccine across a cell membrane. In certain embodiments, the device comprises a lipid coating.

[0028] In another aspect, the present disclosure provides an article comprising:

[0029] a) a reservoir containing a vaccine according to any one of claims 1 to 20, said reservoir being operably connected to a plurality of needles configured to deliver members of a plurality of injections into different injection sites; and

[0030] b) means for applying pressure to said reservoir to push the vaccine through the plurality of needles.

[0031] In yet another aspect, the present disclosure provides an article of manufacture comprising:

[0032] a. a plurality of reservoirs, each reservoir containing a member of a plurality of injections of a vaccine according to any one of claims 1 to 21, wherein each reservoir is operably connected to a needle, wherein needles connected to different reservoirs are configured to deliver members of the plurality of injections to different injection sites;

[0033] b. One or more devices for applying pressure to the reservoir to push the vaccine through the multiple needles.

[0034] In this and previous aspects of the invention, the device may be derived from a variety of sources including, but not limited to, pneumatic, hydraulic, electrical, or mechanical sources.

[0035] In yet another aspect, the present disclosure provides a method of administering an effective dose of a nucleic acid-based vaccine to a fish, the method comprising delivering multiple portions of the effective dose to different injection sites of the fish, wherein the multiple portions are injected substantially simultaneously.

[0036] In certain embodiments, in the methods disclosed herein, the plurality has 2, 3 or 4 members and can be delivered within 60 seconds, or 30 seconds, or 15 seconds. In a certain embodiment, the plurality has 2 members and is delivered within 15 seconds or less. In other embodiments, the plurality has three members and is delivered within 15 seconds or less.

[0037] In certain embodiments of the methods 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 salmon anemia (ISA) virus (ISAV), fish myocarditis virus (PMCV) and fish orthoreovirus (PRV).

[0038] In certain embodiments of the methods of this aspect of the invention, the pathogen is a salmonid alphavirus and the antigen comprises SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the pathogen is PMCV and the antigen comprises SEQ ID NO: 3 or SEQ ID NO:4.

[0039] Also disclosed is a method according to this aspect of the invention, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral neurological necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV) and tilapia lake virus.

[0040] In certain embodiments of the method according to this aspect of the invention, the nucleic acid sequence encoding the antigen is delivered by a heterologous vector. In certain embodiments, the heterologous vector is a plasmid vector. In other embodiments, the heterologous vector is a viral vector.

[0041] Also disclosed is a method according to this aspect of the invention, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator. In certain embodiments, the molecular immunomodulator is an interferon.

[0042] Also disclosed is a method according to this aspect of the invention, wherein the vaccine further comprises an adjuvant.

[0043] Also disclosed are methods according to this aspect of the invention, wherein the vaccine comprises a means for transporting the nucleic acid-based vaccine across a cell membrane. In certain embodiments, the means comprises a lipid coating.

[0044] Also disclosed are methods according to this aspect of the invention, wherein members of the plurality of parts act cooperatively.

[0045] Also disclosed are methods according to this aspect of the invention, wherein the total dose provides for a faster onset of immunity than an effective dose administered as a single injection. DETAILED DESCRIPTION

[0046] In order to better understand the present invention, the following definitions are provided:

[0047] The term "about" as applied to a reference numeral refers to the reference numeral plus or minus 10% of the stated value.

[0048] The term "different injection sites" refers to injection points that are positioned sufficiently apart from each other so that different skin penetrations are made for injection into these injection points. For example, the injection sites may 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, 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 20 mm apart, or about 10 mm to about 20 mm apart, or about 10 mm to about 30 mm apart, or about 10 mm to about 25 mm apart, or about 5 to about 20 mm apart, or about 10 mm to about 20 mm apart, or about 10 mm to about 30 mm apart, or about 10 mm to about 25 mm apart. About 40mm, or about 10mm to about 50mm, or about 10mm to about 60mm, or about 10mm to about 70mm, or about 10mm to about 80mm, or about 10mm to about 90mm, or about 20mm to about 30mm, or about 20mm to about 40mm, or about 20mm to about 50mm, or about 20mm to about 70mm, or about 30mm to about 40mm.

[0049] The term "effective dose" refers to the amount of an antigen that, if administered as a single injection of a given formulation, will provide the desired level of protection against the intended pathogen.

[0050] The term "members of a plurality (plurality) act synergistically" means

[0051] a) if the total dose is substantially equal to the effective dose, the ability of the total dose to elicit a greater protective immune response than an identical formulation containing the effective dose; or

[0052] b) If the total dose is less than the effective dose, the ability of the total dose to elicit the same protective immune response as the same formulation containing the effective dose.

[0053] 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 needs to enter the cell, replicate and be expressed as an antigen. Nucleic acid-based vaccines include, but are not limited to, DNA vaccines and mRNA vaccines. DNA vaccines can be vectorized via a plasmid or via a heterologous viral vector.

[0054] The term "substantially simultaneously" refers to the time interval between the injections of the portions of an effective dose of a vaccine disclosed herein. If the last portion of the vaccine is injected no later than 5 minutes, 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 after the first portion, then the portions are injected substantially simultaneously.

[0055] The term "substantially the same amount of antigen" in the members of the multiple injections means that the amount of antigen in the members of the multiple N injections varies between 1 / 2N and 1.5 / N of the total dose of the antigen. Therefore, if there are two members in the multiple injections, each member in the multiple should preferably contain 1 / 4 to 3 / 4 of the total dose. If there are three members in the multiple, each member in the multiple should preferably contain 1 / 6 to 1 / 2 of the total dose. In the most preferred embodiment, each member in the multiple N injections should contain about 1 / N of the total dose of the antigen. Therefore, if N=2 (two members in the multiple injections), each member in the multiple should contain about 1 / 2 of the total dose. If N=3 (three members in the multiple injections), each member in the multiple should contain about 1 / 3 of the total dose.

[0056] The expression "protection from a pathogen" refers to both the absence (or reduced level) of infection and the absence (or reduced level) of symptoms associated with infection. If the protective titer against a given pathogen is known, protection against the pathogen also means that, following vaccination as described herein, a statistically significant percentage of fish achieve a protective titer.

[0057] The term "total dose" refers to the sum of the doses of the antigen administered to each member of the multiple injections. For example, but not limited to, if the vaccine is administered in three injections, each injection containing 100 ng of a vector containing a nucleotide sequence encoding an antigen, the total dose is 300 ng.

[0058] The expression "total dose is substantially equal to the effective dose" refers to a total dose of at least 90% and not more than 100% of the effective dose. Conversely, the expression "total dose is less than the effective dose" refers to a total dose less than 90% of the effective dose.

[0059] The term "same protective immune response" refers to a response of at least 95% and no greater than 110% of a reference value, the reference value being the immune response elicited by a single injection containing an antigen dose equal to the total dose. Conversely, the term "greater protective immune response" refers to a response greater than 110% of the reference value, unless tissue damage is measured, in which case the term "greater protective immune response" refers to a response less than 95% of the reference value.

[0060] It is believed that administering the same amount of vaccine in several injections has the potential to increase antigen uptake by host cells, thereby leading to increased antigen expression and allowing for sparing of antigen doses, to obtain substantially the same protective immune response as a larger antigen dose (i.e., an effective dose) administered in a single injection, and / or to allow for an improved protective immune response if the total dose of vaccine administered in several injections is the same dose as the effective dose.

[0061] In a first broad aspect, the invention provides a vaccine comprising a nucleotide sequence encoding an antigen derived from a pathogen, which vaccine is used in a method for protecting fish from infection with the pathogen, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein members of the multiple injections are delivered to different injection sites; each member of the multiple injections contains less than an effective dose of the vaccine; the total dose of the vaccine is no greater than an effective dose; and all members of the multiple injections are delivered to the fish substantially simultaneously.

[0062] In one set of preferred embodiments, the administration of the members of the multiple times provides a synergistic protective immune response that is higher than the protective immune response elicited by the same dose of the same vaccine administered as a single effective dose, wherein the two groups of fish (the group treated with the single effective dose and the group treated according to the claims of the present disclosure) are maintained under the same conditions. This may be important because fish are poikilothermic animals and the aquarium water temperature may be important. The temperature itself is not as important as maintaining the two groups under the same conditions.

[0063] Also disclosed is a method of administering a vaccine according to the method disclosed above, wherein the total dose of the vaccine is less than the effective dose.

[0064] The present invention also provides a vaccine comprising a nucleic acid sequence encoding an antigen derived from a pathogen affecting fish, the vaccine being used in a method of actively protecting fish from the pathogen, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein the members of the multiple are delivered to different injection sites; all members of the multiple are delivered to the fish substantially simultaneously, and wherein the members of the multiple act synergistically, or wherein when administered to fish maintained under the same conditions, the total dose administered with the multiple injections provides a faster onset of immunity than the same dose administered with a single injection of the same formulation. Preferably, the members of the multiple act synergistically, or wherein when administered to fish maintained under the same conditions, the total dose administered with the multiple injections provides a faster onset of immunity than the same dose administered with a single injection of the same formulation.

[0065] The immune response can be measured in a variety of ways, depending on the pathogen and knowledge in the art. For example, antibody titers specific to the pathogen can be determined. In other embodiments, the protective immune response can be measured by the percentage of seroconverted animals. This evaluation index is particularly suitable for pathogens for which protective titers have been established.

[0066] In other embodiments, mortality or survival rate may be an appropriate evaluation index for determining synergistic effect. Virus count may also be a suitable evaluation index for determining synergistic effect. Alternatively, the prevalence of infection in at least one organ (i.e., the ratio of virus-positive fish to total fish) at one or more time points may be a suitable evaluation index. The immune response may also be measured by organ or tissue damage (or conversely, the lack of organ or tissue damage) in response to the challenge. The synergistic effect in at least one of these evaluation indicators indicates a synergistic protective immune response.

[0067] When applied to fish maintained under the same conditions, vaccines administered according to these methods can provide an earlier onset of immunity than the same vaccine administered with a single injection. The onset of 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 that do not exhibit viral infection. The earlier post-challenge immune response in the group administered with multiple injections indicates an earlier onset of immunity, even though the corresponding immune responses elicited by single and multiple injections may not be so obvious at a later stage compared to the group administered with a single injection.

[0068] Vaccines suitable for use according to the methods described herein are vaccines in which nucleic acid molecules encoding antigens enter host cells and express the antigens. Such vaccines may be DNA vaccines or mRNA vaccines. Both types of such vaccines are known in the art.

[0069] An mRNA-based vaccine against COVID-19 has been approved for human use. Thus, in certain embodiments, the vaccine comprises an mRNA sequence of a protein derived from a pathogen that causes a disease in fish.

[0070] DNA vaccines are also known. At least one DNA vaccine It has been approved in Europe for the protection of salmon from pancreatic disease caused by salmonid alphaviruses. Consists of a DNA plasmid (pUK-SPDV-poly2#1) dissolved in phosphate buffered saline. No adjuvant or preservative is present. The quantitative and qualitative composition has been properly defined as 'pUK-SPDV-poly2#1 DNA plasmid encoding salmon pancreatic disease virus (SPDV) protein', 5.1μg-9.4μg / 0.05ml dose (101.6μg / ml-188.4μg / ml).

[0071] Thus, in the DNA vaccines according to various embodiments of the present invention, the nucleic acid sequence encoding the antigen is in a heterologous vector. A variety of heterologous vectors are suitable for the present invention and are known in the art. In a non-limiting manner, vectors include viral vectors, plasmid vectors, and circular DNA vectors, also known as doggybone vectors.

[0072] 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), etc. These viruses can be genetically modified to remove portions of the viral genome responsible for replication. Thus, the resulting virus will be infectious to fish cells and suitable for antigen production, but will not be pathogenic.

[0073] Suitable plasmids include, but are not limited to, pUC-based vectors, pVAX vectors, pcDNA vectors, NTC vectors. In a preferred embodiment, the vector is NTC9385R (Nature Technology Corporation) or a variant thereof.

[0074] In other embodiments, the relatively new "dog bone" or DBDNA TM Plasmids can be used as vectors. TM Plasmids and processes for making these plasmids have been described at least in WO2018 033730, WO2016034849, WO2019193361, WO2012017210 and WO2021161051.

[0075] The advantage of this method is that the vector can be synthesized in the cell-free process, so the manufacturing efficiency is improved. The cell-free process preferably involves replicating the amplification template via strand displacement. The synthesis releases a single-stranded DNA, which can be copied into double-stranded DNA using a polymerase. Alternatively, strand displacement can be achieved by supplying DNA polymerase and a separate helicase. The replicating helicase can unwind the double-stranded DNA and promote the advancement of the leading strand polymerase. The resulting double-stranded DNA concatemer is cut enzymatically and joined to form a DNA construct in a dog-bone shape. The dog-bone-shaped vector is composed of a telomerase recognition site, a desired construct (comprising a sequence encoding an antigen, a promoter and a polyA site) and a sequence complementary thereto. Therefore, the complementary parts of the vector hybridize with each other and form a spiral.

[0076] A variety of pathogens are suitable for use as the source of the antigen in the nucleic acid-based vaccine disclosed herein. In certain embodiments, the pathogen is a virus. Suitable viruses may be selected from the group consisting of the following: salmonid alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), fish myocarditis virus (PMCV), fish orthoreovirus (PRV), roundfin fish virus, viral neuronecrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV) and tilapia lake virus. In a particular embodiment, the virus is a salmonid alphavirus. In another embodiment, the virus is PMCV.

[0077] In other embodiments, 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., Edwardsiella sp. sp.), Francisella sp., Pseudomonas sp., Cytophaga sp., Nocardia sp., and Mycobacerium sp.

[0078] Particularly preferred are antigens from intracellular bacterial pathogens, including but not limited to Rickettsia species, Edwardsiella species, Yersinia species, Francisella species, Photorhabdus species, Mycobacterium species, and Nephrobacterium species.

[0079] Surface proteins from viruses and bacteria may be suitable candidates for the nucleic acid-based vaccines disclosed herein.

[0080] In other embodiments, the pathogen is a parasite, such as sea lice. In certain embodiments, the sea lice is selected from the genus Lepeophtheirus or Caligus, and the antigen may be a midgut protein or an immunogenic fragment thereof. See, e.g., U.S. Patent No. 11,167,017.

[0081] Certain vaccines particularly suitable for administration to salmonids, such as Atlantic salmon, comprise a nucleic acid sequence encoding an immunogen that elicits a protective immune response against salmonid alphaviruses. Suitable non-limiting examples of such antigens include, but are not limited to, SEQ ID NO: 1 or SEQ ID NO: 2.

[0082] Other vaccines also suitable for use in salmonids, such as Atlantic salmon, include nucleic acid sequences encoding immunogens that elicit a protective immune response against fish myocarditis virus (PMCV). Suitable non-limiting examples of such antigens include proteins encoded by the ORF-1 of PMCV (e.g., SEQ ID NO: 3), or fragments thereof, including but not limited to SEQ ID NO: 4.

[0083] Still other vaccines suitable for administration to fish such as Atlantic salmon comprise nucleic acid sequences encoding immunogens that elicit a protective immune response against fish myocarditis virus (PMCV) as described above, and immunogens that elicit a protective immune response against salmonid alphaviruses.

[0084] In other embodiments, the pathogen is a parasite, such as, for example, sea lice, particularly sea lice of the genus Myxocytidae, and more particularly sea lice of the species Myxocytidae. Peptides from sea lice have been proposed as potential antigens in vaccines. Some suitable candidates are disclosed in U.S. Patent No. 11,167,017.

[0085] The nucleic acid sequence in a DNA vaccine is generally under the control of a suitable promoter. Promoters suitable for vaccines according to the present invention should be able to initiate transcription in the host organism. In embodiments where the host is salmonids, such as Atlantic salmon, suitable promoters include, but are not limited to, the simian virus 40 early promoter (SV40), the cytomegalovirus immediate early promoter (CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1 alpha promoter (EF1A), the mouse phosphoglycerate kinase 1 promoter (PGK), and the chicken β-actin promoter (CAGG) coupled to the CMV early enhancer.

[0086] The carrier may further comprise a nucleic acid sequence encoding a molecular immunomodulator. Suitable molecular immunomodulators include interferon. It has been previously demonstrated that the nucleic acid sequence encoding salmon interferon delivered in a DNA vaccine enhances antigen-specific immune response. Therefore, in certain embodiments, the molecular immunomodulator is an interferon selected from the group consisting of salmon IFNa, IFNb, IFNb1, IFNa2 and IFNc.

[0087] 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 (e.g., 1005), quaternary amines (such as dimethyldioctadecyl ammonium bromide (hereinafter "DDA")). Complexes comprising saponin, sterols (e.g., cholesterol) and optionally phospholipids have been described in the art. Combinations of CpG oligonucleotides with saponin, CpG with cholesterol, and CpG with alum have been reported to induce synergistic effects.

[0088] In certain embodiments, the vaccine may include a liposomal adjuvant and / or carrier to facilitate transport of the vector across the cell membrane and thereby cause increased expression of the antigen and / or molecular immunomodulator. Suitable non-limiting examples of such liposomal adjuvant / carrier systems are described in, for example, U.S. Patent No. 10,456,459.

[0089] The vaccine according to the present invention may further comprise excipients, such as preservatives, stabilizers, buffers and the like.

[0090] A variety of fish species are suitable for vaccination according to the methods described herein. Suitable species include salmonids (including species of the genus Salmo and Salmon) and sea bass (European sea bass (Dicentrarchus labrax)), as well as warm water fish, including tilapia (Oreochromis niloticus) and giant catfish (striped catfish (PangasiusHypophthalmus)). Other species include whitefish, Arctic charr, Mandarin perch, and largemouth bass. When the fish weight is between about 15 grams and about 200 grams, more preferably between about 40 grams and about 110 grams, the fish can be vaccinated according to the methods disclosed herein.

[0091] As mentioned above, the vaccine according to the invention is delivered in several parts via multiple injections administered substantially simultaneously (the first and last parts are administered within five minutes). Preferably, the last part 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 and no later than 15 seconds, and no later than 5 seconds and no later than 1 second from the first part of the vaccine.

[0092] The exact number of members in a multiple injection is typically 2, 3, 4 or 5, more preferably 2 or 3.

[0093] The volume of the members in multiple injections can be determined by one of ordinary skill 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. It is currently preferred that the members in multiple injections contain substantially the same amount of antigen.

[0094] There are a variety of methods to administer injections substantially simultaneously. In certain embodiments, commercially available fish vaccination devices may be equipped with a multi-needle injection tip, wherein the tips of the needles are configured to be spaced a desired distance apart from one another. Suitable vaccination machines include the NFTT line of products (Pharmaq) and specific models include NFT 20, NFT 25 and NFT 30. NFT 20, NFT 25 deliver vaccines intraperitoneally, but can also be configured to perform intramuscular injections. NFT 30 NFT 30 has a special DNA module that can deliver DNA vaccines intramuscularly into fish tenderloins.

[0095] The machine handles fish sizes from 120mm to 250mm (20g to 150g). Once completed, it sorts the vaccinated fish into three different sizes. In addition, it has a channel for misplaced, undersized or rejected fish.

[0096] Alternatively, the vaccine may be administered manually via a syringe containing one or several needles. See, for example, the Syringes. This product is available in single and dual syringe versions. 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 is available with a replaceable 0.025 ml piston when necessary. The dual 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. When necessary, the 0.05 ml syringe is available with a replaceable 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%. Dose accuracy has been documented to deviate by less than 3%.

[0097] The pressure at which the injection is delivered is not critical and may be derived from a hydraulic, pneumatic, electrical, or mechanical source.

[0098] In general, the exact number of injections (ie, members of a plurality of injections) can be determined experimentally. Preferably, the number is between two and five, more preferably between two and four, and most preferably two or three.

[0099] In one embodiment, there are three members in multiple injections and the antigen comprises a PMCV antigen, such as, for example, SEQ ID NO: 3 or SEQ ID NO: 4. In these embodiments, the vaccine may also contain an immunomodulator, such as an interferon. Preferably, the interferon is IFNb. In another embodiment, there are two members in multiple injections and the antigen comprises a PMCV antigen, such as, for example, SEQ ID NO: 3 or SEQ ID NO: 4. In these embodiments, the vaccine may also contain an immunomodulator, such as an interferon. Preferably, the interferon is IFNb.

[0100] In another embodiment, there are two members of the multiple injections and the antigen comprises a SAV antigen, such as, for example, SEQ ID NO:1 or SEQ ID NO:2.

[0101] The following examples are presented as illustrative embodiments, but should not be considered to limit the scope of the present invention. Many changes, variations, modifications and other uses and applications of the present invention will be apparent to those skilled in the art to which the present invention belongs.

[0102] Examples

[0103] Example 1. Multiple SAV DNA vaccine injections produce lower viral loads and lower clinical scores compared to single injections. Materials and methods: Atlantic salmon juveniles weighing an average of 26 grams were vaccinated in fresh water by intramuscular vaccination with a standard dose of PD DNA vaccine. For one group, the entire 0.05 mL dose was administered as a single injection. For the other group, the 0.05 mL dose was administered as 2 consecutive injections of 0.025 mL each. Fish injected with phosphate buffered saline (PBS) served as negative controls. Fish were kept in the same fish tank throughout the study. Starting from the second day after vaccination, fish were exposed to continuous light to induce smoltification, inducing physiological changes required to prepare for the transfer of fish to seawater. After a 6-week (42 days, 500 degree days) vaccination period, fish were transferred to seawater and exposed to SAV3 by commensal challenge. Fish were introduced into the same fish tank by intraperitoneal injection of infectious SAV3 material to challenge the original fish, so that the vaccinated fish and control fish were exposed to SAV3 in a manner that simulated a natural infection outbreak. For vaccinated fish, 29 to 30 fish were challenged per group, and for negative control fish, 20 fish were challenged. All surviving fish were sampled at the end of 5 weeks (36 days) after the start of the commensal challenge. The fish were weighed and incubated in formalin and Heart and pancreas samples (both important target organs for PD) were obtained on 24-hour day 48 hours. Because no mortality was obtained during the observation period in both vaccinated groups and only a single mortality (5%) was obtained for negative control fish, efficacy was evaluated primarily by evaluating the weight of the heart and pancreas, the severity of tissue damage, and the number of SPDV in the heart and pancreas.

[0104] Statistical analysis of cardiac viral load and pancreatic viral load in the vaccinated group compared with the negative control group was performed by Mann-Whitney test using GraphPad v.8.1.1. Statistical analysis of the weight of the vaccinated group at the end of the challenge period compared to the negative control group was performed by unpaired t-test with Welch's correction using GraphPad v.8.1.1 is underway.

[0105] Quantification of SPDV in heart and pancreatic tissue samples was performed by a probe-based reverse transcriptase quantitative PCR (RT-qPCR) assay. The assay targets the nonstructural protein 1 (nsP1) of SPDV and is able to detect all known salmonid alphaviruses (Hodneland & Endresen, 2006). The analysis was performed by an officially recognized diagnostic laboratory (PHARMAQ Analytiq) authorized in Norway to test for SPDV infection in salmonids.

[0106] At the end of 5 weeks post-challenge (wpc) the severity of tissue damage in surviving fish was analyzed by histopathology using the scoring system described in Graham et al., Journal of fish diseases, Vol. 34, No. 4, 237-286.2011, on a scale of 0-3 (pancreas) and 0-4 (heart). The scoring system is provided in Table 1.

[0107] Table 1. Tissue damage score table

[0108]

[0109]

[0110] The results are in Table 2 (viral counts), Table 3 (severity of tissue damage) and Table 4 (weight).

[0111] Table 2. PCR results of surviving fish at the end of 5 wpc.

[0112]

[0113] The PCR results showed that both single and double injections caused a reduced prevalence of infection and reduced viral load (higher counts), but the prevalence was lower in the double injection group. Heart viral counts in both the single and double injection groups were significantly different from the control group. Pancreatic viral counts in the double injection group were significantly different from the control group. These results suggest that double injections with 2x0.025 ml vaccine are more effective than single injections with 0.05 ml vaccine.

[0114] Table 3. Severity of tissue damage in surviving fish at the end of 5 wpc

[0115]

[0116] The tissue damage of the pancreas and heart of the fish in the single injection group and the double injection group was different from the pancreas and heart damage of the control group, respectively. It is noteworthy that in both the pancreas and the heart, the double injection group tended to show less damage than the single injection group (0.13 vs. 0.41 in the heart and 0.17 vs. 0.31 in the pancreas).

[0117] Table 4. Weights of surviving fish at the time of challenge and at the end of 5 wpc.

[0118]

[0119] The weights of the vaccinated groups 5 weeks after the start of the commensal challenge showed that both vaccinated groups were highly protected from PD-related growth reduction, with significantly higher weights and growth rates compared to the negative control group. No statistically significant differences were observed between the one-injection and two-injection groups.

[0120] Increasing the number of injections from one to two resulted in a decrease in viral load and viral prevalence in both the heart and pancreas five weeks after challenge. The prevalence in the heart and pancreas was reduced more than twofold in the double-injection group compared to the single-injection group, even though the total amount of vaccine in both groups was the same.

[0121] Example 2. Multiple PMCV DNA vaccine injections produce lower viral loads and lower histopathology scores compared to a single injection.

[0122] Atlantic salmon (n=150) with an average weight of 29g were kept in a 500L freshwater (12C) fish tank with a 12:12 light:dark lighting scheme. The fish were fasted for one day and then anesthetized with MS222 (Tricaine, PHARMAQAS), marked as one of five groups (n=30 / group) by clipping the adipose fin or maxilla, and vaccinated intramuscularly with DNA plasmids expressing part of the ORF1PMCV antigen and salmon IFNb as a molecular adjuvant. One of the five groups was given a negative control vaccine (PBS), while the remaining four groups were injected 1, 2, 3 or 4 times with a vaccine containing the PMCV ORF1 coding sequence during the same anesthesia period. The concentration of each group of plasmids was adjusted so that the total amount of plasmids was 10 μg per fish, regardless of the number of injections. Table 5 details the groups at the time of vaccination.

[0123] Table 5. Experimental settings

[0124]

[0125] Seven weeks after immunization, all fish were anesthetized again and injected intraperitoneally with 0.1 ml of kidney homogenate containing infectious PMCV particles. Hearts were then sampled from 15 fish per group at each time point at 3 and 7 weeks after challenge. Hearts were stored at The samples were sent to the diagnostic laboratory PHARMAQ Analytiq (Bergen, Norway) for real-time PCR detection of PMCV RNA. The heart pathology of the heart sampled 7 weeks after the challenge was also examined (PHARMAQ Analytiq, Bergen Norway). The viral counts at three and seven weeks after the challenge are summarized in Table 6 and Table 7, respectively. The histological analysis of the atria is summarized in Table 8.

[0126] Table 6. Heart real-time PCR results for fish challenged with PMCV 3 weeks previously. Negative fish were given a default Ct value of 40.

[0127] Group Number of injections per fish fish (n) Number of PMCV-positive fish Mean ct value (PMCV) 1 1x0.05 ml 15 13 33.37 2 2x0.05ml 15 10 35.38 3 3x0.05ml 15 7 37.51 4 4x0.05ml 15 10 36.23 5 4x0.05ml 15 15 32.03

[0128] Table 7. Heart real-time PCR results for fish challenged with PMCV 7 weeks earlier. Negative fish were given a default Ct value of 40.

[0129] Group Number of injections per fish fish (n) Number of PMCV-positive fish Mean ct value (PMCV) 1 1x0.05 ml 15 10 33.74 2 2x0.05ml 15 10 35.34 3 3x0.05ml 15 8 37.26 4 4x0.05ml 15 10 35.59 5 4x0.05ml 15 15 27.22

[0130] Table 8. Histological analysis of atria. Histological lesions were scored from 0 (normal) to 4 (severe lesions). The number of fish per score per group is indicated.

[0131]

[0132]

[0133] The number of injections increased from 1 to three times, and three weeks after the challenge, the virus count (virus load is lower) of increase and the percentage of negative fish (from two in the group treated with one injection to eight in the group treated with three injections) were found. Seven weeks after the challenge, the number of negative fish in the single, double and four injection groups was 5 in 15, and in the three injection groups, the number of negative fish was 7 in 15. These results show that the vaccine used with multiple injections promotes faster immune initiation. No significant weight difference was found between the one, two, three and four injection groups.

[0134] All publications (both patent publications and non-patent publications) cited in this specification are indicative of the technical level of those skilled in the art to which the invention pertains. All of these publications are incorporated herein in their entirety by reference, to the same extent as if each individual publication was expressly and individually indicated to be incorporated by reference.

[0135] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that many modifications may be made to the illustrative embodiments and other arrangements may be designed without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A vaccine comprising a nucleic acid sequence encoding an antigen derived from a pathogen affecting fish, the vaccine being used in a method for actively protecting fish from the effects of the pathogen, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein a. delivering multiple members to different injection sites; b. each member of said plurality of times contains less than an effective dose of said vaccine; c. The total dose of the vaccine is not greater than the effective dose; as well as d. All members of said plurality of times are delivered to said fish substantially simultaneously.

2. The vaccine of claim 1, wherein the members of the plurality act synergistically.

3. A vaccine according to claim 1 or claim 2, wherein when administered to fish maintained under the same conditions, the total dose provides a faster onset of immunity than an effective dose administered as a single injection.

4. A vaccine comprising a nucleic acid sequence encoding an antigen derived from a pathogen affecting fish, the vaccine being used in a method for actively protecting fish from the effects of the pathogen, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein a. delivering multiple members to different injection sites; b. all members of said plurality of times are delivered to said fish substantially simultaneously, and wherein said members of said plurality act synergistically, or wherein the total dose administered in said plurality of injections provides a more rapid onset of immunity when administered to fish maintained under the same conditions than the same dose administered in a single injection of the same formulation.

5. The vaccine of claim 4, wherein the members of the multiple injections act synergistically, and wherein when administered to fish maintained under the same conditions, the total dose administered as the multiple injections provides a faster onset of immunity than the same dose administered as a single injection of the same formulation.

6. The vaccine according to any one of claims 1 to 5, wherein the total dose of the vaccine is less than the effective dose.

7. The vaccine of any one of claims 1 to 6, wherein the plurality has 2, 3 or 4 members.

8. The vaccine of any one of claims 1 to 7, wherein the multiple injections are delivered within 1 minute.

9. The vaccine of any one of claims 1 to 8, wherein the multiple injections are delivered within 30 seconds.

10. The vaccine of any one of claims 1 to 9, wherein the multiple injections are delivered within 15 seconds.

11. The vaccine of any one of claims 1 to 10, wherein each member of said plurality contains substantially the same amount of said antigen.

12. The vaccine according to claim 11, wherein a. said multiple times having two members and each member of said multiple times having about 1 / 2 of said total dose of said antigen; or b. The plurality has three members and each member of the plurality has about 1 / 3 of the total dose of the antigen.

13. The vaccine according to any one of claims 1 to 12, wherein 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 salmon anemia (ISA) virus (ISAV), fish myocarditis virus (PMCV), fish orthoreovirus (PRV), roundfin fish virus, viral neuronecrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), tilapia lake virus, Piscirickettsia sp., Edwardsiella sp., Yersinia sp., Francisella sp., Photobacterium sp., Mycobacterium sp., Renibacterium sp., Lepeophtheirus sp. sp.) and Caligus sp.

14. The vaccine according to any one of claims 1 to 12, wherein 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 salmon anemia (ISA) virus (ISAV), fish myocarditis virus (PMCV) and fish orthoreovirus (PRV).

15. The vaccine according to claim 13 or 14, wherein: a) the plurality has two or three members and the pathogen is PMCV, or b) The plurality has two members and the pathogen is SAV.

16. The vaccine according to any one of claims 1 to 13, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral neurological necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV) and tilapia lake virus.

17. The vaccine according to any one of claims 1 to 16, wherein the nucleic acid sequence encoding the antigen is delivered by a heterologous vector.

18. The vaccine according to claim 17, wherein the heterologous vector is a plasmid vector or a viral vector.

19. The vaccine according to claim 17 or 18, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator.

20. The vaccine according to claim 19, wherein the molecular immunomodulator is interferon.

21. The vaccine according to any one of claims 1 to 20, further comprising an adjuvant.

22. The vaccine according to claim 21, wherein the adjuvant is selected from the group consisting of: MPLA, CpG-containing oligodeoxyribonucleotides, oligoribonucleotides, saponins, sterols and cationic lipids.

23. The vaccine of any one of claims 1 to 22, wherein the vaccine comprises means for transporting the nucleic acid-based vaccine across a cell membrane.

24. The vaccine of claim 23, wherein the device comprises a lipid coating.

25. An article comprising: a) a reservoir containing a vaccine according to any one of claims 1 to 24, said reservoir being operably connected to a plurality of needles configured to deliver members of multiple injections into different injection sites; as well as b) means for applying pressure to said reservoir to push said vaccine through said plurality of needles.

26. An article comprising: a. a plurality of reservoirs, each reservoir containing a member of a plurality of injections of a vaccine according to any one of claims 1 to 24, wherein each reservoir is operably connected to a needle, wherein the needles connected to different reservoirs are configured to deliver the members of the plurality of injections to different injection sites; b. One or more devices for applying pressure to the reservoir to push the vaccine through the plurality of needles.

27. A method of administering an effective dose of a nucleic acid-based vaccine to a fish, the method comprising delivering multiple portions of the effective dose to different injection sites of the fish, wherein the multiple portions are injected substantially simultaneously.

28. The method of claim 27, wherein members of the plurality act cooperatively.

29. The method of claim 27 or claim 28, wherein when administered to fish maintained under the same conditions, injection of multiple portions of the effective dose into different injection sites of the fish results in faster initiation of immunity than administration of the effective dose as a single injection.

30. A method of administering a total dose of a nucleic acid-based vaccine to a fish, the method comprising delivering multiple portions of the total dose to different injection sites in the fish, wherein the multiple portions are injected substantially simultaneously, wherein the members of the multiple act synergistically, or wherein when administered to fish maintained under the same conditions, the total dose administered as multiple injections provides a faster onset of immunity than the same dose administered as a single injection of the same formulation.

31. The method of claim 31, wherein the members of the plurality act synergistically, or wherein when administered to fish maintained under the same conditions, the total dose administered as the multiple injections provides a faster onset of immunity than the same dose administered as a single injection of the same formulation.

32. The method of any one of claims 27 to 31, wherein the total dose is less than the effective dose of the same vaccine.

33. The method of any one of claims 27 to 32, wherein the plurality has 2, 3 or 4 members.

34. The method of any one of claims 27 to 33, wherein the plurality of portions are delivered within 1 minute.

35. The method of any one of claims 27 to 34, wherein the plurality of portions are delivered within 30 seconds.

36. The method of any one of claims 27 to 35, wherein the plurality of portions are delivered within 15 seconds.

37. The vaccine of any one of claims 27 to 36, wherein each member of the plurality contains substantially the same amount of the antigen.

38. The vaccine according to claim 37, wherein a. said plurality having two members and each member of said plurality having about 1 / 2 of said total dose of said antigen; or b. The plurality has three members and each member of the plurality has about 1 / 3 of the total dose of the antigen.

39. The method of any one of claims 27 to 38, wherein 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 salmon anemia (ISA) virus (ISAV), fish myocarditis virus (PMCV), fish orthoreovirus (PRV), roundfin fish virus, viral neuronecrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), tilapia lake virus, fish Rickettsia species, Edwardsiella species, Yersinia species, Francisella species, Photorhabdus species, Mycobacterium species, Nephrobacterium species, Ichthyophthirius species, and Ichthyophthirius species.

40. The method of any one of claims 27 to 39, wherein 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 salmon anemia (ISA) virus (ISAV), fish myocarditis virus (PMCV) and fish orthoreovirus (PRV).

41. The method of claim 39 or 40, wherein: a) the plurality has two or three members and the pathogen is PMCV, or b) the plurality has two members and the pathogen is SAV.

42. The method of any one of claims 27 to 39, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral neurological necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV) and tilapia lake virus.

43. The method of any one of claims 27 to 42, wherein the nucleic acid sequence encoding the antigen is delivered by a heterologous vector.

44. The method of claim 43, wherein the heterologous vector is a plasmid vector or a viral vector.

45. The method of claim 43 or 44, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator.

46. ​​The method of claim 45, wherein the molecular immunomodulator is an interferon.

47. The method of any one of claims 27 to 46, further comprising an adjuvant.

48. The method of claim 47, wherein the adjuvant is selected from the group consisting of: MPLA, CpG-containing oligodeoxyribonucleotides, oligoribonucleotides, saponins, sterols and cationic lipids.

49. The method of any one of claims 27 to 48, wherein the vaccine comprises a means for transporting the nucleic acid-based vaccine across a cell membrane.

50. The method of claim 49, wherein the device comprises a lipid coating.

Images