BACKGROUND OF THE INVENTION

African horse sickness (AHS) is a severe non-contagious infectious viral disease of equids which is transmitted by Culicoides spp. biting midges and threatens not only sport and companion horses but also working horses in rural communities. The aetiological agent is the African horse sickness virus (AHSV), genus Orbivirus, family Reoviridae. In Africa, AHSV is maintained in large populations of zebra (Equus quagga), whichare thought to be the natural vertebrate reservoir host. Zebra infected with AHSV, rarely display clinical signs of infection yet in susceptible horses the virus has a mortality rate of up to 95%. African horse sickness is endemic to Sub-Saharan Africa; however, the disease incidentally has spread beyond Africa threatening horse populations in Europe and Asia. Due to its severity, AHS is classified as being notifiable by the World Organization for Animal Health (OIE). South Africa and Kenya being the only regions in the world where all nine serotypes of AHSV have been isolated. No effective treatment exists and consequently control of the disease relies on vaccination, control of animal movements and prevention of bites by bloodsucking culicoides midges. Vaccination is mandatory in endemic countries of Southern Africa with the only registered commercially available product being a polyvalent live attenuated viral vaccine (LAV) lacking serotypes 5 and 9, produced by Onderstepoort Biological products (OBP). Immunity against all nine AHSV serotypes is essential in an endemic setting. Due to safety and efficacy concerns and variable immune response to most serotypes with the LAV vaccine, the need for an alternative safe, efficacious new generation vaccine has become imperative. DIVA compliance, the ability to serologically differentiate between naturally Infected and vaccinated horses, is also desirable for sero-surveillance, early detection of localised outbreaks, as well as freedom of movement and trade of horses nationally and internationally.

Vaccine candidates that may hold promise include inactivated, recombinant canarypox and modified Vaccinia Ankara (MVA) viral vectored vaccines, Entry Competent Replication Abortive (ECRA) vaccine strains, previously also referred to as Disabled Infectious Single Cycle (DISC) vaccines, Disabled Infectious Single Animal (DISA) vaccine and VLPs produced in insect cells. Previously, investigators were only able to produce AHSV VP2 in low quantities in insect cells (Aksular et al., 2018), the quantities produced were not viable and too costly for use as a veterinary vaccine product. Many others have tried over the years to produce significant levels of soluble VP2, but have failed. The present invention overcomes this limitation when produced in plants and is the first description of production of soluble VP2 in significant quantities of all nine serotypes, which is easy to produce and purify, highly scalable and cost effective as veterinary vaccine.

The present invention also describes the dose of plant-produced soluble AHSV VP2 of nine distinct serotypes, that will elicit appropriate neutralising antibody titres in animals. Further, the present invention describes the protective immunity conferred by a monovalent AHSV-5 VP2 subunit vaccine candidate when vaccinated animals were challenged with AHSV serotype 5 live virus.

SUMMARY OF THE INVENTION

The present invention relates to a plant-produced African horse sickness virus (AHSV) VP2 fusion protein and to uses of the AHSV VP2 fusion protein in vaccine compositions and/or diagnostic tests. The AHSV VP2 fusion protein, comprises of an AHSV VP2 polypeptide linked to a synthetic peptide. The synthetic peptide comprises or consists of a thrombin cleavage site, a linker, a histidine tag and an endoplasmic reticulum retention signal. It will be appreciated that the AHSV fusion protein may be selected from any one of the nine AHSV serotypes, isolated to date, but does not exclude any additional serotypes that might present itself in future.

In a first aspect of the invention there is provided for a fusion protein comprising or consisting of an African horse sickness virus (AHSV) viral protein 2 (VP2) polypeptide linked to a synthetic peptide, wherein the fusion protein comprises of the formula Xi - X ₂ - X ₃ - X ₄ - X ₅ , Xi is an AHSV VP2 protein, X ₂ is a peptide encoding a thrombin cleavage site, X ₃ is a linker, X ₄ is a histidine tag and X ₅ is an endoplasmic reticulum (ER) retention signal peptide. Further wherein X ₂ - X ₃ - X ₄ - X ₅ is the synthetic peptide.

In a first embodiment of the invention the African horse sickness virus VP2 polypeptide may be selected from the group comprising or consisting of a VP2 polypeptide of the serotype: AHSV-1 , AHSV-2, AHSV-3, AHSV-4, AHSV-5, AHSV-6, AHSV-7, AHSV-8 and AHSV-9.

In a second embodiment of the invention the synthetic peptide comprises or consists of a sequence of SEQ ID NO:19. In a third embodiment of the invention the fusion protein is expressed in and recovered from a plant. It will be appreciated that the plant may be a plant cell, plant part or full plant.

In a fourth embodiment of the invention, the fusion protein has increased expression relative to a wild-type VP2 in a plant cell. Those of skill in the art will appreciate that the addition of the synthetic peptide to the AHSV VP2 protein leads to a surprising increase in the expression of the fusion protein as compared to expression of VP2 without the synthetic peptide.

In a second aspect of the invention there is provided for a nucleic acid encoding the fusion protein described in the first aspect of the invention.

In a third aspect of the invention there is provided for a vaccine composition comprising or consisting of the fusion protein as described in the first aspect of the invention and a pharmaceutically acceptable diluent or excipient. It will be appreciated that the vaccine composition is capable of eliciting a protective immune response against African horse sickness virus.

In one embodiment of the second aspect of the invention the fusion protein is present in an oil in water emulsion vehicle.

In a second embodiment of the second aspect of the invention the vaccine composition contains, consists of or comprises a combination of fusion proteins of different AHSV serotypes. It will be appreciated that the VP2 protein may be selected from any one of the 9 AHSV serotypes, such as AHSV-1 , AHSV-2, AHSV-3, AHSV-4, AHSV-5, AHSV-6, AHSV-7, AHSV-8 and AHSV-9

In a third embodiment of the third aspect of the invention there is provided for the use of the vaccine composition for use in inducing an immune response against African horse sickness virus in a subject.

In a fourth aspect of the invention there is provided for the use of the fusion protein as described in the first aspect of the invention in the manufacture of a vaccine for use in a method of preventing African horse sickness virus infection in a subject, comprising or consisting of administering a therapeutically effective amount of the vaccine to the subject.

In a fifth aspect of the invention there is provided for a method of inducing an immune response against African horse sickness virus in a subject, the method comprising or consisting of administering an immunogenically effective amount of the fusion protein as described in the first aspect of the invention or the vaccine composition as described in the third aspect of the invention to the subject. In a sixth aspect of the invention there is provided for an expression vector comprising or consisting of a nucleic acid encoding any of the fusion proteins described herein.

In a seventh aspect of the invention there is provided for a method of producing the fusion protein as described in the first aspect of the invention in a plant cell. The method of this aspect of the invention comprises or consists of the steps of transforming or infiltrating a plant cell with an expression vector as described herein, expressing the fusion protein in the plant cell, and recovering the fusion protein from the plant cell.

In an eighth aspect of the invention there is provided for a method of detecting the presence of an antibody to an AHSV VP2 antigen in a sample from a subject, wherein the antibody binds to an epitope of the AHSV VP2 antigen, the method comprising or consisting of the steps of cloning a nucleic acid encoding the fusion protein as described in the first aspect of the invention into a vector, infiltrating a plant cell with the vector so as to express the fusion protein in the plant, recovering the fusion protein expressed by the plant, contacting the sample with at least one of the fusion proteins, and detecting the formation of an antibody-antigen complex, wherein the antibody-antigen complex comprises antibodies in the sample bound to one or more of the fusion proteins. It will be appreciated that the formation of an antibody-antigen complex confirms that the subject has been exposed to AHSV or has been vaccinated with a formulation containing an AHSV VP2 antigen. Those of skill in the art will appreciate that the formation of the antibody-antigen complex is detected by either (i) the binding of a labelled secondary antibody to the antibody-antigen complex, or (ii) by the binding of a labelled secondary antigen to the antibody-antigen complex. It will be further appreciated that an antibody-antigen complex will form if the subject has been exposed to African horse sickness virus.

In one embodiment of the invention the sample obtained from the subject is selected from the group comprising or consisting of blood, serum, plasma, saliva, conjunctival fluid, urine and faeces.

In a ninth aspect of the invention there is provided for a device for assaying for the presence of an antibody to AHSV VP2 in a sample from a subject, comprising or consisting of at least one fusion protein comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 and 18, and a means for detecting the formation of an antibody-antigen complex between an antibody in the sample and the at least one fusion protein. It will be appreciated that the means for detecting the formation of an antibody-fusion protein complex may comprise of or consist of a labelled secondary antibody, or a labelled secondary antigen. It will further be appreciated that binding of the labelled secondary antibody or labelled secondary antigen to the antibody-fusion protein complex confirms that the subject has been exposed to African Horse sickness virus.

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 : Schematic diagram of the African Horse Sickness Virus (AHSV) Viral Protein 2 (VP2) fusion proteins of the present invention. VP2 = represents the African Horse sickness virus VP2, TCS = represents the thrombin cleavage site, L = represents the linker, 6xHis = represents the 6x histidine tag and ERRS = represents the endoplasmic reticulum retention signal. The AHSV VP2 fusion protein is indicated by the line above the schematic diagram and the synthetic peptide (SEQ ID NO:19) is shown by the line underneath the schematic diagram.

Figure 2: SDS-PAGE and TEM images of plant produced chimaeric AHSV-1/5 VLPs and AHSV-5 VP2. Six-week-old N. benthmiana (AXT/FT) were coinfiltrated with Agrobacterium AGL-1 harbouring relevant constructs to assemble chimaeric AHSV-1/5 VLPs. (a) lodixanol density gradient fractions 40-35% were collected as well as immobilized metal ion affinity chromatography (IMAC) purified soluble VP2 and separated by 4-12% Bolt Tris-Bis Plus precast gels (Life Technologies), followed by Coomassie blue staining: lane 1 , MW, SeeBlue® Plus2 Pre-stained Protein Standard; lane 2, pEAQ-HT-empty vector expressed in plant leaf tissue; lanes 3-5, chimaeric AHSV-1/5 VLPs fractions 10-12; 40-30% lodixanol; lanes 6-7, IMAC partially purified VP2. Viral capsid proteins VP2 (124 kDa), VP3 (103 kDa), VP5 (58 kDa) and VP7 (38 kDa) are indicated with arrows, (b) VLPs in lodixanol fractions were stained using uranyl acetate and visualised using TEM. Fully assembled VLPs measure ~80 nm (A-C).

Figure 3: Schematic diagram of a) safety and immunogenicity and b) efficacy study designs in IFNAR ^/_ mice.

Figure 4: T-cell responses of mice splenocytes harvested at 28 days after vaccination with different doses of AHSV-1/5 VLPs (1 , 5, 10 pig). Splenocytes stained with CD4- and CD8-specific antibodies and analysed by flow cytometry. The % splenocytes positive for both CD4+ and CD8+ were quantified and presented. Splenocytes were stimulated with PBS buffer or VLPs. Unstained cells served as a negative control. The fold change relative to the negative control of the respective groups was calculated.

Figure 5: T-cell responses of mice splenocytes harvested 28 days after vaccination with different doses of AHSV-5 VP2 ^His (1 , 5, 10 pg). Splenocytes stained with CD4- and CD8-specific antibodies and analysed by flow cytometry. The % splenocytes positive for both CD4+ and CD8+ were quantified and presented. Splenocytes stimulated with PBS buffer or AHSV-5 VP2 ^His . Unstained cells served as a negative control. The fold change relative to the negative control of the respective groups was calculated.

Figure 6: SDS-PAGE of plant produced AHSV serotypes 1 -9 VP2 ^His . Six- weeks-old N. benthamiana (dXT/FT) were co-infiltrated with Agrobacterium AGL-1 harbouring relevant constructs to produce IMAC purified soluble VP2 ^His and separated by 4-12% Bolt Tris-Bis Plus precast gels (Life Technologies), followed by Coomassie blue staining: lane 1 , MW, SeeBlue® Plus2 Pre-stained Protein Standard; lane 2, pEAQ-HT-empty vector expressed in plant leaf tissue; lanes 3-1 1 , VP2 ^His of serotypes 1 -9. Viral capsid proteins VP2 ^His (124 kDa) of all nine serotypes are indicated with arrow.

Figure 7: Images of N. benthamiana AXT/FT plant leaf tissue infiltrated with pEAQ-HT-AHSV-5 VP2 without histidine tag (synthetic peptide) or pEAQ-HT- AHSV-5 VP2 ^His , 5 days after infiltration. Small brown circular dots on harvested leaves (A) are marks of the syringe position of infiltration. Note, that only selected leaves were infiltrated and tagged to simplify harvest (B).

Figure 8: SDS-PAGE of plant produced AHSV-5 VP2 (without the synthetic peptide) or VP2 ^His fusion protein (VP2 including the synthetic peptide of SEQ ID NOU 9). Avian influenza H6 ^His antigen of 37 kDa (tagged with the same C-terminal end containing 6X histidine), produced and purified identical to VP2 ^His , served as positive control. Six weeks old N. benthamiana (AXT/FT) were co-infiltrated with Agrobacterium AGL-1 harbouring relevant constructs to produce IMAC purified fusion proteins and separated by 4-12% Bolt Tris-Bis Plus precast gels (Life Technologies), followed by Coomassie blue staining: lane 1 , MW, SeeBlue® Plus2 Pre-stained Protein Standard; lane 2, pEAQ-HT-empty vector expressed in plant leaf tissue; lanes 3-4, VP2 crude extract and Ni-TED purified, respectively; lanes 4-5, AHSV-1/5 VLPs lodixanol density gradient purified fractions 9-10; lane 6, AHSV-5 VP2 ^His and H6 ^His , both Ni-TED purified.

Figure 9: Immunoblots of denatured AHSV-5 VP2 ^His and H6 ^His as detected by anti-His-HRP (1 :1000). Lane 1 , MW, Western C; lane 3, pEAQ-HT-empty vector expressed in plant leaf tissue; lanes 4-5, AHSV-5 VP2 crude extract and Ni-TED purified, respectively; lanes 6-7, AHSV-1/5 VLPs lodixanol density gradient purified fractions 9-10; lane 10, AHSV-5 VP2 ^His Ni-TED purified and lane 13, H6 ^His , Ni-TED purified. Lanes 2, 8-9 and 11-12 were left open to eliminate spill over and incorrect detection.

Figure 10: ELISA detecting AHSV-5 VP2 ^His and H6 ^His IMAC partially purified antigens. VP2 without histidine tag served as background. Dilutions of 1 :50 to 1 :6400 were prepared and coated on a Maxisorp Nunc-lmmunoplate in triplicate: anti- His-HRP (1 :1000) were used to detect the antigens.

Figure 11 : Amino acid sequence of the AHSV-1 VP2 ^His fusion protein (SEQ ID NO:4).

Figure 12: Amino acid sequence of the AHSV-2 VP2 ^His fusion protein (SEQ ID NO:6).

Figure 13: Amino acid sequence of the AHSV-3 VP2 ^His fusion protein (SEQ ID NO:8).

Figure 14: Amino acid sequence of the AHSV-4 VP2 ^His fusion protein (SEQ ID NQ:10).

Figure 15: Amino acid sequence of the AHSV-5 VP2 ^His fusion protein (SEQ ID NO:2).

Figure 16: Amino acid sequence of the AHSV-6 VP2 ^His fusion protein (SEQ ID NO:12).

Figure 17: Amino acid sequence of the AHSV-7 VP2 ^His fusion protein (SEQ ID NO:14).

Figure 18: Amino acid sequence of the AHSV-8 VP2 ^His fusion protein (SEQ ID NO:16).

Figure 19: Amino acid sequence of the AHSV-9 VP2 ^His fusion protein (SEQ ID NO:18).

Figure 20: Amino acid sequence of the synthetic peptide.

Figure 21 : Transmission electron microscopy (TEM) images of plant produced double chimaeric AHSV-1/5 and AHSV-1/6 VLPs. VLPs in lodixanol fractions were stained using uranyl acetate. Fully assembled VLPs measure ~80 nm.

Figure 22: SDS-PAGE of partially purified AHS VP2 proteins of all nine serotypes. Six weeks old N. benthamiana wild type were infiltrated with Agrobacterium AGL-1 harbouring relevant constructs to produce the individual nine VP2 fusion proteins, represented as serotypes 1 -9. IMAC purified soluble VP2 and separated using a 4-12% Bolt Tris-Bis Plus precast gels (Life Technologies), and stained by Coomassie blue staining: lane MW, SeeBlue® Plus2 Pre-stained Protein Standard; lane pQ, pEAQ-HT-empty vector expressed in plant leaf tissue; lanes 1 -9, IMAC partially purified VP2 fusion proteins of serotypes 1 -9 respectively.

Figure 23: LC-MS/MS characterization of partially purified AHS VP2 proteins of all nine serotypes. Six weeks old N. benthamiana wild type were infiltrated with Agrobacterium AGL-1 harbouring relevant constructs to produce the individual nine VP2 fusion proteins, represented as serotypes 1 -9.

Figure 24: Schematic diagrams of VLP and VP2 immunogens using various prime-boost approaches and validated in IFNAR-/- mice (study designs 1 and 2). Proposed cross-neutralisation were indicated as previously published (von Teichman et al., 2010) in the commercially available LAV vaccine, bottle 1 and 2, which served as positive control vaccine. Serotypes 5 and 9 underlined as these are omitted from the LAV. Irrespective of the combinations, the plant produced VLPs or antigens were formulated as 5 pg per serotype. The commercial LAV vaccine, bottle 1 , was administered as 1 pl per mice (study 1 ) and 0.2 pl per mice (study 2).

Figure 25: ELISA detection and isotyping of mice serum collected when the study was terminated. Serum of the six mice in each group pooled unless otherwise stated. A) IFNAR study 1 : mice were vaccinated with PBS buffer as negative control (group 1 ), the commercial LAV vaccine from OBP (group 2, 4 mice serum pooled, 2 succumbed before the end point), the plant produced VLP/VP2 (group 3) or plant produced VP2 pentamer prime-boost vaccines (group 4) with groups 3 and 4 adjuvanted with Montanide Gel 01. B) IFNAR study 2: mice were vaccinated with Bicine buffer as negative control (group 1 ), the commercial LAV vaccine from OBP (group 2, serum of a single mice, 5 succumbed before the end point), the plant produced polyvalent VP2, serotypes 1 -9 prime-boost vaccine adjuvanted with either Montanide Gel 01 (group 3) or Nanoalum (group 4).

Figure 26: Serum neutralizing test (SNT) titers of mice (mice trial 2) vaccinated with Bicine buffer (negative control), commercial vaccine (bottle 1 only) or plant produced VP2 nonavalent vaccine including all nine serotypes, and adjuvanted with either Montanide Gel 01 or Nanoalum.

SEQUENCE LISTING

The nucleic acid and amino acid sequences listed in the accompanying ST.26 sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand.

In the accompanying sequence listing:

SEQ ID NO:1 - Nucleotide sequence encoding AHSV-5 VP2 ^HIS Fusion Protein

SEQ ID NO:2 - Amino acid sequence of AHSV-5 VP2 ^HIS Fusion Protein

SEQ ID NO:3 - Nucleotide sequence encoding AHSV-1 VP2 ^HIS Fusion Protein

SEQ ID NO:4 - Amino acid sequence of AHSV-1 VP2 ^HIS Fusion Protein

SEQ ID NO:5 - Nucleotide sequence encoding AHSV-2 VP2 ^HIS Fusion Protein

SEQ ID NO:6 - Amino acid sequence of AHSV-2 VP2 ^HIS Fusion Protein

SEQ ID NO:7 - Nucleotide sequence encoding AHSV-3 VP2 ^HIS Fusion Protein

SEQ ID NO:8 - Amino acid sequence of AHSV-3 VP2 ^HIS Fusion Protein

SEQ ID NO:9 - Nucleotide sequence encoding AHSV-4 VP2 ^HIS Fusion Protein

SEQ ID NO:10 - Amino acid sequence of AHSV-4 VP2 ^HIS Fusion Protein

SEQ ID NO:1 1 - Nucleotide sequence encoding AHSV-6 VP2 ^HIS Fusion Protein

SEQ ID NO:12 - Amino acid sequence of AHSV-6 VP2 ^HIS Fusion Protein

SEQ ID NO:13 - Nucleotide sequence encoding AHSV-7 VP2 ^HIS Fusion Protein

SEQ ID NO:14 - Amino acid sequence of AHSV-7 VP2 ^HIS Fusion Protein

SEQ ID NO:15 - Nucleotide sequence encoding AHSV-8 VP2 ^HIS Fusion Protein

SEQ ID NO:16 - Amino acid sequence of AHSV-8 VP2 ^HIS Fusion Protein

SEQ ID NO:17 - Nucleotide sequence encoding AHSV-9 VP2HIS Fusion Protein

SEQ ID NO:18 - Amino acid sequence of AHSV-9 VP2 ^HIS Fusion Protein

SEQ ID NO:19 - Amino acid sequence of the synthetic peptide comprising “a thrombin cleavage site - linker - His tag - ER retention signal”

SEQ ID NQ:20 - Forward primer for amplification of AHSV-5 VP2

SEQ ID NO:21 - Reverse primer for amplification of AHSV-5 VP2 and inclusion of the synthetic peptide

SEQ ID NO:22 - Forward primer for amplification of AHSV-1 VP2

SEQ ID NO:23 - Reverse primer for amplification of AHSV-1 VP2 and inclusion of the synthetic peptide

SEQ ID NO:24 - Forward primer for amplification of AHSV-2 VP2

SEQ ID NO:25 - Reverse primer for amplification of AHSV-2 VP2 and inclusion of the synthetic peptide

SEQ ID NO:26 - Forward primer for amplification of AHSV-3 VP2

SEQ ID NO:27 - Reverse primer for amplification of AHSV-3 VP2 and inclusion of the synthetic peptide

SEQ ID NO:28 - Forward primer for amplification of AHSV-4 VP2 SEQ ID NO:29 - Reverse primer for amplification of AHSV-4 VP2 and inclusion of the synthetic peptide

SEQ ID NO:30 - Forward primer for amplification of AHSV-6 VP2

SEQ ID NO:31 - Reverse primer for amplification of AHSV-6 VP2 and inclusion of the synthetic peptide

SEQ ID NO:32 - Forward primer for amplification of AHSV-7 VP2

SEQ ID NO:33 - Reverse primer for amplification of AHSV-7 VP2 and inclusion of the synthetic peptide

SEQ ID NO:34 - Forward primer for amplification of AHSV-8 VP2

SEQ ID NO:35 - Reverse primer for amplification of AHSV-8 VP2 and inclusion of the synthetic peptide

SEQ ID NO:36 - Forward primer for amplification of AHSV-9 VP2

SEQ ID NO:37 - Reverse primer for amplification of AHSV-9 VP2 and inclusion of the synthetic peptide

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 “African horse sickness” is meant a virus belonging to a group of approximately 9 related but genetically distinct “serotypes”. The virus may also be referred to herein as “African horse sickness virus” or “AHSV”.

AHSV 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. AHSV is transmitted between its hosts by biting midges of the genus Culicoides, causing disease in equids.

Virus protein (VP) 2 is the most variable of the AHSV capsid proteins and contains the epitopes involved in virus neutralisation and serotype determination. Nine distinct serotypes of AHSV have been identified based on neutralisation activity of VP2. Each serotype shows variation that is associated with the geographical origins of the virus.

The fusion proteins and compositions containing the fusion proteins according to the invention may be used to treat AHSV infection or conditions associated with AHSV infection. AHSV can infect equids, specifically horses, mules, donkeys and zebra. The mortality rate in horses is 70 - 95%, mules around 50% and donkeys around 10%.

A fusion protein according to the invention includes amino acid sequence of a VP2 protein, or a derivative thereof fused to a peptide encoding a thrombin cleavage site, a linker and a histidine tag. It will be appreciated by those skill in the art that the VP2 protein or derivative thereof may be selected from any of the AHSV serotypes, but particularly AHSV-1 , AHSV-2, AHSV-3, AHSV-4, AHSV-5, AHSV-6, AHSV-7, AHSV-8, or AHSV-9. It will further be appreciated that the VP2 fusion protein or derivative thereof may be selected from a polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 or a nucleic acid sequence encoding the VP2 fusion protein selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1 1 , SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17 or a sequence having at least 90% sequence identity thereto.

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).

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 of a fusion protein comprising an AHSV VP2 protein, or derivative thereof, linked to a peptide encoding a thrombin cleavage site, a linker and a histidine tag. 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 one embodiment, the VP2 polynucleotide sequences may be “naturally occurring” or “native” that is they are isolated from a natural source rather than artificially produced. Sources of such native polynucleotides may include, biological samples, such as biopsies, blood, mucus, oral, plasma, semen, serum, urine, etc.) obtained from an infected subject or from another source. The VP2 polynucleotide sequences are modified by addition of polynucleotides encoding a thrombin cleavage site peptide, a linker and a histidine tag followed by an endoplasmic reticulum (ER) retention signal peptide.

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 VP2 polypeptide sequences and/or, for instance inserting, deleting or replacing nucleic acids at any position of the nucleic acid molecule encoding a VP2 polypeptide from any AHSV serotype.

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, horses, 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 containing the fusion proteins to subjects suffering from AHSV infection or subjects which are presymptomatic for a condition associated with AHSV 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 equids.

For vaccine formulations, an effective amount of the fusion proteins or compositions containing the fusion proteins 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 containing the fusion proteins 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 containing the fusion proteins 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 AHSV infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in AHSV viremia, inhibition of viral gene expression, delay in development of a pathology associated with AHSV 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 containing the fusion proteins 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. 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 AHSV 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. Neutralising antibodies elicited by plant produced VP2 vaccination can also be used for passive immunity protection or therapeutic.

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.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Cloning of constructs

A construct comprising “AHSV VP2 - a thrombin cleavage site - a two amino acid linker - 6x histidine tag - ER retention signal” was produced by cloning the gene encoding AHSV-5 VP2 into pEAQ-HT. Primers synthesized by Integrated DNA Technologies (IDT, Whitehead Scientific) were designed to produce a fusion gene encoding the fusion protein. The primers included a forward primer having the sequence AAA ACC GGT GCC ACC ATG GCT (SEQ ID NO:20) and a reverse primer having the sequence TCC CTC GAG TCA AAG CTC ATC ATG GTG ACC GCT AGA ACC CCT AGG CAC GAG CTT CTC AGT CTT GGC GAG (SEQ ID NO:21 ). The reverse primer was designed so that in addition to serving as an amplification template it also encoded the synthetic peptide (SEQ ID NO:19) for inclusion in the fusion protein. The fusion protein has the following configuration “AHSV-5 VP2 - thrombin cleavage site - linker - histidine tag - ER retention signal peptide” and is referred to as “AHSV VP2 ^His ”. The gene encoding the fusion protein (SEQ ID NO:1 ) was cloned into the plant expression vector pEAQ-HT.

The AHSV-5 VP2 sequence was obtained from Genbank (Genbank accession number ALM00085.1 ). Consequently, VP2 genes from the other eight AHSV serotypes were obtained and fusion genes encoding fusion proteins for these serotypes were also produced according to the same method, as described in Example 4.

The initial reasoning for adding the synthetic peptide comprising a thrombin cleavage site, a linker, a histidine tag and an endoplasmic reticulum retention signal was in order to assist with purification of the fusion protein. However, the inventors found that the addition of the synthetic peptide led to a surprisingly significant increase in the amount of soluble protein that was produced and recovered.

A schematic representation of the construct showing the full fusion protein and the synthetic peptide is shown in Figure 1 . EXAMPLE 2

Purification of plant-produced VLPs

N. benthamiana /XT/FT plants were grown in a growth room facility maintained at 26-28°C, 16 h day and 8 h dark. Agrobacterium strain AGL-1 (ATCC® BAA-101 TM) harbouring each of the constructs encoding AHSV-1 VP3/VP7, AHSV-5 VP2 and AHSV-5 VP5 proteins, were adjusted to OD ₆ oo = 2 and mixed in a ratio of 2:1 :1 , respectively, for plant infiltration. Leaves were harvested 7-8 days post infiltration in a Bicine buffer (50 mM Bicine, 20 mM NaCI pH 8.4) supplemented with protease inhibitor cocktail (Sigma P2714) and purified using depth filtration, followed by tangential flow filtration (TFF) and filter sterilisation using a 100K MinimateTM TFF Capsule (Pall Life Sciences). A sample of the TFF purified plant lysate containing the VLPs was subjected to lodixanol (Optiprep, Sigma) density gradient ultracentrifugation to quantity the VLPs within 1 ml TFF lysate for subsequent animal studies.

Purification and validation of AHSV-5 VP2 ^His proteins

AGL-1 harbouring the pEAQ-HT-AHSV-5 ^His construct (OD ₆ oo = 1.4) was infiltrated in plant leaf tissue. Five days post infiltration (dpi) fresh leaf material (-100 grams) was harvested using a Matstone juice extractor followed by a 2 min homogenizing step using an Ultra turrax in a Bicine buffer with the addition of 0.1 % (w/v) N-Lauroylsarcosine sodium salt and supplemented with protease inhibitor cocktail in a leaf tissue:buffer ratio of 1 :2. Centrifuge clarified supernatant was subjected to immobilized metal ion affinity chromatography (IMAC), with nickel as the metal ion. The column used was a 5 ml bed volume Ni-TED resin packed in a XK16 column (GE Health). Bound protein was eluted isocratically with 5 bed volumes of imidazole-containing buffer. Eluted fractions were pooled and concentrated using Vivaspin 15 columns (Sartorius, VS15T01 ) in a swing bucket rotor (4 000 g x 60 minutes). The concentrate was dialyzed twice within 16 hours against 2 L PBS buffer (140 mM NaCI, 1.5 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 2.7 mM KCI, pH 7.4) at 4 ^e C before use in the vaccine formulation or in serological tests.

The iodixanol density gradient purified VLPs and IMAC purified soluble VP2 were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE). The assembled VLPs and production of soluble VP2 antigen was confirmed using transmission electron Microscopy (TEM) and LC-MS/MS based peptide sequencing. Protein concentration was determined using the PierceTM Micro BCA protein assay kit (Catalogue # 23235, Thermo Scientific). Production and purification of plant-produced AHSV-1/5 VLPs and AHSV-5 VP2

AHSV-1/5 VLPs and AHSV-5 VP2 ^His (SEQ ID NO:2) were produced in N. benthamiana AXT/FT. The AHSV virion is a triple layered particle formed by the outer capsids (VP2 and VP5), the middle layer (VP7), and the inner shell (VP3, subcore). In this study, chimaeric AHSV-1/5 VLPs displaying VP2 and VP5 of serotype 5 as the outer shell layered on the AHSV serotype 1 core (VP3/VP7), measured 80 nm (Figure 2) and were similar in size to the homogenous AHSV-5 VLPs previously used to vaccinate horses (Dennis et al., 2018). lodixanol-purified VLPs and histidine tagged AHSV-5 VP2 ^His proteins were subjected to SDS PAGE (Figure 2) (a) and the appropriate band size of 124 kDa, representing VP2 ^His , subjected to LC-MS/MS based peptide sequencing. VP2 that forms part of assembled VLPs and VP2 antigens were confirmed by 73-77.4% coverage (133 - 144 unique peptides) and 77 - 86.8% coverage (206 - 235 unique peptides), respectively. Only VP2 assembled in VLPs, or histidine tagged VP2 ^His were subjected to LC-MS/MS based peptide sequencing as VP2 is the major determinant eliciting serotype specific neutralising antibodies.

EXAMPLE 3

Formulation of VLP and VP2 ^His vaccines

D-(+)-Trehalose dihydrate (Sigma- Aldrich) (5% m/v) was added to the TFF purified VLPs and IMAC purified VP2 ^His antigens before filter sterilisation with a 0.45 pM+0.2 pM Sartopore 2 sterile capsule (Sartorius, 5441307H4) using a peristaltic pump. The appropriate filter sterilised VLPs and VP2 ^His were mixed with autoclaved adjuvant (5% Montanide GEL 01 PR, Seppic, France) immediately before vaccination. The binary ethyleneimine (BEI) inactivated virus vaccine (positive control) and PBS (negative control) were formulated similarly. A cytotoxicity study in Vero cells was also conducted to confirm the safety of the plant-produced products.

IFNAR ^A mice study design to test vaccine safety, immunogenicity, and efficacy The study designs for the immunogenicity and efficacy testing of plant- produced AHSV-1/5 VLPs and AHSV-5 VP2 ^His as vaccine candidates, are depicted in Table 1 and Figure 3. In short, 24 and 21 female IFNAR ^/_ mice were used in these two studies, respectively. Initially, the vaccine was formulated as per the approved volume of 300 pl, but it became evident due to the high concentration of purified VLPs and VP2 ^His , that the volume could be reduced to 200 pl and used as such for the booster vaccination. IFNAR ^7- mice vaccination and challenge

IFNa/pR ^7- mice (A129, IFNAR type 1 ) were purchased and imported from B&K Universal, Marshall BioResources and bred at PCDDP with the necessary licence in place. Seven-week-old, female, mice were acclimatised under pathogen-free conditions in the biosafety level 3 (BSL-3) facility for 7 days before vaccination. Feed and water were provided ad libitum. Room temperature and humidity were continuously monitored and kept at 22°C±2 and 55%±15 respectively. Light-dark cycles were set at 12 hours each.

Table 1 : Experimental design of a) safety and immunogenicity and b) efficacy trials of plant produced chimaeric AHSV-1/5 VLPs and soluble AHSV-5 VP2 as vaccine candidates in female IFNAR ^7- mice (n = 24 or n = 21 , respectively). Mice were injected intraperitoneally with 300 pl (primary vaccine) and 200 pl (booster vaccine) of adjuvanted vaccines or PBS buffer as negative control. The AHSV-5 BEI inactivated monovalent vaccine (proprietary to OBP) served as positive control. All vaccines were adjuvanted with 5% Montanide GEL 01 obtained from Seppic (France). IFNAR ^7- mice in the efficacy study (groups 1 -4) were challenged on day 28 with a low cell culture passaged strain of AHSV-5 at 1 .4x10 ⁵ PFU. a) Safety and immunogenicity study b) Efficacy study

Eight groups of IFNAR ^7- mice (n = 3) were vaccinated intraperitoneally with 1 pg, 5 pg or 10 pg plant-produced VLPs or VP2 ^His using a fixed needle syringe (25G diameter, 16 mm length). Five groups of mice (n = 6 for vaccinated; and n = 3 for control groups) were used in the challenge study. For ethical reasons only 3 mice were used in the dose escalating study and efficacy study control groups. Vaccinated mice were prime boost vaccinated (days 0 and 14) with either 10 pg VLPs (group 1 ) or 10 pg VP2 (group 2) or PBS buffer (negative control, group 3) or BEI inactivated 5x10 ⁴ PFU (plaque forming units) (positive control, group 4). All vaccines were adjuvanted with Montanide™ GEL 01 PR. Group 0 was not vaccinated and used as naive control. Groups 1 -4 were challenged 28 days after the primary vaccine. No sera were collected from the mice in the challenge study on day 28 to minimise stress. Challenge with a dose containing 1.4 X 10 ⁵ pfu of AHSV-5 per mouse on day 28 (14 days post booster immunization) was administered subcutaneously as it most closely resembles the route of entry during the natural AHSV infection cycle (Castillo-Olivares et aL, 201 1 ).

Sera collection and clinical signs

All sera collected were heat inactivated at 56°C for 30 minutes before transport to the designated analysis facilities. Clinical scoring was performed as previously described (De la Grandiere et al., 2014). Mice were humanely euthanised when they showed severe clinical signs (weight loss, dehydration, frequent hunching, severe conjunctivitis, or any other condition that prevented food or water intake).

Isolation of solenocytes, antigen stimulation and flow cytometry

Mice were euthanised and their spleens aseptically collected when the study was terminated. Spleen cells were released into incomplete RPMI media by mashing the organs and filtering through 25 mm sterile cell strainers (Fisherbrand™) directly into 50 ml falcon tubes. After red blood cell lysis with RBC lysis buffer (BioLegend) splenocytes were re-suspended in complete RPMI media supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine. Isolated cells were stimulated with either: 1 ) PBS buffer or 2) the relevant antigen or 3) lipopolysaccharides (Escherichia coli produced, Sigma) and technical duplicate 24 well cell culture plates (TPP) and incubated at 37°C in 5% CO2 for 48 hours. Unstained cells served as negative control. After 48 h of incubation, the cells were harvested by centrifugation and resuspended in 0.1% BSA/PBS with the addition of mouse Fc blocker (Biorad) for the cells to be stained.

Following antigen stimulation, cells were recovered to perform surface staining with FITC-labelled anti-CD3, PE anti-mouse CD8a and PerCP/Cyanine5.5 anti-mouse CD4 antibodies (BioLegend 100204, 100708 and 1 16012, respectively; all Biocom Africa). After incubation, the cells were fixed in 3% paraformaldehyde, and then once more centrifuged before resuspending in 0.1% BSA/PBS and transferred to FACS tubes. Samples were analysed with the BD FACSLyric flow cytometer (BD Biosciences) equipped with a 488 nm laser for excitation of CD3-FITC, CD8-PE and, CD4-PerCP-Cy5.5. A lymphocyte gate was used during analysis to capture 15 000 cells. Data were analysed with FCSExpress version 7 (De Novo Software, Pasadena, CA, USA) and GraphPad Prism version 9 (San Diego, CA, USA). Lymphocytes were identified on a forward scatter (FSC) and side scatter (SSC) density plot. To ensure stringent single-cell gating, doublets were excluded using SSC and FSC Height and Width. Single events were gated on the FSC-H vs. FSC-W density plots.

Detection of antibodies against plant-produced VP2 ^His by ELISA

MaxiSorp plates (Thermo Scientific) were coated with 1.25 pg IMAC purified VP2 and incubated overnight at 4°C. Plates were saturated with blocking buffer (PBS- 0.05% Tween 20 and 4% casein hydrolysate). The animal sera diluted in blocking buffer (1 :200 or 1 :500) were added and incubated for 1 h at 37°C. After three washes in PBS-0.05% Tween 20, plates were incubated for 1 h at room temperature with a goat anti-mouse-IgG/IgM-HRP secondary antibody (Sigma) at a 1 :5000 dilution in blocking buffer. Finally, after three washes in PBS-0.05% Tween 20, the reaction was developed with substrate 3,3',5,5'-Tetramethylbenzidine (TMB)(Sigma) and stopped by adding 50 ml of 3 N H2SO4. Results were expressed as optical densities (ODs) measured at 450 nm. Background OD values were obtained from wells containing blocking buffer without serum samples. This background OD was subtracted from the OD measurements of the sample wells.

Serum neutralising tests (SNTs)

A 1 :5 start dilution of each serum was made in PBS + containing 1 % Gentamycin 50. Each serum sample was inactivated at 56°C for 30 min before testing. Twofold serum dilutions (initial dilution 1 :10) were made in duplicate rows in minimum essential medium with 2 g/l NAHCO3, gentamycin sulfate (Gentamycin 50) 0.05 mg/ml and 5% fetal calf serum. Equal volume of virus with a predetermined titre was added and incubated at 37°C in a 5 % CO2 gassed incubator for 1 hour. A Vero cell suspension containing 480 000 cells/ml were added in a volume of 80 pl. Plates were incubated at 37°C in 5 % CO2 and the progress of the cytopathic effect of the virus was recorded daily for up to 4-5 days of incubation. Titres were determined as the reciprocal of the highest serum dilution that provided >50% protection of the cell monolayers.

Statistical analysis

For statistical considerations, statistical difference at P < 0.05 were considered significant in a two tailed Student's t test. Immunogenicity study

Mice were vaccinated in a dose escalating manner with 1 , 5 or 10 pg of either monovalent chimaeric AHSV-1/5 VLPs or monovalent AHSV-5 VP2 ^His vaccine candidates on days 0 and 14, in a prime-boost regime for the initial safety and immunogenicity study. Mice vaccinated with a single dose of plant-produced chimaeric AHSV-1/5 VLPs seroconverted at a 5 pg and 10 pg vaccine dose, within the first 14 days (Table 2). The primary vaccination of AHSV-1/5 VLPs led to seroconversion of 1 :40 (1.6 logic) and 1 :28 (1.45 log™) when vaccinated with 5 and 10 pg AHSV-1/5 VLPs already on day 14, respectively. A booster vaccine was however necessary to elevate the neutralising antibodies (nAbs) to 1 :320 (2.5 log™) on day 28 for all VLP vaccine doses (1 , 5 and 10 pg). Ten micrograms of soluble VP2 ^His per mouse were required to equal this immune response after prime-boost vaccination (Table 2). Mice vaccinated with the positive control vaccine, BE I inactivated AHSV-5, seroconverted within the first 14 days at 1 :44 (1.64 log™) whereafter the titre dropped to 1 :14 (1.15 logic) on day 21 and 0 on day 28. A booster vaccine was not administered due to an adverse effect leading to the death of one of the three mice in the group. Although 1 , 5 and 10 pg adjuvanted chimaeric AHSV-1/5 VLPs resulted in SNTs of 1 :320 (2.5 logic), only 10 pg of soluble VP2 ^His resulted in a similar 1 :320 (2.5 log™) titre. An ELISA using VP2 ^His as antigen (coated with 1 .25 pg VP2) corroborated the SNT data as serum from seroconverted mice resulted in an OD450 detection of >1.20, whilst antibody negative control mice were below 0.36 (Table 2). As mentioned before, the BEI inactivated virus vaccine was not administered as a second dose and therefore resulted in an ELISA detection of 0.17 on day 28. As small quantities of serum were collected from the 20-25 gram IFNAR ^/_ mice, the samples within the same groups were pooled to facilitate analysis, particularly for the SNTs.

Complementary to the nAbs response in mice, harvested splenocytes were stimulated for 48 hours with the respective antigens to measure the cellular immune responses by means of flow cytometry (Figures 3 and 4). Compared to unstimulated PBS controls of the same mice, all the test antigens indicated a measure of CD4+/CD8+ stimulation which is required to induce cell memory. Mice vaccinated with plant-produced AHSV-1/5 VLPs (1 , 5 and 10 pg dose, Figure 4) showed a larger increase in stimulation as compared to the OBP BEI inactivated vaccine as positive control and to a lesser extent for the soluble VP2 ^His vaccinated mice (1 , 5 and 10 pg dose, Figure 5). This suggests a role in cell-mediated immunity contributing to the protection. Table 2: Safety and immunogenicity study: serum neutralizing test (SNT) titres of IFNAR ^7- mice vaccinated with plant produced chimaeric AHSV-1/5 VLPs or AHSV-5 VP2, PBS buffer as negative control and BEI inactivated AHSV-5 monovalent vaccine as positive control. Vaccines and PBS buffer were formulated with 5% Montanide GEL 01 ®. ELISA plates were coated with plant-produced soluble VP2 purified by IMAC* (1 .25 pg per well) followed by a second purification step using Dynabeads** (0.6 pg per well).

Efficacy study in IFNAR-/- mice

The prime-boost vaccine regime of 10 pg was chosen for the subsequent efficacy study as both 10 pg VLPs or 10 pg VP2 ^His resulted in strong seroconversion by day 28 (1 :320, 2.5 log , Table 2). Vaccinated mice (groups 1 -4) were challenged with AHSV5 at 1 .4x10 ⁵ PFU on day 28 and all groups of mice were monitored daily for clinical signs of AHS for the 25-day period after challenge until the planned study termination on day 53. No clinical signs were observed in mice, potentially due to low challenge dose of 1.4x10 ⁵ PFU per mouse, slow onset of viraemia (not determined), or low strain virulence. Nevertheless, protection against AHSV-5 conferred by both the plant-produced adjuvanted VLP and VP2 ^His vaccines correlated strongly with SNTs determined during the immunogenicity study (Table 2) and mice (n = 6) of each group survived until day 25 post challenge when the study was terminated. The negative control group (PBS with adjuvant) succumbed within 8 - 1 1 days after challenge. One of the BEI-vaccinated positive control group mice were euthanized on day 1 1. Viral infection in lungs cells showed a trend of reduced viral load (VLPs<VP2 ^His <BEI inactivated) but were not statistically significant (statistical difference at P < 0.05 for averages obtained with n=3 in a two tailed Student's t test, Table 3. Table 3: Summary table of efficacy study. Clinical protection and survival of IFNAR-/- mice vaccinated with plant produced chimaeric AHSV-1/5 VLPs (Group 1 ) or AHSV-5 VP2 (Group 2), PBS buffer as negative control (Group 3) and OBP BEI inactivated AHSV-5 monovalent vaccine as positive control (Group 4). Vaccines and PBS buffer were formulated with 5% Montanide GEL 01 . Group 0, non -vaccinated, non-challenged but serum samples collected at specific time points. Total RNA (5 pg) extracted from harvested lung tissue were subjected to a type-specific RT-qPCR for AHSV-5. Higher cycle threshold (Ct) values are indicative of virus suppression.

EXAMPLE 4

Production, purification and validation of AHSV1 -9 VP2 ^His proteins

Following on from the successful production of soluble AHSV-5 VP2 ^His and confirmation of immunogenicity of the protein, production of VP2 ^His from the other AHSV serotypes was performed. VP2 proteins from each of the AHSV serotypes were amplified with the primers set out in Table 4. For each AHSV serotype the reverse primer was designed so that in addition to serving as an amplification template it also encoded the synthetic peptide (SEQ ID NO:19) for inclusion in the fusion protein. pEAQ-HT constructs were produced for each of the AHSV serotypes containing the VP2 ^His genes produced in the same manner as the AHSV-5 VP2 ^His construct. The sequences of the genes and the respective proteins are shown in Table 5.

AGL-1 harbouring the pEAQ-HT-AHSV-VP2 ^His constructs for each serotype (OD ₆ OO = 2) individually were infiltrated in plant leaf tissue, two pots per serotype. Five days post infiltration (dpi) fresh leaf material (-100 grams) was harvested using a Matstone juice extractor followed by a 2 min Ultra turrax green juice extraction step in a PBS buffer buffer (140 mM NaCI, 1.5 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 2.7 mM KCI, pH 7.4) at 4 ^e C with the addition of 0.04% (w/v) sodium metabisulfite (Na ₂ S ₂ O ₅ , Sigma 31448) and supplemented with protease inhibitor cocktail in a leaf tissue:buffer ratio of 1 :2. Centrifuge clarified supernatant was subjected to immobilized metal ion affinity chromatography (IMAC), with nickel as the metal ion. Twenty ml of each AHSV VP2 serotype were loaded on an equilibrated 1 ml bed volume Ni-TED resin pre-packed (Protino Ni-TED 2000, Macherey Nagel). Bound protein was eluted isocratically with 5 bed volumes of imidazole-containing buffer. Eluted fractions were pooled and concentrated (100X) to 200 pl using Vivaspin 6 columns (Sartorius, VS0601 ) in a swing bucket rotor (4 000 g x 20 minutes) before subjected to SDS-PAGE (Figure 6). Table 4: Sequences of the forward and reverse primers used to amplify the VP2 genes from each of the nine ASHV serotypes and to introduce nucleic acid sequences encoding the synthetic peptide.

Table 5: Sequences of the AHSV-1 to 9 VP2 ^His constructs.

EXAMPLE 5

Expression of AHSV-5 VP2 as assembled in VLPs, VP2 without histidine tag or VP2 ^His fusion protein containing histidine tag in Nicotiana benthamiana plant leaf tissue.

Production of an African horse sickness VP2 fusion protein

The production of chimaeric AHS virus-like particles (VLPs) in plants as a vaccine candidate was previously described in international publication number WO/2017/182958. Due to the difficulty assembling the triple layer VLPs of some serotypes, the inventors decided to explore the production of a proprietary soluble viral protein 2 (VP2) vaccine candidate. This is due to the fact that VP2 is the main target to elicit virus-neutralising antibodies. The inventors completed an efficacy study, comparing plant produced chimaeric African horse sickness VLPs and VP2 of serotype 5 (AHSV-5) in IFNAR-/- mice. It was demonstrated that both VLPs and VP2 antigen vaccines (prime-boost, 10 pg dose each) elicit the desired neutralizing antibodies to confer protective immunity in IFNAR mice. The ease of production, purification, and concentration of the VP2 ^His fusion protein antigens will underpin a nine-serotype polyvalent vaccine consisting of both VLPs and VP2 ^His for the African continent; or a polyvalent vaccine containing only soluble VP2 ^His of all nine serotypes. Producing mono- and bivalent vaccines to combat serotype specific outbreaks in European countries are not excluded. In addition, the VP2 ^His can equally serve in serotype specific diagnostic test kits and are simultaneously validated.

Purification and validation of AHSV-5 VP2His proteins

As previously described, AGL-1 harbouring the pEAQ-HT-AHSV-5 VP2 or pEAQ-HT-AHSV-5 VP2 ^His construct (ODeoo = 2) were individually infiltrated in Nicotiana benthamiana XT/F plant leaf tissue, two pots per serotype. Five days post infiltration (dpi) fresh leaf material (~20 grams) was harvested using a Matstone juice extractor in a PBS buffer (140 mM NaCI, 1.5 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 2.7 mM KCI, pH 7.4) at 4°C with the addition of 0.04% (w/v) sodium metabisulfite (Na ₂ S ₂ O ₅ , Sigma 31448) and supplemented with protease inhibitor cocktail in a leaf tissue:buffer ratio of 1 :2. Centrifuge clarified supernatant was subjected to immobilized metal ion affinity chromatography (IMAC), with nickel as the metal ion. Twenty ml of each AHSV VP2 (without the synthetic peptide tag), AHSV-5 VP2 ^His and also influenza H6 ^His were loaded on an equilibrated 1 ml bed volume Ni-TED resin pre-packed (Protino Ni-TED 2000, Macherey Nagel). Bound protein was eluted isocratically with 4 bed volumes of imidazole-containing buffer. Eluted fractions were pooled and concentrated (100X) to 200 pl using Vivaspin 6 columns (Sartorius, VS0601 ) in a swing bucket rotor (4 000 g x 20 minutes) before subjected to SDS-PAGE. AHSV-1/5 VLPs were purified using lodixanol density gradient ultracentrifugation as described in Example 2.

Leaves infiltrated with VP2 (non-fusion peptide tagged) (AHSV-5 VP2) shrivelled up and had grey-brown lesions (arrows) whilst leaves infiltrated with pEAQ- HT-AHSV-5 VP2 ^His remained unaffected, dark green and healthy (Figure 7).

RuBisCO, the most abundant protein in plants, contaminated all the partially purified products as visualised with SDS PAGE (Figure 8). AHSV-5 VP2 ^His was produced conservatively estimated as >34-72 mg per kilogram plant leaf tissue. Only AHSV-5 VP2 ^His and H6 ^His were detected using immunoblotting and ELISA (Figure 9 and 10), indicating that the fusion protein assists in the increased production of the fusion protein in planta.

The AHSV-5 VP2 ^His fusion protein was modelled using l-TASSER, Department of Computational Medicine & Bioinformatics, University of Michigan. The modelling indicated the Histidine tag (HHHHHH) was hidden (score of 222323, 0 being buried and 9 highly exposed residue) yet it produced the VP2 ^His fusion protein not only of AHSV-5 but all nine serotypes.

Soluble AHSV-5 VP2 (without the synthetic peptide) produced in plant leaf tissue, resulted in shrivelled up leaves, browning lesions, and faint band on SDS PAGE within five days after infiltration. AHSV-5 VP2 (without the synthetic peptide) was not detected by either immunoblotting or ELISA.

Although avian influenza H6 antigen of 37 kDa was also produced using the same C-terminal end, AHS VP2 is 124 kDa and more than 3.3 fold larger and potentially unlikely to produce a functional VP2 protein that can serve as vaccine candidate. The VP2 fusion protein indeed resulted in protective immunity in IFNAR mice.

The present inventors have studied the use of the C-terminal synthetic peptide fusion with other glycoproteins in order to establish whether this fusion results in an increase of all heterologous glycoproteins expressed in plants. Influenza H5 (44 kDa) and H7 (37 kDa) antigens produced with the same C-terminal fusion resulted in low production of the antigens which could only be detected by immunoblotting. Additionally, attempts to amplify the S1 of the Spike protein of SARS-CoV-2, the same tag was fused either as a N- or C-terminal addition to the protein but resulted in no production of S1. It is thus surprising to see that the addition of the C-terminal fusion to AHSV VP2 leads to a significant increase in the production of soluble AHSV VP2 ^His in plants, as compared to AHSV VP2 (without the C-terminal fusion).

EXAMPLE 6

Production and purification of plant produced VLPs

Wild type N. benthamiana plants were grown in a growth room facility maintained at 26-28°C, 16 h day and 8 h dark. Agrobacterium strain AGL-1 (ATCC® BAA-101 TM) harbouring each of the constructs encoding AHSV-1 VP3/VP7, AHSV-5 VP2 and AHSV-5 VP5 proteins, were adjusted to OD ₆ oo = 1.6 and mixed in a ratio of 2:1 :1 , respectively, for plant infiltration. Double chimaeric AHSV-6 VLPs was assembled by co-expression of constructs encoding AHSV-1 VP3/VP7, AHSV-6 VP2 and AHSV-6 VP5 proteins. Leaves (-100 grams) were harvested 7-8 days post infiltration in a Bicine buffer (50 mM Bicine, 20 mM NaCI pH 8.4) supplemented with protease inhibitor cocktail (Sigma P2714), purified using depth filtration, and concentrated using tangential flow filtration (TFF) 100K Minimate™ TFF Capsule (Pall Life Sciences). A sample of the TFF purified and filter sterilised plant lysate containing the VLPs was subjected to lodixanol (Optiprep, Sigma) density gradient ultracentrifugation as described above to quantity the VLPs within 1 ml TFF lysate for subsequent animal studies.

AHSV VLPs and VP2 antigens were produced in wild type N. benthamiana. The AHSV virion is a triple layered particle formed by the outer capsids (VP2 and VP5), the middle layer (VP7), and the inner shell (VP3, subcore). In this study, double chimaeric AHSV-1/5 VLPs displaying VP2 and VP5 of serotype 5 as the outer shell layered on the AHSV serotype 1 core (VP3/VP7), measuring in the order of 77 to 84 nm (Figure 21 A and 21 B). Similarly, the double chimaeric AHS-1/6 assembled measuring -80 nm but with less abundance (Figure 21 C). The histidine tagged AHS VP2 fusion proteins were subjected to SDS PAGE (Figure 22) and the appropriate band representing VP2 (indicated with an arrow) and subjected to LC-MS/MS based peptide sequencing analysis (Figure 23). The percentage coverage with 95% confidence of each serotype as well as the unique peptides of each serotype are depicted (Figure 23) and clearly demonstrates the distinctiveness of each serotype.

EXAMPLE 7

Purification and validation of AHSV-5 VP2 ^His proteins

AGL-1 harbouring the pEAQ-HT-AHSV-n ^His (n = serotypes 1 -9) constructs (OD ₆ OO = 1 .4) were individually infiltrated in plant leaf tissue. Five days post infiltration (dpi) fresh leaf material (-20-30 grams) was harvested using a Matstone juice extractor followed by a 2 min homogenizing step using an Ultra turrax in a Bicine buffer with the addition of 0.1 % (w/v) N-Lauroylsarcosine sodium salt and supplemented with protease inhibitor cocktail in a leaf tissue:buffer ratio of 1 :2. Centrifuge clarified supernatant was subjected to immobilized metal ion affinity chromatography (IMAC), with nickel as the metal ion (Macherey Nagel Protino® Ni-TED 2000, Catalogue number 745120.25). Bound protein was eluted isocratically with 5 bed volumes of imidazole-containing buffer. Eluted fractions were pooled and concentrated using Vivaspin 6 columns (Sartorius, VS0601 ) in a swing bucket rotor (4 000 g x 15-20 minutes). The concentrate was dialyzed twice within 16 hours against 2 L PBS buffer (140 mM NaCI, 1.5 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 2.7 mM KCI, pH 7.4) at 4 ^e C before use in the vaccine formulation. To coat the enzyme-linked immunosorbent assay (ELISA) plates, VP2 was further purified using Dynabeads™ (Invitrogen, Catalogue number 10104D) to mitigate antibody detection of contaminating plant proteins in serological tests. For the second mice study, plant produced VP2 of all nine serotypes were individually produced as described above with the exception that a bicine buffer was used from extraction throughout until filter sterilisation, and dialysis was substituted with Zeba™ Spin Desalting Columns (7K MWCO, 989892).

The iodixanol-purified density gradient VLPs and IMAC purified soluble VP2 were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE). The assembled VLPs and production of soluble VP2 antigen was confirmed using transmission electron Microscopy (TEM) and LC-MS/MS based peptide sequencing as described above. The SDS-PAGE and LC-MS/MS validation of VP2 antigens were depicted in Figures 22 and 23. Concentration of partially purified VP2 proteins were determined using the Pierce™ Micro BCA protein assay kit (Catalogue # 23235, Thermo Scientific).

EXAMPLE 8

Formulation of VLP and VP2 vaccines

D-(+)-Trehalose dihydrate (Sigma-Aldrich) (15% m/v) was added to the TFF purified VLPs and IMAC purified VP2 antigens before filter sterilisation with a 0.45 pM+0.2 pM Sartopore 2 sterile capsule (Sartorius, 5441307H4) using a peristaltic pump. VLPs and/or VP2 combinations were adjuvanted with 10% Montanide GEL 01 (Seppic, France) or Afrigen Nanoalum immediately before vaccination.

IFNAR ⁷ ' mice vaccination

IFNa/pR ^7- mice (A129, IFNAR type 1 ) were purchased and imported from B&K Universal, Marshall BioResources and bred at the Preclinical Drug Development Platform (PCDDP) in South Africa with the necessary licence in place. Seven-week- old, female, mice were acclimatised under pathogen-free conditions in the biosafety level 3 (BSL-3) facility for 7 days before vaccination. Feed and water were provided ad libitum. Room temperature and humidity were continuously monitored and kept at 22°C±2 and 55%±15 respectively. Light-dark cycles were set at 12 hours each.

In each study, four groups of IFNAR ^/_ mice (n=6) were vaccinated intraperitoneally with 5 pg of each plant produced VLPs/VP2 or VP2 (200 pl formulated vaccine per mice) using a fixed needle syringe (25G diameter, 16 mm length). Mice (study 1 , Table 6) were prime boost vaccinated (days 0 and 14) with PBS buffer as negative control (group 1), the commercial live attenuated vaccine (LAV) of Onderstepoort Biological Products (OBP) as positive control (group 2), pentavalent vaccines consisting of VLPs/VP2 (group 3) or exclusively pentavalent VP2 vaccine (group 4). The vaccines of groups 1 , 3-4 were adjuvanted with 10% Montanide GEL 01 whereas the commercial vaccine contains its own proprietary adjuvant. Sera were collected from the mice on days 0 and 28. All sera collected were heat inactivated at 56°C for 30 minutes before transport to the designated analysis facilities. Mice (study 2, Table 7) were vaccinated as described above apart from the negative control group being vaccinated with bicine buffer (group 1), and the VP2 nine serotype vaccine formulation in bicine buffer (groups 3 and 4) to accommodate the non-compatibility of the Nanoalum to a phosphate buffer. In addition, group 3 and 4 were prime boost vaccinated with 5 pg of each of the nine serotypes (nonavalent) in the primary and booster vaccine, but adjuvanted with either Montanide Gel 01 or Nanoalum, respectively.

Table 6: Design of Mice trial 1

Table 7: Design of Mice trail 2

The use of the commercially available and registered LAV comprise the administration of a trivalent component (serotypes 1 , 3 and 4) followed by a tetravalent preparation (serotypes 2, 6, 7 and 8). In mice trial 1 , the plant produced VLP/VP2 vaccine was designed to emulate the formulation except for the inclusion of serotypes 5 and 9 in the primary vaccine formulation and VP2 of serotype 4 in both primary and booster vaccine (Figure 24). Immunity against AHSV is serotype-specific and there is limited cross-reactivity between certain AHSV serotypes: 1 and 2, 3 and 7, 5 and 8, 6 and 9 (Figure 24). In mice trial 2, a polyvalent VP2 (all nine serotypes) were administered (groups 3 and 4) and adjuvanted with either Montanide Gel 01 or Nanoalum, respectively.

Detection of antibodies against purified plant produced VP2 by ELISA

Mice serum of all six mice were pooled in each independent group at the trial end point (day 28) and used for ELISA and SNT tests. MaxiSorp plates (Thermo Scientific) were coated with 1 pg per well IMAC and Dynabeads purified VP2 (9 independent serotypes) and incubated two days at 4°C. Plates were saturated with 200 pl blocking buffer (PBS-0.05% Tween 20 and 4% casein hydrolysate) and incubated for 90 minutes. The wells were washed thrice with PBS-0.05% Tween 20. Thereafter, mice sera were diluted in blocking buffer (1 :200) were added and incubated for 1 h at room temperature. After three washes in PBS-0.05% Tween 20, plates were incubated for 1 h at room temperature with isotype specific reagents at 1 :1000 (lgG1 or lgG2a; mouse monoclonal antibody Isotyping reagents, Sigma ISO2-1 KT) for 30 minutes. After three washes, a donkey anti-goat-HRP secondary antibody (Donkey anti-goat IgG H&L (HRP) preabsorbed, ab97120, abeam) at a 1 :5000 dilution was added. Finally, after three washes in PBS-0.05% Tween 20, the reaction was developed with 50 pl substrate 3,3',5,5'-Tetramethylbenzidine (TMB from Sigma, T0440-100ml) and stopped by adding 50 pl of 3 N H2SO4. Results were expressed as optical densities (ODs) measured at 450 nm. lgG2a/lgG1 isotyping reflects the balance of Thi-type/Th ₂ -type immune responses in some degree. Higher lgG2a mediates strong cell-mediated cytotoxicity (ADCC) effect and opsonophagocytosis by macrophages. This immune response was observed when mice was vaccinated with the commercially available vaccine, primary vaccine (bottle I, serotypes 1 , 3 and 4, group 2) which is indicative of a stronger bias towards Thi responses for serotype 1 , 3 and 4 (Figure 25) and even serotype 7, probably due to potential cross reactivity of serotypes 3 and 7. In contrast, all mice immunized with plant produced VLP/VP2 or VP2 vaccines formulated in water-in-oil Montanide or Nanoalum adjuvants had lgG1 :lgG2a ratios >1 , indicating that these adjuvants induced primarily Th ₂ -type antibody responses (Figure 25).

Serum neutralising tests (SNTs)

A 1 :5 start dilution of each serum was made in PBS containing 1% Gentamycin 50. Each serum sample was inactivated at 56°C for 30 min before testing. Twofold serum dilutions (initial dilution 1 :10) were made in duplicate rows in minimum essential medium with 2 g/l NAHC03, gentamycin sulfate (Gentamycin 50) 0.05 mg/ml and 5% fetal calf serum. Equal volume of virus with a predetermined titre was added and incubated at 37°C in a 5% CO2 gassed incubator for 1 hour. A Vero cell suspension containing 480 000 cells/ml were added in a volume of 80 pl. Plates were incubated at 37°C in 5 % CO2 and the progress of the CPE of the virus was recorded daily for up to 4-5 days of incubation. Titres were determined as the reciprocal of the highest serum dilution that provided >50% protection of the cell monolayers.

Neutralising antibodies (nAbs) were determined using serum neutralising tests (SNTs) of the mice at the initiation (day 0) and end point (day 28) of the study. It was confirmed that serum collected prior to inoculation (naive mice) was free of AHS antibodies and served as negative control for each individual group. It was demonstrated in mice study 1 that booster vaccination of each serotype is essential, as only serotype 4 that was included in both primary and booster vaccine resulted in titres of 1 :320 (data not shown). In a follow up study 2, where all nine serotypes (nonavalent) were included in both the primary and booster vaccine, the desired neutralising antibodies of 1 :320 were obtained for almost all nine serotypes (Figure 26).

Remarkably, mice vaccinated with the plant produced VP2 antigen nonavalent vaccine (5 pg of each of the nine serotypes) in a prime-boost regime elicited serotype specific antibodies of >112 to all nine serotypes (Figure 26). Thus, the plant produced nonavalent VP2 vaccine resulted in seroconversion to all nine serotypes though superior nAbs were elicited when adjuvanted with Montanide Gel 01 compared to Nanoalum. nAbs titres of mice vaccinated with the AHS VP2 nonavalent vaccine and adjuvanted with Montanide Gel 01 were consistently similar or superior to titres elicited by AHS LAV for each of the nine serotypes on day 28 (end point). Note, that only bottle 1 containing serotypes 1 , 3 and 4 of the commercial vaccine was administered. Mice vaccinated with the LAV bottle 1 incurred adverse effects in the mice, and for humane purposes, bottle 2 (containing serotypes 2, 6, 7 and 8) were not administered. As expected, mice injected with buffer alone remained seronegative.

Statistical analysis

For statistical considerations, statistical difference at P < 0.05 were considered significant in a two tailed Student's t test. REFERENCES

Aksular et al., 2018. A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model. Vaccine doi: 10.1016/j. vaccine.2018.09.065 pii: S0264-410X(18)31341 -0.

Castillo-Olivares et al., 2011 . A modified Vaccinia Ankara Virus (MVA) vaccine expressing African horse sickness virus (AHSV) VP2 protects against AHSV challenge in an IFNAR-/- mouse model. PlosOne 6(1 ): 16503.

De la Grandiere et a!., 2014. Study of the virulence of serotypes 4 and 9 of African horse sickness virus in IRNAR-/-, Balb/C and 129 Sv/Ev mice. Veterinary Microbiology 174: 322-332.