WO2020132770A1 - Virus-like particles (vlp) of the infectious salmon anemia virus (isav) that comprise matrix protein and one or more antigenic proteins of the virus; production method, composition, vaccine and fish feed; recombinant baculovirus; and vaccination kit; and - Google Patents

Abstract

The present invention relates to the field of veterinary medicine, particularly vaccines and animal health in aquaculture. The invention concerns virus-like particles (VLP) formed by molecular complexes comprising proteins of infectious salmon anemia virus (ISAV), the genes of which are expressed in insect cells, and which are used as vaccines together with induced cellular bodies as adjuvants. The invention also concerns pharmaceutical compositions, vaccines, fish feed and a kit, which comprise the VLP, and the use of the VLP to prepare drugs.

Description

Infectious salmon anemia virus (ISAV) virus-like particles (VLPs) comprising the matrix protein and one or more antigenic proteins of said virus; method of obtaining, composition, vaccine and food for recombinant baculovirus fish; and vaccination kit.

Field of the Invention

The present invention relates to the field of veterinary medicine, particularly with vaccines and animal health in the field of aquaculture. The present invention relates to virus-like particles (VLPs), formed by molecular complexes that comprise proteins of the infectious salmon anemia virus (ISAV) whose genes are expressed in insect cells, and which are used as vaccines in conjunction with cell bodies induced as adjuvants. It is also within the scope of the present invention, the pharmaceutical compositions, vaccines, fish feed and kit comprising the VLPs, as well as the use of the VLPs to prepare drugs.

Description of the state of the art

Infectious salmon anemia (ISA) is a multisystemic disease that mainly affects Atlantic salmon (Salmo salar) in captivity and is caused by the infectious salmon anemia virus (ISAV) (Thorud and Djupvik, 1988).

The ISAV virus worldwide has been associated with the appearance of highly deadly outbreaks in the main Atlantic salmon-producing countries, so that between 1988 and 2008 the virus caused production losses in both Norway, Canada, Scotland, United States, Faroe Islands, as well as Chile (Thorud and Djupvik, 1988; Kibenge et al., 2009; Rodger et al., 1998; Bouchard et al., 1999; Rowley et al., 1999; Bouchard et al., 2001 ; Christiansen et al., 2011). In the latest high mortality outbreaks registered in Chile between 2007 and 2008, the Atlantic salmon producing centers in this country registered a decrease in their production of 300,000 tons per year, dropping from 400,000 tons of fish in 2006 to 100,000 tons in 2010 (Asche , F., Hansen, H., Tveteras, R., Centrum, ST 2010. The Salmon Disease Crisis in Chile (Marine Resource Economics, 24: 405-411) with serious economic and social consequences. It is based on these losses that it is imperative to search and find an effective method to control the disease.

The infectious salmon anemia virus (ISAV) is classified as a member of the Orthomyxoviridae family, its genome is of single-stranded RNA of negative polarity, which is divided into 8 segments, it has a membranous covering, of associated cellular origin to surface viral glycoproteins, and has a helical capsid. In the viral particle, each of the 8 RNA segments is associated with multiple nucleoprotein units (NP) and a copy of the RNA-dependent RNA polymerase, which is made up of a heterotimer of the protein subunits of PB1, PB2 and PA, thus forming the ribonucleoprotein complex (RNP). Structurally, the matrix protein (Mx) is found towards the internal face of the lipid covering, whose function is to provide structural support to the viral particles, while the hemagglutinin (HE) and fusion (F) glycoproteins are towards the external face. , which have the function of recognizing the viral receptor in the cell, at the beginning of the infection, and releasing the virus during the generation of a new particle and fusing the viral membrane with that of the endosomes, respectively. The basis for the development of vaccines to protect fish from ISAV infection can be based on the following factors: (i) A relationship has been established between infections of Atlantic salmon with increases in blood IgM levels; (ii) Fish have been detected that carry ISAV but do not develop the disease; and (iii) it has been observed that salmon infected with low pathogenic ISAV, or those that survive one infection, are better able to resist a second infection (Plarre et al., 2005; Cipriano, 2009; Ritchie et al., 2009 ). In addition to the above, the presence of anti-ISAV antibodies has been established in samples of naturally infected wild salmon. Furthermore, in laboratory tests, it was determined that at 6 weeks post infection the fish already generates anti-ISAV antibodies (Kibenge et al., 2002; Cipriano, 2009), and it was determined that the serum of fish surviving an infection by ISAV can be used to neutralize viral infection in vivo, observing that the number of fish killed by ISAV infection decreased after inoculation with sera (Falk & Dannevig, 1995).

This has led to a growing interest in the development of alternative vaccines against ISAV (Gomez-Casado et al., 2011). To date, the vaccines used to counteract ISAV infections are of two types, subunit and inactivated virus, and even when fish are immunized, outbreaks of ISAV continue to occur in Atlantic salmon producing centers. These vaccines seek to decrease the accumulated mortality in fish inoculated and challenged with ISAV, a process that has been associated with an increase in the levels of antibodies and variations in the level of gene expression related to the immune response of fish, such as gamma interferon, Alpha Interferon and Major Histocompatibility Complex I (MHCI) (Lauscher et al., 2011). Some examples of ISA virus subunit vaccines can be described in: AKZO NOBEL NV patent US6471964 (CL 2862-2000; granted), which describes a vaccine developed with a protein structural called SP-1, which was produced in baculovirus; US Patent 7183404 (CL 1399-2000; granted) to AKZO NOBEL NV disclosing the use of an ISAV protein with a mass of 42.8 kDa; US patent 7279167 (CL2355 - 2002) from INTERVET INT. BV, which describes a vaccine developed with a 48 kDa protein from ISAV which was produced in baculovirus; finally, publications WO01 10469 (CL 750 - 2002) OTTAWA HEALTH RESEARCH INST. NOVARTIS AG. and WO0149712 (CL 19-2001) AKZO NOBEL NV, describe several proteins that are declared as antigenic and that can be used as vaccines. Multivalent vaccines for salmon are marketed in Chile, which act on various pathogens, including isolates of the ISA virus isolated in Chile (FAV). Table 1 below includes vaccines registered with the Agricultural and Livestock Service (SAG).

Table 1

Since the existing vaccines have not ensured the production of ISAV-free salmon, a new strategy to be explored by the present invention is the generation of vaccines using virus-like particles (VLP). VLPs are generated by simultaneous expression of the structural proteins of a virus and that when self-assembling are particles that resemble the viral structure, but lack their genetic material. In a healthy individual, this allows an infection to be emulated but without the development of the disease, inducing the immune response against VLPs, which by resembling the structure of the viral agent, are able to protect against a subsequent infection by the virus (Noad & Roy, 2003). In the ISAV study area, there are no works that demonstrate the generation of this type of structure, but in other viral fish systems there are examples in this research area.

The expression of the structural proteins of infectious pancreatic necrosis virus (IPNV) and grouper nerve necrosis virus (GNNV), using the heterologous systems of yeast and bacteria respectively, allowed the generation of particles that have a structure similar to that of the native virus when analyzed by electron microscopy (Lu et al., 2003; Allnutt et al., 2007). In addition to the above, when used as immunogens to vaccinate salmon and grouper, in the case of IPNV and GNNV respectively, the VLPs were able to generate a protective immune response associated with the increase in the levels of antibodies against the virus, which was subsequently related to a decrease in viral load and lower cumulative mortality when fish were challenged with the virus (Lu et al., 2003; Allnutt et al., 2007).

Publication WO2012130723 (CL 2723-2013) describes virus-like immunogenic particles (VLPs) against salmonid alphaviruses (SAVs). VLPs are produced in Baculovirus that presents a vector that comprises a polynucleotide that encodes an alphavirus capsid protein, E3 protein, an E2 envelope protein, 6K protein, and E1 protein. This document describes specific temperature conditions of infection of insect cells by baculovirus to produce alphavirus VLPs. Unlike viral systems that have a protein capsid, which tend to self-assemble their coat proteins into icosahedral structures, ISAV has a membranous coat, which presents a difficulty when developing structures such as VLPs since it is necessary to split the cell membrane containing viral proteins so that virus-like structures are formed. The ISA virus is a negative polarity RNA virus, a member of the Oortomyxovirus family, which is made up of the following six genera: Influenza virus A. Influenza virus B. Influenza virus C, Isavirus. Thogotovirus and Quaraniavirus. The first three viruses cause influenza in vertebrates, including birds, humans, and other mammals. The ISA virus is the only member of the Orthomyxovirus family capable of infecting fish, giving its name to the Isavirus genus. Thogotoviruses are arboviruses, which infect vertebrates and invertebrates, such as ticks and mosquitoes. Quaranjaviruses predominantly infect arthropods and birds and are transmitted between vertebrates by ticks, resembling members of Thogotoviruses.

The ISA virus differs from influenza viruses in that the surface protein Hemagglutinin acetyl esterase (HE) also has an activity similar to neuroaminidase (NA) and has a separate fusion protein, resembling the Paramixoviridae viral family. While influenza viruses have a Hemagglutinin esterase (HA) protein, which also has fusion activity and also has a Neuraminidase (NA) protein. In the state of the art, the formation of influenza virus VLPs is described, by means of the expression of the matrix protein individually or simultaneously to hemagglutinin, neuraminidase and / or nucleoprotein, using heterologous expression systems such as baculovirus / insect cells. This allowed obtaining Influenza VLPs structurally similar to the native virus, added that when used to immunize mice or rats, they allowed the generation of an immune response capable of protecting them from exposure to a lethal dose of the virus (W02002000885, W02005020889, W02007047831, W02009012489, WQ2007130327. W02008148104 or W02007130330). The formation of influenza virus VLPs with the protein hemagglutinin or modified hemagglutinin, expressed in plant cells, has also been described (W02009076778, W02008148104,

W02009009876, WO2010003225, W02010148511) or VLPs formed in mammalian cells that express matrix proteins, hemagglutinin and / or neuraminidase (W02008094195 and WO 2011 102900).

ISAV has several structural differences with influenza virus, which do not make it predictable that the exact same ISAV VLP preparation strategy can be followed as that disclosed in the state of the art for influenza virus VLP.

The main structural difference found between the Influenza and ISA viruses lies in the organization of surface protein domains. It has been described that Influenza virus has the Receptor Destructive Activity or Neuraminidase domain, in a single protein of the same name, on the other hand, it shares in a same polypeptide the receptor binding domain with that of fusion, this protein being called Hemagglutinin . In contrast, the ISA virus has a fusion domain in a single polypeptide called the Fusion protein, and on the other hand, the binding domain and destructive activity of receptor are found on a single polypeptide called Hemagglutinin-Esterase (Cottet et al., 2010).

According to the above, it is not easy to suggest that the influenza virus VLP vaccine strategy, which has been effective in humans, will be functional in salmonids, and it is necessary to demonstrate this by experimenting with recombinant proteins.

There is a publication, WO2013036745 (CL 551-2014), which describes bivalent VLPs, IPNV and ISAV. However, this invention has fundamental differences with the solution proposed in the present invention. WO 2013036745 describes viral particles similar to IPNV, this is a virus with a viral capsid that ISAV HE antigenic epitopes have been inserted into VP2 proteins. On the contrary, the solution proposed in the present invention creates viral particles similar to ISAV, a virus with a membrane that surrounds it. Another difference is that the VLP of the invention incorporates the presentation of more than one complete ISAV antigenic protein, with its protein conformation similar to that found in the virus.

VLP vaccines have advantages in relation to live attenuated or dead virus vaccines since they do not work with infectious material or with possible reversion to infectious viruses. VLP vaccines have the advantage over subunit vaccines in that antigens have a multimeric conformation similar to antigenic proteins within the infecting virus. This allows the vaccinated organism to identify said structure as an infectious virus, neutralize it and be able to generate an immunological memory against infections with the infectious pathogen.

In the present invention, structures that can be described as VLPs are taught by the expression of at least one matrix protein and at least one of its ISAV antigenic proteins. It is also the scope of this Invention Pharmaceutical or vaccine compositions, alternatives to existing ones, comprising VLPs expressing a matrix protein and one or more of the ISAV antigenic proteins. Part of the present invention is the use of the ISAV VLPs of the present invention to immunize a population of virus-free Atlantic salmon. The use of VLP in salmon of the present invention allowed to decrease the mortality accumulated over time after the viral challenge, observing a measurable increase in the levels of anti-ISAV IgM and of the expression of genes related to the response of the immune system. Thus, the invention features an alternative ISAV vaccine to commercially available inactivated and subunit vaccines.

Description of the figures

Figure 1: Characteristics of the Baculovirus vector pBac4 41. The vector has as its skeleton the plasmid pBac4x from Merck, which was used to clone the gene that encodes M1 from ISAV 752_09, for the M2 from ISAV 752_09 low the Polh promoter command, for HE of ISAV 752_09 under the command of the P10 promoter, for Protein Fusion of ISAV 752_09 under the command of the Polh promoter. Vector that has resistance to Ampicillin and an element that allows it to replicate in E. coli and recombine with the baculovirus genome with the BacVector3000 system (Merck).

Figure 2: Characteristics of the Baculovirus vector pBac4 42. The vector has as its skeleton the plasmid pBac4x from Merck, which was used to clone the gene that encodes NP of ISAV 752_09 under the HE promoter of ISAV 901_09 under the command of the P10 promoter, for protein Fusion of ISAV 901 _09 under the command of the Polh promoter. Vector that has resistance to Ampicillin and element that allows it to replicate in E. coli and recombine with the baculovirus genome with the BacVector3000 system (Merck).

Figure 3: Simultaneous expression of fusion proteins (F), hemagglutinin (HE) and matrix (M) of ISAV 901 _09 in Sf9 insect cells. (A) Polyacrylamide gel electrophoresis under denaturing conditions of the total proteins obtained from Sf9 cells, M corresponds to the molecular mass standard Pagerule Unstained Protein Ladder, 1 uninfected Sf9 cells, 2 Sf9 cells infected with control baculovirus and 3 infected Sf9 cells with recombinant baculoviruses. (B) Western-blot of the recombinant proteins expressed in Sf9 cells, 1 purified ISAV virus, 2 Sf9 cells infected with control baculovirus and 3 Sf9 cells infected with recombinant baculovirus.

Figure 4: Electron microscopy of viruses and VLPs obtained by purification with a sucrose cushion and negatively stained. (A) ISAV 901 _09 virus obtained from ASK cells. (B) VLPs obtained by the co-expression of fusion, hemagglutinin and matrix proteins in Sf9 insect cells. In both images the bar represents 100 nm.

Figure 5: Graphic representation of the Kaplan-Meier estimator for the survival of fish vaccinated with VLPs and control fish vaccinated with PBS buffer. The total number of dead fish was counted up to day 80 post-vaccination. The graph compares the mortality of fish vaccinated with VLPs versus fish vaccinated with placebo (PBS). The arrow indicates the point at which the ISAV challenge was performed. Commercial vaccines for the ISA virus have been found to protect no more than 40% in the field and an acceptable vaccine is defined as one that has an RPS of over 50%. In this case, an RPS of 57.3% was obtained with the VLPs. Using two formulations containing the recombinant proteins HE and F of the ISA virus strain 7b, under the same study conditions carried out with VLPs vaccines have obtained RPS of 22 and 37.5%, indicating that VLPs have a higher performance compared to recombinant protein vaccines.

Detailed description of the invention

The invention relates to infectious salmon anemia virus (ISAV) virus-like particles (VLPs) comprising the matrix protein and one or more antigenic proteins of the fish infectious anemia virus. Preferably, the matrix protein is: matrix protein 1 (M 1) and / or matrix protein 2 (M 2). In a preferred embodiment the ISAV virus-like particles comprise one or more of the ISA virus antigenic proteins selected from the following proteins: hemagglutinin (HE), fusion (F) and nucleoprotein (NP). Specifically, the ISAV virus-like particles comprise at least the ISAV antigenic proteins HE, F and the matrix protein M1, as well as, in addition, NP and M2 proteins may be involved, in addition.

Viral proteins, in particular, come from isolates of ISAV isolated in Chile, although they are highly similar to strains described in Scotland, Norway, the United States, Canada, the Faroe Islands, Australia, Ireland, England, Spain, and Japan. Proteins can come from ISAV strains described as virulent as well as avirulent, for example ISAV: ISAV genotypes, HPR00, HPROa, HPROb, HPR20, HPR6, HPR14, HPR36, HPR17, HPR9b, HPR3a, HPR3, HPR4, HPR4b , HPR9, HPR21, HPR10, HPR12, HPR18, HPR31, HPR1 1 a, HPR13, HPR16, HPR30, HPR2d, HPR19, HPR35, HPR2, HPR2c, HPR1, HPR5, HPR34, HPR7a, HPR7b, HPR7c, HPR7e, HPR7e, HPR7e , HPR7i, HPR15a, HPR15b, HPR15c, HPR15d, HPR15e, HPR8, HPR7d, HPR7f, HPR32, HPR33. The invention also relates to a recombinant baculovirus expressing the genes for ISAV proteins, which comprises the nucleic acid encoding the matrix protein and one or more antigenic proteins of the infectious salmon anemia virus. Preferably, the matrix protein is: matrix protein 1 (M 1) and / or matrix protein 2 (M 2). In a preferred embodiment, the recombinant baculoviruses express the genes for the ISAV proteins and comprise nucleic acids encoding one or more antigenic proteins of the infectious fish anemia virus selected from the following proteins: hemagglutinin (HE), fusion (F) and nucleoprotein (NP). Likewise, the VLP particles additionally comprise antigenic proteins from Pisciricketsia salmonis, Renibacterium salmoninarum, Yersinia ruckeri, pancreatic necrosis virus, hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, Piscine reovirus virus, vibriosis, Aeromonas salmonicida. Specifically, recombinant baculoviruses comprise nucleic acids that encode the genes for ISAV antigenic proteins: HE, F, NP, M1, and M2.

In particular, baculoviruses express the genes of virus proteins from isolated ISAV strains in Chile, although they are similar to strains from Scotland, Norway, the United States, Canada, Faroe Islands, Australia, Ireland, England, Japan. Proteins can come from ISAV strains named: ISAV 752_09, ISAV 901_09, ST28 / 97, ST25 / 97, ST27 / 97, 97/09/615, SK-05/144, MR102 / 05, SF83 / 04 , SK 779/06, (Frederick SB Kibenge et al. 2009). The recombinant baculovirus expressing the genes of the ISAV proteins according to the present invention may be that generated with the vector represented in the scheme of Figure 1 or Figure 2.

The invention further relates to a pharmaceutical composition comprising one or more ISAV virus-like particles (VLPs) defined above in the present disclosure. Also within the scope of this invention is a vaccine against the infectious salmon anemia virus (ISAV) comprising one or more ISAV virus-like particles (VLPs) according to the preceding description, a pharmaceutically acceptable carrier and one or more adjuvants. pharmaceutically acceptable in fish.

The adjuvant of the vaccine of the invention can be selected from one or more of the following group: baculovirus lysate, lipoproteins, modified lipoproteins, cell bodies, Montanide, Alumina, water / mineral oil emulsion (W / O), squalene, water / squalene emulsion , Freund 's complete adjuvant ^'s, Freund's incomplete adjuvant, saponin, Quillaja Saponaria, keyhole limpet hemocyanin, nonionic block polymers (NBP), dimethyl dioctadecyl ammonium bromide (DDA). Preferably, the adjuvant is one or more of the following group: baculovirus lysate, lipoproteins, cell bodies, Montanide, water / mineral oil emulsion (W / O). In particular, the vaccine adjuvant is a baculovirus and cell body lysate.

The vaccine against infectious salmon anemia virus (ISAV) according to the invention may additionally contain one or more types of ISAV vaccines. Additional vaccines may be, but are not limited to: ISAV subunit vaccines or inactivated ISAV viruses. Inactivated viruses preferably come from isolates of ISAV isolated in Chile. In other Preferred embodiment vaccines further comprise vaccines against other commercially important fish pathogens in aquaculture.

Additional vaccines are selected from the group of vaccines against: Pisciricketsia salmonis, Renibacterium salmoninarum, Yersinia ruckeri, pancreatic necrosis virus, hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, Piscine reovirus virus, vibriosis, Aero monas salmonicida.

The vaccines of the invention are formulated for administration by oral, immersion, intraperitoneal, intramuscular or parenteral routes. Another alternative of the present invention is a fish food comprising an ISAV VLP comprising the matrix protein and one or more antigenic proteins of the infectious fish anemia virus. Preferably, the matrix protein is: matrix protein 1 (M 1) and / or matrix protein 2 (M 2). In a preferred embodiment the ISAV virus-like particles comprise one or more of the antigenic proteins of the infectious fish anemia virus selected from the following proteins: hemagglutinin (HE), fusion (F) and nucleoprotein (NP). The food may also have other ISAV vaccines or other fish pathogens, such as those detailed above. The invention is related to the use of ISAV virus-like particles (VLPs) according to the previous descriptions, because it serves to prepare a medicine for the protection or treatment of infectious salmon anemia in aquaculture or other aquaculture diseases.

Likewise, the present invention is related to the protection or treatment of fish, because it comprises administering the virus-type particles (VLP) of the present invention to fish. The method consists in the protection or treatment of diseases, such as infectious salmon anemia. The virus-like particles (VLP) of the present invention or vaccines detailed above are administered to salmon or trout. The type of salmon can be: White Corregono (Coregonus albula), Common Lavaret (Coregonus lavaretus), Corégono peled (Coregonus peled), Pink salmon (Oncorhynchus gorbuscha), Keta salmon (Oncorhynchus ketá), Silver salmon or Coho (Oncorhynchus kisutch) , Rainbow trout (Onchorinchus mykiss), Atlantic salmon (Salmo salar), Brown trout (Salmo truttá), Salvelino (Salvelinus alpinus), Brook trout (Salvelinus fontinalis), Lake trout (Salvelinus namaycush), Tímalo

(Thymallus thymallus), king salmon or Chinook (Oncorhynchus tshawytscha).

Finally, the present invention is related to a vaccination kit for fish that comprises a package that contains the ISAV VLPs described in the present invention or the vaccines defined in the present description. The vaccination kit additionally comprises one or more packages with vaccines for other fish pathogens. Vaccines other than ISAV virus-type particles (VLPs) may be vaccines against one or more of the following pathogens: Pisciricketsia salmonis, Renibacterium salmoninarum,

Yersinia ruckeri, pancreatic necrosis virus, hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, Piscine reovirus virus, vibriosis, Aeromonas salmonicida. Also, the kit may comprise packages with subunit ISAV or inactivated virus vaccines.

The invention further relates to an ISAV VLP production method according to the above description because it comprises the steps of: Incorporating one or more recombinant DNA molecules which encode the ISAV matrix protein plus at least one ISAV antigenic protein in a recombinant viral vector. Infect host cells with the vector recombinant. ISAV antigenic proteins can be selected from F, HE and NP proteins and matrix proteins can be selected from M1 and M2. It is a preferred embodiment that the viral vector can be baculovirus, however, it can be other adenoviral, poxyviral, cytomegalovirus, lentiviral, etc. vectors. The host cell may be an insect, yeast, plant, or mammalian cell. In a particularly preferred embodiment, the host cell is an insect cell.

Additionally, the invention relates to an ISAV VLP vaccine production method comprising the steps of: incorporating one or more recombinant DNA molecules which encode the ISAV matrix protein plus, at least one ISAV antigenic protein in a recombinant viral vector; infecting host cells and lysing host cells. The preferred viral vector is a baculovirus and the host cell is an insect cell. Additionally, the VLP vaccine production method comprises an additional step of incorporating an adjuvant into the cell lysate and / or a pharmaceutically acceptable vehicle. The adjuvant is selected from one or more of the following group: baculovirus lysate, lipoproteins, modified lipoproteins, cell bodies, Montanide, Alumina, water / mineral oil emulsion (W / O), squalene, water / squalene emulsion, complete Freund's adjuvant ^' s, incomplete Freund's adjuvant, saponins, Quillaja Saponaria, hemocyanin Keyhole limpet, non-ionic block polymers (NBP), dimethyl dioctadecyl ammonium bromide (DDA).

Examples of preferred embodiment

Example 1: Production and characterization of recombinant proteins of the infectious salmon anemia virus.

Cells and viruses Spodoptera frugiperda Sf9 cells (ATCC CRL-171 1) were maintained as adhered culture in SF-900 II medium (Invitrogen) supplemented with 0.1% fetal bovine serum. ISAV 901 _09 virus was maintained in ASK Atlantic salmon cells (ATCC CRL-2747) in L-15 medium as previously described (www.atcc.org).

ISA virus sequences and transport vector

The sequences of the genes that encode the hemagglutinin (HE), fusion (F) and matrix (Mx) proteins of the Chilean virus ISAV 901_09, were previously obtained in the laboratory (Cottet et al., 201 1). The codogenic use of the four genes was optimized for expression in the Sf9 insect cell line, being biochemically synthesized (GenScript, Piscataway, NJ) and cloned into the pBac4x transfer vector (Merck-Millipore). Recombinant baculoviruses were generated with the BacVector-2000 Triple Cut expression system (Merck-Millipore).

Preparation of the Baculovirus vector pBac4 41 and pBac4 42.

Recombinant baculoviruses were generated in which the sequences of the genes selected as antigens were optimized for expression in insect cells with this system. For this, the sequences of the genes that generate the fusion proteins and hemagglutinin of both Chilean viruses, the ISAV 901 _09 and ISAV 752_09 strains, were synthesized. The GenBank accession numbers of the sequences used in this study are GU830895 to GU830902 for the ISAV752_09 isolate and GU830903 to GU830910 for the ISAV901_09 isolate plus nucleoprotein and matrix, optimizing codogenic use and were subsequently cloned into the transfer vector pBac4x (Cottet L. and cois 2010). This process was carried out by the company GenScript, generating the vectors pBac4 41 (Figure 1) and pBac4 42 (Figure 2).

Recombinant protein characterization The electrophoretic profile of the total proteins obtained from the insect cells infected with the recombinant baculoviruses, was visualized by electrophoresis in 15% w / v polyacrylamide gels under denaturing conditions, following the protocol previously described by King and Posee, ( 1992). While the specific recognition of each protein by Western blot was performed with specific antibodies, according to the method described by Gallagher et al., (2004).

The expression and molecular size of the recombinant proteins were determined, this by electrophoresis in polyacrylamide gels under denaturing conditions (Figure 3A). The image shows the bands corresponding to the fusion, hemagglutinin and matrix proteins, which have an approximate size of 54 kDa, 47 kDa and 21 kDa, respectively. Subsequently, the proteins were characterized by Western blotting (Figure 3B). As can be seen in the image, the proteins obtained have sizes of 50 and 57 kDa for fusion, 41 and 48 kDa for hemagglutinin and 23 kDa for matrix, their migration pattern being similar to that obtained for the three native proteins obtained from ISA virus. purified (Figure 3B).

For the generation of the VLPs, the selection of the proteins to be expressed was carried out for structural reasons, so fusion and hemagglutinin were selected because they are the two proteins on the viral external surface, which makes them the best candidates to be recognized. by the immune system of the fish, while the expression of the matrix protein was carried out since it is the one that stabilizes the viral particles, added to the fact that in convalescent fish from the disease and in mice that have been immunized with ISAV, the presence of antibodies against this protein was detected (Rimstad et al., 201 1). According to the data obtained by SDS-PAGE and Western blot, the molecular mass of the recombinant matrix protein corresponds to approximately 22 kDa, a value similar to that obtained in the same experiment for the protein of viral origin, which was determined in 24 kDa (Figure 3). The result agrees with the molecular mass described in the matrix literature, which varies between 22 and 24 kDa (Rimstad et al., 201 1). A similar result was obtained for the recombinant hemagglutinin and fusion proteins, which present molecular masses similar to those previously described in the literature (Falk et al., 2004; Rimstad et al., 201 1), but unlike the matrix protein, in both surface proteins a double band pattern was obtained when analyzed by Western blot. The molecular mass of the recombinant fusion protein was determined at 49 kDa and 53 kDa, while for hemagglutinin it was 45 kDa and 49 kDa (Figure 3). As they are both membrane proteins, they must follow the secretion route through the endoplasmic reticulum and the Golgi apparatus, organelles in charge of making post-translational modifications, such as incorporating oligosaccharides. Different degrees of glycosylation can lead to the appearance of more than one band associated with the protein of interest, in this case the viral surface proteins. This effect has been previously described in glycoproteins expressed heterologously with the baculovirus / insect cell system (Ghiasi et al., 1994; Ganguly et al., 2010).

Example 2: Preparation of ISAV VLPs

Preparation and purification of viruses and VLPs

ASK and Sf9 cells were infected with ISAV and recombinant baculoviruses, respectively, as explained in Example 1. The purification and partial concentration of both types of particles was performed by a sucrose cushion (Chen et al., 2007), with some modifications. The ASK cells were subjected to 3 cycles of freeze-thaw, Sf9 were not, the medium of both infections was centrifuged at 5,000 xg to eliminate the cellular debris, subsequently, the supernatant was centrifuged at 100,000 xg for 2 hours at 4 ^Q C. The pellet was resuspended in 1.0 mL of 0.2 M pH 7.2 phosphate buffer, and the VLPs were partially purified by passing them through a 30% sucrose buffer in 0.2 M pH 7.2 phosphate buffer, centrifuging at 150,000 xg for 2 hours at 4 ^Q C and finally the obtained sediment was resuspended in 50 pL of phosphate buffer.

Electron microscopy

Viral particles and VLPs were absorbed for 15 minutes directly on gold grids and washed three times in PBS for 1 minute. For negative staining, the grids were incubated for 1 minute in 1% w / v phosphotungstic acid (pH 6.5). The samples were visualized in a Phillips CM100 transmission electron microscope operating at 80 kV. Figure 4 presents the photomicrographs obtained by transmission electron microscopy of the samples corresponding to ISAV isolate 901 _09 from ISAV (A) and the VLPs obtained by co-expression in insect cells (B). In the photograph corresponding to ISAV, membranous structures with spherical and pleiomorphic morphologies can be observed. Their approximate diameters are 20nm and 70nm, respectively, and membrane-associated extensions are observed on their surfaces, which are 8.5nm long and would correspond to the hemagglutinin and fusion glycoproteins (Figure 4A). For its part, in the photomicrograph obtained for the VLPs (Figure 4B), structures with spherical and pleiomorphic morphology, with approximate diameters of 40 nm and 150 nm respectively, can also be observed, and as in the case of the purified virus, the presence of of extensions on the membrane surface, those with a length of 10 nm and that would correspond to the recombinant fusion and hemagglutinin proteins.

The expression of the structural fusion, hemagglutinin and matrix proteins of ISAV 901 _09 using the baculovirus-insect cell system, allowed the generation of this type of virus-like particles (Figure 4B). With mainly pleiomorphic morphology, although some with a spherical shape can be obtained, the ISAV VLPs show similarity in shape to the purified viral particles, while the sizes in both cases vary between 20 and 150 nm with extensions on their surfaces ranging from 8.5 to 10 nm (Figure 4). In the case of the previous characterization of the viral particles of ISAV, the observation by electron microscopy of thin sections of infected tissues, allowed to determine that they have a spherical shape with a size of approximately 100 nm and extensions on the external surface of approximately 10 nm. (Cottet et al 2010). On the other hand, when the same analysis is performed, but using purified viral particles, mainly pleiomorphic structures with sizes ranging from 50 to 140 nm are observed, with surface extensions ranging from 10 to 12 nm (Cottet et al 2010). These latest data coincide with that obtained for the structures generated by the expression of the structural proteins in insect cells (Figure 4B), which would confirm the presence of ISAV virus-like particles.

Example 3: Preparation of vaccines with VLP

Once the generation of the VLPs had been established, the immunological capacity of the proteins expressed in insect cells was checked.

Vaccine formulation

For the vaccine formulation, two flasks were prepared with 50 mL of suspension from the Hi5 insect cell line at a density of 5x10 ⁵ cells / mL, these were infected with the Bac41 recombinant baculovirus at an MOI of 0.1. The infection was maintained for 5 days and the cells were fixed with formaldehyde to a final concentration of 0.01% v / v. From this suspension, the surface proteins were quantified by Western blotting and 1 vaccine formulation was generated. The vaccine formulation was made using the cell lysate of the baculovirus infected insect cells and the adjuvant was added. A control was added in which the cell suspension was replaced by PBS. In unpublished data from these inventors, different adjuvants were tested in fish with VLP vaccines. For the case of the present invention, the adjuvant was used with the best results, however, immunogenicity is shown in all the adjuvants tested.

Example 4: Vaccination of salmon with the immunogenic composition comprising ISAV VLP

Vaccination and challenge

In total, 460 smolt-developing Atlantic salmon were used, which were distributed among the containers of three culture lines, called A, B and C, which consisted of 6, 8 and 4 ponds, respectively. This culture system allowed the recircularization of the water between the ponds connected by line, making it possible to maintain the water conditions among all the fish during the test. In this way, in lines A and C the controls of vaccinated but not challenged fish were maintained, while in line B the vaccinated and challenged fish were maintained. For vaccination, 200 pL of each formulation consisting of the structural fusion proteins, hemagglutinin and matrix of ISAV 901 _09 expressed using the Baculovirus-insect cell system along with apoptotic bodies used as co-adjuvant to four groups of 20 individuals. To determine the effectiveness of the vaccine and as controls, 5 fish vaccinated with the placebo were added per pond. Prior to the challenge with ISAV, an inoculum was obtained from viral infections to the SHK-1 cell line, the supernatant was collected and centrifuged at 3,000 xg for 10 min to eliminate the cell debris in suspension. Cohabitation was used for the challenge, for this 7 unvaccinated fish, which were called Trojans, were infected intraperitoneally with the viral inoculum and deposited in each pond, leaving a density of 32 fish. During the test, daily mortality by pond was recorded, a process that was carried out up to 80 dpv. Cohabitation infection has previously been described in Atlantic salmon as an effective way to emulate infection under natural conditions (Jones et al., 1999; Mikalsen et al., 2005b; Lauscher et al., 201 1), while as placebo PBS was used since in previous vaccination trials in fish, this buffer has been used for this purpose (Thiéry et al., 2006; Lauscher et al., 2011; Munang'andu et al., 2012).

The formulation of the VLP vaccine tested in this work, allowed to decrease the mortality of the immunized and ISAV-challenged salmon (Figure 5). However, of the vaccines licensed for use in Chile, only one company provides information on the amount of antigen used per dose (between 5.4 and 8.1 pg of recombinant proteins expressed in yeast). When comparing the amounts of protein per dose administered with the VLPs, the vaccines developed for this work contain between 2.4% and 4.1% of the total protein declared for the commercial vaccine, and even so, it is able to decrease mortality to levels that its RPS is 57.3%. The problem with this comparison is that the company does not provide data on field trials with the vaccine, therefore, mortalities cannot be related to the concentration of antigen used in vaccines.

It should be noted that the evaluation of the immune response of the vaccine constituted by the VLPs, through the analysis of the expression of genes related to the immune response through qRT-PCR, show that the fish vaccinated with the VLPs are capable of inducing the relative expression of IFN-y, TGF-b and IL-10, but not CD4. This suggests activation of the innate response, probably from wild-type killer cells by increased expression of IFN- and and a regulation of the response by cytokines TGF-b and IL-10, which could explain why it was not observed. adhesions or melanosis in vaccinated fish. While, after the challenge, the fish vaccinated with VLPs show an increase in the relative expression of IFN-g and especially of CD4, which suggests an activation of the acquired cellular immune response that is the result required to obtain a response. optimal against viral infection.

References

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Claims

1. Infectious salmon anemia virus (ISAV) virus-like particles (VLPs) characterized in that it comprises the matrix protein and one or more antigenic proteins of the infectious fish anemia virus.

2. ISAV virus-like particles according to claim 1 characterized in that the matrix protein is matrix protein 1 (M

one ) ·

3. ISAV virus-like particles according to claim 1 characterized in that the matrix protein is matrix protein 2 (M

two).

4. ISAV virus-like particles according to claims 1 to 3 characterized in that the matrix protein is matrix protein 1 (M 1) and matrix protein 2 (M 2).

ISAV virus-like particles according to claims 1-4, characterized in that it comprises one or more of the antigenic proteins of the infectious fish anemia virus that is selected from the following proteins: hemagglutinin (HE), fusion (F ) and nucleoprotein (NP).

6. ISAV virus-like particles according to claims 1-5, characterized in that it comprises ISAV antigenic proteins hemagglutinin (HE), fusion (F) and nucleoprotein (NP).

7. ISAV virus-like particles according to claims 1-6 characterized in that the ISAV matrix and antigenic proteins come from strains isolated in Chile.

8. ISAV virus-like particles according to claim 7 characterized in that the ISAV antigenic proteins are selected from the ISAV 901_09 strain with the GenBank accession numbers of the sequences GU830903 to GU830910.

9. ISAV virus-like particles according to claim 7 characterized in that the ISAV antigenic proteins are selected from the ISAV 752_09 strain with the GenBank accession numbers of the sequences are GU830895 to GU830902.

ISAV virus-type particles according to claims 1-9 characterized in that it additionally comprises antigenic proteins of Pisciricketsia salmonis, Renibacterium salmoninarum, Yersinia ruckeri, pancreatic necrosis virus, hemorrhagic septicemia virus, hematopoietic necrosis virus infectious, Piscine reovirus virus, vibriosis, Aeromonas salmonicida.

1 1. ISAV VLP production method according to claims 1 to 10, characterized in that it comprises the steps of: a. Incorporate one or more recombinant DNA molecules which encode the ISAV matrix protein and at least one ISAV antigenic protein into a recombinant vector.

b. Infect host cells.

12. Method according to claim 1 1, characterized in that the vector is a baculovirus.

13. Method according to claim 1 1, characterized in that the host cell is an insect, yeast, plant cell or mammalian cell.

14. The method according to claim 13, characterized in that the host cell is an insect cell.

15. The method according to claims 1 1 to 14, characterized in that the matrix protein is selected from one or more: matrix protein 1 (M 1) and matrix protein 2 (M 2).

16. Method according to claims 11 to 15, characterized in that the antigenic proteins of the infectious salmon anemia virus are selected from the following proteins: hemagglutinin (HE), fusion (F) and nucleoprotein (NP).

17. Method according to claims 1 to 16, characterized in that the ISAV antigenic proteins are: fusion (F), hemagglutinin (HE) and nucleoprotein (NP).

18. Recombinant baculovirus expressing ISAV proteins, characterized in that it comprises the nucleic acid encoding a matrix protein and one or more antigenic proteins of the infectious salmon anemia virus.

19. Recombinant baculovirus expressing ISAV proteins according to claim 18, characterized in that it comprises the nucleic acid encoding the matrix protein is matrix protein 1 (M 1).

20. Recombinant baculovirus expressing ISAV proteins according to claim 18, characterized in that it comprises the nucleic acid encoding the matrix protein is matrix protein 2 (M 2).

21. Recombinant baculovirus that expresses ISAV proteins according to claims 18 to 20, characterized in that it comprises nucleic acids that encode matrix protein 1 (M 1) and matrix protein 2 (M 2).

22. Recombinant baculovirus expressing ISAV proteins according to claims 18 to 21, characterized in that it comprises the nucleic acid encoding one or more antigenic proteins of the infectious fish anemia virus, which is selected from the following proteins: hemagglutinin ( HE), fusion (F) and nucleoprotein (NP).

23. Recombinant baculovirus expressing ISAV proteins according to claims 18 to 22, characterized in that it comprises the nucleic acid encoding the ISAV antigenic proteins hemagglutinin (HE), fusion (F) and nucleoprotein (NP).

24. Recombinant baculovirus expressing ISAV proteins according to claims 18 to 23, characterized in that it comprises the nucleic acid encoding ISAV antigenic proteins of matrix 1 (M1), matrix 2 (M2), the fusion protein (F ) and hemagglutinin (HE).

25. Recombinant baculovirus expressing ISAV proteins according to claims 18 to 24, characterized in that it comprises nucleic acid encoding ISAV antigenic proteins from strains isolated in Chile.

26. Recombinant baculovirus expressing ISAV proteins according to claim 25, characterized in that the ISAV antigenic proteins are selected from strain ISAV 901 _09 with the GenBank accession numbers of the sequences GU830903 to GU830910.

27. Recombinant baculovirus expressing ISAV proteins according to claim 25, characterized in that the ISAV antigenic proteins are selected from the ISAV 752_09 strain with the GenBank accession numbers of the sequences GU830895 to GU830902.

28. Recombinant baculovirus expressing ISAV proteins according to claims 18 to 27, characterized in that it comprises the scheme represented in Figure 1.

29. Recombinant baculovirus expressing ISAV proteins according to claims 18 to 27, characterized in that it comprises the scheme represented in Figure 2.

30. Pharmaceutical composition, characterized in that it comprises an ISAV VLP as defined in claims 1 to 10, plus one to more pharmaceutically acceptable carriers.

31. Vaccine against infectious salmon anemia virus (ISAV), characterized in that it comprises an ISAV VLP according to claims 1 to 10, one or more pharmaceutically acceptable adjuvants in fish and a pharmaceutically acceptable carrier.

32. Vaccine against the infectious salmon anemia virus (ISAV) according to claim 31, characterized in that the adjuvant is selected from one or more of the following group: baculovirus lysate, lipoproteins, modified lipoproteins, cell bodies, Montanide, alumina, water / mineral oil (W / O), squalene, water / squalene, complete Freund 's adjuvant ^'s, incomplete Freund's, saponin, Quillaja Saponaria, keyhole limpet hemocyanin, nonionic block polymers (NBP), dimethyl dioctadecyl ammonium bromide (DDA).

33. Vaccine against the infectious salmon anemia virus (ISAV) according to claims 31-32, characterized in that the adjuvant is selected from one or more of the following group: baculovirus lysate, cell bodies, Montanide, water emulsion / mineral oil (W / O).

34. Vaccine against the infectious salmon anemia virus (ISAV) according to claims 31-33, characterized in that it also comprises one or more types of ISAV vaccines.

35. Vaccine against the infectious salmon anemia virus (ISAV) according to claim 34, characterized in that it also comprises vaccines selected from: ISAV sub-unit vaccines and ISAV inactivated virus vaccines.

36. Vaccine against the infectious salmon anemia virus (ISAV) according to claims 31-35, characterized in that it also comprises vaccines against other fish pathogens.

37. Vaccine against the infectious salmon anemia virus (ISAV) according to claim 36, characterized in that the vaccines for other fish pathogens correspond to one or more vaccines selected from the group against: Pisciricketsia salmonis, Renibacterium salmoninarum, Yersinia ruckeri, pancreatic necrosis virus, hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, Piscine reovirus virus, vibriosis, Aeromonas salmonicida.

38. Vaccine against the infectious salmon anemia virus (ISAV) according to claims 31-37, characterized in that they are formulated for the oral, immersion, intraperitoneal, intramuscular or parenteral routes.

39. ISAV VLP vaccine production method according to claims 30 to 37, characterized in that it comprises the steps of a. Incorporate one or more recombinant DNA molecules which encode the ISAV matrix protein plus, at least one ISAV antigenic protein in a recombinant viral vector. b. Infect host cells

c. Lysing host cells

40. Method according to claim 39, characterized in that the viral vector is a baculovirus.

41. Method according to claim 39, characterized in that the host cell is an insect cell.

42. Method according to claim 39, characterized in that it comprises an additional step of incorporating an adjuvant to the cell lysate and / or a pharmaceutically acceptable vehicle.

43. Method according to claim 42, characterized in that the adjuvant is selected from one or more of the following group: baculovirus lysate, lipoproteins, modified lipoproteins, cell bodies, Montanide, Alumina, water / mineral oil emulsion (W / O) , squalene, water / squalene, complete Freund 's adjuvant ^'s, Freund's incomplete adjuvant, saponin, Quillaja Saponaria, keyhole limpet hemocyanin, nonionic block polymers (NBP), dimethyl dioctadecyl ammonium bromide (DDA).

44. Fish feed, characterized in that it comprises an ISAV VLP according to claims 1-10.

45. Fish feed, characterized in that it comprises the vaccines of claims 31-38.

46. Use of the ISAV VLPs according to claims 1-10, characterized in that it serves to prepare a medicine for the protection or treatment of infectious salmon anemia in aquaculture.

47. Use of the ISAV VLPs according to claims 1-10, characterized in that it serves to prepare a medicine for the protection or treatment of infectious salmon anemia and other diseases in fish.

48. Use of the ISAV VLPs according to claims 46-47, characterized in that the medications are for the protection and treatment of fish, such as White Corregono (Coregonus albula), Common Lavaret {Coregonus lavaretus), Peleted Corregono (Coregonus peled), Pink salmon (Oncorhynchus gorbuscha), Keta salmon (Oncorhynchus keta), Silver or Coho salmon (Oncorhynchus kisutch), Rainbow trout (Onchorinchus mykiss), Atlantic salmon (Salmo salar), Brown trout (Salmo trutta), Salvelino ( Salvelinus alpinus), Brook trout (Salvelinus fontinalis), Lake trout (Salvelinus namaycush), Grayling (Thymallus thymallus), Royal salmon or Chinook (Oncorhynchus tshawytscha).

49. Vaccination kit for fish, characterized in that it comprises a package comprising the ISAV VLPs of claims 1 to 10 or the vaccines of claims 31-38.

50. Vaccination kit for fish according to claim 49, characterized in that it additionally comprises one or more packages with vaccines for other fish pathogens.

51. Vaccination kit for fish according to claim 49, characterized in that it additionally comprises one or more packages with inactivated ISAV vaccines and ISAV subunit vaccines.

52. Vaccination kit according to claims 49 - 51, characterized in that the vaccinated fish are selected from: White maggot (Coregonus albula), Common lavage {Coregonus lavaretus), Magnet peled (Coregonus peled), Pink salmon (Oncorhynchus gorbuschá) , Keta salmon (Oncorhynchus ketá), Silver or Coho salmon (Oncorhynchus kisutch), Rainbow trout (Onchorinchus mykiss), Atlantic salmon (Salmo salar), Brown trout (Salmo truttá), Salvelino (Salvelinus alpinus), Brook trout (Salvelinus fontinalis), Lake trout (Salvelinus namaycush), Grayling (Thymallus thymallus), king salmon or Chinook (Oncorhynchus tshawytscha).