Chimeric protein comprising a cytokine epitope

Abstract

There is described a chimeric protein comprising a first peptide (for example ColE7 or a functional portion thereof) and first and second segments of a second peptide, wherein said second peptide includes an epitope from a target peptide. The target peptide is selected from the IL-6 family of cytokines. The second peptide is derived from a target peptide in which an N-terminal segment is transposed with a C-terminal segment. The epitope is available for binding by a separate binding partner, for example an antibody or a cell receptor.

Description

[0001] CHIMERIC PROTEIN COMPRISING IL-6 EPITOPE

[0002] Technical Field

[0003] The present invention relates to chimeric proteins comprising or consisting of a first peptide and second peptide, wherein said second peptide includes an epitope from a target peptide and is formed in two segments which create non-native N and C terminals, with a peptide bond or linker joining the native N and C terminals of the second peptide. The target peptide is a member of the IL-6 family of cytokines. Optionally, the chimeric peptide can bind onto a protein which can self-assemble into a virus-like particle (VLP). Optionally, the first peptide segment itself can self-assemble into a virus-like particle (VLP). The chimeric protein can be used in medical or veterinary applications, including as a vaccine. The chimeric protein can also be used within research.

[0004] Background to the Invention

[0005] Signalling of the cytokine Interleukin 31 (IL31) proceeds by initial binding of IL31 to the receptor protein IL3 IRA on a target cell. This initial binding event allows IL31 to also bind a second receptor protein called OSMRbeta. Once these two receptor proteins are brought into close contact, their intracellular domains interact and trigger a signal to achieve the intended outcome.

[0006] IL-31 is known to be involved in both innate and adaptive immunity in tissues such as the skin, the airways and the lung, and the lining of the intestine. Increased levels of IL-31 are associated with a number of diseases, including pruritic diseases such as atopic dermatitis, and also in allergy and inflammatory bowel disease. For example, the signal of IL31 causes the sensation of itchy skin (pruritus). If IL31 binding to its receptor is prevented, no pruritus is experienced. This can be achieved by regular injection of an antibody blocking the receptor. Alternatively, the body can be instructed to make its own antibodies against IL31. This can be achieved by placing IL31 on top of a Virus-Like Particle (VLP) to induce antibody formation. Once this VLP-IL31 construct is injected into a person, the recipient makes antibodies against IL31.

[0007] Virus-like particles (VLPs) are molecules which mimic the structure of viruses, but contain no viral genetic material and are therefore non-infectious. VLPs are formed by the selfassembly of viral structural proteins, typically viral capsid proteins that self-assemble into aparticle resembling a virus. VLPs can be rod-shaped or can be in the form of a hollow sphere. VLP structures can be formed from viral capsid or envelope proteins. Frequently, VLPs are produced using proteins(s) from a single virus type. However, chimeric VLPs can also be created by the assembly of structural proteins from different viruses.

[0008] VLPs are highly immunogenic and can stimulate both humoral and cellular immune responses meaning that VLPs find utility in vaccine use. Several VLP -based vaccines are now commercially available, including malaria vaccines such as Mosquirix™ by GlaxoSmithKline, HPV vaccines such as Cervarix™ by GlaxoSmithKline or Gardasil™ by Merck & Co, and Hepatitis B vaccine such as Sci-B-Vac™ from VBI vaccines.

[0009] Commonly the base VLP is used to present an epitope on its surface in order to stimulate the immune system. Thus, the viral capsid proteins forming the VLP shell are modified genetically to include the epitope peptide sequence. However, inclusion of the epitope peptide sequence can disrupt the ability of the viral capsid or envelope protein to self -assemble. A further disadvantage can be when the epitope is not displayed on the exterior surface of the VLP in such a manner for it to induce a suitable immune response.

[0010] There remains a need for further an improved methods of incorporating epitopes onto the surface of a VLP in a manner which presents the epitope in a manner which facilitates interaction of the epitope with a separate binding partner (such as a cell receptor or antibody).

[0011] One prior art method of creating a VLP to present an epitope externally relies upon the VLP capsid protein being genetically or chemically modified to include the desired epitope. Prior art methods of creating a chimeric protein to present an epitope externally of a VLP have included the epitope being added by its N or C terminus onto the N or C terminus of the capsid protein to form a chimeric construct. However, the chimeric construct so formed may not present the epitope in an optimal spatial orientation to induce antibody formation against the desired epitope. This is especially the case where the tertiary structure of the epitope requires amino acids from distinct segments in the primary sequence, together forming a single conformational epitope, such as one segment residing at the N terminus of the linear primary sequence merging with another segment residing at the C terminus of the epitope. The resulting conformational epitope, therefore, cannot be displayed simply bychoosing one distinct segment of the primary sequence for presentation. Current state-of-the-art technology does not allow for a way to arbitrarily spatially orient an epitope protein displayed by a VLP to present a desired conformational epitope to the surface. There is a need for improved constructs able to present an epitope optimally, either to induce antibody formation against that epitope or alternatively to allow the epitope to interact with a known binding partner (such as a cell receptor).

[0012] Summary of the Invention

[0013] The present invention provides a chimeric protein which comprises a first peptide (for example ColE7 or a functional portion thereof or a virus capsid protein) linked to a chimeric second peptide, wherein said second peptide is formed from first and second segments of a target peptide which is a member of the IL-6 family of cytokines, wherein said target peptide has an epitope, characterised in that the first and second segments are arranged in the second peptide to form non-native N and C terminals, and wherein said second peptide comprises said epitope. The C-terminal of the second peptide is formed from an N-terminal segment of the target peptide. The N-terminal of the second peptide is formed from a C-terminal segment of the target peptide.

[0014] Optionally, the chimeric protein consists of a first peptide linked to a second peptide, wherein said second peptide includes an epitope of a target peptide, wherein said second peptide is formed from C-terminal and N-terminal segments of the target peptide comprising the epitope, characterised in that the C-terminal segment of the second peptide is formed from a N-terminal segment of the target peptide, and the N-terminal segment of the second peptide is formed from a C-terminal segment of the target peptide, and wherein the target peptide is a member of the IL-6 family of cytokines.

[0015] Thus, in the second peptide, a C-terminal portion and an N-terminal portion of the target peptide are transposed relative to their respective locations in the native target peptide. There can be a peptide bond or linker joining the C-terminal amino acid of the target peptide to the N-terminal amino acid in the segments of the target peptide used to form the second peptide.

[0016] In the second peptide, the new N-terminal segment (first segment) can be formed from a segment of the target peptide normally located towards the C-terminal of that peptide. Thisnew N-terminal segment can be joined “upstream” (i.e. on the amino side) of a portion of the target peptide normally located towards the N-terminal of that peptide, which then becomes the new C-terminal segment (second segment) in the second peptide. The final amino acid of that second segment will form the new C-terminus of the second peptide. The first and second segments can be joined together by a peptide bond or by a linker, typically up to 6 amino acids. The sequence of the target peptide is therefore re-arranged to so that the second peptide has artificial or non-native N and C terminals. The re-arrangement substantially maintains the functionality of the epitope, i.e. will maintain its ability to bind to a targeted binding partner by at least 50% relative to the epitope in the wild type target peptide.

[0017] Generally, the second peptide is formed from a linear chain of amino acids. This linear chain of amino acids is desirably folded to form a tertiary structure which is similar to that of the native target peptide. In particular the tertiary structure of the epitope is preserved.

[0018] In some embodiments, the sequence of the target peptide is a simple transposition of C and N-terminal segments of the native target peptide. These segments are joined at the native N and C-terminal segments of the target peptide, optionally via a linker. The second peptide therefore has an N-terminal which is different to the native form of the target peptide and has a C-terminal which is different to the native form of the target peptide.

[0019] Optionally, portions of the target peptide at the N-terminal and / or C-terminal and / or middle section can be omitted in the formation of the second peptide provided that, a section of the target peptide is transposed towards the N-terminal side of the second peptide.

[0020] In some embodiments, the new N-terminal segment (first segment) of the second peptide is formed from a C-terminal segment of the target peptide joined by a peptide bond or linker to a section of the original N-terminal portion of the target peptide. The new C-terminal portion of the second peptide is therefore formed from a middle section of the target peptide. In this embodiment, the first and second segments are joined together by a peptide bond or linker (which may be the same or different to any linker used to form the new N-terminal segment (first segment) of the second peptide.Optionally, in all embodiments, the first and second segments can be joined together by a peptide bond or a linker.

[0021] Optionally, formation of the epitope formed in the second peptide relies on a combination of both the first and second segments of the second peptide. In other words, the epitope is formed by amino acids from both segments forming the second peptide.

[0022] Desirably, the first and second segments of the second peptide together form a tertiary structure which is substantially similar to that of the native epitope of the target peptide, preferably which is substantially identical to that of the native epitope of the target peptide. Tertiary folding of peptides and protein can be analysed by various means, including modelling software such as Alphafold (see Jumper et al., Nature 596:583-589, 2021). For example, the 3D folding of the second peptide epitope can be within a root-mean-square deviation tolerance of no more than 1.5 A of the native peptide epitope (in the target peptide) when the structure of the epitope in the native peptide and the epitope of the chimeric protein are superimposed.

[0023] The fusion of the first peptide to the second peptide allows the epitope to be presented in an outward facing orientation, so that the epitope is available for binding by a separate binding partner, for example an antibody or a cell receptor.

[0024] Optionally, the first peptide is selected from an at least functional portion of a bacterial colicin, for example can be a functional portion of ColE7 from E. coli, or can be a functional portion of ColE2, ColE8 or ColE9. The first peptide can also be a functional portion of a bacterial ribonuclease, for example can be Barnase (for example from Bacillus amyloliquefaciens) or a functional portion thereof, can be a functional portion of a member of the Spycatcher family of proteins. Optionally, the first peptide can be a capsid or envelope protein and self-assemble directly.

[0025] Optionally, the functional portion of the bacterial colicin can be mutated to decrease or prevent its activity. Exemplary modifications are shown in SEQ ID Nos: 14, 16, 18 and 20 (ColE7, ColE2, ColE8 and ColE9, respectively) which each have the following mutations relative to the wild type sequence: R95A, E99A and H126A. Optionally SEQ ID No: 14can be modified to omit the C-terminal Serine residue, which essentially acts as a linker residue.

[0026] Optionally, the first peptide does not form a first peptide-first peptide dimer. This has the benefit of allowing greater spacing around the fusion ligand of interest, thereby avoiding steric hindrance.

[0027] Optionally, the first peptide has a natural binding partner. For example, ColE7 binds to Im7, ColE2 binds to Im2, ColE8 bind to Im8, ColE9 binds to Im9, Bamase binds to Barstar etc. The term “functional portion” with respect to the first peptide refers to a portion of the wild type protein which retains its ability to still bind to its natural binding partner.

[0028] Optionally, the first peptide can bind to a modified capsid or envelope protein, for example as described in WO2024 / 018188A1.

[0029] Optionally, the second peptide is formed from first and second segments which together include at least 70% of the wild type sequence of the target peptide divided at a location (to form new N and C terminals) which is selected to ensure that the epitope of the second peptide is presented in an outward facing orientation in the chimeric protein of the invention.

[0030] Optionally, the second peptide includes at least 80%, for example at least 85%, for example at least 90% of the wild type sequence of the target peptide. The percentage amount of the target peptide included in the second peptide is dependent on the tertiary structure to form the epitope.

[0031] Optionally a linker can be used between the first and second segments forming the second peptide. Optionally, there may be a further linker conjoining C and N terminal portions of the target peptide to form the first segment (new N terminal segment) of the second peptide.

[0032] Optionally, the second peptide can be linked to either the N terminal of the first peptide or to the C terminal of the first peptide. Optionally, there is a linker between the first peptide and the second peptide.

[0033] Optionally, the chimeric protein can consist of (from N to C terminal):First peptide (optional linker 1) First Segment (2nd peptide) (optional linker 2) Second Segment (2nd peptide); or

[0034] or

[0035] First Segment (2nd peptide) (optional linker 2) Second Segment (2nd peptide) (optional linker 1) First Peptide.

[0036] In a second aspect, the present invention provides a VLP having a capsid comprising a chimeric protein as described above. Optionally, the first peptide can be a capsid or envelope protein and self-assemble directly. Optionally, the first peptide can bind to a modified capsid or envelope protein, for example as described in WO2024 / 018188A1.

[0037] In a third aspect of the invention, the invention provides a polynucleotide which encodes the chimeric protein described above.

[0038] In addition, the invention also encompasses a polynucleotide which specifically hybridizes under stringent conditions to the polynucleotide encoding the chimeric protein. For the purposes of the present specification, hybridisation under stringent hybridisation conditions means remaining hybridised after washing with 0.1 *SSC, 0.5% SDS at a temperature of at least 68° C, as described by Sambrook et al (Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press).

[0039] Optionally, the polynucleotide of the invention has a nucleotide sequence which expresses a protein with at least 85% sequence identity to one of SEQ ID Nos. 22 to 34. Optionally, the polynucleotide of the invention encodes a protein having a sequence identity to SEQ ID Nos. 22 to 34 which is more than 85%, for example which is 90%, 95%, 98% or even more.

[0040] In a fourth aspect, the present invention provides a vector comprising a polynucleotide according to the invention as described above. Optionally, the vector can be an expression vector expressing, or overexpressing, said chimeric protein.

[0041] In a fifth aspect, the present invention provides a host cell transformed with a polynucleotide according to the invention or a vector according to the invention. A cell culture comprising one or more host cells according to the invention is also encompassed.In a sixth aspect, the present invention provides a process of producing a chimeric protein according to the invention, said method comprising:

[0042] a) Identifying a target peptide comprising an epitope of interest;

[0043] a) Identifying a division site in the target peptide wherein said division site is within an unstructured region or is within a Glycine bend region of the target peptide, and thereby defining N- and C-terminal segments of the target peptide;

[0044] b) Transposing the native C and N terminal segments of the target peptide to form a second peptide;

[0045] c) Forming a chimeric polynucleotide comprising an in-frame sequence encoding both first and second peptides;

[0046] d) Expressing said chimeric protein from said chimeric polynucleotide.

[0047] The “division site” is any suitable site within the target peptide at which the peptide can be split without the disruption of secondary structure elements (such as alpha helices; beta sheets; binding sites to metal ions etc). Examples of suitable divisions sites are an unstructured region and / or a Glycine bend region of the target peptide.

[0048] Optionally, once the division site has been identified, the first and second segments are identified and the process can optionally further include a step of structural modelling of the chimeric protein chain so as to identify any requirement for linker amino acids to be inserted between the segments of the chimeric second peptide. For example, the first segment (forming the new N-terminus) of the second peptide can be formed from a C terminal portion of the target peptide linked to an N-terminal portion of the target peptide, optionally using a linker. The second segment of the second peptide, (forming the new C-terminus) can then be linked to the first segment, optionally using a linker Insertion of any additional linker amino acids preferably achieves a Root-Mean-Square Deviation of less than 1.5 Angstrom between the superimposed structures of the native structure of the epitope in the target peptide and the final resulting epitope formed after the chimeric joining of both peptide segments in the chimeric second peptide.

[0049] In a seventh aspect, the present invention provides a process for the production of a viruslike particle (VLP), said method comprising:

[0050] a) A first polynucleotide encoding a chimeric protein according to the invention and expressing said chimeric protein;b) Allowing said chimeric protein to self-assemble into a VLP.

[0051] In an alternative embodiment, the present invention further provides a process for the production of a virus-like particle (VLP), said method comprising:

[0052] a) A first polynucleotide encoding a chimeric protein according to the invention and expressing said chimeric protein, wherein said first peptide of said chimeric protein comprises a binding site;

[0053] b) Admixing said chimeric protein with a VLP -forming protein, wherein said VLP- forming protein comprises a binding partner for the first peptide binding site, to form a VLP-chimeric protein complex and allowing complex to assemble into a VLP; or

[0054] c) Providing a VLP -forming protein having a binding partner for the first peptide binding site, and allowing said VLP -forming protein to self-assemble into a VLP and then admixing said VLP with the chimeric protein and allowing said chimeric protein to bind thereto.

[0055] The chimeric protein and a VLP comprising the chimeric protein can be used to cause a biological response, for example as a consequence of the epitope of the second peptide binding to a cell receptor. For example, the epitope can bind to a B-cell and induce an immunogenic response leading to the production of antibodies against the epitope. In a further aspect, the present invention provides an immunogenic composition comprising the chimeric protein or VLP according to the present invention.

[0056] The chimeric protein is therefore suitable for use as a medicament. The VLP comprising the chimeric protein is also suitable for use as a medicament

[0057] Optionally, the medicament can be for use in the prevention and / or treatment of inflammatory diseases, cancer, neurodegenerative, metabolic, cardiovascular, renal, gastrointestinal, respiratory, dermatologic, endocrine, rheumatological, or autoimmune diseases. In particular, the medicament can be for use in the prevention and / or treatment of pruritic diseases such as atopic dermatitis, and also in allergy and inflammatory bowel disease.Brief Description of the Figures

[0058] Figure 1: Model showing the IL3 IRa-receptor binding-epitope on IL31, shaded black, shown from two different angles (A: left, B: centre), and as simplified schematic (C). The native amino- and carboxyl-terminus of the entire IL31 protein are indicated. The model illustrates that the receptor-binding-epitope (shaded black) is composed of helixcomponents derived from spatially separate parts of the protein.

[0059] Figure 2: Schematic illustration of a conventional end-to-end fusion from the C-terminus of the VLP-scaffold protein to the N-terminus of native IL31. The epitope shown in black is not presented on the exterior of the fusion.

[0060] Figure 3: Schematic illustration showing the conventional end-to-end fusion protein of Figure 2 attached to a VLP. The epitope of interest (shown in black) is located away from the surface, making recognition by the immune system difficult.

[0061] Figure 4: Schematic illustration of the second peptide according to the invention.

[0062] Figure 5: Schematic illustration of a chimeric protein according to the invention in which the second peptide has been divided into two segments, which (in the embodiment illustrated) is attached to the first peptide at the C terminus of the first peptide.

[0063] Figure 6: Schematic illustration showing the chimeric protein according to the invention attached to a VLP. The epitope of interest (shown in black) is facing away from the VLP core, facilitating recognition by the immune system.

[0064] Figure 7: Identification of unstructured domains in IL31, designed Unstructured Region (USR)l and 2, respectively, which are located on the opposite surface relative to the IL3 IRA-binding interface (grey shaded). Top: ribbon model structure, Bottom: primary sequence of hIL31, indication location of helices, the IL31RA binding interface (grey font) and the USR 1 and USR2 regions. The grey arrow denotes Glycine29 (G29) which was also identified as potential divisional site.

[0065] Figure 8: Schematic illustration of relevant features in plasmids designed to express: (A) the novel hIL13 (plasmid 78488), (B) cIL31 (plasmid 78457), (C) hIL31 (plasmid 79252)and (D) cIL31 (plasmid 79449) chimeric proteins of the invention. A T7 promoter drives expression of the VLP scaffold protein which consists of linker-facilitated fusion of the Im7 protein to the N and C termini of the HBc capsid peptide. The second peptide consists of a functional portion of ColicinE7 (designated ColE7), fused directly to an inverted human IL13 (A), (C) or inverted canine IL31 (B), (D). The T7 promoter generates a single mRNA transcript where the second open reading frame is translation-initiated via an internal ribosomal binding site (RBS). Linker sequences are indicated by white spaces. Histidine tags (HAHEHRHDHE) are indicated by HDE.

[0066] Figure 9A: Expression and partial purification of the novel plasmid 78488 comprising an ORF for a chimeric protein wherein the second peptide is derived from human IL-31. Plasmids were transfected into a BL21 / DE3 E. coli strain and induced with 0.3 mM IPTG at 16C for 3h. Data shown are denaturing reducing SDS PAGE analyses of cytosolic fractions obtained by cell disruption, centrifugation at 10,000g and filtration through 0.2 pm (labelled ‘Cyt’) and VLP partially purified by IMAC (labelled IMAC), respectively. White arrowheads denote the epitope proteins, black arrowheads denote the VLP scaffold protein.

[0067] Figure 9B: Expression and partial purification of the novel plasmid 78457 comprising an ORF for a chimeric protein wherein the second peptide is derived from canine IL-31.

[0068] Plasmids were transfected into a BL21 / DE3 E. coli strain and induced with 0.3 mM IPTG at 16C for 3h. Data shown are denaturing reducing SDS PAGE analyses of cytosolic fractions obtained by cell disruption, centrifugation at 10,000g and filtration through 0.2 pm (labelled ‘Cyt’) and VLP partially purified by IMAC (labelled IMAC), respectively. White arrowheads denote the epitope proteins, black arrowheads denote the VLP scaffold protein.

[0069] Figure 10: Plots confirming the ability of the chimeric constructs to trigger the generation of antibodies which will react with the wild-type native cytokine. Left: Mice were dosed with chimeric human IL13 placed on the outside of a Virus-Like Particle (clone 78488) by subcutaneous injection + booster shot. Thereafter the sera from mice (n = 5) were tested for the presence of anti-IL13 antibodies by ELISA, using commercially sourced human IL 13 produced in mammalian cells as coated antigen on the ELISA plates. Right: Mice were dosed with chimeric cIL31 placed on the outside of a Virus-Like Particle (clone 78457) bysubcutaneous injection + booster shot. Thereafter the sera from mice (n = 6) were tested for the presence of anti-IL31 antibodies by ELISA, using commercially sourced canine IL31 produced in mammalian cells as coated antigen on the ELISA plates. Data represent endpoint anti -human IL 13 IgG titres and group median (horizontal bar).

[0070] Figure 11: Sucrose density gradient ultracentrifugation of hIL 13 -graft -bearing VLPs (clone 78488), and cIL13 graft bearing VLPs (clone 78457). Data shown are reducing denaturing SDS PAGE of the cytosolic fractions shown above after overnight centrifugation at 130,000g in a linear sucrose gradient from 10-60% sucrose. Only large particles re-equilibrate into the high-density fractions. Both graft bearing VLP’s equilibrate into the 50% and 60% interface layers, confirming formation of intact large nanoparticles.

[0071] Figure 12: Expression and partial purification of the novel plasmids 79252 (A) and 79449 (B) each comprising an ORF for a chimeric protein wherein the second peptide is derived from canine IL-31. Plasmids were transfected into a BL21 / DE3 E. coli strain and induced with 0.3 mM IPTG at 16C for 3h. Purification was using POROS HQ anion exchange chromatography, followed by immobilized metal affinity (IMAC) chromatography. Arrows indicate scaffold (28.4) and epitope (79252; 31.2 kDa, 79449; 32.3 kDa).

[0072] Figure 13: Antibody titre (A) and quantification (B) ELISAs for female Sprague Dawley rats were dosed with 0.18 mg / mL 79252 VLP.

[0073] Figure 14: % Inhibition in isolated IgG following IgG isolation from plasma from female Sprague Dawley rats were dosed with 0.18 mg / mL 79252 VLP using the Melon™ Gel protocol.

[0074] Figure 15: Antibody titre (A) and quantification (B) ELISAs for female C57B16 mice were dosed with 79449 VLP with 2% Alhydrogel adjuvant at days 21 and 35 post-dosage.

[0075] Detailed Description of the Invention

[0076] The protein, polynucleotides and vectors encoding the protein, compositions comprising the protein, expression system and methods of the present invention are now described in further detail.As used herein, the term "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other.

[0077] As used herein, terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but includes the general class of which a specific example may be used for illustration.

[0078] As used herein, the term "comprising" is to be construed as encompassing both "including" and "consisting of, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention.

[0079] As used herein the term “peptide” refers to a polymer composed of amino acids joined by peptide bonds and does not refer to a specific length of the polymer. A "peptide bond" is a covalent bond between two amino acids in which the a-amino group of one amino acid is bonded to the a-carboxyl group of the other amino acid. The peptide can be modified, for example by glycosylation, amidation, carboxylation, phosphorylation, or the like. The modification can be in vitro or in vivo. Amino acid chains with a length of less than approximately 100 amino acids are generally considered within the art to be "peptides", but both "polypeptides", and "proteins" are included within the definition of "peptides" as used herein.

[0080] The terms “amino acid sequence” and “peptide sequence” are used interchangeably. All amino acid or peptide sequences, unless otherwise designated, are written from the amino terminus (N-terminus) to the carboxy terminus (C -terminus). Each amino acid sequence can be optionally modified by inclusion of an initial “Met” residue.

[0081] For convenience of nomenclature, this application refers to a “protein”. However, the designation of “protein” is not intended to suggest any information regarding the size or relative size of the polymer concerned.

[0082] As used herein, when applied to an amino acid sequence, “conservative substitution” refers to the substitution of one amino acid residue with another amino acid residue having a side chain with similar physical and chemical properties. For example, conservative substitution may be conducted among amino acid residues having a hydrophobic side chain (e.g., Met,Ala, VaL, Leu, and He), amino acid residues having a neutral hydrophilic side chain (e.g., Cys, Ser, Thr, Asn, and Gin), amino acid residues having an acidic side chain (e.g., Asp and Glu), amino acid residues having a basic side chain (e.g., His, Lys, and Arg), or amino acid residues having an aromatic side chain (e.g., Trp, Tyr and Phe). It is known in the art that a conservative substitution generally does not cause a significant change in the conformational structure of a protein, and thus can retain the biological activity of the protein.

[0083] The term "polynucleotide" refers to a polymer of nucleic acid, for example, DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric polynucleotide molecule comprising one of these polynucleotides alone or in combination. The term “nucleic acid” is used interchangeably with the term “polynucleotide”. Each polynucleotide sequence can optionally be modified by inclusion of a start codon “atg” and / or one or more “stop” codons e.g. taa.

[0084] The term "vector" as used herein is well-known in the art and refers to a genetic construct to facilitate the handling of a target polynucleotide. A vector can be used to transport the target genetic construct into a suitable host cell. A vector typically contains each of the necessary elements required for transcription the target genetic construct. Optionally, the vector facilitates the translation of the target genetic construct into a polypeptide.

[0085] Expression of said polynucleotide or vector comprises transcription of the polynucleotide into a translatable mRNA. Usually, a vector comprises regulatory sequences ensuring initiation of transcription. Other elements which are responsible for the initiation of transcription, such as regulatory elements, may also be present. The vector may also comprise transcription termination signals downstream of the target polynucleotide. The vector may comprise further genes such as marker genes, which allow for the selection of the vector in a suitable host cell and under suitable conditions. A vector can also include elements (for example an origin of replication) required for replication of the vector, whether independently of host DNA or otherwise.

[0086] As used herein, the term “operably linked” or equivalent expressions refers to the connecting a polynucleotide to a second polynucleotide in a functional manner so that the polynucleotides are able to interact with each other in the manner intended. For example,the polynucleotides can be positioned and orientated for transcription to be initiated from a promoter.

[0087] When applied to an amino acid sequence (or a nucleic acid sequence), “percent sequence identity” refers to a percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those of a reference sequence, relative to the amino acid (or nucleic acid) residues in the candidate sequence during sequence alignment, and if necessary, after introducing gaps to maximize the number of identical amino acids (or nucleic acids). Sequence alignments and determination of sequence identity are well-known in the art. A conservative substitution of amino acid residue may or may not be considered as an identical residue. Percent sequence identity of amino acid (or nucleic acid) sequences can be determined by aligning sequences through tools disclosed in the art. A person skilled in the art may use the default parameters of the tools or adjust the parameters appropriately according to the needs of the alignment, for example by choosing an appropriate algorithm. The percentage identity between two polypeptide sequences may be readily determined by programs such as BLASTp which is freely available at http: / / blast.ncbi.nlm.nih.gov and is described by Altschul et al. 1990 (J Mol Biol 215: 403-10). The " Blast 2” sequences algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250) can also be used. Typically, the percentage sequence identity is calculated over the entire length of the sequence. The percentage identity of the resulting optimal global alignment is calculated from the ratio of the number of aligned bases to the total length of the alignment (i.e. the alignment length including both matches and mismatches) multiplied by 100.

[0088] As used herein, the terms "identity" and "identical" and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules, or between two protein molecules.

[0089] An “isolated” material has been artificially altered from its natural state. If an “isolated” substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally occurring in a living animal is not isolated but may be considered “isolated” if the polynucleotide or peptide is sufficiently isolated from the materials with which it coexists in its native state and exists in a sufficiently pure state. In some embodiments, the polynucleotide or peptide are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ionexchange chromatography or reverse phase HPLC).

[0090] The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.

[0091] As used herein, the term “immunogenic" means that the chimeric protein or VLP of the invention (optionally in the form of a composition for ease of administration) can elicit an immune response in a subject. For example, the chimeric protein or VLP of the invention (optionally in the form of a composition for ease of administration) may be capable of generating an antibody response and / or a non-antibody based immune response in a subject.

[0092] As noted above, the present invention provides a chimeric protein which comprises or consists of a first peptide (for example ColE7) and a chimeric second peptide, wherein said second peptide is formed from C-terminal and N-terminal segments of a target peptide, wherein said target peptide is a member of the IL-6 family of cytokines and has an epitope, characterised in that the relative positions of the C-terminal segment and the N-terminal segment are transposed in the second peptide relative to the target peptide. Thus, the C-terminal of the second peptide is formed from an N-terminal segment of the target peptide. The N-terminal of the second peptide is formed from a C-terminal segment of the target peptide.

[0093] Thus, the N and C terminal segments of the target peptide present in the second peptide are transposed (swopped over positionally) and joined (optionally via a linker). Thus, in the second peptide, the C-terminal segment of the target peptide is joined “upstream” of the N-terminal segment of the target peptide. Optionally, there may be deletions from one or both of the N and C terminal segments of the target peptide (particularly where such amino acid segments do not form part of the epitope of interest). For example, there may be a deletion from the N-terminal end of the target peptide so that the “N-terminal segment” references a segment of the target peptide which is closer to the N-terminus than the “C-terminal segment” of the target peptide. Likewise, there may be a deletion from the C-terminal end of the target peptide so that the “C-terminal segment” references a segment of the targetpeptide which is closer to the C-terminus (in the target peptide) than the “N-terminal segment”. Optionally or additionally amino acids between the native N and C terminus segments of the target peptide may be omitted in the second peptide.

[0094] The first peptide can be linked to either the first segment or the second segment of the second peptide.

[0095] Thus, the first peptide is linked to either the new N-terminal (first segment) of the second peptide or is linked to the new C-terminal (second segment) of the second peptide.

[0096] Optionally, the chimeric protein may also comprise a linker between the first peptide and the second peptide. Suitable linkers include S (Serine), A (alanine), SD, SG, GGS, GGG, GGGS (SEQ ID No: 75), GSGGS (SEQ ID No: 76), or GSGGGS (SEQ ID No: 77). Other examples might be GG, GS, GSG, GSGG (SEQ ID No: 78), GGGSG (SEQ ID No: 79), and the like. Alternatively, the first peptide and the second peptide are joined by a peptide bond.

[0097] In one embodiment, the second peptide can have the following structure:

[0098] X-[First Segment]-[Linker]-[Second Segment]-Y wherein X is the N-terminal of the second peptide;

[0099] wherein Y is the C-terminal of the second peptide, and

[0100] wherein the First Segment and Second Segment represent portions of the target peptide; and

[0101] wherein Linker represents a linking sequence of amino acids or a peptide bond.

[0102] In the target peptide, the Second Segment would be located “upstream” of the First Segment, i.e. would be towards the N-terminal of the target peptide.

[0103] Formation of the first and second segments of the second peptide creates new N and C terminals for the second peptide (relative to its native form in the target peptide). The second peptide is linked via its new N terminal or via its new C terminal to the first peptide.

[0104] Thus, the chimeric protein may be in the form:

[0105] first peptide-linker-N’ second peptide segment-linker-second peptide segment-C’; orN’ second peptide segment-linker-second peptide segment-C’- linker-first peptide wherein N’ is the non-native N terminal of the second peptide;

[0106] wherein C’ is the non-native C terminal of the second peptide; and

[0107] wherein “linker” is a linking sequence of amino acids or is a peptide bond, and each linker is the same or different.

[0108] Referring to Fig. 4, which is a schematic illustration of the second peptide, 1, it can be seen that the peptide is formed from two segments 2, 3 which can be designated “first” and “second” segments for ease of reference. Each segment 2, 3 includes an epitope section 5, 6 (shaded in black) which contributes to formation of the epitope. Thus, the epitope is formed from the combination and interaction of sections 5, 6. Within the second peptide 1, the original carboxy terminus (or C terminal segment) of the target peptide is labelled as “C”. Within the second peptide 1, the original amino terminal (or N terminal segment) of the target peptide is labelled as “N”. A linker 4 joins the carboxy terminal segment of the target peptide to the amino terminal segment of the target peptide to form second peptide 1. The linker 4 can be a peptide bond. A “division site” has been identified and new carboxy and amino terminals (designated as C’ and N’ in Fig. 4) are located either side of the division site. Compared to Fig. 1C, the epitope in the second peptide of Fig. 4 is now outwardly presented (see Fig. 6) and its orientation facilitates recognition of the epitope by specific binding partners, such as antibodies or cell receptors.

[0109] The first peptide acts as a carrier to the second peptide and ensures that the epitope is presented so that the epitope is presented in the desired orientation (see Fig. 5).

[0110] Optionally, the first peptide is selected from an at least functional portion of a bacterial colicin, for example can be a functional portion of ColE7 from E. coli. It is known in the art that ColE7, ColE2, ColE8, ColE9 are highly homologous and thus the first peptide can likewise be any one of ColE2, ColE8, or ColE9, or can be a functional portion of ColE2, ColE8 or ColE9. The first peptide can also be a functional portion of a bacterial ribonuclease, for example can be Barnase (for example from Bacillus amyloliquefaciens) or a functional portion thereof, can be a functional portion of a member of the Spycatcher family of proteins.Optionally, the first peptide may have the ability to bind specifically to a known binding partner. For example, the first peptide can be a functional portion of ColE7 and its binding partner can be derived from Im7. For example, ColE2 binds to Im2, ColE8 bind to Im8, ColE9 binds to Im9, Barnase binds to Barstar etc. For example, the first peptide can be Barnase and its binding partner can be Barstar (or vice versa). Other examples of suitable first peptides and their binding partners include a version of the SpyCatcher series of proteins plus SpyTag; or any artificially designed peptide pairs which show high binding affinity (Kd < 10-12) to each other. The term “functional portion” with respect to the first peptide refers to a portion of the wild type protein which retains its ability to still bind specifically to its natural binding partner. The first peptide can be a functional portion of the wild type protein, for example can be a truncation of the wild type protein which retains its ability to bind to its natural binding partner. Optionally, the first peptide can be truncated by deletion of the N-terminal portion of the wild type protein.

[0111] Optionally, the binding partner of the first peptide can be located onto the surface of a VLP. For example, the binding partner can be a part of a chimeric virus capsid protein or can be chemically attached to the VLP.

[0112] The chimeric protein of the invention therefore includes a fusion of the first peptide to a site other than the native N-terminus of the target peptide. This configuration allows the epitope of the second peptide to be presented in an outward facing orientation, so that the epitope is available for binding by a separate binding partner, for example an antibody or a cell receptor.

[0113] The target peptide is a peptide from the IL-6 family of cytokines having an epitope of interest. The tertiary structure of this epitope is maintained in the second peptide so that function is substantially retained. Typically, the position of the epitope of interest within the native tertiary structure of the second peptide would compromise or prevent the epitope being presented in an immunogenic manner within a simple end-to-end chimeric construct as known in the art.

[0114] Suitably the second peptide may be biologically active in humans and / or in non-human mammals (such as dogs). Suitably the second peptide may cause antibody production such that the antibodies formed in situ cause a biological response, for example by binding tocytokines or receptors thereof. Such a biological response may treat, ameliorate or prevent a disease or disorder in a subject.

[0115] The target peptide is selected from the IL-6 family of cytokines. These cytokines are characterised by a 4-helix structure. The IL-6 family of cytokines is a group of cytokines including IL-31, IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatinM (OSM), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and IL-27. Optionally, the second peptide is IL31, for example is human IL31 or canine IL31. Optionally, the target peptide is IL13, for example is human IL13 or canine IL13.

[0116] In one embodiment of the present invention, the first peptide is selected from ColE7, ColE2, ColE8 or ColE9 (or a functional portion thereof, which can optionally be mutated to reduce its activity) and the second peptide is selected from the IL-6 family of cytokines.

[0117] In one embodiment of the present invention, the first peptide is selected from ColE7, ColE2, ColE8 or ColE9 (or a functional portion thereof, which can optionally be mutated to reduce its activity) and the second peptide is selected from human IL31, canine IL31 and human IL13.

[0118] In one embodiment of the present invention, the first peptide is ColE7 or a functional portion thereof (which can optionally be mutated to reduce its activity) and the target peptide is selected from the IL-6 family of cytokines.

[0119] In one embodiment of the present invention, the first peptide is ColE7 or a functional portion thereof (which can optionally be mutated to reduce its activity and the target peptide is selected from human IL31, canine IL31 and human IL13.

[0120] In one embodiment of the present invention, the first peptide is ColE2 or a functional portion thereof (which can optionally be mutated to reduce its activity) and the target peptide is selected from the IL-6 family of cytokines.

[0121] In one embodiment of the present invention, the first peptide is ColE2 or a functional portion thereof (which can optionally be mutated to reduce its activity and the target peptide is selected from human IL31, canine IL31 and human IL13.In one embodiment of the present invention, the first peptide is ColE8 or a functional portion thereof (which can optionally be mutated to reduce its activity) and the target peptide is selected from the IL-6 family of cytokines.

[0122] In one embodiment of the present invention, the first peptide is ColE8 or a functional portion thereof (which can optionally be mutated to reduce its activity and the target peptide is selected from human IL31, canine IL31 and human IL13.

[0123] In one embodiment of the present invention, the first peptide is ColE9 or a functional portion thereof (which can optionally be mutated to reduce its activity) and the target peptide is selected from the IL-6 family of cytokines.

[0124] In one embodiment of the present invention, the first peptide is ColE9 or a functional portion thereof (which can optionally be mutated to reduce its activity and the target peptide is selected from human IL31, canine IL31 and human IL13.

[0125] Exemplary sequences for the first segment of the second peptide (i.e., C-terminal segment of the target peptide) can comprise or consist of one of the following:

[0126]

[0127] Optionally, the first segment of the second peptide can comprise a sequence having at least 80% sequence identity to any one of SEQ ID Nos: 1 to 4, for example at least 85% forexample at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of any one of SEQ ID Nos: 1 to 4.

[0128] Exemplary sequences for the second segment of the second peptide (i.e., N-terminal segment of the target peptide) can comprise or consist of one of the following:

[0129]

[0130] Optionally, the second segment of the second peptide can comprise a sequence having at least 80% sequence identity to any one of SEQ ID Nos: 5 to 8, for example at least 85% for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of any one of SEQ ID Nos: 5 to 8.

[0131] Optionally, the second peptide can comprise a sequence having at least 80% sequence identity to either of SEQ ID Nos: 9 to 13, for example at least 85% for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of any one of SEQ ID Nos: 9 to 13.

[0132] Optionally, the first peptide can comprise or consist of the sequence (ColE7 functional portion, including mutations R95A, E99A and H126A):

[0133] ESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRK KFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKATSFALHHEKPISQNGGVYDMDNISVVTPKRAIDIHRGKS (SEQ ID No: 14). Optionally SEQ ID No: 14 can be modified to omit the C-terminal Serine residue, which essentially acts as a linker residue.

[0134] Optionally, the first peptide can comprise or consist of the sequence (ColE2 functional portion, including mutations R95A, E99A and H126A):

[0135] ESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKNFDDFRK KFWEEVSKDPDLSKQFKGSNKTNIQKGKAPFARKKDQVGGAERFALHHDKPISQD GGVYDMNNIRVTTPKRAIDIHRGK (SEQ ID No: 16).

[0136] Optionally, the first peptide can comprise or consist of the sequence (ColE8 functional portion, including mutations R95A, E99A and H126A):

[0137] ESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKNFDDFRRK FWEEVSKDPELSKQFNPGNKKRLSQGLAPRARNKDTVGGARSFALHHDKPISQDG GVYDMDNLRITTPKRAIDIHRGQ (SEQ ID No: 18).

[0138] Optionally, the first peptide can comprise or consist of the sequence (ColE9 functional portion, including mutations R95A, E99A and H126A):

[0139] ESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKSFDDFRKA VWEEVSKDPELSKNLNPSNKSSVSKGYSPFTPKNQQVGGAKVYALHHDKPISQGG EVYDMDNIRVTTPKRAIDIHRGK (SEQ ID No: 20).

[0140] Each of these sequences can include a His-tag at the C-terminal, optionally attached vis a linker, such as GGG. Exemplary sequences are set out in SEQ ID Nos: 15, 17, 19 and 21.

[0141] Optionally, the first peptide can comprise a sequence having at least 80% sequence identity to SEQ ID Nos: 14 to 21, for example at least 85% for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of SEQ ID Nos: 14 to 21.

[0142] Optionally SEQ ID No: 14 can be modified to omit the C-terminal Serine residue, which essentially acts as a linker residue.

[0143] Optionally, the chimeric protein of the invention has a sequence which comprises the sequence of one of SEQ ID Nos: 22 to 34 or a functional variant thereof.SEQ ID No: 22 (First peptide based on mutated functional portion of ColE7-Second peptide based on hIL13)

[0144] ESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRK

[0145]

[0146] QFNGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVS GCSAIEKTQRMLSGFCPHK

[0147] SEQ ID No: 23 (First peptide based on mutated functional portion of ColE7-Second peptide based on hIL13)

[0148] ESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRK

[0149]

[0150] QFNGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVS GCSAIEKTQRMLSGFCPHKS

[0151] A variant of this sequence including a His tag is SEQ ID No: 30.

[0152] SEQ ID No: 24 (First peptide based on mutated functional portion of ColE7-Second peptide based on cIL31) ESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRK KFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKATSFALHHEKPISQN GGVYDMDNISVVTPKRAIDIHRGKSSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIE QLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGGGGAPTHQL PPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT

[0153] A variant of this sequence including a His tag is SEQ ID No: 31.

[0154] SEQ ID No: 25 (First peptide based on mutated functional portion of ColE2-Second peptide based on cIL31) ESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKNFDDFRK KFWEEVSKDPDLSKQFKGSNKTNIQKGKAPFARKKDQVGGAERFALHHDKPISQD GGVYDMNNIRVTTPKRAIDIHRGKSSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIE QLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGGGGAPTHQL PPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT

[0155] A variant of this sequence including a His tag is SEQ ID No: 32.SEQ ID No: 26 (First peptide based on mutated functional portion of ColE8-Second peptide based on cIL31) ESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKNFDDFRRK FWEEVSKDPELSKQFNPGNKKRLSQGLAPRARNKDTVGGARSFALHHDKPISQDG GVYDMDNLRITTPKRAIDIHRGQSSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQL DKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGGGGAPTHQLPPS DVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT

[0156] A variant of this sequence including a His tag is SEQ ID No: 33.

[0157] SEQ ID No: 27 (First peptide based on mutated functional portion of ColE9-Second peptide based on cIL31) ESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKSFDDFRKA VWEEVSKDPELSKNLNPSNKSSVSKGYSPFTPKNQQVGGAKVYALHHDKPISQGG EVYDMDNIRVTTPKRAIDIHRGKSSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQL DKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGGGGAPTHQLPPS DVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT

[0158] A variant of this sequence including a His tag is SEQ ID No: 34.

[0159] SEQ ID No: 28 (First peptide based on mutated functional portion of ColE7-Second peptide based on hIL31) ESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRK KFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKATSFALHHEKPISQN GGVYDMDNISVVTPKRAIDIHRGKSKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAY LKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDL ALKSLTSGAGGRPSDDVQKIVEELQSLSKMLLKDVEEE

[0160] A variant of this sequence including a His tag is SEQ ID No: 29.

[0161] Optionally, the functional variant maintains the tertiary structure of the epitope to within a root-mean-square deviation of no more than 1.5 A of the native tertiary structure in the target peptide, for example within 1.2A of the native tertiary structure or even less.

[0162] Optionally, the variant has a sequence identity of at least 80%, for example at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of any one of SEQ ID Nos: 22 to 34. The person skilled in the art would understandthat a mutation to an amino acid sequence can include insertions, deletions or substitutions as compared to the wild type sequence. Such mutations can be introduced to further enhance biological response. Alternatively, such mutation may have no effect on the function of the protein, i.e., its ability to interact with binding partner to cause a biologic effect.

[0163] Optionally a His tag or other suitable purification tag can conveniently be included at either the N-terminal or at the new C-terminal of the chimeric protein. The purification tag can be linked to the chimeric protein via a linker, for example as described above for linking the peptides of the chimeric protein. Examples include SEQ ID Nos: 29 to 34.

[0164] Generally, formation of the epitope relies on a combination of both the first and second segments of the second peptide. In other words, the epitope is formed by interaction between amino acids from both the first and second segments of the second peptide.

[0165] Desirably, the first and second segments of the second peptide form a tertiary structure which is substantially similar to that of the native epitope, preferably which is substantially identical to the tertiary structure of the native epitope. In the present invention, the native tertiary folding of the second peptide is preserved to within a root-mean-square deviation tolerance of no more than 1.5 A of the native peptide epitope.

[0166] Tertiary folding of peptides and proteins can be analysed by various means, including modelling software such as Alphafold (see Jumper et al., Nature 596:583-589, 2021). Root-mean-square-deviation (RMSD) can be analysed using freeware (such as iCn3D at NCBI.com). For example, the 3D folding of the second peptide epitope can be within a root-mean-square deviation tolerance of no more than 1.5 A, for example within 1.4A, of the target peptide epitope.

[0167] The second peptide is formed from a combination of first and second segments derived from a target peptide. The position at which the target peptide is divided into the 2 segments is herein termed a “division site” though it should be understood that expression of the chimeric protein will be via expression of a chimeric polynucleotide so that no physical cutting or cleavage of the target peptide is required. The division site is desirablyselected to ensure that the epitope of interest is presented in an outward facing orientation when part of the chimeric protein. The division site can be selected from:

[0168] i) An unstructured region (USR) of the target peptide; or

[0169] ii) A glycine residue located at the protein surface of the native tertiary structure of the target peptide and in the loop at the end of an alpha helix or beta sheet.

[0170] The term “Unstructured Region” as used herein refers to a portion of the peptide which, in the tertiary structure do not form well-structured three-dimensional fold, for example are not part of an alpha helix or beta sheet configuration. A USR (also termed an “intrinsically disordered region” in the art) is often characterized by the presence of a larger proportion of small and hydrophilic amino acids and proline residues. Such regions will commonly fail to yield identifiable structures in x-ray crystallography or NMR structure studies. An “unstructured Region” as used herein will fail to yield structural elements with a high probability of prediction (termed pLDDT) when using modelling programs such as Alphafold.

[0171] Optionally, the division site is located immediately upstream of an Unstructured Region (USR). According the first amino acid of the USR may then conveniently form the new N-terminal of the second peptide.

[0172] Once the division site has been selected, it is then possible to create a polynucleotide encoding the chimeric protein of the invention. Polynucleotide sequences corresponding to each segment of the second peptide (i.e. either side of the division site of the target peptide) can positioned at an end of a polynucleotide sequence encoding the first peptide, such that the whole chimeric peptide is expressed as a single entity. Optionally linkers can be inserted between the polynucleotide sequence for either or each segment of the second peptide and the polynucleotide sequence for the first peptide. Generally, these linkers will be relatively short and will ensure that expression of the whole chimeric protein can occur without disruption. Suitable linkers include S (serine), A (alanine), SD, SG, GGS, GGG, GGGS (SEQ ID No: 75), GSGGS (SEQ ID No: 76), or GSGGGS (SEQ ID No: 77). Other examples might be GG, GS, GSG, GSGG (SEQ ID No: 78), GGGSG (SEQ ID No: 79), and the like. Alternatively, no linker may be required and the peptides can be joined by a peptide bond.Optionally, a coding sequence for a purification tag, such as a His tag can be added to the polynucleotide such that the tag is operably linked to the chimeric peptide. Optionally, a His tag can be added to the N-terminal end of the first peptide optionally via a linker. A suitable linker is as described above, for example GGG.

[0173] Optionally, the first peptide is attached immediately upstream of the USR identified in the second peptide and which form the new N-terminal residue of the second peptide.

[0174] In one example, considering cIL31 as the target peptide, the wild type (target peptide) sequence (excluding the signal peptide) is:

[0175] APTHQLPPSDVRI< IILELQPLSRGLLEDYQI< I< ETGVPESNRTLLLCLTSDSQPPRLNS SAILPYFRAIRPLSDI< NIIDI< IIEQLDI< LI< FQHEPETEISVPADTFECT< SFILTILQQFSA CLESVFKSLNSGPQ (SEQ ID No: 82)

[0176] Analysis according to the present invention identifies two unstructured regions: USR1: SDSQ (SEQ ID No: 83) and USR2: FQHEP (SEQ ID No: 84), both underlined in the sequence above.

[0177] The two terminal amino acids of the target peptide Pro-Gin are deleted from the C-terminus of the target peptide. A chimeric second peptide can be formed using a division site immediately upstream of USR1, so creating a first segment formed from USR1 to the truncated C-terminal end of the target peptide. This first segment is re-arranged ahead of the original N-terminal Alanine of the target peptide and connected thereto using a linker (for example three glycines). The second peptide sequence so formed is (linker is shown in lower case):

[0178] SDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKS FILTILQQFSACLESVFKSLNSGgggAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKE TGVPESNRTLLLCLT (SEQ ID No: 12).

[0179] The serine residue at the start of USR1 in the target peptide is now the new N’-terminus of the second peptide.The second peptide can then be linked to the first peptide (a functional portion of ColE7) via a peptide bond. Optionally, a His tag (HAHEHRHDHE, SEQ ID No: 89) is added to the N-terminal of the first peptide via a linker, such as GGG, to give the chimeric protein sequence (His tag show underlined):

[0180] HAHEHRHDHEHGGGESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIAN KLRDKEFKSFDDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSG KATSFALHHEKPISQNGGVYDMDNISVVTPKRAIDIHRGKSSDSQPPRLNSSAILPYF RAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVF KSLNSGGGGAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT

[0181] (SEQ ID No: 31)

[0182] In alternative embodiments an alternative purification tag can be used and / or the location of the His tag or alterative purification tag can be the C-terminal of the chimeric peptide (optionally via a linker).

[0183] In another example, considering hIL31 as the target peptide, the wild type (target peptide) sequence (excluding the signal peptide) is:

[0184] ASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVE EEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF QDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT (SEQ ID No: 91)

[0185] Analysis according to the present invention identifies an unstructured region: USR1:

[0186] KGVL (SEQ ID No: 94) underlined in the sequence above. This is immediately after the end of the first helix.

[0187] The initial sequence of 32 amino acids is deleted:

[0188] ASHSGPSTSVLFLFCCLGGWLASHTLPVRLL (SEQ ID No: 93).

[0189] The five terminal amino acids of the target peptide QQATT (SEQ ID No: 92) are deleted from the C-terminus of the target peptide. A chimeric second peptide can be formed using a division site immediately upstream of USR1, so creating a first segment formed from USR1 to the truncated C-terminal end of the target peptide. This first segment is re-arranged aheadof the original N-terminal Alanine of the target peptide and connected thereto using a linker (for example two glycines). The second peptide sequence so formed is (linker is shown in lower case):

[0190] KGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQD APETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAggRPSDDVQKIVEELQSL SKMLLKDVEEE (SEQ ID No: 13).

[0191] The lysine residue at the start of USR1 in the target peptide is now the new N’ -terminus of the second peptide.

[0192] The second peptide can then be linked to the first peptide (truncated mutated ColE7) via a peptide bond. Optionally, a His tag (HAHEHRHDHE, SEQ ID No: 89) is added to the N-terminal of the first peptide via a linker, such as GGG, to give the chimeric protein sequence (His tag show underlined):

[0193] HAHEHRHDHEHGGGESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIAN KLRDKEFKSFDDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSG KATSFALHHEKPISQNGGVYDMDNISVVTPKRAIDIHRGKSKGVLVSQNYTLPCLS PDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHEC KRFILTISQQFSECMDLALKSLTSGAGGRPSDDVQKIVEELQSLSKMLLKDVEEE

[0194] (SEQ ID No: 29)

[0195] In alternative embodiments an alternative purification tag can be used and / or the location of the His tag or alterative purification tag can be the C-terminal of the chimeric peptide (optionally via a linker).

[0196] In another example, considering hIL13 as the target peptide, the wild type (target peptide) sequence (excluding the signal peptide) is:

[0197] MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQ KAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQ FSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN (SEQ ID No: 85)An unstructured region USR1: AGQFSSL is identified (shown underlined; SEQ ID No: 86).

[0198] A division site immediately upstream of USR1 is determined, so creating a first segment formed from USR1 to the C-terminal end of the target peptide as shown above. This segment is re-arranged to become the new N-terminal segment (first segment) of the second peptide.

[0199] The initial N-terminal region of the target peptide:

[0200] MHPLLNPLLLALGLMALLLTTVIALTCLGGFASP (SEQ ID No: 87) is deleted. The first segment is linked directly via a peptide bond upstream of the remaining target peptide N-terminal segment:

[0201] GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCS AIEKTQRMLSGFCPHK (SEQ ID No: 5), which forms the second segment of the second peptide in this construct.

[0202] Finally, the new N-terminus, consisting of USR1 AGQFSSL (SEQ ID No: 86), and which is unstructured, thereby serving as a linker sequence, is shortened and its hydrophobicity reduced by omitting GQF, thereby resulting in the new N-terminus ASSL (SEQ ID No: 88).

[0203] The second peptide sequence so formed is (first and second segments are linked directly via a peptide bond):

[0204] ASSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN-GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCS AIEKTQRMLSGFCPHK (SEQ ID No: 9)

[0205] The alanine residue at the start of USR1 in the target peptide is now the new N’ -terminus of the second peptide.

[0206] A linker, such as “SG”, can be used before a suitable purification tag, such as a His tag. The sequence including the linker and His tag (HAHEHRHDHE, SEQ ID No: 89) would then be:ASSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFNGPVPPSTALRELIEELVNITQNQ KAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKSGHAH EHRHDHEH (SEQ ID No: 11).

[0207] In alternative embodiments an alternative purification tag can be used and / or the location of the His tag or alterative purification tag can be the N-terminal of the chimeric peptide (optionally via a linker).

[0208] In an alternative embodiment, the target peptide N-terminal segment:

[0209] GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCS AIEKTQRMLSGFCPHKVS (SEQ ID No: 90), is used to form the second segment of the second peptide, but is modified by deletion of the penultimate valine to produce the second segment sequence:

[0210] GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCS AIEKTQRMLSGFCPHKS (SEQ ID No: 6).

[0211] The chimeric second peptide so formed (with the first and second peptides linked directly via a peptide bond) is therefore:

[0212] ASSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN-GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCS AIEKTQRMLSGFCPHKS (SEQ ID No: 10).

[0213] A linker, such as “G”, can be used before a suitable purification tag, such as a His tag. The sequence including the linker and His tag (HAHEHRHDHE, SEQ ID No: 89) would then be:

[0214] ASSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFNGPVPPSTALRELIEELVNITQNQ KAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKSGHAH EHRHDHEH (SEQ ID No: 30).

[0215] In alternative embodiments an alternative purification tag can be used and / or the location of the His tag or alterative purification tag can be the N-terminal of the chimeric peptide (optionally via a linker).In a second aspect, the present invention provides a VLP comprising a chimeric protein as described above. The VLP of the present invention is able present the epitope of the second peptide on its surface. The epitope of the second peptide is therefore presented for example to the immune system to induce antibody formation. Optionally, the first peptide can be a core, capsid or envelope protein and can self-assemble directly. Optionally, the first peptide can bind to a modified protein of the VLP, for example as described in WO2024 / 018188A1. In this embodiment, the VLPs of the invention can include a binding molecule which can attach specifically to the first peptide of the chimeric protein, and therefore attach the second peptide to the viral capsid proteins forming the VLP.

[0216] VLPs are known in the art and VLPs have been created from Hepatitis B Virus (HBV), human papillomavirus (HPV), and hepatitis E virus (HEV). However, other viruses can be used to create VLPs, including Parvoviridae, Retroviridae (e.g. HIV), Flaviviridae, Paramyxoviridae and bacteriophages (e.g. Qβ, AP205). Plant viruses including tobacco mosaic virus (TMV) can also be used. Optionally, the viral protein forming the VLP is a mammalian hepatitis viral protein. For example, the viral protein forming the VLP can be a human Hepatitis B viral protein. Optionally, the viral protein forming the VLP is from an icosahedral virus such as Tomato Aspermy Virus (TASV).

[0217] In one embodiment of the present invention, the VLP comprises the core protein (HBc) from a Hepatitis B virus (HBV) which self-assembles to form an empty icosahedral shell. Optionally, the VLP comprises the small Hepatitis B surface antigen (HBsAg). Optionally, the VLP comprises a chimeric form of an HBV protein, for example a chimeric form of HBc or HBsAg. Optionally the VLP comprises a chimeric viral protein which comprises or is derived from Im7 Im2, Im8 or Im9, which can optionally be modified (for example to include an F to L mutation) as in SEQ ID Nos: 53 to 56. In one embodiment the VLP comprises a chimeric HBV protein (such as human HBc) which comprises Im7, Im2, Im8 or Im9 or a modified form thereof (see for example SEQ ID Nos: 57 to 65). In one embodiment the VLP comprises a chimeric HBV protein (such as human HBc) which comprises or is derived from Im7 (see, for example, SEQ ID No: 58). Where the VLP comprises Im7, the first peptide of the invention can be a functional portion of ColE7 or any other protein which can bind specifically to Im7.Optionally, the VLP is a chimeric protein having at least 80% sequence identity to either one of SEQ ID Nos: 57 to 65. Optionally, the VLP is a chimeric protein having at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to either one of SEQ ID Nos: 57 to 65. Optionally, the chimeric protein can be expressed with an initial N-terminal Met amino acid.

[0218] Optionally, the VLP is a chimeric protein having at least 80% sequence identity to either one of SEQ ID Nos: 57, 58 or 62. Optionally, the VLP is a chimeric protein having at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to either one of SEQ ID Nos: 57, 58 or 62. Optionally, the chimeric protein can be expressed with an initial N-terminal Met amino acid.

[0219] Where the VLP comprises Im7 (e.g. SEQ ID No. 57, 58 or 62), the first peptide of the invention can be ColE7 or a functional (optionally mutated) portion thereof (e.g. SEQ ID No: 14 or 15) which can bind specifically to Im7.

[0220] Where the VLP comprises Im2 (e.g. SEQ ID No. 59 or 63), the first peptide of the invention can be ColE2 or a functional (optionally mutated) portion thereof (e.g. SEQ ID No: 16 or 17) which can bind specifically to Im2.

[0221] Where the VLP comprises Im8 (e.g. SEQ ID No. 60 or 64), the first peptide of the invention can be ColE8 or a functional (optionally mutated) portion thereof (e.g. SEQ ID No: 18 or 19) which can bind specifically to Im8.

[0222] Where the VLP comprises Im9 (e.g. SEQ ID No. 61 or 65), the first peptide of the invention can be ColE9 or a functional (optionally mutated) portion thereof (e.g. SEQ ID No: 20 or 21) which can bind specifically to Im9.

[0223] The VLP of the invention stably displays the epitope of the second peptide on its surface. The VLP will comprise a plurality of subunits and in some embodiments each subunit will comprise an epitope of the second peptide. In other embodiments, steric restrictions may require that only a proportion of the subunits will comprise an epitope of the second peptide. The ability of the subunits to self-assemble into a VLP will be retained.In a third aspect of the invention, the invention provides a polynucleotide which encodes the chimeric protein described above.

[0224] Optionally the polynucleotide encodes a chimeric protein which comprises a first segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 1 to 4.

[0225] Optionally the polynucleotide encodes a chimeric protein which comprises a second segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 5 to 8.

[0226] Optionally the polynucleotide encodes a chimeric protein which comprises a first segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 1 to 4., and further comprises a second segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 5 to 8.

[0227] Optionally the polynucleotide encodes a chimeric protein which comprises a first peptide comprising or consisting of any one of SEQ ID Nos: 14 to 21. Optionally, the sequence encoded for the first peptide can be a variant of SEQ ID No: 14 in which the terminal Serine residue is omitted.

[0228] Optionally the polynucleotide encodes a chimeric protein which comprises a first peptide comprising or consisting of SEQ ID No: 14 or SEQ ID No: 15. Optionally, the sequence encoded for the first peptide can be a variant of SEQ ID No: 14 in which the terminal Serine residue is omitted.

[0229] Optionally the polynucleotide encodes a chimeric protein of SEQ ID Nos: 22 to 34 or a functional variant thereof. Optionally, the variant has a sequence identity of at least 80%, for example at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of any one of SEQ ID Nos: 22 to 34.

[0230] Optionally, the polynucleotide of the invention has a nucleotide sequence which expresses a protein with at least 80% sequence identity to one of SEQ ID Nos: 22 to 34. Optionally, the polynucleotide of the invention encodes a polypeptide having a sequence identity to SEQ ID Nos: 22 to 34which is more than 80%, for example which is 85%, 90%. 95%, 98% or even more sequence identity to one of SEQ ID Nos: 22 to 34.In addition, the invention also encompasses a polynucleotide which specifically hybridizes under stringent conditions to the polynucleotide encoding the chimeric protein of the present invention (for example a chimeric protein of SEQ ID Nos. 22 to 34 or a functional variant thereof. Optionally, the variant has a sequence identity of at least 80%, for example at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% of any one of SEQ ID Nos: 22 to 34). For the purposes of the present specification, hybridisation under stringent hybridisation conditions means remaining hybridised after washing with 0.1 x SSC, 0.5% SDS at a temperature of at least 68° C, as described by Sambrook et al (Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press).

[0231] It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same protein as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the proteins are to be expressed.

[0232] The polynucleotide of the invention may consist of DNA or RNA. The polynucleotide may be single-stranded or double-stranded. The polynucleotide may include synthetic or modified nucleotides. Several different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the invention as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or life span of polynucleotides of interest.

[0233] Optionally the polynucleotide according to the invention can comprises a promoter to drive expression of the proteins encoded thereby. The promoter can be an inducible promoter. One suitable promoter is the T7 promoter, but other promoters can also be used depending upon the host cell selected for expression of the polynucleotide. Optionally, the polynucleotide can include a tag or other marker to assist preparation and selection of a vector comprising the polynucleotide as is known within the art.Optionally, the polynucleotide of the invention comprises a nucleotide sequence which with at least 80% sequence identity to one of SEQ ID Nos: 35 to 47. Optionally, the polynucleotide of the invention has a sequence identity of at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to one of SEQ ID Nos: 35 to 47. Optionally, each sequence can be modified to include a start codon (“ATG”).

[0234] Optionally, in addition to expression of the chimeric protein of the invention, the polynucleotide can also express a viral protein able to self-assembly to form a VLP.

[0235] Optionally, the viral protein can comprise a binding partner for the first peptide of the chimeric protein. For example, the binding partner can be a sequence of any one of SEQ ID Nos: 53 to 56 (Im7, Im2, Im8 and Im9 respectively, each including one point mutation).

[0236] Optionally, the polynucleotide can also include a coding sequence for a VLP which comprises Im7 or a functional variant thereof. An exemplary polynucleotide is SEQ ID No: 66 or SEQ ID No: 67 or 71. Other examples include any one of SEQ ID Nos: 68 to 70 and 72 to 74.

[0237] Optionally, the polynucleotide can also include a coding sequence for a VLP which comprises Im2, or a functional variant thereof, for example SEQ ID No: 59 or 63. An exemplary polynucleotide for a HBc-Im2 VLP is SEQ ID No: 68 or 72.

[0238] Optionally, the polynucleotide can also include a coding sequence for a VLP which comprises Im8, or a functional variant thereof, for example SEQ ID No: 60 or 64. An exemplary polynucleotide for a HBc-Im8 VLP is SEQ ID No: 69 or 73.

[0239] Optionally, the polynucleotide can also include a coding sequence for a VLP which comprises Im9, or a functional variant thereof, for example SEQ ID No: 61 or 65. An exemplary polynucleotide for a HBc-Im9 VLP is SEQ ID No: 70 or 74.

[0240] Optionally, the polynucleotide of the invention comprises a nucleotide sequence which with at least 80% sequence identity to one of SEQ ID Nos: 66 to 74. Optionally, the polynucleotide of the invention has a sequence identity of at least 85%, for example at least90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to SEQ ID Nos: 66 to 74.

[0241] Optionally, the polynucleotide of the invention has a nucleotide sequence which expresses a protein with at least 80% sequence identity to one of SEQ ID Nos: 57 to 61 in relation to the VLP forming peptide able to bind the chimeric protein of the present invention. For example, the VLP forming peptide can be a chimeric peptide comprising a peptide able to self-assemble into a VLP operably linked (optionally via a linker) to a peptide which a is binding partner for the first peptide of the chimeric protein of the invention. The peptide able to self-assemble into a VLP can be based on HBc capsid protein or can be based on the capsid protein of a cucumovirus such as Tomato Aspermy Virus (TASV). The peptide which a is binding partner for the first peptide of the chimeric protein of the invention can be selected from SEQ ID Nos: 53 to 56. Optionally the linker is based on SEQ ID No: 81. Optionally, the VLP forming peptide can include a purification tag, such as a His tag. For example, the His tag can be linked to the N-terminal via a linker, such as the linker of SEQ ID No: 80. Optionally, the polynucleotide of the invention encodes a polypeptide having a sequence identity to SEQ ID Nos: 57 to 61 which is more than 80%, for example which is 85%, 90%. 95%, 98% or even more sequence identity to one of SEQ ID Nos: 57 to 61.

[0242] In a fourth aspect, the present invention provides a vector comprising a polynucleotide according to the invention as described above. Optionally, the vector can be an expression vector expressing, or overexpressing, said chimeric protein as described above, for example a chimeric protein of SEQ ID Nos: 22 to 34 or a functional variant thereof. Optionally, the variant has a sequence identity of at least 80%, for example at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to any one of SEQ ID Nos: 22 to 34.

[0243] Optionally, the vector comprises a polynucleotide which encodes a chimeric protein comprising a first segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 1 to 4.

[0244] Optionally vector comprises a polynucleotide which encodes a chimeric protein comprising a second segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 5 to 8.Optionally vector comprises a polynucleotide which encodes a chimeric protein comprising a first segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 1 to 4, and further comprises a second segment of the second peptide comprising or consisting of any one of SEQ ID Nos: 5 to 8.

[0245] Optionally vector comprises a polynucleotide which encodes a chimeric protein comprising a first peptide comprising or consisting of any one of SEQ ID Nos: 14 to 21. Optionally, the sequence encoded can be a variant of SEQ ID No: 14 in which the terminal Serine residue is omitted.

[0246] Optionally vector comprises a polynucleotide which encodes a chimeric protein comprising a first peptide comprising or consisting of SEQ ID No: 14. Optionally, the sequence encoded can be a variant of SEQ ID No: 14 in which the terminal Serine residue is omitted.

[0247] Optionally, the vector of the invention comprises a nucleotide sequence with at least 80% sequence identity to one of SEQ ID Nos: 35 to 47. Optionally, the nucleotide sequence has a sequence identity of at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to one of SEQ ID Nos: 35 to 47.

[0248] Optionally, the vector further comprises a nucleotide sequence with at least 80% sequence identity to any one of SEQ ID Nos. 66 to 74. Optionally, the nucleotide sequence has a sequence identity of at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to SEQ ID Nos: 66 to 74.

[0249] Optionally, the nucleotide sequence can encode a purification tag, such as a His tag, operably linked to the VLP forming peptide. For example, the His tag can be linked to the N-terminal via a linker, such as the linker of SEQ ID No: 80.

[0250] Optionally, the vector further comprises a nucleotide sequence with at least 80% sequence identity to any one of SEQ ID Nos. 62 or 63. Optionally, the nucleotide sequence has a sequence identity of at least 85%, for example at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or even 99% sequence identity to SEQ ID Nos: 62 or 63.

[0251] The vector according to the present invention refers to a vehicle into which a polynucleotide encoding the chimeric protein can be operably inserted for enabling thechimeric protein to be expressed. The vector can be used to transform, transduce, or transfect (which terms are used interchangeably herein) a host cell, such that the genetic elements carried by the vector are expressed in the host cell. A variety of vectors are available. The vector may comprise a variety of elements that control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a signal sequence, one or more marker genes, a selection element, a reporter gene, and a transcription termination sequence. Further, the vector may also comprise an origin of replication. The vector may also comprise a component that facilitates the vector to enter cells, including, but not limited to, viral particle, liposome, or protein shell.

[0252] For example, suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages such as λ bacteriophage or M13 bacteriophage, animal viruses, and the like.

[0253] In some embodiments, the vector systems include mammalian, bacterial, and yeast systems, and will include plasmids such as, but not limited to, ‘pENDO-2’ and other vectors available from the laboratory or commercially available vectors. Suitable eukaryotic vectors include vectors having a 2 micron or centromeric origin of replication. Suitable vectors may include plasmid or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Suitable vectors include pET28 and pET32, for example.

[0254] The present invention thus provides an expression vector comprising the polynucleotide according to the invention. The expression vector of the present invention can be prepared by subcloning the polynucleotide as described above into the expression vector by any conventionally known genetic engineering method. The type of expression vector that can be used in the present embodiment is not particularly limited, and examples thereof include any expression vector suitable for heterologous gene expression in eukaryotes or prokaryotes and able to drive expression of the chimeric protein.

[0255] Where the vector is an expression vector, the polynucleotide according to the invention will include a promoter to drive expression of the proteins encoded thereby. The promoter can be an inducible promoter. One suitable promoter is the T7 promoter, but other promoters can also be used depending upon the host cell selected for expression of the polynucleotide.Optionally, in addition a polynucleotide for expression of the chimeric protein of the invention, the vector can also comprise a polynucleotide to express a viral protein able to self-assembly to form a VLP. Optionally, the viral protein can comprise a binding partner for the first peptide of the chimeric protein. Optionally, the same promoter is used to drive expression of both the viral protein and the chimeric protein of the invention.

[0256] In one embodiment the vector is formed using plasmid DU78331 of SEQ ID No. 48 or a functional variant thereof having at least 80% sequence identity thereto, for example which has 85%, 90%. 95%, 98% or 99% sequence identity thereto.

[0257] Exemplary plasmids according to the present invention include:78488 (SEQ ID No: 49), 78457 (SEQ ID No: 50), 79449 (SEQ ID No: 51) and 79252 (SEQ ID No: 52). The present invention comprises a vector having at least 80% sequence identity to any one of SEQ ID Nos: 49 to 52, for example having at least 85%, 90%. 95%, 98% or 99% sequence identity to any one of SEQ ID Nos: 49 to 52.

[0258] A vector comprising a polynucleotide encoding the chimeric protein may be introduced into a host cell for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art.

[0259] In a fifth aspect, the present invention provides a host cell transformed with a polynucleotide according to the invention or a vector according to the invention. A cell culture comprising one or more host cells according to the invention is also encompassed. The host cell is transfected with a vector according to the invention and is then cultured under suitable conditions.

[0260] The host cell according to the present invention refers to a cell into which an exogenous polynucleotide and / or a vector (as described above) are introduced. Amino acid sequences of the chimeric protein of the present application may be converted to corresponding DNA coding sequences using genetic engineering techniques well known in the art. Due to the degeneracy of genetic code, the transformed DNA sequences may not be completely identical, while the encoded protein sequences remain unchanged.Suitably the processes may further comprise transfecting the one or more vectors comprising the nucleic acids into the or each host cell. Suitably prior to culturing the or each host cell. Suitably transfection may take place by any suitable method such as electroporation, microinjection, particle delivery, chemical mediated endocytosis, calcium phosphate co- precipitation, or liposome mediated delivery.

[0261] Host cells suitable for cloning or expressing the vectors of the present invention include prokaryotic, yeast or eukaryotic cells. Prokaryotic cells suitable for use in the present invention include E. coli (for example E. coli DH5a and BL21de3).

[0262] The host cell is transformed with the above-mentioned expression or cloning vector that can produce the chimeric protein, and then cultured in a conventional nutrient medium, which is suitable for inducing promoters, selecting transformed cells, or amplifying genes encoding target sequences after being modified.

[0263] The host cells used to produce the chimeric protein of the present invention can be cultured in a variety of media known in the art. The media may also comprise any other necessary additives known in the art in a suitable concentration. The conditions of the media, such as temperature, pH and the like are those selected previously for expression of host cells, which are well known to those of ordinary skill.

[0264] Generally, care is taken to culture the host cells under conditions to facilitate protein expression and may include culturing the host cells under optimum growth conditions. Selection of the optimum growth conditions will vary depending on the host cell being used.

[0265] In another aspect of the present invention, the present invention provides a recombinant cell or recombinant microorganism which contains the polynucleotide or vector according to the invention as described above. Thus, the recombinant cells or recombinant microorganisms according to the present invention can express the chimeric protein of the present invention. The invention further relates to a recombinant host cell comprising the polynucleotide, or the vector as described above. The polynucleotide or vector of the present invention, which is present in the host cell, may either be integrated into the genome of the host cell, or it may be maintained extra-chromosomally. Once the polynucleotide or vector has beenincorporated into the appropriate “host cell”, the host cell is maintained under conditions suitable for high level expression of the polynucleotide or vector.

[0266] The transformed host cells can be grown according to methodology known in the art to achieve cell growth.

[0267] Optionally, once expressed, the chimeric protein can be purified according to standard procedures of the art. Mention may be made of affinity columns, column chromatography, such as size exclusion chromatography (SEC), gel electrophoresis, ammonium sulphate precipitation and the like. The protein of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the protein may be by any conventional means such as, for example, preparative chromatographic separations.

[0268] The present invention further provides a method for producing a chimeric protein as described above, wherein the method comprises the following steps of suitably culturing a recombinant host cell comprising and expressing a polynucleotide encoding the chimeric protein according to the invention or a vector encoding the chimeric protein according to the invention. The polynucleotide can include a sequence according to any one of SEQ ID Nos: 35 to 47 or a functional variant thereof. The polynucleotide can express a chimeric protein having a sequence of any one of SEQ ID Nos: 22 to 34.

[0269] In a sixth aspect, the present invention provides a process of producing a chimeric protein according to the invention, said method comprising:

[0270] b) Identifying a target peptide comprising an epitope of interest;

[0271] c) Identifying a division site in the target peptide wherein said division site is within an unstructured region or is within a Glycine bend region of the target peptide, and thereby defining N- and C-terminal segments of the target peptide;

[0272] d) Transposing the native C and N terminal segments of the target peptide to form a second peptide;

[0273] e) Forming a chimeric polynucleotide comprising an operably linked sequence encoding both first and second peptides;

[0274] f) Expressing said chimeric protein from said chimeric polynucleotide.Identification of the division site creates new N and C terminals for the second peptide (relative to the target peptide). The second peptide is linked via its new N terminal or via its new C terminal to the first peptide.

[0275] Thus, the chimeric protein may be in the form:

[0276] first protein-(optional linker)-N’ second peptide segment- linker-second peptide segment-C’; or

[0277] N’ second peptide segment-linker-second peptide segment-C’ -(optional linker)-first peptide wherein N’ is the non-native N terminal of the second peptide; and

[0278] wherein C’ is the non-native C terminal of the second peptide.

[0279] Optionally, the process may further comprise a step of recovering or purifying the chimeric protein, for example, from the host cells.

[0280] Optionally, the N’ second peptide segment is formed from a portion of the target peptide close to its C terminal linked to an N-terminal portion.

[0281] Any suitable method for recovery or purification of the chimeric protein can be used, for example centrifugation, filtration, or chromatography.

[0282] In a seventh aspect, the present invention provides a process for the production of a viruslike particle (VLP), said method comprising:

[0283] a) A first polynucleotide encoding a chimeric protein according to the invention and expressing said chimeric protein;

[0284] b) Allowing said chimeric protein to self-assemble into a VLP.

[0285] In an alternative embodiment, the present invention further provides a process for the production of a virus-like particle (VLP), said method comprising:

[0286] a) A first polynucleotide encoding a chimeric protein according to the invention and expressing said chimeric protein, wherein said first peptide of said chimeric protein comprises a binding site;

[0287] b) Admixing said chimeric protein with a viral protein, wherein said viral protein comprises a binding partner for the first peptide binding site and is able to selfassemble into a VLP.Optionally, the process may further comprise a step of recovering the VLP comprising the chimeric protein. Generally, the VLPs self-assemble into particle prior to the recovery step. Optionally, the VLPs can be recovered from host cells, and the step of recovery can include disrupting the host cells. Alternatively, the host cells may secrete the VLPs into the culture solution. Options for disrupting the host cells can include known methods, for example homogenisation, sonication, or freeze-thaw cycles. Any suitable method for recovery or purification of the VLPs can be used, for example centrifugation, filtration, or chromatography.

[0288] The chimeric protein and a VLP comprising the chimeric protein can be used to cause a biological response, for example as a consequence of the epitope binding to a cell receptor. For example, the epitope can bind to a B-cell and induce an immunogenic response leading to the production of antibodies against the epitope. In a further aspect, the present invention provides an immunogenic composition comprising the chimeric protein or VLP according to the present invention.

[0289] The chimeric protein is therefore suitable for use as a medicament. The VLP comprising the chimeric protein is also suitable for use as a medicament.

[0290] Optionally, the medicament can be for use in the prevention and / or treatment of inflammatory diseases, cancer, neurodegenerative, metabolic, cardiovascular, renal, gastrointestinal, respiratory, or autoimmune diseases. In particular, the medicament can be for use in the prevention and / or treatment of pruritic diseases such as atopic dermatitis, and also in allergy and inflammatory bowel disease.

[0291] The terms “therapy” “therapeutic” “treatment” or “treating” refer to preventing, reducing, ameliorating or eliminating one or more signs, symptoms, or effects of a disease or condition. " Treatment," or “therapy” as used herein thus includes any treatment of a disease in a mammal, for example a human or dog. The term “treatment “ or “therapy and includes: (a) preventing the disease from occurring in a subject at risk of acquiring the disease but has not yet been diagnosed as having the disease (b) preventing the disease from occurring in a subject known to be predisposed to the disease or; (c) inhibiting the disease, in other words slowing or preventing the further development of the disease; and (d) relieving the disease, in other words causing signs, symptoms, or effects of the disease to be improved.The “administration” of an agent to a subject includes any route of introducing or delivering to a subject the agent to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, intraocularly, ophthalmically, parenterally (intravascularly, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another, including by a medical professional. The terms “individual,” “subject,” and “patient” are used interchangeably, and refer to any individual subject with a disease or condition in need of therapy, suitably in need of therapy by treatment with the present invention.

[0292] The subject may be a human or animal, for example primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or bovine, and the like.

[0293] Thus, the present invention further provides an immunogenic composition comprising the chimeric protein or VLP of the invention. The immunogenic composition may be a vaccine.

[0294] Optionally, the immunogenic composition may further comprise one or more adjuvants or pharmaceutically acceptable excipients.

[0295] The immunogenic composition can be formulated as a solid or as a liquid. A liquid formulation can optionally be an injectable liquid formulation.

[0296] The present invention further provides a method of treating a subject having a disease, said method comprising administering an effective amount of a chimeric protein as described above or a VLP comprising the chimeric protein, to the subject. In further aspect, the present invention further provides a method of manufacturing a medicament for the treatment of a disease, the medicament comprising an effective amount of a chimeric protein as described above or a VLP comprising the chimeric protein. The chimeric protein or VLP may in the form of an immunogenic composition.

[0297] Preferred or alternative features of each aspect or embodiment of the invention apply mutatis mutandis to each other aspect or embodiment of the invention (unless the context demands otherwise).All documents referred to herein are incorporated by reference. Any modifications and / or variations to described embodiments that would be apparent to one of skill in art are hereby encompassed. Whilst the invention has been described herein with reference to certain specific embodiments and examples, it should be understood that the invention is not intended to be unduly limited to these specific embodiments or examples.

[0298] The following examples are provided to further illustrate the invention but are not intended to limit the scope of the invention in any manner.

[0299] Examples

[0300] Example 1:

[0301] Plasmids as illustrated schematically in Fig. 8 A to D were created using plasmid DU78331 (SEQ ID No: 48). Plasmid 79105 is the parent vector, based on a pET scaffold. A T7 promoter drives expression of both the VLP scaffold protein (designated Hcl) and the epitope protein, consisting of ColicinE7 (designated C7) fused directly to human IL31 (hIL31). The T7 promoter generates a single mRNA transcript where the second open reading frame is translation-initiated via an internal ribosomal binding site (RBS). A purification tag (designated HDE) is placed at the C-terminus of the construct.

[0302] The novel plasmids 78488 (see SEQ ID No: 49; Fig. 8A) and 78457 (see SEQ ID No: 50; Fig. 8B), respectively, are designed analogously, except that the C7-hIL31 ORF is replaced by a novel ORF encoding the chimeric protein of the invention (78488: ColE7-human IL13 and 78457: ColE7- canine IL31).

[0303] The novel plasmid 79252 (SEQ ID No: 52) is designed analogously, with the ORF for the chimeric protein of the invention being as set out in SEQ ID No: 47.

[0304] The novel plasmid 79499 (SEQ ID No: 51) (Fig. 8D) is analogous to 78457, but has kanamycin resistance (not shown in Figure 8).

[0305] The chimeric second peptide of 78488 (SEQ ID No: 23) is based on the target peptide human IL13.For convenience a His tag can be added to the C-terminal of the second peptide via a linker, which in this construct is G, see SEQ ID No: 30. In alternative embodiments an alternative purification tag can be used and the location of the His tag or alterative purification tag can be the N-terminal of the chimeric peptide (optionally via a linker).

[0306] The chimeric second peptide of 78457 (SEQ ID No: 24) is based on the target peptide canine IL31.

[0307] Optionally, a purification tag, such as a His tag can be added to the chimeric peptide.

[0308] Optionally, a His tag can be added to the N-terminal end of the first peptide optionally via a linker.

[0309] In 78457, the second peptide can be linked to the first peptide (mutated, truncated ColE7) via a peptide bond. Optionally, a His tag is added to the N-terminal of the first peptide in 78457 via a linker, which in this construct is GGG, to give the chimeric protein sequence (His tag show underlined):

[0310] HAHEHRHDHEHGGGESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIAN KLRDKEFKSFDDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSG KATSFALHHEKPISQNGGVYDMDNISVVTPKRAIDIHRGKSSDSQPPRLNSSAILPYF RAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVF KSLNSGGGGAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT

[0311] (SEQ ID No: 31)

[0312] In alternative embodiments an alternative purification tag can be used and the location of the His tag or alterative purification tag can be the C-terminal of the chimeric peptide (optionally via a linker).

[0313] The same construct was used in each of plasmids 79252 and 79449.

[0314] Plasmids 78488, 78457, 79252 and 79449 each containing a chimeric protein according to the invention were expressed in E.coli. Plasmids were transfected into a BL21 / DE3 E.coli strain and induced with 0.3 mM IPTG at 16C for 3h.Plasmids were transfected into a BL21 / DE3 E.coli strain and induced with 0.3 mM IPTG at 16C for 3h.

[0315] Example 2

[0316] Plasmids 78488 and 78457 were expressed in E. coli as described above in Example 1. Fig.

[0317] 9 shows denaturing reducing SDS PAGE analyses of cytosolic fractions obtained by cell disruption, centrifugation at 10,000g and filtration through 0.2 pm, respectively. White arrowheads denote the chimeric proteins, black arrowheads denote the VLP scaffold protein. The SDS PAGE results shown in Fig. 9 demonstrate that the chimeric proteins of 78488 (see Fig. 9A) and 78457 (see Fig. 9B) each co-purify at 1:1 stoichiometric ratio together with the VLP scaffold protein, demonstrating that the folding of ColicinE7 and Im7 are intact, which confer binding of the scaffold protein and epitope protein, respectively.

[0318] Example 3

[0319] E. coli cytosol harbouring either a chimeric protein including human IL 13 (78488) or a chimeric protein including canine IL31 (78457) were subjected to density gradient analysis (see Fig. 11 for details). After 16h, the chimeric protein band (white arrow), as well as the VLP scaffold band (black arrow), have equilibrated into the 40% and 50% high density fractions, demonstrating the successful formation of high-molecular weight nanoparticles.

[0320] Example 4: Immunogenicity

[0321] VLPs harbouring either conventional end-to-end fusion of hIL31 (termed “linear”) or VLPS comprising the chimeric proteins according to the invention were administered to mice. Female C57B16 / j mice (n = 5 per group) were dosed subcutaneously with purified VLPs, either bearing conventional C7-fused hIL31, or inventive IL13 protein (78488), or inventive IL31 protein (78457). One week after a booster dose, the serum of the recipient was collected and tested via ELISA for the emergence of hl3-specific antibodies or hIL31-specific antibodies, as appropriate. Analysis of the resulting blood for antibodies specific for canine IL31 (see Figure 10) demonstrates that the chimeric proteins are able to induce antibody titres to the same level as conventional end-to-end fusion IL31. This demonstrates that the surface structure of the graft version is entirely preserved, as predicted by the structural folding overlay).Example 5: Purification

[0322] Plasmids 79252 and 79449 were expressed in E. coli as described above in Example 1. Fig.

[0323] 12 shows purification of the cytosolic fraction using POROS HQ anion exchange chromatography, followed by immobilized metal affinity (IMAC) chromatography. Arrows indicate scaffold (28.4) and epitope (79252; 31.2 kDa, 79449; 32.3 kDa).

[0324] The results shown in Fig. 12 demonstrate that the chimeric proteins of 79252 (Fig. 12A) and 79449 (Fig. 12B) each co-purify together with the VLP scaffold protein, demonstrating that the folding of ColicinE7 and Im7 are intact, which confer binding of the scaffold protein and epitope protein, respectively.

[0325] Example 6: Immunogenicity

[0326] Female Sprague Dawley rats were dosed with 0.18 mg / mL 79252 VLP.

[0327] Antibody titre and quantification ELISAs were performed (see Figure 13 A and B). Briefly, ELISA 96 well plates were coated with 50 pL of IL-31 (0.5 pg / mL) and left to incubate overnight at 4°C. IL-31 solution was discarded and the plates were washed with PBS + 0.5% Tween 20. Plates were blocked with 5% BSA in PBS + 0.5% Tween 20. Plasma samples were prepared by diluting into 0.5% BSA + PBS + 0.5% Tween 20 to 1:200, 1:600, 1: 1800, 1:3600, 1:7200 and 1: 14400). Additional runs were performed to perform further dilutions at 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:162000, 1:324000, 1:648000, if required. A standard curve of human IL-31 antibody was prepared at 0.1 -0.0009 pg / mL. 100 pL of standards and sample dilutions were aliquot into designated wells and incubated for 2hours at RT. Following incubation, the plates were washed with washing buffer (PBS + 0.5% Tween 20). Secondary antibodies (anti-Rabbit HRP and anti- rat-HRP) were prepared at 1:10,000 in dilution buffer and 100 pL was added to respective wells and incubated for 1 hour at RT. Plates were washed and then 100 pL of TMB substrate was added to each well. After 15-30 minutes, the reaction was stopped with 50 pL 0.2M sulfuric acid. Absorbance readings for each well were read at 450 nm using a microplate reader. Background cut off was determined by taking an average of all the control blank samples + 2 standard deviations. The lowest dilution antibodies detected above the cutoff was selected as the antibody titre. Initial analysis shows that 79252 elicited a robust anti-hIL-31 antibody response in rats, with antibodies detected following the prime / boost dosing regimen.Percentage Inhibition in isolated IgG following IgG isolation from plasma using the Melon™ Gel protocol (see Figure 14). Briefly, hIL-31 was prepared at a working concentration of 5 pg / mL in bicarbonate buffer and ELISA plates were coated with 50 pL hIL-31 per well and incubated overnight at 4 °C. Plates were washed three times with wash buffer (PBS + 0.5% Tween 20) and blocked with 200 pL blocking buffer (5% BSA in PBS + 0.5% Tween 20) for 1 hour at RT.

[0328] During the blocking step, antibody standards and sample dilutions were prepared in PBS, ensuring all solutions were well mixed. Positive controls included serial dilutions (3-0.18 pg / mL) of anti-IL-31 antibody, and a maximum-response control consisting of IL-31 with IL-31RA.

[0329] After blocking, plates were washed three times. PBS (75 pL) was added to designated wells for the maximum-response control, followed by addition of antibodies and sample dilutions (75 pL). All binding steps were performed for 2 hours at RT with gentle shaking (300 rpm), followed by four washes.

[0330] IL-31RA (1.5 pg / mL) was then added (75 pL) to the appropriate wells and incubated for 2 hours at RT with shaking before washing. Anti-His-HRP (1:5000) was applied for 1 hour at RT, plates were washed four times, 100 pL of TMB substrate was then added to each well. After 15-30 minutes, the reaction was stopped with 50 µL 0.2M sulfuric acid. Absorbance readings for each well were read at 450 nm using a microplate reader. The results are shown in Figure 14. Purified IgG from animals treated with 79252 showed near complete inhibition of IL31 / IL31RA binding, approaching the level of the anti-IL31 positive control, confirming that dosing induced a functionally relevant antibody response by Day 35.

[0331] Example 7: Immunogenicity

[0332] Female C57B16 mice were dosed with 79449 VLP with 2% Alhydrogel adjuvant.

[0333] Antibody titre and quantification ELISAs were performed. Briefly, ELISA 96 well plates were coated with 50 pL of cIL-31 (0.5 pg / mL) and left to incubate overnight at 4°C. cIL-31 solution was discarded and the plates were washed with PBS + 0.5% Tween 20. The plates were blocked with 5% BSA in PBS + 0.5% Tween 20. Plasma samples wereprepared by diluting into 0.5% BSA + PBS + 0.5% Tween 20, to 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400,1:12800 and 1:25600. Following Day 22 analysis, selected Day 35 mouse plasma samples with high titre were diluted to 1:600, 1: 1800, 1: 5400, 1: 16200, 1:48600, 1:145800 and 1:437400). A standard curve of canine monoclonal IL-31 antibody (Lokivetmab) was prepared at 0.0001 to 0.03 pg / mL. 100 pL of standards and sample dilutions were aliquot into designated wells and incubated for 2 hours at RT. Following incubation, the plates were washed with washing buffer (PBS + 0.5% Tween 20).

[0334] Secondary antibodies (anti-canine-HRP and anti-mouse-HRP) were prepared at 1:10,000 in dilution buffer and 100 pL was added to respective wells and incubated for 1 hour at RT. Plates were washed and then 100 pL of TMB substrate was added to each well. After 15-30 minutes, the reaction was stopped with 50 pL 0.2M sulfuric acid. Absorbance readings for each well were read at 450 nm using a microplate reader.

[0335] Background cut off was determined by taking an average of all the control samples + 2 standard deviations. The lowest dilution antibodies detected above the cutoff was selected as the titre result.

[0336] The results are shown in Figure 15: (A). Antibody titre (B) Antibody concentration at days 21 and 35 post-dosage. Initial analysis shows that 79449 induced an anti-cIL-31 antibody response in mice, with antibodies detected following the prime / boost dosing regimen.

[0337] Example 8: Other First Peptide options

[0338] Example 1 can be adapted by using the constructs of SEQ ID Nos 32, 33 and 34 in place of the sequence of SEQ ID No: 31 in plasmid 78457 or plasmid 79449.

[0339] These plasmids can be further revised by replacing the ORF1 sequence of SEQ ID No: 58 with the sequence of SEQ ID No: 64, 65 or 66 respectively. Thus, the new plasmid will contain either SEQ ID No: 32 and SEQ ID No: 64, or will contain SEQ ID No: 33 and SEQ ID No: 65 or will contain SEQ ID No: 34 and SEQ ID No: 65.

[0340] Expression in E. coli as described above in Example 1 will lead to the formation of immunogenic VLPs.

Claims

CLAIMS1. A chimeric protein which comprises a first peptide linked to a second peptide, wherein said second peptide includes an epitope of a target peptide, wherein said second peptide is formed from C-terminal and N-terminal segments of the target peptide comprising the epitope, characterised in that the C-terminal segment of the second peptide is formed from a N-terminal segment of the target peptide, and the N-terminal segment of the second peptide is formed from a C-terminal segment of the target peptide, and wherein the target peptide is a member of the IL-6 family of cytokines.

2. The chimeric protein as claimed in Claim 1 wherein the epitope is formed by interaction between amino acids from both the first and second segments of the second peptide.

3. The chimeric protein as claimed in either of Claims 1 and 2 wherein said second peptide is selected from IL-31, IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and IL-27.

4. The chimeric protein as claimed in Claim 3 wherein said target peptide is IL13.

5. The chimeric protein as claimed in Claim 4 wherein said first segment of the second peptide comprises a sequence with at least 80% sequence identity to SEQ ID No: 1 or 2 and / or wherein the second segment of the second peptide comprises a sequence with at least 80% sequence identity to SEQ ID No: 5 or 6.

6. The chimeric protein as claimed in Claim 3 wherein said second peptide is human or canine IL-31.

7. The chimeric protein as claimed in Claim 6 wherein said first segment of the second peptide comprises a sequence with at least 80% sequence identity to SEQ ID No: 3 or 4 and / or wherein the second segment of the second peptide comprises a sequence with at least 80% sequence identity to SEQ ID No: 7 or 8.

8. The chimeric protein as claimed in any one of Claims 1 to 7 wherein said first peptide is a bacterial colicin, a bacterial ribonuclease or a member of the Spycatcher group of proteins, or a functional portion thereof.

9. The chimeric protein as claimed in Claim 9 wherein said first peptide is a bacterial colicin or a functional portion thereof.

10. The chimeric protein as claimed in Claim 9 wherein first peptide is selected from ColE7, ColE2, ColE8 or ColE9, or a functional portion thereof, optionally mutated to reduce activity.

11. The chimeric protein as claimed in any one of Claims 1 to 8 wherein said first peptide is ColE7 or Barnase, or is a functional portion thereof, optionally mutated to reduce activity.

12. The chimeric protein as claimed in either one of Claims 10 and 11, wherein the first peptide is ColE7 or a functional portion thereof, optionally mutated to reduce activity, and the second peptide is selected from human IL31, canine IL31 and human IL13.

13. The chimeric protein as claimed in any one of Claims 9 to 12 wherein the first peptide comprises a sequence with at least 80% sequence identity to any one of SEQ IDNos: 14 to 21.

14. The chimeric protein as claimed in any one of Claims 1 to 13 wherein the chimeric peptide has a sequence which comprises the sequence of any one of SEQ ID Nos: 22 to 34 or a functional variant thereof, wherein the functional variant maintains the tertiary structure of the epitope to within a root-mean-square deviation of no more than 1.5 A of the native tertiary structure.

15. The chimeric protein as claimed in Claim 14 wherein the functional variant has a sequence identity of at least 80% to at least one of SEQ ID Nos: 22 to 34.

16. The chimeric protein as claimed in any one of Claims 1 to 15 wherein the second peptide is divided into the first and second segments at a division site selected from: a. An unstructured region (USR) of the second peptide; orb. A glycine residue located at the protein surface of the native tertiary structure of the second peptide and in the loop at the end of an alpha helix or beta sheet.

17. The chimeric protein as claimed in any one of Claims 1 to 16, wherein said chimeric protein further includes a linker:a. between the second segment of the second peptide and the first peptide, or b. between the first segment of the second peptide and the first peptide.

18. The chimeric protein as claimed in Claim 17 wherein the linker is selected from A, S, SD, GGS, GGG, GGGS, GSGGS, GG, GS, GSG, GSGG, and GGGSG.

19. A VLP having a capsid comprising a chimeric protein as claimed in any one of Claims 1 to 18.

20. The VLP as claimed in Claim 19 wherein, the first peptide is a protein that can selfassemble into a VLP.

21. The VLP as claimed in Claim 19 or Claim 20 which includes a binding molecule which binds specifically to the first peptide of the chimeric protein.

22. The VLP as claimed in Claim 21 wherein the viral protein forming the VLP is a mammalian hepatitis viral protein.

23. The VLP as claimed in Claim 22 wherein the viral protein forming the VLP is a human Hepatitis B viral protein.

24. The VLP as claimed in Claim 21 wherein the viral protein forming the VLP is a viral protein forming an icosahedral viral capsid.

25. The VLP as claimed in Claim 24 wherein the viral protein forming the VLP is from Tomato Aspermy Virus (TASV)26. The VLP as claimed in Claim 25 wherein the viral protein forming the VLP comprises or is derived from Im7 and the first peptide of the chimeric protein is an at least functional portion of ColE7 or any other protein which can bind specifically to Im7.

27. The VLP as claimed in any one of Claims 20 to 26 which comprises a sequence of any one of SEQ ID Nos: 53 to 65 or a functional variant thereof.

28. A polynucleotide which encodes the chimeric protein as claimed in any one of Claims 1 to 18.

29. The polynucleotide as claimed in Claim 28 which comprises a nucleotide sequence which expresses a protein with at least 80% sequence identity to any one of SEQ ID Nos: 22 to 34.

30. The polynucleotide as claimed in either one of Claims 28 and 29 which comprises a nucleotide sequence with at least 80% sequence identity to any one of SEQ ID Nos: 35 to 47.

31. The polynucleotide as claimed in Claim 30 which comprises the nucleotide sequence of least one of SEQ ID Nos: 35 to 47.

32. The polynucleotide as claimed in any one of Claims 29 to 31 which also includes a polynucleotide encoding a viral protein able to self-assembly to form a VLP.

33. The polynucleotide as claimed in Claim 32 wherein the viral protein comprises a binding partner for the first peptide of the chimeric protein.

34. The polynucleotide as claimed in Claim 33 which comprises a nucleotide sequence with at least 80% sequence identity to any one of SEQ ID Nos: 66 to 74.

35. A vector comprising a polynucleotide as claimed in any one of Claims 28 to 34.

36. A host cell transformed with a polynucleotide as claimed in any one of Claims 28 to 34 or a vector as claimed in Claim 35.

37. A cell culture comprising at least one host cell as claimed in Claim 36.

38. A process of producing a chimeric protein as claimed in any one of Claims 1 to 18, said method comprising:a. Identifying a target peptide comprising an epitope of interest;b. Identifying a division site within the target peptide, wherein the division site is any point where the peptide can be split without disrupting formation of secondary structure elements, and thereby defining N- and C-terminal segments of the target peptide;c. Transposing the native C and N terminal segments of the target peptide to form a second peptide;d. Forming a chimeric polynucleotide comprising an operably linked sequence encoding both first and second peptides;e. Expressing said chimeric protein from said chimeric polynucleotide.

39. The method as claimed in Claim 38, wherein the division site is selected from an unstructured region and / or a Glycine bend region of the second peptide.

40. The method as claimed in either one of Claims 38 and 39 further including a step of structural modelling of the chimeric protein, to identify any requirement for a linker between the first peptide and the second peptide.

41. The method as claimed in Claim 40, wherein the step of structural modelling shows a Root-Mean-Square Deviation of less than 1.5 Angstrom between the superimposed structures of the epitope in the target peptide and the epitope in the chimeric protein.

42. A process for the production of a virus-like particle (VLP) as claimed in Claim 20, said method comprising:i. Providing a first polynucleotide encoding a chimeric protein according any one of Claims 1 to 19, wherein said first peptide can self-assemble into a VLP and expressing said chimeric protein;ii. Allowing said chimeric protein to self-assemble into a VLP.

43. A process for the production of a virus-like particle (VLP) as claimed in any one of Claims 21 to 27, said method comprising:i. Providing a first polynucleotide encoding a chimeric protein as claimed in any one of Claims 1 to 19 and expressing said chimeric protein, wherein said first peptide of said chimeric protein comprises a binding site; ii. Admixing said chimeric protein with a VLP -forming protein, wherein said VLP -forming protein comprises a binding partner for the first peptide binding site.

44. An immunogenic composition comprising the chimeric protein as claimed in any one of Claims 1 to 18 or a VLP as claimed in any one of Claims 19 to 27.

45. The chimeric protein as claimed in any one of Claims 1 to 18 for use as a medicament.

46. The chimeric protein as claimed in any one of Claims 1 to 18 for use as a medicament for the prevention and / or treatment of inflammatory diseases, cancer, neurodegenerative, metabolic, cardiovascular, renal, gastrointestinal, respiratory, or autoimmune diseases. In particular, the medicament can be for use in the prevention and / or treatment of pruritic diseases such as atopic dermatitis, and also in allergy and inflammatory bowel disease.

47. The VLP as claimed in any one of Claims 19 to 27 for use as a medicament.

48. The VLP as claimed in any one of Claims 20 to 28 for use as a medicament for the prevention and / or treatment of inflammatory diseases, cancer, neurodegenerative, metabolic, cardiovascular, renal, gastrointestinal, respiratory, or autoimmune diseases. In particular, the medicament can be for use in the prevention and / or treatment of pruritic diseases such as atopic dermatitis, and also in allergy and inflammatory bowel disease.

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