Sedoreoviridae vaccines based on truncated VP2 capsid proteins

Abstract

The present technology relates to vaccines for the prevention or treatment of diseases resulting from an infection with a virus belonging to the Reoviridae family as well as their manufacture. The virus may belong to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Equine Encephalosis virus (EEV), and Bluetongue virus (BTV).

Description

[0001] Sedoreoviridae vaccines based on truncated VP2 capsid proteins

[0002] FIELD OF THE TECHNOLOGY

[0003] The present technology relates to vaccines for the prevention or treatment of diseases resulting from an infection with a virus belonging to the Reoviridae family as well as their manufacture. The virus may belong to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Equine Encephalosis virus (EEV), and Bluetongue virus (BTV).

[0004] BACKGROUND OF THE TECHNOLOGY VP2 is a key structural protein of viruses in the Orbivirus genus, part of the Reoviridae family (which can also be referred to as Sedoreoviridae family). Notably found in viruses such as Epizootic Hemorrhagic Disease Virus (EHDV) and Bluetongue Virus (BTV), VP2 plays a critical role in viral assembly and host cell entry. This protein forms the outer capsid layer, facilitating protection of the viral genome and mediating interactions with host cells. Its antigenic properties also make it a target for vaccine development and serological diagnostics, highlighting its significance in managing viral infections in livestock and wildlife.

[0005] To date, seven different serotypes of EHDV have been identified, which have been detected in North and South America, Africa, Asia, Oceania, southern Europe, and the Middle East. The serotype nomenclature ranges from 1 to 2 and from 4 to 8, as genetic studies have confirmed that serotype 3, originally described in Nigeria, was actually serotype 1. This notifiable pathogen has a significant range of hosts, being capable of infecting various species of domestic ungulates, primarily cattle, in which most outbreaks have been observed. However, it has also been noted that the virus can infect sheep, goats, pigs, alpacas, and yaks. Among its wild hosts, deer are the most affected, although it also affects other ruminant species, many of which are threatened to varying degrees, and thus the impact of this disease on wild populations could have significant repercussions.

[0006] Regarding the countries of the Mediterranean basin, before the year 2000, there were few reports on the presence of EHDV. The first report dates to 1951 when a disease similar to Blue Tongue (BT) was described in cattle and sheep in Israel. The situation of EHDV in the Mediterranean changed after the year 2000. In 2001, clinical cases were described in cattle in Israel, identifying EHDV as the cause of the outbreak, but no information was available on the serotype. In 2004, an outbreak caused by EHDV-6 (strain 318) was reported in Morocco. The same serotype was responsible for another outbreak in the same country two years later (OIE, 2006a). That same year, in 2006, EHDV-6 caused outbreaks in Algeria and Tunisia and a year later in Turkey (OIE, 2006b; OIE, 2006c). Also in 2006, clinical cases were described in Israeli cattle (OIE, 2006d). The first affected herd was reported on August 28, 2006, and the virus spread from this herd southward towards the Dead Sea, and northward along the Jordan Valley. Overall, the disease was reported in 83 dairy herds located in 78 rural localities and in 22 beef herds, most during the first 8 weeks and some more until the third week of November. Unlike other outbreaks in the Mediterranean, EHDV-7 was identified as the responsible serotype. That same year suspected clinical cases of EHDV were described in Jordan. Therefore, between 2006 and 2007, outbreaks caused by EHDV infection were reported in Algeria, Tunisia, Morocco, Israel, Jordan, and Turkey. Two different serotypes were involved, serotype 6 (EHDV 318) and serotype 7. Both isolates were pathogenic and capable of causing disease in cattle. There was no information available on the possible origin of the two strains. In the late 1980s, EHDV-6 was identified in Sudan, Bahrain, and Oman, and it is likely that this serotype remained in the region until the recent outbreaks. In contrast, the only place where EHDV-7 was evidenced besides Israel and Jordan is Australia. Currently, EHD is considered endemic in the United States, Australia, and certain regions of Africa and Asia. However, Europe remained free of EHD until the first cases of EHDV-8 were observed in Sicily, Sardinia, and southern Spain in 2022.

[0007] Like BTV, the transmission of EHDV is primarily through vectors of the genus Culicoides, biting midges that are very present in tropical and temperate regions around the world. Additionally, this type of transmission results in a marked seasonality of EHD cases, with the peak prevalence observed between July and October (EFSA, 2009). However, the global increase in temperatures has influenced the activity of these insects, extending the period of mosquito presence throughout the year, thus affecting seasonality, and favoring the spread of Culicoides carrying the virus to geographic regions with traditionally colder climates such as Canada or the northern United States, and they could even reach Nordic regions. On the other hand, some studies demonstrate the capability of EHDV-1 transmission via oral and fecal routes in white-tailed deerorthrough direct contact in the absence of vectors, which can be concerning in cattle as the herd size increases the risk of pathogen transmission through these routes, although it is not possible to determine the epidemiological relevance of these other infection routes. Transplacental transmission was demonstrated by isolating EHDV-2 (Ibaraki) from internal organs of aborted fetuses. No reports of EHDV isolation in semen were found.

[0008] It is believed that the origin of the EHDV-8 cases in Spain came from Tunisia, where the presence of the same serotype had already been reported, which had not been isolated since 1982 when it was described in Australia. As the EFSA warned in its 2009 report, the spread of the virus through arthropod vectors in air masses from Africa is the most likely main entry route of the virus to Europe, as was the case with the arrival of BTV-2 in Sardinia. Thus, on November 10, 2022, the first detection of the virus in Europe (Sardinia) was reported, and only 8 days later, on November 18, 2022, the first cases in Spain were detected in cattle, in the provinces of Cadiz (Campo de Gibraltar municipality) and Seville (El Pedroso municipality). It was in this same locality in Seville where the first case of this disease in deer (in Spain) was diagnosed on June 23, 2023. Since then, outbreaks have been reported in Andalusia and Extremadura, reaching the north of the country in early September 2023 (according to Agricultural Ministry of Spain data).

[0009] Given the socioeconomic repercussions that EHD could potentially have in Europe, it is imperative to establish a series of measures to help control the transmission and spread of the virus. Among all possible control measures, once the disease is introduced in Spain, the implementation of vaccination campaigns against EHDV would be recommended. In Japan, two vaccines have been developed and commercialized: one monovalent attenuated (EHDV-2) and another bivalent inactivated (EHDV-2 and bovine ephemeral fever virus). In the United States, where EHDV-1 and -6 are endemic, autogenous inactivated vaccines are often used for disease control in white-tailed deer breeders, although there is no data on their effectiveness (EFSA, 2009). Currently, the efficacy of a subunit vaccine against EHDV-2 capsid proteins is being studied. Preliminary results in mice and cattle are promising with very wide safety margins (Sunwoo et al., 2020). Finally, a team of researchers from the United Kingdom has developed a subunit vaccine against various capsid proteins, conferring immunity to inoculated individuals against EHDV-1, -2, and -6 (Alshaikhahmed and Roy, 2016). What is clear is that if EHD continues to progress through Europe, a DIVA vaccine, effective against multiple serotypes and safe, will be necessary, allowing differentiation between naturally infected and vaccinated individuals.

[0010] Although a vaccine against EHDV is available in Spain, this is based on inactivated virus. For this reason, it would be impossible to determine the difference between vaccinated and infected animals, which complicates epidemiological studies and eradication efforts, as well as preventing the export of live cattle and possibly meat products to disease-free countries. Therefore, it would be necessary the development of a commercial subunit vaccine based on a single protein, combined with a diagnostic method based on other structural immunogenic protein of the virus like VP7 (there are commercial diagnostic tests based on this protein)

[0011] SUMMARY OF THE TECHNOLOGY

[0012] The present technology addresses the above needs and provides a fusion protein comprising: (a) a truncated VP2 capsid protein, wherein the truncated VP2 capsid protein is derived from a VP2 capsid of a virus belonging to the Reoviridae family (also referred as Sedoreoviridae family); and

[0013] (b) a trimerization domain, wherein the trimerization domain is a continuous domain.

[0014] Hence, the present technology provides a fusion protein comprising:

[0015] (a) a truncated VP2 capsid protein, wherein the truncated VP2 capsid protein is of a virus belonging to the Reoviridae family; and

[0016] (b) a trimerization domain, wherein the trimerization domain is a continuous domain.

[0017] The present invention further provides a nucleic acid sequence encoding the fusion protein.

[0018] Further, the invention provides a pharmaceutical composition comprising the fusion protein, or the nucleic acid sequence.

[0019] The invention further provides a vaccine comprising the fusion protein, or the nucleic acid sequence. BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1. 3D predicted structure comparison between BTV and EHDV viruses and their respective sequences defining the different protein domains (Hub domain in grey; Hairpin domain in italic; Antigenic domain in bold; Body domain underlined)

[0021] Figure 2. 3D predicted structure of the wild-type VP2 protein of EHDV (labeled " Full-length VP2 EHDV-8") and the fusion protein of the present invention (labeled " VP2t EHDV-8 Mod"). The trimerization domain includes a GGGS linker at the C-terminus followed by a polyhistidine tag.

[0022] Figure 3. PCR gels of Bacmids bands. (A) Bac VP2-EHDV-8 wells: 1 MW, 2 Bac VP2-EHDV-8 PCR1, 3 Negative control PCR1, 4 Empty well, 5 MW, 6 Bac VP2-EHDV-8 PCR2, 7 Negative control PCR2, 8 Empty well. (B) Bac VP2t-EHDV-8 Mod wells: 1 MW, 2 Bac VP2 t-EHDV-8 Mod PCR1, 3 Negative control PCR1, 4 Empty well, 5 MW, 6 Bac VP2 t-EHDV-8 Mod PCR2, 7 Negative control PCR2, 8 Empty well.

[0023] Figure 4. Analysis by PCR of viral passages. (A) Baculovirus VP2-EHDV-8 pl. (B) Baculovirus VP2t-EHDV-8-FD4-His pl.

[0024] Figure 5. Protein expression in sf9-RVN cells SDS-PAGE analysis. (A) VP2-EHDV-8 and VP2t-EHDV-8 Mod proteins expression in insect cells analyzed by Coomassie blue staining. (B) Detection by Western blot of proteins VP2-EHDV-8 and VP2t-EHDV-8 Mod expressed in insect cells using an anti-Histidine monoclonal antibody. Red arrows indicate the recombinant protein.

[0025] Figure 6. Protein expression detected in infected pupae extracts. (A) Protein VP2-EHDV-8 expression analyzed by SDS gels and Coomassie blue staining. (B) Protein VP2t-EHDV-8 Mod expression analyzed by SDS gels and Coomassie blue staining. Red arrows indicate the recombinant protein. Figure 7. Purification of pupae-derived recombinant VP2 proteins and calculation of yields of purified full-length and modified protein versions obtained per liter of pupae extract. Proteins were quantified by densitometry of the gels with a BSA curve.

[0026] Figure 8. FPLC-SEC analysis supporting the presence of trimeric structures in both VP2 molecules expressed. Trimers are indicated as expected trimer elution according to column calibration.

[0027] Figure 9. Recognition of the pupae-derived VP2 proteins by sera from naturally infected bovines with EHDV or BTV. Sera from BTV infected animals were unable to recognize by ELISA the EHDVVP2 protein.

[0028] Figure 10. Quantification of neutralizing antibodies generated after mice immunization at 28 days post prime, 14 days post boost and 18 days post challenge.

[0029] Figure 11. Percentage of mice survival after EHDV-8 challenge.

[0030] Figure 12. (A) RNAemia in mice at different days after challenge. (B) Presence of viremia in mice at different days after challenge.

[0031] Figure 13. Protein expression in sf9-RVN cells SDS-PAGE analysis. (A) Coomassie blue stain. Wells: 1 MW, 2 VP2-BTV3 protein, 3 VP2t-BTV3 Mod protein and 4 Empty well. (B) Detection by Western blot using an anti-Histidine monoclonal antibody. Wells: 1 MW, 2 VP2-BTV3 protein, 3 VP2t-BTV3 Mod protein and 4 Empty well. Arrows indicate recombinant protein.

[0032] Figure 14. Protein solubility in sf9-RVN cells SDS-PAGE analysis. (A) Coomassie blue stain. Wells: 1 MW, 2 Total extract VP2-BTV3 protein, 3 Soluble extract VP2-BTV3 protein 4 Insoluble extract VP2-BTV3 protein, 5 Negative protein, 6 Total extract VP2t-BTV3 Mod protein, 7 Soluble extract VP2t-BTV3 Mod protein 8 Insoluble extract VP2t-BTV3 Mod protein (B) Detection by Western blot using an anti-Histidine monoclonal antibody. Wells:: 1 MW, 2 Total extract VP2-BTV3 protein, 3 Soluble extract VP2-BTV3 protein 4 Insoluble extract VP2-BTV3 protein, 5 Negative protein, 6 Total extract VP2t-BTV3 Mod protein, 7 Soluble extract VP2t-BTV3 Mod protein 8 Insoluble extract VP2t-BTV3 Mod protein. Arrows indicate soluble fraction of both recombinant proteins.

[0033] Figure 15. Purification process of VP2-Mod. SDS-PAGE with Coomassie blue stain. TF Total extract; SF soluble extract; PF insoluble extract, Input fraction loaded into IMAC resin. FT nonbound fraction into resin; Emix elution from IMAC resin.

[0034] Figure 16. Experimental in vivo design and results. Panel A. Mice vaccination protocol. Panel B. Sera neutralization assay. Panel C. Survival proportions. Panel D. RNAemia levels analysis. Panel E. Viremia levels analysis. The " ALGENEX" group was vaccinated with VP2-Mod protein.

[0035] DESCRIPTION

[0036] The inventors have identified specific domains within the VP2 capsid protein of a virus belonging to the Reoviridae family (also referred as Sedoreoviridae family) which are not critical for the immunogenicity of the VP2 capsid protein. Advantageously, when these domains are deleted and / or replaced with a recombinant trimerization domain, production yields of the fusion protein are significantly increased, and the protein also acquires the trimerization conformation needed to induce neutralizing antibodies. Specifically, the inventors have identified that the deletion of the hairpin domain of the VP2 protein and the replacement of this discontinuous trimerization domain with a continuous trimerization domain result in increased production yields of the fusion protein. Preferably, the continuous trimerization domain is smaller than the discontinuous trimerization domain of the wild-type VP2 capsid protein.

[0037] Definitions

[0038] All terms as used herein, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the description and claims unless an otherwise expressly set out definition provides a broader definition. Throughout the description and claims the term "comprise" and variations of the term (e.g., "comprising", "having", "including", "containing"), are not intended to exclude othertechnical features, additives, components, or steps. Furthermore, the term "comprise" and variations of the term encompasses the case of "consisting of". Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

[0039] In this specification and claims, the use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0040] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0041] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0042] If the term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. For instance, the term "about" means the indicated value ± 1% of its value, or the term "about" means the indicated value ± 2% of its value, or the term "about" means the indicated value ± 5% of its value, the term "about" means the indicated value ± 10% of its value, or the term "about" means the indicated value ± 20% of its value, or the term "about" means the indicated value ± 30% of its value; preferably the term "about" means exactly the indicated value (± 0%). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Units, prefixes, and symbols are denoted in their " Systeme International de Unites" (SI) accepted form.

[0043] Numeric ranges are inclusive of the numbers defining the range.

[0044] Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited by specifying the full literature reference in the text, or by naming the author and the publication year (e.g., " Zhang etal. 2016") ora reference number and by specifying the corresponding full literature reference in the " References" section.

[0045] The body domain (which may be comprised in the truncated VP2 capsid protein of the present technology) may comprise both a-helices and p-sheets. It is a globular region with surface exposed to dissolvent. The body domain forms the bulk of the protein and is involved in the structural stability of the VP2 capsid protein of a virus belonging to the Reoviridae family (also referred as Sedoreoviridae family). The body domain corresponds to positions 163-190 and 408-849 of SEQ ID NO: 1. In some embodiments, the body domain additionally includes the residue corresponding to position 162 of SEQ ID NO: 1 (i.e., the body domain corresponds to positions 162-190 and 408-849 of SEQ ID NO: 1).

[0046] The antigenic domain comprises serotype-specific epitopes and is responsible for binding to receptors. The antigenic domain corresponds to positions 191-407 of SEQ ID NO: 1. Preferably, the body domain and the antigenic domain both belong to the same virus belonging to the Reoviridae family.

[0047] The trimerization domain comprised in the fusion protein of the present technology may comprise p-barrel motifs in parallel and antiparallel conformation and may comprise two a- helices. The trimerization domain is involved in the formation of trimers. In the context of immunogenic proteins, the trimerization domain is believed to be necessary for the correct conformation of the protein to induce neutralizing antibodies. In the context of the invention, the term "discontinuous trimerization domain" will be understood as referring to a trimerization domain in which the nucleic acid encoding sequence for the trimerization domain is spread across different regions of the coding sequence, possibly interrupted by nucleic acid stretches encoding for other domains. In the wild-type VP2 capsid protein, the amino acid sequence encoding for the trimerization domain is interrupted by the amino acid sequence encoding for a hairpin domain and for a body domain comprising an antigenic domain. The terms "continuous trimerization domain" will be understood as a trimerization domain in which the amino acid sequence encoding for the trimerization domain is uninterrupted. This trimerization domain may include at the C-terminus end a linker for increase accessibility of a potential tag incorporated to the molecule that facilitate the purification of the fusion protein.

[0048] A hairpin domain generally extends from the body domain and may be between trimerization domain and body domain. The hairpin domain may comprise two a-helices joined by two |3-sheets. The fusion protein according to the present technology does not comprise a hairpin domain.

[0049] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure. Furthermore, the present technology covers all possible combinations of particular aspects and embodiments described herein.

[0050] The figures and the experimental part / examples are only given to further illustrate the technology and should not be interpreted or construed as limiting the scope of the technology and / or of the appended claims in any way, unless explicitly indicated otherwise herein.

[0051] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the technology. Modifications and variation of the above-described embodiments of the technology are possible without departing from the technology, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the technology may be practiced otherwise than as specifically described.

[0052] The fusion protein of the present technology

[0053] In a first aspect, the present technology provides a fusion protein comprising (i) a truncated VP2 capsid protein, and (ii) a continuous trimerization domain. In some embodiments, the truncated VP2 capsid protein consists of an antigenic domain and a body domain.

[0054] In one embodiment according to the technology, the amino acid sequence of the fusion protein comprises or consists of, from / V-terminus to C-terminus:

[0055] (i) optionally, a purification tag (can be fused via a peptide linkertothe / V-terminus of the truncated VP2 capsid protein);

[0056] (ii) the truncated VP2 capsid protein;

[0057] (iii) a peptide linker;

[0058] (iv) the continuous trimerization domain; and

[0059] (v) optionally, a purification tag (can be fused via a peptide linkertothe C-terminus of the continuous trimerization domain).

[0060] In one embodiment according to the technology, the amino acid sequence of the fusion protein consists of or consists essentially of, from / V-terminus to C-terminus:

[0061] (i) a purification tag (can be fused via a peptide linker to the / V-terminus of the truncated VP2 capsid protein);

[0062] (ii) the truncated VP2 capsid protein;

[0063] (iii) a peptide linker; and

[0064] (iv) the trimerization domain.

[0065] In one embodiment according to the technology, the amino acid sequence of the fusion protein consists of or consists essentially of, from / V-terminus to C-terminus:

[0066] (i) the truncated VP2 capsid protein;

[0067] (ii) a peptide linker;

[0068] (iii) the trimerization domain; and (iv) a purification tag (can be fused via a peptide linker to the C-terminus of the continuous trimerization domain).

[0069] In one embodiment according to the technology, the amino acid sequence of the fusion protein consists of or consists essentially of, from / V-terminus to C-terminus:

[0070] (i) the truncated VP2 capsid protein;

[0071] (ii) a peptide linker; and

[0072] (iii) the trimerization domain, wherein, optionally, the fusion protein may be fused to other peptide sequences at the / V-terminus and / or C-terminus.

[0073] The truncated VP2 capsid protein comprised in the fusion protein of the present technology was originally a VP2 capsid protein from a virus belonging to the Reoviridae family (also referred as Sedoreoviridae family) which has been modified to lack the hairpin domain and discontinuous trimerization domain (i.e., the hub domain). Preferably, the VP2 capsid protein is from a virus belonging to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), Bluetongue virus (BTV), Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV) and Mitchell River virus (MRV). Preferably, the virus belongs to the Orbivirus genus and is selected from Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and Bluetongue virus (BTV).

[0074] The term "truncated" will be understood as a polypeptide that has been shortened through the deletion of one or more amino acids from its / V-terminus and / or C-terminus, or from any part of its amino acid sequence. In some embodiments, the amino acid sequence of the truncated VP2 capsid protein consists of an amino acid sequence corresponding to positions 163-849 of SEQ ID NO: 1. For example, the amino acid sequence of the truncated VP2 capsid protein may consist of residues 163-849 of SEQ ID NO: 1 or residues 163-849 of the BTV-serotype 3 VP2 capsid protein depicted in Figure 1. In some embodiments, the truncated VP2 capsid protein consists of an antigenic domain and a body domain. Other domains of the VP2 capsid protein, such as the hairpin domain and the hub domain (i.e., the native discontinuous trimerization domain), have been deleted. A fusion protein comprising the truncated VP2 capsid protein will not comprise the hairpin or hub domain of the wild-type VP2 capsid protein. In addition to the amino acid sequence corresponding to positions 163-849 of SEQ ID NO: 1, the amino acid residue corresponding to position 162 of SEQ ID NO: 1 may be included in the fusion protein. In other words, in some embodiments, the amino acid sequence of the truncated VP2 capsid protein consists of an amino acid sequence corresponding to positions 162-849 of SEQ ID NO: 1. In some embodiments, at least the residues corresponding to positions 1-161 and 850-970 of SEQ ID NO: 1 have been removed.

[0075] In the context of the technology, the truncated VP2 capsid protein is derived from a full-length VP2 capsid protein of a virus belonging to the Reoviridae family, preferably of a virus belonging to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and Bluetongue virus (BTV). Hence, the truncated VP2 capsid protein comprised in the fusion protein of the present technology was originally a VP2 capsid protein from a virus belonging to the Reoviridae family (also referred as Sedoreoviridae family) which hairpin domain and trimerization domain have been deleted, as described herein. Preferably, the VP2 capsid protein is from a virus belonging to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), Bluetongue virus (BTV), Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV) and Mitchell River virus (MRV). Preferably, the virus belongs to the Orbivirus genus and is selected from Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and Bluetongue virus (BTV). Even more preferably the virus is EHDV or BTV. In an even more preferred embodiment, the virus is EHDV serotype 8 or BTV serotype 3.

[0076] In one embodiment, the body domain comprised in the fusion protein of the present technology corresponds to the body domain of the VP2 capsid protein of a virus belonging to the Reoviridae family, as described herein, with the difference that the first amino acid of the body domain comprised in the fusion protein of the present technology is a methionine. In embodiments where a purification tag (and optionally a peptide linker connecting the purification tag to the N-terminus of the truncated VP2 capsid protein) is included at the N- terminus, the purification tag and / or peptide linker may be inserted between the N-terminal methionine and the rest of the coding sequence.

[0077] SEQ ID NO: 6 and SEQ ID NO: 24 have 24.4% sequence identity. Thus, in some embodiments, the truncated VP2 capsid protein comprised in the fusion protein of the present technology comprises or consists of a sequence as defined in SEQ ID NO.: 6 or SEQ ID NO.: 24, or a sequence with at least 20% (e.g., at least 21%, at least 22%, at least 23%, or at least 24%) sequence identity to SEQ ID NO.: 6 or SEQ ID NO.: 24.

[0078] In a preferred embodiment, the truncated VP2 capsid protein comprised in the fusion protein of the present technology comprises or consists of a sequence as defined in SEQ ID NO.: 6 or 7, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 6 or 7, or a sequence with one or more amino acid difference with SEQ ID NO.: 6 or 7, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference. Hence, in a preferred embodiment, the fusion protein of the present technology comprises a sequence as defined in SEQ ID NO.: 6 or 7, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 6 or 7, or a sequence with one or more amino acid difference with SEQ ID NO.: 6 or 7, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference.

[0079] In a preferred embodiment, the truncated VP2 capsid protein comprised in the fusion protein of the present technology comprises or consists of a sequence as defined in SEQ ID NO.: 24 or 25, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 24 or 25, or a sequence with one or more amino acid difference with SEQ ID NO.: 24 or 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference. Hence, in a preferred embodiment, the fusion protein of the present technology comprises a sequence as defined in SEQ ID NO.: 24 or 25, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 24 or 25, or a sequence with one or more amino acid difference with SEQ ID NO.: 24 or 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference. In the context of the technology, it will further be understood that the fusion protein may comprise:

[0080] An antigenic domain of a VP2 capsid protein

[0081] A body domain of a VP2 capsid protein; and

[0082] A continuous trimerization domain.

[0083] The trimerization domain may be located at the C-terminus of the truncated VP2 capsid protein. In the wild-type VP2 capsid protein, the " Hub" domain comprises a discontinuous trimerization domain.

[0084] The different VP2 domains, such as the antigenic domain, the body domain, the hairpin domain or the hub domain (trimerization domain) can be identified by assays and bioinformatics tools well-known in the field. For example, the different VP2 capsid protein domains can be identified using structure visualization software, such as Swisspdbviewer. In the context of the technology, the VP2 capsid protein from EHDV can be divided in the same four domains as described for VP2 from BTV (Bisset et al. (2024). From that approach, the following domains of a wild-type VP2 capsid protein could be determined: a discontinuous trimerization domain (e.g., amino acids 1-49; amino acids 121-162; amino acids 850-970, see SEQ ID NO.: 1 or 2); a hairpin domain (e.g., amino acids 50-120, see SEQ ID NO.: 1 or 2); a body domain (e.g., amino acids 163-190 and 408-849, see SEQ ID NO.: 1 or 2) and an antigenic domain (e.g., amino acids 191-407, see SEQ ID NO.: 1 or 2). One can also identify the corresponding positions in other VP2 capsid proteins by aligning the sequences as done in the Examples below.

[0085] In one embodiment according to the technology, the fusion protein has: a truncated VP2 capsid protein (amino acids 2-689 in SEQ ID NO.: 4); a linker, which is preferably a GS linker, (amino acids 690-699 in SEQ ID NO.: 4, see also SEQ ID NO.: 9); and continuous trimerization domain, such as a FD4 domain (amino acids 700-766 in SEQ ID NO.: 4, see also SEQ ID NO.: 8). In one embodiment according to the technology, the fusion protein has: a truncated VP2 capsid protein (amino acids 2-688 in SEQ ID NO.: 23); a linker, which is preferably a GS linker, (amino acids 689-700 in SEQ ID NO.: 23, see also SEQ ID NO.: 26); and continuous trimerization domain, such as a FD4 domain (amino acids 701-767 in SEQ ID NO.: 23, see also SEQ ID NO.: 8).

[0086] In some embodiments, the fusion protein comprises an antigenic domain and body domain of a VP2 capsid protein, and a trimerization domain, preferably linked to the body domain via a peptide linker.

[0087] The truncated VP2 capsid protein comprised in the fusion protein of the present technology preferably comprises or consists of a sequence as defined in SEQ ID NO.: 6 or as defined in SEQ ID NO.: 7, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 6 and / or with SEQ ID NO.: 7 or a sequence with one or more amino acid difference with SEQ ID NO.: 6 and / or with SEQ ID NO.: 7, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference. Alternatively, the truncated VP2 capsid protein comprised in the fusion protein of the present technology preferably comprises or consists of a sequence as defined in SEQ ID NO.: 24 or as defined in SEQ ID NO.: 25, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 24 and / or with SEQ ID NO.: 25 ora sequence with one or more amino acid difference with SEQ ID NO.: 24 and / or with SEQ ID NO.: 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference.

[0088] SEQ ID NO: 10 and SEQ ID NO: 28 have 29.4% sequence identity. Thus, in some embodiments, the antigenic domain comprised in the fusion protein of the present technology comprises or consists of a sequence as defined in SEQ ID NO.: 10 or SEQ ID NO.: 28, or a sequence with at least 25% (e.g., at least 26%, at least 27%, at least 28%, or at least 29%) sequence identity to SEQ ID NO.: 6 or SEQ ID NO.: 24.

[0089] The antigenic domain comprised in the fusion protein of the present technology preferably comprises or consists of a sequence as defined in SEQ ID NO.: 10, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 10 or a sequence with one or more amino acid difference with SEQ ID NO.: 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference. Alternatively, the antigenic domain comprised in the fusion protein of the present technology preferably comprises or consists of a sequence as defined in SEQ ID NO.: 28, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 28 or a sequence with one or more amino acid difference with SEQ ID NO.: 28, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference.

[0090] In one embodiment, the fusion protein of the present technology does not comprise a sequence as defined in SEQ ID NO.: 3. In one embodiment, the fusion protein of the present technology does not comprise a sequence as defined in SEQ ID NO.: 27. In one embodiment, the fusion protein of the present technology does not comprise a sequence as defined in SEQ ID NO.: 3 and / or SEQ ID NO.: 27.

[0091] The continuous trimerization domain comprised in the fusion protein of the present technology is not the discontinuous trimerization domain of the wild-type VP2 capsid protein, i.e., not the native trimerization domain present in the wild-type VP2 capsid protein commonly known as the hub domain. Preferably, the continuous trimerization domain comprised in the fusion protein of the present technology is not the discontinuous trimerization domain of the wild-type VP2 capsid protein of EHDV serotype 8 or BTV serotype 3. The discontinuous trimerization domain present in the wild-type VP2 capsid protein is preferably replaced by a continuous trimerization domain, such as a continuous trimerization domain selected from the list consisting of T4 fibritin domain orfoldon (FD4), isoleucine zipper trimerization domain (e.g., GCN4-based), and collagen XVIII homotrimerization domain. The continuous trimerization domain according to the present technology is more preferably a T4 fibritin domain orfoldon (FD4). According to the present technology, the T4 fibritin domain or foldon (FD4) preferably comprises or consist of SEQ ID NO.: 8, ora sequence with at least 80% identity, such as at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 97% identity with SEQ ID NO.: 8. In the context of the present technology, the degree of identity between two amino acid sequences can be determined by conventional methods, for example, by means of standard sequence alignment algorithms known in the state of the art, such as, for example BLAST (Altschul S. F. et al. 1990).

[0092] The amino acid sequence of the continuous trimerization domain may consist of less than 200, 150 or 100 amino acids.

[0093] In the context of the technology, the term "smaller" refers to the amino acid length of the continuous trimerization domain compared to the discontinuous trimerization domain of the wild-type VP2 capsid protein. For example, the continuous trimerization domain may consist of fewer amino acids than the discontinuous trimerization domain, leading to a shorter polypeptide chain.

[0094] The skilled person will understand that any linkers that generates flexibility between the continuous trimerization domain according to the technology and the truncated VP2 capsid protein according to the technology can be used. Preferably, the linker is a peptide linker. The peptide linker may be less than 40 amino acids in length (e.g., less than 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 amino acids in length) The peptide linker may be 3-40, 5-40, 5-30, 5-20 or 5-15 amino acids in length. The peptide linker may be a flexible linker or rigid linker. A flexible linker may consist of one or more glycine and / or serine residues (e.g., a so-called " GS linker"). The flexible linker may consist solely of a rigid linker, e.g., (EAAAK)n (n=l-3). Other possible peptide linkers can be selected from one of a (G4S)n (n= 1-5) linker, (GS)n (n=l-10) linker, a GGGGGG linker (SEQ ID NO.: 11), a GGGGGGGG linker (SEQ ID NO.: 12), a GGGGGGSGGGGS linker (SEQ ID NO.: 26), and a (EAAAK)n (n=l-3) linker. Preferably, the linker is a (G4S)n (n= 1-5) linker, more preferably GGGGSGGGGS (SEQ ID NO: 9). Alternatively, the linker is a GG(G4S)n (n= 1-5) linker, more preferably GGGGGGSGGGGS linker (SEQ ID NO.: 26).

[0095] The purification tag may be any purification tag known in the art that allows the fusion protein to be isolated from a mixture using affinity chromatography, such as a polyhistidine tag (" His- Tag"). The purification tag may be fused to the fusion protein via a peptide linker, such as a GS linker. The linker may consist of GGGS (SEQ ID NO: 13).

[0096] In one embodiment according to the technology, the fusion protein comprises or, alternatively, consists of a sequence as defined in SEQ ID NO.: 4 or as defined in SEQ ID NO.: 5, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 4 and / or with SEQ ID NO.: 5 or a sequence with one or more amino acid difference with SEQ ID NO.: 4 and / or with SEQ ID NO.: 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference.

[0097] In one embodiment according to the technology, the fusion protein comprises or, alternatively, consists of a sequence as defined in SEQ ID NO.: 22 or as defined in SEQ ID NO.: 23, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 22 and / or with SEQ ID NO.: 23 or a sequence with one or more amino acid difference with SEQ ID NO.: 22 and / or with SEQ ID NO.: 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference.

[0098] In some embodiments, the fusion protein of the present technology (also referred to as "the protein of the present technology") comprises:

[0099] (i) a truncated VP2 capsid protein derived from a VP2 capsid protein of a virus belonging to the Reoviridae family, wherein the truncated VP2 capsid protein comprises an antigenic domain and a body domain or, alternatively, consists of SEQ ID NO.: 6 or SEQ ID NO.: 24, or a sequence with at least 20%, preferably at least 22%, more preferably at least 23%, even more preferably at least 24% sequence identity with SEQ ID NO.: 6 and / or with SEQ ID NO.: 24;

[0100] (ii) a trimerization domain which comprises or, alternatively, consists of SEQ ID NO.: 8, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 8, or a sequence with one or more amino acid difference with SEQ ID NO.: such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid difference; (iii) optionally, a linker, which is preferably a peptide linker, such as a GS linker, more preferably comprising or consisting of SEQ ID NO.: or SEQ ID NO.: 26, wherein optionally the linker links the truncated VP2 capsid protein defined in (i) with the trimerization domain defined in (ii).

[0101] In one preferred embodiment, the fusion protein of the present technology (also referred to as "the protein of the present technology") comprises:

[0102] (i) a truncated VP2 capsid protein derived from a VP2 capsid protein of a virus belonging to the Reoviridae family, wherein the truncated VP2 capsid protein comprises an antigenic domain and a body domain or, alternatively, consists of SEQ ID NO.: 6 or SEQ ID NO.: 7, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 6 and / or with SEQ ID NO.: 7 or a sequence with one or more amino acid difference with SEQ ID NO.: 6 and / or with SEQ ID NO.: 7, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference;

[0103] (ii) a trimerization domain which comprises or, alternatively, consists of SEQ ID NO.: 8, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 8, or a sequence with one or more amino acid difference with SEQ ID NO.: such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid difference;

[0104] (iii) optionally, a linker, which is preferably a peptide linker, such as a GS linker, more preferably comprising or consisting of SEQ ID NO.: 9, wherein optionally the linker links the truncated VP2 capsid protein defined in (i) with the trimerization domain defined in (ii).

[0105] In another preferred embodiment, the fusion protein of the present technology (also referred to as "the protein of the present technology") comprises:

[0106] (i) a truncated VP2 capsid protein derived from a VP2 capsid protein of a virus belonging to the Reoviridae family, wherein the truncated VP2 capsid protein comprises an antigenic domain and a body domain or, alternatively, consists of SEQ ID NO.: 24 or SEQ ID NO.: 25, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 24 and / or with SEQ ID NO.: 25 ora sequence with one or more amino acid difference with SEQ ID NO.: 24 and / or with SEQ ID NO.: 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid difference;

[0107] (ii) a trimerization domain which comprises or, alternatively, consists of SEQ ID NO.: 8, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 8, or a sequence with one or more amino acid difference with SEQ ID NO.: such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid difference;

[0108] (iii) optionally, a linker, which is preferably a peptide linker, such as a GS linker, more preferably comprising or consisting of SEQ ID NO.: 26, wherein optionally the linker links the truncated VP2 capsid protein defined in (i) with the trimerization domain defined in (ii).

[0109] In one embodiment, the fusion protein of the present technology does not comprise the hub or trimerization domain of VP2 wt (wild type), preferably does not comprise the hub or trimerization domain of VP2wt of EHDV serotype 8, represented by amino acids 1-49, 121-162 and 850-970 of SEQ ID NO.: 1.

[0110] In one embodiment, the fusion protein of the present technology does not comprise the hub or trimerization domain of VP2 wt and also does not comprise the hairpin domain of VP2 wt. In one preferred embodiment, the fusion protein not comprise the hub or trimerization domain of VP2wt of EHDV serotype 8, represented by amino acids 1-49, 121-162 and 850-970 of SEQ ID NO.: 1 and also does not comprise the hairpin domain of VP2wt of EHDV serotype 8, represented by SEQ ID NO.: 3. In one preferred embodiment, the fusion protein not comprise the hub or trimerization domain of VP2wt of BTV serotype 3, represented by amino acids 1-49, 121-162 and 850-959 of SEQ ID NO.: 14 and also does not comprise the hairpin domain of VP2wt of BTV serotype 3, represented by SEQ ID NO.: 27.

[0111] The nucleic acid sequence, vector, bacmid, and baculovirus of the present technology In one further aspect, the present technology provides a nucleic acid sequence encoding the fusion protein. In a further aspect, the present technology provides a vector comprising the nucleic acid sequence. The vector can also be referred to as "transfer vector". The skilled person will recognize and select suitable vectors forthe expression of fusion proteins. In one embodiment according to the technology, the fusion protein is expressed under the control of a suitable promoter, such as a polyhedrin promoter.

[0112] In a further aspect, the present technology provides a bacmid comprising the nucleic acid sequence and / or the vector, preferably wherein the bacmid comprises a mini Tn7-replicon. The term "mini Tn7-replicon" is well-known in the art and will be understood as sequence needed for incorporation of foreign sequences into an empty bacmid.

[0113] As used herein, a "bacmid" refers to a plasmid construct which contains the DNA sequence sufficient for generating a baculovirus when transfected into a cell or insect.

[0114] The transfer vector and / or bacmid may be derived from any of the commercially available baculovirus expression systems " Bac-to-Bac®" (invitrogen™), " BacPAKTM" (ClontechTM), " FlashBACTM" (Oxford Expression TechnologiesTM), " BacuVanceTM" (GenScriptTM), " Bac-N-Blue DNATM" (invitrogenTM), " BaculoDirectTM" (invitrogenTM), " BacVector®" 1000, 2000, 3000 (Novagen®), " DiamondBacTM" (Sigma-Aldrich®) or " BaculoGoldTM" (BD biosciencesTM).

[0115] In the context of the present technology, any technology using transposition or homologous recombination can be used. In one embodiment according to the present technology, the bac-to-bac technology can be used to obtain the baculovirus by transposition. The "bac-to-bac technology" is well-known in the art and is generally understood as a method used for the production of recombinant proteins, particularly in insect cells. It involves the use of a baculovirus expression system, which utilizes the baculovirus AcMNPV (Autographa californica multiple nucleopolyhedrovirus) to deliver and express foreign genes in host cells, such as insect cells including but not limited to Sf9 or Sf21.

[0116] In a further aspect, the technology provides a baculovirus comprising the nucleic acid sequence and / or the vector and / or the bacmid. In a preferred embodiment, the baculovirus is derived from AcMNPV (Autographa californica nuclear polyhedrosis virus) or BmNPV (Bombyx mori nucleopolyhedro virus).

[0117] The baculovirus expression vector system (BEVS) is a well-established method for the production of recombinant proteins, for example proteins to be used as vaccines, therapeutic molecules or diagnostic reagents. With its potential for over-expression and rapid speed of development, the BEVS is one of the most attractive choices for producing recombinant proteins for any purpose. The most employed baculovirus vector used in industry for recombinant protein expression is based on Autographa californica multinuclear polyhedrosis virus (AcMNPV) with Spodoptera frugiperda 9 (5 / 9) or 21 (5 / 21) insect cells as suitable expression hosts (Nettleship, J. E., Assenberg, R., Diprose, J. M., Rahman-Huq, N., Owens, RJ. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J. Struct. Biol. 2010, 172, 55-65), as well as Trichoplusia ni (T. ni) insect larvae as living biofactories (Gomez-Casado E, Gomez-Sebastian S, Nunez MC, Lasa-Covarrubias R, Martinez-Pulgarin S, Escribano JM. Insect larvae biofactories as a platform for influenza vaccine production. Protein Expr Purif. 79: 35-43. 2011). Since the BEVS was developed in the 80's (Smith, G. E., M. D. Summers, and MJ. Fraser. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3: 2156-21 65), hundreds of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Recently, new baculovirus vectors have been described. For instance, WO 2012 / 168493 and WO 2012 / 168492 relate to recombinant DNA elements forthe expression of recombinant proteins in insects and insect cells. Specifically, WO 2012 / 168492 relates to recombinant baculovirus, transfer vectors, and bacmids suitable for use in the present technology.

[0118] In the context of the technology, any conventional baculovirus can be used forthe expression of the fusion protein. For example, any improved baculovirus can be used, such as toTop-Bac. The term " Top-Bac" is well-known in the art and generally refers to a refined version of the bac-to-bac technology used for the expression of recombinant proteins in e.g., insect cells via a baculovirus system. The host cell and the insect of the present technology

[0119] In a further aspect, the present technology provides a host cell comprising the nucleic acid sequence and / or the vector and / or the bacmid and / or the baculovirus of the present technology. In one preferred embodiment according to the technology, the host cell is an insect cell, preferably a Spodoptera frugiperda 9 (Sf9), 21 (Sf21), Bm5 or BmE-SWU insect cell.

[0120] Ina further aspect the technology also provides an insect comprising the nucleic acid sequence and / or the vector and / or the bacmid and / or the baculovirus and / or the host cell. In a preferred embodiment according to the technology, the insect is a pupa or a larva. For instance, WO2017 / 046415 describes the use of a pupa. In one embodiment according to the technology, the pupa belongs to the order Lepidoptera, preferably to the genus Trichoplusia, Rachiplusia or Bombix mori, even more preferably to the genus Trichoplusia and to the species Trichoplusia ni.

[0121] In the context of the technology, when the pupa belongs to the genus Trichoplusia or Rachiplusia, a AcMNPV baculovirus is preferably used. In the case the pupa belongs to the genus Bombix mori, a BmNPV baculovirus is preferably used.

[0122] The method of the present technology

[0123] In a further aspect, the present technology provides a method for producing a fusion protein, wherein the method comprises the use of the host cell and / or the use of the insect to express the fusion protein, the extraction and optionally the purification of the fusion protein by conventional means, preferably by affinity purification using a tag, more preferably by affinity purification using a polyhistidine tag.

[0124] In one embodiment according to the technology, the method comprises the use of an extraction buffer comprising Tris, NaCI, Arginine, Imidazole, and Tween at a pH of about 7 and 8. In a preferred embodiment, the protein extraction can be performed in optimized extraction buffer comprising or consisting of Tris 20 mM, NaCI 300 mM, Arginine 50 mM, Imidazole 50 mM, Tween 0,05% pH 7.5 using a colloidal mill.

[0125] The method of the present technology may comprise the steps of a) Baculovirus generation;

[0126] b) Protein expression;

[0127] c) Protein purification;

[0128] d) Protein characterization; and / or

[0129] e) Protein formulation in vaccines.

[0130] In one embodiment, the generation of a baculovirus comprises synthesizing the encoding sequence for fusion protein according to the technology. The codon usage of the VP2 capsid protein encoding genes can be optimized fortheir expression in insect cells (OptimumGene™-Codon Optimization algorithm). The sequences can contain adequate flanking regions to facilitate their cloning in pFastBacl donor plasmid. Once the donor plasmids with the wildtype VP2 capsid protein or the fusion protein genes are obtained, the bacmids for the generation of the different baculoviruses can be prepared in E. coli DHIOBac bacteria containing a mini Tn7-replicon. Then, the transfection of the bacmids in the regulatory Sf9-RVN Glycobac cells (SIGMA-ALDRICH, USA) can be performed. The viral stock can be amplified to generate a high titer stock. The baculovirus obtained can be titrated by a standard plaque protocol in Sf21. The virus titer can be determined as plaque-forming units (pfu).

[0131] In one embodiment, the protein expression step comprises or consists of the following steps: Once the recombinant baculoviruses (rBV) are generated expressing fusion proteins, these can be tested in insect cells and experiments can be carried out to determine the best condition for fusion protein expression in Trichoplusia ni as living biofactories.

[0132] Different rBV doses between 5000-50000 pfus / insect, insect incubation temperature from 23 to 28 °C and infection times from 3 to 6 days can be tested. Pupae infected at every condition (5g of pupae biomass) can be mechanically homogenized using as extraction buffer 80 mL of Tris 20 mM, NaCI 300 mM, Arginine 50 mM, Tween 800.5 % pH 7.5.

[0133] 1 mL of pupae extract can be taken from each condition and centrifuged at 13000xg 15 min at 4°C to split samples in soluble and insoluble fractions. Protein expression and solubility can be checked using SDS-PAGE and Coomassie blue staining. Bands corresponding to the fusion protein can be submitted to densitometry and relative quantification can be carried out with ImageLab software analysis.

[0134] In one embodiment, the protein purification step comprises or consists of the following steps: once pupae batches are generated using the advantageous expression conditions, purification of the fusion protein can be performed using the His tag in C -terminal. Briefly, protein extraction can be performed in optimized extraction buffer, Tris 20 mM, NaCI 300 mM, Arginine 50 mM, Imidazole 50 mM, Tween 0.05% pH 7.5 using a colloidal mill. Insect biomasses (62.5g of pupae) can be homogenized in IL of extraction buffer, followed by high pressure homogenization with 1 cycle at 300 bars. A centrifugation step can be performed to remove the insoluble fraction, and the soluble extract can be clarified using depth filtration. IMAC chromatography using Histrap Crude FF resin can be performed, and protein elution can be achieved using a 500 mM Imidazole concentration. SDS-PAGE analysis can help to select protein fractions that can be later dialyzed in PBS IX pH 7.4.

[0135] In one embodiment, the protein characterization step comprises or consists of the following steps: protein concentrations can be measured using SDS-PAGE and band densitometry using BSA curve as reference. Purity of the proteins can be measured by SDS-PAGE and Coomassie blue staining using band densitometry of gels. Gels can be analyzed using ImageLab software from Bio-Rad. Protein trimerization, necessary for inducing neutralization antibodies, can be assessed by FPLC-SEC using Superose 6 16 / 300 from Cytiva, equilibrated in PBS IX pH 7.4, previously calibrated with gel filtration HMW calibration kit.

[0136] As described above, the method of the present technology is optimized for the production of a fusion protein. Hence, the method and cells of the present technology can be used for the production of increased protein yields of the fusion protein for use as a medicament and / or for use in a method of treating and / or preventing a disease related to a virus belonging to Reoviridae family, preferably related to a virus belonging to the Orbivirus genus, such as Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV), Mitchell River virus (MRV), Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV) more preferably related to Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV) and / or for use in a method of reducing the incidence or severity of at least one clinical symptom of Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV), Mitchell River virus (MRV), Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV), preferably of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV).

[0137] The present technology thus provides a fusion protein directly obtained by the methods of the present technology.

[0138] The uses of the present technology

[0139] In a further embodiment, the present technology provides the use of cells and / or the insect for the expression of a fusion protein.

[0140] The composition of the present technology

[0141] In a further aspect, the present technology provides a composition comprising the fusion protein or the nucleic acid sequence. In a preferred embodiment, the composition is a pharmaceutical composition. It will be understood that the composition may further comprise pharmaceutically acceptable carriers, adjuvants, preservatives, stabilizers, or combinations thereof. The pharmaceutical composition may be a vaccine composition.

[0142] The medical application of the present technology

[0143] In a further aspect, the present technology provides a vaccine comprising the fusion protein, the nucleic acid sequence, or the composition of the present technology. In a preferred embodiment, the vaccine comprises or consists of more than one fusion protein as described herein. Preferably, each of the fusion proteins comprised in the vaccine comprises an antigenic domain from a VP2 capsid protein belonging to a different serotype of the same virus, e.g., such as a serotype selected from the list consisting of serotype 1, 2, 4, 5, 6, 7, and 8. Hence in one embodiment, the vaccine and / or composition of the present technology comprises more than one fusion proteins as described herein, wherein the fusion proteins comprise antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of the same virus, e.g., such as serotypes 1, 2, 4, 5, 6, 7, and / or 8. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of EHDV. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of AHSV. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of EEV. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of BTV. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of EHDV, such as serotypes 1, 2, 4, 5, 6, 7, and / or 8. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of AHSV, such as serotypes 1 to 9. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of EEV, such as serotypes 1 to 7. For instance, the vaccine and / or composition of the present technology may comprise fusion proteins comprising antigenic domains and / or body domains from VP2 capsid proteins belonging to different serotypes of BTV, such as serotypes 1 to 27. In another embodiment, the vaccine comprises or consists of more than one fusion protein as described herein, wherein at least one fusion protein comprises an antigenic domain and / or body domain from a VP2 capsid protein belonging to EHDV, preferably to EHDV serotype 8. In another embodiment, the vaccine comprises or consists of more than one fusion protein as described herein, wherein at least one fusion protein comprises an antigenic domain and / or body domain from a VP2 capsid protein belonging to BTV, preferably to BTV serotype 3.

[0144] In a further embodiment, the vaccine may alternatively, or additionally, comprise or consists of more than one fusion protein, each fusion protein comprising an antigenic domain from a VP2 capsid protein belonging to a different virus, wherein the virus belongs to the Reoviridae family, preferably from a virus selected from the list comprising or consisting of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and Bluetongue virus (BTV), more preferably Epizootic Hemorrhagic disease virus (EHDV) and Bluetongue virus (BTV). Hence, the vaccine and / or composition of the present technology may comprise more than one fusion protein as described herein, wherein the fusion proteins comprise antigenic domains and / or body domains from VP2 capsid proteins belonging to different virus, such as Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV), Mitchell River virus (MRV), EHDV, AHSV, EEV and / or BTV. In some embodiments, the vaccine and / or composition of the present technology comprises:

[0145] (i) a first fusion protein comprising:

[0146] a truncated VP2 capsid protein, wherein the VP2 capsid protein is of EHDV and wherein the truncated VP2 capsid protein consists of an antigenic domain and a body domain; and

[0147] a trimerization domain, wherein the trimerization domain is a continuous domain; and

[0148] (ii) a second fusion protein comprising:

[0149] a truncated VP2 capsid protein, wherein the VP2 capsid protein is of BTV and wherein the truncated VP2 capsid protein consists of an antigenic domain and a body domain; and

[0150] a trimerization domain, wherein the trimerization domain is a continuous domain.

[0151] The first and second fusion proteins may be in accordance with any embodiment described above. The truncated VP2 capsid protein of the first fusion protein may consist of SEQ ID NO: 6 or 7, or a sequence with at least 80% sequence identity to SEQ ID NO: 6 or 7, and the truncated VP2 capsid protein of the second fusion protein may consist of SEQ ID NO: 24 or 25, or a sequence with at least 80% sequence identity to SEQ ID NO: 24 or 25. The trimerization domain of the first and second fusion proteins may be a T4 fibritin domain, foldon (FD4), isoleucine zipper trimerization domain, or a collagen XVIII homotrimerization domain, preferably a T4 fibritin domain or foldon (FD4), more preferably a T4 fibritin domain which comprises or consists of SEQ ID NO.: 8, or a sequence with at least 80% sequence identity to SEQ ID NO.: 8. The first fusion protein may be in accordance with any embodiment described above, wherein the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6 or 7, and the second fusion protein may be in accordance with any embodiment described above, wherein the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 24 or 25.

[0152] In one embodiment, the vaccine further comprises an adjuvant, preferably an oil adjuvant or an aqueous adjuvant, more preferably Freund's adjuvant or any other commercial adjuvant such as Montanide adjuvant. The skilled person will recognize that any adjuvant adequate for ruminant species can be used in the context of the present technology.

[0153] The present technology also provides the fusion protein, the nucleic acid sequence, the composition, or the vaccine of the present technology for use as a medicament.

[0154] The technology also provides the fusion protein, the nucleic acid sequence, the composition or the vaccine of the present technology for use in a method of treating and / or preventing a disease related to a virus belonging to Reoviridae family, preferably related to a virus belonging to the Orbivirus genus, more preferably related to Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV), Mitchell River virus (MRV), Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV), even more preferably belonging to EHDV, AHSV, EEV and / or BTV. The disease may preferably be Epizootic Hemorrhagic disease, African horse sickness, equine encephalosis and / or Bluetongue.

[0155] In a preferred embodiment, the fusion protein, the nucleic acid sequence, the composition or the vaccine of the present technology is administered intramuscularly. In some embodiments, the subject is administered a first dose that delivers about 20 to 150 micrograms of the fusion protein to the subject. The subject may also receive a booster dose of about 20 and 150 micrograms about 2 to 3 weeks after the first dose. In one embodiment, the subject to be treated is a ruminant mammal (e.g., cattle, deer or sheep).

[0156] The technology also provides a fusion protein, the nucleic acid sequence, the composition or the vaccine of the present technology for use in a method of reducing the incidence or severity of at least one clinical symptom of Orungo virus, Palyam virus, Peruvian horse sickness virus, Wad Medani virus, Wongorr virus, Yunnan orbivirus, Yunnan orbivirus (YUOV), Mitchell River virus (MRV), Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV), preferably of EHDV, AHSV, EEV and / or BTV.

[0157] In one embodiment, the subject receiving the vaccine should not show clinical symptoms and the virus load should be reduced in peripheral blood and in organs compared to a subject not receiving the vaccine according to the technology. Also, the subject should not spread viruses to the environment. In this context, the reduction of virus load is very advantageous as to avoid dissemination of the virus to other subjects.

[0158] The present technology also provides the fusion protein, the nucleic acid sequence, the composition or the vaccine of the present technology for use in a method of reducing the incidence or severity of at least one clinical symptom of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalitis virus (EEV), and / or Bluetongue virus (BTV), wherein the clinical symptoms are selected from loss of appetite, loss of fear of people, weakness, excessive salivation, rapid pulse, rapid respiration rate, fever, lying in bodies of water to reduce body temperature, unconsciousness, blue tongue, head swelling, neck swelling, sloughing or breaking of hooves, lameness, shock, death, and combinations thereof.

[0159] Any treatment or prevention of a disease related to EHDV disclosed herein or method of reducing the incidence or severity of at least one clinical symptom of EHDV disclosed herein may involve a fusion protein in accordance with an embodiment described above that comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6 or 7. Any treatment or prevention of a disease related to BTV disclosed herein or method of reducing the incidence or severity of at least one clinical symptom of BTV disclosed herein may involve a fusion protein in accordance with an embodiment described above that comprises a sequence with at least 80% sequence identity to SEQ ID NO: 24 or 25. A vaccine and / or composition of the present technology that comprises a first and second fusion protein as described above may be used to treat or prevent a disease related to EHDV and / or a disease related to BTV. A vaccine and / or composition of the present technology that comprises a first and second fusion protein as described above may be used in a method of reducing the incidence or severity of at least one clinical symptom of EHDV and / or at least one clinical symptom of BTV.

[0160] Items of the present invention

[0161] The present invention also provides the following items that are combinable with any one of the embodiments described above.

[0162] [1] A fusion protein comprising:

[0163] (a) a truncated VP2 capsid protein, wherein the VP2 capsid protein is of a virus belonging to the Reoviridae family and wherein the truncated VP2 capsid protein consists of an antigenic domain and a body domain; and

[0164] (b) a trimerization domain, wherein the trimerization domain is a continuous domain.

[0165] [2] The fusion protein according to item [1], wherein the amino acid sequence of the fusion protein comprises or consists of, from / V-terminus to C-terminus:

[0166] (i) optionally, a purification tag;

[0167] (ii) the truncated VP2 capsid protein of (a);

[0168] (iii) a peptide linker;

[0169] (iv) the trimerization domain of (b); and

[0170] (v) optionally, a purification tag;

[0171] wherein, optionally, the purification tag of (i) is fused via a peptide linker to the N-terminus of the truncated VP2 capsid protein and / or the purification tag of (v) is fused via a peptide linker to the C-terminus of the trimerization domain.

[0172] [3] The fusion protein according to item [1] or [2], wherein the virus belongs to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Equine encephalosis virus (EEV), and Bluetongue virus (BTV), wherein, optionally, the truncated VP2 capsid protein consists of SEQ ID NO: 6 or 7, or a sequence with at least 80% sequence identity to SEQ ID NO: 6 or 7.

[0173] [4] The fusion protein according to any one of items [l]-[3], wherein the trimerization domain is a T4 fibritin domain, foldon (FD4), isoleucine zipper trimerization domain, or a collagen XVIII homotrimerization domain, preferably a T4 fibritin domain or foldon (FD4), more preferably a T4 fibritin domain which comprises or consists of SEQ ID NO.: 8, or a sequence with at least 80% sequence identity to SEQ ID NO.: 8.

[0174] [5] The fusion protein according to any one of items [1] to [3], wherein the fusion protein further comprises a peptide linker between the truncated VP2 capsid protein of (a) and the trimerization domain of (b), wherein, optionally:

[0175] the peptide linker is less than 40 amino acids in length; and / or

[0176] the peptide linker is a flexible linker or rigid linker, wherein, further optionally:

[0177] the flexible linker consists of one or more glycine and / or serine residues; and / or the rigid linker consists of (EAAAK)n (n=l-3).

[0178] [6] The fusion protein according to any one of items [1] to [5], wherein the fusion protein comprises or consists of a sequence as defined in SEQ ID NO.: 4 or as defined in SEQ ID NO.: 5, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 4 and / or with SEQ ID NO.: 5.

[0179] [7] A nucleic acid sequence encoding the fusion protein according to any one of items [1] to [6],

[0180] [8] A pharmaceutical composition comprising the fusion protein according to any one of items [1] to [6], or the nucleic acid sequence according to item [7],

[0181] [9] A vaccine comprising the fusion protein according to any one of items [1] to [6], or the nucleic acid sequence according to item [7],

[0010] The vaccine according to item [9], wherein the vaccine comprises more than one fusion protein according to any one of items [1] to [6], wherein each fusion protein comprises an antigenic domain from a VP2 capsid protein belonging to

[0182] (a) a different serotype of the same virus; and / or

[0183] (b) a different virus, wherein the virus belongs to the Reoviridae family, preferably from a virus selected from the list comprising or consisting of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and Bluetongue virus (BTV), more preferably Epizootic Hemorrhagic disease virus (EHDV) and Bluetongue virus (BTV).

[0184]

[0011] The fusion protein according to any one of items [1] to [6], the nucleic acid sequence according to item [7], the pharmaceutical composition according to item [8], or the vaccine according to item [9] or

[0010] for use as a medicament.

[0185]

[0012] The fusion protein according to any one of items [1] to [6], the nucleic acid sequence according to item [7], the pharmaceutical composition according to item [8], or the vaccine according to item [9] or

[0010] for use in a method of treating and / or preventing a disease related to a virus belonging to the Reoviridae family, preferably related to a virus belonging to the Orbivirus genus, more preferably related to Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV).

[0186]

[0013] The fusion protein, nucleic acid sequence, pharmaceutical composition or vaccine for use according to item

[0012] , wherein the fusion protein, nucleic acid sequence, pharmaceutical composition or vaccine is administered intramuscularly, wherein, optionally, the subject is administered a first dose that delivers about 20 to 150 micrograms of the fusion protein to the subject, and, further optionally, the subject receives a booster dose of about 20 and 150 micrograms about 2 to 3 weeks after the first dose.

[0187]

[0014] The fusion protein according to any one of items [1] to [6], the nucleic acid sequence according to item [7], the pharmaceutical composition according to item [8], or the vaccine according to item [9] or

[0010] for use in a method of reducing the incidence or severity of at least one clinical symptom of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV).

[0188]

[0015] The fusion protein, nucleic acid sequence, pharmaceutical composition or vaccine for use according to item

[0014] , wherein the clinical symptom is loss of appetite, loss of fear of people, weakness, excessive salivation, rapid pulse, rapid respiration rate, fever, lying in bodies of water to reduce body temperature, unconsciousness, blue tongue, head swelling, neck swelling, sloughing or breaking of hooves, lameness, shock, death, or combinations thereof.

[0189] Sequences

[0190] SEQ ID NO.: 1: VP2-EHDV-8 wild type MDSVEFAILNTTQRPDESVIYDYLASVWTRVYKDELEGKMEHLVNANTIELTRGEARNVFERTLSDEYKISFPDA INYGIMRYDDEHEKYNEKRLMLAESLKPTGEYEILLRTSVKHQRIKPEYGAQNARISFSFSGGTLRIHSKFVESL KFDWNYEKEDCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEIT NREPYKDEQTKIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLE AKGKEMKIARTNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDS PIYRGVMLYATEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGRE VKTNWMDGYPYEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAG FVKLPDYYDKLIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTK QQRSARFNELVAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNM

[0191] 11 S SRKFS SLI VSMI I KAYGDDRVKEI SNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKI SEVRVAEKVEDMLPL VFFQALLMSENQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVL PKVLDFWMNVSFEKEALHTIKYVSEQYTKNIMCSYCGGNELVASFVLPITHPDRGFIVIIITTEDVTNSNAEAIA KSRFSDVAKYIKGIVHISATREGTARVRGGGGIKSRVLDKWLGTRFQLVQIKMGENVFENHELVTKLMN

[0192] SEQ ID NO.: 2: VP2-EHDV-8 wild type-His Tag MDSVEFAILNTTQRPDESVIYDYLASVWTRVYKDELEGKMEHLVNANTIELTRGEARNVFERTLSDEYKISFPDA INYGIMRYDDEHEKYNEKRLMLAESLKPTGEYEILLRTSVKHQRIKPEYGAQNARISFSFSGGTLRIHSKFVESL KFDWNYEKEDCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEIT NREPYKDEQTKIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLE AKGKEMKIARTNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDS PIYRGVMLYATEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGRE VKTNWMDGYPYEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAG FVKLPDYYDKLIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTK QQRSARFNELVAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNM 11 S SRKFS SLI VSMI I KAYGDDRVKEI SNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKI SEVRVAEKVEDMLPL VFFQALLMSENQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVL PKVLDFWMNVSFEKEALHTIKYVSEQYTKNIMCSYCGGNELVASFVLPITHPDRGFIVIIITTEDVTNSNAEAIA KSRFSDVAKYIKGIVHISATREGTARVRGGGGIKSRVLDKWLGTRFQLVQIKMGENVFENHELVTKLMNHHHHH HHH

[0193] SEQ ID NO.: 3: VP2-EHDV-8 wild type hairpin domain ELTRGEARNVFERTLSDEYKISFPDAINYGIMRYDDEHEKYNEKRLMLAESLKPTGEYEILLRTSVKHQRI

[0194] SEQ ID NO.: 4: VP2mod sequence MCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQT KIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLEAKGKEMKIAR TNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDSPIYRGVMLYA TEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGREVKTNWMDGYP YEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPDYYDK LIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTKQQRSARFNEL VAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNMIISSRKFSSL IVSMIIKAYGDDRVKEISNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKISEVRVAEKVEDMLPLVFFQALLMSE NQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVLPKVLDFWMNV SFEKEALHTIKYVSGGGGSGGGGSHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDGQ AYVRKDGEWVLLSTFL

[0195] SEQ ID NO.: 5: VP2mod sequence-His Tag MCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQT KIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLEAKGKEMKIAR TNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDSPIYRGVMLYA TEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGREVKTNWMDGYP YEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPDYYDK LIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTKQQRSARFNEL VAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNMIISSRKFSSL IVSMIIKAYGDDRVKEISNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKISEVRVAEKVEDMLPLVFFQALLMSE NQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVLPKVLDFWMNV SFEKEALHTIKYVSGGGGSGGGGSHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDGQ AYVRKDGEWVLLSTFLGGGSHHHHHH

[0196] SEQ ID NO.: 6: truncated VP2 capsid protein consisting of the body domain and antigenic domain CDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQTK IQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLEAKGKEMKIART NTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDSPIYRGVMLYAT EKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGREVKTNWMDGYPY EAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPDYYDKL IKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTKQQRSARFNELV AEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNMIISSRKFSSLI VSMIIKAYGDDRVKEISNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKISEVRVAEKVEDMLPLVFFQALLMSEN QWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVLPKVLDFWMNVS FEKEALHTIKYVS

[0197] SEQ ID NO.: 7: truncated VP2 capsid protein consisting of the body domain and antigenic domain with an N-terminal Met MCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQT KIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLEAKGKEMKIAR TNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDSPIYRGVMLYA TEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGREVKTNWMDGYP YEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPDYYDK LIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTKQQRSARFNEL VAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNMIISSRKFSSL IVSMIIKAYGDDRVKEISNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKISEVRVAEKVEDMLPLVFFQALLMSE NQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVLPKVLDFWMNV S FEKEALHTIKYVS

[0198] SEQ ID NO.: 8: Trimerization domain FD4 HKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0199] SEQ ID NO.: 9: Linker

[0200] GGGGSGGGGS

[0201] SEQ ID NO.: 10: VP2 protein antigenic domain KQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQTKIQASQGPRLNELKKNVFSRRYGLKVKYLE RLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLEAKGKEMKIARTNTEYYRGARTRFYNVLKQNLVKTTDEIRN I RGVSNEKAGS I LAAVLLVSACDS KKRAI WYDDDS P I YRGVMLYATEKLGCVYYGLRKRFTWS I RST

[0202] SEQ ID NO.: 11: Linker

[0203] GGGGGG

[0204] SEQ ID NO.: 12: Linker

[0205] GGGGGGGG SEQ ID NO.: 13: Linker

[0206] GGGS

[0207] SEQ ID NO.: 14: VP2-BTV-3 wild type ( NET2023 segment 2 GenBank: OR603993.1) (VP2 complete BTV-serotype 3 as shown on the left side of Figure 1)

[0208] MEELVI PVI SRQFDKKLVGRYEYVI ELAEPEQDEWTNHDVTQI PDRRMFDVAQQGI REAI I YKPLDNDGEVLPRI LDMSIACYDMRKTMMKKEGVDFVSNTRWLEWMIQDSMDVQPLRVQMKEDHSTVQYDMFSAKVHIDSRKADTTSYH AIAVEAKAERKCCHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQ LGDNAYDKFLRGLVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKD EKDLSVKFQFKLDEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKL GDVYHTLRSKYEWSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYP DDVEITTKFNEDRYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMF NARVKPCDIELAERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYS KLLRRRDADLDYSSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVH MYDRFFEKRRVLRSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNI SYEHRQWSIPIILYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTIS NGGVQTSIVSTKALLYEMYLSSICGGYSEGVLWYLPITHPVKCLVALEVSDALVGADVRIDKIRRRFPLSAKHLK GIVQISVHPNRTFSVTTCGIVRHKVCKKTLLKHRCDVILLQTPGYVFGNDELLTKLLNI*

[0209] SEQ ID NO.: 22: VP2mod sequence-His Tag MCHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQLGDNAYDKFLR GLVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKDEKDLSVKFQFK LDEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKLGDVYHTLRSKY EWSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYPDDVEITTKFNE DRYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMFNARVKPCDIEL AERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYSKLLRRRDADLD YSSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVHMYDRFFEKRRV LRSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNISYEHRQWSIPI ILYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTISNGGVQTSIVST KALLYEMYLSSICGGGGGGSGGGGSHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDG QAYVRKDGEWVLLSTFLGGGSHHHHHH

[0210] SEQ ID NO.: 23: VP2mod sequence MCHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQLGDNAYDKFLR GLVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKDEKDLSVKFQFK LDEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKLGDVYHTLRSKY EWSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYPDDVEITTKFNE DRYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMFNARVKPCDIEL AERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYSKLLRRRDADLD YSSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVHMYDRFFEKRRV LRSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNISYEHRQWSIPI ILYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTISNGGVQTSIVST KALLYEMYLSSICGGGGGGSGGGGSHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDG QAYVRKDGEWVLLSTFL

[0211] SEQ ID NO.: 24: truncated VP2 capsid protein consisting of the body domain and antigenic domain CHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQLGDNAYDKFLRG LVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKDEKDLSVKFQFKL DEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKLGDVYHTLRSKYE WSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYPDDVEITTKFNED RYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMFNARVKPCDIELA ERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYSKLLRRRDADLDY SSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVHMYDRFFEKRRVL RSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNISYEHRQWSIPII LYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTISNGGVQTSIVSTK ALLYEMYLSSIC

[0212] SEQ ID NO.: 25: truncated VP2 capsid protein consisting of the body domain and antigenic domain with an N-terminal Met MCHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQLGDNAYDKFLR GLVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKDEKDLSVKFQFK LDEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKLGDVYHTLRSKY EWSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYPDDVEITTKFNE DRYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMFNARVKPCDIEL AERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYSKLLRRRDADLD YSSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVHMYDRFFEKRRV LRSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNISYEHRQWSIPI ILYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTISNGGVQTSIVST KALLYEMYLSSIC

[0213] SEQ ID NO.: 26: Linker

[0214] GGGGGGSGGGGS

[0215] SEQ ID NO.: 27: VP2-BTV-3 wild type hairpin domain

[0216] DVAQQGIREAIIYKPLDNDGEVLPRILDMSIACYDMRKTMMKKEGVDFVSNTRWLEWMIQDSMDVQPLRVQ SEQ ID NO.: 28: VP2 protein antigenic domain QTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQLGDNAYDKFLRGLVQLRVSGTTPAKIRDEVAALDVIRDNW IRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKDEKDLSVKFQFKLDEKFSPNDPERNVI FTSKTHRTNEDRFY VLLIIAASDTNNGRVWWSNPYPCLRGALIAAECKLGDVYHTLRSKYEWSVRPTYKPRDLEREREKYV

[0217] EXAMPLE 1 - fusion protein comprising a truncated VP2 capsid protein of EHDV Materials and experimental methods

[0218] 1- Protein design and sequences

[0219] The VP2 capsid protein from EHDV 8 is a 970 amino acids protein that oligomerize in trimer structures generating a complex of 300 kDa approximately. The available complete sequence of the VP2 capsid protein from EHDV serotype 8 (Genbank accession no. OP897266.1 derived from virus isolate EHDV-82022. TE.50459.1.2) was used to synthesize different versions of the encoding gene (SEQ ID NOs: 2 and 5).

[0220] Based on the information available from the equivalent protein of BTV (belonging to the same virus family), VP2 monomer could be divided into 4 different domains: a terminal present in both proteins; amino acids 121-162 and 850-970, see SEQ ID NO.: 1 and 2), this domain is the less exposed region and could be involved in trimer formation. Hairpin domain (amino acids 50-120, see SEQ ID NO.: 1 and 2) could be the responsible with VP5 interaction. The body domain (amino acids 163-190 and 408-849, see SEQ ID NO.: 5), the immunogenic domain (amino acids 191-407, see SEQ ID NO.: 5).

[0221] To reduce the VP2 protein size and complexity, theoretically facilitating its production by recombinant DNA technologies, the strategy followed consisted in removing the Hub domain (i.e., the discontinuous trimerization domain) and hairpin domains, and add an external, continuous trimerization domain the T4 fibritin domain or foldon (FD4). Trimerization of VP2 is necessary to induce neutralizing antibodies. As a result of these deletions and additions, a protein of 776 amino acids was designed, including a linker between VP2 and FD4 domains as well as a purification tag connected via a GGGS linker, see SEQ ID NO.: 5. Both genes were synthesized including a His tag encoding sequence at 3' end of the genes to facilitate purification processes of produced proteins. VP2-EHDV-8:

[0222] MDSVEFAILNTTQRPDESVIYDYLASVWTRVYKDELEGKMEHLVNANTIELTRGEARNVFERTLSDEYKISFPDA INYGIMRYDDEHEKYNEKRLMLAESLKPTGEYEILLRTSVKHQRIKPEYGAQNARISFSFSGGTLRIHSKFVESL KFDWNYEKEDCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEIT NREPYKDEQTKIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLE AKGKEMKIARTNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDS PIYRGVMLYATEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGRE VKTNWMDGYPYEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAG FVKLPDYYDKLIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTK QQRSARFNELVAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNM

[0223] 11 S SRKFS SLI VSMI I KAYGDDRVKEI SNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKI SEVRVAEKVEDMLPL VFFQALLMSENQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVL PKVLDFWMNVSFEKEALHTIKYVSEQYTKNIMCSYCGGNELVASFVLPITHPDRGFIVIIITTEDVTNSNAEAIA KSRFSDVAKYIKGIVHISATREGTARVRGGGGIKSRVLDKWLGTRFQLVQIKMGENVFENHELVTKLMNHHHHH HHH* (SEQ ID NO: 2)

[0224] VP2t-EHDV-8 Mod:

[0225] MCDHTNWDVHYDMIQQGTIMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQT KIQASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLEAKGKEMKIAR TNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVSACDSKKRAIWYDDDSPIYRGVMLYA TEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQTFMTRIPYFDLNQEEGDSIYKWNLVALGREVKTNWMDGYP YEAYAEDDEDQVLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPDYYDK LIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTKQQRSARFNEL VAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNMIISSRKFSSL IVSMIIKAYGDDRVKEISNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKISEVRVAEKVEDMLPLVFFQALLMSE NQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVLPKVLDFWMNV SFEKEALHTIKYVSGGGGSGGGGSHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDGQ AYVRKDGEWVLLSTFLGGGSHHHHHH*(SEQ ID NO: 5)

[0226] 2- Baculovirus generation

[0227] The encoding sequences for these proteins (VP2-EHDV-8 and VP2t-EHDV-8 Mod) were synthesized by the company GenScript. The codon usage of the VP2 encoding genes were optimized fortheir expression in insect cells (OptimumGene™-Codon Optimization algorithm). Both sequences contained the adequate flanking regions to facilitate their cloning in pFastBacl donor plasmid. Once the donor plasmids with the complete VP2 or modified VP2 genes were obtained, the bacmids for the generation of the different baculoviruses were prepared in E. coli DHIOBac bacteria (Invitrogen, San Diego, CA, USA) containing the mini Tn7-replicon. Then, the transfection of the bacmids in the regulatory Sf9-RVN Glycobac cells (SIGMA-ALDRICH, USA) was performed. The viral stock was amplified to generate a high titer stock. The baculovirus obtained were titrated by a standard plaque protocol in Sf21 cells (Invitrogen, San Diego, CA, USA). Virus titer was determined as plaque-forming units (pfu).

[0228] 3- Protein expression

[0229] Once the recombinant baculoviruses (rBV) were generated expressing each VP2 version, those were tested in insect cells and experiments were carried out to determine the best condition for recombinant protein expression in Trichoplusia ni as living biofactories.

[0230] Different rBV doses between 5000-50000 pfus / insect, insect incubation temperature from 23 to 28°C and infection times from 3 to 6 days were tested. Pupae infected at every condition (5g of pupae biomass) were mechanically homogenized using as extraction buffer 80 mL of Tris 20 mM, NaCI 300 mM, Arginine 50 mM, Tween 800.5 % pH 7.5.

[0231] 1 mL of pupae extract was taken from each condition and centrifuged at 13000xg 15 min at 4°C to split samples in soluble and insoluble fractions. Protein expression and solubility were checked using SDS-PAGE and Coomassie blue staining. Bands corresponding to the recombinant proteins were submitted to densitometry and relative quantification was carried out with ImageLab software analysis.

[0232] 4- Protein purification

[0233] Once pupae batches were generated using the best expression condition for each version, purification of the two recombinant VP2 proteins was performed using the His tag in C-terminal present in both proteins. Briefly, protein extraction was performed in optimized extraction buffer, Tris 20 mM, NaCI 300 mM, Arginine 50 mM, Imidazole 50 mM, Tween 0.05% pH 7.5 using a colloidal mill. Insect biomasses (62.5g of pupae) were homogenized in IL of extraction buffer, followed by high pressure homogenization with 1 cycle at 300 bars. A centrifugation removed insoluble fraction, and soluble extract was clarified using depth filtration. IMAC chromatography using Histrap Crude FF resin was performed, and protein elution was achieved using a 500 mM Imidazole concentration. SDS-PAGE analysis helped to select protein fractions that were later dialyzed in PBS IX pH 7.4.

[0234] 5- Protein characterization

[0235] Protein concentrations were measured using SDS-PAGE and band densitometry using BSA curve as reference. Purity of the proteins were measured by SDS-PAGE and Coomassie blue staining using band densitometry of gels. Gels were analyzed using ImageLab software from Bio-Rad.

[0236] Protein trimerization, necessary for inducing neutralization antibodies, was checked by FPLC-SEC using Superose 616 / 300 from Cytiva, equilibrated in PBS IX pH 7.4, previously calibrated with gel filtration HMW calibration kit.

[0237] 6- Protein formulation in vaccines

[0238] EHDV recombinant subunit vaccines were formulated using Freund's adjuvant. Briefly, 2 mL of VP2 complete or VP2 truncated and modified at 0.6 mg / mL were mixed with 2 mL of complete Freund's adjuvant, (for the first dose) and incomplete Freund's adjuvant (for second dose) using vigorous agitation for 5 min. Emulsion state was checked overnight. Each formulation contained 0.3 mg / mL of antigen.

[0239] 7- Mice immunization and challenge

[0240] Three groups of male IFNAR(- / -) mice (n=5) were intraperitoneally immunized following a homologous prime-boost regime separated 4 weeks and consisting of 100 pL of placebo or 30 pg / dose of complete VP2 or truncated and modified VP2 proteins. Animals were subcutaneously challenged with a lethal dose of EHDV-8 (100 PFU) two weeks post-boost. Sera of immunized and control animals were collected three weeks post-prime and two weeks post-boost for the analysis of the neutralizing antibody response. After virus challenge, mice were daily examined for survival and clinical signs, and blood samples were collected at 3, 5, 7, 10 and 14 d.p.i. for the analysis of viremia and RNAemia by plaque assay in Vero cells and RT-qPCR, respectively. Results

[0241] 1- Protein design

[0242] To reduce the size of the VP2 maintaining the immunogenic domain, we used as a model to define the EHDV VP2 protein domains the same protein derived from bluetongue virus which shows very similar structure (Figure 1). A complete deletion of trimerization domain and hairpin domain was done. Removing these domains, hydrophobic amino acids that could remain exposed to solvent were eliminated, avoiding the propensity of aggregation. However, removing the trimerization domain, the trimerization was not possible and, for that reason, an external, continuous trimerization domain, T4 fibritin domain or foldon (FD4), was added, including a linker between truncated VP2 capsid protein and the FD4 domain, to increase flexibility among domains. To visualized both proteins, Swiss-PdbViewer was used (see Figure 2).

[0243] 2-. Baculovirus generation

[0244] The gene sequences designed were synthesized and used to obtain the corresponding recombinant baculoviruses, using the Bac-to-Bac system (Invitrogen), based on the transposition of the sequence of interest from the pFastBac vector to the baculoviral DNA. The donor vectors were transformed in DHIOBac E coli competent cells which contains a helper plasmid expressing the transposase jointly with a bacmid with the baculovirus genome carrying a prokaryotic replication origin and the mini Tn7-replicon.

[0245] The bacmids generated were isolated and checked by PCR to confirm the correct genes transposition. Two PCR reactions designed with specific primers to detect either, the whole transposition cassette (PCR 1) or the expression cassette (PCR 2) were performed, an empty bacmid without gene transposition was used as negative control. Expected size bands were obtained in both PCRs (Table 1 and Figure 3).

[0246] Table 1: PCR bands size

[0247] Bacmid name PCR 1 size band (bp) PCR 2 size band (bp) Bac VP2-EHDV-8 5240 3130

[0248] Bac VP2t-EHDV-8 Mod 4630 2530

[0249]

[0250] To generate the recombinant baculoviruses (rBV) to express the corresponding protein, the purified bacmids DNAs were transfected to sf9-RVN cells. The cell supernatants harvested from transfected cells contains the passages 0 generated (pO). These pO baculoviruses were used as inoculum for an amplification round in the same cell line, generating the corresponding passages 1 viruses (pl). The pl viruses were titrated with a plaque assay protocol. (Table 2)

[0251] Table 2: Baculovirus titer

[0252] BV name Passage Titer (pfu / ml)

[0253] Bac VP2-EHDV-8 pl RVN 6,4E+08

[0254] Bac VP2t-EHDV-8 Mod pl RVN 4,lE+08

[0255]

[0256] The rBVs passages were also analyzed by PCR using the same two sets of primers for PCR 1 and PCR 2 used with the bacmids and the bacmids DNA as positive control of PCR amplification. As shown in Figure 4 bands of the expected sizes (the same of bacmids) were obtained for all viruses' passages with both PCR reactions.

[0257] The rBVs passages were analyzed for protein expression in sf-9 RVN cells. To perform this analysis, infected cell cultures at 0.1 multiplicity of infection using the pl of rBVs were collected, spun down and the pellets were lysed with RIPA buffer. The lysates were loaded into SDS-PAGE and gels stained by Coomassie blue. Results are shown in Figure 5.

[0258] 3- Protein expression in pupae

[0259] Good expression level was achieved for full-length VP2 in all the conditions tested, however, the protein remains mostly insoluble usingTris 20 mM, NaCI 300 mM, Arginine 50 mM, Tween 801%, pH 7.5 as extraction buffer. The condition selected for protein expression was 50,000 PFUs / insect, 23°C and 6 days of incubation.

[0260] An increase in protein expression was observed in VP2 truncated and modified molecule, compared to the full-length VP2, as shown in figure 5. The best expression condition for VP2 truncated and modified was 50,000 PFUs / insect, 23°C and 5 days. This modified protein showed higher percentages in the soluble fraction, facilitating its purification, with respect to the native VP2 protein in pupae extracts (Figure 7).

[0261] 4- Purification of the two VP2 protein versions and trimers formation

[0262] Full-length wild-type VP2 could be purified, however, protein losses were observed during protein extraction, despite of optimizing extraction buffer to improve protein solubility, and during protein load into IMAC resin. After protein conditioning in PBS IX pH 7.4, protein concentration was stablished at 0.6 mg / mL, with a purity of more than 80%. The final yield was as low as 6 mg per Liter of pupae extract (O.lmg of protein / g of pupae) due to the losses previously mentioned (Figure 7 C).

[0263] Truncated and modified VP2 molecule could be purified easier than VP2 complete, with 10 times more yield (60 mg per Liter of pupae extract) and similar levels of purity (more than 85%) (Figure 6 C). As previously described, due to the mutations, an increase of total amount and protein solubility was achieved. The final protein concentration was stablished at a 0.8 mg / mL.

[0264] Protein trimerization was checked using size exclusion chromatography with Superose 6 10 / 300 from cytiva previously calibrated. Analysis of the wild-type full-length VP2 revealed a principal peak with a residence time of 14.5 mL that could correspond to trimer of VP2, and a peak of proteins with lower molecular weight that could be protein monomers or protein degradation due to the width of this elution peak, Figure 8A.

[0265] On the other hand, SEC analysis of truncated and modified VP2 reveals multiple protein populations, however, principal peak at retention time of 14.85 min could be considered as trimeric population, and in less concentration ratio the 17.14 min peak corresponding to monomer fraction, Figure 8B.

[0266] 5- Recombinant VP2 proteins induced neutralizing antibodies and protected against a virulent EHDV challenge vaccinated model animals

[0267] The recombinant VP2 proteins were analyzed by ELISA for their recognition by sera from bovines naturally infected by EHDV or BTV viruses. As shown in Figure 9, only sera from animals suffering EHDV infection recognized the two recombinant proteins derived from EHDV but not by sera derived from BTV infected, discarding cross reactivity between VP2 proteins from different origin

[0268] Three groups (n=5) of mice were submitted to a prime-boost vaccination regime. Two of them were vaccinated intramuscularly with 30 pL vaccine containing 30 pg of every VP2 protein, adjuvanted with Freund's adjuvant (complete in the first vaccination and incomplete with the second). Second vaccination was administered 4 weeks after the first vaccination. A third group were used as control group, vaccinated with the adjuvant without any VP2 protein. All mice were challenged with the homologous virulent EHDV-8 virus (100 PFUs / mice) two weeks after the second immunization.

[0269] High titers of neutralizing antibodies were detected after the prime vaccination with both VP2 proteins. These antibody levels were increased after the boost immunization and challenge (Figure 10). No significant differences were observed in the induction of neutralizing antibody titers among the two VP2 versions used to formulate the vaccines.

[0270] After virulent virus lethal dose challenge, 100% of vaccinated mice with every VP2 protein survived to infection without any clinical symptom. In contrast, none of the mice belonging to the control group survived after 7 days post-challenge (Figure 11).

[0271] Neither viremia nor RNAemia were detected in groups vaccinated with VP2 molecules (Figure 12), indicating that both protein versions were able to confer a solid protection against EHDV.

[0272] 6- CLUSTAL 0(1.2.4) multiple sequence alignment

[0273] Annealing obtained using CLUSTAL OMEGA (1.2.4) multiple sequence alignment from EMBL-EBL The sequences used for the annealing are noted below.

[0274] Legend:

[0275] Hub domain in grey.

[0276] Hairpin domain in italic.

[0277] Antigenic domain in bold Body domain underlined.

[0278] VP2-EHDV2 VP2-EHDV7 VP2-EHDV1 VP2-EHDV6 VP2-EHDV8 VP2-EHDV4 VP2-EHDV5 MESLDIIVFHGEEGFNNVYAYDYLLALNTKQAGNKDWTKITSLVNAETVELIQAPAIELL 60

[0279] VP2-EHDV2 SYESKEDYGVI FPDALSIGIRRYDWRRAEYSKNQSVERSNGIISSEDAFEELIRSSSANV 120 VP2-EHDV7 SYESKMGYDII FPDALSIGIRRYDWRHAKNSKDPAKGEEQGLVTSEGAFEELMRSSSSNT 120 VP2-EHDV1 RQNGRDGYKLII PEALSTGIIRYENKTKGA - QSEVELENQLRSSIRHQ 105 VP2-EHDV6 DRAIGDEYRINFPDAVNYGIMRYDNEHERYNERGVILL — EHVKPVGEYEIMLRTSIGHQ 118 VP2-EHDV8 ERTLSDEYKESFPDAENYGEMRTYDDEHEKYNEKREMLA — ESLKPTGEYE ELERT SVKHQ 118 VP2-EHDV4 TKAGSPDYRITVPSALNYGIRRYDMKKEKEKSKGGIGAGRELWMSEKDFESLMLTDSNNQ 120 VP2-EHDV5 TREGSADYRITVPSAINYGIRRYDMKKTRDRRNTTREKEGDCWVSEKDYETLMLTDLGNQ 120

[0280] VP2-EHDV2 KLKT — DFSGGRIHHELSYCDIHTDATIAETIEIDAHNNEEEGCFHGENTWYNHLLTES 178 VP2-EHDV7 178 VP2-EHDV1 0G 165 VP2-EHDV6 OG 178 VP2-EHDV8 IT 178 VP2-EHDV4 ID 180 VP2-EHDV5 KIKTGVDYTNRRIRIQLQQLDILMHPKFVETLSVWFCDEKDQCDHTRSDIAYNRVLHGD 180

[0281]

[0282] VP2-EHDV2 NYIGSGTCYDLGDHIQLRTIGDVGPRPRDHVDVL-GRTHPRGEKHIIRRYGG-DEIKTLT 236 VP2-EHDV7 ICIGSGTCYDLDNHIQIKTIGEVSSYARDHTDVL-GRFHPQGEKRISRRYQG-NEIKMLS 236 VP2-EHDV1 INVGTGTCYDLSSRLKLRVIGDVDRHRRSMQNVL-GRVIHTGDPKIINRVNQIGSQQFID 224 VP2-EHDV6 LMIGSGTCYDLLKQLELIVIGEIKPSTRERQNVITRQMI PIGTPEITNREPYKNKQTKIQ 238 VP2-EHDV8 IMIGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQTKIQ 238 VP2-EHDV4 LHIMAGTCFDIDQSIQLNIIGEVGVNRHERQLAL-GGVQRVGDKI FTKRFQTADQQKVLH 239 VP2-EHDV5 LHIMAGTCYDLAQNIQLNIIGEVGVNAHDRQLAL-GGVLRRGEKI FTSRVQNVAHQKVIR 239

[0283] VP2-EHDV2 TSMSPDEFELKKKILNGDVAIGVEKRNLIKYSNEILQLDDIAASWIRSQNANDLEKIVAL 296 VP2-EHDV7 VSITPADFDLKKRI FNGDIALEVEKRNLLQYDDEILQMDRIAEKWIDNQSADDSDKIITL 296 VP2-EHDV1 RAIGPDKFELKREI FDRLKALDVDVRKVIREEEASAELDEMGRRWMRDQNVNIVNDIIQS 284 VP2-EHDV6 AALGPRVNELKREI FSGKYGLEVKHVARLLDDPLITRLDVIAEEWMQRQSDEKVDELCDL 298 VP2-EHDV8 ASQGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNL 298 VP2-EHDV4 MTVASNLYPEKAALYDGQRGASVKVKEGFELDAESRALDEIAIEWKRHPTGARTSDILRL 299 VP2-EHDV5 AGTPSDLFLEKVALFNGERGADVKTREGIAPSAETIALDDIAAKWKQHPTDEYTRDIIRL 299

[0284] VP2-EHDV2 LERLGEKDQKVEPH — NSNDIRERFRRKLLQNLQK — TDGEIRNIRNYHQQDATKRFAAV 352 VP2-EHDV7 LEEIGRKDKRIEPV — NSEDMRNRFKQQLVINLRK — TDGEVRNIRDNQRQGKPKRLATV 352 VP2-EHDV1 LVKKGSRSEKLAHR — NEQGMQARFRRTIATNLRDQRQGKEVLNIRGTRDQPEEKKFAAV 342 VP2-EHDV6 LEAKGKQIKMAGTSADYCKKARSRLHGVLKANLVK — TTDEIGNIRAVRNENAGSILAAV 356 VP2-EHDV8 LEAKGKEMKIARTNTEYYRGARTRFYNVLKQNLVK — TTDEIRNIRGVSNEKAGSILAAV 356 VP2-EHDV4 CTRKGKREGYTDPQ — GAERLHARLLQNFSRFLGA — NTQEIRNIRAE-DKSDGEI FAAT 354 VP2-EHDV5 CKAKGRWSGYTEPS — NSDAMDSRLEGNLNRYLKN — VTQEIQNIRSE-NKGAGPTLAAA 354

[0285] VP2-EHDV2 LIVTMCDTMNRAIWGDNRFKLVRGVYEYAKYRMGSIYYSMRTDVTWQLRTSYVD — ACPR 410 VP2-EHDV7 LMITVCDVLKRAIWANDRFELVRGTYTYARCRLGSVYNAMWKDMAWQLRPAYKD — SCPR 410 VP2-EHDV1 LLMTGCDIVERAIWSNEETAILRGLYAYAHDKLGCVYRAMKKDFVWSIRPTYTD — RCAG 400 VP2-EHDV6 LWSACDSRKRAIWYNDDSPIYRGIMLYATEKFGSVYYGLRRRFMWSIRPTYVD — SCPR 414 VP2-EHDV8 LLVSACDSKKRAIWYDDDSPIYRGVMLYATEKLGCVYYGLRKRFTWSIRSTYVD — ECRK 414 VP2-EHDV4 LMVITSDSMERTIWSEEKYSVLRGIYLYCREKLGPIYYTLRKDFRWRIRDRYTDRTKCWN 414 VP2-EHDV5 LIIITCDSLERTIWDNEAHSVLRGIYLYGQEKLGSVYYTLRKDFRWSIRERYTDRSTCWR 414

[0286] VP2-EHDV2 ICDRRRYIMQRYDYFSLNRETGDSIYKWDVGDIRG-GKKTSRWEGWPYKSIEDEEE-DEE 468 VP2-EHDV7 ICDRRKYIMQRYDYFSLSREIGDTIYKWDVKNLRG-DEKTNREMGWLYKAEEDEDE-DED 468 VP2-EHDV1 VCDRKRTIMVREDYFDLQREENDSVYKWIITEWDKNDVIISAKNGYLYSKYSGEDE — DD 458 VP2-EHDV6 VCDRRQTFMTRI PYFDLNQEEGDSIYKWNLEPILR-DVKTTRMDGYPYEAYDGDDE — DA 471 VP2-EHDV8 VCDRRQTFMTRI PYFDLNQEEGDSIYKWNLVALGR-EVKTNWMDGYPYEAYAEDDE — DQ 471 VP2-EHDV4 VCDQRQYFMNRYNYYDLRLEAGNTIYKWKVEKIEQ-EAKTNQDEGYPYKKFDGEEEDDPD 473 VP2-EHDV5 VCDRKQYFMARYNYYGLRLEAGDTIYKWEIKKMDL-EKETTSEEGYPYSKYEGAEEDDDD 473 VP2-EHDV2 VLIHDFDEDKYTQYMQRVIQGPWVEKDGIGVLMKEEAAIELFDFTRDAYVDEAGFLRLPA 528 VP2-EHDV7 TIIHDFDEDKYTEYMQRVIQGPWVEKDGIEILMKEGTAIEKFDFAQDAYVDEAGFLRLPP 528 VP2-EHDV1 ILVHEIDDRLYTAMIDRILINGWIEKEGLSQIIKEEVRLESFDFTKDAYVDEAGFLVLPE 518 VP2-EHDV6 GLVHDIDQRKYREMIQRIIDNEWQEKDGIATIILDVGGIEKYDFTKDAYIDEAGFIKLPD 531 VP2 -EHDV8 VLVHDIDSRKYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPD 531 VP2-EHDV4 VIVHSIDAEKYNTFMQRIINNGWQEKDGIGQIMEERSGIENFYFTKDAYIDDRGFLVLPP 533 VP2-EHDV5 TIVHNIDGAKYSAFLQRVINNGWQEKDGIGQIMEERSGLENFQFTKDAYIDDRGFLVLPQ 533 VP2-EHDV2 YYNKTIKSSLYESSFKIRRVEIMHGKKADPWTKKTNDELKKENEMWLLPLPTWDKTLCL 588 VP2-EHDV7 YYNKLIKSSLYESSFRIRRIDIKKNKEADPWIQKTEDEVKKENEMWLLPLRSILDRALCF 588 VP2-EHDV1 YYDRVIASNIYDCKFKISRVSITSSSNDDPWDKKTADSIIDEQCLWKIPLPNIIDVRPCF 578 VP2-EHDV6 YYGKQIRSKLYGYSFEITRVSITASKTEDPWHQKTGGKLINEGELWRVPLDNIIDVTQCL 591 VP2-EHDV8 YYDKLIKSTMYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCL 591 VP2-EHDV4 YYDKKIKSTLYQSSFHVRRVTITSPGTEDPWVIKTADKEISEERTWLLPLDHIVDTVPCL 593 VP2-EHDV5 YYEKRIKSTLYCSEFCIRRVAITSRGTDDPWVRKTADKEVTEEDTWLLPLDHIIDTVPCF 593 VP2-EHDV2 TGNILSTIKQEQSARFTAIIEALKKEKEIVKRKYTR-DDTYTCPMLNVLNYTGYRQRRFI 647 VP2-EHDV7 TGNILSTARQEQSARFTAIIDALKKDENWRRKYSR-NDGYTCPTLNVLGYTGYRQRRFV 647 VP2-EHDV1 RGDLLTSNSQEYSKRFSGIIDELKKDKEIYDDFIPVQEGVRPCVQGHVCRYAFYRQKLTI 638 VP2-EHDV6 SGRAISDAKQKRSVRFDELLEEDE - DGENGECVQKYVLGKIRKKLFTRV 639 VP2-EHDV8 SGSSVSNTKQQRSARFNELVAE - DGEDKDCVQKYVLTKIRNETFIRV 637 VP2-EHDV4 RGQGLSDVEQQTSGRFNEIINDIKKDEQLKAFFPDEINEEDYCPAKTVLKYVSLRKRIHV 653 VP2-EHDV5 RGQILSNQKQPVSARFGEIIQELKLDDYNEKILQDKIQGEDYCPTKIILGYVSLRKRMKV 653 VP2-EHDV2 FSILKNHLPKDLLMEVYQDPDEEYDPHDYTDCMGKEEVLVGMRSI FEVILYLIHLGFENQ 707 VP2-EHDV7 FSILKNHLPKDVLISMYPEEEVEYDPRDYTDCNGREEVLTRMKSIFDWLYLIQMGFEKQ 707 VP2-EHDV1 FTILKRYYPIERILELTDEEDYEYNLYLDKECYKKESLILNLRSIFSLICFLIDFGYEGR 698 VP2-EHDV6 FSILKWYFPTEYIDSLIGEDEYVYDTEMFNDIFNEDEFINEQQSLSSMVLSMIIR - 694 VP2-EHDV8 YSILKWYYPTTLIDELIGEDEYEYHAEEYSDTFGEQNMIISSRKFSSLIVSMIIK - 692 VP2-EHDV4 FSVLKWFMPVERLREFIGSDDYEYVGEDHTQVFQLEQEIERRHSYTSIVLYVIELGYERI 713 VP2-EHDV5 FSILKWYMPVSRIEEYIGSEEYKYTTERYDQVFGMEHEIEIRKSYTSVILYIIELGYELQ 713 VP2-EHDV2 ITTYSEEEIRVIKHR - MIKKEHRDGIVDILAPNFSRIIRENEKMIKIEKYEDL 759 VP2-EHDV7 ISTLSEEDIRILKQR - MVGGESRNEVLQNLAPNFLRIIKAGEKTADAMKNEDL 759 VP2-EHDV1 EITRGEDEYLK - I FNEINYGGHARKEAINKYFPQFYQRLM - RVRTSENIEDL 748 VP2-EHDV6 — AYKDEQVDELRNSTTYLYRMTERRGKEREAFLKKSMPKFYAKIL - KVREAEKVEDI 749 VP2-EHDV8 — AYGDDRVKEISNSVTLLYRLLEYKGKEREEYLKRIMPKFYAKIS - EVRVAEKVEDM 747 VP2-EHDV4 VNMYTDEQIRVLQEI LRSNEEGSRNDWIQKNLPCFYECFK - KVPLAKKVEDV 764 VP2-EHDV5 VRTYTDEEIRVLEEV LRTNDNEVRTLWFQKNLPAFFSKLS - ALPKAKIVEDL 764 VP2-EHDV2 LPMYFYQALVLSNEMIYENMNKSHPMLMFCDNRVRIVPVQTNSWRKSVPLLSTLFILKYY 819 VP2-EHDV7 LPMYFYQALILSNEIIYEDANKSHPMLMLCEKRVRIVPVRTNFWLKWPLLSVLHIMKYH 819 VP2-EHDV1 LPLAFYQALLLSDPCTDNSEKSSHPLILFCQDKVRWPIRTATQERGLPLLCCIHI FKFH 808 VP2-EHDV6 LPFIFLQALLTSIDHQKLDKRVSLPFFLFCKEEERIIPVSFKDSLIPLPLLQVLHIMRFH 809 VP2-EHDV8 LPLVFFQALLMSENQWVMDKRVSLPLILFCKDQERIVPISLKSNMISSPLLQLLHIMRFH 807 VP2-EHDV4 VPLVLYQALVLSTYPRAANQNKSHPLFLFEKDKLRIVPINTSNQRGDYELMRGLYIMRFH 824 VP2-EHDV5 I PLVLYQALIMSIYPRAANQNRSHPFFLFEEGKLRIVPLKTANHRGDYELMRCLHVMRFH 824 VP2-EHDV2 VP2-EHDV7 VP2-EHDV1 VP2-EHDV6 VP2-EHDV8 VP2-EHDV4 VP2-EHDV5

[0287]

[0288] VP2-EHDV2 VP2-EHDV7 VP2-EHDV1 VP2-EHDV6 VP2-EHDV8 VP2-EHDV4 VP2-EHDV5

[0289]

[0290] VP2-EHDV2 YGEGDIKCNVLDKFCCGKKTKIIRVRLNGKVYANPEIISKLMN 982

[0291] VP2-EHDV7 YGEGNVKCSILEKFCCGKKTKIVRVKLNGKVYANPEIISKLMN 982

[0292] VP2-EHDV1 HHSGNIQARILEKVFFEHKWHIVQVKLNGKI FENHELITKLMN 971

[0293] VP2-EHDV6 N 972

[0294] VP2-EHDV8 970 VP2-EHDV4 IMCENIQGRQLKKWLGHKMELLHIKFPGRVFENHEIITKLVN 987

[0295] VP2-EHDV5 ILCENLKGKRLNKWMGHKMELLHVKFPGRVFENHEWTKLVN 987

[0296] Serotypes used in the annealing above:

[0297] EHDV-1 VP2. UNIPROT C8TE59 strain New jersey MEDINLTIFSDDALPHAVWDDYVIAIKRNPNGTFLEPHQMYDRYTQEFLQGKARDVLRQNGRDGYKLIIPEALS TGIIRYENKTKGAQSEVELENQLRSSIRHQRVKPRMDEAHRKLQI ELRGGQILLHPRIAESIEFSIISKENATCSHTPVNCAYEVLLSGGINVGTGTCYDLSSRL KLRVIGDVDRHRRSMQNVLGRVIHTGDPKIINRVNQIGSQQFIDRAIGPDKFELKREIFD RLKALDVDVRKVIREEEASAELDEMGRRWMRDQNVNIVNDIIQSLVKKGSRSEKLAHRNE QGMQARFRRTIATNLRDQRQGKEVLNIRGTRDQPEEKKFAAVLLMTGCDIVERAIWSNEE TAILRGLYAYAHDKLGCVYRAMKKDFVWSIRPTYTDRCAGVCDRKRTIMVREDYFDLQRE ENDSVYKWIITEWDKNDVIISAKNGYLYSKYSGEDEDDILVHEIDDRLYTAMIDRILING WIEKEGLSQIIKEEVRLESFDFTKDAYVDEAGFLVLPEYYDRVIASNIYDCKFKISRVSI TSSSNDDPWDKKTADSIIDEQCLWKIPLPNIIDVRPCFRGDLLTSNSQEYSKRFSGIIDE LKKDKEIYDDFIPVQEGVRPCVQGHVCRYAFYRQKLTIFTILKRYYPIERILELTDEEDY EYNLYLDKECYKKESLILNLRSIFSLICFLIDFGYEGREITRGEDEYLKIFNEINYGGHA RKEAINKYFPQFYQRLMRVRTSENIEDLLPLAFYQALLLSDPCTDNSEKSSHPLILFCQD KVRWPIRTATQERGLPLLCCIHIFKFHPGLQMRKKELEDDIKKTLPAIFDYWIELEMKR LDTGDRLRTRAQMVELYYSTNCGGSYETLNFVFPIVHPNKGFIACVISSKGGMGALNEDD VRRRFRRIQSSIQGIFSISIDEEMEIQLHHSGNIQARILEKVFFEHKWHIVQVKLNGKIF ENHELITKLMN ( SEQ ID NO.: 15 )

[0298] EHDV-2 VP2. UNIPROT C8TE77 strain Alberta MEEIFFSVIDSSQLIPKQLYENYPWIDVGHRKNEGRLPVERLEDKHTIELIQAEARNLF SYESKEDYGVIFPDALSIGIRRYDWRRAEYSKNQSVERSNGIISSEDAFEELIRSSSANV KLKTDFSGGRIHHELSYCDIHTDATIAETIEIDAHNNEEEGCFHGENTWYNHLLTESNY IGSGTCYDLGDHIQLRTIGDVGPRPRDHVDVLGRTHPRGEKHIIRRYGGDEIKTLTTSMS PDEFELKKKILNGDVAIGVEKRNLIKYSNEILQLDDIAASWIRSQNANDLEKIVALLERL GEKDQKVEPHNSNDIRERFRRKLLQNLQKTDGEIRNIRNYHQQDATKRFAAVLIVTMCDT MNRAIWGDNRFKLVRGVYEYAKYRMGSIYYSMRTDVTWQLRTSYVDACPRICDRRRYIMQ RYDYFSLNRETGDSIYKWDVGDIRGGKKTSRWEGWPYKSIEDEEEDEEVLIHDFDEDKYT QYMQRVIQGPWVEKDGIGVLMKEEAAIELFDFTRDAYVDEAGFLRLPAYYNKTIKSSLYE SSFKIRRVEIMHGKKADPWTKKTNDELKKENEMWLLPLPTWDKTLCLTGNILSTIKQEQ SARFTAIIEALKKEKEIVKRKYTRDDTYTCPMLNVLNYTGYRQRRFIFSILKNHLPKDLL MEVYQDPDEEYDPHDYTDCMGKEEVLVGMRSIFEVILYLIHLGFENQITTYSEEEIRVIK HRMIKKEHRDGIVDILAPNFSRIIRENEKMIKIEKYEDLLPMYFYQALVLSNEMIYENMN KSHPMLMFCDNRVRIVPVQTNSWRKSVPLLSTLFILKYYAGWRKREETIEDDIRTVWPHL TRYWLDVEFQRREIADGTVIRMQPLKTHLSTYCSYMSEVYSFALPIVHPTKGIIAVGVIP DVI SNAQGFS I I KQRFYS I DRYVHARVI LRI QKDGSVNVYGEGDI KCNVLDKFCCGKKTK IIRVRLNGKVYANPEIISKLMN ( SEQ ID NO.: 16)

[0299] EHDV-4 VP2. UNIPROT C8TE95 strain IbAr 33853 MESLDVIVFHGDDGFDKIFVSDYLIALNTHQGGNKDWSKITSLVGANTLELLQTPATGIL TKAGSPDYRITVPSALNYGIRRYDMKKEKEKSKGGIGAGRELWMSEKDFESLMLTDSNNQ KIKAGVDFTGRRVQIQLQQTDVLIHPRMLETLSIITVGVGKKGCDHTRSDVMYERVYQGD LHIMAGTCFDIDQSIQLNIIGEVGVNRHERQLALGGVQRVGDKIFTKRFQTADQQKVLHM TVASNLYPEKAALYDGQRGASVKVKEGFELDAESRALDEIAIEWKRHPTGARTSDILRLC TRKGKREGYTDPQGAERLHARLLQNFSRFLGANTQEIRNIRAEDKSDGEIFAATLMVITS DSMERTIWSEEKYSVLRGIYLYCREKLGPIYYTLRKDFRWRIRDRYTDRTKCWNVCDQRQ YFMNRYNYYDLRLEAGNTIYKWKVEKIEQEAKTNQDEGYPYKKFDGEEEDDPDVIVHSID AEKYNTFMQRIINNGWQEKDGIGQIMEERSGIENFYFTKDAYIDDRGFLVLPPYYDKKIK STLYQSSFHVRRVTITSPGTEDPWVIKTADKEISEERTWLLPLDHIVDTVPCLRGQGLSD VEQQTSGRFNEIINDIKKDEQLKAFFPDEINEEDYCPAKTVLKYVSLRKRIHVFSVLKWF MPVERLREFIGSDDYEYVGEDHTQVFQLEQEIERRHSYTSIVLYVIELGYERIVNMYTDE QIRVLQEILRSNEEGSRNDWIQKNLPCFYECFKKVPLAKKVEDWPLVLYQALVLSTYPR AANQNKSHPLFLFEKDKLRIVPINTSNQRGDYELMRGLYIMRFHYGFKARRNDLEADLLS VIKKVWEYWERLSSDPVALDDARRIRKELIKAHIYGYCGIIQTAATFVLPITSPKKGFIV LAISQDIVKLADLQNVIRAQYHDIANYIIGICGVTLGRQGHMEAIMCENIQGRQLKKWL GHKMELLHIKFPGRVFENHEIITKLVN ( SEQ ID NO.: 17 )

[0300] EHDV-5 VP2. UNIPROT C8TEA4 strain CSIRO 157 MESLDIIVFHGEEGFNNVYAYDYLLALNTKQAGNKDWTKITSLVNAETVELIQAPAIELL TREGSADYRITVPSAINYGIRRYDMKKTRDRRNTTREKEGDCWVSEKDYETLMLTDLGNQ KIKTGVDYTNRRIRIQLQQLDILMHPKFVETLSVWFCDEKDQCDHTRSDIAYNRVLHGD LHIMAGTCYDLAQNIQLNIIGEVGVNAHDRQLALGGVLRRGEKIFTSRVQNVAHQKVIRA GTPSDLFLEKVALFNGERGADVKTREGIAPSAETIALDDIAAKWKQHPTDEYTRDIIRLC KAKGRWSGYTEPSNSDAMDSRLEGNLNRYLKNVTQEIQNIRSENKGAGPTLAAALIIITC DSLERTIWDNEAHSVLRGIYLYGQEKLGSVYYTLRKDFRWSIRERYTDRSTCWRVCDRKQ YFMARYNYYGLRLEAGDTIYKWEIKKMDLEKETTSEEGYPYSKYEGAEEDDDDTIVHNID GAKYSAFLQRVINNGWQEKDGIGQIMEERSGLENFQFTKDAYIDDRGFLVLPQYYEKRIK STLYCSEFCIRRVAITSRGTDDPWVRKTADKEVTEEDTWLLPLDHIIDTVPCFRGQILSN QKQPVSARFGEIIQELKLDDYNEKILQDKIQGEDYCPTKIILGYVSLRKRMKVFSILKWY MPVSRIEEYIGSEEYKYTTERYDQVFGMEHEIEIRKSYTSVILYIIELGYELQVRTYTDE EIRVLEEVLRTNDNEVRTLWFQKNLPAFFSKLSALPKAKIVEDLIPLVLYQALIMSIYPR AANQNRSHPFFLFEEGKLRIVPLKTANHRGDYELMRCLHVMRFHYGFKARRHELEADVLD VIGKIWKYWDAMACDMVALDDTRRVRNELTKAHMYTYCGWRTAAIFVLPITNPEKGFIA LIISQKGANLSFLRDWRARFNDVEKYLIGMCGVWGEGGHIESILCENLKGKRLNKWM GHKMELLHVKFPGRVFENHEWTKLVN ( SEQ ID NO.: 18 )

[0301] EHDV-6 VP2. UNIPROT C8TEB3 strain CSIRO 753 MESVEFAIINKQQQPDESIIYDYLTTIQTRTYDEKLRGKVEHMINKDTIELTQGEARNFL DRAIGDEYRINFPDAVNYGIMRYDNEHERYNERGVILLEHVKPVGEYEIMLRTSIGHQRI KPEYGTQNARITFLFSGGNLRIHSKFVESLKFEIVNYETEDCDHTRWKINYDMILEGGLM IGSGTCYDLLKQLELIVIGEIKPSTRERQNVITRQMIPIGTPEITNREPYKNKQTKIQAA LGPRVNELKREIFSGKYGLEVKHVARLLDDPLITRLDVIAEEWMQRQSDEKVDELCDLLE AKGKQI KMAGT SADYCKKARS RLHGVLKANLVKTTDEI GNI RAVRNENAGS I LAAVLWS ACDSRKRAIWYNDDSPIYRGIMLYATEKFGSVYYGLRRRFMWSIRPTYVDSCPRVCDRRQ TFMTRIPYFDLNQEEGDSIYKWNLEPILRDVKTTRMDGYPYEAYDGDDEDAGLVHDIDQR KYREMIQRIIDNEWQEKDGIATIILDVGGIEKYDFTKDAYIDEAGFIKLPDYYGKQIRSK LYGYSFEITRVSITASKTEDPWHQKTGGKLINEGELWRVPLDNIIDVTQCLSGRAISDAK QKRSVRFDELLEEDEDGENGECVQKYVLGKIRKKLFTRVFSILKWYFPTEYIDSLIGEDE YVYDTEMFNDIFNEDEFINEQQSLSSMVLSMIIRAYKDEQVDELRNSTTYLYRMTERRGK EREAFLKKSMPKFYAKILKVREAEKVEDILPFIFLQALLTSIDHQKLDKRVSLPFFLFCK EEERIIPVSFKDSLIPLPLLQVLHIMRFHPGEERRRKQVGAEVKEILPKLLDFWFNLAYD RKALNAVEHISEQYVKNIVCSYCGGNEWASFILPITHPEKGFIWITTTEDVANSNAEA I VKS RFGEVAKYI KGWHI S I TQDGVARVRGGGGI KS RI LEKWLGTRFQLVQI KMGENV FENHELVTKLMN ( SEQ ID NO.: 19 )

[0302] EHDV-7 VP2. UNIPROT C9QNL4 strain CSIRO 775 MEEIFFSVIDSTKQIPKQLYDGYPWIDIGRRKGEGKLNVEHLQDKHTIELVQAEARELF SYESKMGYDIIFPDALSIGIRRYDWRHAKNSKDPAKGEEQGLVTSEGAFEELMRSSSSNT KLKSDFKGEQIHHELSFCDVYVNATIAETLEINAHNNEKDNCFHGEDTILYNHLLTEAIC IGSGTCYDLDNHIQIKTIGEVSSYARDHTDVLGRFHPQGEKRISRRYQGNEIKMLSVSIT PADFDLKKRIFNGDIALEVEKRNLLQYDDEILQMDRIAEKWIDNQSADDSDKIITLLEEI GRKDKRIEPVNSEDMRNRFKQQLVINLRKTDGEVRNIRDNQRQGKPKRLATVLMITVCDV LKRAIWANDRFELVRGTYTYARCRLGSVYNAMWKDMAWQLRPAYKDSCPRICDRRKYIMQ RYDYFSLSREIGDTIYKWDVKNLRGDEKTNREMGWLYKAEEDEDEDEDTIIHDFDEDKYT EYMQRVI QGPWVEKDGI El LMKEGTAI EKFDFAQDAYVDEAGFLRLP P YYNKLI KS S LYE SSFRIRRIDIKKNKEADPWIQKTEDEVKKENEMWLLPLRSILDRALCFTGNILSTARQEQ SARFTAIIDALKKDENWRRKYSRNDGYTCPTLNVLGYTGYRQRRFVFSILKNHLPKDVL ISMYPEEEVEYDPRDYTDCNGREEVLTRMKSIFDWLYLIQMGFEKQISTLSEEDIRILK QRMVGGESRNEVLQNLAPNFLRIIKAGEKTADAMKNEDLLPMYFYQALILSNEIIYEDAN KSHPMLMLCEKRVRIVPVRTNFWLKWPLLSVLHIMKYHAGWRTREEPTEEDIRTVWPYL TKYWLNLEFKRKEIIDPTMIRIHPMNTHLSTYCNCMSEVYSFALPIVHPAKGIIIVGIVP HAVSNAQGFS I LKQRFS S I VRYVHACVLLKVQEDGHVQVYGEGNVKCS I LEKFCCGKKTK IVRVKLNGKVYANPEIISKLMN ( SEQ ID NO.: 20 ) EHDV-8 VP2. GenBank: OP897266.1

[0303] strain: 2022. TE.50459.1.2. Sardinia & Andalusia MDSVEFAILNTTQRPDESVIYDYLASVWTRVYKDELEGKMEHLV ANTIELTRGEARNVF ERTLSDEYKISFPDAINYGIMRYDDEHEKYNEKRLMLAESLKPTGEYEILLRTSVKHQRI KPEYGAQNARISFSFSGGTLRIHSKFVESLKFDWNYEKEDCDHTNWDVHYDMIQQGTIM IGSGTCYDLLKQLELIWGDIRLSTQERRHLITQQIIPVGTPEITNREPYKDEQTKIQAS QGPRLNELKKNVFSRRYGLKVKYLERLQDDPIITRLDEIAEEWMRRQSGEKIEELCNLLE AKGKEMKIARTNTEYYRGARTRFYNVLKQNLVKTTDEIRNIRGVSNEKAGSILAAVLLVS ACDSKKRAIWYDDDSPIYRGVMLYATEKLGCVYYGLRKRFTWSIRSTYVDECRKVCDRRQ TFMTRIPYFDLNQEEGDSIYKWNLVALGREVKTNWMDGYPYEAYAEDDEDQVLVHDIDSR KYSTMIQRVIDHGWSEKDGISTIISDIGGIEKYDFTKDAYIDEAGFVKLPDYYDKLIKST MYGFSFKITRVSITSSKTNDPWHKKTADNLISEMELWRVPLDNVIDVTQCLSGSSVSNTK QQRSARFNELVAEDGEDKDCVQKYVLTKIRNETFIRVYSILKWYYPTTLIDELIGEDEYE YHAEEYSDTFGEQNMI I SSRKFSSLIVSMI IKAYGDDRVKEI SNSVTLLYRLLEYKGKER EEYLKRIMPKFYAKISEVRVAEKVEDMLPLVFFQALLMSENQWVMDKRVSLPLILFCKDQ ERIVPISLKSNMISSPLLQLLHIMRFHPGKMRRATALGADVKEVLPKVLDFWMNVSFEKE ALHTIKYVSEQYTKNIMCSYCGGNELVASFVLPITHPDRGFIVIIITTEDVTNSNAEAIA KSRFSDVAKYIKGIVHISATREGTARVRGGGGIKSRVLDKWLGTRFQLVQIKMGENVFE NHELVTKLMN ( SEQ ID NO.: 1 )

[0304] EXAMPLE 2 - fusion protein comprising a truncated VP2 capsid protein of BTV Materials and experimental methods

[0305] 1- Protein design and sequences

[0306] The VP2 capsid protein from BTV-3 is a 959 amino acids protein that oligomerize in trimer structures generating a complex of 300 kDa approximately. The available complete sequence of the VP2 capsid protein from BTV serotype 3 (Genbank accession no. OR603993.1 derived from virus isolate BTV-3 / NET2023 segment 2) was used to synthesize different versions of the encoding gene (SEQ ID Nos.: 21 and 22).

[0307] Based on the protein of BTV information available (Zheng et al 2016) VP2 monomer of BTV-serotype 3 could be divided into 4 different domains as depicted in Figure 1.

[0308] To reduce the VP2 protein size and complexity, theoretically facilitating its production by recombinant DNA technologies, the strategy followed consisted in removing the Hub domain (i.e., the discontinuous trimerization domain) and hairpin domains, and adding an external, continuous trimerization domain, and a T4 fibritin domain or foldon (FD4). Trimerization of VP2 is necessary to induce neutralizing antibodies. As a result of these deletions and additions, a protein of 777 amino acids was designed, including a linker between VP2 and FD4 domains as well as a purification tag connected via a GGGS linker (see SEQ ID NO.: 22). The wild-type control also included a His tag sequence at the C-terminus to facilitate purification (see SEQ ID NO: 21).

[0309] VP2-BTV-3:

[0310] MEELVI PVI SRQFDKKLVGRYEYVI ELAEPEQDEWTNHDVTQI PDRRMFDVAQQGI REAI I YKPLDNDGEVLPRI LDMSIACYDMRKTMMKKEGVDFVSNTRWLEWMIQDSMDVQPLRVQMKEDHSTVQYDMFSAKVHIDSRKADTTSYH AIAVEAKAERKCCHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQ LGDNAYDKFLRGLVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKD EKDLSVKFQFKLDEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKL GDVYHTLRSKYEWSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYP DDVEITTKFNEDRYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMF NARVKPCDIELAERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYS KLLRRRDADLDYSSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVH MYDRFFEKRRVLRSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNI SYEHRQWSIPIILYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTIS NGGVQTSIVSTKALLYEMYLSSICGGYSEGVLWYLPITHPVKCLVALEVSDALVGADVRIDKIRRRFPLSAKHLK GIVQISVHPNRTFSVTTCGIVRHKVCKKTLLKHRCDVILLQTPGYVFGNDELLTKLLNIGGGSHHHHHH (SEQ ID NO: 21)

[0311] VP2t BTV-3 Mod:

[0312] MCHVRTEVWNSWRNHLFNTAQESCYTFKQTYELIVNSERLSTEEEFRVGAPQFHTIQRNHRMQLGDNAYDKFLR GLVQLRVSGTTPAKIRDEVAALDVIRDNWIRGSFDRSHIKSLELCRLLSSIGRKMVNMEEEPKDEKDLSVKFQFK LDEKFSPNDPERNVI FTSKTHRTNEDRFYVLLI IAASDTNNGRVWWSNPYPCLRGALIAAECKLGDVYHTLRSKY EWSVRPTYKPRDLEREREKYWGRVNLFDLEGEPATKVIHWEYELISPTYSVSNHKGNHCDLYPDDVEITTKFNE DRYREMIQSVIDDGWDQKNLKMYKILEEEGNPLLYDLEKDIKLDSQSQWFPSYYNKWTHAPMFNARVKPCDIEL AERKNDDPFVKRTLKPIKADCVDLLRYHMSHYYDLRPSLKGVSLSNKQTSSGIHQALAQDDLYSKLLRRRDADLD YSSPCPIITNYFLLEKFHILILTIMEKHYWELDDSEDVYEFPKIDASAFKVDGTLYDISQTIVHMYDRFFEKRRV LRSIDESRWILHLIRISQGRERLEVIERFFPNYGKAMRQRDFKRVKDVMFLNFLPFFFLTGDNISYEHRQWSIPI ILYADKLRILPVEVGAYYNRFGVTCILELLNFFPSYEKREEKLEEDIVSCADAIVNFYLQTTISNGGVQTSIVST KALLYEMYLSSICGGGGGGSGGGGSHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVGSGSGGYIPEAPRDG QAYVRKDGEWVLLSTFLGGGSHHHHHH (SEQ ID NO: 22)

[0313] 2- Baculovirus generation The encoding sequences for these proteins (VP2-BTV-3 and VP2t BTV-3 Mod) were synthesized by the company GenScript. The codon usage of the VP2 encoding genes were optimized fortheir expression in insect cells (OptimumGene™-Codon Optimization algorithm). Both sequences contained adequate flanking regions to facilitate their cloning in pFastBacl donor plasmid. Once the donor plasmids with the complete VP2 or modified VP2 genes were obtained, the bacmids for the generation of the different baculoviruses were prepared in E. coli DHIOBac bacteria (Invitrogen, San Diego, CA, USA) containing the mini Tn7-replicon. Then, the transfection of the bacmids in the regulatory Sf9-RVN Glycobac cells (SIGMA-ALDRICH, USA) was performed. The viral stock was amplified to generate a high titer stock. The baculovirus obtained were titrated by a standard plaque protocol in Sf21 cells (Invitrogen, San Diego, CA, USA). Virus titer was determined as plaque-forming units (pfu).

[0314] 3- Protein expression

[0315] Once the recombinant baculoviruses (rBV) were generated expressing each VP2 version, those were tested in insect cells and experiments were carried out to determine the best condition for recombinant protein expression in Trichoplusia ni as living biofactories.

[0316] Different rBV doses between 5000-50000 pfus / insect, insect incubation temperature from 23 to 28°C and infection times from 3 to 6 days were tested. Pupae infected at every condition (5g of pupae biomass) were mechanically homogenized using extraction buffer 80 mL of Tris 50 mM, NaCI 300 mM, PMSF ImM, DTT ImM, Arginine 50 mM, Tween 800.5 % pH 7.5.

[0317] 1 mL of pupae extract was taken from each condition and centrifuged at 14000xg 10 min at 4°C to split samples in soluble and insoluble fractions. Protein expression and solubility were checked using SDS-PAGE and Coomassie blue staining. Bands corresponding to the recombinant proteins were submitted to densitometry and relative quantification was carried out with ImageLab software analysis.

[0318] 4- Protein purification

[0319] Once pupae batches were generated using the best expression condition recombinant VP2 modified protein was purified using the His tag in C-terminal present in its sequence. Briefly, protein extraction was performed in optimized extraction buffer, Tris 20 mM, NaCI 300 mM, Arginine 50 mM, Imidazole 50 mM, Tween 0.05% pH 7.5 using a colloidal mill. Insect biomass (62.5g of pupae) was homogenized in IL of extraction buffer, followed by high pressure homogenization with 1 cycle at 300 bars. A centrifugation removed insoluble fraction, and soluble extract was clarified using depth filtration. IMAC chromatography using Histrap Crude FF resin was performed, and protein elution was achieved using a 500 mM Imidazole concentration. SDS-PAGE analysis helped to select protein fractions that were later dialyzed in Tris-HCI lOmM, NaCI 150mM, pH 8.2.

[0320] 5- Protein characterization

[0321] Protein concentrations were measured using SDS-PAGE and band densitometry using BSA curve as reference. Purity of the proteins were measured by SDS-PAGE and Coomassie blue staining using band densitometry of gels. Gels were analyzed using ImageLab software from Bio-Rad.

[0322] Protein trimerization, necessary for inducing neutralization antibodies, was checked by FPLC-SEC using Superose 616 / 300 from Cytiva, equilibrated in PBS IX pH 7.4, previously calibrated with gel filtration HMW calibration kit.

[0323] 6- Protein formulation in vaccines

[0324] VP2-modified BTV-3 recombinant subunit vaccine was formulated using Freund's adjuvant. Briefly, 2 mL of VP2 complete or VP2 truncated and modified at 0.6 mg / mL were mixed with 2 mL of complete Freund's adjuvant, (for the first dose) and incomplete Freund's adjuvant (for second dose) using vigorous agitation for 5 min. Emulsion state was checked overnight. Each formulation contained 0.3 mg / mL of antigen.

[0325] 7- Mice immunization and challenge

[0326] Two groups of male IFNAR(- / -) mice (n=5) were subcutaneous immunized following a homologous prime-boost regime separated 4 weeks and consisting of 150 pL of placebo or 30 pg / dose of truncated and modified VP2 protein. Animals were subcutaneously challenged with a lethal dose of BTV-3 (100 PFU) two weeks post-boost. Sera of immunized and control animals were collected three weeks post-prime and two weeks post-boost for the analysis of the neutralizing antibody response. After virus challenge, mice were daily examined for survival and clinical signs, and blood samples were collected at 3, 5, 7, 10 and 14 d.p.i. for the analysis of viremia and RNAemia by plaque assay in Vero cells and RT-qPCR, respectively.

[0327] Results

[0328] Higher production affusion protein when compared to control

[0329] The bacmids generated were isolated and checked by PCR to confirm the correct genes transposition. Two PCR reactions designed with specific primers to detect either, the whole transposition cassette (PCR 1) or the expression cassette (PCR 2) were performed, an empty bacmid without gene transposition was used as negative control. Expected size bands were obtained in both PCRs.

[0330] To generate recombinant baculoviruses (rBV) to express the corresponding protein, the purified bacmids DNAs were transfected to sf9-RVN cells. The cell supernatants harvested from transfected cells contain the passages 0 generated (pO). These pO baculoviruses were used as inoculum for an amplification round in the same cell line, generating the corresponding passages 1 viruses (pl). The pl viruses were titrated with a plaque assay protocol.

[0331] The rBVs passages were also analyzed by PCR using the same two sets of primers for PCR 1 and PCR 2 used with the bacmids and the bacmids DNA as positive control of PCR amplification. Bands of the expected sizes (the same as bacmids) were obtained for all viruses passages with both PCR reactions.

[0332] The rBVs passages were analyzed for protein expression in sf-9 RVN cells. To perform this analysis, pl of rBVs were collected, spun down and the pellets were lysed with RIPA buffer. The lysates were loaded into SDS-PAGE and gels analyzed by Coomassie blue stain and Western blot using an anti-Histidine monoclonal antibody. Results are shown in Figure 13.

[0333] An increase in protein expression was observed in VP2 truncated and modified molecule, compared to the full-length VP2, as shown in Figure 13. This modified protein showed similar soluble behavior, with respect to the native VP2 protein however, the increase in protein expression facilitates the purification and permits the industrialization of the molecule (Figure 14).

[0334] Truncated and modified VP2 molecule is purified at 30 mg per Liter of pupae extract and similar levels of purity (more than 85%) (Figure 15). The final protein concentration was established at 0.7 mg / mL.

[0335] Recombinant VP2 proteins induced neutralizing antibodies and protected against a virulent BTV challenge vaccinated model animals

[0336] Two groups (n=5) of mice were submitted to a prime-boost vaccination regime. One of them were vaccinated subcutaneous with 150 pL vaccine containing 30 pg of VP2-modified protein, adjuvanted with Freund's adjuvant (complete in the first vaccination and incomplete with the second). Second vaccination was administered 3 weeks after the first vaccination. A second group were used as control group, vaccinated with the adjuvant without any VP2 protein. All mice were challenged with the homologous virulent BTV-3 virus (10 PFUs / mice) 20 days after the second immunization (see Figure 16A).

[0337] High titers of neutralizing antibodies were detected after the prime vaccination with VP2 protein (see Figure 16B).

[0338] After virulent virus lethal dose challenge, 100% of vaccinated mice with VP2-modified protein survived to infection without any clinical symptom. In contrast, none of the mice belonging to the control group survived after 7 days post-challenge (see Figure 16C).

[0339] Neither viremia nor RNAemia were detected in groups vaccinated with VP2-modified molecule (see Figures 16D, E), indicating that truncated protein was able to confer a solid protection against BTV-3. References

[0340] Sun Young Sunwoo, Leela E. Noronha, Igor Morozov, Jessie D. Trujillo, In Joong Kim, Erin E. Schirtzinger, Bonto Faburay, Barbara S. Drolet, Kinga Urbaniak, D. Scott McVey, David A. Meekins, Mitchell V. Palmer, Velmurugan Balaraman, William C. Wilson, and Juergen A. Richt. Evaluation of A Baculovirus-Expressed VP2 Subunit Vaccine for the Protection of White-Tailed Deer (Odocoileus virginianus) from Epizootic Hemorrhagic Disease, Vaccines 2020, 8, 59; doi:10.3390 / vaccines8010059.

[0341] Luis Jimenez-Cabellol, Sergio Utri I la-Trigol, Julio Benavides-Silvan2, Juan Anguita3,4, Eva Ca Ivo-Pi nil la 1, Javier Ortego. I FN AR(- / -) Mice Constitute a Suitable Animal Model for Epizootic Hemorrhagic Disease Virus Study and Vaccine Evaluation. Int. J. Biol. Sci. 2024, Vol.

[0342] 20. 3076-3093

[0343] Luis Jimenez-Cabello, Sergio Utri lla-Trigo, Gema Lorenzo, Javier Ortego and Eva Calvo-Pinilla. Epizootic Hemorrhagic Disease Virus: Current Knowledge and Emerging Perspectives.

[0344] Microorganisms 2023, 11, 1339. https: / / doi.org / 10.3390 / microorganismsll051339

[0345] Sara L. Bissett,l Polly Roy. Impact of VP2 structure on antigenicity: comparison of BTV1 and the highly virulent BTV8 serotype. J Virol. 2024, 98 Issue 10. 1-18. doi:10.1128 / jvi.00953-24

[0346] Sun Young Sunwoo, Leela E Noronha, Igor Morozov, Jessie D Trujillo, In Joong Kim, Erin E Schirtzinger, Bonto Faburay, Barbara S Drolet, Kinga Urbaniak, D Scott McVey, David A Meekins, Mitchell V Palmer, Velmurugan Balaraman, William C Wilson, Juergen A Richt. Evaluation of A Baculovirus-Expressed VP2 Subunit Vaccine for the Protection of White-Tailed Deer (Odocoileus virginianus) from Epizootic Hemorrhagic Disease. Vaccines 2020, 8, 59; doi:10.3390 / vaccines8010059

[0347] Kinda Alshaikhahmed, Polly Roy. Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates. Vaccine 2016, 34, 1103; doi:10.1016 / j.vaccine.2015.12.069

[0348] Altschul S. F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3):403-10 Scientific Opinion on Epizootic Hemorrhagic Disease. EFSA Journal. 2009, 7 (12): 1418. https: / / doi. Org / 10.2903 / j.efsa.2009.1418

Claims

CLAIMS1. A fusion protein comprising:(a) a truncated VP2 capsid protein, wherein the VP2 capsid protein is of a virus belonging to the Reoviridae family and wherein the truncated VP2 capsid protein consists of an antigenic domain and a body domain; and(b) a trimerization domain, wherein the trimerization domain is a continuous domain.

2. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein comprises or consists of, from / V-terminus to C-terminus:(i) optionally, a purification tag;(ii) the truncated VP2 capsid protein of (a);(iii) a peptide linker;(iv) the trimerization domain of (b); and(v) optionally, a purification tag;wherein, optionally, the purification tag of (i) is fused via a peptide linker to the N-terminus of the truncated VP2 capsid protein and / or the purification tag of (v) is fused via a peptide linker to the C-terminus of the trimerization domain.

3. The fusion protein according to claim 1 or 2, wherein the virus belongs to the Orbivirus genus, such as Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Equine encephalosis virus (EEV), and Bluetongue virus (BTV), wherein, optionally:(i) the truncated VP2 capsid protein consists of SEQ ID NO: 6 or 7, or a sequence with at least 80% sequence identity to SEQ ID NO: 6 or 7, or(ii) the truncated VP2 capsid protein consists of SEQ ID NO: 24 or 25, ora sequence with at least 80% sequence identity to SEQ ID NO: 24 or 25.

4. The fusion protein according to any one of claims 1-3, wherein the trimerization domain is a T4 fibritin domain, foldon (FD4), isoleucine zipper trimerization domain, or a collagen XVIII homotrimerization domain, preferably a T4 fibritin domain or foldon (FD4), more preferably a T4 fibritin domain which comprises or consists of SEQ ID NO.: 8, or a sequence with at least 80% sequence identity to SEQ ID NO.: 8.

5. The fusion protein according to any one of claims 1 to 4, wherein the fusion protein further comprises a peptide linker between the truncated VP2 capsid protein of (a) and the trimerization domain of (b), wherein, optionally:the peptide linker is less than 40 amino acids in length; and / orthe peptide linker is a flexible linker or rigid linker, wherein, further optionally:the flexible linker consists of one or more glycine and / or serine residues; and / or the rigid linker consists of (EAAAK)n (n=l-3).

6. The fusion protein according to any one of claims 1 to 5, wherein the fusion protein comprises or consists of:(a) a sequence as defined in SEQ ID NO.: 4 or as defined in SEQ ID NO.: 5,(b) a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 4 and / or with SEQ ID NO.: 5,(c) a sequence as defined in SEQ ID NO.: 22 or as defined in SEQ ID NO.: 23, or (d) a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with SEQ ID NO.: 22 and / or with SEQ ID NO.: 23.

7. A nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 6, or the nucleic acid sequence according to claim 7.

9. A vaccine comprising the fusion protein according to any one of claims 1 to 6, or the nucleic acid sequence according to claim 7.

10. The vaccine according to claim 9, wherein the vaccine comprises more than one fusion protein according to any one of claims 1 to 6, wherein each fusion protein comprises an antigenic domain from a VP2 capsid protein belonging to(a) a different serotype of the same virus; and / or(b) a different virus, wherein the virus belongs to the Reoviridae family, preferably from a virus selected from the list comprising or consisting of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and Bluetongue virus (BTV), more preferably Epizootic Hemorrhagic disease virus (EHDV) and Bluetongue virus (BTV).

11. The fusion protein according to any one of claims 1 to 6, the nucleic acid sequence according to claim 7, the pharmaceutical composition according to claim 8, or the vaccine according to claim 9 or 10 for use as a medicament.

12. The fusion protein according to any one of claims 1 to 6, the nucleic acid sequence according to claim 7, the pharmaceutical composition according to claim 8, or the vaccine according to claim 9 or 10 for use in a method of treating and / or preventing a disease related to a virus belonging to the Reoviridae family, preferably related to a virus belonging to the Orbivirus genus, more preferably related to Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV).

13. The fusion protein, nucleic acid sequence, pharmaceutical composition or vaccine for use according to claim 12, wherein the fusion protein, nucleic acid sequence, pharmaceutical composition or vaccine is administered intramuscularly, wherein, optionally, the subject is administered a first dose that delivers about 20 to 150 micrograms of the fusion protein to the subject, and, further optionally, the subject receives a booster dose of about 20 and 150 micrograms about 2 to 3 weeks after the first dose.

14. The fusion protein according to any one of claims 1 to 6, the nucleic acid sequence according to claim 7, the pharmaceutical composition according to claim 8, or the vaccine according to claim 9 or 10 for use in a method of reducing the incidence or severity of at least one clinical symptom of Epizootic Hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and / or Bluetongue virus (BTV).

15. The fusion protein, nucleic acid sequence, pharmaceutical composition or vaccine for use according to claim 14, wherein the clinical symptom is loss of appetite, loss of fear of people, weakness, excessive salivation, rapid pulse, rapid respiration rate, fever, lying in bodies of water to reduce body temperature, unconsciousness, blue tongue, head swelling, neck swelling, sloughing or breaking of hooves, lameness, shock, death, or combinations thereof.

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