Vaccine, method of production, use for treating and preventing piscine orthoreovirus capsid proteins

Abstract

The present invention relates to recombinant proteins, encoding nucleic acids, a vector, transformed microorganism and host cell, a vaccine composition intended for salmon farming, and use for preventing and treating infections caused by Piscine orthoreovirus (PRV). In particular, the present invention relates to a vaccine based on recombinant proteins expressed in a baculovirus / insect cell system and corresponding to the structural proteins in the first and second layer of the viral capsid. This vaccine can be administered intraperitoneally, reducing the viral load of Piscine orthoreovirus in salmon infected with this virus.

Description

[0001] VACCINE, PRODUCTION METHOD, USE FOR TREATMENT AND PREVENTION OF PISCINE ORTHOREOVIRUS CAPSID PROTEINS.

[0002] FIELD OF INVENTION

[0003] The present invention relates to the field of veterinary medicine, especially to the prevention and treatment of viral infections in aquaculture breeding environments. In particular, the present invention relates to recombinant proteins, encoding nucleic acids, vectors, transformed microorganisms, and expression cells, vaccine compositions, and uses thereof for preventing and treating Piscine orthoreovirus (PRV) infections in farmed fish. In particular, virus-like particles (VLPs) formed by said recombinant proteins of the Piscine orthoreovirus (PRV) virus, nucleic acid molecules cloned into baculoviruses and subsequently expressed in Sf9 insect cells, where the proteins co-expressed in insect cells are administered to gamefish, especially salmon, in the form of an intraperitoneal vaccine.

[0004] PRIOR ART

[0005] In recent years, Chile has stood out internationally in the area of ​​aquaculture, positioning itself as the second largest producer of Atlantic salmon and rainbow trout in the world, after Norway. However, the salmon industry has been affected by various factors that have caused great economic losses, one of the most important being infectious diseases. These diseases became the first and second cause of mortality in Atlantic salmon in 2020 and 2021, respectively, with figures reaching 22.7% in 2020 and 19.3% in 2021 (National Fisheries and Aquaculture Service. (2019). Sanitary Report on Salmon Farming in Marine Centers 1 o Semester 2021. Retrieved from Among infectious diseases, Piscine Orthoreovirus (PRV) has been classified as an emerging pathogen and its prevalence in Chilean salmon farms has been shown to be up to 46% (Godoy, MG, Kibenge, MJ, Wang, Y., Suarez, R., Leiva, C., Vallejos, F., & Kibenge, FS (2016). “First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV". Virology Journal, 13(1), 98-113).

[0006] Piscine orthoreovirus is a non-fusogenic virus belonging to the Reoviridae family, Orthoreovirus genus, characterized as a viral particle with two concentric protein layers (internal and external capsid), 80 nm in diameter. HIV has a non-enveloped icosahedral morphology and produces inclusion bodies in infected cells (Malik, MS, Bjargen, H., Dhamotharan, K., Wessel, O., Koppang, EO, Di Cicco, E., ... & Rimstad, E. (2019). Erythroid progenitor cells in atlantic salmon (salmo salar) may be persistently and productively infected with piscine orthoreovirus (prv). Viruses, 11(9), 824). Its genome is made up of 10 double-stranded RNA (dsRNA) segments, which code for at least 11 proteins (Kibenge, MJ, Iwamoto, T., Wang, Y., Morton, A., Godoy, MG, & Kibenge, FS (2013).“Whole-genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate sub-genotypes”. Virology Journal, 10(1), 230-250). Being a reovirus, its genome can be subdivided into 3 large segments (L1 , L2 and L3, Large) that code for the Lambda (X) proteins, 3 medium segments (M1 , M2 and M3, Medium) that code for the Miu (p) proteins and 4 small segments (S1 , S2, S3 and S4, Small) that code for the Sigma (o) proteins. The coding assignments and protein characteristics of PRV were determined by the similarity to mammalian reovirus (MRV) sharing the same nomenclature (Palacios, G., Lovoll, M., Tengs, T., Hornig, M., Hutchison, S., Hui, J., Kongtorp, R., Savji, N., Bussetti, A., Solovyov, A., Khstoffersen, A., Christopher, C., Street, C., & Lipkin, WI (2010).“Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus”. PLoS ONE, 5(7), e11487; Kibenge, M. J., Iwamoto, T., Wang, Y., Morton, A., Godoy, M. G., & Kibenge, F. S. (2013). “Whole- genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate sub-genotypes”. Virology Journal, 10(1), 230-250), ver tabla 1 , Figura 1 de Markussen et al 2013 (Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar), Markussen T, Dahle MK, Tengs T, Lovoll M, Finstad 0W, et al. (2013) Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar). PLOS ONE 8(7): e70075. https: / / doi.org / 10.1371 / journal.pone.0070075).Currently, it is known that the M2, S1 and S4 segments code for the μ1, σ3 and y*1 proteins respectively, which make up the external capsid of the virus, and therefore can be the target proteins in the search for an immune response against PRV since they are the most exposed proteins that are generally the most antigenic.

[0007] PRV infection can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, erythrocyte inclusion body syndrome (EIBS) in Coho salmon, and HSMI-like disease with anemia in rainbow trout. Recently, association with jaundice syndrome in Chinook salmon and proliferative browning syndrome (PBS) in brown trout has also been reported (Di Cicco, E., Ferguson, H.W., Kaukinen, K.H., Schulze, A.D., Li, S., Tabata, A., ... & Miller, K.M. (2018). The same strain of Piscine orthoreovirus (PRV-1) is involved in the development of different, but related, diseases in Atlantic and Pacific Salmon in British Columbia. Facets, 3(1), 599-641 ; Kuehn, R., Stoeckle, B.C., Young, M., Popp, L., Taeubert, J.E., Pfaffl, M.W., & Geist, J. (2018).Identification of a piscine reovirus-related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline. PLoS One, 13(10), e0206164). Despite the multiple syndromes associated with PRV infection, it does not necessarily cause mortality, but its effect on the heart and red blood cells can weaken the infected fish and make it less tolerant to stress and hypoxic conditions (Lund, M., Krudtaa Dahle, M., Timmerhaus, G., Alarcon, M., Powell, M., Aspehaug, V., ... & Jorgensen, S. M. (2017). Hypoxia tolerance and responses to hypoxic stress during heart and skeletal muscle inflammation in Atlantic salmon (Salmo salar). PLoS One, 12(7), e0181109). Since PRV is almost ubiquitous in farmed Atlantic salmon and HSMI outbreaks are common in Norway, the virus is considered a major cause of loss due to the poor quality of fish after overcoming the infection.To date, three subtypes or genotypes of PRV have been identified in salmonids: PRV-1 in Atlantic salmon, Chinook salmon and rainbow trout, PRV-2 in Coho salmon (Di Cicco, E., Ferguson, HW, Kaukinen, KH, Schulze, AD, Lita, S. Miller, K. A., & K. ... (2018). The same strain of Piscine orthoreovirus (PRV-1) is involved in the development of different, but related, diseases in Atlantic and Pacific Salmon in British Columbia Facets, 3(1), 599-641 Palacios, G., Lovoll, M., Teng, T. Huison, Huison, M., Hug. J., Kongtorp, R., Savji, N., Bussetti, A., Solovyov, A., Kristoffersen, A., Christopher, C., Street, C., & Lipkin, WI (2010 “Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus” ONE, 157). PRV-3 in rainbow trout, Coho salmon and brown trout (Olsen, AB, Hjortaas, M., Tengs, T., Hellberg, H., & Johansen, R. (2015).“First Description of a New Disease in Rainbow Trout (Oncorhynchus mykiss (Walbaum)) Similar to Heart and Skeletal Muscle Inflammation (HSMI) and Detection of a Gene Sequence Related to Piscine Orthoreovirus (PRV)”. PLoS ONE, 10(7), e0131638; Takano, T., Nawata, A., Sakai, T., Matsuyama, T., Ito, T., Kurita, J., Terashima, S., Yasuike, M., Nakamura, Y., Fujiwara, A., Kumagai, A., & Nakayasu, C. (2016). “Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus”. PLoS ONE, 11(10), e0165424; Kuehn, R., Stoeckle, B. C., Young, M., Popp, L, Taeubert, J. E., Pfaffl, M. W., & Geist, J. (2018). Identification of a piscine reovirus- related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline. PLoS One, 13(10), e0206164).To date, only infections associated with PRV genogroup 1 (PRV-1) and 3 (PRV-3) have been reported in Chile. PRV-1 infection can cause heart and skeletal muscle inflammation (HSMI) in Atlantic salmon, rainbow trout, and Coho salmon, and jaundice syndrome in Chinook salmon, whereas PRV-3 infection has been linked to pathological lesions in the heart in rainbow trout and an HSMI-like pathology in Coho salmon termed HSMI-like (Olsen, A.B., Hjortaas, M., Tengs, T., Hellberg, H., & Johansen, R. (2015). “First Description of a New Disease in Rainbow Trout (Oncorhynchus mykiss (Walbaum)) Similar to Heart and Skeletal Muscle Inflammation (HSMI) and Detection of a Gene Sequence Related to Piscine Orthoreovirus (PRV)". PLoS ONE, 10(7), e0131638).

[0008] Clinical signs of HSMI in fish include abnormal swimming behavior, decreased appetite, anorexia, swimming with or against the current in an orderly manner and close to the net, dark appearance, and a certain degree of lethargy. HSMI produces an inflammation of all the structures of the heart of the fish, generating a myocarditis that causes pain in the specimens which leads to reduced mobility (Kongtorp, R., Taksdal, T., & & Lyngoy, A. (2004b). “Pathology of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar”. Diseases of Aquatic Organisms, 59, 217-224; Godoy, MG, Kibenge, MJ, Wang, Y., Suarez, R., Leiva, C., Vallejos, F., & Kibenge.FS (2016). “First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV". Virology Journal, 13(1), 98-113).Following primary PRV-1 infection, a persistent phase develops, which can last the entire life of farmed salmon and has been linked to the formation of black spots on salmon fillets (Bjargen, H., Wessel, O., Fjelldal, PG, Hansen, T., Sveier, H., Saebo, HR, ... & Koppang, EO (2015). Piscine orthoreovirus (PRV) in red and melanized foci in white muscle of Atlantic salmon (Salmo salar). Veterinary Research, 46(1), 1-12), a product quality problem that causes significant economic losses for producers. A study conducted in Chile by reference laboratories of the National Fisheries Service SERNAPESCA.

[0009] (https: / / www.salmonexpert.cl / archivo / laboratorios-de-referencia-de-sernapesca-entregaron- resultados-de-enfermedades-en-salmnidos / 1248400), found PRV present in 64% of the Atlantic salmon farming centers analyzed, with a prevalence of 46%. Therefore, the economic damage that PRV infection can cause is considerable. In any case, mortality due to HSMI usually ranges between 0-20%, but its morbidity is close to 100%, reducing productivity and fish welfare.

[0010] Regarding the productive stage affected by Piscine Orthoreovirus, it has been described that the infection is detectable in different stages of salmon growth, having been described that specimens as young as those in the pre-smolt stage (in freshwater) are already affected by PRV-1, showing a significant prevalence and high viral loads. The prevalence of PRV in early stages of production has become a great concern because these same infected specimens will serve as vectors to transport the virus with them when they are transported in tankers (wellboats) (L. voll, M., Wiik-Nielsen, J., Grove, S., Wiik-Nielsen, CR, Kristoffersen, AB, Faller, R., ... & Tengs, T. (2010). A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS). Virology Journal, 7(1), 1-7).The freshwater phase of salmonids represents the middle of the salmonid production cycle, lasting approximately three years. The quality of the smolts emerging from this phase will determine their subsequent performance in the fattening centers, so controlling PRV-1 in the initial stages of the production process is essential to avoid significant economic losses in later stages of salmon growth.

[0011] Due to the aforementioned points, the development of a vaccine to prevent PRV infection becomes vitally important to avoid losses in the production chain. However, the development of these has been highly limited due to the difficulty of propagating PRV in vitro. To date, several attempts to propagate this virus have been reported, for example, Mikalsen, A.B., Haugland, O., Rode, M., Solbakk, I.T., & Evensen, O. (2012). Atlantic salmon reovirus infection causes a CD8 T cell myocarditis in Atlantic salmon (Salmo salar).

[0012] L.). PLoS One, 7(6), e37269), reported the propagation of PRV in a cell line up to the fourth passage of infection, not being sufficient for the generation of viral progeny at the scale necessary for the formulation of vaccines (Mikalsen, AB, Haugland, O., Rode, M., Solbakk, IT, & Evensen, O. (2012). Atlantic salmon reovirus infection causes a CD8 T cell myocarditis in Atlantic salmon (Salmo salar L). PLoS One, 7(6), e37269) industrially. Other authors have attempted to develop a PRV vaccine using inactivated virus obtained from ex vivo cultures in nucleated erythrocytes (primary culture), however, this technique is expensive and there is no evidence to demonstrate a sufficient level of protection in farmed fish (Finstad, O., Dahle, M., Lindholm, T., Nyman, I., Lovoll, M., Wallace, C., Olsen, C.M., Storset, A. K., & Rimstad, E. (2014). “Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes”. Veterinary Research, 45(1), 35-48; Wessel, 0., Haugland, 0., Rode, M., Fredriksen, B., Dahle, M., & Rimstad, E. (2018). “Inactivated piscine orthoreovirus vaccine protects againts heart and skeletal muscle inflammation in Atlantic salmon.” Journal of Fish Diseases, 41 (9), 1411-1419). On the other hand, studies have reported the design of plasmid-based DNA vaccines expressing the structural and non-structural proteins of PRV and the use of salmonid Alphavirus replicons, however, these do not achieve sufficient levels of protection and have intrinsic difficulties for their scaling (Haatveit, HM, Hodneland, K., Braaen, S., Hansen, EF, Nyman, IB, Dahle, MK, Frost, P., & Rimstad, E. (2018). “DNA vaccine expressing the non-structural proteins of Piscine orthoreovirus delay the kinetics of PRV infection and induces moderate protection against heart and skeletal muscle inflammation in Atlantic salmon (Salmo salar)”. Vaccine, 36(50), 7599-7608) at an industrial level.

[0013] One solution to the aforementioned problems has been the development of so-called virus-like particles (VLPs). This structure has been successfully replicated in a wide range of viruses (Sarkar, B., Islam, S.S., Zohora, U.S., & Ullah,

[0014] MA (2019). Virus-like particles-A recent advancement in vaccine development. The Microbiological Society of Korea, 55(4), 327-343), for example, the development of such a vaccine that protects against the infectious pancreatic necrosis virus, IPNV, has been reported in salmon. For this virus, VLPs composed of the IPNV capsid proteins VP2 and VP3 have been generated in a subunit vaccine capable of inducing lymphocyte proliferation, mobilizing CD4+ lymphocytes, IgM+ and IgT+ cells, and inducing the type 1 interferon response (Martinez-Alonso, S., Vakharia, VN, Saint-Jean, SR, Perez-Prieto, S., & Tafalla, C. (2012). Immune responses elicited in rainbow trout through the administration of infectious pancreatic necrosis virus-like particles. Developmental & Comparative Immunology, 36(2), 378- 384), achieving a decrease in the mortality rate from 77% to 56% (Shivappa, RN, McAllister, PE, Edwards, GH, & Santi, N. (2005).Using a baculovirus insect / larvae. Dev. Biol, 121, 165-174). These antecedents demonstrate that this technology is promising for application in salmon viruses.

[0015] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses, but lack the viral genome, allowing antigens to be presented to the immune system without the risk of infection, reversion, or replication that occurs with other types of vaccines such as attenuated vaccines. These virus-like particles have been applied not only as prophylactic and therapeutic vaccines, but also as vehicles for drug and gene delivery and, more recently, as tools in nanobiotechnology (Schwarz B, Illchida M, Douglas T. Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology. Adv Virus Res. 2017; 97: 1-60. doi: 10.1016 / bs.aivir.2016.09.002. Epub 2016 Nov 8. PMID: 28057256; PMCID: PMC5384350.), so their application can go beyond just protection against the virus for which they were designed, providing a wide range of uses for it.

[0016] Based on the above with the examples of IPNV and ISAV and considering that the proteins Lambda 1 (λ1) and 2 (λ2), Sigma 2 (σ1) and 3 (σ3) and Miu 1 ( .1), are responsible for the assembly of the minimal icosahedral viral particle that allows the insertion of sigma 1, which has been described as the most antigenic protein of this virus and added to the fact that it has been previously reported that the structural proteins of Avian Reovirus (ARV) (Avian homolog of MRV, model for the study of PRV) spontaneously self-assemble when co-expressed, thus forming virus-like particles, the use of VLPs turns out to be the most appropriate strategy to achieve the generation of a subunit vaccine against PRV.Unlike inactivated virus vaccines, subunit or recombinant protein vaccines are composed of antigenic subunits that are protein in nature and retain the antigen's ability to induce immunogenicity against a specific pathogen.

[0017] The production of these proteins can be achieved through different methods, such as heterologous systems that usually use the most antigenic viral protein(s), however, the most common protein expression systems have major problems. For example, the expression of eukaryotic proteins in prokaryotic systems has the disadvantage that the protein will not be glycosylated, which can significantly alter its immunogenic properties (Khow, O., & Suntrarachun, S. (2012). Strategies for production of active eukaryotic proteins in bacterial expression system. Asian Pacific journal of tropical biomedicine, 2(2), 159-162). On the other hand, if the proteins are expressed in yeast systems, they will be hyperglycosylated (Malissard, M., Zeng, S., & Berger, E.G. (1999). The yeast expression system for recombinant glycosyltransferases.Glycoconjugate journal, 16(2), 125- 139), which, as in the previous point, will affect the immunogenic properties of the antigen. Thus, a mammalian cell-based expression system appears to be the best alternative; however, these systems require the use of fetal bovine serum, among other components, which significantly increases the production cost, and the fact that these systems are difficult to scale in terms of production.

[0018] Due to the above, the ideal system for producing a vaccine with structural and immunogenic characteristics as similar as possible to the WT virus must have a vector that allows the synthesis of a large amount of recombinant protein for the generation of VLPs, which must be of good quality, in structural terms, so that they can generate an immune response as strong as that triggered by natural infection. The baculovirus / insect cell system is a tool capable of meeting these conditions. This system is established in eukaryotic cells and under strong expression promoters. In this way, large quantities of correctly folded proteins can be obtained, with post-translational modifications, such as glycosylation, which is very common in viral surface proteins.The efficiency with which baculoviruses generate recombinant proteins can reach 50% of the infected cell's dry weight or 30% of the insect's weight, reflecting their high yield. Given these characteristics, developing PRV VLPs composed of the proteins that assemble the inner and outer capsids and are responsible for stimulating the host's immune system is highly interesting. The MultiBac® system (Geneva Biotech) was used in the present invention. The baculovirus expression system is carried out in lepidopteran cells (Sf9 or S21); these cells do not require animal-derived supplements, minimizing the risk of co-culturing opportunistic pathogens and reducing production costs compared to systems based on mammalian cells.On the other hand, the baculoviruses used for protein expression have a narrow host range that includes only a few Lepidoptera species, so they do not pose a danger to vaccinated species.

[0019] Thus, while there are no vaccines on the market against PRV, current treatment alternatives propose, for example, the use of functional foods rich in polyunsaturated acids, a treatment that manages to reduce the inflammation of the lesions typical of salmon infected with HSMI (Martin, SA, & Król, E. (2017). Nutrigenomics and immune function in fish: new insights from omics technologies. Developmental & Comparative Immunology, 75, 86-98). Components of these functional diets are also capable of being ligands that compete with the virus for binding to its cellular receptors; or improving the composition of the lipid membrane; and promote cell signaling by inducing transcription factors (Madhun, AS, Isachsen, CH, Omdal, LM, Einen, ACB, Bjorn, PA, Nilsen, R., & Karlsbakk, E. (2016). Occurrence of salmonid alphavirus (SAV) and piscine orthoreovirus (PRV) infections in wild sea trout Salmo trutta in Norway.Diseases of Aquatic Organisms, 120(2), 109-113). However, due to their nature, these foods do not provide immunity against PRV.

[0020] Thus, there is a need for vaccines for the treatment and prevention of PRV in gamefish, and therefore, antigens that allow the preparation of said vaccines are also necessary, these antigens being able to be recombinant proteins that can be used in isolation in a vaccine composition, or in conjunction, to prepare an effective and safe VLP against PRV, preferably PRV-1, and nucleic acid molecules that encode them and allow the preparation of vectors, transformed microorganisms, and complementarily, host cells that in turn allow the preparation of vaccine compositions comprising them in isolation or in conjunction.

[0021] Brief Description of the Figures

[0022] Figure 1. Recombinant viral vector used to generate recombinant baculoviruses. The recombinant viral vector is based on the plasmid pFastBac-1™ (Thermo Fisher) into which the genes encoding the six PRV proteins of interest have been inserted under the control of the polyhedrin promoter. The recombinant viral vector has an ampicillin resistance gene for replication in E. coli. GOI corresponds to the following six PRV proteins: σ1, σ2, σ3, λ1, λ2, and .1. Each recombinant viral vector has only one recombinant gene.

[0023] Figure 2. Expression of PRV inner and outer capsid proteins in Sf9 cells infected with recombinant baculoviruses. Western blot of recombinant proteins expressed in Sf9 cells infected with recombinant baculoviruses. 1. Protein extract of uninfected Sf9 cells. 2. Protein extract of cells infected with wild-type baculoviruses. 3. Protein extract of cells infected with recombinant baculoviruses containing a PRV gene.

[0024] Figure 3. Heterologous expression of PRV proteins in Sf9 cells infected with recombinant baculovirus. Indirect immunofluorescence of uninfected Sf9 cells (mock), infected with wild-type baculovirus (WT) and infected with recombinant baculoviruses containing the aforementioned genes (Bac-PRV).

[0025] Figure 4. Infection kinetics in fish vaccinated with the VLP-adjuvant formulation. Quantification of viral titer from the anterior kidney at 3, 4, and 5 weeks postinfection in fish vaccinated and challenged with PRV-1. A control group vaccinated with PBS was included. Statistical analysis was performed using ANOVA. **p<0.005; ***p<0.0005.

[0026] Figures 5A-5E Calibration curves. Fig. 5A ELISA standard curve of o1 . Fig. 5B ELISA standard curve of a2. Fig. 5C ELISA standard curve of o3. Fig. 5D ELISA standard curve of 1 . Fig. 5E ELISA standard curve of λ2.

[0027] Figure 6 Transmission electron microscopy for VLP visualization. Transmission electron microscopy analysis of virus-like particles formed by λ1, σ2, λ2, σ1, σ3 and 1. The purified particles were adsorbed onto nickel grids and stained with 1% w / v uranyl acetate. Size 2048. Exp. 1s, Mag. 57000x, Spot 5, HT 200 kV, pixel size 460.9 pm, Fov 943.9 nm, defocus -38 μm, scale 100 nm.

[0028] Detailed Description of the Invention

[0029] The present invention relates to a recombinant protein as antigens against PRV, preferably PRV-1 (genotype 1), selected from one of the following 6 proteins having a sequence based on the 6 structural proteins of the internal and external capsid of the PRV viral particle: a) recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, b) recombinant protein σ2 of amino acid sequence SEQ ID No.: 2, c) recombinant protein σ3 of amino acid sequence SEQ ID No.: 3, d) recombinant protein λ 1 of amino acid sequence SEQ ID No.: 10, e) recombinant protein λ2 of amino acid sequence SEQ ID No.: 11, or f) recombinant protein 1 of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity.

[0030] Preferably, the recombinant protein is the recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity.

[0031] Preferably, the recombinant protein is the recombinant σ2 protein of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity.

[0032] Preferably, the recombinant protein is the recombinant σ3 protein of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity.

[0033] Preferably, the recombinant protein is the recombinant protein λ1 of amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity.

[0034] Preferably, the recombinant protein is the recombinant protein λ2 of amino acid sequence SEQ ID No.: 11, or a variant thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity. Preferably, the recombinant protein is the recombinant protein μ1 of amino acid sequence SEQ ID No.: 13, or a variant thereof, having the same antigen functionality with high similarity to PRV wild type and more than 90% sequence identity similarity, preferably more than 95% sequence identity similarity, even more preferably more than 99% sequence identity similarity.

[0035] Furthermore, viral proteins were obtained from PRV strains detected in tissues obtained from salmon farms in Chile and belonging to genogroup 1 (PRV-1), which has been detected not only in Chile but also in Scotland, Norway, and the Faroe Islands, among others. The present invention also relates to nucleic acid molecules encoding each of the aforementioned / described recombinant proteins (σ1, σ2, σ3, λ1, λ2, λ), and selected from one of the following 6 nucleic acid molecules: a) nucleic acid molecule of nucleic acid sequence SEQ ID No.:13 encoding recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, b) nucleic acid molecule having nucleic acid sequence SEQ No.: 14 encoding recombinant protein σ2 of amino acid sequence SEQ ID No.: 2, c) nucleic acid molecule having nucleic acid sequence SEQ No.: : 15 encoding the recombinant protein σ3 of amino acid sequence SEQ ID No.: 3, d) nucleic acid molecule having the nucleic acid sequence SEQ No.: 18 encoding the recombinant protein λ1 of amino acid sequence SEQ ID No.: 10, e) nucleic acid molecule having the nucleic acid sequence SEQ No.: 17 encoding the recombinant protein λ2 of amino acid sequence SEQ ID No.: 11, or f) nucleic acid molecule having the nucleic acid sequence SEQ No.: 16 encoding the recombinant protein μ1 of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0036] Preferably, the nucleic acid molecule is selected from the nucleic acid sequence SEQ ID No.:13 encoding the recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0037] Preferably, the nucleic acid molecule is selected from the nucleic acid molecule sequence having the nucleic acid sequence SEQ No.: 14 encoding the recombinant σ2 protein of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity. Preferably, the nucleic acid molecule is selected from the nucleic acid molecule sequence having the nucleic acid sequence SEQ No.: 15 encoding the recombinant σ3 protein of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0038] Preferably, the nucleic acid molecule is selected from the nucleic acid molecule sequence having the nucleic acid sequence SEQ No.: 18 encoding the recombinant protein λ1 with amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0039] Preferably, the nucleic acid molecule is selected from the nucleic acid molecule sequence having the nucleic acid sequence SEQ No.: 17 encoding the recombinant protein λ2 with amino acid sequence SEQ ID No.: 11, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0040] Preferably, the nucleic acid molecule is selected from the nucleic acid molecule sequence having the nucleic acid sequence SEQ No.: 16 encoding the recombinant protein μ d1e amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0041] The present invention also relates to a recombinant viral vector for producing antigenic recombinant protein against Piscine orthoreovirus (PRV), preferably Piscine orthoreovirus genotype 1 (PRV-1), which can be selected from an adenovirus vector, a lentiviral vector, a cytomegalovirus vector, a baculovirus vector, and preferably, said baculovirus.The recombinant viral vector comprises the Thermo Fisher pFastBac-1 plasmid with the Baculoviral Polyhedrin promoter, one of the 6 aforementioned / described nucleic acid molecules or variants thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, having the ability to express one of the 6 aforementioned / described proteins or variants thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity and a nucleic acid sequence having ampicillin resistance ((https: / / tools.thermofisher.com / content / sfs / manuais / bactobac man. pdf), to perform cloning and subsequent selection with said antibiotic.

[0042] Preferably, the vector comprises the plasmid pFastBac-1 from Thermo Fisher, the nucleic acid sequence SEQ ID No.: 13 encoding the recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a nucleic acid sequence having ampicillin resistance.

[0043] Preferably, the vector comprises the plasmid pFastBac-1 from Thermo Fisher, the nucleic acid sequence SEQ ID No.: 14 encoding the recombinant σ2 protein of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a nucleic acid sequence having ampicillin resistance.

[0044] Preferably, the vector comprises the plasmid pFastBac-1 from Thermo Fisher, the nucleic acid sequence SEQ ID No.: 15 encoding the recombinant σ3 protein of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a nucleic acid sequence having ampicillin resistance.

[0045] Preferably, the vector comprises the plasmid pFastBac-1 from Thermo Fisher, the nucleic acid sequence SEQ ID No.:17 encoding the recombinant protein λ1 of amino acid sequence SEQ ID No.: 11, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a nucleic acid sequence having ampicillin resistance.

[0046] Preferably, the vector comprises the plasmid pFastBac-1 from Thermo Fisher, the nucleic acid sequence SEQ ID No.: 13 encoding the recombinant protein λ2 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a nucleic acid sequence having ampicillin resistance.

[0047] Preferably, the vector comprises the plasmid pFastBac-1 of Thermo Fisher, the nucleic acid sequence SEQ ID No.: 16 encoding the recombinant protein pl of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a nucleic acid sequence having ampicillin resistance. Likewise, the present invention relates to a microorganism transformed with the aforementioned / described vector selected from an adenovirus, cytomegalovirus, baculovirus and the like, and preferably relates to transformed baculovirus comprising a vector which in turn comprises one of the 6 nucleic acid molecules encoding the 6 aforementioned / described recombinant proteins.

[0048] The transformed baculoviruses have registration number LMBP14885CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity; registration number LMBP14888CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant protein σ2 of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity; Belgian Coordinated Collections of Microorganisms (BCCM) accession number LMBP14889CB of August 29, 2023, having the capacity to express the recombinant σ3 protein of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity; Belgian Coordinated Collections of Microorganisms (BCCM) accession number LMBP14886CB of August 29, 2023, having the capacity to express the recombinant λ1 protein of amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity; Belgian Coordinated Collections of Microorganisms (BCCM) accession number LMBP14883CB of August 29, 2023, having the ability to express the recombinant protein λ2 of amino acid sequence SEQ ID No.: 11, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity; or Belgian Coordinated Collections of Microorganisms (BCCM) accession number LMBP14885CB of August 29, 2023, having the ability to express the recombinant protein JLX1 of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity. Furthermore, the present invention relates to a host cell to be infected with the transformed microorganism described above and which can be selected from insect cells, yeast cells, plant cells and mammalian cells depending on the transformed microorganism comprising the recombinant viral vector into which each of the nucleic acid molecules expressing the 6 PRV-1 proteins mentioned / described above have been separately inserted. Preferably, the host cell is an insect cell, and even more preferably it is the sf9 cell.

[0049] Preferably, a host cell comprising the transformed baculovirus of accession number LMBP14885CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0050] Preferably, a host cell comprising the transformed baculovirus of accession number LMBP14888CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant σ2 protein of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0051] Preferably, a host cell comprising the transformed baculovirus of accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant σ3 protein of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0052] Preferably, a host cell comprising the transformed baculovirus of accession number LMBP14886CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant protein λ1 of amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0053] Preferably, a host cell comprising the transformed baculovirus of accession number LMBP14883CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant protein λ2 of amino acid sequence SEQ ID No.: 11, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity. Preferably, a host cell comprising the transformed baculovirus of accession number LMBP14884CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to express the recombinant protein ,1 of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity.

[0054] The present invention also relates to a vaccine composition for preventing or treating PRV in gamefish wherein the vaccine composition may comprise one or more of the aforementioned 6 recombinant proteins and vetehnahamente acceptable excipients, and optionally, one or more vetehnahamente acceptable adjuvants.

[0055] Preferably, the vaccine composition comprises the recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a veterinarily acceptable excipient, and optionally, one or more veterinarily acceptable adjuvants.

[0056] Preferably, the vaccine composition comprises the recombinant σ2 protein of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a veterinarily acceptable excipient, and optionally, one or more veterinarily acceptable adjuvants.

[0057] Preferably, the vaccine composition comprises the recombinant σ3 protein of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a veterinarily acceptable excipient, and optionally, one or more veterinarily acceptable adjuvants.

[0058] Preferably, the vaccine composition comprises the recombinant λ1 protein of amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a veterinarily acceptable excipient, and optionally, one or more veterinarily acceptable adjuvants.

[0059] Preferably, the vaccine composition comprises the recombinant λ2 protein of amino acid sequence SEQ ID No.: 11, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a veterinarily acceptable excipient, and optionally, one or more veterinarily acceptable adjuvants.

[0060] Preferably, the vaccine composition comprises the recombinant μ1 protein amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity, preferably more than 95% sequence identity, even more preferably more than 99% sequence identity, and a veterinarily acceptable excipient, and optionally, one or more veterinarily acceptable adjuvants.

[0061] Preferably, the virus-like particle (VLP) vaccine composition having the aforementioned 6 self-assembled recombinant proteins, wherein said recombinant proteins have a structure like that of the inner and outer capsid proteins of PRV genogroup 1 (PRV-1), each of the host cells comprising the transformed baculoviruses comprising a vector each comprising the 6 nucleic acid molecules encoding each of the aforementioned 6 recombinant proteins, wherein the baculoviruses have deposit number LMBP14886CB dated August 29, 2023, deposit number LMBP14883CB dated August 29, 2023, deposit number LMBP14884CB dated August 29, 2023, deposit numbers LMBP14885CB dated August 29, 2023, deposit LMBP14888CB dated August 29, 2023, deposit numbers LMBP14889CB dated August 29, 2023,and each baculovirus separately expresses each of the 6 nucleic acid molecules encoding each of the 6 recombinant proteins, and also comprises veterinarily acceptable excipients, and optionally, one or more veterinarily acceptable adjuvants, and where said one or more veterinarily acceptable adjuvants are selected from one or more of Montanide®, Alumina, water / oil emulsion (WO), water / oil / water emulsion (WOW), complete Freund's adjuvant, incomplete Freund's adjuvant, saponins, dimethyl dioctadecyl amino bromide (DDA). See Fig. 1.,

[0062] Preferably, the VLP vaccine composition comprises host cell lysate with each of the 6 aforementioned baculoviruses comprising the vector of Figure 1, and more preferably, said host cell is an insect cell, and even more preferably, said insect cell is an sf9 cell.

[0063] The present vaccine composition can be formulated to be administered intraperitoneally, by bathing or immersion, orally in food, cutaneously, or by administration through the anal pore.

[0064] The present vaccine composition is selected from a vaccine composition formulated for water-in-oil emulsion, or oil-in-water emulsion, or their preparative variations. The formulation may also be through nanoparticles or microparticles that coat or nucleate the antigen, or the latter may coat the nano- or microstructures. Even more preferably, the vaccine composition is a formulated emulsion comprising 70% of the Montanide® adjuvant and one or more of the 6 aforementioned / described recombinant proteins, in an amount ranging from 0.5 µg to 1.5 µg per dose.

[0065] The present invention also relates to a method for treating or preventing PRV infection in gamefish, comprising administering the aforementioned vaccine composition to said fish. The present invention also relates to a method for treating or preventing Heart and Skeletal Muscle Inflammation (HSMI) caused by PRV-1 infection in farmed fish, comprising administering the aforementioned vaccine composition to said fish.

[0066] Furthermore, the present invention also relates to the use of the aforementioned vaccine composition / deschta, which is useful in the preparation of a medicament useful for treating or preventing a PRV infection in gamefish. In particular, the present invention relates to a vaccine composition comprising a PRV-1 VLP, which is useful for preparing a medicament useful for treating or preventing a PRV-1 infection in gamefish, wherein the VLP is as mentioned above. The present invention also relates to a vaccine composition comprising PRV-1 VLP, which is useful for preparing a medicament useful for treating or preventing skeletal and cardiac muscle inflammation (HSMI) caused by PRV-1 infection.

[0067] Chance fish are selected from salmonids. Preferably, said salmonids may be selected from one or more of the following groups: Coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar), and Rainbow trout (Oncorhynchus mykiss).

[0068] The present invention also relates to a method for producing the virus-like particles (VLPs) of PRV-1 as mentioned above, comprising the following steps: a) incorporating nucleic acid molecules expressing each of the following 6 PRV proteins: recombinant protein σ1 of amino acid sequence SEQ ID No.: 1, recombinant protein σ2 of amino acid sequence SEQ ID No.: 2, recombinant protein σ3 of sequence SEQ ID No.: 3, recombinant protein λ1 of amino acid sequence SEQ ID No.: 10, recombinant protein λ2 of amino acid sequence SEQ ID No.: 11, recombinant protein JLL1 of amino acid sequence SEQ ID No.: 12 or a variant thereof, having the same antigen functionality with high similarity to PRV wild type, containing the most antigenic protein of the virus (σ1) and more than 90% sequence identity similarity, preferably more than one 95% sequence identity similarity,even more preferably more than 99% sequence identity similarity, in a recombinant viral vector that can be selected from an adenoviral, lentiviral, cytomegaloviral vector, among others; b) obtaining a transformed microorganism having the vector obtained in step a), where said transformed microorganism can be selected from an adenovirus, a cytomegalovirus, a baculovirus, among others, and where preferably said transformed microorganism is a baculovirus, and even more specifically said baculovirus is selected from baculovirus registration LMBP14886CB dated August 29, 2023, baculovirus registration LMBP14883CB dated August 29, 2023, baculovirus registration LMBP14884CB dated August 29, 2023, baculovirus registration LMBP14885CB dated August 29, 2023, baculovirus registration LMBP14888CB dated August 29, 2023 and baculovirus registration LMBP14889CB dated August 29, 2023,and c) infecting host cells with the transformed microorganism obtained in step b) for replication of said recombinant viral vector, where said host cell can be selected from insect cells, yeast cells, plant cells, mammalian cells, among others. Preferably, said host cell is an insect cell, even more preferably it is an SF9 insect cell.

[0069] Examples

[0070] Example 1: Production and characterization of recombinant PRV-1 proteins PRV-1 sequences and transfer vectors

[0071] The sequence of the S4 segment (SEQ ID No.: 15) of PRV genotype 1 was obtained from tissue of affected fish on a farm in Chile. The S4 segment was used to perform phylogeny of the isolate and determine its genotype. All computational genomics analyses were performed with the bioinformatics package found on the EXPASY server. The sequences were compared with those of homologous proteins of Avian and Mammalian Reoviruses available in GenBank according to Table 2. With the nucleotide information, it was determined that the isolate corresponded to Chilean PRV called CGA280-05 (GenBank access KC795565-KC795574 at https: / / www.ncbi.nlm.nih.gov / ), which presented 100% similarity with the virus detected in the tissue that was analyzed. The rest of the S, M and L segments that code for the proteins σ1 (SEQ ID No.: 1), σ2 (SEQ ID No.: 2), σ3 (SEQ ID No.: 3), λ1 (SEQ ID No.: 10), λ2 (SEQ ID No.: 11) and .1 (SEQ ID No.: 12) were used based on said isolate, and the access codes to each of the sequences are detailed in the following table:.

[0072] Table 1. GenBank access codes of the segments used for the generation of the transfer vectors obtained from Genscript. The S1 and M2 segments were synthesized by GenScript in a pUC57 vector (Addgene) according to the aforementioned / described GenBank sequence. From this pUC57 vector, the coding sequence of the segments was excised using the restriction enzymes BamHI and EcoRI and then cloned into a pFastBad™ transfer vector. Finally, the remaining 4 segments were codon optimized for expression in the Sf9 insect cell line and pFastBad™ transfer vectors with the optimized ORFs were biochemically synthesized by GenScript Co. (USA). The codon-optimized nucleic acid molecules are selected from SEQ ID No.: 14 (segment S2), SEQ ID No.: 15 (segment S4), SEQ ID No.: 17 (segment L2) and SEQ ID No.: 18 (segment L1).

[0073] Table 2: Accession number of proteins from mammalian reovirus (MRV) and avian reovirus (ARV) homologous to Reovirus Piscine

[0074] The six PRV nucleic acid molecules used to generate the VLPs were cloned under the polyhedrin (PH) promoter in the pFastBad™ vector. The bacmids were generated in the bacterium DHIOMultibac® (Geneva Biotech).

[0075] Generation of baculoviruses

[0076] Using the pFastBad™ vectors (Thermo) containing synthetic structural nucleic acid molecules of PRV genotype 1 encoding the recombinant protein σ1 having amino acid sequence SEQ ID No.: 1, recombinant protein σ2 having amino acid sequence SEQ ID No.:2, recombinant protein <J3 teniendo secuencia de amino ácidos SEQ ID No.:3, proteína recombinante λ1 teniendo secuencia de amino ácidos SEQ ID No.: 10, proteína recombinante λ2 teniendo secuencia de amino ácidos SEQ ID No.: 11 y proteína recombinante teμn1iendo secuencia de amino ácidos SEQ ID No.: 12, bajo el control del promotor de polihedrina y junto la bacteria DHIOMultibac® (Geneva Biotech).

[0077] The cloned plasmids were transformed into chemocompetent bacteria E. coli DHIOMultiBac by heat shock by incubating them on ice for 30 minutes, 45 seconds at 42°C and 5 minutes on ice. Then, they were incubated for 4 hours at 37°C with shaking in “super optimal Broth with catabolite repression (SOC)” medium (Qiao-Yang Sun, Ling-Wen Ding, Liang-Liang He, Yong-Bin Sun, Jun-Li Shao, Ming Luo, Zeng-Fu Xu, Culture of Escherichia coli in SOC medium improves the cloning efficiency of toxic protein genes, Analytical Biochemistry, Volume 394, Issue 1 , 2009, 144-146, D0l:10.1016 / j.ab.2009.07.023).Subsequently, they were seeded on LB-agar plates with 50 pg / mL of Kanamycin (Roche), 7 pg / mL of Gentamicin (Thermo Scientific), 10 pg / mL of Tetracycline (Sigma-Aldrich), IPTG (40 pg / mL), and X-gal (80 pg / mL) to finally be incubated for 48 hours at 37°C and by sectioning white colonies (recombinant) differentiated from blue colonies (non-recombinant) known as “Blue-White screening”, recombinant colonies were selected and grown overnight in LB medium (Qiao-Yang Sun, Ling-Wen Ding, Liang-Liang He, Yong-Bin Sun, Jun-Li Shao, Ming Luo, Zeng-Fu Xu, Culture of Escherichia coli in SOC medium improves the cloning efficiency of toxic protein genes, Analytical Biochemistry, Volume 394, Issue 1 , 2009,144-146,.

[0078] D0l:10.1016 / j.ab.2009.07.023) with the aforementioned antibiotics, and the bacmids were subsequently purified by alkaline lysis. The recombinant bacmids were tested by PCR using the Phusion DNA polymerase enzyme (Thermo Scientific), using 5 ng of template DNA and primers specific for the M13 promoter using the following thermal profile: Hot-start of 30 seconds at 98°C, followed by 30 cycles of 98°C for 10 seconds, 55°C for 40 seconds and 72°C for 3 minutes with a final extension of 72°C for 5 minutes. The PCR products were resolved by electrophoresis on a 1% w / v agarose gel. The bacmids that possessed the gene of interest were transfected into Sf21 cells, seeded at a density of 1x10 5 cells / cm 2, using the X-tremeGENE transfection reagent (Roche) at a ratio of 8:1 (transfection reagent: pg of DNA) in 100 pL of SF900II SFM medium. The transfection was incubated at 28°C until the appearance of cytopathic effect or 72 hours maximum, at which time the supernatant containing the transformed Baculoviruses was harvested.

[0079] To amplify the obtained baculoviruses, Sf9 cells were seeded at a density of 1x10 6 cells / mL in 50 mL of SF900II SFM medium, which were infected with 1 mL of supernatant and incubated for 72 hours at 28°C with shaking. To harvest the supernatant, the culture was centrifuged at 500 g for 5 minutes and the supernatant was recovered for subsequent titration by TCIDso.

[0080] Characterization of recombinant proteins

[0081] To analyze the expression of the 6 recombinant proteins mentioned above / described, Sf9 cells were infected at a multiplicity of infection (MOI) of 1 and incubated at 28°C with shaking for 72 hours or until 80% cell death was observed. At the end of the incubation, the culture was centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in 0.1 mM polymethylsulfonyl fluoride (PMSF) in PBS, and the cells were then mechanically lysed. The protein extract was quantified by the Bradford method by measuring its absorbance at 595 nm. Ten pg of total protein was analyzed by SDS-PAGE on a 12% polyacrylamide separating gel. To immunodetect proteins, the gel contents were transferred to a PVDF (Polyvinylidene fluoride) membrane for 60 minutes at 70V.The membrane was incubated in blocking solution (Tween20 0.1% v / v; Sigma; BSA 5% w / v in 1X PBS) overnight at 4°C, then washed 3 times with T-PBS (Tween20 0.1% w / v in PBS) and incubated for 1 hour with primary antibody at a 1:1000 dilution for recombinant proteins α1, σ2 and σ3 and 1:10000 for recombinant proteins λs1, λ2 and μ1. At the end of the incubation, it was washed 3 times with T-PBS and incubated for 1 hour with secondary antibody anti-ra£>£> / t conjugated to HRP (Sigma) at a 1:4000 dilution in blocking solution. Finally, the presence of PRV proteins was analyzed by revealing the membranes on radiographic plates using chemiluminescence.

[0082] As seen in Figure 2, when insect cells were infected with the transformed baculovirus containing the nucleic acid molecule encoding recombinant protein σ1, a protein of approximately 35 kDa was detected. When cells infected with the recombinant baculovirus containing the nucleic acid molecule encoding recombinant protein σ2 were analyzed, a protein of approximately 45 kDa was detected. When cells infected with the recombinant baculovirus containing the nucleic acid molecule encoding recombinant protein σ3 were analyzed, a protein of approximately 37 kDa was detected. When cells infected with the recombinant baculovirus containing the nucleic acid molecule encoding recombinant protein 1 were analyzed, a protein of approximately 70 kDa was detected.When cells infected with the recombinant baculovirus containing the nucleic acid molecule encoding the recombinant λ1 protein were analyzed, a protein of approximately 130 kDa was detected. Finally, when cells infected with the recombinant baculovirus containing the nucleic acid molecule encoding the recombinant λ2 protein were analyzed, a protein of approximately 130 kDa was detected. All these weights are consistent with previous literature (Kibenge, M.J., Iwamoto, T., Wang, Y., Morton, A., Godoy, M.G., & Kibenge, F.S. (2013). “Whole-genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate subgenotypes”. Virology Journal, 10(1), 230-250).

[0083] The expression of the 6 recombinant proteins mentioned above / described was also confirmed by indirect immunofluorescence of Sf9 cells infected with the recombinant baculoviruses. At 72 hpi, cells were washed 3 times with PBS (2 minutes each time) and fixed with 4% v / v formaldehyde in PBS for 30 minutes. The fixed cells were washed 3 times with PBS and permeabilized with 0.3% v / v Triton X-100 (Calbiochem) in PBS for 10 minutes. The plates were then blocked for 1 hour with blocking buffer (3% w / v BSA and 0.1% v / v Triton X-100 in PBS). After blocking, cells were incubated for 1 hour with the primary antibody PRV (GeneScript) diluted 1:500 in blocking buffer. After this, the cells were washed 3 times with PBS to be incubated for 1 hour with secondary antibody anti-rabbit Alexa Fluor 594 (Life Technologies) diluted 1:500 in 0.5 pg / mL 4', 6- diamino-2-phenylindole (DAPI, Life Technologies) in blocking buffer.Finally, the covers were washed 4 times with PBS and mounted on coverslips with 1,4-diazabicyclo[2.2.2]octane (DABCO, Merck Millipore). The samples were observed in a Zeiss LSM 800 confocal microscope. As shown in Figure 3, all proteins of interest were detected in the infected cells.

[0084] For the generation of virus-like particles, these recombinant proteins have structural reasons, with σ2 and λ1 being the inner core proteins that allow the assembly of the outer core which is formed by σ3μ.1 The λ2 protein interacts with both cores and allows the association of σ1 to the VLP, which is the most antigenic protein of PRV.

[0085] Assessment of antigen expression

[0086] The expression of each of the 6 recombinant proteins mentioned / described above was determined by ELISA. To do this, a 100 L aliquot of insect cell lysate infected with the recombinant baculoviruses was adsorbed onto 96-well ELISA plates for 2 hours at room temperature. At the end of the incubation, the supernatant was discarded and the plate was washed 3 times with PBS (Phosphate Buffered Saline) and then incubated with BSA blocking solution (Bovine Serum Albumin 5% in PBS) for 2 hours at room temperature. After blocking, the antibody was washed 3 times with PBS and incubated with the primary antibody at a 1:1000 dilution for recombinant proteins σ1, σ2 and σ3 and 1:10000 for recombinant proteins λ1, λ2 and μ1 prepared in blocking solution. The antibody was then discarded, washed 3 times with PBS and incubated for 1 hour with an HRP (HorseRadish Peroxidase) conjugated secondary antibody (anti-rabbit) at a 1:8000 dilution in blocking solution.Finally, the mixture was washed again with PBS and TMB (3,3',5,5'-Tetramethylbenzidine) was added, leaving the reaction in the dark for 10 minutes. The reaction was stopped with 1 M HCl and the absorbance was read at 450 nm. The concentration of the 6 recombinant proteins mentioned / described above was determined by comparing the absorbance with a standard curve of pure protein.

[0087] Example 2: Preparation of vaccines with VLPs

[0088] Antigen production

[0089] To obtain the particulate antigen (VLPs), Sf29 cells were infected with the six baculoviruses at a MOI (Multiplicity of Infection) of 1. After 3 days, cells were collected and used. Then, a method for quantifying the 6 aforementioned / described recombinant proteins by ELISA was implemented, covering the bottom of the wells with the six purified recombinant proteins (expressed in E. coli) mentioned above or with the synthetic peptides (“Core Shell 2” peptide, SEQ ID No.: 4; “Core Shell 3” peptide, SEQ ID No.: 5; “Core Shell 1” peptide, SEQ ID No.: 6; “Turret 2” peptide, SEQ ID No.: 7; “Turret 3” peptide, SEQ ID No.: 8; “Turret 1” peptide, SEQ ID No.: 9), material used to obtain hyperimmune serum in rabbits. Thanks to this semi-quantitative method, it was determined that the co-expression of proteins through baculovirus coinfection is possible.

[0090] Purification of empty viral particles by sucrose gradient and PEG 8000 precipitation For the purification of empty subviral particles, 1.25x10 7High Five cells in 25 mL of Express Five medium supplemented with 18 mM L-Glutamine (Sigma) and 100 ug / mL penicillin / streptomycin (Gibco). Subsequently, co-infections were performed with the recombinant baculoviruses, bac-A1 having accession No. LMBP14886CB dated August 29, 2023, bac-λ2 having accession No. LMBP14883CB dated August 29, 2023, bac-pl having accession No. LMBP14884CB dated August 29, 2023., bac-σ1 having accession No. LMBP14888CB dated August 29, 2023., bac-σ2 having accession No. LMBP14888CB dated August 29, 2023 and bac-σ3 having accession No. LMBP14889CB dated August 29, 2023, using an MOI of 1 for each. After 72 hours post infection, the cell suspension was centrifuged for 10 min at 2000 xg to pellet the cells, then cell lysis was performed using 1 mL of TNN buffer (50 mM Tris-HCI, pH 8.0, 150 mM NaCI and 1% v / v of NP40), discarding cell debris by centrifugation at 2000 xg for 15 min.Then, 1 mL of supernatant from the previous step was applied to a discontinuous gradient of 30-50% w / v sucrose in PBS prepared in a 1.5 mL tube, 250 uL each phase. The gradients were centrifuged at 23,500 xg for 2 hours at 4°C. After centrifugation, 200 uL of the interface fraction of 30% w / v and 50% w / v of the sucrose gradient was collected and resuspended in 4 mL of 15% w / v PEG 8000 (polyethylene glycol) prepared in SM buffer (0.1 M NaCl; 50 mM Ths HCl pH 7.4; 10 mM Mg2SÜ4); the resulting suspension was incubated on ice overnight to promote the precipitation of empty viral particles. After the precipitation time, the suspension was centrifuged at 6000 g for 1 hour at 4°C and the resulting pellet was resuspended in 40 uL of PBS.

[0091] Transmission electron microscopy

[0092] Transmission electron microscopy using negative contrast staining with uranyl acetate was used to visualize the previously purified empty particles. A 400 mesh nickel grid coated with formvar film (polyvinyl formaldehyde in 1,2 dichloroethane chloroform) was deposited on 5 uL of empty particle suspension and left to stand for 1 minute. After washing three times with PBS, the sample was contrasted with 1% w / v uranyl acetate for 1 min. Once dry, the sample was viewed in a Philips CM12 transmission electron microscope (TEM) at 100 kV.

[0093] In order to determine the formation of empty viral particles by co-expression of recombinant proteins having amino acid sequence SEQ ID No.: 10, λ2 having amino acid sequence SEQ ID No.:11, μ1 having amino acid sequence SEQ ID No.: 12, a1 having amino acid sequence SEQ ID No.:1, σ2 having amino acid sequence SEQ ID No.:2 and σ3 having amino acid sequence SEQ ID No.:3, purification of viral particles and subsequent analysis by electron microscopy for visualization was carried out. Figure 5 shows the electron micrographs corresponding to the purification of VLP particles, generated by co-expression of the aforementioned / described recombinant proteins. Figure 5 shows the formation of particles with an icosahedral appearance with a diameter similar to that of reoviral particles.

[0094] Formulation of prototype vaccine

[0095] The vaccine prototypes were formulated using established protocols for oil-based vaccines, emulsions formulated with 70% Montanide® (Seppic) adjuvant. The antigen corresponds to a minimum of 0.5 pg and a maximum of 1.5 pg of recombinant σ1 protein as a normalizing protein per dose.

[0096] Example 3: Vaccination for determination of safety and efficacy

[0097] Safety of the vaccine prototype

[0098] To determine whether the vaccine is safe for injection into salmonids, a trial was conducted in trout. This trial included 34 groups of 20 trout each (Groups: 23 vaccine doses and 1 saline control) which were vaccinated intraperitoneally with 0.1 mL of each formulation. The results showed that the fish did not suffer adverse effects or mortality due to the experimental formulation.

[0099] Vaccination and challenge

[0100] To evaluate the effectiveness, 300 fish of 33 ± 3.3 g were used, divided into 3 groups of 50 fish with pond replica at a density of 9.9 kg / m 3. After acclimatization, the fish were vaccinated with 0.1 mL of each of the two experimental formulations containing the six PRV proteins according to the paragraph "Prototype vaccine formulation" or with saline as a control, and were kept in freshwater for 600 ATU (Accumulated Thermal Units). The smoltification process was carried out over 5 weeks until adaptation was achieved under seawater conditions at 12 ° C. Subsequently, at 600 ATU post-vaccination, the fish were challenged with PRV genotype 1 inoculum intraperitoneally and mortality was recorded until 5 weeks post-challenge, at which time the trial was terminated. Tissue samples were collected at weeks 3, 4 and 5 weeks post-challenge to evaluate the PRV viral load in vaccinated and challenged fish in order to assess the effect of vaccination on the viremia developed in vaccinated and non-vaccinated fish.To quantify viral titer, total RNA was extracted from the anterior kidney using the FlavoPrep kit according to the manufacturer's instructions. Using the template RNA, the number of copies of PRV segment 1 per mg of tissue was determined using the Genesig Piscine Reovirus kit (Primerdesign).

[0101] The formulations tested in this trial (Figure 4) showed concentration-dependent protection against PRV, with the formulation with the highest protein concentration being the most effective, decreasing the number of PRV copies per milligram of tissue by about 16 times compared to the unvaccinated control. Standard curves for protein quantification by ELISA

[0102] Since pure PRV proteins were available, standard curves were made for the viral proteins σ1, σ2, σ3, λ1, λ2 and μ1, the data are shown below:

[0103] Table 3. Standard curve data for recombinant protein σ1

[0104] Table 4. Standard curve data for recombinant σ2 protein

[0105] Table 5. Standard curve data for recombinant σ3 protein Table 6. Standard curve data for recombinant protein λ1 Table 7. Standard curve data λ2

Claims

MODIFIED CLAIMS received by the International Bureau on July 12, 2025 1. Recombinant baculoviruses having the capacity to contain each ORF and produce one of the following 6 recombinant antigenic proteins, useful for treating or preventing Piscine Orthoreovirus (PRV) infections, preferably Piscine Orthoreovirus genotype 1 (PRV-1) in gamefish: a) recombinant antigenic protein of amino acid sequence SEQ ID No.:1, b) recombinant antigenic protein of amino acid sequence SEQ ID No.:2, c) recombinant antigenic protein of amino acid sequence SEQ ID No.:3, d) recombinant antigenic protein of amino acid sequence SEQ ID No.:10, e) recombinant antigenic protein of amino acid sequence SEQ ID No.:11 f) recombinant antigenic protein of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same functionality and more than 90% identity with respect to one of the sequences described in a), b), c), d), e) or f).

2. Recombinant baculovirus of claim 1 comprising one of the following nucleic acid molecules with the following sequences: a) nucleic acid molecule of nucleic acid sequence SEQ ID No 13 encoding the recombinant antigenic protein of amino acid sequence SEQ ID No.:1, b) nucleic acid molecule of nucleic acid sequence SEQ ID No. 14 encoding the recombinant antigenic protein of amino acid sequence SEQ ID No.:2, c) nucleic acid molecule of nucleic acid sequence SEQ ID No. 15 encoding the recombinant antigenic protein of amino acid sequence SEQ ID No.:3, d) nucleic acid molecule of nucleic acid sequence SEQ ID No. 18 encoding the recombinant antigenic protein of amino acid sequence SEQ ID No.:10, e) nucleic acid molecule of nucleic acid sequence SEQ ID No. 17 encoding the recombinant antigenic protein of amino acid sequence SEQ ID No.:11 f) nucleic acid molecule of nucleic acid sequence SEQ ID No. 19 encoding the recombinant antigenic protein of amino acid sequence SEQ ID No.: 16, or variants thereof, having the same functionality and more than 90% identity with respect to one of the sequences described in a), b), c), d), e) or f).

3. The recombinant baculovirus of any of claims 1 or 2 selected from one of the following 6 baculoviruses: i) a recombinant baculovirus with accession number LMBP14885CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein o1 of amino acid sequence SEQ ID No.: 1, or a vahante of the same, having the same functionality and more than 90% sequence identity; (i) a recombinant baculovirus with accession number LMBP14888CB of 29 August 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 02 of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, (iii) a recombinant baculovirus with accession number LMBP14889CB of 29 August 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 03 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, iv) a recombinant baculovirus is the transformed baculovirus of accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein O3 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, v) recombinant baculovirus of accession number LMBP14886CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein X1 of amino acid sequence SEQ ID No.: : 10, or a variant thereof, having the same functionality and more than 90% sequence identity, vi) a recombinant baculovirus with accession number LMBP14884CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein p1 of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity.

4. Host cell having the capacity to produce recombinant antigenic proteins useful for treating or preventing Piscine Orthoreovirus (PRV) infections, preferably Piscine Orthoreovirus genotype 1 (PRV-1) in gamefish, wherein said host cell is infected with a recombinant baculovirus having the capacity to produce one of the following 6 recombinant antigenic proteins: a) recombinant antigenic protein of amino acid sequence SEQ ID No.:1, b) recombinant antigenic protein of amino acid sequence SEQ ID No.:2, c) recombinant antigenic protein of amino acid sequence SEQ ID No.:3, d) recombinant antigenic protein of amino acid sequence SEQ ID No.:10, e) recombinant antigenic protein of amino acid sequence SEQ ID No.:11 or f) recombinant antigenic protein of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% identity with one of the sequences of a), b), c), d), e) or f), and where said recombinant baculovirus is selected from: i) a recombinant baculovirus with registration number LMBP14885CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce recombinant protein o1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity; (i) a recombinant baculovirus with accession number LMBP14888CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 02 of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, (iii) a recombinant baculovirus with accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 03 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, (iv) a recombinant baculovirus with accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the ability to produce the recombinant protein o3 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, v) recombinant baculovirus of accession number LMBP14886CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein X1 of amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same functionality and more than 90% sequence identity, vi) a recombinant baculovirus of accession number LMBP14884CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein p1 of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity.

5. The host cell of claim 4 wherein said host cell is selected from one of insect cells, yeast cells, plant cells or mammalian cells.

6. The host cell of claim 5 wherein said host cell is an insect cell.

7. The host cell of claim 6 wherein said insect cell is the sf9 cell.

8. Virus-like particle (VLP) vaccine composition for preventing or treating PRV in gamefish comprising host cell lysate, wherein said host cell comprises the following 6 recombinant baculoviruses: recombinant baculovirus registration number LMBP14886CB dated August 29, 2023., ¡i) recombinant baculovirus registration number LMBP14883CB dated August 29, 2023, iü) recombinant baculovirus registration number LMBP14884CB dated August 29, 2023, V) recombinant baculovirus registration number LMBP14885CB dated August 29, 2023, V) recombinant baculovirus registration number LMBP14888CB dated August 29, 2023, v¡) recombinant baculovirus registration number LMBP14889CB dated August 29, 2023 and veterinarily acceptable excipients, and where said recombinant baculoviruses having the capacity to produce one of the following 6 recombinant antigenic proteins: a) recombinant antigenic protein of amino acid sequence SEQ ID No.: 1, b) recombinant antigenic protein of amino acid sequence SEQ ID No.: 2, c) recombinant antigenic protein of amino acid sequence SEQ ID No.: 3, d) recombinant antigenic protein of amino acid sequence SEQ ID No.: 10, e) recombinant antigenic protein of amino acid sequence SEQ ID No.: 11 or f) recombinant antigenic protein of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same functionality and more of 90% sequence identity with one of the sequences a), b), c), d), e) or f).

9. The virus-like particle (VLP) vaccine composition of claim 8 further comprising one or more veterinarily acceptable adjuvants.

10. The virus-like particle (VLP) vaccine composition of claim 8 wherein said one or more veterinarily acceptable adjuvants are selected from one or more of Montanide®, Alumina, water / oil (WO) emulsion, water / oil / water (WOW) emulsion, Freund's complete adjuvant, Freund's incomplete adjuvant, saponins, dimethyl dioctadecyl amino bromide (DDA).

11. The virus-like particle (VLP) vaccine composition of claim 8 wherein said host cell lysate is insect cell lysate.

12. The virus-like particle (VLP) vaccine composition of claim 11 wherein said insect cell lysate is sf9 cell lysate.

13. The virus-like particle (VLP) vaccine composition of any of claims 8 to 12, wherein said composition is a formulation for administration intraperitoneally, by bathing or immersion, orally in food, cutaneously, or by administration through the anal pore.

14. The virus-like particle (VLP) vaccine composition of any of claims 8 to 13 wherein said vaccine composition is a vaccine composition formulated for water-in-oil or oil-in-water emulsion or preparative variations thereof, or of nanoparticles or microparticles that coat or nucleate the antigen, or that coat the nano or micro structures.

15. The virus-like particle (VLP) vaccine composition of claim 10 comprising 70% v / v of the Montanide® adjuvant and each of the following 6 recombinant antigenic proteins; a) recombinant antigenic protein o1 of amino acid sequence SEQ ID No.: 1, b) recombinant antigenic protein o2 of amino acid sequence SEQ ID No.: 2, c) recombinant antigenic protein o3 of amino acid sequence SEQ ID No.: 3, d) recombinant antigenic protein X1 of amino acid sequence SEQ ID No.: 10, e) recombinant antigenic protein X2 of amino acid sequence SEQ ID No.: 11, and f) recombinant antigenic protein p.1 of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same antigen functionality with high similarity to PRV wild type, and more than 90% sequence identity similarity with one of the sequences a), b), c), d), e) or f),, in an amount in the range of 0.5 pg to 1.5 pg, per dose.

16. A method for treating or preventing a PRV infection in gamefish, comprising administering to said gamefish a virus-like particle (VLP) vaccine composition comprising the following 6 recombinant antigenic proteins: a) recombinant antigenic protein of amino acid sequence SEQ ID No.:1, b) recombinant antigenic protein of amino acid sequence SEQ ID No.:2, c) recombinant antigenic protein of amino acid sequence SEQ ID No.:3, d) recombinant antigenic protein of amino acid sequence SEQ ID No.: 10, e) recombinant antigenic protein of amino acid sequence SEQ ID No.: 11 and f) recombinant antigenic protein of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same functionality and more than 90% sequence identity with one of the sequences of a), b), c), d), e) or f), where said 6 recombinant antigenic proteins are produced by one of the following recombinant baculoviruses having the capacity to produce one of said 6 recombinant antigenic proteins: i) a recombinant baculovirus with accession number LMBP14885CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce recombinant protein 01 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than one 90% sequence identity; (i) a recombinant baculovirus with accession number LMBP14888CB of 29 August 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 02 of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, (iii) a recombinant baculovirus with accession number LMBP14889CB of 29 August 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 03 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, iv) a recombinant baculovirus is the transformed baculovirus of accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein O3 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, v) recombinant baculovirus of accession number LMBP14886CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein X1 of amino acid sequence SEQ ID No.: : 10, or a variant thereof, having the same functionality and more than 90% sequence identity, vi) a recombinant baculovirus with accession number LMBP14884CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein p1 of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity.

17. A method for treating or preventing skeletal and cardiac muscle inflammation (HSMI) caused by PRV-1 infection in gamefish, comprising administering to said gamefish a virus-like particle (VLP) vaccine composition comprising the following 6 recombinant antigenic proteins: a) recombinant antigenic protein of amino acid sequence SEQ ID No.:1, b) recombinant antigenic protein of amino acid sequence SEQ ID No.:2, c) recombinant antigenic protein of amino acid sequence SEQ ID No.:3, d) recombinant antigenic protein of amino acid sequence SEQ ID No.:10, e) recombinant antigenic protein of amino acid sequence SEQ ID No.:11 and f) recombinant antigenic protein of amino acid sequence SEQ ID No.: 12, or variants thereof, having the same functionality and more than 90% sequence identity with one of the sequences of a), b), c), d), e) or f), and where said 6 recombinant antigenic proteins are produced by one of the following recombinant baculoviruses having the capacity to produce one of said 6 recombinant antigenic proteins: i) a recombinant baculovirus with registration number LMBP14885CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce recombinant protein o1 of amino acid sequence SEQ ID No.: 1, or a variant thereof, having the same functionality and more than 90% sequence identity;. (i) a recombinant baculovirus with accession number LMBP14888CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 02 of amino acid sequence SEQ ID No.: 2, or a variant thereof, having the same functionality and more than 90% sequence identity, (iii) a recombinant baculovirus with accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein 03 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity, (iv) a recombinant baculovirus with accession number LMBP14889CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the ability to produce the recombinant protein o3 of amino acid sequence SEQ ID No.: 3, or a variant thereof, having the same functionality and more than 90% sequence identity. (v) recombinant baculovirus of accession number LMBP14886CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein X1 of amino acid sequence SEQ ID No.: 10, or a variant thereof, having the same functionality and more than 90% sequence identity, (vi) a recombinant baculovirus of accession number LMBP14884CB of August 29, 2023 of the Belgian Coordinated Collections of Microorganisms (BCCM), having the capacity to produce the recombinant protein p.1 of amino acid sequence SEQ ID No.: 12, or a variant thereof, having the same functionality and more than 90% sequence identity.

18. The method of any of claims 16 or 17 wherein said farmed fish are selected from salmonids such as salmon or trout.

19. The method of any of claims 16 or 17 wherein said salmon are selected from Coho Salmon (Oncorhynchus kisutch), Atlantic Salmon (Salmo salar") and Rainbow Trout (Oncorhynchus mykiss).

20. Use of the virus-like particle (VLP) vaccine composition of any of claims 8 to 15 for preparing a medicament useful for treating or preventing a PRV infection in minnows.

21. Use of the virus-like particle (VLP) vaccine composition of any of claims 8 to 15 for preparing a virus-like particle (VLP) vaccine useful for treating or preventing a PRV-1 infection in gamefish.

22. Use of the virus-like particle (VLP) vaccine composition of any of claims 8 to 15 because it serves to prepare a virus-like particle (VLP) vaccine useful for treating or preventing skeletal and cardiac muscle inflammation (HSMI) caused by PRV-1 infection in farmed fish.

23. The use of any of claims 20 to 22 wherein said game fish are selected from salmonids such as salmon or trout.

24. The use of claim 23 wherein said salmons are selected from Coho Salmon (Oncorhynchus kisutch), Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss).

25. Method for preparing a vaccine composition of virus-like particles (VLPs) of Piscine Orthoreovirus genotype 1 (PRV-1) that assemble the 6 recombinant proteins with a structure like those of the internal and external capsid of PRV-1, comprising the following steps: a) incorporating nucleic acid molecules that express each of the following 6 recombinant antigenic proteins of PRV: a.1) recombinant antigenic protein o1 of amino acid sequence SEQ ID No.: 1, a.2) recombinant antigenic protein o2 of amino acid sequence, SEQ ID No.: 2, a.3) recombinant antigenic protein o3 of amino acid sequence SEQ ID No.: 3, a.4) recombinant antigenic protein X1 of amino acid sequence SEQ ID No.: 10, a.5) recombinant antigenic protein X2 of amino acid sequence SEQ ID No.: 11, a.6) recombinant antigenic protein p.1 of amino acid sequence SEQ ID No.: 12, a.7) recombinant antigenic protein p.2 of amino acid sequence SEQ ID No.: 13, a.8) recombinant antigenic protein p.3 of amino acid sequence SEQ ID No.: 14, a.9) recombinant antigenic protein p.4 of amino acid sequence SEQ ID No.: 15, a.10) recombinant antigenic protein p.5 of amino acid sequence SEQ ID No.: 16, a.11) recombinant antigenic protein p.6 of amino acid sequence SEQ ID No.: 17, a.12) recombinant antigenic protein p.7 of amino acid sequence SEQ ID No.: 18, a.13) recombinant antigenic protein p.8 of amino acid sequence SEQ ID No.: 19, a.14) recombinant antigenic protein p.9 of amino acid sequence SEQ ID No.: 20, a.15) recombinant antigenic protein p.10 of amino acid sequence SEQ ID No.: 21, a.16) recombinant antigenic protein p.11 of amino acid sequence SEQ ID No.: 22, a.17) recombinant antigenic protein p.12 of amino acid sequence of amino acids SEQ ID No.: 12 or a variant thereof, having the same antigen functionality with high similarity to PRV wild type, and more than 90% sequence identity similarity with one of the sequences of a.1), a.2), a.3), a.4), a.5) and .a.6), in a recombinant viral vector is a baculovirus vector; b) obtaining a transformed microorganism is a baculovirus selected from: b.1) baculovirus registration LMBP14886CB dated August 29, 2023, b.2) baculovirus registration LMBP14883CB dated August 29, 2023, b.3) baculovirus registration LMBP14884CB dated August 29, 2023., b.4) baculovirus registration LMBP14885CB dated August 29, 2023, b.5) baculovirus registration LMBP14888CB dated August 29, 2023, and b.6) baculovirus registration LMBP14889CB of August 29, 2023, and c) infecting host cells with the transformed microorganism obtained in step b) for the replication of said recombinant viral vector, where said host cell is selected from one of: insect cells, yeast cells, plant cells, mammalian cells.

26. The method of claim 25 wherein in step c), said host cell is an insect cell, including an sf9 insect cell.

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