Extracellular assembly of virus like particles

Abstract

The present invention relates to in vitro methods for the extracellular production of African Horse Sickness Virus (AHSV) virus like particles (VLPs). AHSV virus like particles per se and to methods of preventing African Horse Sickness. The invention also relates to use of AHSV VLPs in the prevention of African Horse Sickness.

Description

[0001] EXTRACELLULAR ASSEMBLY OF VIRUS LIKE PARTICLES

[0002] BACKGROUND OF THE INVENTION

[0003] The African horse sickness virus (AHSV) genome consists of 10 segments of linear double-stranded RNA, encoding seven structural and five non-structural proteins. The virion is non-enveloped and is composed of three distinct protein layers. The inner core is formed by VP3, and the outer core layer is formed by the protein VP7, which is the group-specific antigen used in ELISA-based diagnostics. VP7 assembles into trimers that attach to the VP3 surface. In native virions, these two layers enclose the subcore, which comprises 10 dsRNA segments together with the transcription complex, to form a stable icosahedral core particle around 78 nm in diameter. The outer capsid is composed of globular trimers of protein VP5, surrounded by trimeric spikes of protein VP2, which is the protein containing the antigenic determinants that induce serotype-specific neutralizing antibodies.

[0004] African horse sickness (AHS) is an infectious, non-contagious illness of horses, which is frequently fatal in susceptible hosts. It is recognized as one of the most lethal viral diseases of horses worldwide. The disease is caused by a number of distinct serotypes of African horse sickness virus (AHSV), a group of non-enveloped isometric dsRNA viruses from the genus Orbivirus, family Reoviridae, the virus being transmitted by biting midges of the Culicoides genus. The virus is endemic to sub- Saharan Africa. South Africa is one of the few countries where all nine serotypes of the virus have been isolated. However, the virus occasionally escapes its endemic range and outbreaks of AHS have previously extended to countries in North Africa, the Middle East, South-West Asia and India and the Mediterranean region. Additionally, climate change is believed to be contributing to the northward migration of the midge vector, which has resulted in international awareness that AHS-free countries with milder climate conditions are increasingly at risk of outbreaks of the disease. An AHS outbreak that occurred in the western Mediterranean countries between 1987 and 1991 , has reinforced this concern.

[0005] Disease control in South Africa has largely been effected by immunization with live-attenuated vaccines. The currently used live-attenuated vaccine is supplied in two polyvalent vials containing three and four AHSV serotypes each, however neither AHSV-5 nor AHSV-9 is included in the vaccine. Although the live-attenuated vaccine is currently the best option in the fight against AHS, its use has raised concerns with regard to possible reversion to virulence, gene segment re-assortment between outbreak and vaccine strains, and the absence of its ability to Differentiate between Infected and Vaccinated Animals (DIVA). Most importantly, the live- attenuated vaccine is not licensed or authorised for use outside of the African subcontinent.

[0006] AHS outbreaks result in large economic losses to the equine industry. There is thus a need to develop new, safe, efficacious and cost-effective DIVA vaccines which would primarily address the concerns of the South African equestrian community, as well as being acceptable prophylactic or rapid response vaccines in the European and other emerging outbreak contexts.

[0007] Due to raised international awareness and local dissatisfaction with the current vaccine, AHSV research has focussed in recent years on the development of recombinant vaccines based on selected antigenic AHSV proteins, particularly the outer capsid proteins VP2 and VP5. Baculovirus expression systems and poxvirus vectors have been used to produce vaccines that induce protective immunity against various AHSV antigens.

[0008] Disadvantages inherent to these types of vaccines include firstly, that recombinant soluble antigens are generally poorly immunogenic and require potent adjuvants or repeated boost inoculations to enhance immunogenicity; secondly, that pre-existing immunity against the viral vector may compromise vaccine efficacy. Virus-like particles (VLPs) that mimic the structure of intact virions, on the other hand, provide an attractive alternative vaccine platform. Sharing certain key characteristics with live viruses, VLPs are safe non-replicating protein assemblies with the advantage of being highly immunogenic, as epitopes are displayed in ordered repetitive arrays on the particle surface.

[0009] Such vaccines present no risk of reversion to virulence nor of dsRNA segment re-assortment with wild virus strains because they do not contain viral RNA or non-structural proteins, which also makes it possible to distinguish between vaccinated and infected animals using molecular diagnostic techniques.

[0010] Over recent years, the use of plant systems to express recombinant viral structural proteins, with the resulting assembly of VLPs, has become increasingly popular as the method is both cost-effective and free from the risk of contaminating animal pathogens.

[0011] Conventional thinking has previously dictated that spontaneous self-assembly of multimeric protein complexes like VLPs requires all the component proteins to be simultaneously co-expressed within a particular host cell system. To the best of our knowledge, no previous evidence exists of spontaneous self-assembly of VLPs from their protein constituents outside of a living system. Here we report the in vitro expression and extracellular assembly of complete AHSV VLPs.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to in vitro methods for extracellular production of African Horse Sickness Virus (AHSV) virus like particles (VLPs) and to uses of said VLPs.

[0014] In a first aspect of the invention there is provided for an in vitro method of producing an African Horse Sickness Virus (AHSV) virus like particle (VLP), the method comprising, providing four nucleic acid sequences each encoding one of the AHSV VP2, VP3, VP5 and VP7 structural proteins; cloning the nucleic acid sequences into at least two, at least three or four expression vectors adapted to express the structural proteins in a host cell; transforming at least two separate host cell populations with the expression vectors, with the proviso that no single host cell population is transformed such that it contains expression vectors encoding all four of the AHSV VP2, VP3, VP5 and VP7 structural proteins and such that the four structural proteins are expressed in the at least two separate host cell populations; expressing the structural proteins in the host cell populations; recovering the expressed structural proteins from the host cell populations; and co-incubating the recovered expressed structural proteins together, such that the expressed structural proteins self-assemble to form AHSV VLPs.

[0015] It will be appreciated that the AHSV structural proteins may be selected from structural proteins from different AHSV serotypes, resulting in the production of chimaeric VLPs that may be used in the production of a multivalent vaccine composition.

[0016] It will be further appreciated that one or more nucleic acid sequences may be cloned into a single expression vector. For example, two of the nucleic acid sequences could be cloned into one expression vector and two nucleic acid sequences could be cloned into a second expression vector. Provided that each nucleic acid is capable of being expressed as an independent protein. Alternatively, each nucleic acid sequence could be individually cloned into its own expression vector.

[0017] In one embodiment of the invention the host cell populations may be combined after the step of expressing the structural proteins but before the step of recovering the expressed structural proteins. In an alternative embodiment of the invention the expressed structural proteins are first recovered from their respective host cell populations and then combined.

[0018] It will be appreciated that incubation of the recovered structural proteins at 4 °C for between 20 to 48 hours, specifically either 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 hours, allows the proteins to mature properly and to assemble into VLPs.

[0019] In one embodiment of the invention the step of transforming at least two separate host cell populations with the expression vectors may be performed as follows:

[0020] (i) transforming a first host cell population with an expression vector encoding a first structural protein and transforming a second host cell population with at least one expression vector encoding the remaining three structural proteins;

[0021] (ii) transforming a first host cell population with an expression vector encoding a first structural protein, transforming a second host cell population with a second vector encoding a second structural protein and transforming a third host cell population with at least one expression vector encoding the remaining two structural proteins;

[0022] (iii) transforming a first host cell population with an expression vector encoding a first structural protein, transforming a second host cell population with an expression vector encoding a second structural protein, transforming a third host cell population with an expression vector encoding a third structural protein and transforming a fourth host cell population with an expression vector encoding a fourth structural protein; or

[0023] (iv) transforming a first host cell population with at least one expression vector encoding two of the structural proteins and transforming a second host cell population with at least one expression vector encoding the remaining two structural proteins.

[0024] It will be appreciated that the step of transforming is performed on at least two host cell populations, since co-transformation and co-expression of all of the structural proteins in a single host cell population would not provide evidence of extracellular assembly of the VLPs. The inventors however contend that co transformation of a single cell population with expression vectors encoding the four structural proteins, co-expression of the structural proteins, recovery and subsequent incubation of the structural proteins for between 20 to 48 hours at 4 °C would also result in extracellular assembly of the VLPs.

[0025] In a fourth embodiment of the invention the nucleotide sequences may be codon optimised sequences.

[0026] Those of skill in the art will recognise that the host cell may be selected from any suitable cell used for the expression of proteins such as the group consisting of plant cells, insect cells, mammalian cells, algal cells, yeast cells or bacterial cells. Preferably, the host cell is a plant cell and most preferably the plant cell is a Nicotiana benthamiana cell.

[0027] In one embodiment of the invention the four structural proteins are all from a single AHSV serotype. In an alternative embodiment at least one of the structural proteins is from a first AHSV serotype and wherein at least one of the structural proteins is from a second AHSV serotype. In such an alternative embodiment the resultant VLP will be a chimaeric VLP comprising structural proteins from different AHSV serotypes.

[0028] In a second aspect of the invention there is provided for an African Horse Sickness Virus (AHSV) virus like particle (VLP), that is produced according to an method comprising the steps of:

[0029] (i) providing four nucleotide sequences each encoding one of the AHSV VP2, VP3, VP5 and VP7 structural proteins;

[0030] (ii) cloning the four nucleotide sequences encoding the AHSV VP2, VP3, VP5 and VP7 structural proteins into at least two expression vectors adapted to express the structural proteins in a host cell;

[0031] (iii) transforming at least two separate host cell populations with the expression vectors, with the proviso that no single host cell population is transformed such that it contains expression vectors encoding all four of the AHSV VP2, VP3, VP5 and VP7 structural proteins and such that all four of the structural proteins are expressed in the at least two separate host cell populations;

[0032] (iv) expressing the structural proteins in the host cell populations;

[0033] (v) recovering the expressed structural proteins from the host cell populations; and

[0034] (vi) incubating the recovered expressed structural proteins together, such that the expressed structural proteins self-assemble to form the AHSV VLPs.

[0035] In a further aspect of the invention there is provided for a method of preventing African Horse Sickness in a subject, the method comprising administering a therapeutically effective amount of the AHSV VLP produced according to the method of the invention to a subject. The subject being is selected from the group consisting of horses, mules, donkeys and zebras.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0038] Figure 1 : After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous Optiprep™ gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Leaves were singly infiltrated with the 4 AHSV serotype 5 recombinant Agrobacterium strains and then homogenized together - fractions 6 (lane 1) and 7 (lane 2) are shown. The location of the AHSV viral proteins VP2 (SEQ ID NO:1), VP3 (SEQ ID NO:2), VP5 (SEQ ID NO:3) and VP7 (SEQ ID NO:4) are indicated to the right of the gel, while the molecular weight marker sizes are shown on the left.

[0039] Figure 2: After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous Optiprep™ gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Leaves co-infiltrated with the AHSV-5 VP3, AHSV-5 VP5 and AHSV-5 VP7 recombinant Agrobacterium strains were homogenized together with leaves infiltrated with the AHSV-5 VP2 recombinant Agrobacterium strain - fractions 6 (lane 3), 7 (lane 2) and 8 (lane 1) are shown. The location of the AHSV viral proteins VP2, VP3, VP5 and VP7 are indicated to the right of the gel, while the molecular weight marker sizes are shown on the left.

[0040] Figure 3: Gradient fraction 7 of the homogenate obtained from the leaves singly infiltrated with the 4 AHSV serotype 5 recombinant Agrobacterium strains was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates. Scale bars, 200 nm.

[0041] Figure 4: Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP3, AHSV-5 VP5 and AHSV-5 VP7 Agrobacterium strains and the leaves separately infiltrated with the AHSV-5 VP2 recombinant Agrobacterium strain was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates. Scale bars, 200 nm.

[0042] Figure 5: After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous Optiprep™ gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Leaves co-infiltrated with the AHSV-5 VP3, AHSV-4 VP5 (SEQ ID NO:6) and AHSV-5 VP7 Agrobacterium strains were homogenized together with leaves infiltrated with the AHSV-4 VP2 (SEQ ID NO:5) recombinant Agrobacterium strain - fractions 6 (lane 1), 7 (lane 2) and 8 (lane 3) are shown. The location of the AHSV viral proteins VP2, VP3, VP5 and VP7 are indicated to the left of the gel, while the molecular weight marker sizes are shown on the right.

[0043] Figure 6: After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous Optiprep™ gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Lane 1 shows the results from leaves co-infiltrated with the AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 Agrobacterium strains , while in lane 2, leaves co-infiltrated with the AHSV-5 VP3 and AHSV-5 VP7 Agrobacterium strains were homogenized together with leaves co-infiltrated with the AHSV-4 VP2 and AHSV-4 VP5 recombinant Agrobacterium strain - fraction 7 is shown in each case. The location of the AHSV viral proteins VP2, VP3, VP5 and VP7 are indicated to the right of the gel, while the molecular weight marker sizes are shown on the left.

[0044] Figure 7: Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 Agrobacterium strains and the leaves separately infiltrated with the AHSV-4 VP2 recombinant Agrobacterium strain was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates.

[0045] Figure 8: Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 Agrobacterium strains was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates and demonstrating that AHSV-5 VP2 will assemble with AHSV-4 VP5 to form the outer capsid layer.

[0046] Figure 9: Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP3 and AHSV-5 VP7 Agrobacterium strains and the leaves separately co-infiltrated with the AHSV-4 VP5 and AHSV-4 VP2 recombinant Agrobacterium strains was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates, however far fewer fully assembled VLPs were observed when leaves co-infiltrated with the AHSV-5 VP3 and AHSV-5 VP7 Agrobacterium strains were homogenized together with leaves co infiltrated with the AHSV-4 VP2 and AHSV-4 VP5 recombinant Agrobacterium strains. Scale bars, 200 nm.

[0047] Figure 10: Amino acid sequence of the AHSV5 VP2 protein (SEQ ID

[0048] NO:1).

[0049] Figure 11 : Amino acid sequence of the AHSV5 VP3 protein (SEQ ID

[0050] NO:2).

[0051] Figure 12: Amino acid sequence of the AHSV5 VP5 protein (SEQ ID

[0052] NO:3).

[0053] Figure 13: Amino acid sequence of the AHSV5 VP7 protein (SEQ ID

[0054] NO:4).

[0055] Figure 14: Amino acid sequence of the AHSV4 VP2 protein (SEQ ID

[0056] NO:5).

[0057] Figure 15: Amino acid sequence of the AHSV4 VP5 protein (SEQ ID

[0058] NO:6).

[0059] Figure 16: Codon optimised nucleotide sequence encoding the AHSV5 VP2 protein (SEQ ID NO:7).

[0060] Figure 17: Codon optimised nucleotide sequence encoding the AHSV5 VP3 protein (SEQ ID NO:8).

[0061] Figure 18: Codon optimised nucleotide sequence encoding the AHSV5

[0062] VP5 protein (SEQ ID NOS).

[0063] Figure 19: Codon optimised nucleotide sequence encoding the AHSV5

[0064] VP7 protein (SEQ ID NO:10).

[0065] Figure 20: Codon optimised nucleotide sequence encoding the AHSV4 VP2 protein (SEQ ID NO:11).

[0066] Figure 21 : Codon optimised nucleotide sequence encoding the AHSV4 VP5 protein (SEQ ID NO:12).

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] 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.

[0069] 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.

[0070] 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.

[0071] The present invention relates to the production of African horse sickness virus-like particles (AHSV VLPs) by separately expressing or expressing in different combinations in plants the 4 individual AHSV capsid proteins (VP2, VP3, VP5 and VP7) which make up the virus particle and then mixing the clarified plant lysates to allow assembly of the AHS VLPs in vitro. The inventors have shown that following this approach the formation of serotype specific VLPs is possible, further this method also allows for the production of chimeric AHSV VLPs by mixing the capsid proteins from different African horse sickness virus (AHSV) serotypes to make particles. The present invention allows for the production of a multivalent VLP vaccine that provides protection against multiple serotypes of AHSV.

[0072] This invention allows manufacturers to rapidly produce multi-capsid protein component viruses from available stocks. The individual proteins may be produced at large scale and stored long term. Then once an outbreak of AHS virus occurs VLPs can be produced in vitro by combining the relevant proteins from the relevant serotype. Additionally, chimeric particles may be produced by the methods disclosed herein in order to produce vaccines that protect against multiple serotypes. The methods also ensure that variable expression is managed. Variable expression occurs when multiple vectors are transformed into a single plant. If one of the expression vectors does not produce the intended target protein, then there is no way of knowing this. This could also result in incomplete formation of the capsid particles. The present invention solves that problem directly by allowing the amounts of each protein to be determined prior to the structural proteins being combined in order to produce a AHSV VLP. Preferably, in order to facilitate the formation of VLPs in vitro, the structural proteins may be combined in a ratio of 1 :1 :1 :1 or a ratio of 1 :1 :2:1 or a ratio of 2:1 :2:1 of the structural proteins VP2:VP3:VP5:VP7.

[0073] This invention gives a manufacturer additional control in the assembly of a candidate chimaeric AHS vaccine. It enables the improved assembly of the AHS chimaeric VLPs, by mixing different combinations of structural proteins from different serotypes of AHSV. The value proposition is therefore the additional control that the method provides the manufacturer and the possibility of using the method within a quality management system towards the production of AHS vaccine.

[0074] By “African horse sickness” or “AHS” is meant the disease itself. The virus is referred to herein as “African horse sickness virus” or “AHSV” belongs to a group of approximately 9 related but genetically distinct “serotypes”.

[0075] AHSV is a double stranded ribonucleic acid (dsRNA) virus that causes an infectious, non-contagious disease of equids. It is classified as an Orbivirus in the family Reoviridae. The virus is transmitted by biting midges of the Culicoides species.

[0076] The AHS virion is an icosahedral, non-enveloped particle, composed of three concentric layers surrounding the segmented double-stranded RNA genome. The AHS virion has been reported to be approximately 80 nm in diameter. The subcore, composed of structural protein VP3, encloses 10 linear genome segments and enzymatic minor proteins VP1 , VP4 and VP6. The subcore is covered by a layer of VP7 trimers forming the core particle. The core is surrounded by the outermost layer composed of structural proteins VP5 and VP2, with VP2 being the neutralizing antigen and serotype determinant. There are nine known serotypes of AHSV and all are present within South Africa and most parts of sub-Saharan Africa.

[0077] The AHSV VLPs and compositions according to the invention may be used to treat or prevent AHSV infection or conditions associated with AHSV infection. By “condition associated with AHSV infection” is meant any condition, disease or disorder that has been correlated with the presence of an existing AHSV infection and includes secondary effects.

[0078] AHSV infects equid species, such as horses, donkeys, mules and zebra, amongst others. The mortality rate in horses, the most susceptible species, can be up to 95% while donkeys and mules generally develop milder disease. Zebras are considered the natural vertebrate host of AHSV and rarely exhibit clinical signs of infection. Respiratory and circulatory functions are impaired in diseased animals and result in oedema of subcutaneous and intermuscular tissues, of lungs and haemorrhages of serosal surfaces. These animals also exhibit pyrexia and loss of appetite.

[0079] According to the method of the present invention the AHSV VLPs may be produced by either:

[0080] (ii) transforming a first host cell population with an expression vector encoding a first structural protein, transforming a second host cell population with a second vector encoding a second structural protein and transforming a third host cell population with at least one expression vector encoding the remaining two structural proteins;

[0081] (iii) transforming a first host cell population with an expression vector encoding a first structural protein, transforming a second host cell population with an expression vector encoding a second structural protein, transforming a third host cell population with an expression vector encoding a third structural protein and transforming a fourth host cell population with an expression vector encoding a fourth structural protein; or

[0082] (iv) transforming a first host cell population with at least one expression vector encoding two of the structural proteins and transforming a second host cell population with at least one expression vector encoding the remaining two structural proteins.

[0083] The structural proteins may be expressed as set out in cell populations transformed according to Table 1 .

[0084] Table ! : Transformation of cell populations with different combinations of AHSV structural proteins. It will be appreciated that an AHSV VLP made according to the method of the invention may be a chimaeric AHSV VLP wherein the capsid proteins are produced from different AHSV serotypes. Preferably, the VP3, VP5 and VP7 structural proteins are from one AHSV and the VP2 structural protein is from a second AHSV sertotype.

[0085] 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).

[0086] The terms “nucleic acid” or “nucleic acid molecule” encompass both ribonucelotides (RNA) and deoxyribonucleotides (DIMA), 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.

[0087] 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.

[0088] 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 one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. 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.

[0089] 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).

[0090] 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.

[0091] 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 VP2, VP3, VP5 and / or VP7 polypeptides of the invention and regulatory sequences are connected in such a way as to permit expression of the 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 or vectors can be used for the purposes of expressing the VP2, VP3, VP5 and / or VP7 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.

[0092] 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 VP2, VP3, VP5 and / or VP7 polypeptides 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 VP2, VP3, VP5 and / or VP7 polypeptides. 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.

[0093] The AHSV VLPs 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 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.

[0094] 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 AHSV VLPs or vaccine composition to a subject. The pharmaceutically acceptable carrier can be suitable for intramuscular, intraperitoneal, intravenous, subcutaneous, 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.

[0095] Suitable formulations or compositions to administer the AHSV VLPs and compositions to subjects who are to be prophylactically treated for an African horse sickness infection, who are suffering from an African horse sickness infection or subjects which are presymptomatic for a condition associated with African horse sickness 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.

[0096] As used herein the term “subject” includes wild and domestic ruminants, equids or any specified target animal

[0097] For vaccine formulations, an effective amount of the AHSV VLPs 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 AHSV VLPs 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 AHSV VLPs produced according to the method of the invention invention may be provided in a kit, optionally with a carrier and / or an adjuvant, together with instructions for use.

[0098] 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 an African horse sickness infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in viremia, inhibition of viral gene expression, delay in development of a pathology associated with the African horse sickness 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.

[0099] The dosage of any of the AHSV VLPs produced according to the methods 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, the African horse sickness infection being treated 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.

[0100] By “immunogenically effective amount” is meant an amount effective, at dosages and for periods of time necessary, to achieve a desired immune response. The desired immune response may include stimulation or elicitation of an immune response, for instance a T or B cell response.

[0101] 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 an African horse sickness 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.

[0102] 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 AHSV VLPs 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.

[0103] 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.

[0104] 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).

[0105] 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.

[0106] A “VLP” or “virus-like particle” refers to the capsid-like structure which results from the assembly of AHSV VP2, VP3, VP5 and VP7 polypeptides. These particles are antigenically and morphologically similar to native AHS virus virions but do not include viral genetic material; accordingly, these particles are not replicating nor infectious.

[0107] The invention also relates in part to a method of eliciting an immune response in a subject comprising administering to a subject in need thereof a prophylactically effective amount of the AHSV VLPs produced according to the method of present invention.

[0108] The following example is offered by way of illustration and not by way of limitation.

[0109] EXAMPLE

[0110] Recombinant Agrobacterium AGL1 strains, each carrying one of the AHSV serotype 5 (AHSV-5) capsid protein genes (VP2 (SEQ ID NO:7), VP3 (SEQ ID NO:8), VP5 (SEQ ID NO:9) or VP7(SEQ ID NO:10)) were each separately cloned into the pEAQ HT™ expression vector. Additionally, AHSV serotype 4 (AHSV-4) capsid protein genes (VP2 (SEQ ID NO:11) and VP5 (SEQ ID NO:12)) were each separately cloned into the pEAQ HT™ expression vector. The recombinant vectors were used to infiltrate the leaves of five-week-old N. benthamiana plants as described below. Plants were either infiltrated individually with each Agrobacterium strain (VP2 (serotype 4 or 5), VP3 (serotype 5), VP5 (serotype 5) or VP7 (serotype 5)) or co-infiltrated with strains carrying the VP3 (serotype 5), VP5 (serotype 4 or 5) and VP7 (serotype 5) pEAQ constructs.

[0111] Constructs

[0112] A consensus gene sequence for each of the AHSV-5 viral capsid proteins VP2, VP3, VP5, and VP7 and for the AHSV-4 viral capsid proteins VP2 and VP5 was obtained by aligning sequences (7-12 available) for these genes listed in GenBank, using CLC Mainbench bioinformatics software (Qiagen Bioinformatics, Aarhus, Denmark). Consensus sequences were codon optimized for expression in N. benthamiana and synthesized by GenScript Biotech Corporation (Nanjing, China) with flanking Age I and Xho\ restriction enzyme sites. Restriction enzyme cloning was used to insert the genes into the pEAQ-HT expression vector obtained from George Lomonossoff, John Innes Centre, UK (Sainsbury et at., 2009), to produce pEAQ- AHS5-VP2, pEAQ-AHS5-VP3, pEAQ-AHS5-VP5, pEAQ-AHS5-VP7, pEAQ-AHS4- VP2 and pEAQ-AHS4-VP5. The AHSV-5 and AHSV-4 plasmid constructs were electroporated into Agrobacterium radiobacter AG L1-ATCC BAA-101 as described previously (Maclean et at., 2007), and recombinant clones were selected at 27 °C on Luria-Bertani (LB) media plates containing 25 pg / rnL carbenicillin and 50 pg / rnL kanamycin.

[0113] Transient expression in plants

[0114] Expression of the AHSV-4 and AHSV-5 capsid proteins was achieved by agroinfiltration of 5-week-old N. benthamiana plants. Agrobacterium transformants each carrying one of the AHSV capsid protein genes were subcultured and grown overnight with agitation at 27 °C in Luria-Bertani broth (LBB) base supplemented with 50 pg / mL kanamycin, 20 pM acetosyringone and 2 mM MgS04. The cultures were diluted in resuspension solution (10 mM MES, pH 5.6, 10 mM MgCh, 100 pM acetosyringone) to the desired optical density and incubated for 1 h at 22 °C to allow for expression of the vir genes. For single infiltrations, each AHSV-4 or AHSV-5 Agrobacterium recombinant suspension was diluted to OD6oo = 0.5 or 1 .0, while co infiltration suspensions contained two or three AHSV-4 or AHSV-5 recombinants. Plants were grown at 22-25 °C under 16-h / 8-h light / dark cycles. Agrobacterium suspensions were infiltrated into the leaf intercellular spaces using either a blunt- ended syringe or a vacuum infiltrator, applying a vacuum of 100 kPa.

[0115] Purification and Western Blots

[0116] At 4 days post infiltration leaves from plants individually infiltrated with strains carrying AHSV-5 VP2, AHSV-5 VP3, AHSV-5 VP5 or AHSV-5 VP7 were harvested and homogenized together in two volumes of Bicine buffer pH 8.4 containing 1 x Complete Mini, EDTA-free protease inhibitor cocktail (Roche) - PI buffer. Alternatively, leaves co-infiltrated with strains carrying AHSV-5 VP3, AHSV-5 VP7 and either AHSV-4 VP5 or AHSV-5 VP5 were homogenized together with leaves infiltrated with the Agrobacterium strain carrying the pEAQ-AHSV-4 VP2 or with AHSV-5 VP2 construct in two volumes of PI buffer. The homogenates were incubated at 4 °C for 60 min with gentle agitation and then filtered through four layers of Miracloth™ (Merck). The crude plant filtrates were clarified by centrifugation at 13000 rpm for 20 min at 4 °C in a JAM rotor (Beckman) and the supernatants incubated for 20 - 48 hours at 4 °C. The crude homogenates were then again clarified by centrifugation at 13 000 rpm for 20 min and the supernatants overlaid onto 9 ml of an Optiprep™ (Sigma-Aldrich) 20% to 40% step gradient (3 ml of each gradient in 10% incrementing steps) and centrifuged at 32 000 rpm for 2 hours at 4 °C in a SW 32Ti rotor (Beckman).

[0117] Fractions (500 mI) collected from the bottom of the tubes were electrophoresed on a 10% SDS-polyacrylamide gel, followed by Coomassie blue staining (Figure 1 and 2). Fractions of plant extracts prepared from AFISV serotype 5 individually infiltrated leaves homogenized together are shown in Figure 1 . Fractions of plant extracts prepared from leaves co-infiltrated with AFISV-5 VP3, AFISV-5 VP5 and AFISV-5 VP7, homogenised together with plant extract from leaves infiltrated with AFISV-5 VP2 alone are shown in Figure 2. TEM carried out on fraction 7 of both preparations (individual infiltration of separate plants with each of the capsid proteins and mixing of co-infiltrated VP3, VP5 and VP7 and individually infiltrated VP2) showed fully assembled VLPs (Figure 3 and 4, respectively).

[0118] Fractions of plant extracts prepared from leaves co-infiltrated with AFISV-5 VP3, AHSV-4 VP5 and AFISV-5 VP7 homogenised together with plant extract from leaves infiltrated with AHSV-4 VP2 alone are shown in Figure 5. Fractions of plant extracts prepared from leaves co-infiltrated with AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 are shown in Figure 6, lane 1 , while fractions of plant extracts prepared from leaves co-infiltrated with AHSV-5 VP3 and AHSV-5 VP7 homogenised together with plant extract from leaves co-infiltrated with AHSV-4 VP2 and AHSV-4 VP5 are shown in Figure 6, lane 2. TEM carried out on fraction 7 of all three preparations comprising either mixing of co-infiltrated AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 and individually infiltrated VP2 of AHSV-4, or co-infiltrated AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7, or mixing of co-infiltrated AHSV- 5 VP3 and AHSV-5 VP7 and co-infiltrated AHSV-4 VP2 and AHSV-4 VP5 showed fully assembled VLPs (Figure 7, 8 and 9) respectively.

[0119] Additionally, in order test the propensity of AHSV-5 VP2 to be accommodated on a core containing AHSV-4 VP5, co-infiltrations of a single plant were performed with AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7, conversely single plant co-infiltrations were also performed to test the propensity of AHSV-4 VP2 to be accommodated on a core containing AHSV-5 structural proteins, namely AHSV-4 VP2, AHSV-5 VP3, AHSV-5 VP5 and AHSV-5 VP7.

[0120] Table 2: Summary of the protein combinations used thus far and the outcome in terms of yield and complete VLP formation

[0121] REFERENCES

[0122] Maclean, J., Koekemoer, M., Olivier, A., Stewart, D., Hitzeroth, I., Rademacher, T., Fischer, R. et al. (2007) Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV- 16L1 gene variants and different cell-compartment localization. J. Gen. Virol. 88, 1460-1469.

[0123] Sainsbury, F., Thuenemann, E.C. and Lomonossoff, G.P. (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J. 7, 682-693.

Claims

An in vitro method of producing an African Horse Sickness Virus (AHSV) virus like particle (VLP), the method comprising: (i) providing four nucleotide sequences each encoding one of the AHSV VP2, VP3, VP5 and VP7 structural proteins; (ii) cloning the nucleotide sequences into at least two expression vectors adapted to express the structural proteins in a host cell; (iii) transforming at least two separate host cell populations with the expression vectors, with the proviso that no single host cell population is transformed such that it contains expression vectors encoding all four of the AHSV VP2, VP3, VP5 and VP7 structural proteins and such that the four structural proteins are expressed in the at least two separate host cell populations;  (iv) expressing the structural proteins in the host cell populations; (v) recovering the expressed structural proteins from the host cell populations; and (vi) incubating the recovered expressed structural proteins together, such that the expressed structural proteins self-assemble to form AHSV VLPs.The method of claim 1, wherein the host cell populations are combined after the step of expressing the structural proteins and before the step of recovering the expressed structural proteins.The method of claim 1, wherein the host cell populations are not combined after the step of expressing the structural proteins and wherein the expressed structural proteins are combined after the step of recovering the expressed structural proteins.The method of claim 1, wherein step (iii) is selected from: (i) transforming a first host cell population with an expression vector encoding a first structural protein and transforming a second host cell population with at least one expression vector encoding the remaining three structural proteins;(ii) transforming a first host cell population with an expression vector encoding a first structural protein, transforming a second host cell population with a second vector encoding a second structural protein and transforming a third host cell population with at least one expression vector encoding the remaining two structural proteins;(iii) transforming a first host cell population with an expression vector encoding a first structural protein, transforming a second host cell population with an expression vector encoding a second structural protein, transforming a third host cell population with an expression vector encoding a third structural protein and transforming a fourth host cell population with an expression vector encoding a fourth structural protein; or(iv) transforming a first host cell population with at least one expression vector encoding two of the structural proteins and transforming a second host cell population with at least one expression vector encoding the remaining two structural proteins.The method of claim 1, wherein the nucleotide sequences are codon optimised sequences.The method of claim 1, wherein the host cell is selected from the group consisting of plant cells, insect cells, mammalian cells, algal cells, yeast cells or bacterial cells.The method of claim 6, wherein the host cell is a plant cell.The method of claim 7, wherein the plant cell is a Nicotiana benthamiana cell.The method of claim 1, wherein the structural proteins are all from a single AHSV serotype.The method of claim 1, wherein at least one of the structural proteins is from a first AHSV serotype and wherein at least one of the structural proteins is from a second AHSV serotype.

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