WO2007052238A2 - Chimeric antigens and vaccines - Google Patents

Abstract

The invention provides a chimeric antigen which includes an orbivirus VP7 polypeptide with a foreign peptide inserted into a top domain region of said orbivirus VP7 polypeptide. The chimeric antigen in accordance may be used for treating or preventing a disease such as foot and mouth disease, African horsesickness, or bluetongue disease. The invention also extends to a method of inducing an immune response in a subject by administering an effective amount of an immunogenic composition comprising the chimeric antigen and a pharmaceutically acceptable carrier or diluent to the subject thereby to elicit or induce said immune response.

Description

CHIMERIC ANTIGENS AND VACCINES

THIS INVENTION relates to THIS INVENTION relates to the technical field of immunology. More particularly, this invention relates to a nucleic acid vector, an antigen display system, a vaccine, a method for presenting or displaying an antigen, a method for increasing the solubility of a desired peptide, polypeptide, protein or protein multimer, a method of inducing an immunogenic response in a host, a method for producing an antigen display unit, a method of increasing the solubility of a desired peptide, and a recombinant DNA molecule.

BACKGROUND OF THE INVENTION

The aetiological agent of African horsesickness, a highly infectious noncontagious disease of equines, is African horsesickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae. The molecular biology, structure and morphology of the virus are very similar to that of bluetongue virus (BTV), the orbivirus prototype. Orbiviruses include ten double-stranded (ds)RNA genome segments packaged in a highly ordered icosahedral core particle including two major proteins, VP3 and VP7, and also including three minor structural proteins, VP1 , VP4 and VP6, with enzymatic functions related to the transcription and processing of virus mRNA. The core is surrounded by an outer capsid layer, comprised of two viral proteins VP2 and VP5, of which VP2 is the serotype-specific antigen able to induce a protective humoral immune response.

Structural analysis has revealed that VP7 is a major constituent of the BTV core and that 260 tripod-like shaped VP7 trimers, forming a T=13 lattice, are located on a VP3 scaffold of 12 closely bonded decamers. The 3-dimensional structure of the top domain of AHSV VP7 trimers has been confirmed by X-ray crystallography to a resolution of 2.6 A. The results indicate that the VP7 monomer is composed of two distinct domains - a top domain composed of amino acids 121 -249 folded into an anti- parallel β-sandwich, and a lower domain containing both the first 120 amino acids at the N-terminus and the last 99 residues at the C-terminus arranged in the form of nine α- helices with extended loops in-between.

The two domains are twisted anti-clockwise in the trimer so that the top domain of one monomer rests on the lower domain of the three-fold related subunit, primarily the C-terminal region. Each VP7 has a short C-terminal arm, which may tie trimers together during capsid formation. Deletion of five amino acids at the C-terminus of BTV VP7 abolishes core-like particle (CLP) formation after co-expression of VP7 and VP3, presumably due to lack of trimer-thmer interactions.

A unique feature of AHSV VP7 is that, in contrast to BTV VP7, it is a highly hydrophobic and insoluble protein. When AHSV VP7 is expressed in virus-infected cells or by means of a baculovirus expression system, the hydrophobic VP7 trimers spontaneously aggregate into large, flat, hexagonal disc-shaped crystals with a dimension of up to about 6 μm. The pool of soluble VP7 trimers in infected cells is therefore very small and this has traditionally been considered to be a problem in their use in particulate or sub-unit vaccines.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided a chimeric antigen, the chimeric antigen including an orbivirus VP7 polypeptide or part thereof with a foreign peptide inserted into a top domain region of said orbivirus VP7 polypeptide, or part thereof.

The term "orbivirus" refers generally to African horsesickness virus or bluetongue virus.

The term "foreign", as used herein, refers to peptides, or proteins derived from any organism or species including sequences derived from disease-causing organisms, as well as peptides encoded by synthetic nucleic acids. The use of this term also includes peptides or proteins derived from structural or non-structural proteins of AHSV or BTV or any of the other oribiviruses. According to a further aspect of the invention, there is provided a method of making a chimeric antigen, the method including the step of inserting a foreign peptide into a top domain of an orbivirus VP7 polypeptide, or part thereof.

According to another aspect of the invention, there is provided an immunogenic composition comprising the chimeric antigen of the invention, and a pharmaceutically acceptable carrier or diluent.

According to a further aspect of the invention, there is provided a method of inducing an immune response in a subject, the method comprising the step of administering an effective amount of the immunogenic composition of the invention to the subject thereby to elicit or induce said immune response.

In other words, the invention provides a method of prophylactic treatment or method of inducing a protective immune response in a subject, the method comprising administering an immunologically effective amount of a chimeric antigen according to the invention to the subject.

The foreign peptide may be a peptide or epitope from any organism, and may include peptides or epitopes from disease-causing agents, such as a viral, bacterial, fungal, or parasitic disease-causing agents. However, it is within the scope of the invention that any suitable immunogen, antigen, epitope, or other desired proteinaceous compound may be suitable for inclusion in the antigen display system of the invention, whether synthetically manufactured, or derived from an organism or biological material. The foreign peptide may be a polypeptide or protein.

The foreign peptide may be inserted into the top domain of the orbivirus VP7 polypeptide by providing an isolated nucleic acid sequence encoding the foreign peptide and inserting it into the appropriate region of the VP7 gene sequence encoding the top domain, or a region proximal thereto, thereby providing a recombinant nucleic acid molecule encoding the chimeric VP7 antigen of the invention.

Accordingly, the invention further comprises a recombinant DNA molecule which includes a nucleic acid sequence encoding an orbivirus VP7 polypeptide or part thereof and a nucleic acid sequence encoding a foreign peptide such that the foreign peptide is inserted into a top domain of said orbivirus VP7 polypeptide, or part thereof, when the gene encoding the recombinant VP7 polypeptide is expressed and translated.

The invention includes, as another aspect thereof, a nucleic acid vector including a nucleic acid sequence encoding an orbivirus VP7 polypeptide or part thereof, the nucleic acid sequence having at least one cloning site inserted therein at a position corresponding to, or proximal to, a top domain of the encoded VP7 polypeptide when the gene encoding the recombinant VP7 polypeptide is expressed and translated.

A single orbivirus VP7 protein may combine with two other VP7 proteins to form a VP7 protein trimer. The orbivirus VP7 trimers may aggregate to form VP7 protein particles. The VP7 orbivirus protein may be African horsesickness virus (AHSV) VP7 or bluetongue virus (BTV) VP7. More particularly, the VP7 protein may be the VP7 protein of AHSV serotype 9.

The invention extends to a purified or isolated AHSV VP7 polypeptide comprising at least one of the amino acid sequence of SEQ. ID. NO. 7, a fragment thereof; and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

The AHSV VP7 polypeptide may be encoded by an isolated nucleic acid sequence selected from at least one of the nucleotide sequence of SEQ. ID. NO. 1 , the complement thereof, and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

The insertion site or sites of the AHSV VP7 protein vector may be provided by inserting multiple nucleic acid cloning sites between the nucleic acid codons corresponding to amino acids 144 and 145 of the VP7 protein, between amino acids 177 and 178 of the VP7 protein, between amino acids 200 and 201 of the VP7 protein, or between any one or more combinations of such codons. However, it is to be understood that other suitable cloning sites within the top domain region may also be suitable for insertion of foreign peptides. More specifically, the AHSV VP7 polypeptide or protein may be encoded by an isolated nucleic acid sequence comprising at least one of the nucleotide sequences selected from SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ ID. No. 5, SEQ. ID. NO. 6, the complement thereof, and nucleic acid sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology, to such nucleic acid sequences.

The encoded AHSV VP7 polypeptide may have substantial sequence homology with at least one of the polypeptides selected from SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11 , or SEQ. ID. NO. 12, fragments thereof, and polypeptide sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

The nucleic acid sequence of the VP7 vector may have inserted therein a foreign nucleic acid sequence encoding a desired foreign peptide, thereby forming a recombinant nucleic acid sequence in accordance with the invention. The recombinant nucleic acid may produce a chimeric protein or antigen following expression of the gene and translation of the mRNA thereof.

The foreign nucleic acid sequence may comprise of the nucleic acid sequences selected from SEQ. ID. NO. 17, SEQ. ID. NO. 18, SEQ. ID. NO. 19, SEQ. the complement thereof, fragments thereof, and nucleic acid sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

Multiple copies of the foreign nucleic acid sequence or sequences may be inserted into any one or more of the cloning sites provided in the nucleic acid vector sequences.

Multiple copies of the same foreign nucleic acid sequence, or multiple copies of different foreign nucleic acid sequences may be inserted into the nucleic acid vector sequences. The copies may be inserted in tandem. In addition, tandem copies may be inserted in more than one of the multiple cloning sites of the vector. For example, cloning site 144/145 may have inserted therein a nucleic acid sequence encoding, in tandem, two copies of a first antigen, while site 177/178 in the same vector may have a single copy of a second antigen.

The nucleic acid vector may operably be linked to a cis control element, such as a promoter.

The nucleic acid vector may include a prokaryotic or eukaryotic origin of replication.

The nucleic acid vector may include nucleic acid sequences encoding selection attributes, such as antibiotic resistance selection attributes, or other marker genes.

According to a further aspect of the invention, there is provided an antigen presentation system for presenting a desired antigen to the immune system of a subject, the antigen presentation system including: any one or more of the nucleic acid vectors of the invention, said nucleic acid vector having at least one of the foreign nucleic acid sequences mentioned herein inserted into at least one of the cloning sites provided in the nucleic acid vector.

According to a further aspect of the invention, there is provided a purified chimeric protein or antigen, the protein or antigen including: a protein sequence encoded by at least one of the nucleic acid vectors of the invention; and a foreign peptide sequence inserted into a top domain of the protein sequence encoded by the nucleic acid vector.

According to a further aspect of the invention, there is provided a vaccine composition which comprises: a chimeric protein or antigen of the invention including a peptide derived from a disease-causing agent; and a suitable pharmaceutically acceptable carrier or adjuvant. According to another aspect of the invention, there is provided an antigen display unit comprising a trimer of the orbivirus VP7 polypeptide of the invention having inserted into a top domain thereof a foreign peptide or epitope.

It is to be understood that the antigen display unit of the invention may include trimers of the VP7 protein as well as associative structures comprised of complexes of two, three, or any higher level of associated or aggregated VP7 trimers.

The invention extends further to any one or more of the purified chimeric proteins or antigen display units of the invention, in crystalline or semi-crystalline form.

According to another aspect of the invention, there is provided a eukaryotic or prokaryotic host cell including the nucleic acid vector of the invention. The nucleic acid vector may have inserted therein a nucleic acid sequence encoding a desired foreign polypeptide or protein.

It is to be understood by someone skilled in the field of the invention that prophylactic treatment of an animal against a disease-causing agent may also be accomplished by providing a DNA vaccine composition including any one or more of the isolated recombinant nucleic acid sequences of the invention, under control of a suitable promoter, the recombinant nucleic acid sequence having included therein a nucleic acid sequence coding for a peptide derived from, or similar to, a peptide encoded by the disease-causing agent.

According to yet another aspect of the invention, there is provided a method of increasing the solubility of a desired peptide, the method including the steps of: providing an isolated nucleic acid sequence encoding an orbivirus VP7 polypeptide of the invention, or part thereof; inserting an isolated nucleic acid sequence encoding the desired peptide into a region of the nucleic acid sequence of the orbivirus VP7 gene encoding a top domain loop or a region proximal to a top domain loop of the structural protein, thereby forming a recombinant nucleic acid molecule; and causing the resultant recombinant nucleic acid molecule to be expressed as a chimeric protein or antigen. The chimeric protein or antigen may be subjected to at least one of sonification or repeated freeze/thaw cycles.

According to a further aspect of the invention, there is provided a method of increasing the immunogenicity of a desired peptide, the method including the steps of: providing a nucleic acid sequence encoding an orbivirus VP7 polypeptide of the invention, or part thereof; inserting a nucleic acid sequence encoding the desired peptide into the orbivirus VP7 encoding nucleic acid sequence to form a recombinant nucleic acid sequence; and causing the resulting recombinant nucleic acid sequence to be expressed in a suitable expression system to produce a chimeric protein or antigen; and subjecting the chimeric protein or antigen to at least one of sonification or repeated freeze/thaw cycles.

According to yet another aspect of the invention, there is provided a method of eliciting an immune response in an subject, the method including the steps of: providing a nucleic acid sequence encoding the orbivirus VP7 polypeptide of the invention, or part thereof; inserting a nucleic acid sequence encoding the desired foreign polypeptide into the orbivirus VP7 nucleic acid sequence to form a recombinant nucleic acid sequence; causing the resultant recombinant nucleic acid sequence to be expressed in a suitable expression system to produce a chimeric protein or antigen; and introducing the resultant chimeric protein or antigen into the body of the subject thereby to elicit an immune response.

The method may include a further step of introducing one or more booster doses or shots of the chimeric protein into the body of the animal at suitable periods following the initial introduction of the chimeric protein.

Such periods may include a first booster shot administered between 9 and 16 days following initial introduction into the animal, and a second booster shot administered between 18 and 32 days following initial introduction of the chimeric protein into the animal. The nucleic acid sequences and recombinant nucleic acid sequences of the invention may be expressed in eukaryotic or prokaryotic cells, preferably in insect cells. More specifically, the nucleic acid sequences may be expressed as recombinant baculoviruses in insect cells, such as insect cells derived from Spodoptera frugiperda. However, it is within the contemplation of the invention that any suitable expression system may be used to produce the chimeric proteins of the invention.

The chimeric protein may be complexed with or attached to an adjuvant, immunogen, or other product suitable for eliciting or promoting an immune response against the chimeric protein or desired peptide.

The chimeric protein may be purified prior to introduction into the body of the animal.

According to yet another aspect of the invention, there is provided a method of administering antigens into the body of a subject, the method including the steps of: providing a nucleic acid sequence encoding the orbivirus VP7 polypeptide of the invention, or part thereof; inserting a nucleic acid sequence encoding a desired antigen or foreign peptide into the orbivirus VP7 nucleic acid sequence to form a recombinant nucleic acid sequence; causing the resultant recombinant nucleic acid sequence to be expressed in a suitable expression system to produce a chimeric antigen; and administering into the body of the subject a whole cell fraction or crude lysate containing a sufficient amount of the chimeric antigen to elicit at least a humoral immune response in the subject.

According to a further aspect of the invention, there is provided a substance or composition for use in a method of treating or preventing a disease in a subject, the substance or composition comprising a chimeric protein or antigen in accordance with the invention, and said method comprising administering an effective amount of said substance or composition to said subject. According to a further aspect of the invention, there is provided use of a chimeric protein or antigen in accordance with the invention in the manufacture of a medicament or preparation for treating or preventing a disease caused by a disease- causing agent.

According to a further aspect of the invention, there is provided use of a recombinant DNA molecule in accordance with the invention in the manufacture of a medicament or preparation for treating or preventing a disease caused by a disease- causing agent.

According to another aspect of the invention, there is provided a method for producing a chimeric protein or chimeric antigen, which method comprises growing a host cell or organism containing the recombinant DNA molecule in accordance with the invention, such that the DNA molecule is expressed by the host cell or organism, and isolating the expressed chimeric protein or chimeric antigen.

The disease may be selected from, but is not limited to, foot and mouth disease, African horsesickness, bluetongue disease.

According to a further aspect of the invention, there is provided a kit for producing a chimeric protein or antigen in accordance with the invention, the kit including: a plasmid vector having inserted therein an isolated nucleic acid orbivirus VP7 sequence of the invention, or part thereof; and instructions for the insertion and/or expression of a desired polypeptide into the plasmid vector.

The invention includes, as another aspect thereof, antibodies produced against an antigen delivered or displayed using the system, methods, or antigens of the invention. The antibodies may be polyclonal antibodies, monospecific polyclonal antibodies, or monoclonal antibodies.

The foreign peptide or polypeptide may be an epitope, an antigen, a hapten, an immunogen, a structural protein, a non-structural protein, or a polypeptide having an enzymatic function. More particularly, the polypeptide may be selected from at least one of HIV proteins, foot-and-mouth disease virus antigens, green fluorescent protein, enhanced green-fluorescent protein, one of the influenza virus proteins and/or orbiviral structural or non-structural proteins, e.g. such as from AHSV or BTV.

The invention extends further to an orbivirus VP7 polypeptide including a non-polar residue located within about 20 residues of the C-terminal end of the protein substituted by a polar residue. More specifically, the invention extends to an AHSV VP7 L345R polypeptide having a leucine residue at position 345 mutated to an arginine residue. The invention includes a polypeptide sequence selected from at least one of amino acid SEQ. ID. No. 15, a fragment thereof, and polypeptides having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

The invention includes a polynucleotide sequence selected from at least one of nucleotide SEQ. ID. NO. 13, the complement thereof, fragments thereof, and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

According to another aspect of the invention, there is provided an AHSV VP7 L345R , having inserted into a top domain thereof a cloning site. The invention extends thus to include a polypeptide selected from at least one of amino acid SEQ. ID. No. 16, a fragment thereof, and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

The invention extends also to an isolated polynucleotide sequence selected from at least one of nucleic acid sequence SEQ. ID. NO. 14, the complement thereof, a fragment thereof, and nucleic acid sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control or apply. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods and examples disclosed herein are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following description, sequence listings and drawings.

The invention will now be described, by way of non-limiting example, with reference to the following examples, sequence listings and accompanying drawings.

DRAWINGS

In the drawings:

Figure 1 shows a model of the three-dimensional structure of VP7 obtained using the PyMOL program;

Figure 2 shows SDS-PAGE analysis of recombinant VP7 proteins in insect cells. C: Molecular markers; Lane 1 : Mock infected cells; Lane 2: Wild-type baculovirus infected cells; Lane 3: Cells infected with recombinant baculoviruses that express wild- type AHSV VP7; Lane 4: Cells expressing VP7-177; Lane 5: Cells expressing VP7-200;

Figure 3 (3A to 3F) show sucrose density gradient analysis of total cell lysates of Sf9 insect cells infected with the different insertion vector constructs listed in Table 1. Cells were harvested 72 hours after infection, lysed in buffer containing NP40 as detergent, layered onto 50-70% sucrose gradients and centrifuged for 14 hours at 30 000 rpm in an SW 50.1 rotor in a Beckmann ultracentrifuge. The gradients were fractionated by drop collection from the bottom and an equal portion of each fraction, including the pellet analyzed by SDS-PAGE. VP7-wild-type is shown in 3A, the insertion mutant proteins with single insertions VP7-200, VP7-177 and VP7-144 in 3B, 3C and 3D. The insertion mutant VP7 protein with the double insertion VP7-144-200 is shown in Figure 3E and with the triple insertion VP7-144-177-200 in Figure 3F. Figure 4 (4A to 4F) 4A shows a light microscopic view of the needle-like structures formed in cells infected with recombinant baculoviruses expressing VP7-200.

Figures 4B to 4F show Scanning Electron Microscopy photographs of the particulate structures in cells infected with recombinant baculoviruses expressing VP7 derivatives. Figure 4B shows the hexagonal crystals formed by wild-type VP7, Figures 4C and 4D show similar looking structures formed by VP7-177 and VP7-200 respectively. Figures 4E and 4F show the particles from protein VP7-144-177-200 at both a lower magnification (Figure 4E) and a higher magnification (Figure 4F).

Figures 5 (5A to 5D) shows sucrose density gradient analyses of the total cell lysates of Sf9 insect cells infected with L345R mutated WT AHSV VP7 and AHSV VP7- 200 constructs. Cells were harvested 72 hours after infection, lysed in buffer containing NP40 as detergent, layered onto 50-70 % sucrose gradients and centhfuged for 14 hours at 30 000 rpm in an SW 50.1 rotor in a Beckmann ultracentrifuge. The gradients were fractionated by drop collection from the bottom and an equal portion of each fraction, including the pellet, analyzed by SDS-PAGE. The VP7-WT is shown in Figure 5A, mutated VP7 in Figure 5B, the VP7-177 control in Figure 5C and the mutated VP7- 177 in Figure 5D.

Figure 6 shows a trimerization assay of VP7 wild-type and protein VP7-177. Boiled as well as unboiled samples of the soluble fractions of AHSV VP7-L345R and VP7-177-L345R were loaded onto a 10% polyacrylamide gel and run for 2 hours at 110kV. The first lane shows molecular weight size marker. The positions of the trimers and monomers are as indicated.

Figure 7 shows sucrose gradient analyses of four different constructs expressed in Sf9 insect cells, viz. P4 (SEQ. ID. NO. 17) and P5 (SEQ. ID. NO. 18) inserted, respectively into site 144 of pFB-VP7-144, and the P5 (SEQ. ID. NO. 18) and P6 (SEQ. ID. NO. 19) peptides inserted into site 177 of pFB-VP7-177.

Figure 8 shows a graph of the amount of fluorescence of each fraction containing VP7-eGFP following sucrose gradient analysis. Figure 9 shows a graph of soluble VP7-eGFP production and formation of insoluble aggregates. Soluble VP7-eGPF in the medium represents cells that have broken up.

Figure 10 shows a graph of differences between long (18 hour) and short (1 hour) periods of centhfugation on particle distribution for VP7-eGFP.

Figure 11 shows the results of sucrose fractionations of VP7mt177 containing the P6 and P7 inserts.

Figure 12 shows a representation of the antibody responses against VP7mt177 containing the P6 insert.

Figure 13 shows a representation of the antibody responses against VP7mt177 containing the P7 insert

Figure 14 shows a representation of antibody responses against a crude lysate, soluble fraction from the crude lysate, and the particulate fraction from the crude lysate of Sf9 insect cells expressing VP7mt177 with the P6 insert.

Figure 15 shows an immune blot of an NS1 fusion protein containing an VP2 insert that overlaps with P4 against guinea pig serum raised against VP7-144-P4 NS1.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the use of a structural orbiviral protein, VP7, as an antigen for the presentation of epitopes or other desired peptides, so as to elicit an immune response from host animals against such epitopes or peptides. Frequently, epitopes, immunogens or haptens are not soluble, severely impairing their use as vaccine candidates when used by themselves. Conjugating such epitopes, immunogens, or haptens to carrier molecules facilitates the generation of antibodies against such molecules, but is frequently unsuccessful due to poor or inadequate display of such molecules to the host immune system. The Inventors, in considering the use of chimeric AHSV VP7 proteins as a general purpose vaccine delivery system, considered three different antigen display strategies. Firstly, the use of a core-like particle (CLP) strategy based on the co- expression of chimeric VP7 trimers with major core protein VP3 was considered. However, due to the intrinsic insolubility of AHSV VP7, the yield of such CLPs is extremely low and the strategy is not viable unless VP7 trimer solubility can be improved. Secondly, the use of the VP7 crystalline particles formed during VP7 trimer aggregation was also considered. However, due to the large size of some of the VP7 crystals, such crystals are unlikely to induce an effective humoral immune response. The third strategy, which is the strategy pursued by the Inventors hereof, is to use soluble chimeric AHSV VP7 trimers with or without associated small particles comprised of complexes of two, three, or more aggregated trimers for immune display. However, the inherent insolubility of the AHSV VP7 posed a significant barrier to the use of AHSV VP7 protein in antigen delivery, display, or presentation.

The decision to use this strategy was based on the assumption that the conformational constraints of a trimer will provide some of the advantages of particle display while the very small size of the display system and its larger flexibility may provide the best exposure of peptide inserts to the aqueous environment. This advantage may also apply to small particles comprised of a small number of aggregated trimers.

Central to all of the different display strategies is, however, the solubility of AHSV VP7 trimers and the question to what degree this solubility can artificially be manipulated by inserting peptides in the VP7 top domain. To investigate this the Inventors have inserted a range of small and larger peptides of different hydrophobicity into the VP7 top domain. The range of peptides include immunologically important domains on proteins 1 D of foot and-mouth-disease virus (FMDV), VP2 of AHSV and gp41 of human immunodeficiency virus (HIV). The Inventors also explored the contribution that a leucine residue in position 345 of the AHSV9 VP7 sequence makes to VP7 insolubility, as it was hypothesized that this residue may have an effect on AHSV VP7 solubility. Surprisingly, the Inventors have now found that AHSV VP7 solubility can be manipulated within a range of between 2.5% to about 70% of the total amount of AHSV VP7 expressed. These effects appear to be dependent on the peptide that is inserted, as well as the site of insertion into the VP7 protein. Even more surprisingly, the Inventors have found that a leucine-345 substitution to arginine can result in a large increase in AHSV VP7 solubility.

EXAMPLE 1 :

MATERIALS AND METHODS

Viruses and cells

Baculoviruses were propagated in Spodoptera frugiperda (Sf9) cells, maintained in suspension or monolayer cultures at 27°C using Grace's or Sf900 medium supplemented with 10% (v/v) foetal calf serum (FCS) and antibiotics. Recombinant baculoviruses were generated by means of the BAC-to-BAC™ baculovirus expression system (Life Technologies). Bacmid DNA was transfected into Sf9 cells using Cellfectin® reagent (Life Technologies). Cells were incubated at 27°C until evidence of baculovirus infection was obtained (about 3 days), after which virus-containing supernatants were collected and stored at 4°C until further use. Pure recombinant viral stocks were prepared from single plaques using established procedures. A recombinant plaque-purified baculovirus, capable of expressing the AHSV-9 VP7 gene was available at the start of the investigation. As used herein, the term "insertion mutant" refers to an AHSV VP7 protein into which the multiple cloning sites encoded by the nucleic acid sequences P1 , P2, and/or P3 have been inserted. In addition, use of the term "chimeric protein" refers to an AHSV VP7 protein or insertion mutant protein into which a foreign epitope has been inserted in accordance with the invention.

Modified AHSV-9 VP7 constructs

Vector constructs: Plasmid pBR-VP7 with the complete coding sequence of the AHSV-9 VP7 gene was used to clone the VP7 gene into pFastBac to obtain pFastBac-VP7-WT. This was used for PCR modifications to generate five different pFastBac (pFB)-VP7 vector constructs with different small multiple cloning sites at codon positions corresponding to amino acids 144, 177 and 200 of the encoded VP7 protein. The five different vector constructs that were prepared are listed in Table 1 and included three single insertion site vectors (pFB-VP7-144, pFB-VP7-177 and pFB-VP7- 200), a double insertion vector (pFB-VP7-144-200) and a triple vector (pFB-VP7-144- 177-200). The number refers to the VP7 gene codon immediately downstream of which an 18 nucleotide DNA fragment containing the restriction enzyme (RE) sites listed in Table 1 were inserted. These DNA fragments encode six amino acid peptides P1 , P2 or P3, as listed in Table 1. The corresponding five pFB-VP7 vectors including pFB- VP7-WT were then used to prepare bacmid DNA which was used to generate the recombinant baculoviruses by transfection into Sf9 cells. All plasmid constructs were sequenced at several stages of the construction process to ensure that no additional mutations were introduced.

Vector constructs with amino acid substitutions at site 345 of AHSV9:

The leucine 345 codon was substituted with a codon that encodes arginine in the nucleotide sequences that encode VP7-WT and VP7-177. A PCR strategy was designed to create a Sma\ site at nucleotide position 1047 to 1052, which resulted in the required codon change. It also resulted, however, in a substitution of codon 344 by changing it from a valine to an alanine codon. Since both of these are nonpolar hydrophobic amino acids, this mutation should not affect the hydrophilic character of that site. The modified genes were sequenced and used to generate the respective baculovirus recombinants producing a modified AHSV VP7 L345R protein.

VP7 constructs with peptides P4 (SEQ. ID. NO. 17), P5 (SEQ. ID. NO. 18) and P6 (SEQ. ID. NO. 19) inserted into sites 144 and 177:

P4 (SEQ. ID. NO. 17) insert: The sequence encoding a proposed neutralisation- specific domain of AHSV located within amino acids 377-401 of VP2 of AHSV-9 was amplified from the original cDNA clone adding suitable restriction enzyme sites at the flanking region. This fragment was inserted into site 144 of pFB-VP7-144 and then used to generate the recombinant baculovirus. P5 (SEQ. ID. NO. 18) insert: A sequence encoding the ELLELDKWASLW peptide was obtained from annealed complementary oligonucleotide sequences (Life Technologies), inserted into the multiple cloning sites of pFB-VP7-144 and pFB-VP7-177 and expressed as baculovirus recombinants. This peptide contains the so-called ELDKWA epitope located at amino acids 671-677 of HIV gp41. P6 (SEQ. ID. NO. 19) insert: A sequence encoding the cluster of B-cell epitopes located on the 1 D gene of FMDV (aa 129-164) was amplified using a plasmid containing a part of the genome of FMDV vaccine strain SAT2/ZIM7/83 as template. The PCR product was cloned into site 177 of pFB-VP7-177 and the corresponding recombinant baculovirus was generated.

Separation of soluble and particulate VP7 by means of sucrose density centrifugation:

Chimeric VP7 proteins were expressed in Sf9 cells seeded in a single 75cm² flask and harvested 48 or 72 hours post-infection by dislodging the cells from the surface of the flask and centhfuging them at 1500rpm in a Beckman SW55Ti rotor. The collected cell pellet from each monolayer was resuspended in 800 μl lysis buffer (0.01 M STE with 0.5% Nonidet P40) and cells were incubated on ice for 30 minutes and dounced before being layered on a discontinuous 50-70% (w/v) sucrose gradient in 0.01 M STE. The gradient was centrifuged in the Beckman SW55Ti rotor at 30 OOOrpm for 18 hours. Fractions of 500μl were collected from the bottom volumes, resulting in a total of 10-11 fractions. The pellet from the gradient was resuspended in the same volume 0.01 M STE. Fractions were either stored at -20^°C or immediately analysed by SDS-PAGE. Ten microlitres of each fraction, including the pellet, was analyzed on a 12% denaturing polyacrylamide gel. The gels were stained with Coomassie blue and the protein content of specific bands quantified using the Sigma Gel™ software program (Jandel Scientific). Fractions containing the particulate VP7 proteins were, where necessary, diluted 6-fold with 5OmM Tris-HCI pH 8.0, 5OmM NaCI, and recovered by centrifugation for 45min at 5000 rpm for electron microscopic analysis.

Analysis of protein complexes by scanning electron microscopy

Samples of purified AHSV VP7 protein were fixed in phosphate buffered 2.5% formaldehyde/0.1 % glutaraldehyde at room temperature for 30 min. The fixed particulate protein samples were filtered onto a 0.2 μm nylon filter, washed three times in 0.075 M Na₂HPO₄ and dehydrated by successive treatment in 30%, 50%, 70%, 90% and 100% ethanol. The treatment with 100% ethanol was repeated three times after which the filters were air-dried, mounted onto a stub, and spatter-coated with gold- palladium a few atoms thick followed by a layer of carbon. The stub was viewed at 5.0 kV in a Jeol scanning electron microscope. RESULTS

Construction of VP7 display vectors.

Crystallographic analysis of the top domain of AHSV VP7 protein indicated the presence of four hydrophilic, surface exposed β-loops that connect the β-sheets in the top domain. These loops include an "RGD" motif located on a highly flexible amino acid loop, spanning amino acids 175-180. These hydrophilic loops were targeted as peptide insertion sites. To identify the most suitable sites within these loops, a model of the three-dimensional structure of the top domain of VP7 was obtained with the PyMOL computer program (Fig 1A). From this model, amino acids 144-145, 177-178 and 200- 201 were identified as putatively being the most suitable of these possible insertion sites. A linear presentation of the insertion sites is shown in Fig 1 B. At each of the insertion sites an 18 nucleotide DNA fragment, containing three unique restriction enzyme sites, encoding either peptide P1 or P2 (Tablei ), was inserted immediately downstream of codon positions 144, 177, or 200 of the VP7 gene. This generated three single insertion site vectors (pFB-VP7-144, pFBVP7-177 and pFB-VP7-200), one dual insertion vector (pFB-VP7-144-200) and a triple insertion vector (pFB-VP7-144-177- 200). The position into which the different peptides P1 , P2 or P3 were inserted into the VP7 top domain is as indicated in Table 1.

The insertion mutant VP7 proteins containing the amino acids encoded by the multiple cloning site nucleotides were expressed in Sf9 insect cells and analysed by SDS-PAGE. The results are shown in Fig. 2. A unique protein band, corresponding to the expected size of about 39 kDa, was synthesised for each of the insertion mutant VP7 proteins. The expressed insertion mutant VP7 proteins with double and triple peptide inserts showed a distinct size difference from that of the VP7 control (results not shown). The insertions did not affect the level of VP7 expression and were normally expressed as the most abundant protein with levels of approximately 1 -2 mg VP7 for each batch of 2 x 10⁷ Sf9 cells.

Solubility of the insertion mutant VP7 proteins.

To determine the effect of these small peptide inserts on VP7 solubility and trimer-thmer aggregation, the cellular extracts containing the different VP7 insertion mutant proteins were analysed by sucrose gradient density analysis. A typical result, indicating the sucrose gradient sedimentation pattern of the VP7 proteins after high speed centrifugation, is shown in Fig. 3. The position of the VP7 insertion mutant proteins are indicated by arrows. In most cases the positions of the VP7 insertion mutant proteins were confirmed by Western blot analyses (results not shown).

A typical gradient fractionation pattern of the insertion mutant VP7 proteins indicated that some VP7 proteins sedimented in a position at the top of the gradient together with the bulk of the soluble cellular and baculovirus proteins (fractions 8-9 in Figure 3). These fractions are referred to as the soluble protein fractions and were postulated to contain the soluble VP7 trimers. The particulate proteins are either in the pellet, or in fractions in the lower part of the gradient. In a few exceptional cases the proteins were distributed over several different fractions. The sedimentation profiles of the particulate and soluble VP7 proteins reflected a combination of isopycnic and zonal centrifugation conditions. The soluble proteins sedimented under rate-zonal centrifugation conditions. The larger VP7 particles on the other hand appeared to have a much lower density than free protein and sedimented through the gradient until density equilibrium was reached. This is evident from gradients shown in Figs 3C and 3F in which the sucrose concentration was increased in order to prevent the larger particles from sedimenting to the pellet, as was the case in Figs 3A and 3B. The proteins in fraction 3 in Fig. 3C, and fractions 2 and 3 in Fig. 3F therefore do not represent particles of a particular size, but rather particles of a similar density.

To determine this density, an extract of fusion protein VP7-144-177-200 proteins was subjected to isopycnic centrifugation in the presence of density marker beads. The results (not shown) indicated that the VP7 protein particles sedimented in a range with an approximate buoyant density of 1.11 to 1.33 g/ml. Fusion proteins VP7- 144 and VP7144-200 (Figs 3D and 3E) appeared to contain a significant number of VP7 proteins in fractions 4 to 7. These do not represent soluble proteins but may well provide an indication of very small trimer aggregates which would suggest that, depending on the hydrophobicity of the trimers, there is some spread of differently sized particles ranging from very small trimer-thmer aggregates to the large particles that are seen. Ordinary light microscopy did not provide evidence of the needle-like structures that are normally seen in VP7-WT baculovirus infected insect cells and, in this case, in VP7-200 expressing cells. To investigate what particles could be detected, the Inventors employed electron-microscopy of sucrose gradients of Sf9 insect cells infected with the different recombinant baculoviruses. A typical preparation of VP7-WT, VP7-177 and VP7-200 particles, examined at low magnification in a Scanning Electron Microscope (SEM) revealed large numbers of intact and fragmented flat, hexagonal structures with a diameter of up to 6 μm (as shown in Figs 4B, 4C and 4D). The particles from the VP7- 177 and VP7-200 gradients were largely indistinguishable from the VP7-WT particles, except that in the case of VP7-177 the larger particles often exhibited a distinct rosette type of morphology indicating some reduced stability of the particles. The particulate fractions from the VP7-177 gradient were also associated with large numbers of smaller particles of an unspecific size which were assumed to represent small VP7-177 particles. There was no indication of any recognisable large particulate structures in either the soluble or the particulate fractions of VP7-144 and VP7-144-200. The particles from the VP7-144-177-200 gradients were clearly distinguishable from those of VP7-200 and VP7-177, as may be seen in Fig. 4F. The particles had a rough surface with a diameter of between 6 and 10 μm. The particles were invariably distorted and often had a thick "cookie" type appearance with different layers piled on top of each other. Fig. 4E shows a number of such "cookie-like" particles packed next to one another.

When the cells expressing the VP7-144-177-200 protein were investigated by light microscopy they were also found to lack the distinct needle-shaped structures observed in cells expressing either wild-type AHSV VP7, or proteins VP7-177 or VP7- 200 (Fig 4A). The needle-like structures were only observed when the VP7 proteins formed the smooth hexagonal structures that can be seen in Figs 4B to 4D.

Analysis of the proteins in the soluble fraction confirmed that insertion mutant protein VP7-144 is predominantly soluble with only a relatively small particulate fraction. This is in contrast to insertion mutant protein VP7-200 that only has a very small soluble fraction and insertion mutant protein VP7-144-177-200 which appeared to be even less soluble than VP7-WT. To determine the relative amount of VP7 protein present in either the soluble or particulate fractions, the Coomassie-stained gel profiles were scanned using the Sigma Gel™ software package. The amount of soluble VP7 was expressed as a percentage of the total insertion mutant VP7 in a cell lysate at 72 hours after infection. At this late stage of infection there appears to be no further increase in VP7 synthesis and this provided the most consistent comparative results. The results are summarized in Table 2.

Surprisingly, the Inventors have found that in each case, when either P1 or P2 was inserted in the top domain in one or more of the different sites, there was a marked increase in VP7 solubility. The largest increase was observed when P1 was inserted into site 144 (66% solubility). When P2 was inserted into site177 of VP7-WT the solubility is increased from 8% to 25% as compared to 13% when the same peptide was inserted into site 200. The insertion of P1 and P2 in, respectively, the 144 and 200 sites of VP7-WT resulted in an insertion mutant VP7 protein with a solubility of 41 %. However, when this insertion mutant protein is further modified by inserting P3 into site 177, there is an almost complete loss of all trimer solubility. A large increase in trimer hydrophobicity appears to have occurred and the trimers aggregate almost immediately after synthesis into large, highly distorted particles (Fig. 4F). In addition, no needle-like structure were seen in infected cells.

Effect of a single amino acid substitution on VP7 solubility

It was also investigated whether a leucine-345 substitution to a more hydrophilic amino acid could enhance AHSV VP7 solubility. Accordingly, in both VP7- WT and VP7-177, the leucine residue at position 345 was substituted by an arginine residue. The mutated proteins AHSV VP7-L345R and VP7-177-L345R were expressed in insect cells and analysed by sucrose density analysis to detect any changes in VP7 solubility. The results are shown in Fig 5.

Surprisingly, the L345R mutation was found to have a very large effect on AHSV VP7 solubility. The largest proportional effect was on VP7-WT solubility, in which total solubility of the protein increased from about 8% to more than 40%. The solubility of fusion protein VP7-177 was also increased from about 25% to more than 45%. To investigate if the L345R mutation or its combination with the six amino acid insertion at site 177 disrupted the AHSV VP7 thmeric structure, the different VP7 proteins were analysed under nondenaturing conditions (Fig. 6). The results indicated that under nondenaturing conditions the VP7-L345R and VP7-177-L345R were present as trimers, indicating that trimer formation was not affected by the mutation or the P2 insertions into sites 177. The Inventors found no evidence of dimers in the insertion mutant VP7 proteins that were analysed.

The Inventors also investigated the VP7-144-177-200 fusion protein for protein trimehzation. Although the lack of a suitable soluble fraction complicated the assay, the results confirmed that the insertion mutant proteins that aggregated into the distorted VP7 particles, took the form of a trimer (results not shown).

The effect on VP7 vector solubility after the insertion of a range of small peptides.

The different AHSV, HIV and FMDV peptides that were inserted into the VP7 insertion sites represent important immunological domains on the corresponding viral proteins. The peptides are described in the sequence listings and in Table 1. Peptide P4 (SEQ. ID. NO. 17) overlaps a region associated with the induction of neutralising antibodies against AHSV. Peptide P5 (SEQ. ID. NO. 18) contains the ELDKWA epitope flanked on each side by 3 additional amino acids that correspond to those in the transmembrane protein gp41 of HIV-1 subtype B. Peptide P6 (SEQ. ID. NO. 19): contains the cluster of well-known immuno-dominant B-cell epitopes with flanking regions from the 1 D protein of FMDV. The P4 (SEQ. ID. NO. 17) and P5 (SEQ. ID. NO. 18) peptides were respectively inserted into site 144 of pFB-VP7-144 and the P5 (SEQ. ID. NO. 18) and P6 (SEQ. ID. NO. 19) peptides into site 177 of pFB-VP7-177 (Table 1 ). The four different constructs were expressed in insect cells and analysed by sucrose gradient density analysis. The results are shown in Fig. 7. The insertion of the hydrophilic FMDV peptide (P6 (SEQ. ID. NO. 19)) increased the solubility of the VP7- 177 protein construct from about 25% to more than 50%. The insertion of the AHSV peptide (P4 (SEQ. ID. NO. 17)) slightly reduced the solubility of VP7-144 from 66% to just less than 50%. The insertion of the ELDKWA peptide (P5 (SEQ. ID. NO. 18)) on the other hand caused a large reduction in the solubility of both VP7-177 and VP7-144. The effect on the latter was the most pronounced, reducing solubility from 66% to close to 5%. The effect on VP7-177 was less with a reduction from 25% to about 10%. The results indicate that the effect of small peptides on solubility is both peptide specific and site dependent. DISCUSSION The optimised display of epitopes to the aqueous environment is a key factor in all recombinant vaccine strategies. A common strategy for improving the immune display of small peptides is to constrain them to mimic protein substructures such as virus-like particles or other multimeric protein structures. The Inventors have demonstrated that the insertion of certain small peptides into the top domain of major core protein VP7 of AHSV results in an increase in the concentration of soluble VP7 trimers when the insertion mutant protein is expressed in insect cells. The effect can most likely be ascribed to a reduction in the hydrophobicity of the trimers, resulting in a reduction in the tendency of hydrophobic trimers to aggregate. This modification is affected by the hydrophilicity of the inserted peptide and the site of insertion in the VP7 top domain. The solubility of the insertion mutant VP7 trimers suggests that the protein is correctly folded with the inserted peptide optimally exposed to the aqueous environment.

Although the use of chimeric viral capsid proteins to present peptides to the immune system has been explored for a large number of different viral proteins, the possibility of using the building blocks of such particles, the soluble protein oligomers, has not received the same attention. These strategies nevertheless have certain aspects in common and the physical constraints placed on the oligomehzation of a protein are not unlike those placed on the protein subunits in the formation of larger particles. If this implied rigidity of the polymeric structure is viewed together with the benefits of a very small, nano-sized display unit, an oligomer display strategy may well have several advantages over the more conventional particle display system. Because not all VP7 structures that form trimers will necessarily form CLPs (because the constraints placed on CLP formation are much more rigid than on trimer formation), the constructs of the invention may make it possible to display foreign peptides on the top domain of trimers that could not be displayed on chimeric CLPs. The success of such a strategy is, however, entirely dependent on the solubility of the chimeric VP7 trimers.

This has raised the question of how VP7 trimerization and solubility are affected by the insertion of different peptides in the VP7 top domain. As shown herein, the Inventors have engineered some of the hydrophilic loops in the VP7 top domain shown in Fig 1 as peptide insertion sites. Different combinations of three restriction enzyme sites were subsequently inserted at codon positions 144, 177, and 200 in the VP7-WT gene, generating three mono-insertion vectors, one dual insertion vector and one triple insertion vector (Table 1 ), as discussed hereinbefore.

The six amino acid peptides (P1 , P2 or P3) that were inserted at these different sites all enhanced VP7 solubility, but to very different degrees depending on the peptide, its insertion site, and how many sites were modified simultaneously in the same protein sequence. The only exception to this observation was VP7-144-177-200 which became less soluble than VP7-WT with a soluble fraction of less than 3%. In this case, the simultaneous insertion of three different peptides at three different insertion sites in the VP7 top domain appears to have overly distorted the structure, resulting in a large increase in thmer-trimer aggregation, and the formation of relatively low density protein particles with a distorted and irregular shape.

At the other extreme, the insertion of P1 into site 144 resulted in an increase in the soluble trimer fraction to close to 70% of the total VP7 at 72 hours post-infection. The simultaneous insertion of P1 in site 144 and P2 into site 200 (VP7-144-200) kept the solubility close to 40%. The site of insertion is important, as shown by the result that when P2 was inserted into site 177, the solubility of the insertion mutant VP7 is at least twice that shown when P2 was inserted into site 200.

The insertion of larger, immunologically important peptides into these sites confirmed the observation that both the site and the nature of the insert were important in modifying the hydrophobicity and solubility of chimeric VP7 trimers. The insertion of peptide P6 (SEQ. ID. NO. 19) into VP7-177 increased the solubility of the corresponding insertion mutant protein to more than 50%, whereas the insertion of peptide P5 (SEQ. ID. NO. 18) into the same top domain insertion site reduced solubility to about 10%. This was further confirmed by the observation that, while the insertion of peptide P4 (SEQ. ID. NO. 17) reduced fusion protein VP7-144 solubility from approximately 70% to a more modest 50%, the insertion of P5 (SEQ. ID. NO. 18) into the same site reduced solubility to 5%, which is even less than VP7-WT.

The particles that formed in the case of the VP7-200, VP7-177 and VP7-144- 177-200 insertion mutant proteins were all analyzed by electronmicroscopy. Examination of the gradient purified particulate structures revealed that, in the case of the VP7-200 and VP7-177 constructs, the insertion mutant proteins aggregated into the typical flat, disc-shaped, usually hexagonal crystals of up to 6 μm in diameter and 200 nm thick that have been previously described. These crystals are formed of flat sheets of VP7 trimers with each sheet presumed to represent a double layer of VP7 trimers with their hydrophobic bottom domains located on an operatively internal face, away from the aqueous surroundings. The hydrophobic interactions between these lower or bottom domains appear to keep the layers together. The space between these layers most likely account for the relatively low density of these particles that have been observed by the Inventors.

In the case of the VP7-144-177-200 and other insoluble chimeric proteins with large inserts (results not shown), the surface of the layers is distorted and several of the layers pile up on top of one another, resulting in some of the distorted particulate structures that were observed.

The most likely variables that affect the solubility of VP7 chimers are, firstly, the change in hydrophilicity in the VP7 top domain associated with the insertion of each of the different peptides and, secondly, constraints-related differences determined by the location of the insertion sites and the length of the peptide. These site-specific constraints can affect the folding of the inserted peptides. It appears that such differences in constraints can be predicted by viewing the location of the insertion sites in Fig 7.

In Fig. 1A it is evident that when the three VP7 monomers interact to form a trimer that the location of the 200 top domain insertion sites in each of the VP7 monomers are grouped very closely together in the resultant trimer. This suggests the possibility of some steric hindrance when peptides are inserted into each of the 200 sites of each insertion mutant VP7 monomer, as the peptides will have to compete for the limited available space in the immediate vicinity of these sites, once in trimehc form. This could reduce the exposure of hydrophilic amino acids inserted into this site and reduce the hydrophilic effect of these insertions. It is also clear from Fig. 1A that this steric hindrance does not apply to either the three 144 insertion sites, nor the three 177 insertion sites, as these sites are stehcally situated further apart in a trimer and the peptides inserted into these sites are therefore much less likely to suffer from space constraints. A constraint problem may, however, arise by the simultaneous insertion of peptides into sites 144 and 177 in a single VP7 peptide, as the 144 and 177 sites are (as seen in Fig. 1A) are stehcally very close together which could result in the same distortions as have been proposed for insertion site 200. The results presented herein appear to confirm some of these predictions.

The insertion of hydrophilic peptides into site 200 resulted in only a very minor increase in VP7 solubility whereas the insertion of such small peptides into sites 144 and 177 resulted in a substantial increase in solubility. Even when both sites 144 and 200 were modified, the resultant insertion mutant protein was largely soluble because sites 144 and 200 are far apart. However, when P3 (a very hydrophilic peptide) was inserted into site 177 of pFB-VP7-144-200 the protein was almost completely insoluble. Sites 144 and 177 are very close together and the simultaneous insertion of P1 and P3 may have caused distortions that affect the display of charged amino acids in the VP7 loops in the VP7 top domain, causing the insolubility and distortions that were observed.

Although the hydrophilicity of the inserted peptide is important, its effect is not immediately predictable from hydrophilicity predictions because the insertion site will play a large role in determining how the overall hydrophilicity of the VP7 top domain is affected by the insertion. Therefore, even though P1 is less hydrophilic than P2 and P3 respectively, its insertion into site 144 improves the overall VP7 solubility to a larger extent than when the more hydrophilic P2 is inserted into site 200. It is even more difficult to predict the effect of inserting larger peptides into the different sites because it is not known how these peptides are normally folded and what space constraints are involved. The best characterized of the peptide inserts is the 36 amino acid P6 (SEQ. ID. NO. 19) peptide inserted into site 177. This insertion increased VP7 solubility to more than 50%. This peptide is largely hydrophilic and forms a natural loop on 1 D of FMDV. Surprisingly, the fusion of this loop with the flexible amino acid loop 175 to 180 of the VP7 top domain previously appears to be have resulted in a significant increase in hydrophilicity and VP7 solubility. Equally surprising was the effect of insertion of the AHSV VP2 peptide P4 (SEQ. ID. NO. 17). This peptide has previously been shown to be insoluble and non- immunogenic when it was expressed in insect cells. However, after inserting it into site 144 of VP7, it appears to have become soluble. The insertion of peptide P5 (SEQ. ID. NO. 18), on the other hand, reduced the solubility in both insertion sites but much more so in site 144 than in site 177. The reason for that is not entirely clear, although a plot of the change in hydrophobicity clearly indicated that in both cases the hydrophilic domains at sites 144 and 177 are interrupted by hydrophobic domains. This may well have increased the overall trimer hydrophobicity, resulting in increased trimer-trimer aggregation.

The results presented herein also show that the solubility of VP7 and the VP7-177 protein may be further enhanced by single amino acid changes, as shown by the fact that the leucine-345 in the ninth C-terminal helix of the bottom domain contributes to AHSV VP7 insolubility. When this leucine residue was change to an arginine residue by site-directed mutagenesis in both wildtype VP7 as well as VP7-177, there was a surprisingly large increase in solubility to approximately 40% and 48% respectively.

Since the C-terminal residues of VP7 play an important role in trimer stability, it was necessary to confirm that the mutations of the invention did not affect trimehzation. A trimerization assay was performed similar to that which has been described, but it was evident that neither the C-terminal modification, nor the modifications at the 177 and 200 sites, of the VP7 top domain affected the ability of VP7 to form trimers. The most likely explanation for the increase in solubility is therefore that the mutation increased the overall hydrophilicity. In the case of BTV VP7, the interaction between different trimers is non-specific, involving a set of hydrophobic residues believed to form a thin hydrophobic band around the lower domain. There is very limited contact area at the three fold interfaces indicating that the trimer-trimer interaction is relatively weak. The trimer-trimer interaction may thus be affected by relatively specific changes in hydrophobicity. By decreasing the hydrophobic effect that stabilizes the trimer-trimer interaction, the trimer concentration threshold at which the trimers will aggregate is increased and more trimers are present in solution. The Inventors have found that it is possible to manipulate the equilibrium between AHSV VP7 soluble trimers, on the one hand, and particle formation, on the other hand, in a cell. This has important applications in the development of recombinant vaccines based on the presentation of small peptides as chimerical VP7 trimers to the immune system, as discussed in the further examples below. In the case of AHSV, the neutralization-specific outer capsid protein VP2 has been the target of most recombinant vaccine strategies. However, the insolubility of insect cell-produced VP2 proteins remains the main stumbling block to using VP2 peptides and epitopes as particulate vaccines by themselves. The possibility of using epitopic domains of AHSV VP2, rather than the full-length VP2, has also been explored and a number of putative neutralization-specific domains have been located between amino acid residues 253 and 413 on VP2 of AHSV. However, when these peptides are expressed in either bacterial or insect cells they are again largely insoluble and non-immunogenic. The Inventors have now shown that one of these peptides can be displayed in a soluble form when presented on the top domain of soluble VP7 trimers. The option of using chimeric trimers for immune display is also not confined to small peptides and the results shown in the following examples indicates that inserting even a full-length protein (viz. the 220 amino acid eGFP protein) into site 177 of the VP7 protein resulted in an immunogenic, green fluorescing, chimeric protein with a large soluble fraction trimer fraction.

TABLE 1 :

Modifications to generate: VP7 pFB insertion vectors; AHSV VP7 with L345R mutations; VP7 chimerae with immunologically important peptides. Numbers 144, 177 and 200 in the VP7 refer to the VP7 gene codon immediately downstream of which a multiple cloning site was inserted. The DNA inserts encode one of peptides P1 , P2 or P3. The peptides involved in all the different modifications are: P1 : aa "PGQFLQ" encoded by DNA fragment with RE sites Sma\, EcoR\ and Xho\; P2: aa"KLSRVD" encoded by DNA fragment with RE sites Hind\\\, Xba\ and Sa/I; P3: aa"LQRPAR" encoded by DNA fragment with RE sites BssHW, Stu\ and PsM ; P4: (SEQ. ID. NO. 17): aa'OPNHDTWKNHVKDIRERMQKEQSAN" (aa377-401 ) of VP2 of AHSV-9; P5: (SEQ. ID. NO. 18): aa" ELLE LD KWASLW" (aa668-679) of gp41 of HIV-1 subtype B; P6: (SEQ. ID. NO. 19): aa 'RYNGECKYTQQSTAIRGDRAVLAAKYANTKHKLPST" (aa129-164) of 1 D of

FMDV vaccine strain SAT2/ZIM7/83.

Table 2: The percentage of VP7 insertion mutant protein in the soluble protein

% Chimeric VP7 protein in the soluble

VP7-based protein fraction at 72hpi

VP7 Wildtype 8.2 ± 1.5

VP7-177 (P2-insert) 25.4 ± 2.8

VP7-144 (P1 insert) 66.2 ± 3.8

VP7-200 (P2 insert) 13.2 ± 1.8

VP7-144-200 (P1.P2 insert) 41.0 ± 4.6

VP7-144-177-200 (P1 , P2, P3 insert) 1.5 ± 0.5

EXAMPLE 2:

MATERIALS AND METHODS

Construction of various VP7-eGFP fusion proteins:

VP7-177-eGFP: A full-length eGFP protein (220 amino acids) was inserted into site

177 of the VP7-177 top domain.

VP7-C-eGFP: The eGFP protein was attached to the C-terminal of VP7.

VP7-177-eGFP-truncC: The eGFP protein was inserted into site 177 of the VP7 top domain, but with the C-terminal half deleted immediately after insertion of the eGFP.

Optimizing time of harvesting

Two separate suspension cultures of 5 x 10⁷ Sf9 cells were infected 12 hours apart with recombinant baculovirus expressing VP7-eGFP. About 1 x 10⁶ cells were collected at 18 hours post infection and every representative time 6 hours thereafter, up to, and including, 90 h.p.i. Cells were collected by centhfugation at 20Og for 5 minutes, after which the TC100 medium in which the cells were growing was separated and kept, and the cells were resuspended in 1 ml STE (0.01 M NaCI, 0.01 M Tris, 0.05M EDTA) containing protease inhibitors (Pepstatin and Pefabloc). The medium and cells were frozen and kept at -80⁰C until all representative samples were collected. Each sample of cells was treated with NP40 for 30 minutes, the cells lysed mechanically by douncing, and collected by centrifugation for 10 minutes at 16,20Og (benchtop centrifuge) to separate soluble and insoluble components. The insoluble component (pellet) was re- suspended in 1 ml STE (0.01 M NaCI, 0.01 M Tris, 0.05M EDTA). Thereafter, fluorescent measurements of each component (soluble, insoluble, medium) were taken for each representative sample. To account for background fluorescence, samples were also taken pre-infection, as well as at 0 h post infection (i.e. immediately after infection).

Small scale sucrose gradient analysis

For general analysis 5 x 10⁷ cells expressing VP7-eGFP were collected 48 h.p.i. by centrifugation at 5000 rpm for 5 minutes, after which the supernatant was discarded. The cells were re-suspended in 400μl STE (0.01 M NaCI, 0.01 M Tris, 0.05M EDTA) containing 0.5% non-ionic detergent (Triton X100 or NP40) and protease inhibitors (Pepstatin and Pefabloc, maximum amounts recommended by manufacturer) and left on ice for 30 minutes. The cells were then lysed mechanically by douncing. A discontinuous sucrose gradient was prepared from bottom to top containing equal volumes (4.8 ml total) of 70%, 65%, 60%, 55% and 50% sucrose, upon which all of the cell lysate was loaded. The sucrose gradients were then centrifuged using a Beckman SW55 rotor at 40 OOOrpm (151 00Og) for 18 hours. About 19-21 fractions of approximately 250μl (descending density) each were collected, which were then analyzed by fluorometery as well as by SDS-PAGE.

Large scale sucrose gradients

For large scale purification 1 x 10⁸ Sf9 cells expressing VP7-eGFP were collected 48 h.p.i. by centrifugation at 5000 rpm for 5 minutes, with the supernatant being discarded. The cells were re-suspended in 4 ml STE (0.01 M NaCI, 0.01 M Tris, 0.05M EDTA) containing 0.5% non-ionic detergent (Triton X100 or NP40) as well as protease inhibitors (Pepstatin and Pefabloc, maximum amounts recommended by manufacturer) and left on ice for 30 minutes. The cells were then lysed mechanically by douncing. A discontinuous sucrose gradient was prepared from bottom to top containing equal volumes (20 ml total) of 70%, 65%, 60%, 55% and 50% sucrose, upon which all of the cell lysate was loaded. The sucrose gradients were then centrifuged using a Beckman SW28 rotor at 20 OOOrpm (53 00Og) for 18 hours. Approximately 30- 33 fractions of approximately 750μl each (descending density) were collected, which were then analysed by fluorometery as well as by SDS-PAGE.

Harvesting and partial purification of VP7-eGFP proteins

Cells were harvested at either 48 h.p.i. or at any time after 60 h.p.i. by low speed centhfugation at 5000 rpm for 5 minutes. Cells were resuspended in 0.01 M NaCI STE buffer containing 0.5% detergent (either Triton X100 or NP40), left on ice for 30 minutes, and then dounced 20 times on ice.

RESULTS

Solubility and fluorescence of eGFP inserted into the top domain of AHSV VP7

The use of eGFP as an insert to characterise some of the properties of chimeric VP7 proteins was postulated to hold several advantages. Fluorescence of chimeric VP7-eGFP could provide evidence that the inserted protein is correctly folded and exposed to its aqueous environment. It is also a non-invasive method of quantifying chimeric VP7 expression levels at different times after infection and makes it possible to track the assembly of chimeric VP7 proteins into particles, thereby allowing one to quantify the ratio of soluble versus particulate protein throughout the infection cycle.

Chimeric protein VP7-177-eGFP was expressed by means of a baculovirus recombinant. In this fusion protein eGFP is positioned on the highly flexible amino acid loop 175-180 in the protein top domain. Expression of the protein was confirmed by SDS-PAGE and by means of Western blot (not shown) using an anti-eGFP antibody (N- terminal, SIGMA). Surprisingly, the chimeric protein VP7-eGFP was fluorescent, indicating that the eGFP had retained its conformational and functional integrity, even after having been inserted medially into AHSV VP7.

To determine the ratio of soluble versus particulate VP7-177-eGFP, the cells infected with the baculovirus recombinants were harvested at 72 hours after infection and analysed by sucrose gradient density analysis as indicated under the Materials and Methods section for this example above. The relative amount of fluorescence of each fraction was calculated, together with the relative amount of VP7-177-eGFP protein calculated from Coomassie stained gels. The result is shown in Figure 8.

The largest proportion of VP7-177-eGFP fluorescence (green) is associated with the soluble fractions 25-28. The stability of this soluble fraction, which constituted about 54% of the total VP7 chimeric protein expressed at 72 h.p.i., was tested under a range of different conditions that included different salt concentrations, as well as freeze drying. Surprisingly, the Inventors found that in all cases tested, the trimers remained soluble and did not aggregate. Apart from the soluble fraction, there were, however, also distinct, but small, particulate peaks at fractions 6-13 and 18-22 respectively. The chimeric VP7 protein values (blue) match these positions in the gradient but not in the same relative amount. The soluble fraction appeared to be proportionally much smaller and the soluble fraction was not much more than about 20% of the total VP7 expressed.

Due to the apparent differences between the fluorescence and the protein profiles, the Inventors explored the possibility that the VP7-eGFP in trimer form displayed more fluorescence per unit of VP7 protein than the aggregated form of the protein. To quantify these differences in fluorescence, the amount of VP7-eGFP in each fraction in Figure 8 was correlated with fluorescence readings for each fraction. These calculations showed an approximate 7-fold increase in fluorescence in the trimeric form of VP7-eGFP as compared to the aggregate form. This difference can be ascribed to a "masking" effect of the particulate form of the protein due to the tendency of the hydrophobic VP7 trimers to aggregate in layers that are packed on top of one another. The fluorescence per unit protein value may well represent a proportional indicator of exposure on the surface of either soluble trimers or trimer aggregates which is informative about immune display of such an insert. These results prompted further investigation of the dynamics of aggregation of chimeric VP7 proteins.

The dynamics of particle formation and aggregation at different times after infection. To investigate the assembly process of VP7-eGFP during expression, samples were harvested 18 h.p.i. and every 6 hours thereafter, and separated into three components namely: (i) VP7-eGFP released into the cell culture medium; (ii) VP7-eGFP present in the soluble fraction of the cells; and (iii) VP7-eGFP in the particulate fraction. Cells were separated from the medium by low speed centrifugation and cell lysates were separated into soluble and insoluble fractions by means of high speed centrifugation. The fluorescence in each of the soluble and particulate fractions was determined. In view of the previous observation that the particulate VP7-eGFP fluoresces about 7X less than the soluble VP7-eGFP, all particulate values were multiplied by a factor of 7 in order to make comparisons of the relative amount of VP7- eGFP protein in the soluble and particulate fractions at different times after infection. The results are shown in Fig. 9

The results in Fig. 9 indicate the following: The percentage of soluble VP7 (pink) reaches a maximum by about 48 h.p.i. Most of the VP7 synthesized beyond that point aggregates to form particles. The relative amount of particles (blue) increases from about 30 h.p.i. and reaches a maximum at about 48 h.p.i. with only a small relative further increase. The apparent decline in the percentage soluble protein after 48 hours is due to cell lysis. As the cellular membrane is disrupted, the soluble protein is released into the cell medium. This is apparent from the increase in fluorescence in the medium after 48 h.p.i (black). The particles, on the other hand, remain cell- and cell debris-associated and no such decline is observed (blue). The combined soluble VP7- eGFP chimeric protein (green) reaches a maximum by about 48 h.p.i. The Inventors have found that, in order to ensure maximum solubility, soluble proteins should therefore not be harvested after 48 h.p.i. Particles on the other hand, are best harvested after 48 h.p.i. and until as late as 72 h.p.i.

DISCUSSION

The aggregation of soluble chimeric VP7 into protein aggregates is concentration dependant. Over and above a maximum concentration, the tendency of trimers to aggregate will increase proportionally. This threshold concentration is probably linked to the hydrophobicity of the chimeric trimers and may therefore be different for every different chimeric construct that is made. In chimeric VP7-eGFP this threshold level was reached at 48 hours after infection and also just before cellular lysis starts depleting the amount of soluble protein that can be recovered. The fluorescent eGFP in the medium does not reflect an active process of protein trafficking out of the cell but rather a collapse of the cellular membrane and cell death that will release the soluble protein (and very small particles) into the medium. This set of events can most likely be extrapolated to apply to any chimeric VP7 protein, although the threshold may be reached sooner depending on the hydrophobicity of the chimeric trimers. The results suggest that the best time for harvesting cells with the aim of isolating soluble proteins is in the order of 48 h.p.i. The results in Fig. 9 show that harvesting cells at 72 h.p.i yielded only 40% of the possible maximum soluble protein, relative to cells harvested at 48 h.p.i.

To further investigate this and confirm these results, the experiments were repeated by harvesting cells at 36 and 48 h.p.i and analysing the distribution into soluble and particulate fractions by means of sucrose gradient density centhfugation. Two centrifugation conditions at 150 00Og for either 1 hour or 18 hours were used in this analysis. These differences were introduced to obtain evidence about the size and the density of the particles aggregates. The fluorescence in the different fractions was calculated and the results are shown in Figure 10.

The particulate fraction fluorescence values shown in Figure 8 have not been corrected and will therefore underestimate the amount of particulate protein by a factor of about 7. The data at 36 and 48 h.p.i is, on the other hand, directly comparable to the fluorescence data at 72 h.p.i (green) in Figure 1. The results of the solubility experiments indicate that at 36 h.p.i most of the VP7-eGFP appears to be soluble. At 48 h.p.i. the proportion of particulate VP7-eGFP shows an increase leading up to the results shown in Fig. 1 at 72 h.p.i. The 20% soluble VP7-eGFP at 72 h.p.i is therefore a significant underestimation of the total amount of soluble chimeric VP7 that could be recovered.

The differences between the long (18 hour) and the short (1 hour) periods of centrifugation (shown in Fig. 10) indicated that the particles are a mixture of large and small particles. The large insoluble particles move very quickly through the density gradient until the position of equilibrium density has been reached (fractions 3-5 of the gradient). It is evident that at 48 h.p.i and after 1 hour centhfugation a significant proportion of chimeric VP7 has already reached density equilibrium, which is reflective of the large size of the particles. However, after 18 hours the peak size has increased substantially, reflective of an equal portion of much smaller particles that required much more time to reach density equilibrium. These results provide evidence that the particulate fraction consists of a mixture of large and small particles.

Immune response against VP7-177-eGFP

The increased fluorescence of soluble chimeric VP7-eGFP as compared to the chimeric VP7-eGFP particles suggest that the trimers have an advantage with respect to the immune display of peptide inserts. In order to confirm this, guinea pigs were injected with soluble, particulate and mixed cellular fractions of chimeric VP7-177- eGFP. These results confirmed an immune-specific immune response to the soluble VP7-eGFP. The Inventors have, however, shown by Western blot analysis that the immune serum against mixed particulate-soluble eGFP is able to recognize a chimeric NS1 -eGFP protein, indicating an immune specific response against eGFP (not shown). These results confirm a previous result (shown hereinafter) that a very good neutralization-specific immune response was obtained against a soluble chimer of VP7 having inserted therein a small FMDV peptide.

Evidence that VP7-177-eGFP is a trimer

In order to confirm that VP7-177-eGFP is a trimer and to determine how trimehzation affects VP7 solubility, a chimeric protein was constructed that lacked the C-terminal half of the VP7 protein. This was postulated to eliminate the ability of chimeric VP7 to form trimers. This construct, VP7-177-eGFP-truncC, has eGFP inserted into site 177 of the VP7 top domain, but has amino acids 178-349 deleted immediately downstream of the inserted eGFP. This elimination was predicted to prevent the formation of trimers, since this would eliminate two of the major α-helices involved in trimer formation. The protein was expressed by means of a recombinant baculovirus and sucrose gradient density analysis indicated that the protein was almost completely soluble with almost all fluorescence recovered from the top of the gradient. There was, however, a difference in migration pattern of soluble VP7-177-eGFP and VP7-177-eGFP-truncC. The truncated chimeric protein migrated significantly slower, more or less in agreement with it being a monomer and not a trimer, as is the case for full-length VP7-177-eGFP.

The presence/absence of trimers and monomers was then investigated by means of a trimerization assay. This assay is based on analysing the soluble fractions of VP7-177-eGFP and VP7-177-eGFP-truncC under denaturing (boiled) and non- denaturing (not boiled) electrophoresis conditions and assaying the size of the different proteins by means of a Western blot with eGFP serum.

Evidence that the insertion site is important for solubility

To investigate the effect of insertion site on solubility, the Inventors also prepared a chimeric VP7 fusion protein in which the eGFP was attached to the C- terminal site of VP7. This protein, VP7-C-eGFP, was expressed as a recombinant baculovirus. Good expression and fluorescence was obtained, but on sucrose gradient density analysis, the chimeric protein was found to be completely insoluble. This result indicates that the site of insertion is key to the solubility of the VP7-eGFP fusions and that different sites may reflect very different outcomes.

Evidence that the VP7-eGFP fusion proteins will elicit a good immune reponse.

The Inventors isolated crude, soluble and particulate fractions of VP7-eGFP from cell lysates. The crude fraction contained a mixture of soluble and particulate VP7- eGFP and was expected to provide the best set of options for eliciting an immune response when introduced into a test animal. The Inventors injected these constructs into guinea pigs and analysed the sera. The Inventors found that the serum against the crude chimeric protein isolate gives a good immune response against eGFP - the antibodies generated recognize a fusion product of NS1 with eGFP (not shown). The protein is therefore displayed correctly by the VP7 trimer.

EXAMPLE 3:

MATERIALS AND METHODS Expression and purification of chimeric VP7 proteins containing FMDV inserts-177-P6 (SEQ. ID. NO. 19) (as before) and VP7-177-P7 were constructed, where peptides P6 (SEQ. ID. NO. 19) and P7 represent the following:

P6 (SEQ. ID. NO. 19): aa 'RYNGECKYTQQSTAIRGDRAVLAAKYANTKHKLPST" (aa129-164) of 1 D of FMDV vaccine strain SAT2/ZIM7/83. This epitope contains a cluster of immunodominant epitopes on the 1 D protein of FMDV. The soluble trimer fraction was generally more than 60%. When this peptide is inserted into site 177, it is recognized by FMDV antiserum, indicating that there is some antibody recognition of the inserted peptide.

P7: The full-length 1 D protein of FMDV: This protein includes the P6 (SEQ. ID. NO. 19) epitope. When this protein was expressed in Sf9 cells it was expressed to high levels (results not shown), but it did not appear to have a soluble fraction and all of the protein appeared to be particulate. The VP7-177-P7 fusion protein (VPI inserted into VP7 at site 177) is, however, recognized by FMDV antiserum, indicating that the protein has domains that are recognized by antibodies in the serum.

Cells and Viruses

Sf9 insect cells were infected at an m.o.i. of 5 pfu/cell with recombinant baculoviruses expressing vector protein without any insert (VP7-177), the chimeric protein VP7-177-P6, or the chimeric protein VP7-177-P7.

Protein expression

Cells infected with the various recombinant baculoviruses were harvested at 72 h post infection. Expression was confirmed by gel electrophoresis on 12% SDS- polyacrylamide gels stained with Coomassie blue, followed by Western blot analysis using anti-FMDV vaccine strain SAT2/ZIM7/83 as well as anti-AHSV9 antiserum (not shown).

Protein fractionation

For immunization purposes, three different fractions of each of the fusion proteins were prepared: (i) a crude extract comprising a combination of both soluble and particulate proteins; (ii) a soluble fraction only; and (iii) a particulate fraction only. The crude extracts were prepared by resuspending 2 x 10⁸ infected cells in 0.01 M STE, incubation on ice for 30 min and mechanical disruption by douncing 20 times. The nuclear fraction was removed by low speed centrifugation at 1500 rpm for 3 min and rinsed once. Expression of the chimeric protein and its concentration in the lysate was estimated from the band intensity after SDS-PAGE analysis and Coomassie Blue staining. To prepare the soluble and particulate fractions, lysates were prepared in 0.01 M STE with 0.5% Nonidet P40, incubated on ice, mechanically dounced and the nuclear fraction removed as before. Lysates were then loaded onto 40-70% discontinuous sucrose gradients in 0.01 M STE. Gradients were centrifuged in an SW28 rotor at 20 000 rpm for 16 h. Fractions of 1 ml each were collected using a dialysis pump and analyzed directly on a 12% SDS polyacrylamide gel (not shown). The gels were stained and the protein content of the specific band quantified by the Sigma Gel™ software program (Jandel Scientific). The top fractions of the gradient (fractions 21 -27) representing soluble chimeric protein, and the lower fractions (fractions 5-16) representing protein in a particulate form, were pooled respectively. The size of the pools are as indicated in Fig. 11. The fractions were dialyzed individually overnight against 0.01 M STE with three buffer changes, freeze-dhed and resuspended in 1 x PBS. All proteins were stored at -7O⁰C prior to immunizations.

Immunization procedure

Groups of female guinea pigs were injected intra-muscularly with either a crude-, soluble- or particulate sample of VP7-177, VP7-177-P6 or VP7-177-P7. Two to three animals were immunized with 40 μg of the respective protein samples in 250 μl 1 x PBS emulsified in an equal volume of Freund's complete adjuvant. Two boosts with identical samples were administered at two weekly intervals. Serum samples were obtained from each animal pre-vaccination, 24 hr before each booster injection and two weeks after the final boost.

Detection of antibody responses to FMDV

A sandwich enzyme-linked immunoabsorbent assay (ELISA) was used to detect antibodies in serum to the FMDV. Briefly, 96-well plates coated with rabbit antibodies against FMDV vaccine strain SAT2/ZIM7/83 were used to trap tissue- cultured virus, diluted 1/50. Guinea pig test serums collected at different times pre-and post-vaccination were serially diluted and antibodies detected with peroxidase-labelled anti-guinea pig conjugate. Titres were expressed as the serum dilution that yielded absorption values three times above pre-immunization serum.

The presence of antibodies mediating FMDV neutralizing activity was determined in a virus-neutralization assay. In a 96-well plate, two-fold dilutions of the test sera were added to 100 TCID₅₀ units of FMDV vaccine strain SAT2/ZIM7/83. The mixture of serum dilutions and virus was incubated at 37°C for 1 hr and then adsorbed to IBRS2 cells. Cells were incubated for 3 days until a cytopathic effect (CPE) was observed. The serum neutralization titre was defined as the dilution that resulted in a 50% reduction in CPE.

RESULTS

Preparation of soluble protein fractions

Sf9 cells were infected with recombinant baculoviruses expressing chimeric proteins VP7-177-P6 and VP7-177-P7. Cells were harvested at 72 h.p.i. and the crude, soluble and particulate protein fractions prepared as described. The particulate and soluble fractions were obtained from sucrose gradients as described and the specific fractions collected were as indicated in Fig. 11 from the VP7-177-P6 and the VP7-177- P7 gradients. The relative amount of chimeric protein in the different fractions shown in Fig. 11 was estimated from the band intensity after SDS-PAGE analysis and Coomassie Blue staining.

Immune response:

All animals vaccinated with the VP7-177 or VP7-177-VP7 proteins had undetectable naive anti-FMDV antibody levels. From the results it is clear that the lack of detectable anti-FMDV antibodies against the crude or other fractions of the fusion protein with the full-length 1 D inserted into VP7 (VP7-177-P7) was an indication that large insoluble VP7 chimeric particles without any soluble fraction do not appear to induce a good antibody immune response. As is evident from Fig. 11 , this fusion protein did not contain any detectable soluble protein and the so-called "soluble fraction" from the crude lysate (see above) contained only soluble cellular proteins but no soluble chimeric VP7-177-P7 products. Since no antibodies to FMDV could be detected by ELISA for these samples, virus neutralization assays were not performed for these samples.

In contrast (referring now to Figures 12, 13 and 14), animals vaccinated with chimeric protein VP7-177-P6, containing only the epitope sequence, elicited very strong antibody responses, both in ELISA and virus-neutralization assays. The most significant virus neutralization antibodies were induced using a soluble preparation of the chimeric protein, as well as the crude protein inoculums. Indeed, low anti-FMDV antibody titres could already be detected after the first boost in two of four animals injected with a crude preparation of the protein. This response increased significantly after the second boost, with the highest ELISA response monitored at a serum dilution of 1/4033 and the highest neutralization titer at 1/456. This is highly significant, as a 1/100 neutralization titre in bovine is considered sufficient to provide protection against FMDV. Very strong ELISA signals were also obtained from animals injected with a soluble preparation of the protein, ranging from a 1/1238 to 1/2095 serum dilution after the second boost. Interestingly, ELISA titers decreased after the third boost in those animals where significant antibodies had already been produced following the second boost. This may be due to antibody depletion, following the strong results obtained from the second boost.

On average, the animals injected with soluble VP7-177-P6 elicited the strongest virus neutralization titers. Although one or two animals injected with a particulate preparation of the protein produced antibodies as monitored by ELISA, these antibodies could not neutralize the FMDV virus.

Animal trials

While the results indicate that the full-length FMDV 1 D displayed as a fusion protein in the VP7-177-P7 vector was not highly successful in its ability to elicit antibodies in vaccinated animals due to possible aggregation, the presentation of the immunodominant epitope as a fusion protein with VP7-177-P6 elicited a very strong antibody response as measured by both ELISA and by a virus neutralization assay. This indicates that the epitope is correctly displayed by the VP7 trimer display system. These preliminary results indicate that the most promising candidate for eliciting neutralization antibodies is an immunogen prepared using soluble trimeric fractions of the protein as an epitope display unit. The use of the crude extract, which contains a mixture of both soluble trimeric fusion proteins and a range of differently sized particles also gave a good immune response. The Inventors have previously found that in the case of fusion proteins where there is a large soluble fraction, such as in VP7-177-P6 , there is also a range of small to very small particles with almost no large particles. These small particles may represent anything from about 3 trimers to aggregates containing more than 1000 trimers. In the case of VP7-177-P7 there appeared to be very few small particles or trimer aggregates and the majority of the particles appeared to be in the order of 6 μm or even larger. It is within the contemplation of the invention that the immune responses obtained herein may be further enhanced, if necessary, by the addition of diluents, carriers, adjuvants, or other suitable vaccine carriers required to produce vaccine compositions.

As such, it may well be possible that the small to very small particles or trimer aggregates are adequate in inducing a weak to satisfactory immune response, as shown by the results obtained with particulate VP7-177-P6. The Inventors therefore do not exclude the possibility that the trimer aggregates or particles are able to induce a good immune response. However, the results as presented herein show that a reduction in the size of the particles or trimer aggregates is accompanied by an improved display of the antigen, as well as by an enhanced immune response. The soluble nano-sized trimer particles or antigen display units are therefore considered to be the best display system for foreign epitopes or antigens whereas the large hexagonal crystals or large trimer aggregates are probably less suitable for immune display than the smaller trimer aggregates.

Example 4:

Guinea pigs were immunized to evaluate the immune response against the following VP7 fusion proteins:

The VP7 fusion protein (VP7-177-P6) with the FMDV P6 peptide (SEQ. ID. NO. 19) was inserted into site 177. This is the same fusion protein used in the experiments described in Example 3.

A VP7 fusion protein (VP7-144-P4) with a short AHSV P4 peptide (SEQ. ID. NO. 17) was inserted into site 144. MATERIALS AND METHODS

Peptide inserts and sequences

P6 (SEQ. ID No. 19): aa 'RYNGECKYTQQSTAIRGDRAVLAAKYANTKHKLPST" (aa129-164) of 1 D of FMDV vaccine strain SAT2/ZIM7/83. This epitope contains a cluster of immunodominant epitopes on the 1 D protein of FMDV.

P4 (SEQ. ID No. 17): aa'OPNHDTWKNHVKDIRERMQKEQSAN" (aa377-401 ) of the VP2 protein of AHSV-9. This sequence is part of a domain that has previously been shown to be serotype-specific in an immune blot with AHSV-9 serum and contains a possible neutralization domain of VP2.

Cells and Viruses

Sf9 insect cells were infected at an m.o.i. of 5 pfu/cell with recombinant baculoviruses expressing the chimeric protein VP7-177-P6, or the chimeric protein VP7- 144-P4.

Protein expression

Cells infected with the various recombinant baculoviruses were harvested at 48 h post infection. Expression was confirmed by gel electrophoresis on 12% SDS- polyacrylamide gels stained with Coomassie blue, followed by Western blot analysis using anti-FMDV vaccine strain SAT2/ZIM7/83 as well as anti-AHSV9 antiserum which both gave positive reactions (not shown).

Protein preparation

For immunization with VP7-177-P6, two protein samples were prepared namely (i) a crude extract comprising a combination of soluble and particulate proteins and (ii) a fraction containing soluble protein. For immunization with VP7-144-P4, only a soluble fraction was prepared.

The crude extract was prepared by resuspending 2 x 10⁸ infected cells in 0.01 M STE with 0.5% Nonidet P40, incubation on ice for 30 min and mechanical disruption by douncing 20 times. The nuclear fraction was removed by low speed centrifugation at 1500 rpm for 3 min and rinsed once. The lysate was then dialised overnight against 0.01 M STE with three buffer changes, freeze-dried and resuspended in 1 x PBS. Expression of the chimeric protein was confirmed and its concentration in the lysate was estimated from the band intensity after SDS-PAGE analysis and Coomassie Blue staining.

To prepare the soluble fractions for both VP7-177-P6 and VP7-144-P4, lysates were prepared in 0.01 M STE with 0.5% Nonidet P40, incubated on ice, mechanically dounced and the nuclear fraction removed as before. Lysates were then loaded onto 50-70% discontinuous sucrose gradients in 0.01 M STE. Gradients were centrifuged in an SW28 rotor at 22 000 rpm for 20 h. Seventeen fractions of 2 ml each were collected using a dialysis pump and analyzed directly on a 12% SDS polyacrylamide gel (not shown). The gels were stained with Coomassie Blue to determine the protein content ofeach fraction. The top fractions of the gradient (fractions 11 -14) representing soluble chimeric protein were pooled, dialyzed overnight against 0.01 M STE with three buffer changes, freeze-dried and resuspended in 1 x PBS. All proteins were stored at -7O⁰C prior to immunizations.

Immunization procedure

Groups of female guinea pigs were injected intra-muscularly with either crude- or soluble samples of VP7-177-P6 or the soluble sample of VP7-144-P4. Four animals were immunized with 40 μg of the respective protein samples in 250 μl 1 x PBS emulsified in an equal volume of either Freund's complete or incomplete adjuvant as detailed in the Table 3 below. A single boost using either incomplete Freunds or no adjuvant was administered after 21 days. Serum samples were obtained from each animal pre-vaccination, hours before each booster injection and three weeks after the boost.

Table 3 Immunization protocol and serum analyses

Detection of antibody responses to FMDV

A sandwich enzyme-linked immunoabsorbent assay (ELISA) was used to detect antibodies in serum to the VP7-FMDV fusion protein. Briefly, 96-well plates coated with rabbit antibodies against FMDV vaccine strain SAT2/ZIM7/83 were used to trap tissue-cultured virus, diluted 1/50. Guinea pig test serums collected at different times pre-and post-vaccination were serially diluted, added to the ELISA plates. Antibodies detected with peroxidase-labelled anti-guinea pig conjugate. Titres were expressed as the serum dilution that yielded absorption values three times above pre- immunization serum.

The presence of antibodies mediating FMDV neutralizing activity was determined in a virus-neutralization assay. In a 96-well plate, two-fold dilutions of the test sera were added to 100 TCID₅₀ units of FMDV vaccine strain SAT2/ZIM7/83. The mixture of serum dilutions and virus was incubated at 37°C for 1 hr and then adsorbed to IBRS2 cells. Cells were incubated for 3 days until a cytopathic effect (CPE) was observed. The serum neutralization titre was defined as the dilution that resulted in a 50% reduction in CPE.

Detection of antibody responses to AHSV

The presence of AHSV-9 VP2-specific antibodies in serum was detected by Western blot analyses. Immune reactions were tested against a baculovirus-expressed NS1 fusion protein containing an AHSV-9 VP2 domain that overlaps the P4 insert, as well as against VP7-144-P4. The serum was also analysed in a virus neutralization assay for the presence of antibodies that could mediate AHSV-9 neutralization in a plaque-neutralization assay. Two-fold dilutions of the test sera were added to 100 PFU units of AHSV-9 virus, incubated at 37°C for 1 hr and then adsorbed to CER cells in 6- well plates. Cells were incubated for 5 days until plaques were visible, stained and monitored for a decrease in the number of plaques compared to non-neutralised virus- infected controls. RESULTS

Immune response against VP7-177-P6

The results of a previous animal trial suggested that this fusion protein could elicit very strong immune response against the displayed FMDV peptide. This trial was conducted in order to confirm the previous results, to extend the immunizations to more animals and to get a preliminary idea about the contribution of adjuvants to the immune response. The ELISA and virus neutralization titres of the sera collected at 21 and 42 days after immunization with the different VP7-177-P6 immunogens are summarized in Table 4.

Table 4 Immune responses of individual animals against soluble or crude extracts of VP7-177-P6

CF = complete Freunds adjuvant ; IF = incomplete Freunds ; None = no adjuvant

An excellent immune response was obtained against the soluble VP7-177-P6 fusion protein. The average ELISA titre of the 42 days serum was in the order of more than 1/7600 (injection with CF and boost with IF) with one animal having an ELISA titer of 1/12800 even before the booster injection. The boosters did, however, have a significant effect, resulting in titres of more than 1/12800 in the case of at least two animals. These very high ELISA titers correlated very well with high virus-neutralization titers. These titres averaged at about 1/561 at 42 days after immunization, with individual tritres as high as 1/1149. These very high neutralization titres against animals that had been immunized with the soluble protein extract of VP7-177-P6 were not obtained when the animals were injected with the crude VP7-177-P6 fusion protein. This is seen by average neutralization triters of 1/33 and ELISA tritres of 1/ 280 against the crude protein preparation. Even with CF adjuvant, the crude preparation appears to be a less efficient immunogen.

Immune response against VP7-144-P4

The VP7-144-P4 fusion protein with amino acids 377-401 of VP2 from AHSV contained a large and significant soluble component which was used to induce an immune response in guinea pigs as outlined in group 4 in Table 3.

The immune sera obtained were tested by a AHSV neutralization assay and found to contain no neutralizing antibodies. It was then tested in an immune blot assay against an NS1 fusion protein which contained an AHSV VP2 sequence that overlapped the P4 sequence. The result is shown in Figure 15. The result clearly shows a strong VP2 insert specific immune response. This result confirms that the VP7 soluble trimer, with P4 inserted into site 144, induced antibodies against the 35 amino acid VP2 insert.

The results confirm that both the P4 and P6 inserts induced strong insert specific immune responses when these peptides were displayed on the surface of soluble VP7 trimers. The response against the FMDV insert was particularly impressive with ELISA titres of > 1/12800 and neutralization titres of up to 1/1149 under conditions where a titre of 1/45 is considered a good positive and a 1/100 neutralization titer in bovine is considered sufficient to provide protection against FMDV. The high ELISA titres corresponded very well with the neutralization titres. The response against the crude was not nearly as good as against the soluble extract. Positive ELISA antibody titers were only evident in two animals, and only after they had received a primary and booster injection of the crude material. Furthermore, these antibodies were not neutralizing, as indicated by the low virus neutralization titres reported from these sera.

Results also confirmed the importance of using an appropriate adjuvant. On average, a better immune response was elicited where complete adjuvant was used as an adjuvant for the primary injection and incomplete Freunds adjuvant for the boost. Nonetheless, even when Incomplete Freunds adjuvant was used for the primary immunization and no adjuvant for the boost, positive virus neutralization titers were obtained for all three animals.

The VP2 insert also induced a good antibody immune response. This result is quite significant because it has previously been attempted to induce antibodies against this peptide without the VP7 immune display strategy. However, due to the fact that the peptide was insoluble, no peptide-specific antibodies were raised. However, in this experiment the normally insoluble P4 peptide was presented to the immune system as part of a soluble VP7 fusion protein with peptide P4 inserted into site 144 of the VP7 top domain. Under these conditions we were for the first time able to raise peptide P4 specific antibodies. We were also able to test if such antibodies were able to neutralize the virus. The results confirmed that, if there are any neutralizing epiptopes located on the P4 peptide, these epitopes are likely to be conformational rather than linear epitopes.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

Claims

Claims

1. A chimeric antigen which includes an orbivirus VP7 polypeptide, or part thereof, and a foreign peptide inserted into the top domain region of said orbivirus VP7 polypeptide.

2. A chimeric antigen as claimed in claim 1 comprising a VP7 protein sequence selected from at least one of amino acid sequences of SEQ. ID. NO. 7, a fragment thereof, and polypetides having at least 60% homology thereto.

3. A chimeric antigen as claimed in claim 1 wherein the orbivirus VP7 polypeptide is an African horsesickness virus (AHSV) VP7 or a bluetongue virus VP7 polypeptide.

4. A chimeric antigen as claimed in claim 3 wherein the VP7 protein of AHSV has a leucine residue at position 345 mutated to an arginine residue, the VP7 protein selected from at least one of SEQ. ID. NO. 15, a fragment thereof, and polypeptides having at least 60% to 80% homology thereto.

5. A chimeric antigen as claimed in any one of claims 1 to 4 wherein the foreign peptide is selected from at least one of the group consisting of an immunogen, an antigen, an epitope, a hapten, a structural protein, a non-structural protein, a polypeptide having an enzymatic function and a proteinaceous compound.

6. A chimeric antigen as claimed in any one of claims 1 to 5 wherein the chimeric antigen is complexed with an adjuvant for eliciting an immune response against the chimeric antigen.

7. A method of making a chimeric antigen, the method including the step of inserting a foreign peptide into a top domain of an orbivirus VP7 polypeptide, or part thereof.

8. A method as claimed in claim 7, wherein the VP7 polypeptide is an AHSV or BTV VP7 polypeptide.

9. A method as claimed in claim 7 or claim 8 wherein the step of inserting the foreign peptide into the top domain includes: isolating a nucleic acid sequence encoding the foreign peptide, and inserting the nucleic acid sequence into a predetermined region of an isolated nucleic acid encoding the top domain of the VP7 polypeptide , or a region proximal thereto, thereby to produce a recombinant nucleic acid molecule encoding the chimeric antigen as claimed in any one of claim 1 to 6.

10. A recombinant DNA molecule which includes a first nucleic acid sequence encoding an orbivirus VP7 polypeptide or part thereof and a second nucleic acid sequence encoding a foreign peptide such that the recombinant DNA molecule encodes a chimeric antigen as claimed in any one of claims 1 to 6.

11. A recombinant DNA molecule as claimed in claim 10 wherein the first nucleic acid sequence is selected from at least one of the nucleotide sequence of SEQ. ID. NO. 1 , the complement thereof, and DNA sequences having at least 60% to 80 % homology thereto.

12. A recombinant DNA molecule as claimed in claim 10 or claim 11 wherein the first nucleic acid sequence is selected from at least one of SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ ID. No. 5, SEQ. ID. NO. 6, the complement thereof, and nucleic acid sequences having at least 60% to homology thereto.

13. A recombinant DNA molecule as claimed in any one of claims 10 to 12 wherein the second nucleic acid sequence is selected from at least one of SEQ. ID. NO. 17, SEQ. ID. NO. 18, SEQ. ID. NO. 19, the complement thereof, and nucleic acid sequences having at least 60% homology thereto.

14. A nucleic acid vector including a nucleic acid sequence encoding an orbivirus VP7 polypeptide or part thereof, the nucleic acid sequence having at least one cloning site therein at a position corresponding to, or proximal to, the top domain of the encoded VP7 polypeptide when translated.

15. A nucleic acid vector as claimed in claim 14 wherein the cloning site is between codons encoding amino acids 144 and 145, between amino acids 177 and 178, and between amino acids 200 and 201 of the VP7 polypeptide, the VP7 polypeptide being an AHSV VP7 polypeptide.

16. A nucleic acid vector as claimed in claim 14 or claim 15 wherein the nucleic acid sequence is operably linked to a cis control element.

17. An immunogenic composition comprising the chimeric antigen of any one of claims 1 to 6, and a pharmaceutically acceptable carrier or diluent.

18. A method of inducing an immune response in a subject, the method including a step of administering an effective amount of the immunogenic composition of claim 17 to a subject thereby to induce said immune response.

19. A method as claimed in claim 18 which includes administering two booster doses of the immunogenic composition into the body of the subject wherein a first booster dose is administered between 9 and 16 days following initial administration of the immunogenic composition into the subject, and a second booster dose is administered between 18 and 32 days following initial administration of the immunogenic composition.

20. A method of increasing the solubility of a foreign peptide, the method including the steps of: providing an isolated nucleic acid sequence encoding an orbivirus VP7 polypeptide, or part thereof; inserting an isolated nucleic acid sequence encoding the foreign peptide into a region of the orbivirus VP7 nucleic acid sequence encoding a top domain loop or a region proximal to a top domain loop of the VP7 polypeptide, thereby forming a recombinant DNA molecule; and causing the resultant recombinant DNA molecule to be expressed as a chimeric antigen of any one of claims 1 to 6.

21. A method of increasing the immunogenicity of a foreign peptide, the method including the steps of: providing an isolated nucleic acid sequence encoding an orbivirus VP7 polypeptide, or part thereof; inserting an isolated nucleic acid sequence encoding the foreign peptide into a region of the orbivirus VP7 nucleic acid sequence encoding a top domain of the VP7 polypeptide, thereby forming a recombinant DNA molecule; and causing the resultant recombinant DNA molecule to be expressed as a chimeric antigen of any one of claims 1 to 6.

22. A method as claimed in claim 21 including a further step of subjecting the chimeric antigen to at least one of sonification or repeated freeze/thaw cycles.

23. An antigen presentation system for presenting an antigen to an immune system of a subject, the antigen presentation system including: a nucleic acid vector as claimed in any one of claims 14 to 16, said nucleic acid vector having at least one foreign nucleic acid sequence inserted into at least one cloning site of the nucleic acid vector.

24. An antigen presentation system comprising a trimer of a VP7 polypeptide having inserted into a top domain thereof a foreign peptide or epitope.

25. A host cell including the recombinant DNA molecule of any one of claims 10 to 13.

26. A method for producing a chimeric antigen, which method comprises growing a host cell containing the recombinant DNA molecule of any one of claims 10 to 13, such that the DNA molecule is expressed by the host cell, and isolating the expressed chimeric antigen.

27. A substance or composition for use in a method of treating or preventing at least one of foot and mouth disease, African horsesickness and bluetongue disease in a subject, said substance or composition comprising a chimeric antigen of any one of claims 1 to 6, and said method comprising administering an effective amount of said substance or composition.

28. Use of a chimeric antigen of any one of claims 1 to 6 in the manufacture of a medicament for treating or preventing at least one of foot and mouth disease, African horsesickness and bluetongue disease.

29. A method of prophylactic treatment of at least one of foot and mouth disease, African horsesickness disease and bluetongue disease, the method comprising administering an immunologically effective amount of a chimeric antigen of any one of claims 1 to 6 to a subject.

30. An isolated polypeptide selected from at least one of the polypeptide sequences SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11 , SEQ. ID. NO. 12, SEQ. ID. NO. 15, SEQ. ID. NO. 16, functional fragments thereof, and polypeptides having at least 60% homology thereto.

31. An isolated polynucleotide sequence selected from at least one of nucleotide sequences SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ ID. No. 5, SEQ. ID. NO. 6, SEQ. ID. NO. 13, SEQ. ID. NO. 14, the complement thereof, and sequences having at least 60% homology thereto.