WO2017029619A1

WO2017029619A1 - Synthetic btv vp2 multiepitope peptide vaccine

Info

Publication number: WO2017029619A1
Application number: PCT/IB2016/054925
Authority: WO
Inventors: Albertha René VAN ZYL; Ann Elizabeth Meyers; Edward Peter Rybicki
Original assignee: University Of Cape Town
Priority date: 2015-08-18
Filing date: 2016-08-17
Publication date: 2017-02-23
Also published as: GB201514652D0

Abstract

This invention relates to a second generation, plant-produced synthetic bluetongue virus (BTV) candidate vaccine. The vaccine comprises a fusion protein, comprising of a synthetic BTV VP2 multiepitope polypeptide which is fused to a signal peptide which induces the formation of protein bodies in plant cells. The invention specifically relates to the fusion proteins described herein, methods of producing the fusion proteins in plant cells and pharmaceutical compositions comprising the fusion proteins. More specifically, the invention relates to a BTV vaccine comprising the fusion proteins of the invention and which allow for the distinction between infected and vaccinated animals (DIVA).

Description

SYNTHETIC BTV VP2 ULTIEPITOPE PEPTIDE VACCINE

BACKGROUND OF THE INVENTION

The synthetic BTV VP2 multiepitope fusion protein of the present invention is a plant-produced particulate vaccine which can be used in animals to protect them against more than one different BTV serotype. The vaccine has a high potential for commercialisation as it involves the production of only one recombinant protein which could be used as a vaccine to target multiple BTV serotypes. The fusion protein of the invention comprises a synthetic BTV VP2 multiepitope peptide which presents virus-neutralising antibody-binding epitopes in protein bodies. The fusion proteins of the invention may be used to protect subjects against several BTV serotypes in a single pharmaceutical composition.

The BTV structural protein VP2 is the major immunogenic determinant of BTV. The present inventors have developed a novel BTV vaccine which presents a non-serotype-specific synthetic BTV VP2 multiepitope polypeptide to the immune system of a subject and presents it in a particulate form. This significantly increases the extent of the immune response in a subject. The inventors have fused a gene sequence which encodes a signal sequence called Zera® to a plant codon-optimised synthetic BTV VP2 multiepitope gene sequence.

Zera® is a signal peptide sequence generated from the maize γ-zein sequence (Figure 1) which has been described as being sufficient to induce retention of recombinant proteins in protein bodies called StorPro® organelles (ERA Biotech, Spain). This phenomenon has been shown with the fusion of several different proteins fused to Zera® including E7 (Whitehead et al., 2014), enhanced green fluorescent protein (eGFP) (Rybicki laboratory), DsRed (Joseph et al., 2012), enhanced cyan fluorescent protein (eCFP) (Torrent et al., 2009a), human growth hormone (hGH) (Llompart et al., 2010), calcitonin and epidermal growth factor (EGF) (Torrent et al., 2009b), xylanase (Llop-Tous et al., 2011). The formation of protein bodies allows for better accumulation of the fusion proteins within a plant host cell and it also simplifies the purification process of the fusion protein as the product is particulate. The fusion protein of the invention is also considerably more immunogenic than the synthetic BTV VP2 multiepitope polypeptide alone.

The particulate fusion proteins of the invention are considered excellent immunogens as they lack the viral genome and they have also been shown to stimulate both the humoral and cellular arms of the immune system. Further, the Zera® signal peptide is also believed to have an adjuvanting effect (Whitehead et al., 2014). There are a number of other advantages to using particles as vaccine candidates. Their administration precludes the co-administration of an adjuvant for the induction of a strong antibody response, thus reducing the vaccine dose costs. In the case of animal vaccines, the Zera® sequence can be used as a marker for vaccination (Liu et al., 2012), allowing for the distinction between infected and vaccinated animals (DIVA) - this is a very important requirement in areas affected by disease outbreaks, such as in the EU where vaccination has taken place.

The fusion proteins of the present invention are also safe to produce, and further the production method is relatively cost effective. The recombinant vaccine of the invention should abrogate the need for high biosafety levels during manufacture. This is due to the fact that production of a recombinant vaccine in plants will negate the requirement for high biosafety levels for handling, thus significantly lowering the risks and the cost of its production compared to present live attenuated or whole- virus killed vaccines. The use of a recombinant protein, as opposed to live virus, also accelerates the production rate since the protein bodies can be made transiently in plants within 3 days of infiltration.

The existing vaccine against BTV which is used in South Africa consists of field strains of BTV attenuated through serial passage in embryonated chicken eggs and BHK-21 cells. The vaccine of consists of 3 bottles (A, B and C) each containing 5 different serotypes (A - 1 , 4, 6, 12 and 14; B - 3, 8, 9, 10 and 11 ; C - 2, 5, 7, 13 and 19). The existing vaccine does not represent all of the BTV serotypes circulating in South Africa, which total 22. The reason for this is that the missing serotypes (serotypes 15, 16, 18, 22-26) do not cause severe pathogenicity in sheep.

The current production process using eggs and cell culture is costly, and the inclusion of so many different serotypes also adds substantially to the cost of manufacturing the existing vaccine. The use of live virus also makes it difficult to differentiate infected from vaccinated animals (DIVA).

In the case of the synthetic BTV VP2 multiepitope candidate vaccine, its success obviates the need for multiple serotype-specific vaccination. In other words, a single vaccine could be used to protect a subject against multiple BTV serotypes.

The present invention describes production of synthetic BTV VP2 multiepitope fusion proteins, which are capable of forming protein bodies, the protein bodies of which are particulate in nature. Protein bodies generated according to the present invention present a number of BTV-specific epitopes which are recognised across several BTV serotypes. SUMMARY OF THE INVENTION

The present invention relates to fusion proteins comprising a synthetic BTV VP2 multiepitope polypeptide fused to a maize γ-zein peptide, nucleic acids encoding the fusion proteins, vaccine compositions comprising the fusion proteins and methods for producing the fusion proteins.

According to a first aspect of the invention there is provided for a fusion protein comprising a synthetic BTV VP2 multiepitope polypeptide or a derivative thereof which is linked to a maize γ-zein peptide sequence or a derivative thereof. In some embodiments of the invention the derivative of the multiepitope polypeptide may include the BTV VP2 multiepitope sequence as herein described and additional epitopes that provide immunity against additional BTV serotypes. It will be appreciated by those in the art that the BTV multiepitope polypeptide and maize γ- zein peptide may be optionally linked to each other via a linker peptide.

In one embodiment of the invention the fusion protein comprising the BTV multiepitope polypeptide-linker-maize γ-zein peptide has an amino acid sequence as set forth in SEQ ID NO:20.

In one embodiment of the invention the synthetic BTV VP2 multiepitope polypeptide or its derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:22 or an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:22. Another embodiment of the invention includes a polynucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:22 or a polynucleotide encoding an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:22.

In another embodiment of the invention the maize γ-zein peptide sequence or derivative thereof comprises a sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:2.

It will be appreciated by those of skill in the art that the fusion protein of the invention is capable of forming a protein body, due to the properties of the zein peptide. The protein body formed from the fusion protein of the invention presents the synthetic BTV VP2 multiepitope polypeptide to the immune system, consequently eliciting an immune response. Accordingly, it will further be appreciated that the protein bodies of the invention are particulate in nature and are thus effective immunogens for eliciting immune responses against BTV.

In one embodiment of the invention the fusion protein is expressed in and recovered from a plant or plant cell. It will however be appreciated that any suitable host cell may be used to express the fusion proteins of the invention.

A second aspect of the invention provides for nucleic acid molecules encoding the fusion proteins of the invention. In some embodiments, the nucleic acid molecules of the invention may be operably linked to regulatory sequences in such a way as to permit gene expression thereof when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be in the form of vectors or expression constructs that can be transformed or transfected into host cells for expression. It will be appreciated by those of skill in the art that any suitable vector can be used for this purpose.

It will be appreciated that expression vectors comprising the nucleic acid molecules encoding the fusion proteins and expression cassettes comprising the nucleic acid molecules encoding the fusion proteins also fall within the scope of an embodiment of the invention.

A further aspect of the invention provides for a vaccine composition comprising the fusion proteins of the invention and a pharmaceutically acceptable diluent or excipient, wherein the vaccine composition is capable of eliciting a protective immune response against bluetongue virus. Preferably the protective immune response is a cellular or humoral immune response.

In one embodiment the fusion protein may be present in an oil in water emulsion vehicle.

In one embodiment the vaccine composition is for use in inducing an immune response against bluetongue virus.

The invention also provides for a DNA vaccine composition comprising a a cassette containing a promoter and a polynucleotide encoding the fusion protein of the invention. Alternatively, the DNA vaccine may comprise the expression vectors described herein. The cassette or expression vector containing the nucleic acid molecules encoding the fusion proteins of the invention may be mixed together with pharmaceutically acceptable diluents or excipients. The nucleic acid molecules encoding the fusion proteins are preferably linked to regulatory sequences that allow for the expression of the fusion proteins in a cell. Preferably, the cell is an animal cell. A further aspect of the invention relates to the use of the fusion proteins described herein in the manufacture of a vaccine for use in a method of preventing biuetongue virus infection in a subject, the method comprising administering a therapeutically effective amount of the vaccine to the subject.

According to yet another aspect of the present invention there is provided for a method of producing the fusion proteins of the invention in a plant or plant cell, wherein the method comprises the steps of:

(i) transforming or infiltrating a plant cell with an expression vector which contains a nucleic acid molecule of the invention;

(ii) expressing the fusion protein in the plant cell; and

(iii) recovering the fusion protein from the plant cell.

It will be appreciated that the method of producing the fusion protein may be either by transient expression of the protein or by stably expressing the protein in the plant cell.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1: Zera® signal sequence (339 bases (SEQ ID NO:1); 113 amino acids (SEQ ID NO:2).

Figure 2: Alignment of 8 VP2 amino acid sequences for BTV-1 (SEQ ID NO:4), BTV-2 (SEQ ID NO:5), BTV-6 (SEQ ID NO:6), BTV-8 (SEQ ID NO:7), BTV-10 (SEQ ID NO:8), BTV-13 (SEQ ID NO:9), BTV-17 (SEQ ID NO:10) and BTV-23 (SEQ ID NO:11) showing a) region of the 2 most likely B cell epitopes (solid line boxed areas) predicted across full length VP2 sequences (AA 1 to 414 on VP2 sequences) as well as b) 8 AA homologous region included in the epitope.

Figure 3: Multiepitope encoding nucleotide sequence (54 bp) (SEQ ID NO: 12) and amino acid sequence (18 amino acids) (SEQ ID NO:3).

Figure 4: Synthesised 41 base pair oligonucleotides to assemble the BTV VP2 multiepitope construct by PCR (underlined text - epitope sequence).

Figure 5: Universal alpha (a) helix linker sequence (underlined text).

Figure 6: Schematic of the synthetic construct (Zera® - a-helix linker - VP2ep) after assembly PCR. Figure 7: Zera®-linker-VP2 multiepitope fusion nucleotide sequence (SEQ ID NO:19).

Figure 8: pEAQ-Zera®-1-VP2ep plasmid map.

Figure 9: Zera®-VP2ep detected in the pellet after purification using a- VP2R (a), BTV-8 sheep serum (b) and BTV-10 guinea pig produced serum (c).

Figure 10: Zera®-VP2ep detected in the crude extract after probing with 1 : 2000 BTV-8 sheep serum.

Figure 11 : Serum titration of Zera®-VP2ep vaccinated mice.

Figure 12: A TEM of a leaf section infiltrated with only infiltration medium at 17000x magnification. B TEM of a leaf section showing electron dense Zera®- VP2ep protein bodies (PB) at 3 dpi (10000x magnification). CPT: chloroplast; CW: cell wall; ER: endoplasmic reticulum; CYT: cytoplasm.

Figure 13: A schematic of the BTV particle showing the four structural proteins, the transcriptase complex and the dsRNA genome (Mertens et al., 2004).

Figure 14: Zera® in frame with Nicotiana spp. codon optimised BTV VP2 multi-epitope (379-1116).

Figure 15: Western blot analysis of Zera® BTV VP2 multi-epitope (38.7 kDa) expression of in N. benthamiana extracted on day 3. Nitrocellulose membrane probed with rabbit anti-BTV-8 VP2 (lane 1-2) and anti-Zera® (lane 3-4) primary antibody. Negative controls (lane 1 and 3) plants infiltrated with pEAQ-HT Zera® BTV-2 VP2 multi-epitope (lane 2 and 4).

Figure 16: Showing the BTV-VP2 multiepitope expressed in N benthamiana over time. On the left hand side is the Zera® antibody showing bands at 3 and 5 dpi, however BTV1 , BTV4 and BTV6 did not show appropriate bands of the correct size. The negative control used was empty pRIC3.0 (-).

Figure 17: EM of the BTV-VP2 multiepitope infiltrations (sections). Protein bodies are indicated by the black arrows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

By "bluetongue" or "BT" is meant a virus belonging to a group of approximately 26 related but genetically distinct "serotypes". The virus may also be referred to herein as "bluetongue virus" or "BTV".

BTV is a double stranded ribonucleic acid (dsRNA) virus that causes an insect-borne, infectious non-contagious disease of both domesticated and wild ruminants; it is the type species of the genus Orbivirus that is classified into the family Reoviridae. Reoviridae is one of the largest families of virus that includes major human pathogens, such as rotavirus, as well as pathogens of insects, reptiles, fish, plants and fungi. Orbiviruses differ from other members of the Reoviridae family in that they can multiply in both arthropod and vertebrate cells, causing severe disease and high mortality. BTV is transmitted between its hosts by Culicoides spp., causing disease in ruminants worldwide.

Virus protein (VP) 2 is the most variable of the BTV capsid proteins and contains the antibody-binding epitopes involved in virus neutralisation and serotype determination (De aula et al., 2000, Huismans and Erasmus, 1981). Twenty six distinct serotypes of BTV have been identified based on neutralisation activity of VP2 as well as with BTV specific real time reverse transcriptase polymerase chain reaction (RT-PCR). Each serotype shows variation that is associated with the geographical origins of the virus from around the world. Molecular studies on BTV isolates from different geographic regions have further divided BTV into two major topotypes, namely the eastern and western lineages (Maan et al., 2012, Maan et al., 2010).

The BTV genome is a double-stranded circular dsRNA surrounded by a protein capsid. BTV can replicate in both wild and domestic ruminants as well as some species of deer. Replication takes place in both the host and the Culicoides insect vector. BTV virions are complex three-layered icosahedral structures that are ~80 nanometer (nm) in diameter. The virions are composed of a core of ten segments of dsRNA encapsulated by seven structural proteins (four major and three minor proteins) that are arranged into three distinct layers (Figure 13).

The three minor proteins (viral protein (VP) 1 , VP4 and VP6) are enclosed by the subcore that is made up of VP3. The core-surface layer consists of VP7. The outer capsid is composed of major proteins VP2 and VP5 which is laid onto the foundation provided by the core. The minor proteins together with the genomic RNA form the virus replication complex, whereas the four major proteins make up the capsid of the virus. In addition to the structural proteins BTV has four non-structural (NS) proteins (NS1, NS2, NS3 and NS3a) which are involved in virus replication and assembly in BTV-infected cells.

The fusion proteins and compositions according to the invention may be used to treat BTV infection or conditions associated with BTV infection. BTV can infect all known species of domestic and wild ruminants. Severe disease usually occurs in the fine-wool and mutton breeds of sheep as well as some species of deer. BTV infection of cattle, goats and wild ruminant species is mostly asymptomatic or subclinical. In BTV endemic areas BTV-infected sheep develop only mild or no obvious disease. The blue tongue after which the disease is named is seen only in serious clinical cases.

Onset of the disease in sheep is typically characterised by high fever lasting 5-7 days. Clinical signs of disease can include fever, depression, excessive salivation, nasal discharge, facial oedema, hyperaemia and ulceration of the oral mucosa, coronitis, lameness and death. Abortion can occur in pregnant animals as well as teratogenic defects in calves. The severity of clinical disease and mortality rate is influenced by the breed and age of the animal as well as the virus strain that causes the infection. In acute phases of BT, clinical signs in sheep are mainly associated with damage to microvascular endothelial cells.

After recovery from BT animals may suffer from a number of long-lasting secondary effects, such as reductions in milk production, weight gain, wool break and temporary infertility.

Pathogenesis of BTV infection is similar in sheep and cattle as well as other species of ruminants. After an animal gets infected with BTV, through the bite of a Culicoides vector, the virus will travel to the regional lymph node where initial replication takes place. The virus then spreads throughout the body to a variety of tissues, where replication occurs mainly in mononuclear phagocytic and endothelial cells.

Viraemia is cell associated and can be prolonged in domestic ruminants. During viraemia BTV is associated with all blood cells, but late in the course of infection the virus is mostly associated with the erythrocytes. The longer lifespan of erythrocytes facilitate prolonged infection of ruminants, as well as the infection of the haematophagous insect vectors that feed on viraemic ruminants (Barratt-Boyes et al., 1995). Infectious virus can co-circulate for several weeks with high neutralising antibody titres, the maximum period of viraemia in sheep is about 50 days and in cattle about 100 days.

By "condition associated with BTV infection" is meant any condition, disease or disorder that has been correlated with the presence of an existing BTV infection, and includes secondary effects, such as reductions in milk production, weight gain, wool brake and temporary infertility.

A compound according to the invention includes, without limitation, a fusion protein including the amino acid sequence of a synthetic BTV2 VP2 multiepitope polypeptide, or a derivative thereof fused to a maize γ-zein signal peptide sequence or a derivative thereof.

A "protein," "peptide" or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation).

An "antigen" is a compound, composition, or substance that can stimulate the production of antibodies and/or a CD4+ or CD8+ T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. An "epitope" refers to a site on an antigen, including chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Accordingly, "multiepitope" refers to a molecule or polypeptide comprising more than one epitope or a site which is antigenic for more than one antibody.

The terms "nucleic acid" or "nucleic acid molecule" encompass both ribonucelotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single- stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non- naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By "cDNA" is meant a complementary or copy DNA produced from an RNA template by the action of RNA- dependent DNA polymerase (reverse transcriptase).

Accordingly, a "cDNA clone" refers to a duplex DNA sequence which is complementary to an RNA molecule of interest, and which is carried in a cloning vector. The term "complementary" refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus "complementary" to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

In some embodiments, a fusion protein of the invention may include, without limitation, a polypeptide including an amino acid sequence substantially identical to the amino acid sequence a fusion protein the synthetic BTV2 VP2 multiepitope polypeptide, or derivative thereof, linked to a γ-zein signal peptide or a derivative thereof. Another embodiment of the invention includes, without limitation, nucleic acid molecules encoding the aforementioned fusion protein.

As used herein a "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of the expressed fusion protein or of the polypeptide encoded by the nucleic acid molecule. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

Alternatively, or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re- anneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridisation conditions would be hybridisation carried out for 18 hours at 65°C with gentle shaking, a first wash for 12 min at 65°C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65°C in Wash Buffer B (0.1% SDS; 0.5% SSC).

In an alternative embodiment of the invention, the fusion proteins may be prepared by, for instance, inserting, deleting or replacing amino acid residues at any position of the synthetic BTV2 VP2 multiepitope polypeptide sequences and/or, for instance inserting, deleting or replacing nucleic acids at any position of the nucleic acid molecule encoding the synthetic BTV2 VP2 multiepitope polypeptide.

Those skilled in the art will appreciate that polypeptides, peptides or peptide analogues can be synthesised using standard chemical techniques, for instance, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques known in the art. Polypeptides, peptides and peptide analogues can also be prepared from their corresponding nucleic acid molecules using recombinant DNA technology.

In some embodiments, the nucleic acid molecules of the invention may be operably linked to other sequences. By "operably linked" is meant that the nucleic acid molecules encoding the fusion proteins of the invention and regulatory sequences are connected in such a way as to permit expression of the fusion proteins when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that any vector can be used for the purposes of expressing the fusion proteins of the invention.

The term "recombinant" means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when used in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species.

The term "vector" refers to a means by which polynucleotides or gene sequences can be introduced into a cell. There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term "cassette" refers to a polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequences encoding the fusion proteins of the invention. A cassette generally comprises a gene sequence inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the polynucleotide or gene sequences. In other embodiments, the vector provides the regulatory sequences for the expression of the fusion protein. In further embodiments, the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. "Regulatory sequences" include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.

The fusion proteins or compositions of the invention can be provided either alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any carrier, such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.

As used herein a "pharmaceutically acceptable carrier" or "excipient" includes any and all antibacterial and antifungal agents, coatings, dispersion media, solvents, isotonic and absorption delaying agents, and the like that are physiologically compatible. A "pharmaceutically acceptable carrier" may include a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the fusion protein or vaccine composition to a subject. The pharmaceutically acceptable carrier can be suitable for intramuscular, intraperitoneal, intravenous, oral or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for the preparation of sterile solutions. The use of media and agents for the preparation of pharmaceutically active substances is well known in the art. Where any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is not contemplated. Supplementary active compounds can also be incorporated into the compositions.

Suitable formulations or compositions to administer the fusion proteins and compositions to subjects suffering from BTV infection or subjects which are presymptomatic for a condition associated with BTV infection fall within the scope of the invention. Any appropriate route of administration may be employed, such as, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, topical, or oral administration.

As used herein the term "subject" includes both wild and domestic ruminants.

For vaccine formulations, an effective amount of the fusion proteins or compositions of the invention can be provided, either alone or in combination with other compounds, with immunological adjuvants, for example, aluminium hydroxide dimethyldioctadecylammonium hydroxide or Freund's incomplete adjuvant. The fusion proteins or compositions of the invention may also be linked with suitable carriers and/or other molecules, such as bovine serum albumin or keyhole limpet hemocyanin in order to enhance immunogenicity.

In some embodiments, the fusion proteins or compositions according to the invention may be provided in a kit, optionally with a carrier and/or an adjuvant, together with instructions for use.

An "effective amount" of a compound according to the invention includes a therapeutically effective amount, immunologically effective amount, or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of BTV infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in BTV viremia, inhibition of viral gene expression, delay in development of a pathology associated with BTV infection, stimulation of the immune system, or any other method of determining a therapeutic benefit. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

The dosage of any of the fusion proteins or compositions of the present invention will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the composition. Any of the compositions of the invention may be administered in a single dose or in multiple doses. The dosages of the compositions of the invention may be readily determined by techniques known to those of skill in the art or as taught herein.

By "immunogenically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve a desired immune response, including a cellular and/or humoral response. The desired immune response may include stimulation or elicitation of an immune response, for instance a T cell response.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result, such as prevention of onset of a condition associated with BTV infection. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

Dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the judgment of the person administering or supervising the administration of the fusion proteins or compositions of the invention. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single dose may be administered, or multiple doses may be administered over time. It may be advantageous to formulate the compositions in dosage unit forms for ease of administration and uniformity of dosage.

The term "preventing", when used in relation to an infectious disease, or other medical disease or condition, is well understood in the art, and includes administration of a composition which reduces the frequency of or delays the onset of symptoms of a condition in a subject relative to a subject which does not receive the composition. Prevention of a disease includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.

The term "prophylactic or therapeutic" treatment is well known to those of skill in the art and includes administration to a subject of one or more of the compositions of the invention. If the composition is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

Toxicity and therapeutic efficacy of compositions of the invention may be determined by standard pharmaceutical procedures in cell culture or using experimental animals, such as by determining the LD₅₀ and the ED₅₀. Data obtained from the cell cultures and/or animal studies may be used to formulating a dosage range for use in a subject. The dosage of any composition of the invention lies preferably within a range of circulating concentrations that include the ED₅₀ but which has little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.

It is understood that the synthetic BTV2 VP2 multiepitope polypeptides may be encapsulated in the protein body formed by the Zera® portion of the fusion protein. It appears that the recombinant fusion proteins associate with each other through hydrophobic interactions (8 repeats of the PPVHL domain as well as disulphide bond formation by the 6 cysteine residues embedding the hydrophobic domain). It would appear that interactions between the Zera domains and the ER membrane induces budding off of the protein bodies. It is surmised that the protein bodies of the invention induce an immune response which results in the protein bodies being broken up and consequently resulting in the immune system being exposed to the encapsulated synthetic BTV2 VP2 epitope polypeptides.

The invention also relates in part to a method of treating an infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the fusion proteins or compositions of the present invention.

The following example is offered by way of illustration and not by way of limitation.

EXAMPLE 1

Using 8 full-length (approximately 2.8 kb) BTV VP2 amino acid sequences, obtained from GenBank, representing BTV serotypes 1 , 2, 6, 8, 10, 13, 17 and 23 were aligned with each other (Figure 1). The most likely antibody-binding epitopes were predicted using the software program, COBEpro (http://scratch.proteomics.ics.uci.edu ) as depicted below in Figure 2A.

The two most conserved discontinuous antigenic regions across the 8 BTV VP2's were selected for the epitope based vaccine design. An 18 amino acid synthetic multiepitope sequence was designed by fusing consensus amino acid sequences of these 2 regions together as well as another amino acid sequence representing the greatest region of homology across all 8 BTV serotype VP2 sequences (Figure 2B). The corresponding nucleotide sequence (consisting of 54 nucleotides) encoding the multiepitope was derived using Nicotiana sp. codon- optimisation preferences (Figure 3). The multiepitope-encoding DNA sequence was further modified to contain 5' Mlu\ and 3' Xmal restriction sites to facilitate cloning. Two fragments consisting of 41 bp each were designed and synthesized with 12 complementary base pairs to allow fusion of the fragments during assembly PCR to yield the complete epitope construct (Figure 4). The first fragment (SEQ ID NO: 13) contained the MM restriction site and the second fragment (SEQ ID NO: 14) contained the Xmal restriction site.

The Zera® sequence (SEQ ID NO:1) (Figure 1) was PCR amplified from the pUC18 vector (obtained from ERA Biotech) using the primers Zera-FP 5' GCA CCG GTA TGA GGG TGT TGC TCG TT 3' (SEQ ID NO: 17) containing an ^gel restriction site and Zera-RP 5' GCC CAT GGC TGG CAC GGG CTT GGA T 3' (SEQ ID NO: 18) containing a Nco\ restriction site..

The Zera® encoding sequence was fused to the multiepitope encoding sequence using DNA encoding a universal a-helix linker (5' CCA TGG GAA GCG GCG GCG AAA ACG CGT 3') (SEQ ID NO: 16) flanked by Λ/col (5') and Mlu\ (3') restriction sites (Figure 5), inserted so as to be translationally in the same reading frame as the Zera and poiyepitope reading frames. The universal a-helix linker is known to effectively separate 2 domains of a protein. A synthetic polynucleotide construct was thus created using assembly PCR comprising Zera® - a-helix linker - VP2ep (Figure 6, SEQ ID NO: 19).

The assembled Zera®-VP2ep DNA construct contained Age\ and Xmal restriction enzyme sites after PCR to facilitate cloning into the pEAQ-HT vector (Figure 7). The resulting pEAQ-HTZera®-VP2ep construct (Figure 8) was sequenced and transformed into A. tumefaciens LBA4404.

For agroinfiltration, 10 ml cultures of the recombinant pEAQ-HTZera®-VP2ep were grown up in LB containing magnesium sulphate (2 mM), rifampicin (50 pg/ml) and kanamycin (30 g/ml) at 27 °C overnight with agitation at 200 rpm. A 10th of the volume was transferred to 100 ml induction medium (LB, 10 mM MES, pH 5.6) containing the same concentration of antibiotics as well as 20 μΜ acetosyringone. The culture was incubated overnight at 27 °C with agitation at 200 rpm and then centrifuged at 4000 rpm to pellet the cells. The cell pellet was resuspended in 5 ml infiltration medium (10 mM MES, 10 mM MgCI₂, 3% sucrose, pH 5.6) supplemented with 200 μΜ acetosyringone and incubated at room temperature for 2 h. The culture was diluted to an OD₆₀o of 0.5 and syringe-infiltrated into the abaxial surfaces of six- week-old N. benthamiana plants. At 3 dpi the leaves were either frozen at -80 °C or used for density gradient centrifugation. Harvested leaves were immediately cut up into fine pieces and homogenized thoroughly in five volumes of ice cold buffer PBP3 (100 mM Tris pH8, 50 mM KCI, 6 mM MgCI₂, 10 mM EDTA and 0,4 M NaCI) with 10% sucrose and 1 x Complete Mini, EDTA-free protease inhibitor cocktail (Roche). The crude plant extract was incubated at 4 °C with shaking, after which it was clarified through four layers of Miracloth™ (Merck). The crude extract was centrifuged at 4000 rpm for 10 min at 4 °C. The clarified crude plant sap was overlayed onto 5 ml of a 42% sucrose cushion (prepared in buffer PBP3) and centrifuged for 2 h at 79 000 x g in a SW 32 Ti Rotor (Beckman). The pellet was resuspended in 300 μΙ buffer PBP3 containing 10% sucrose. The resuspended pellet was washed seven times by centrifugation at 6000 rpm for 5 min, collecting the supernatant (SNT and w1 to w6) and resuspending the pellet in 500 μΙ PBP3 buffer containing 10% sucrose. The wash fractions and the final pellet were analysed on dotblots.

Protein detection was carried out by dotblot. For the dotblot, 5 μΙ of the Zera®-VP2ep wash fractions and the final pellet were dropped onto nitrocellulose membranes and left to dry completely. The membranes were probed with 1 :2000 dilutions of ct-VP2R (rabbit raised antibody against E. coli expressed wild type VP2), BTV-8 sheep serum (containing antibodies to BTV-8 VLPs) and BTV-10 guinea pig produced serum (containing antibodies to BTV-10 virus). The membranes were subsequently probed with a 1 :5000 dilution of anti-rabbit alkaline phosphatase- conjugated secondary antibody (Sigma-Aldrich #A3687), 1 :10000 dilution of anti- goat/sheep alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich #A8062) or 1 :5000 dilution of alkaline phosphatase-conjugated anti-guinea-pig antibody (Sigma-Aldrich #A5062), respectively. Detection was performed with 5- bromo-4-chloro-3-indoxyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) phosphatase substrate (BCIP/NBT 1 -component, KPL).

The dotblots (Figure 9) using the different sera indicate that the majority of Zera®-VP2ep remains insoluble with little to no protein detected in the wash fractions. Zera®-VP2ep is insoluble and very stable. The purified protein can be partially denatured with DTT or SDS to analyse with SDS-PAGE. On western blots multiple bands at -15, ~35 and ~70 kDa were observed, the 35 and 70 kDa bands of which constitute oligomerised fusions of Zera®-VP2ep that did not denature completely. However, the crude extract was sufficiently denatured and a single band at -15 kDa was detected on a western blot (Figure 10). The insolubility of the Zera®-VP2ep fusion protein could not be predicted: this may be an advantage, as insoluble antigens work well in providing a "depot effect" for slow leakage of antigen into the circulation for long-term immune stimulation.

Further, it was surprising that the synthetic VP2 multiepitope was detected with the BTV-10 serum as well as the sheep serum and rabbit-derived antibodies. The putative epitopes were predicted using in-silico methods and the regions included within the sequence were selected based on similarity to the BTV-8 VP2. This could indicate that a vaccine based on this peptide might be more cross-reactive and protective than was first thought.

Zera®-VP2ep together with BTV-8 VLPs were tested for their ability to elicit an antibody response in Balb/c mice (AEC# 011-016). The mouse study was performed in duplicate (exp 1 and exp 2).

Each experiment consisted of a total of 20 mice that were divided into 4 groups containing 5 mice each.

Group 1 : Inoculated with BTV-8 VLP vaccine

Group 2: Inoculated with BTV-8 VLP vaccine with Freund's adjuvant

(Incomplete)

Group 3: Inoculated with Zera®-VP2ep vaccine

Group 4: Inoculated with PBS negative control

Three days before vaccination all the mice were pre-bled and serum was harvested in order to determine baseline antibody levels. At day 0 all the mice were vaccinated subcutaneously with 10 pg of the appropriate antigen with/without Freund's adjuvant (Incomplete) and at day 28 the mice received booster vaccinations with 10 pg antigen. The experiments ended at day 56 with the collection of blood from the mice via cardiac puncture. The serum obtained from these animal experiments was analysed by ELISA.

A 96-well Maxisorp® microtitre plate (Nunc) was coated with 100 pL/well (1pg made up to 100 pL in coating buffer [10mM Tris, pH 8.5]) of total soluble wild type BTV-8 VP2 produced in E. coli and incubated overnight at 4 °C. The plates were blocked for 2 hours at room temperature with blocking buffer (5% non-fat dry milk in 1x TBS, pH7.5 [50 mM Tris, 150 mM NaCI]) after which it was washed 4x with 1x TST buffer (1xTBS [pH 7.5], 0.05% Tween®20).

Sera from mice vaccinated with the same vaccine were pooled (5 mice / vaccine) for analysis. Sera were diluted in blocking buffer in a 4-fold series in triplicate ranging from a 1 :50 dilution to 1:51200. Mouse sera from the mice vaccinated with PBS served as a negative control. Positive control wells contained sheep serum produced against BTV-8 VLPs (Thuenemann et al., 2013) and blank wells with no antibody were included for background control. A volume of 100 μΙ_ of the diluted sera was added to each well and incubated for 2 hours at room temperature after which the plates were washed 4x with 1x TST buffer. A 100 pL volume of goat anti-mouse IgG alkaline phosphatase conjugate (1 :10000, Sigma) and monoclonal anti-goat/sheep IgG alkaline phosphatase conjugate (1 :10000, Sigma) diluted in blocking buffer was added to wells containing mouse or sheep serum, respectively and the plates incubated for 1 hour at 37 °C. Plates were washed 4x with 1x TBS (pH 9) buffer and 200 pL SIGMAFAST™ p-Nitrophenyl phosphate (pNPP, Sigma) was added to each well. The plates were developed in the dark for 30 minutes after which the absorbance was read at 405 nm on a BIO-TEK® Powerwave XS microtitre plate reader.

The ELISA results indicate that serum produced against the predicted VP2 multiepitope fused to Zera® can bind to the full length wild type VP2 and that a BTV- specific immune response is observed when compared to the PBS vaccinated mice (Figure 11).

Leaves infiltrated with the above construct were also sectioned and embedded in resin for TEM. The pEAQ-HTZera®-VP2ep construct was cultured as described previously and syringe infiltrated into the abaxial spaces of six-week old N. benthamiana plants. At 3 dpi a whole leaf was picked from the infiltrated plant and a 3cm x 3cm piece was cut out with a scalpel blade in the presence of 2.5% gluteraldehyde (25% gluteraldehyde diluted in 0.1 M phosphate buffer [pH 7.4]). The leaf sample was soaked in 2.5% gluteraldehyde for 6 hours after which it was cut into 1mm x 3mm fragments, also in the presence of 2.5% glutaraldehyde. The leaf fragments were left in 2.5% glutaraldehyde overnight at 4 °C. The following morning the leaf fragments were washed 3 times, 5 minutes for each wash, in 0.1 M phosphate buffer (pH 7.4). The leaf fragments were fixed for one hour in one part 2% osmium tetroxide and one part 0.2 M phosphate buffer (pH 7.4) after which it was washed twice for 5 minutes each with 0.1 M phosphate buffer (pH 7.4) followed with two washes of 5 m in each with water.

After washing the leaf fragments were sequentially dehydrated. The leaf fragments were incubated for 5 minutes each in 30%, 50%, 70%, 80%, 90% and 95% ethanol. The fragments were incubated for 10 minutes in 100% ethanol; this step was repeated twice. After the ethanol dehydration series the leaf fragments were further dehydrated by 10 minute incubation in 100% acetone, repeated twice. The leaf fragments were mixed overnight in 1 :1 acetone; Spurr's resin.

The next morning half of the 1 :1 acetone:Spurr's resin mixture was removed and replaced with 100% Spurr's resin to yield a 1 :3 acetone:Spurr's resin mixture. The sample was mixed for four hours at room temperature, after which the acetone/resin mixture was removed and replaced with 100% Spurr's resin. The leaf fragments were incubated in 100% Spurr's resin for three days at 4 °C. The 100% Spurr's resin was replaced with fresh resin and incubated for four hours at room temperature after which the resin was replaced again and incubated overnight at room temperature. The following morning the samples were embedded and incubated for 24 hours at 60 °C.

The embedded leaf samples were cut into ultrathin sections with a diamond knife and collected onto copper grids. The copper grids were stained with uranyl acetate for 10 minutes after which they were washed five times, 15 seconds each, with water. The grids were blotted dry and transferred to lead citrate for 10 minutes after which the grids were washed with water and blotted dry. Grids were viewed using the Technai G2 transmission electron microscope.

TEM of the leaf sections expressing Zera®-VP2ep (Figure 12B) show the presence of electron dense protein bodies in the cytoplasm that are about 0.36 pm in size). The negative control (Figure 12A) leaves infiltrated with infiltration medium) shows these are absent.

EXAMPLE 2

Thirteen further BTV VP2 multiepitope sequences were subsequently designed from an analysis of 270 BTV VP2 sequences from GenBank. These sequences were used to create 13 consensus epitope sequences representing all the BTV serotypes. The multiepitope polypeptide (SEQ ID NO:22), encoded by the polynucleotide sequence of SEQ ID NO:21 , was assembled by universal spacer sequences between each epitope which was then fused to Zera® (Figure 14) as in Example 1 and synthesised by GenScript.

The Zera® BTV VP2 multiepitope gene was successfully cloned into the plant expression vectors pEAQ-HT and pRIC3.0 to yield pEAQ-HT-Zera-BTV-VP2-multi- epitope and pRIC3.0-Zera-BTV-VP2-multi-epitope, respectively. Six-week-old N. benthamiana plants were infiltrated with recombinant Agrobacterium strains LBA 4404 at an OD₆₀₀ of 0.50. For the pEAQ-HT construct, total plant protein was extracted on day 3 post infiltration and separated using SDS-PAGE. The proteins were then transferred to nitrocellulose membrane and probed with rabbit anti-BTV-8 VP2 and anti-Zera® primary antibody. The Zera® BTV-2 VP2 multi-epitope protein was successfully expressed and detected using the anti-Zera® antibody (Figure 15, lane 4). The theoretical size was expected to be 38.7 kDa, however the protein was seen to run at between 50-60 kDa. However the sheep anti-BTV-8 and rabbit anti-BTV-8 VP2 failed to detect the Zera® BTV-2 VP2 multi-epitope protein (Figure 15, lane 2).

For the pRIC3.0 construct, the pRIC3.0-Zera-BTV-VP2-multiepitope was also successfully expressed in plants (Figure 16). Zera® VP2 multi-epitope expression in both expression vectors was scaled up for purification of protein bodies and EM analysis. Figure 17 shows the results of an EM analysis of the protein bodies obtained through infiltration of N. benthamiana with the vectors encoding the BTV- VP2 multiepitope-Zera polypeptide of SEQ ID NO:22.

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Claims

1. A fusion protein comprising a synthetic BTV VP2 multiepitope polypeptide or a derivative thereof linked to a maize γ-zein peptide sequence or a derivative thereof, wherein the BTV multiepitope polypeptide and maize γ-zein peptide are optionally linked via a linker.

2. The fusion protein of claim 1 , wherein the synthetic BTV VP2 multiepitope polypeptide or the derivative thereof comprises an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:22 or an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:3 or SEQ ID NO:22.

3. The fusion protein of claim 1 or 2, wherein the maize γ-zein peptide sequence or derivative thereof comprises a sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:2.

4. The fusion protein of any one of claims 1 to 3, wherein the fusion protein forms a protein body.

5. The fusion protein of any one of claims 1 to 4, wherein the fusion protein is expressed in and recovered from a plant.

6. A nucleic acid encoding the fusion protein of any one of claims 1 to 5.

7. A vaccine composition comprising a fusion protein of any one of claims 1 to 5 and a pharmaceutically acceptable diluent or excipient, wherein said vaccine composition is capable of eliciting a protective immune response against bluetongue virus.

8. The vaccine composition of claim 7, wherein the fusion protein is present in an oil in water emulsion vehicle.

9. The vaccine composition of claims 7 or 8, containing a combination of fusion proteins of different serotypes.

10. The vaccine composition of any one of claims 7 to 9, for use in inducing an immune response against different serotypes of bluetongue virus.

11. Use of a fusion protein of any one of claims 1 to 4 in the manufacture of a vaccine for use in a method of preventing bluetongue virus infection in a subject, comprising administering a therapeutically effective amount of the vaccine to the subject.

12. An expression vector comprising the nucleic acid of claim 6.

13. A DNA vaccine composition comprising a nucleic acid encoding the fusion

protein of any one of claims 1 to 4 including a suitable promoter sequence and a pharmaceutically acceptable diluent or excipient, wherein the nucleic acid is operably linked to the promotor sequence, and optionally linked to other regulatory sequences that allow for the expression of the fusion protein in a cell.

14. The DNA vaccine composition of claim 14, wherein the cell is an animal cell.

15. A method of producing a fusion protein of any one of claims 1 to 5 in a plant, the method comprising the steps of:

(i) transforming or infiltrating a plant cell with the expression vector of claim 13;

(ii) expressing the fusion protein in the plant cell; and

(iii) recovering the fusion protein from the plant cell.