Il31-VLP and medical uses thereof

Abstract

The present invention relates to virus-like particles (VLPs) decorated with IL31 polypeptides or functional fragments thereof (IL31-VLPs), and optionally additional functional molecules, via a high affinity and versatile protein attachment system which allows for modular, interchangeable decoration of VLPs. The present invention further relates to processes of producing the IL31-VLPs including production methods taking place within single cells, and uses of the IL31-VLPs in research, diagnosis and as medicaments such as vaccines for use in prevention and / or treatment of diseases.

Description

[0001] IL31-VLP AND MEDICAL USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to virus-like particles (VLPs) decorated with IL31 polypeptides or functional fragments thereof (IL31-VLPs), and optionally additional functional molecules, via a high affinity and versatile protein attachment system which allows for modular, interchangeable decoration of VLPs. The present invention further relates to processes of producing the IL31-VLPs including production methods taking place within single cells, and uses of the IL31-VLPs in research, diagnosis and as medicaments such as vaccines for use in prevention and / or treatment of diseases.

[0004] BACKGROUND TO THE INVENTION

[0005] Virus-like particles (VLPs)

[0006] Virus-like particles (VLPs) are molecules that closely resemble viruses, but contain no viral genetic material. They are formed from viral structural proteins, such as viral capsid proteins that, when individually expressed, self-assemble into a particle. Most VLPs look like hollow ‘nano-footballs’ where the entire surface of the football is made up by many copies of a single self-assembled protein. For production purposes this means that production of one single protein is sufficient to generate a big nano-football type VLP structure.

[0007] This has been exploited in medicine. The most common use of VLPs is as vaccines. The basic mechanism behind this is that mammals have evolved immune sensing mechanisms to recognise highly repetitive patterns seen on viral capsids as intruders. These patterns are still present in VLPs, which contain repetitive, high density displays of viral surface proteins, but the harmful viral genome is removed. This is the form of the VLP used as the vaccine against human papillomavirus (HPV) which causes cervical cancer. There are currently a selection of commercially available HPV vaccines of this type such as Cervarix by GlaxoSmithKline along with Gardasil and Gardasil-9, produced by Merck & Co.

[0008] Further developments of VLPs for use as vaccines involve tethering of other agents to the VLP shell. In this case, the VLP shell serves to present an additional agent as an ‘epitope’ to the immune system. In some cases, the viral capsid proteins forming the VLP shell can be modified to directly incorporate the epitope for display through genetic fusion. However, this approach commonly leads to impaired VLP assembly and large proteins routinely cause VLP instability. Further, this approach cannot be used if the agent is not protein-based. Certain COVID19 vaccines that are under development use this form of VLP, where the spike protein from the coronavirus is directly fused to a viral capsid protein forming a VLP shell from an unrelated virus.

[0009] VLP attachment means

[0010] A further alternative is to assemble the VLP and then use attachment means to secure the agent to the VLP shell. Such VLPs with additional attachment means may be termed ‘compound VLPs’. Compound VLPs may be manufactured by methods such as chemical crosslinking, reactive unnatural amino acids, or the use of binding proteins such as the SpyTag / SpyCatcher system, to covalently attach the desired agent to the viral capsid proteins forming the shell.

[0011] The latter method allows the attachment of other non-protein epitopes to the VLP, but requires a complicated production process and cannot yet be used for any agent. In addition, some desired proteins are simply too large to attach to the VLP shell using current attachment means, while some complex epitopes comprising multimers with numerous components that must be separately linked together require multiple steps of chemical crosslinking to achieve.

[0012] The current binding proteins which are used as attachment means, such as in the SpyTag / SpyCatcher system, have further issues in that the binding between the proteins whilst being strong requires formation of a covalent bond and therefore does not occur instantly but requires time for the reactants to fuse, and can result in VLP-shell aggregation depending on which agent is attached to the shell. In addition, using of SpyTag / SpyCatcher as fusion partners does not simultaneously provide a potent chaperone domain to assist correct folding of the client fused proteins.

[0013] For VLPs used in clinical human or veterinary applications, regulators classify VLPs as “biological” active drug intermediates (ADI’s). “Biologic” drugs are produced in living cells, followed by purification according to a regulator-approved process. Each cell line (regardless whether bacterial / plant / yeast / insect / mammalian) used for the production is minutely characterized so as to guarantee long-term stability of the ADI and stored under highly specified conditions as a so-called “Master Cell Bank” (MCB). If a VLP requires two (or even more) proteins to assembled, for example where proteins are used to attach an epitope to the VLP shell, then currently one MCB is required for each protein component of the drug and both require a separate purification process, each requiring separate characterisation procedures, as both are classed as “critical drug intermediates”. Also, a separate qualitycontrol release of required for each critical drug intermediate, as well as the final ADI, multiplying manufacturing cost. In addition to these complexities, the production of the epitope must be established from scratch for each epitope. Financially, the most efficient type of production cell is bacteria, and most commonly E.coli. However, many proteins do not assume their native shape when produced in E.coli but must be re-folded into their proper form from a denatured state as part of the purification process, which results in huge drop of overall yield and significantly adds to process complexity.

[0014] As a result of all these shortcomings, the production process of compound VLP-type drugs which attempt to attach agents such as epitopes to the viral capsid proteins is complex and expensive. This has limited wide-spread exploration of VLP applications to fields where inexpensive mass production could make them more competitive. Consequently, this has also limited the exploration of VLP-based IL31 applications, such as VLP-based IL31 vaccines.

[0015] IL31 and its use in VLP-based vaccines to treat itching conditions

[0016] IL31 is a key cytokine driving itching, and is implicated in a variety of inflammatory skin conditions such as atopic dermatitis which can be chronic and debilitating. Accordingly, IL31 vaccines and anti-IL31 antibodies have been suggested as therapies to treat itching-related conditions (Bachman et al. 2018). However, an effective IL31 vaccine has not yet been developed.

[0017] VLP-based vaccines against canine IL31 have been attempted for treating atopic dermatitis in dogs, wherein sulfhydryl (SH) groups were introduced into IL31 which were then chemically crosslinked using succinimidyl 6-((beta-maleimidopropionamido)hexanoate (SMPH) to surface lysines on VLPs derived from cucumber mosaic virus containing a universal T-cell epitope (Bachman et al., 2018).

[0018] Equine IL31 again crosslinked using SMPH to a VLP derived from cucumber mosaic virus containing a tetanus toxoid universal T-cell epitope has also been used as a vaccine against insect bite hypersensitivity in horses, which is a common seasonal pruritic allergic dermatitis occurring upon insect bites (Olomski et al., 2019).

[0019] Whilst it is possible to chemically crosslink IL31 to VLPs as described by Bachman et al. (2018) and Olomski et al. (2019), which might overcome any issues of impaired VLP assembly or VLP instability, such crosslinking techniques still suffer from the drawbacks discussed above, namely the necessary use of separate cell lines to produce the individual components that are to be crosslinked (e.g. the VLP shell and the IL31 polypeptide). The current IL31-VLP based structures as noted above have a high burden of setting up of a separate production process for each component part to then be coupled into a VLP, often result in improper folding of the IL31 protein and especially when expressed in bacterial hosts commonly used for protein production, suffer from potential VLP-shell aggregation, and the cumbersome assembly of multimeric proteins through multi-step crosslinking processes.

[0020] Use of bacterial toxin and inhibitor pairs to couple IL31 to VLPs

[0021] It would therefore be desirable to provide compound VLPs with the modular ability to display functional molecules such as IL31 , including non-proteins, proteins that experience difficulty in folding within E.coli, and complex and large proteins such as multimers. It would further be desirable to produce such compound VLPs by a much simpler process requiring the use of only one cell-line, and in which the VLP shell and the agent to be displayed can auto-assemble within the cell line, ideally without VLP-shell aggregation. Some of these features can be provided by the use of bacterial toxin and inhibitor protein binding pairs to attach desired functional molecules such as epitopes to VLPs (as described in WO / 2022 / 112790). In essence, this technology is based on a pair of binding proteins comprising a bacterial toxin and its inhibitor, one of which is attached to a VLP shell e.g. a Hepatitis B viral capsid (HBc) protein, the other of which is attached to a desired agent e.g. I L31 , which agent can thus be attached to the VLP shell by the binding of the bacterial toxin to its inhibitor.

[0022] However, even with this technology, the attachment of a specific desired functional molecule such as IL31 to the binding protein (e.g. a bacterial toxin or its inhibitor) of this modular style VLP introduces the problem of unpredictable fusion protein behaviour.

[0023] It would be desirable to provide an optimised version of a modular style VLP structure which is designed specifically to express I L31 , a critical mediator in many immune diseases.

[0024] One or more aspects of the present invention are aimed at solving one or more of the above- mentioned problems.

[0025] SUMMARY OF THE INVENTION

[0026] In a first aspect of the invention, there is provided a virus-like particle (VLP) comprising:

[0027] One or more viral capsid protein,

[0028] One or more pairs of binding proteins, each pair of binding proteins comprising a first binding protein and a second binding protein, wherein the pair of binding proteins comprises a bacterial toxin and its inhibitor,

[0029] One or more functional molecules, wherein at least one of the functional molecules is an IL31 polypeptide or a functional fragment thereof, wherein each viral capsid protein is attached to a first binding protein, wherein each functional molecule is attached to a second binding protein via a linker, and wherein the first and second binding proteins are capable of binding to each other.

[0030] In one embodiment, the one or more viral capsid proteins are homodimeric proteins.

[0031] In one embodiment, the IL31 polypeptide or a functional fragment thereof is human IL31 or a functional fragment thereof, suitably wherein the IL31 polypeptide or a functional fragment thereof is human IL31 or a functional fragment thereof, suitably wherein the IL31 polypeptide comprises a sequence 70% or greater identity to SEQ ID NO: 11 or 12.

[0032] In one embodiment, each linker comprises at least 8 amino acids, suitably between 8 and 30 amino acids, suitably 8 amino acids, 24 amino acids or 28 amino acids.

[0033] In one embodiment, the linker is structurally disordered, or flexible.

[0034] In one embodiment, the linker comprises at least one region that is rich in glycine and serine residues, optionally with a majority of residues being glycine and serine, further optionally consisting of glycine and serine residues.

[0035] In a further embodiment, the linker comprises more than one such region, suitably the linker comprises a first region and a second region that is rich in glycine and serine residues, optionally with a majority of residues being glycine and serine, further optionally consisting of glycine and serine residues.

[0036] In a further embodiment, the linker further comprises a third region, optionally a cleavage site, further optionally a protease cleavage site, yet further optionally a TEV protease cleavage site having a sequence according to SEQ ID NO: 52.

[0037] In a further embodiment, the linker comprises the following structure from N-to-C terminus: the first region, the third region, and the second region.

[0038] In a further embodiment, the or each region that is rich in glycine and serine residues comprises a sequence selected from: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, suitably selected from SEQ ID NO: 4, 7 or 8. .

[0039] In a further embodiment, the linker comprises a sequence with 70% or greater identity to SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO: 34.

[0040] In one embodiment, the first binding protein is the bacterial toxin inhibitor, and the second binding protein is the bacterial toxin. In one embodiment, the bacterial toxin inhibitor is selected from Im7, Im8, Im9, Im2, and Barstar.

[0041] In one embodiment, the bacterial toxin is a bacterial nuclease, optionally the bacterial nuclease is selected from: ColE7, C0IE8, ColE9, ColE2, and Barnase.

[0042] In one embodiment, the bacterial toxin and its inhibitor are ColE7 and Im7, optionally the amino acid sequence of Im7 comprises the following substitution: F41 Lwith reference to SEQ ID NO: 78, and / or the amino acid sequence of ColE7 comprises one or more of the following substitutions: Arg538Ala, Glu542Ala, and His569Ala with reference to SEQ ID NO: 41 , suitably wherein Im7 comprises a sequence with 70% or greater identity to SEQ ID NO: 36 and / or wherein ColE7 comprises a sequence with 70% or greater identity to SEQ ID NO: 1.

[0043] In one embodiment, the viral capsid protein is a Hepatitis B capsid (HBc) protein, optionally a Hepatitis B capsid (HBc) protein of 70% or greater identity to SEQ ID NO: 42.

[0044] In one embodiment, the first binding protein is inserted into the major immunodominant region of the Hepatitis B capsid protein, suitably which is located between amino acids 75 to 81 of SEQ ID NO: 42, optionally the first binding protein is inserted between amino acid residues 76 and 80 of the major immunodominant region of the Hepatitis B capsid protein according to SEQ ID NO: 42.

[0045] In one embodiment, the Hepatitis B capsid protein comprises an amino acid sequence with the following amino acid deletions: E77 and D78 with reference to SEQ ID NO: 42.

[0046] Suitably wherein the Hepatitis B capsid protein comprises a sequence according to SEQ ID NO: 35.

[0047] In one embodiment, the VLP further comprises a secondary linker, suitably wherein each viral capsid protein is attached to a first binding protein via a secondary linker, suitably wherein the secondary linker comprises a sequence according to SEQ ID NO: 13.

[0048] In one embodiment, each viral capsid protein attached to a first binding protein comprises a sequence having at least 70% identity to SEQ ID NO: 3.

[0049] In one embodiment, the first binding protein comprises a chemical modification, optionally the chemical modification is attached to a further functional molecule. In one embodiment, the chemical modification is an alkane having an amine group, optionally a 1-10 carbon alkane having an amine group, optionally the chemical modification is selected from DEAE or octylamine. In one embodiment, the one or more further functional molecules may be the same or different, optionally each functional molecule is selected from: a protein or non-protein antigen or an epitope thereof, an antigen binding protein, or a fluorescent molecule.

[0050] In one embodiment, the antigen binding protein is selected from an antibody or binding fragment thereof, an antibody mimetic, and an aptamer, optionally the antigen binding protein is an antibody, further optionally an antibody which is capable of binding an antigen of interest.

[0051] In one embodiment, the non-protein antigen is selected from a: sugar, lipid, carbohydrate, or small molecule chemical.

[0052] In one embodiment, the protein antigen is derived from an infectious agent, optionally the protein antigen is derived from a virus, bacterium, fungus, protozoan, archaeon, optionally the protein antigen is derived from a virus, further optionally the virus is selected from: Adeno- associated virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, Hantaan virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus, Human herpesvirus, Human immunodeficiency virus, Human papillomavirus, Human parainfluenza, Human respiratory syncytial virus, Human rhinovirus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Japanese encephalitis virus, Polyomavirus, Kunjin virus, Lassa virus, Measles virus, Molluscum contagiosum virus, Mumps virus, Nipah virus, Poliovirus, Rabies virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sapporo virus, Sindbis virus, Toscana virus, Uukuniemi virus, Varicella-zoster virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, West Nile virus, Yellow fever virus, Zika virus, yet further optionally the protein antigen is derived from a coronavirus, yet further optionally from SARS- CoV-2, yet further optionally the protein antigen is the whole or part of a spike protein derived from SARS-CoV-2, or the whole or part of a nucleocapsid protein derived from SARS-CoV-2.

[0053] In one embodiment, the protein antigen is derived from a non-infectious agent, optionally the protein antigen is derived from an inflammatory molecule, a molecule causing degenerative changes in nervous, cartilage or bone tissue, or a molecule causing worsening of a neoplastic disease, further optionally the protein antigen is an inflammatory molecule selected from a chemokine, cytokine, or protease, yet further optionally the protein antigen is selected from: IL1 , IL2, II3, II4, IL5, II6, II7, IL8, IL9, IL10, IL11 , IL12, IL13, IL17, IL31 , IL33, TNFa, TNF0, IFNa, IFNp, IFNy, G-CSF, GM-CSF, M-CSF, erythropoietin, and TGFp, yet further optionally the protein antigen is the whole or part of IL13, IL17 or IL33. In one embodiment, the fluorescent molecule is selected from: GFP, EBFP, EBFP2, Azurite, GFPuv, T-saphhire, Cerulean, CFP, mCFP, mTurquoise2, CyPet, mKeima-red, tagCFP, AmCyanl , mTFP1, midoriishi cyan, turboGFP, tagGFP, emerald, azami green, ZsGreenl , YFP, tagYFP, EYFP, topaz, venus, mCtrine, Ypet, turboYFP, ZsYellowl , Kusabira Orange, mOrange, allophycocyanin, mkO, RFP, turboRFP, tdTomato, tagRFP, dsRed, mStrawberry, turboFP602, asRed2, J-red, R- phycoerythrin, B-phycoerythrin, mCherry, HcRed, Katusha, P3, peridin chlorophyll, mKate, turboFP635, mPlum, mRaspberry, optionally the fluorescent molecule is GFP.

[0054] In one embodiment, each functional molecule is an IL31 polypeptide or a functional fragment thereof.

[0055] In a second aspect, there is provided a functional fusion protein comprising an IL31 polypeptide or a functional fragment thereof fused via a linker to a binding protein wherein the binding protein is a bacterial toxin.

[0056] Any of the embodiments in relation to the first aspect apply equally in relation to the second aspect.

[0057] In one embodiment, the functional fusion protein further comprises an affinity purification tag.

[0058] In one embodiment, the functional fusion protein comprises a sequence having at least 70% identity to SEQ ID NO: 43, 44, 45, 46, 47, 48, 49 or 50. In one embodiment, the functional fusion protein comprises a sequence having at least 70% identity to SEQ ID NO: 47, 48 or 49.

[0059] In a third aspect, there is provided one or more nucleic acids encoding the functional fusion protein according to the second aspect of the invention. Suitably wherein the nucleic acid comprises a sequence having at least 70% identity to SEQ ID NO: 59-66, suitably wherein the nucleic acid comprises a sequence having at least 70% identity to SEQ ID NO: 63, 64 or 65.

[0060] In a fourth aspect, there is provided one or more vectors comprising the one or more nucleic acids of the third aspect of the invention. Suitably wherein the vector comprises a sequence having at least 70% identity to SEQ ID NO: 27-32, 57 or 58, suitably wherein the vector comprises a sequence having at least 70% identity to SEQ ID NO: 31 , 32 or 57.

[0061] In a fifth aspect, there is provided a host cell comprising the one or more nucleic acids of the third aspect, or the one or more vectors of the fourth aspect of the invention.

[0062] In a sixth aspect, there is provided a host cell comprising one or more vectors, the one or more vectors comprising a first nucleic acid encoding a viral capsid protein attached to a first binding protein, and a second nucleic acid encoding an IL31 polypeptide or a functional fragment thereof attached via a linker to a second binding protein; wherein the first and second binding proteins are capable of binding to each other.

[0063] In one embodiment, the first nucleic acid is selected from a sequence having at least 70% identity to SEQ ID NO: 16, and the second nucleic acid is selected from a sequence having at least 70% identity to SEQ ID NO: 59-66. Suitably wherein the second nucleic acid is selected from a sequence having at least 70% identity to SEQ ID NO: 63, 64, or 65.

[0064] In a seventh aspect, there is provided a process of producing a virus-like particle (VLP) in a single host cell comprising:

[0065] (a) Providing a host cell comprising one or more vectors, wherein the one or more vectors comprise:

[0066] (i) a first nucleic acid encoding a viral capsid protein attached to a first binding protein; and

[0067] (ii) a second nucleic acid encoding an IL31 polypeptide or a functional fragment thereof attached via a linker to a second binding protein;

[0068] (b) Culturing the host cell under conditions suitable to express proteins from the first and second nucleic acids;

[0069] (c) Forming VLPs from the proteins.

[0070] In one embodiment, the method further comprises a step of recovering the virus-like particles (VLPs), and optionally a step of formulating the VLPs.

[0071] In a eighth aspect, there is provided a process of producing a virus-like particle (VLP), comprising:

[0072] (a) Providing a first host cell comprising one or more vectors, wherein the one or more vectors comprise a first nucleic acid encoding a viral capsid protein attached to a first binding protein;

[0073] (b) Providing at least one further host cell comprising one or more vectors, wherein the one or more vectors comprise a second nucleic acid encoding an IL31 polypeptide or a functional fragment thereof attached via a linker to a second binding protein;

[0074] (c) Culturing the host cells under conditions suitable to express proteins from the first and second nucleic acids;

[0075] (d) Recovering the proteins; (e) Mixing the proteins to form virus-like particles.

[0076] In one embodiment, the method further comprises a step of recovering the virus-like particles (VLPs), and optionally a step of formulating the VLPs.

[0077] In a ninth aspect, there is provided an immunogenic composition comprising a VLP according to the first aspect of the invention.

[0078] In a tenth aspect, there is provided a VLP according to the first aspect, or an immunogenic composition according to the ninth aspect of the invention, for use as a medicament, optionally wherein the medicament is a vaccine.

[0079] In a eleventh aspect, there is provided a VLP according to the first aspect, or an immunogenic composition according to the ninth aspect of the invention, for use in the prevention and / or treatment of I L31 -mediated conditions, suitably of immune-mediated conditions. For example dermatological conditions, allergic pathologies, and onco-haematological conditions, optionally wherein the dermatological condition involves itching, further optionally wherein the dermatological condition that involves itching is dermatitis.

[0080] In a twelfth aspect, there is provided a VLP according to the first aspect, or an immunogenic composition according to the ninth aspect of the invention, for use in research, or in the diagnosis of a disease.

[0081] In a thirteenth aspect, there is provided a method of generating IL31 antibodies, the method comprising using a VLP according to the first aspect, or an immunogenic composition according to the ninth aspect of the invention in the immunisation of an animal to generate anti-IL31 antibodies and recovering said antibodies, optionally further formulating the recovered antibodies.

[0082] Advantages of the invention

[0083] The invention offers a number of significant advantages over the prior art.

[0084] The present invention provides a particular structure in which an IL31 polypeptide, or a functional fragment thereof, is linked via a linker to the pair of bacterial toxin-inhibitor binding proteins on the virus-like-particle (VLP). The inventors have found that the use of the linkers allows a significantly increased practical usability of the fusion of the toxin / inhibitor pair previously shown to enable the simplified production of decorated VLPs. The inventors have found that the linker that is used to attach the I L31 polypeptide or a functional fragment thereof to the bacterial toxin or its inhibitor can be adapted and optimised such that the properties of length, flexibility and sequence identity that allow for efficient expression and importantly also proper post-translational folding of said protein. Furthermore, the invention provides the advantages of being able to efficaciously purify a functional fusion protein comprising an IL31 or a functional fragment thereof and a bacterial toxin (e.g. ColE7) or its inhibitor (e.g. Im7) using an affinity tag (e.g. a poly-histidine tag) that is also present on the fusion protein. This is by virtue of said fusion protein being able to effectively bind to the corresponding material (e.g. immobilised metals such as Ni2+or Co2+) that is used for purification of said affinity tag. Advantageously, the linker does not sterically occlude an affinity tag that is present on said fusion protein from binding to its affinity partner.

[0085] Therefore the present invention provides a more efficient way of producing a VLP which displays IL31 or a functional fragment thereof with its correct native folding by using an appropriate linker within the structure, thereby opening up the possibility of commercially viable production of such VLPs for use as vaccines in the treatment of a range of IL31- mediated diseases. Such diseases, as explained above, are wide-ranging and can be highly detrimental to both humans and animals, and thus far do not have a suitable treatment which addresses the underlying causative agent of IL31.

[0086] Features and embodiments of the above aspects are described further under headed sections below. Any feature or embodiment may be combined with any aspect in any workable combination.

[0087] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0088] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as ‘a’, ‘an’ and ‘the’ are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0089] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0090] The present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0091] 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. Suitably any nucleic acid molecule or protein molecule herein may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any given reference sequence identified by SEQ ID herein. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequence” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).

[0092] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881- 90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31 ; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10. The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the "help" section for BLAST™ . For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.

[0093] For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.

[0094] The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated.

[0095] The term “operably linked”, “operably connected” or equivalent expressions as used herein refer to the arrangement of various nucleic acid elements relative to each other such that the elements are functionally connected and are able to interact with each other in the manner intended.

[0096] 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. "T reatment," or “therapy” as used herein thus includes any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0097] 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.

[0098] 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. For the purposes of the present disclosure, 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.

[0099] DETAILED DESCRIPTION OF THE INVENTION

[0100] Linker

[0101] The present invention relates to VLPs which display IL31 as a functional molecule for use in various application such as immunogenic compositions for treatment or prevention of diseases. The VLPs of the invention attach IL31 or a functional fragment thereof by using a combination of a pair of binding proteins, comprising a first binding protein and a second binding protein, and at least one linker to ensure correct folding and display of the polypeptide.

[0102] Suitably the IL31 polypeptide, or a functional fragment thereof or indeed any other functional molecule, is attached to the VLP via a linker. Suitably the IL31 polypeptide, or a functional fragment thereof or indeed any other functional molecule, is attached to the second binding protein via a linker. Suitably this may be referred to as the primary linker. Further linkers may also be used in the VLP structure as is described below.

[0103] Suitably the linker is a peptide. Suitably the linker is a peptide of any number of amino acids, suitably between 8 to 30 amino acids in length. Suitably the linker is at least 8 amino acids. Suitably the linker is at least 22 amino acids in length. Suitably the linker is between 22 to 28 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments the linker is 24 amino acids in length. In some embodiments the linker is 28 amino acids in length.

[0104] Suitably the linker is flexible, and / or structurally disordered. Suitably, a flexible or structurally disordered linker lacks a well-defined three-dimensional structure, allowing it to adopt multiple conformations. This flexibility is typically attributed to the absence of rigid secondary or tertiary structural elements, such as alpha helices or beta sheets. Suitably therefore, a flexible of structural disordered linker lacks alpha helices or beta sheets. In the context of connecting a fusion protein, a flexible linker provides freedom of movement and spatial flexibility between the component domains of a fusion protein, allowing said domains to move freely and independently in a spatially unrestricted manner. This property is advantageous for optimising the functional properties of the fusion protein, as it can accommodate variations in distance and orientation between the linked domains, enhancing the overall flexibility and adaptability of the overall molecular structure of a fusion protein. This may contribute to enabling spatially unrestricted access to parts of the fusion protein, such as spatially unrestricted access to a tag that may also be present on the fusion protein, which may in turn allow for efficient purification of the said fusion protein.

[0105] Suitably the linker is rich in glycine and serine residues. Suitably a majority of the residues in the linker are glycine and serine. Suitably at least 60%, 70%, 80%, 90%, 95% of the residues are glycine or serine. Suitably, the linker consists of serine and glycine residues.

[0106] Suitably the linker comprises at least one region. Suitably the region is rich in glycine and serine residues. Suitably a majority of the residues in the region are glycine and serine residues. Suitably at least 60%, 70%, 80%, 90%, 95% of the residues are glycine or serine. Suitably the region may consist of glycine and serine residues.

[0107] In some embodiments, the linker may consist only of a first region.

[0108] In some embodiments, the linker may further comprise a second region. Suitably the second region is rich in glycine and serine residues. Suitably a majority of the residues in the second region are glycine and serine residues. Suitably at least 60%, 70%, 80%, 90%, 95% of the residues are glycine or serine. Suitably the second region may consist of glycine and serine residues.

[0109] Suitably, if the linker comprises both the first and second regions, the first and second regions may be the same, i.e. may comprise the same amino acid sequence, or may be different i.e. may comprise a different amino acid sequence. In some embodiments the first and second regions are identical. In some embodiments the first and second regions are non-identical.

[0110] Suitably the first region may comprise a sequence according to GGGSSGSG, SEQ ID NO: 4 [GS1], or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 08%, 99% identity thereto. Suitably the first and / or second region, if present, may comprise a sequence according to GGGSSGSG, SEQ ID NO: 4 [GS1], or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 08%, 99% identity thereto.

[0111] Suitably the first and / or second region, if present, may comprise a sequence according to SGGGSG (SEQ ID NO: 6), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 08%, 99% identity thereto.

[0112] Suitably the first and / or second region, if present, may comprise a sequence according to SGGGSGGGS, SEQ ID NO: 7 [GS2], or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 08%, 99% identity thereto.

[0113] Suitably the first and / or second region, if present, may comprise a sequence according to SGGGSGGGSGGGS, SEQ ID NO: 8 [GS2v2], or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 08%, 99% identity thereto.

[0114] Suitably at least one of the first or second regions comprises a sequence according to SEQ ID NO: 4 [GS1], and at least one of the first or second regions comprises a sequence according to SEQ ID NO: 6, SEQ ID NO: 7 [GS2] or SEQ ID NO: 8 [GS2v2], Suitably the first region comprises a sequence according to SEQ ID NO: 4 [GS1], and the second region comprises a sequence according to SEQ ID NO: 7 [GS2] or SEQ ID NO: 8 [GS2v2], or vice versa.

[0115] In one embodiment the linker comprises a first region having a sequence according to SEQ ID NO: 4. In one embodiment the linker consists of a first region having a sequence according to SEQ ID NO: 4.

[0116] In one embodiment the linker comprises a first region having a sequence according to SEQ ID NO: 4 [GS1] and a second region having a sequence according to SEQ ID NO: 6 [GS2],

[0117] In one embodiment the linker comprises a first region having a sequence according to SEQ ID NO: 4 [GS1] and a second region having a sequence according to SEQ ID NO: 7 [GS2],

[0118] In one embodiment the linker comprises a first region having a sequence according to SEQ ID NO: 4 [GS1] and a second region having a sequence according to SEQ ID NO: 8 [GS2v2],

[0119] Suitably the linker may further comprise a third region. Suitably the third region is located between the first and second regions. Suitably the third region may comprise a functional site. Suitably the third region may comprise a cleavage site, or may comprise a site allowing chemical modification of the linker. In one embodiment, the linker comprises a third region which comprises a cleavage site. Suitably a protease cleavage site. Suitably the protease cleavage site may be capable of being cleaved by any protease, such as Tabacco Etch Virus (TEV) protease which is capable of cleaving the sequence ENLYFQ(S / G / A / M / C / H) (SEQ ID NO: 52), HRV3C which is capable of cleaving the sequence LEVLFQGP (SEQ ID NO: 53), Factor Xa which is capable of cleaving the sequence l(E / D)GR (SEQ ID NO: 54), Thrombin which is capable of cleaving the sequence LVPRGS (SEQ ID NO: 55), or Enterokinase capable of cleaving DDDDK (SEQ ID NO: 56). Suitably wherein residues separated by 7’ indicate an alternative amino acid residue at that position.

[0120] In some embodiments, the protease cleavage site is a TEV protease cleavage site. Suitably, in such embodiments, the third region may comprise a sequence according to ENLYFQ(S / G / A / M / C / H) (SEQ ID NO: 52), or ENLYFQG (SEQ ID NO: 5), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 08%, 99% identity thereto.

[0121] Suitably the linker may comprise the following structure: N terminus-[first region]-C terminus.

[0122] Suitably the linker may comprise the following structure: N terminus-[first region]-[second region]-C terminus.

[0123] Suitably the linker may comprise the following structure: N terminus-[first region]— [third region]-C terminus.

[0124] Suitably the linker may comprise the following structure:

[0125] N terminus-[first region]— [third region]-[second region]-C terminus.

[0126] In one embodiment, the linker may comprise a sequence according to GGGSSGSG (SEQ ID NO: 4), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In one embodiment, the linker may consist of said sequence.

[0127] In one embodiment, the linker may comprise a sequence according to GGGSSGSGENLYFQGSGGGSG (SEQ ID NO: 68) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In one embodiment, the linker may consist of said sequence.

[0128] In one embodiment, the linker may comprise a sequence according to GGGSSGSGENLYFQGSGGGSGGGS (SEQ ID NO: 33), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In one embodiment, the linker may consist of said sequence.

[0129] In one embodiment, the linker may comprise a sequence according to GGGSSGSGENLYFQGSGGGSGGGSGGGS (SEQ ID NO: 34), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In one embodiment, the linker may consist of said sequence. In one embodiment, the linker may comprise a sequence according to GGGSSGSG (SEQ ID NO: 4), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In one embodiment, the linker may consist of said sequence.

[0130] Suitably the first region is directly attached to the second binding protein, suitably the first region is contiguous with the second binding protein. Suitably the N terminus of the first region is directly attached to the C terminus of the second binding protein, suitably the N terminus of the first region is contiguous with the C terminus of the second binding protein.

[0131] Suitably the first region is directly attached to a functional molecule, suitably the first region is contiguous with the functional molecule, suitably with an IL31 polypeptide or a functional fragment thereof. Suitably the C terminus of the first region is directly attached to the N terminus of the functional molecule, suitably an IL31 polypeptide or a functional fragment thereof. Suitably the C terminus of the first region is contiguous with the N terminus of the functional molecule, suitably an IL31 polypeptide or functional fragment thereof.

[0132] Suitably the first region is directly attached to a second region, suitably the first region is contiguous with the second region. Suitably the C terminus of the first region is directly attached to the N terminus of the second region, suitably the C terminus of the first region is contiguous with the N terminus of the second region.

[0133] Suitably the first region is directly attached to a third region, suitably the first region is contiguous with the third region. Suitably the C terminus of the first region is directly attached to the N terminus of the third region, suitably the C terminus of the first region is contiguous with the N terminus of the third region.

[0134] Suitably the first region is directly attached to both the second binding protein and the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the first region is contiguous with both the second binding protein and the functional molecule, which is suitably an IL31 polypeptide of functional fragment thereof. Suitably the N terminus of the first region is directly attached to the C terminus of the second binding protein, and the C terminus of the first region is directly attached to the N terminus of the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the N terminus of the first region is contiguous with the C terminus of the second binding protein, and the C terminus of the first region is contiguous with the N terminus of the functional molecule which is suitably an IL31 polypeptide or functional fragment thereof.

[0135] Suitably the first region is directly attached to both the second binding protein and the second region. Suitably the first region is contiguous with both the second binding protein and the second region. Suitably the N terminus of the first region is directly attached to the C terminus of the second binding protein, and the C terminus of the first region is directly attached to the N terminus of the second region. Suitably the N terminus of the first region is contiguous with the C terminus of the second binding protein, and the C terminus of the first region is contiguous with the N terminus of the second region.

[0136] Suitably the first region is directly attached to both the second binding protein and the third region. Suitably the first region is contiguous with both the second binding protein and the third region. Suitably the N terminus of the first region is directly attached to the C terminus of the second binding protein, and the C terminus of the first region is directly attached to the N terminus of the third region. Suitably the N terminus of the first region is contiguous with the C terminus of the second binding protein, and the C terminus of the first region is contiguous with the N terminus of the third region.

[0137] Suitably the second region is directly attached to the first region, suitably the second region is contiguous with the first region. Suitably the N terminus of the second region is directly attached to the C terminus of the first region, suitably the N terminus of the second region is contiguous with the C terminus of the first region.

[0138] Suitably the second region is directly attached to the third region, suitably the first region is contiguous with the third region. Suitably the N terminus of the first region is directly attached to the C terminus of the third region, suitably the N terminus of the first region is contiguous with the C terminus of the third region.

[0139] Suitably the second region is directly attached to the functional molecule, suitably to an IL31 polypeptide or a functional fragment thereof. Suitably the second region is contiguous with the functional molecule, suitably with the IL31 polypeptide or functional fragment thereof. Suitably the C terminus of the second region is directly attached to the N terminus of the functional molecule. Suitably the N terminus of the IL31 polypeptide or functional fragment thereof.

[0140] Suitably the second region is directly attached to both the first region and the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the second region is contiguous with both the first region and the functional molecule, which is suitably an IL31 polypeptide or functional fragment thereof. Suitably the N terminus of the second region is directly attached to the C terminus of the first region, and the C terminus of the second region is directly attached to the N terminus of the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the N terminus of the second region is contiguous with the C terminus of the first region, and the C terminus of the second region is contiguous with the N terminus of the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the second region is directly attached to both the third region and the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the second region is contiguous with both the third region and the functional molecule, which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the N terminus of the second region is directly attached to the C terminus of the third region, and the C terminus of the second region is directly attached to the N terminus of the functional molecule which is suitably an IL31 polypeptide or a functional fragment thereof. Suitably the N terminus of the second region is contiguous with the C terminus of the third region, and the C terminus of the second region is contiguous with the N terminus of the functional molecule which is suitably an IL31 polypeptide or functional fragment thereof.

[0141] Suitably the third region is directly attached to the first region, suitably the third region is contiguous with the first region. Suitably the N terminus of the third region is directly attached to the C terminus of the first region, suitably the N terminus of the third region is contiguous with the C terminus of the first region.

[0142] Suitably the third region is directly attached to the second region, suitably the third region is contiguous with the second region. Suitably the C terminus of the third region is directly attached to the N terminus of the second region, suitably the C terminus of the third region is contiguous with the N terminus of the second region.

[0143] Suitably the third region is directly attached to a functional molecule, suitably the third region is contiguous with the functional molecule, suitably with an IL31 polypeptide or a functional fragment thereof. Suitably the C terminus of the third region is directly attached to the N terminus of the functional molecule, suitably an IL31 polypeptide or a functional fragment thereof. Suitably the C terminus of the third region is contiguous with the N terminus of the functional molecule, suitably an IL31 polypeptide or a functional fragment thereof.

[0144] Suitably the third region is directly attached to both the first region and the functional molecule, suitably with an IL31 polypeptide or a functional fragment thereof. Suitably the third region is contiguous with both the first region and with the functional molecule, suitably with an IL31 polypeptide or a functional fragment thereof. Suitably the N terminus of the third region is directly attached to the C terminus of the first region, and the C terminus of the third region is directly attached to the N terminus of the functional molecule, suitably with an IL31 polypeptide or a functional fragment thereof region. Suitably the N terminus of the third region is contiguous with the C terminus of the first region, and the C terminus of the third region is contiguous with the N terminus of the functional molecule, suitably with an IL31 polypeptide or a functional fragment thereof. Suitably the third region is directly attached to both the first region and the second region, suitably the third region is contiguous both the first region and the second region. Suitably the N terminus of the third region is directly attached to the C terminus of the first region, and the C terminus of the third region is directly attached to the N terminus of the second region. Suitably the N terminus of the third region is contiguous with the C terminus of the first region, and the C terminus of the third region is contiguous with the N terminus of the second region.

[0145] Suitably the linker may be fused directly or indirectly to the second binding protein and directly or indirectly to the functional molecule, preferably to an IL31 polypeptide or a functional fragment thereof. Suitably therefore each functional molecule, preferably the or each IL31 polypeptide, or a functional fragment thereof, may be fused directly or indirectly to the second binding protein via the linker.

[0146] Pair of Binding Proteins

[0147] The present invention relates to VLPs which make use of a pair of binding proteins to form a bridge which can attach a functional molecule of interest, preferably IL31 or a functional fragment thereof, to the viral capsid proteins forming the VLP shell.

[0148] The pair of binding proteins may be covalently bound or non-covalently bound.

[0149] Suitably the pair of binding proteins are non-covalently bound. Suitably the pair of binding proteins are bound quasi-covalently. Suitably the pair of binding proteins are bound by any non-covalent type of bonding such as: electrostatic interactions, hydrogen bonds, van der Waals interactions or hydrophobic interactions. However, in some embodiments, the pair of binding proteins are not bound by hydrophobic bonding.

[0150] Alternatively, the pair of binding proteins may be covalently bound. Suitably the pair of binding proteins may be bound by any covalent type of bonding.

[0151] Suitably the pair of binding proteins comprises one net positively charged protein and one net negatively charged protein. Suitably the first binding protein comprises a net negative charge. Suitably the second binding protein comprises a net positive charge.

[0152] Advantageously, the first binding protein having a net negative charge increases stability of the VLP and reduced aggregation or clumping.

[0153] In one embodiment, therefore, the pair of binding proteins are bound non-covalently by electrostatic interactions.

[0154] Suitably, in any case, the pair of binding proteins are bound with high affinity. Suitably the pair of binding proteins are bound with a Kd in the femtomolar to picomolar range. Suitably with a Kd of between: 10fM to 10pM, 10fM to 1 pM, 10fM to 0.1 pM, 10fM to 0.01 pM, 1fM to 1 pM, 1 fM to 0.1 pM, 1fM to 0.01pM.

[0155] Advantageously, high affinity binding between the proteins means that the VLP is more stable.

[0156] Suitably the pair of binding proteins have low homology to proteins of the subjects which may be treated with the VLP. Suitably the pair of binding proteins have low homology to human proteins. Suitably the pair of binding proteins have low homology with the tertiary structure of any human proteins.

[0157] Advantageously, low homology with human proteins means that the binding proteins themselves are is less likely to stimulate an off-target immune reaction.

[0158] Suitably the pair of binding proteins do not contain any disulphide bonds.

[0159] Suitably the pair of binding proteins are not glycosylated.

[0160] Suitably each of the proteins in the pair of binding proteins is relatively small in size. Suitably each of the proteins in the pair of binding proteins comprises a relatively short sequence length. Suitably each of the proteins in the pair of binding proteins comprises a length of between 84 - 134 amino acids. Suitably each of the proteins in the pair of binding proteins comprises a length of less than 135 amino acids.

[0161] Advantageously, the lack of disulphide bonds, lack of glycosylation, and small size means that the binding proteins are easier to produce in bacterial cells such as E.coli.

[0162] Suitably the pair of binding proteins comprises a bacterial toxin and its corresponding inhibitor or antitoxin. Suitably the first binding protein of the VLP is a bacterial toxin inhibitor. Suitably the second binding protein of the VLP is a bacterial toxin.

[0163] Suitable bacterial toxin and inhibitor pairs are: a colicin and colicin immunity protein. Suitably ColE7 and Im7, C0IE8 and Im8, ColE9 and Im9, ColE2 and Im2, or Barnase and Barstar. Suitably the bacterial toxin and inhibitor pair comprises a bacterial nuclease and its inhibitor. Suitably the first binding protein is the inhibitor and the second binding protein is the bacterial nuclease. Suitable bacterial nuclease and inhibitor pairs are: ColE7 / lm7 and Barnase / Barstar.

[0164] In one embodiment, the pair of binding proteins is ColE7 and Im7, wherein the first binding protein is Im7 and the second binding protein is ColE7.

[0165] In one embodiment, the pair of binding proteins is Barnase and Barstar, wherein the first binding protein is Barstar and the second binding protein is Barnase. Suitably the first or second binding protein may be the wild-type proteins, or they may be modified. Suitably the first or second binding proteins may be modified to improve their function as a binding protein in the context of the VLP of the invention. Suitable modifications may include: insertions, deletions, substituents, truncations, reversals, repeats, or the like in the amino acid sequence encoding the protein.

[0166] Suitably, any property of the toxin (second binding protein) detrimental to either the host cell and I or the recipient organism intended for VLP administration is neutralized by targeted modifications.

[0167] Suitably the first or second binding proteins may comprise one or more amino acid substitutions. Suitably the amino acid substitutions may increase the binding affinity between the first and second binding proteins. Suitably the amino acid substitutions may remove undesirable disulphide bonds from the first and / or second binding proteins.

[0168] Suitably the first binding protein may comprise one or more amino acid substitutions.

[0169] In an embodiment where the first binding protein is Barstar, suitably the first binding protein may comprise wild type Barstar, suitably comprising the sequence: MNKAVINGEQIRSMSDLHQTLKKELALPEYYGENLDALWDCLTGWVEYPLVLEWRQFEQS KQLTENGAESVLQVFREAKAEGCDITIILS (SEQ ID NO: 39) or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity thereto, or a fragment thereof. Suitably the amino acid sequence of Barstar comprises one or more of the following substitutions: C40A, C82A, and I87E with reference to SEQ ID NO: 39. Suitably the amino acid sequence of Barstar may comprise all of the following substitutions: C40A, C82A, and I87E with reference to SEQ ID NO: 39. Suitably therefore the amino acid sequence of Barstar comprises: at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with KKAVINGEQIRSISDLHQTLKKELALPEYYGENLDALWDALTGWVEYPLVLEWRQFEQSKQ LTENGAESVLQVFREAKAEGADITIELS (SEQ ID NO: 37), or a fragment thereof.

[0170] In an embodiment where the first binding protein is Im7, suitably the first binding protein may comprise wild type Im7, suitably comprising the sequence: MELKNSISDYTEAEFVQLLKE IEKENVAATDDVLDVLLEHFVKITEHPDGTDLIYYPSDNRDDSPEGIVKEIKEWRAANGKPGF KQG (SEQ ID NO: 78), or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity thereto, or a fragment thereof. Suitably the amino acid sequence of Im7 comprises the following substitution: F41 L with reference to SEQ ID NO: 78. Suitably therefore the amino acid sequence of Im7 comprises: at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with ELKNSISDYTEAEFVQLLKEIEKENVAATDDVLDVLLE HFVKITEHPDGTDLIYYPSDNRDDSPEGIVKEIKEWRAANGKPGFKQ (SEQ ID NO: 36), or a fragment thereof.

[0171] Suitably the second binding protein may comprise one or more amino acid substitutions. Suitably the amino acid substitutions in the amino acid sequence of the second binding protein may increase the negative charge of the second binding protein.

[0172] In an embodiment where the second binding protein is Barnase, suitably the second binding protein may comprise the wild-type Barnase, suitably comprising the sequence: MLFSTAAKT DTSSHKAHTEAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSI GGDIFSNREGKLPAKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYKTFTKMR (SEQ ID NO: 40), or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity thereto, or a fragment thereof. Suitably the amino acid sequence of Barnase comprises the following substitution: E73Wwith reference to SEQ ID NO: 40. Suitably the amino acid sequence of Barnase comprises: at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVA PGKSIGGDIFSNREGKLPGKSGRTWRWADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFT KIR (SEQ ID NO: 38), or a fragment thereof.

[0173] In an embodiment where the second binding protein is ColE7, suitably the second binding protein may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with the wild-type ColE7, suitably comprising the sequence: MSGGDGRGHNSGAHNTGGNINGGPTGLGGNGGASDGSGWSSENNPWG GGSGSGVHWGGGSGHGNGGGNSNSGGGSNSSVAAPMAFGFPALAAPGAGTLGISVSGE ALSAAIADIFAALKGPFKFSAWGIALYGILPSEIAKDDPNMMSKIVTSLPAETVTNVQVSTLPL DQATVSVTKRVTDWKDTRQHIAVVAGVPMSVPVVNAKPTRTPGVFHASFPGVPSLTVSTV KGLPVSTTLPRGITEDKGRTAVPAGFTFGGGSHEAVIRFPKESGQKPVYVSVTDVLTPAQV KQRQDEEKRLQQEWNNAHPVEVAERNYEQARAELNQANKDVARNQERQAKAVQVYNSR KSELDAANKTLADAKAEIKQFERFAREPMAAGHRMWQMAGLKAQRAQTDVNNKKAAFDA AAKEKSDADVALSSALERRKQKENKEKDAKAKLDKESKRNKPGKATGKGKPVNNKWLNNA GKDLGSPVPDRIANKLRDKEFKSFDDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKAPK TRTQDVSGKRTSFELHHEKPISQNGGVYDMDNISVVTPKRHIDIHRGK (SEQ ID NO: 41), or a fragment thereof. Suitably the amino acid sequence of ColE7 comprises one or more of the following substitutions: Arg538Ala, Glu542Ala, and His569Ala with reference to SEQ ID NO: 41. Suitably the amino acid sequence of ColE7 may comprise all of the following substitutions: Arg538Ala, Glu542Ala, and His569Ala with reference to SEQ ID NO: 41. Suitably therefore the amino acid sequence of ColE7 comprises: at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with: ESKRNKPGKA TGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRKKFWEEVSKDPELSKQF SRNNNDRMKVGKAPKTRTQDVSGKATSFALHHEKPISQNGGVYDMDNISVVTPKRAIDIHR GKS (SEQ ID NO: 1), or a fragment thereof.

[0174] Suitably the first or second binding proteins may be truncated, suitably at either the N-terminus or the C-terminus or both the N- and C-termini.

[0175] Suitably the second binding protein is truncated, suitably at the N-terminus.

[0176] In an embodiment where the second binding protein is ColE7, suitably the whole or a part of the ColE7 protein may be used as the second binding protein. Suitably only a part of the ColE7 protein is used as the second binding protein. Suitably the ColE7 protein is truncated, suitably so that it only comprises the catalytic domain of ColE7. Suitably the second binding protein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with the catalytic domain of ColE7, suitably comprising the sequence: ESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFR KKFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKATSFALHHEKPISQNGGVY DMDNISWTPKRAIDIHRGKS (SEQ ID NO: 1).

[0177] In an embodiment where the second binding protein is Barnase, suitably the whole or a part of the Barnase protein may be used as the second binding protein. Suitably only a part of the Barnase protein is used as the second binding protein. Suitably the Barnase protein is truncated, suitably so that it only comprises the catalytic domain of Barnase. Suitably the second binding protein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with the catalytic domain of Barnase, suitably comprising the sequence: AQVINTFDGVADYLQTYHKLPDNYITKSEAQAL GWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWRWADINYTSGFRNSDRILYSSD WLIYKTTDHYQTFTKIR (SEQ ID NO: 38).

[0178] Suitably the viral capsid proteins exist as homodimers. Suitably therefore the VLP comprises one or more viral capsid protein homodimers.

[0179] Suitably, the viral capsid protein may be a hepatitis B viral capsid protein (HBc). Suitably therefore the VLP comprises one or more hepatitis B viral capsid protein homodimers.

[0180] Suitably the first binding protein is attached to a viral capsid protein. Suitably each viral capsid protein is attached to a first binding protein. Suitably the first binding protein is fused into the immunodominant region of the viral capsid protein, as explained elsewhere herein for HBc. Suitably therefore the viral capsid protein may be regarded as a capsid fusion protein. Suitably therefore the VLP comprises one or more capsid fusion proteins. Suitably therefore the VLP comprise one or more capsid fusion protein homodimers.

[0181] Suitably, each capsid fusion protein may comprise a sequence according to SEQ ID NO: 3. Suitably each capsid fusion protein may comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 3 or a fragment thereof. Suitably the capsid fusion protein may consist of a sequence according to SEQ ID NO: 3.

[0182] In one embodiment, each capsid fusion protein comprises a hepatitis B viral capsid protein fused to binding protein Im7. In such an embodiment, each capsid fusion protein may comprise a sequence according to SEQ ID NO: 3. Suitably each capsid fusion protein may comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ I D NO: 3 or a fragment thereof. Suitably the capsid fusion protein may consist of a sequence according to SEQ ID NO: 3.

[0183] In another embodiment, the capsid fusion protein comprises a hepatitis B viral capsid protein fused to binding protein Barstar.

[0184] Suitably, the binding protein may comprise a chemical modification, optionally wherein the chemical modification may be attached to a further functional molecule.

[0185] Fusion proteins

[0186] In accordance with an aspect of the present invention, there is provided a functional fusion protein comprising an IL31 polypeptide or a functional fragment thereof, fused via a linker to a binding protein wherein the binding protein is a bacterial toxin.

[0187] Suitably, the functional fusion protein may comprise a sequence according to any of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50, preferably SEQ ID NO: 47, 48 or 49. Suitably the functional fusion protein may comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50, preferably SEQ ID NO: 47, 48 or 49, or a fragment thereof. Suitably the functional fusion protein may consist of a sequence according to SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50, preferably SEQ ID NO: 47, 48 or 49.

[0188] In one embodiment, the functional fusion protein may comprise IL31 polypeptide or a functional fragment thereof fused to binding protein ColE7. In such an embodiment, the functional fusion protein may comprise a sequence according to SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50, preferably SEQ ID NO: 47, 48 or 49. Suitably the functional fusion protein may comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50, preferably SEQ ID NO: 47, 48 or 49 or a fragment thereof. Suitably the functional fusion protein may consist of a sequence according to SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50, preferably SEQ ID NO: 47, 48 or 49.

[0189] In another embodiment, the functional fusion protein may comprise IL31 polypeptide or a functional fragment thereof fused to binding protein Barnase or Barstar.

[0190] Suitably the functional fusion protein further comprises an affinity purification tag, suitably located at the N terminal end of the protein. Suitably the affinity purification tag may be a polyhistidine tag, suitably a hexa-histidine tag, or a tag comprising the sequence: HAHEHRHDHEHGGGS (SEQ ID NO: 2) or any other common tag used for binding proteins or for protein purification.

[0191] Suitably, each of the fusion proteins comprises one or more linkers. Suitably the linkers are located between the protein coding sequences in the VLP. Suitably a linker is located between the functional molecule, preferably IL31 or a functional fragment thereof, and the second binding protein as is described above. Suitably therefore the functional fusion protein comprises a linker. This linker may be referred to as a primary linker, as described hereinabove.

[0192] In particular, suitably the functional fusion protein comprises a linker selected from: GGGSSGSG (SEQ ID NO: 4) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; GGGSSGSGENLYFQGSGGGSG (SEQ ID NO: 68) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; GGGSSGSGENLYFQGSGGGSGGGS (SEQ ID NO: 33) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and GGGSSGSGENLYFQGSGGGSGGGSGGGS (SEQ ID NO: 34) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In one embodiment, the functional fusion protein comprises a linker having a sequence consisting of GGGSSGSG (SEQ ID NO: 4). In one embodiment, the functional fusion protein comprises a linker having a sequence consisting of GGGSSGSGENLYFQGSGGGSGGGS (SEQ ID NO: 33). In one embodiment, the functional fusion protein comprises a linker having a sequence consisting of GGGSSGSGENLYFQGSGGGSGGGSGGGS (SEQ ID NO: 34).

[0193] Suitably a secondary linker may also be located at the N and C terminus of the first binding protein, suitably to link it to the viral capsid protein. Suitably therefore each viral capsid protein is attached to a first binding protein via a secondary linker. Suitably therefore the secondary linker may be located between the viral capsid protein and the Im7 first binding protein, at the N and C terminus of the Im7 binding protein. Suitably therefore the secondary linker may be located between the Hepatitis B viral capsid protein and the Im7 first binding protein, at the N and C terminus of the Im7 binding protein, suitably as is provided in SEQ ID NO: 3, for example. Suitably the secondary linker is between 5 to 50 amino acids in length. Suitably each secondary linker is 5, 10, 15, 20, 21 , 25, 30, 35, 40 amino acids in length. Suitably each secondary linker is 9, 10 or 11 amino acids in length. Suitably each secondary linker comprises the sequence: GGGGSGGGGS (SEQ ID NO: 13).

[0194] Chemical modifications

[0195] Suitably the first binding protein may comprise additional modifications. Suitably the first binding protein may comprise chemical modification. Suitably Im7 may comprise chemical modification.

[0196] Suitably the chemical modification is capable of binding to a functional molecule. Suitably therefore the first binding protein may bind to additional functional molecules, in addition the IL31 attached to the second binding protein. Suitably the chemical modification is capable of covalently binding to a functional molecule. In one example, the functional molecule bound to the chemical modification may be a fluorescent molecule. Other suitable functional molecules are described elsewhere herein.

[0197] Suitably the chemical is attached to the first binding protein by non-covalent binding. Suitably the chemical is attached to the first binding protein by electrostatic and / or hydrophobic bonding.

[0198] Suitable chemical modifications include alkanes having an amine group. Suitably the alkane may have any chain length. Suitably the alkane is a lower alkane. Suitably the alkane may have a chain length of between 1 and 10 carbons. Suitably the alkane may have a chain length of between 4 and 8 carbons. Suitably the alkane may be branched.

[0199] Suitably, the length of the carbon chain and the length of branched substitutions on the amine group are chosen such as to allow either irreversible attachment to the protein or reversible attachment, dependent on the desired application. In one embodiment, the chemical is attached irreversibly to the first binding protein. Suitably, in such an embodiment, conferring irreversible binding, the alkane has eight carbon atoms and a terminal nitrogen (octylamine). In another embodiment, the chemical is attached reversibly to the first binding protein. Suitably, in such an embodiment, allowing reversible binding the alkane has 4 carbon atoms in a branched structure (diethylethanolamine). Suitably the first binding protein may be chemically modified at one or more sites, suitably at one or more amino acids. Suitably the first binding protein is chemically modified at one amino acid.

[0200] In one embodiment, the first binding protein is chemically modified with DEAE.

[0201] In one embodiment, the first binding protein is chemically modified with octylamine.

[0202] Suitably, in such embodiments, the first binding protein may be Im7.

[0203] Suitably, modification with DEAE allows the first binding protein to be purified. Suitably purification by chromatography. Suitably by ion-exchange chromatography.

[0204] Suitably modification with octylamine allows the first binding protein to directly bind to a functional molecule.

[0205] In one embodiment, the chemical modification of the binding protein occurs within the host cell. Suitably by post-translational modification. In another embodiment, the chemical modification of the binding protein occurs outside of the host cell. Suitably by means of a chemical reaction. Suitably by means of a non-enzymatically catalyzed non-covalent attachment.

[0206] Viral Capsid Protein

[0207] The present invention relates to VLPs which comprise one or more viral capsid protein homodimers, the viral capsid protein homodimers self-assemble into the VLP shell, to which IL31 polypeptides or functional fragments thereof, and other functional molecules, can then be attached using the protein binding pair and / or chemical modification as discussed above.

[0208] Suitably, the viral capsid proteins may be selected from any suitable viral capsid proteins, for example: Hepatitis B viral capsid protein, Hepatitis C capsid protein, HPV capsid protein, AAV capsid protein, HIV capsid protein, influenza capsid protein, Newcastle diseases virus capsid protein, Nipah virus capsid protein, Woodchuck hepatitis B viral capsid protein.

[0209] In one embodiment, the viral capsid protein exists in the VLP as a homodimeric viral capsid protein. Suitably comprised of two viral capsid protein monomers. Suitably wherein each of the viral capsid proteins is identical.

[0210] In one embodiment of any of the aspects, the or each viral capsid protein is a Hepatitis B viral capsid protein (HBc). Suitably therefore the VLP comprises one or more Hepatitis B viral capsid protein homodimers. Suitably therefore the VLP comprises one or more identical Hepatitis B viral capsid protein homodimers, suitably comprised of two Hepatitis B viral capsid protein monomers. Suitably each viral capsid protein is attached to a first binding protein. Suitably therefore each viral capsid protein displays a first binding protein.

[0211] Suitably each viral capsid protein is modified to display a first binding protein. Suitably each viral capsid protein is fused to a first binding protein. Suitably each viral capsid protein is modified to display a first binding protein by fusing the first binding protein to the viral capsid protein. Suitably each viral capsid protein is modified to display a first binding protein by inserting the first binding protein into the viral capsid protein. Suitably the first binding protein is inserted into the major immunodominant region of the viral capsid protein. Suitably the first binding protein is fused to the major immunodominant region of the viral capsid protein. Suitably wherein the major immunodominant region is located between (and including) amino acids 75 to 81 of the viral capsid protein, the first binding protein is inserted between amino acid residues 76 and 80 of the major immunodominant region of the viral capsid protein. Suitably the first binding protein is inserted between amino acid residues 76 and 79 of the major immunodominant region of the viral capsid protein. Suitably the first binding protein is inserted between amino acid residues 77 and 79 of the major immunodominant region of the viral capsid protein. Suitably the first binding protein is inserted between amino acid residues 77 and 78 of the major immunodominant region of the viral capsid protein.

[0212] Suitably each viral capsid protein may comprise further modifications. Suitable modifications may include: insertions, deletions, substituents, truncations, reversals, repeats, or the like in the amino acid sequence encoding the protein. Suitably the viral capsid protein may comprise further modifications in the major immunodominant region. Suitably such modifications aid the insertion of the first binding protein into the viral capsid protein. Suitably the viral capsid protein may comprise amino acid deletions. Suitably the viral capsid protein may comprise amino acid deletions in the major immunodominant region. Suitably the viral capsid protein may comprise amino acid deletions in the major immunodominant region which remove negatively charged amino acids.

[0213] In one embodiment, each viral capsid protein of the homodimer, is a hepatitis B capsid protein, suitably a wild type hepatitis B capsid protein comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with the amino acid sequence: MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEH CSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCL TFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQS PRRRRSQSRESQC (SEQ ID NO: 42), or a fragment thereof.

[0214] In one embodiment, each viral capsid protein of the homodimer, is a hepatitis B capsid protein and comprises the following amino acid deletions: E77 and D78 with reference to SEQ ID NO: 42. Suitably the amino acid sequence of each hepatitis B capsid protein comprises: at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREAL ESPEHCSPHHTALRQAILCWGELMNLATWVGSNL[X]PASRELWSYVNVNMGLKIRQLLW FHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV (SEQ ID NO: 35) wherein [X] indicates the position of an insertion, suitably of an amino acid insertion, suitably of an amino acid sequence encoding the first binding protein.

[0215] In one embodiment, each viral capsid protein, of the homodimer, is a hepatitis B capsid protein, comprising a first binding protein inserted within the major immunodominant region thereof, wherein the major immunodominant region is located between amino acids 75 to 81 of SEQ ID NO: 42, suitably the first binding protein is inserted between residues 76 and 80 with reference to SEQ ID NO: 42, and further comprising the following amino acid deletions: E77 and D78 with reference to SEQ I D NO: 42. Suitably the first binding protein is inserted between residues 76 and 79 with reference to SEQ ID NO: 42 and further comprising the following amino acid deletions: E77 and D78 with reference to SEQ ID NO: 42

[0216] Optionally, each viral capsid protein of the homodimer, may comprise one or more amino acid substitutions. Optionally each hepatitis B capsid protein may comprise a substitution at position F97 of SEQ ID NO: 42. Suitably wherein the substitution is F97L. Suitably this substitution was found to accelerate protein folding.

[0217] Optionally, each viral capsid protein of the homodimer, may comprise a truncation, suitably at the N or C terminus. Optionally the hepatitis B capsid protein may comprise a truncation at the C terminus. Suitably the truncation removes the C terminal end of the polypeptide up to residue V149 of SEQ ID NO: 42, as shown according to SEQ ID NO: 35. In one embodiment, the hepatitis B capsid protein comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 35, or a fragment thereof.

[0218] Optionally each viral capsid protein, of the homodimer, may comprise a C terminal addition, suitably an addition of one or more amino acids at the C terminal end of the polypeptide. Suitably, said addition may comprise a plurality of histidine residues. Suitably six histidine residues are added at the C-terminal end.

[0219] In one embodiment, each viral capsid protein of the homodimer attached to a first binding protein comprising a sequence according to SEQ ID NO: 3, or having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, identity thereto, or a functional fragment thereof. In one embodiment, each viral capsid protein of the homodimer attached to a first binding protein consists of SEQ ID NO: 3.

[0220] IL31 Polypeptide

[0221] The present invention relates to VLPs which are able to display an IL31 polypeptide or a functional fragment thereof on their surface by virtue of the protein binding pair.

[0222] Suitably each pair of binding proteins is attached to at least one IL31 polypeptide or a functional fragment thereof. Suitably each pair of binding proteins may be attached to more than one IL31 polypeptide or a functional fragment thereof. Suitably in each case, the IL31 polypeptide or a functional fragment thereof is attached to the pair of binding proteins via a linker as described hereinabove.

[0223] Suitably each pair of binding proteins is attached to one IL31 polypeptide or a functional fragment thereof. Suitably, in such an embodiment, the IL31 polypeptide or a functional fragment thereof is attached to the second binding protein in accordance with the first aspect. Suitably the I L31 polypeptide or a functional fragment thereof is attached to the second binding protein via the linker, suitably the primary linker, as described hereinabove. Suitably therefore each viral capsid protein homodimer comprises two IL31 polypeptides or a functional fragment thereof attached thereto, suitably attached to each second binding protein via a linker, as described hereinabove.

[0224] Suitably the IL31 polypeptide or a functional fragment thereof may be a wild type IL31 polypeptide or a functional fragment thereof derived from any species. Suitably it is a wild type IL31 polypeptide or a functional fragment thereof derived from a mammal, suitably the IL31 polypeptide or a functional fragment thereof is a murine or human IL31 polypeptide. In one embodiment the I L31 polypeptide or a functional fragment thereof is a human IL31 polypeptide or a functional fragment thereof. Optionally the IL31 polypeptide or a functional fragment thereof may be modified, suitably it may comprise one or more amino acid modifications which may be insertions, deletions, substitutions, truncations, rearrangements and the like.

[0225] Suitably the IL31 polypeptide comprises an amino acid sequence according to SEQ ID NO: 9 or 10 or 11 or 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, identity thereto, or a functional fragment thereof.

[0226] In one embodiment, the IL31 polypeptide may consist of an amino acid sequence according to SEQ ID NO: 9 or 10 or 11 or 12.

[0227] Suitably, reference throughout this document to a “fragment thereof” of IL31 should be taken as a reference to a functional fragment of IL31 . Suitably a ‘functional fragment’ may mean a section, domain, part or sub-section of the complete amino acid sequence of the polypeptide which is capable of performing the same or similar biological functions as the wild type polypeptide. Suitably a functional fragment of IL31 therefore is capable of eliciting an immune response in a subject, suitably therefore the functional fragment thereof is an antigenic fragment of IL31. In some cases a functional fragment may be a truncated version of the wild type polypeptide, suitably which be truncated at the C terminal or N terminal end or both ends.

[0228] Suitably the IL31 polypeptide or a functional fragment thereof may comprise a truncation at the N-terminal end thereof. Suitably the truncation at the N-terminal end may comprise removal of one or more amino acids from the wild type polypeptide sequence, suitably removal of between 2-5 amino acids from the N-terminal end. Suitably removal of 3 amino acids at the N-terminal end. Suitably removal of RLL at the N-terminal end.

[0229] Suitably the IL31 polypeptide or a functional fragment thereof may comprise a truncation at the C-terminal end thereof. Suitably the truncation at the C-terminal end may comprise removal of one or more amino acids from the wild type polypeptide sequence, suitably removal of between 2-5 amino acids from the C-terminal end. Suitably removal of 3 amino acids at the C-terminal end. Suitably removal of AAT at the C-terminal end.

[0230] In one embodiment, the IL31 polypeptide or a functional fragment thereof may comprise a truncation at both the N-terminal and C-terminal ends, suitably of around 3 amino acids.

[0231] Suitably such an embodiment of the IL31 polypeptide comprises an amino acid sequence according to SEQ ID NO: 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, identity thereto, or a functional fragment thereof.

[0232] In one embodiment, the IL31 polypeptide thereof may consist of an amino acid sequence according to SEQ ID NO: 12, or a functional fragment.

[0233] In one embodiment, the IL31 polypeptide or a functional fragment thereof may comprise a truncation at only the N-terminal end, suitably of around 3 amino acids.

[0234] Suitably such an embodiment of the IL31 polypeptide comprises an amino acid sequence according to SEQ ID NO: 11 , or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, identity thereto, or a functional fragment thereof.

[0235] In one embodiment, the IL31 polypeptide thereof may consist of an amino acid sequence according to SEQ ID NO: 11 or a functional fragment.

[0236] Suitably VLPs of the invention may comprise one or more further functional molecules, in addition to the or each IL31 polypeptide or a functional fragment thereof. Suitably the further functional molecules may be of the same type or different types. For example, each pair of binding proteins may be attached to any combination of IL31 polypeptides or a functional fragment thereof plus one or more further functional molecules such as one or more antigens, antigen binding proteins, or flourescent molecules. Suitable further functional molecules are described below.

[0237] Further Functional Molecules

[0238] The present invention relates to VLPs which are able to display IL31 polypeptides or a functional fragment thereof and optionally various further functional molecules on their surface by virtue of the protein binding pair or by virtue of chemical modifications to the first binding protein.

[0239] Suitably each pair of binding proteins is attached to at least one IL31 polypeptide or a functional fragment thereof as described above. Suitably each pair of binding proteins may be attached to more than one functional molecule. Suitably the functional molecules may be of the same type or different types. For example, each pair of binding proteins may be attached to any combination of I L31 polypeptide or a functional fragment thereof and a further functional molecule selected from: one or more antigens, antigen binding proteins, or fluorescent molecules.

[0240] Suitably each chemical modification may be attached to one functional molecule. Suitably, in such an embodiment, a further functional molecule may be attached to the first binding protein via the chemical modification. Suitably in such embodiments, the further functional molecule is a non-protein antigen or epitope thereof, or a fluorescent molecule.

[0241] Suitably, in some embodiments, there may be more than one functional molecule per pair of binding proteins. Suitably a first functional molecule may be attached to the first binding protein via a chemical modification, and suitably a second functional molecule may be attached to the second binding protein wherein the second functional molecule is an IL31 polypeptide or a functional fragment thereof.

[0242] Alternatively, a first and second functional molecule may be attached to the second binding protein. Suitably wherein at least one of said functional molecules is an IL31 polypeptide or a functional fragment thereof.

[0243] Suitable further functional molecules may include: protein or non-protein antigens; antigen binding proteins such as antibodies or binding fragments thereof, antibody mimetics, and aptamers; fluorescent molecules.

[0244] Suitable antigens may include the whole or part of an antigen. Suitably the antigen may be a subunit or monomer of an antigen. Suitably the functional molecule may be an epitope of an antigen. Suitably the use of an antigen as a functional molecule produces a VLP which is capable of stimulating an immune response to the antigen. Suitably this is useful as a vaccine.

[0245] Suitably the antigen may be a protein or non-protein antigen. Suitable non-protein antigens may include sugars, lipids or carbohydrates, or small molecule chemicals to which an immune response is desired, or who need to be detected, such as nicotine, cocaine, or other exogenous toxins.

[0246] Suitably the antigen may be a self or non-self antigen relative to the subject intended to be treated with the VLP. Suitably the antigen may be a human or non-human antigen.

[0247] Suitably the antigen may be derived from the causative agent in a disease or disorder. Suitably the causative agent may be self or non-self.

[0248] Suitably a non-self causative agent may be an infectious agent. Suitably therefore the antigen may be derived from an infectious agent such as a virus, bacterium, fungus, protozoan, archaeon.

[0249] Suitably the antigen may be derived from a virus selected from: Adeno-associated virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, Hantaan virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus, Human herpesvirus, Human immunodeficiency virus, Human papillomavirus, Human parainfluenza, Human respiratory syncytial virus, Human rhinovirus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Japanese encephalitis virus, Polyomavirus, Kunjin virus, Lassa virus, Measles virus, Molluscum contagiosum virus, Mumps virus, Nipah virus, Poliovirus, Rabies virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sapporo virus, Sindbis virus, Toscana virus, Uukuniemi virus, Varicella-zoster virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, West Nile virus, Yellow fever virus, Zika virus.

[0250] Suitably the antigen may be derived from a bacterium selected from: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae , Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes , Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella enterica subsp. enterica , Salmonella typhi, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis .

[0251] In one embodiment the antigen is derived from a coronavirus, suitably from SARS-CoV-2. Suitably the antigen is the whole or part of a spike protein derived from SARS-CoV-2, or the whole or part of a nucleocapsid protein derived from SARS-CoV-2.

[0252] In one embodiment, therefore, the functional molecule is part of a spike protein derived from SARS-CoV-2. Suitably the receptor binding domain.

[0253] In another embodiment, therefore, the functional molecule is part of a nucleocapsid protein derived from SARS-CoV-2. Suitably the C-terminus.

[0254] Suitably a self-causative agent may be a non-infectious agent. Suitably therefore the antigen may be derived from a non-infectious agent such as an inflammatory molecule, or a molecule causing degenerative changes in nervous (such as beta-amyloid), cartilage or bone tissue, or a molecule causing worsening of a neoplastic disease.

[0255] Suitably the antigen may be an inflammatory molecule or a molecule causing degenerative changes or a molecule conducive to a neoplastic disease which is a causative agent in a disease or disorder. Suitably the molecule may operate in humans or in non-human mammals. Suitably the molecule may cause a disease or disorder in a specific species.

[0256] Suitable inflammatory molecules may include chemokines or cytokines, or proteases. Suitable chemokines or cytokines may include: interleukins, tumour necrosis factors, interferons, and colony stimulating factors. Suitable chemokines or cytokines may include: IL1 , IL2, II3, II4, IL5, II6, II7, IL8, IL9, IL10, IL11 , IL12, IL13, IL17, IL31 , IL33, TNFa, TNF0, IFNa, IFNp, IFNy, G-CSF, GM-CSF, M-CSF, erythropoietin, and TGFp. Suitable proteases may include ADAMTS4, ADAMTS5. Suitably the antigen is an interleukin or a protease. Suitably the antigen is I L13, I L17 or I L33 or a functional fragment thereof.

[0257] In one embodiment, therefore, the further functional molecule may be IL13, IL17 or IL33 or a functional fragment thereof.

[0258] Suitable molecules which case degenerative changes in nervous tissue or worsening of neoplastic diseases may include: ADAMTS4 / 5, angiogenesis factors, or factors allowing escape of tumours such as galectin proteins.

[0259] References to any antigens herein may equally refer to an epitope of said antigen.

[0260] Suitable antigen binding proteins such as antibodies for use as a further functional molecule are capable of binding an antigen of interest. Suitably the use of an antigen binding protein such as an antibody as a further functional molecule produces a VLP which is capable of binding to an antigen. Suitably this is useful for detecting an antigen, or for targeting the VLP to an antigen.

[0261] An antigen of interest may be any of those listed above. For example, an antigen of interest may be from a disease causing agent such as a virus, bacterium, fungus, protozoan, or archaeon. Alternatively, an antigen of interest may be from a non-infectious agent, for example, a cell surface receptor.

[0262] Suitably the antibody may be capable of binding to an antigen from a virus, bacterium, fungus, protozoan, archaeon as listed above. Suitable viruses may be selected from, for example: Adeno-associated virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, Hantaan virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus, Human herpesvirus, Human immunodeficiency virus, Human papillomavirus, Human parainfluenza, Human respiratory syncytial virus, Human rhinovirus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Japanese encephalitis virus, Polyomavirus, Kunjin virus, Lassa virus, Measles virus, Molluscum contagiosum virus, Mumps virus, Nipah virus, Poliovirus, Rabies virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sapporo virus, Sindbis virus, Toscana virus, Uukuniemi virus, Varicella-zoster virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, West Nile virus, Yellow fever virus, Zika virus. In one embodiment, the further functional molecule is an antibody capable of binding to an antigen from a coronavirus. In one embodiment, the antibody is capable of binding to an antigen from SARS-CoV-2.

[0263] Suitable bacteria may be selected from: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae , Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes , Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella enterica subsp. Enterica , Salmonella typhi, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis .

[0264] Suitably in such an embodiment, the VLP may be targeted to a particular virus. Suitably targeted to bind to a particular virus. Suitably the VLP may therefore be used for detecting the presence of a virus. Further details on this use are provided elsewhere.

[0265] Suitably the antigen binding protein such as an antibody may be capable of binding to an antigen from a cell surface receptor. Suitably the cell surface receptor may be an ion-channel linked receptor, a G-protein coupled receptor, or an enzyme-linked receptor. Suitably the cell surface receptor is selected from: 5-HT receptor, nAch-receptor, Zinc-activated ion channel, GABAA receptor, Wnt-family member receptors, co-receptors contained in lipid rafts, T-cell and T-cell co-receptors, B-cell receptors and B-cell costimulatory molecules, Glycine receptor, AM PA receptor, Kainate receptor, NMDA receptor, Glutamate receptor, ATP-gated channel, PIP2 gated channel, Erb receptor, GDNF receptor, NP receptor, trk receptor, toll-like receptor, GABAB receptor, GBPCR class A, B, C, D, E, or F.

[0266] Suitably in such an embodiment, the VLP may be targeted to a particular cell. Suitably targeted to bind to a particular cell. Suitably the VLP may be used to deliver cargo to a cell. Further details on this use are provided elsewhere. Suitable antibodies may include IgG, IgM, IgE, IgA, IgD antibodies. Suitably, the antibody is an IgG antibody. Suitably IgG subclasses include lgG1 , lgG2, lgG3 and lgG4.

[0267] Suitable further antigen binding proteins may include antibody binding fragments or antibody mimetics which perform the same function as an antibody. Suitably they are also capable of binding an antigen of interest. Suitably the use of an antibody binding fragment or mimetic as a further functional molecule also produces a VLP which is capable of binding to an antigen. Suitably this is useful for detecting an antigen, or for targeting the VLP to an antigen as described above.

[0268] Suitable antibody binding fragments may include: Fab, monospecific or bispecific F(ab)2, F(ab’)2, monospecific or bispecific diabody, nanobody, ScFv, ScFv-Fc, F(ab)3.

[0269] Suitable antibody mimetics may include affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, nanCLAMPs.

[0270] Suitably the use of a fluorescent molecule as a further functional molecule produces a VLP which is visible. Suitably this is useful for labelling, especially when combined with a further functional molecule which can bind to an antigen, for example antibodies or binding fragments thereof, antibody mimetics, or aptamers.

[0271] Suitable flourescent molecules may include: GFP, EBFP, EBFP2, Azurite, GFPuv, T-saphhire, Cerulean, CFP, mCFP, mTurquoise2, CyPet, mKeima-red, tagCFP, AmCyanl , mTFP1, midoriishi cyan, turboGFP, tagGFP, emerald, azami green, ZsGreenl , YFP, tagYFP, EYFP, topaz, venus, mCtrine, Ypet, turboYFP, ZsYellowl , Kusabira Orange, mOrange, allophycocyanin, mkO, RFP, turboRFP, tdTomato, tagRFP, dsRed, mStrawberry, turboFP602, asRed2, J-red, R-phycoerythrin, B-phycoerythrin, mCherry, HcRed, Katusha, P3, peridin chlorophyll, mKate, turboFP635, mPlum, mRaspberry.

[0272] Suitably the flourescent molecule is GFP or any modified form of GFP.

[0273] In one embodiment, the or each further functional molecule, present in addition to the IL31 polypeptide or a functional fragment thereof, is IL13, IL17, IL33, the receptor binding domain of SARS Cov-2 spike protein, or the C-terminus of the SARS Cov-2 nucleocapsid protein, or a functional fragment thereof.

[0274] Nucleic Acids & Vectors

[0275] The present invention relates to nucleic acids encoding component protein parts which form the VLP, and vectors comprising said nucleic acids which may be used in host cells to produce VLPs. Suitably the invention relates to, and makes use of, a first nucleic acid encoding a viral capsid protein attached to a first binding protein. Suitably the first nucleic acid may encode a fusion protein comprising the viral capsid protein fused to a first binding protein. Suitably the viral capsid protein may be a hepatitis B capsid protein. Suitably this may be known as the ‘capsid fusion protein’.

[0276] Suitably the first nucleic acid may comprise a sequence according to SEQ ID NO: 16. Suitably the first nucleic acid may comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 16. Suitably the first nucleic acid may consist of a sequence according to SEQ ID NO: 16.

[0277] Suitably the invention relates to, and makes use of, a second nucleic acid encoding a functional molecule attached to a second binding protein. Suitably the second nucleic acid may encode a fusion protein comprising the functional molecule fused to a second binding protein. Suitably this may be known as the ‘functional fusion protein’.

[0278] In some alternative embodiments, the second nucleic acid may encode only a functional molecule.

[0279] In one embodiment, the second nucleic acid encodes a functional molecule attached to a second binding protein. In one embodiment, the second nucleic acid encodes a functional molecule fused to a second binding protein.

[0280] Suitably the second nucleic acid may comprise a sequence according to SEQ ID NO: 59, 60, 61 , 62, 63, 64, 65, or 66, preferably SEQ ID NO: 63, 64 or 65. Suitably the second nucleic acid may comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 59, 60, 61 , 62,

[0281] 63, 64, 65 or 66, preferably SEQ ID NO: 63, 64 or 65, or a fragment thereof. Suitably the second nucleic acid may consist of a sequence according to SEQ ID NO: 59, 60, 61 , 62, 63,

[0282] 64, 65, or 66 preferably SEQ ID NO: 63, 64 or 65. Suitably the functional molecule is an IL31 polypeptide, or a functional fragment thereof.

[0283] In some embodiments, the invention relates to, and makes use of a further nucleic acid encoding a second functional molecule. Suitably this further nucleic acid may be known as the fourth nucleic acid. In some embodiments, this may occur when the second nucleic acid already encodes a first functional molecule attached to a second binding protein. Suitably in such embodiments, the first binding protein is chemically modified.

[0284] In some embodiments, the invention relates to, and makes use of, a further nucleic acid encoding a second binding protein attached to a third binding protein. Suitably this further nucleic acid may be known as the third nucleic acid. Suitably the third binding protein is a protein capable of binding to an antigen binding protein such as an antibody. Suitably the third binding protein may be protein G, for example.

[0285] Suitably the first, second, and third binding proteins are defined elsewhere herein. However, suitably the first binding protein may be a bacterial toxin inhibitor and the second binding protein may be a bacterial toxin. Suitably the third binding protein may be an antibody binding protein.

[0286] In some embodiments, the invention may make use of the first and second nucleic acids.

[0287] In some embodiments, the invention may make use of the first, second and third nucleic acids.

[0288] In some embodiments, the invention may make use of the first, second and fourth nucleic acids.

[0289] In some embodiments, the invention may make use of the first and third nucleic acids.

[0290] In some embodiments, the invention may make use of the first, second, third, and fourth nucleic acids.

[0291] Suitably the first, second, third and fourth nucleic acids described herein may be provided as one contiguous nucleic acid sequence, or may be provided as a plurality of separate nucleic acid sequences. References to the first, second, third, and fourth nucleic acids include embodiments where plurality of nucleic acid sequences may be used to encode the same proteins as the first, second, third, and fourth nucleic acids.

[0292] Suitably the nucleic acids may comprise one or more expression elements to aid in expression of the proteins encoded thereon.

[0293] Suitable expression elements include promoters, operators, enhancers, activators, repressors, 5’llTRs, 3’llTRs, introns, IRES, etc.

[0294] Suitably each of the nucleic acids comprises one or more expression elements which ensure equal expression of the proteins encoded thereon. Suitably each of the nucleic acids comprises a promoter which ensures equal expression of the proteins encoded therein. Suitably the promoter may comprise one or more modifications which adapt the level of expression therefrom. Suitably the promoter may comprise one or more mutations. Suitably the or each nucleic acid described herein is operably linked to a promoter.

[0295] Suitable promoters may be selected from: CMV-IE, EF1a, SV40, PGK1 , CAG, human beta actin, T7, TetR / TetA, T7lac, SP6, LP1 , TTR, CK8, Synapsin, Glial fibrillary acidic protein (GFAP), CaMKII, TBG, and albumin promoter. Suitably each nucleic acid may be linked to the same promoter or a different promoter.

[0296] Suitably each nucleic acid may be linked to the same promoter. Suitably therefore each nucleic acid may be expressed at the same time. Suitably each nucleic acid may be linked to a T7 promoter, optionally with one or more modifications to ensure equal expression levels of the proteins encoded by the nucleic acids.

[0297] Suitably each nucleic acid may be linked to a different promoter. Suitably therefore each nucleic acid may be expressed at different times. Suitably the or each nucleic acid may be independently expressed. Suitably expression of each nucleic acid may be induced at different times. Suitably therefore the or each promoter may be an inducible promoter. Suitably which may be induced by contacting the promoter with a suitable inducer, at a concentration effective to induce expression therefrom. In one embodiment, the first nucleic acid sequence may be linked to a first promoter and the second nucleic acid may be linked to a second promoter. Suitably the first promoter may be a T7 promoter to modified T7 promoter as described herein. Suitably the second promoter may be a TetR / TetA promoter.

[0298] In one embodiment, the T7 promoter operably linked to the second nucleic acid is modified. Suitably the T7 promoter operably linked to the second nucleic acid is modified to reduce the expression level of the functional molecule attached to a second binding protein encoded thereon. Suitably the T7 promoter is modified by a point mutation. Suitably the T7 promoter may comprise any of the following modifications in the nomenclature according to Konczal et al, PloS One 2019: 1 C, 1T, 2T, 5A, 8G, 4C, or any combination thereof, wherein the parent sequence is: agcataat (SEQ ID NO: 67). Suitably the T7 promoter comprises a sequence according to SEQ ID NO: 67. In such an embodiment, suitably the T7 promoter operably linked to the first nucleic acid is not modified.

[0299] Suitably therefore the first nucleic acid expresses the viral capsid protein attached to a first binding protein at the same level as the second nucleic acid expresses the IL31 polypeptide or other functional molecule attached to a second binding protein, or at the same level as the third nucleic acid.

[0300] Suitably the first nucleic acid is selected from a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 16 or a fragment thereof, and suitably the second nucleic acid is selected from a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 59-66 or a fragment thereof. Suitably wherein the second nucleic acid is selected from a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 63, 64, or 65. In one embodiment, the first nucleic acid comprises a sequence according to SEQ ID NO: 16. In one embodiment, the second nucleic acid comprises a sequence according to one of SEQ ID NOs: 59-66, suitably one of SEQ ID NO: 63, 64, or 65.

[0301] Suitably therefore the capsid fusion protein is expressed at a 1 :1 level compared to the functional fusion protein, or the functional molecule.

[0302] Suitably the nucleic acids may be comprised on one or more vectors. Suitably wherein the vector comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 26, 27-32, 57 or 58, or a fragment thereof. Suitably wherein the vector comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 26, 31 , 32 or 57 or a fragment thereof. Suitably the first, second, third, and / or fourth nucleic acids may be comprised on one vector. Alternatively, first, second, third, and / or fourth nucleic acids may be comprised on multiple vectors. In one embodiment, the first nucleic acid may be comprised on one vector and the second nucleic acid may be comprised on another vector.

[0303] In one embodiment, the first nucleic acid is comprised on a first vector, suitably the vector having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 26 or a fragment thereof. In one embodiment, the first nucleic acid is comprised on a first vector, wherein the vector comprises SEQ ID NO: 26.

[0304] In one embodiment, the second nucleic acid is comprised on a second vector, suitably the vector of having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to any of SEQ ID NO: 27-32, 57 or 58, or a fragment thereof. In one embodiment, the second nucleic acid is comprised on a second vector, suitably the vector of having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to any of SEQ ID NOs: 31 or 32, or 57, or a fragment thereof. In one embodiment, the second nucleic acid is comprised on a second vector wherein the vector comprises any of SEQ ID NO: 27-32, 57 or 58, suitably any of SEQ ID NOs: 31 or 32, or 57.

[0305] Alternatively, in one embodiment, the first and second nucleic acids may be comprised on the same vector.

[0306] In one embodiment, the first nucleic acid and the third nucleic acid are comprised on the same vector. Suitably the one or more vectors may be comprised in one or more host cells. Suitably the one or more vectors may be comprised in a single host cell. Alternatively the one or more vectors may be comprised in a plurality of host cells in any combination.

[0307] Suitably in the process of the seventh aspect, the first, second and / or third nucleic acids may be comprised on one vector or on a first and second vector, or on a first, second and third vector respectively. In one embodiment of the process of the seventh aspect, the first and second nucleic acids are comprised on one vector. Suitably the or each vector is present in the single host cell. In one embodiment of the process of the seventh aspect, the first and second nucleic acids are comprised on a single vector. In one embodiment of the process of the eighth aspect, the first and second nucleic acids are comprised on two different vectors. Suitably the first nucleic acid may be comprised on a first vector according to SEQ ID NO: 26. Suitably the second nucleic acid may be comprised on a second vector selected from any of SEQ ID NOs: 27 to 32, 57 or 58, preferably SEQ ID NOs: 31 or 32, or 57. Suitably any workable combination of first and second vectors may be used in the single host cell.

[0308] Suitably in the process of the eighth aspect, the first and second nucleic acids are comprised on a first and second vector respectively. Suitably the third nucleic acid may be comprised on a second vector together with the second nucleic acid or alone. Alternatively the third nucleic acid may be comprised on a third vector. Suitably the first vector is present in the first host cell and the second and / or third vector is present in a second host cell. Alternatively, the third vector may be present in a third host cell. In one embodiment of the process of the eighth aspect, suitably the first vector is of SEQ ID NO: 26, and the second vector is of SEQ ID NO: 31 or 32, 57 or 58. Suitably any workable combination of first and second vectors may be used in the two host cells.

[0309] Suitably, the one or more vectors may further comprise the third and / or fourth nucleic acids. In one embodiment, the one or more vectors may further comprise both a third nucleic acid encoding a second binding protein attached to a third binding protein, and a fourth nucleic acid encoding a further functional molecule.

[0310] Suitably the further third and / or fourth nucleic acids may be comprised on a vector in the first or second host cells. Suitably the further third and / or fourth nucleic acids may be comprised on the same vector as the first and / or second nucleic acids, or on different vectors. Suitably the third and / or fourth nucleic acids may both be comprised on a third vector. Alternatively, the third and / or fourth nucleic acids may be comprised on a third and a fourth vector respectively. Suitably the third and / or fourth vector may be present in the first or second host cells. Alternatively, the third and / or fourth vector may be present in a third host cell. Alternatively, the third vector may be present in a third host cell and the fourth vector may be present in a fourth host cell.

[0311] Any suitable vector may be used for the chosen host cell / s. Suitable host cells are discussed below. Suitably the vector is selected from: a plasmid, a cosmid, a phage, a virus, an artificial chromosome. Suitably the or each vector is a plasmid.

[0312] Suitable plasmid vectors for a host E.coli cell may include, for example: pALTER-Ex1 , pALTER-Ex2, pBAD / His, pBAD / Myc-His, pBAD / glll, pCal-n, pCal-n-EK, Cal-c, pCal-Kc, pcDNA 2.1 , pDUAL, pET-3a-c, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET- 16b, pET-17b, pET-19b, pET-20b(+), pET-21a-d(+), pET-22b(+),pET-23a-d(+), pET-24a-d(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a-c(+), pET-29a-c(+), pET-30a-c(+), pET-31 b(+), pET-32a-c(+), pET-33b(+), pET-34b(+) , pET-35b(+), pET-36b(+), pET-37b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a-c(+), pET-42a-c(+), pET-43a-c(+), pETBIue-1 , pETBIue-2, pETBIue-3, pGEMEX-1 , pGEMEX-2, pGEX-1 IT, pGEX-2T, pGEX-2TK, pGEX-3X, pGEX-4T, pGEX-5X, pGEX-6P, pHAT10 / 11 / 12, pHAT20, pHAT-GFPuv, pKK223-3, pLEX, pMAL-c2X, pMAL-c2E, pMAL-c2G, pMAL-p2X, pMAL-p2E, pMAL-p2G, pProEX HT, pPROLar.A, pPROTet.E, pQE-9, pQE-16, pQE-30 / 31 / 32, pQE-40, pQE-60, pQE-70, pQE-80 / 81 / 82L, pQE- 100, pRSET, pSE280, pSE380, pSE420, pThioHis, pTrc99A, pTrcHis, pTrcHis2, pTriEx-1 , pTriEx-2, pTrxFus.

[0313] In one embodiment, the vector used is pET-Duet.

[0314] Suitable plasmid vectors for a host mammalian cell may include: the pSV and the pCMV series of vectors.

[0315] In one embodiment, the vector used is pcDNA5D. In one embodiment, host mammalian cells are HEK293 cells or CHO cells or derivatives thereof.

[0316] Suitably if more than one vector is used, it is the same vector.

[0317] Suitably the vector may comprise a variety of other functional nucleic acid sequences, such as one or more selectable markers, one or more origins of replication, multiple cloning sites and the like.

[0318] Process of Producing VLPs

[0319] The present invention further relates to processes for the production of VLPs. Two different processes are described herein, one is a single cell process, the other is a process which takes place in at least two cells and requires mixing of component parts to form the VLP. In accordance with the seventh aspect of the invention, there is provided a single cell process of producing a VLP.

[0320] In accordance with the eighth aspect of the invention, there is provided a multiple cell process of producing a VLP.

[0321] 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 coprecipitation, or liposome mediated delivery.

[0322] Suitably culturing the host cells under conditions to express the proteins comprises culturing the host cells under optimum growth conditions. Suitably the optimum growth conditions will vary depending on the host cell being used.

[0323] Suitably the host cell may be selected from any bacterium, yeast, insect cell or human cell. Suitably the host cell is a bacterial host cell. Suitably the host cell is selected from E.coli, B.subtilis, Caulobacter crescentus, Rodhobacter sphaeroides, Pseudoalteromonas haloplanktis, Shewa nella sp. Strain Ac 10, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Halomonas elongate, Chromohalobacter salexigens, Streptomyces lividans, Streptomyces griseus, Nocardia lactamdurans, Mycobacterium smegmatis, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevi bacterium lactofermentum, Bacillus brevis, Bacillus megaterium, Bacillus licheniformis, Bacillus amyloliquefacien, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri.

[0324] In one embodiment, the host cell is E.coli. Suitably the E.coli strain is selected from BL21 , Iemo21 , NiCo21 , NEB Express, Shuffle, T7 Express, BLR, HMS174, Tuner, Origami2, Rosetta2, m15.

[0325] In one embodiment, the host cell further comprises one or more nucleic acids encoding dsbC and erv1 P, suitably wherein said nucleic acids are heterologous, suitably under the control of an inducible promoter. In one such embodiment, the E.coli strain is BL21 (DE3) where the additional genes regulating disulfide formation, dsbC and erv1 P, are integrated genomically. Suitably, the genomic integration is within the recAX locus. Alternatively, the genes may be expressed from transfected expression construct such as from a transfected vector. In one embodiment, the E.coli strain may comprise the CyDisCo system as described in Gqciarz et al. Microb Cell Fact (2017) 16:108. In an alternative embodiment, the host cell is a human cell, such as a HEK293T cell.

[0326] Suitably optimum growth conditions comprise culturing at a temperature of 15-25°C. Suitably optimum growth conditions comprise culturing in a medium compatible with bioprocess applications for medicines intended for use in humans, such as chemically defined medium. Suitably optimum growth conditions comprise culturing in an aerated culture medium.

[0327] Suitably the host cells are cultured to a high density. Suitably to a density ODeoo of 4-20.

[0328] Suitably culturing the host cells under conditions to express the proteins may also comprise inducing the host cells to express the proteins. Suitably inducing the host cells may comprise addition of an inducer into the culture medium, or the creation of certain inducive conditions within the culture medium such as acid / alkali pH, heat shock, hypoxia or the like. Suitably the inducer or inducive condition stimulates transcription of the nucleic acids. Suitably an inducer or inducive condition does so by stimulating an inducible expression control sequence within the nucleic acids. Suitably the inducible expression control sequence may be an inducible promoter. Suitable inducers include isopropyl-p-d-thiogalactoside (IPTG) for lactose driven promoters or tetracycline for tetracycline - regulated promoters.

[0329] Suitably the host cells are induced to express the proteins once the culture has reached the optimal density described above. Suitably the host cells are induced to express the proteins during logarithmic growth.

[0330] Suitably the concentration of proteins may be varied by adjusting the concentration of an inducer or altering the inducive conditions to which the host cells are exposed.

[0331] Suitably the culturing step takes between 4 - 24 hours.

[0332] Suitably the host cells are induced to express the proteins after 2-6h of culturing or when an OD of 6-8 has been achieved.

[0333] In a further aspect of the invention, there is provided a cell culture comprising one or more host cells of the invention and a culture medium. Suitably a plurality of said cells.

[0334] Alternatively, the process may not be conducted within one or more cells, and may be conducted in a cell-free system. Suitably in the process of the seventh aspect, step (a) is conducted within a host cell, to ensure proper production of the VLP shell. However, suitably step (b) may occur outside of a host cell, in a cell free system.

[0335] Suitably the processes may further comprise a step of recovering the VLPs. Suitably recovering the VLPs from the host cells. Suitably after the VLPs have been formed. Suitably recovering the VLPs may comprise disrupting the host cells. Alternatively, the host cells may secrete the VLPs into the culture solution. Suitably disrupting the host cells may be carried out by any suitable method such as homogenisation, sonication, or freeze-thaw.

[0336] Recovery of the VLPs may take place by any suitable method such as filtration, pull-down, centrifugation, or chromatography.

[0337] Suitably, in an embodiment where the binding protein comprises a chemical modification, suitably the recovery and purification of VLPs takes place by chromatography. Suitably involving a sequence of steps including mixed mode (hydrophobic interaction and size exclusion) chromatography, anion exchange chromatography, and ultrafiltration. Suitably by anion exchange chromatography. Suitably when anion exchange chromatography is used to recover the VLPs, the VLP may comprise chemical modification, suitably in such an embodiment the first binding protein of the VLP is modified with DEAE. Suitably the DEAE molecules can bind to the chromatography column.

[0338] Suitably, in the process of the eighth aspect, step (d) comprises recovering the proteins. Suitably recovering the proteins from the host cells. Suitably recovering the proteins may be performed by similar techniques. Suitably recovering the proteins may comprise disrupting the host cells as above. Alternatively, the host cells may secrete the proteins into the culture solution.

[0339] Suitably the VLPs form by self-assembly, suitably automatic self-assembly. Suitably once the component proteins are mixed, either within a single host cell as per the seventh aspect or outside of a cell as per the eighth aspect, they will assemble to form VLPs.

[0340] In respect of the single cell process of the seventh aspect, suitably the step of culturing the host cell further comprises culturing under conditions such that the proteins expressed from the first and second nucleic acids, or from any further nucleic acids, bind to each other.

[0341] In some embodiments, after the culturing step the first binding protein may be chemically modified. Suitably therefore the method may comprise a step of recovering the proteins, and subsequently chemically modifying the first binding protein. Suitably these steps take place after step (b) but prior to step (c).

[0342] In some embodiments, the one or more vectors may further comprise a further (fourth) nucleic acid encoding a further functional molecule. Suitably in such embodiments, the first binding protein is chemically modified. Suitably the further functional molecule binds to the chemical modification. Suitably in such embodiments, the host cell is cultured under conditions to express the proteins from the first, second and fourth nucleic acids. In some embodiments, the one or more vectors may further comprise a third nucleic acid encoding a second binding protein attached to a third binding protein. Suitably in such embodiments, the host cell is cultured under conditions to express the proteins from the first and third nucleic acids. Suitably in such an embodiment, the second nucleic acid may be present, and may encode only a functional molecule. Suitably in such an embodiment, the functional molecule is an antigen binding protein.

[0343] In some embodiments, the host cell may be cultured under conditions so as to express proteins from the first, second, third and fourth nucleic acids.

[0344] In one embodiment, the second nucleic acid encodes only a functional molecule. Suitably, in such an embodiment, the first binding protein is chemically modified, or the third nucleic acid is present.

[0345] In one embodiment, the second nucleic acid encodes a functional molecule, suitably an IL31 polypeptide, attached to a second binding protein. Suitably in such an embodiment, the first binding protein may or may not be chemically modified.

[0346] In one embodiment, step (c) of the seventh aspect comprises each first binding protein binding to each second binding protein. In an alternative embodiment, step (c) comprises each first binding protein binding to a functional molecule, suitably via a chemical modification. In one embodiment, step (c) comprises both of these steps.

[0347] In respect of the multiple cell process of the eighth aspect, suitably during the culturing step the first binding protein may be chemically modified. Suitably therefore the conditions for culturing the first host cell are such that the first binding protein is chemically modified. Suitably such chemical modification of the first binding protein may take place post-translationally. Alternatively, the method may comprise a step of chemically modifying the first binding protein. Suitably this step takes place after step (d) but prior to step (e).

[0348] In some embodiments, the one or more vectors may further comprise a further (fourth) nucleic acid encoding a further functional molecule. Suitably the fourth nucleic acid may be comprised on a vector in the first or second host cells, or may be comprised on a vector in a third host cell. Suitably in such embodiments, the first binding protein is chemically modified. Suitably in such embodiments, the host cells are cultured under conditions to express the proteins from the first, second and fourth nucleic acids.

[0349] In some embodiments, the one or more vectors may comprise a third nucleic acid encoding a second binding protein attached to a third binding protein. Suitably in such embodiments, the host cells are cultured under conditions to express the proteins from the first, and third nucleic acids.

[0350] Suitably in such an embodiment, the second nucleic acid if present encodes only a functional molecule. Suitably in such an embodiment, the functional molecule is an antigen binding protein.

[0351] In some embodiments, the host cells may be cultured under conditions so as to express proteins from the first, second, third and fourth nucleic acids.

[0352] In one embodiment, step (e) comprises each first binding protein binding to each second binding protein. In an alternative embodiment, step (e) comprises each first binding protein binding to each functional molecule, suitably via a chemical modification. In one embodiment, step (e) comprises both of these steps.

[0353] In one embodiment, step (e) further comprises mixing under conditions such that the proteins bind to each other. Suitably step (e) comprises mixing host cell supernatants or host cell lysates. Suitably mixing the first host cell supernatant or lysate with the further host cell(s) supernatant or lysate. Suitably the mixing is such that the ratio of the binding proteins confers an even stoichiometric concentration. Suitably the mixing is such that the ratio of first host cell supernatant or lysate to further host cell(s) supernatant or lysate is about 1 :1. Suitably the mixing step takes place at room temperature, suitably around 18-22°C. Suitably mixing takes place for between 15 minutes to 2 hours, suitably between 20 minutes and 1 hour, suitably between 25 minutes and 45 minutes, suitably for about 30 minutes.

[0354] Immunogenic Composition

[0355] The present invention further relates to an immunogenic composition comprising the VLP of the invention.

[0356] Suitably the immunogenic composition may be a vaccine.

[0357] Suitably the immunogenic composition may further comprise one or more adjuvants. Suitable adjuvants include: mineral salts, emulsions, microorganism derived adjuvants, carbohydrates, cytokines, particulates or tensoactive compounds.

[0358] Suitable mineral salts include: adjumer, alhydrogel, aluminium hydroxide, aluminum phosphate, aluminium potassium sulphate, amorphous aluminium hydroxyphosphate sulfate (AAHSA), aluminium salts in general, calcium phosphate, Rehydragel HPA, or Rehydragel LV. Suitable emulsions include: Freund’s complete, Freund’s incomplete, montanide ISA720, montanide ISA 51 , montanide incomplete, Ribi, TiterMax, AF03, AS03, MF59, speed, SPT, or squalene.

[0359] Suitable microorganism derived include: cholera toxin or mutants thereof, cholera toxin subunit B, CpG DNA, LTR 192G, MPL, Bordella pertussis components, E.coli heat labile toxin, CTA1-DD gene fusion protein, Etx B subunit, lipopolysaccharides, flagellin, Corynebacterium derived P40, LTK72, MPL-SE, or Ty particles.

[0360] Suitably the immunogenic composition may further comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients may include stabilizers, fillers, preservatives, diluents, nutrients, antioxidants, antimicrobial agents, buffers, solvents, inactivating agents, purifiers, emulsifiers, surfactants and the like.

[0361] Suitable excipients may be selected from, for example: monosodium glutamate, sucrose, D- mannose, D-fructose, dextrose, human serum albumin, potassium phosphate, plasdone C, anhydrous lactose, microcrystalline cellulose, polacrilin potassium, magnesium stearate, cellulose acetate phthalate, alcohol, acetone, castor oil, sodium chloride, benzethonium chloride, formaldehyde, ascorbic acid, hydrolyzed casein, sodium bicarbonate, sodium carbonate, glutaraldehyde, 2-phenoxyethanol, polysorbate 80 (Tween 80), neomycin, polymyxin B sulfate, bovine serum albumin, neomycin sulfate, polymyxin B, yeast protein, streptomycin sulfate, ammonium thiocyanate, rice protein, lactose, formalin, amino acid supplement, phosphate-buffered saline solution, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, yeast DNA, deoxycholate, phosphorothioate linked oligodeoxynucleotide, dibasic dodeca hydrate, monobasic dehydrate, L-histidine, sodium borate, sodium taurodeoxycholate, ovalbumin, sorbitan trioleate, sodium citrate dehydrate, citric acid monohydrate, kanamycin, barium, hydrocortisone, egg proteins, cetyltrimethylammonium bromide (CTAB), octoxynol-10 (TRITON X-100), a-tocopheryl hydrogen succinate, gentamicin sulfate, monobasic sodium phosphate, dibasic sodium phosphate, cetyltrimethlyammonium bromide, and p-propiolactone, Thimerosal, a-tocopheryl hydrogen succinate, hydrolyzed porcine gelatin, arginine, dibasic potassium phosphate, monobasic potassium phosphate, protamine sulfate , sodium metabisulphite, Vero cell protein, CRM197 protein, vitamins, bovine calf serum, urea, succinate buffer, isotonic saline solution, phenol, M-199 medium, chicken protein, polygeline, chlortetracycline, dextran, Dulbecco’s Modified Eagle Medium, magnesium sulfate, ferric (III) nitrate, L-cystine, L-tyrosine, sorbitol, xanthan, water, EDTA, dioleoyl phosphatidylcholine (DOPC), 3-O-desacl4’monophosphoryl lipid A (MPL), QS-21 , and cholesterol.

[0362] In one embodiment, the excipients may be arginine, glutamine and trehalose. Suitably the immunogenic composition is formulated as a fluid, suitably as a liquid. Suitably the excipients and additives are selected such that the formulation is a liquid. Suitably an injectable liquid.

[0363] The term “Immunogenic" means that a VLP or an immunogenic composition comprising the VLP of the invention is capable of eliciting an immune response in a subject. Suitably a potent and preferably a protective immune response in a subject. Thus, the VLP or an immunogenic composition comprising the VLP of the invention may be capable of generating an antibody response in a subject and / or a non-antibody based immune response in a subject. Suitably this may be referred to as its immunogenic activity.

[0364] As set out in the Examples, the inventors have demonstrated that an immunogenic composition comprising the VLPs of the invention exhibit immunogenic activity that is comparable, if not improved, compared with a control vaccine. However, surprisingly, the inventors have found that a vaccine comprising the VLPs of the invention elicited an immunogenic response that was quicker and then more sustained and consistent as compared to a control vaccine. Therefore, the VLPs of the invention show immunogenic activity that is well suited to therapeutic use as a medicament.

[0365] Suitably the immunogenic activity of the VLP or an immunogenic composition comprising the VLP of the invention may be determined by the amount of antibodies present in a subject after administration of the VLP or an immunogenic composition comprising the VLP of the invention i.e. antibody production. Suitably the amount of antibodies which bind to the antigen of the VLP. Suitably the amount of antibodies present in a subject after administration of the VLP or an immunogenic composition comprising the VLP of the invention, i.e. antibody production, is sustained and consistent over a period of time. Suitably the immunogenic activity of the VLP or an immunogenic composition comprising the VLP of the invention may be determined by the amount of antibodies present in a subject after administration of the VLP or an immunogenic composition comprising the VLP of the invention over a given period of time, i.e. antibody production over a given period of time. Suitable periods of time are outlined below. By amount of antibodies it is meant the titre or concentration thereof. Suitably the concentration of antibodies in sera.

[0366] Suitably a VLP or an immunogenic composition comprising the VLP of the invention may sustain immunogenic activity for at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, or at least 100 days or more in a subject. Suitably a VLP or an immunogenic composition comprising the VLP of the invention may sustain immunogenic activity for at least 110 days, at least 120 days, at least 130 days, at least 140 days, at least 150 days, at least 160 days, at least 170 days, at least 180 days, at least 190 days, at least 200 days, at least 210 days, at least 220 days, at least 230 days, at least 240 days, at least 250 days, at least 260 days, at least 270 days, at least 280 days, at least 290 days, at least 300 days or more in subject.

[0367] Suitably a VLP or an immunogenic composition comprising the VLP of the invention may sustain immunogenic activity for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks days or more in a subject. Suitably a VLP or an immunogenic composition comprising the VLP of the invention may sustain immunogenic activity for at least at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks or more in a subject.

[0368] Suitably a VLP or an immunogenic composition comprising the VLP of the invention may sustain immunogenic activity for at least for at least 1 year, at least 2 years at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years or at least 10 years or more in a subject. Suitably, a VLP or an immunogenic composition comprising the VLP of the invention may sustain immunogenic activity for at least 10 years, for at least 15 years, for at least 20 years, for at least 25 years, for at least 30 years, for at least 35 years, for at least 40 years, for at least 45 years, for at least 50 years or more in a subject. .

[0369] Suitably wherein immunogenic activity may refer to immunogenic antibody production. Suitably antibody production at a concentration which is immunogenic. Suitably antibody production at a concentration in sera which is immunogenic. Suitably at a concentration of between 1-20p.g / ml, 1-18p.g / ml, 1-16p.g / ml, 1-14p.g / ml, 1-12p.g / ml, 2-18p.g / ml , 2-16p.g / ml , 2- 14p.g / ml, 2-12p.g / ml, or 2 - 10 p.g / ml in sera for example.

[0370] The skilled reader, on considering the information set out in the Examples, will recognise that the VLPs or the immunogenic compositions of the invention exhibit immunogenic activity that makes them well suited to therapeutic use in the manner described in this specification.

[0371] Medical Uses

[0372] The present invention further relates to use of the VLP or the immunogenic composition comprising the VLP as a medicament. Suitably for use in therapy, or in the prevention and / or treatment of a disease. In further aspect, the present invention further provides a method of treating a subject having a disease or preventing a disease in a subject, comprising administering an effective amount of a VLP according to the first aspect or an immunogenic composition thereof according to the ninth aspect, to the subject.

[0373] In further aspect, the present invention further provides a method of manufacturing a medicament for the treatment or prevention of a disease, the medicament comprising an effective amount of a VLP according to the first aspect or an immunogenic composition thereof according to the ninth aspect.

[0374] Suitably the disease may be selected from: an infectious disease, cancer, an autoimmune disease, a cardiovascular disease, a metabolic disease, an inflammatory disease, a neurological disease, or rheumatological degenerative disease, or an addiction.

[0375] Suitable infectious diseases include: viral, bacterial, fungal, or protozoan infections.

[0376] Suitable viral infections include: COVID-19, SARS, MERS, influenza, common cold, respiratory syncytial virus infection, adenovirus infection, parainfluenza virus infection, norovirus infection, rotavirus infection, astrovirus infection, measles, mumps, rubella, chickenpox, shingles, roseola, smallpox, fifth disease, chikungunya virus infection, HPV infection, Hepatitis A, B, C, D or E, warts, herpes, molluscum contagiosum, ebola, lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, polio, viral meningitis, viral encephalitis, rabies, zika virus infection, west nile virus infection, HIV / AIDS, Hantavirus infection, HPS.

[0377] Suitable bacterial infections include: urinary tract infections, cystitis, impetigo, bacterial food poisoning, campylobacteriosis, C. difficile infection, bacterial cellulitis, MRSA, CRPA, VRSA, sepsis, erysipelas, necrotising fasciitis, bacterial folliculitis, gonorrhoea, chlamydia, syphilis, mycoplasma genitalium, bacterila vaginosis, pelvic inflammatory disease, tuberculosis, whooping cough, Haemophilus influenzae disease, pneumonia, bacterial meningitis, lyme disease, cholera, botulism, tetanus, anthrax, Cryptosporidiosis, Diphtheria, E. coli infection, Legionnaires Disease, Leptospirosis, Listeriosis, salmonella infections, Shigellosis gastroenteritis, Staphylococcal infections, Streptococcal infections, TSS, typhoid fever, Yersenia infection.

[0378] Suitable cancers include: breast cancer, liver cancer, lung cancer, pancreatic cancer, brain cancer, prostate cancer, bowel cancer, rectal cancer, bone cancer, leukemia, bladder cancer, cervical cancer, endometrial cancer, eye cancer, retinoblastoma, ewing sarcoma, gallbladder cancer, head and neck cancer, kaposi’s sarcoma, kidney cancer, laryngeal cancer, mesothelioma, myeloma, lymphoma, ovarian cancer, oesophageal cancer, mouth cancer, nasopharyngeal cancer, nose and sinus cancer, skin cancer, sarcoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, penile cancer, vulval cancer.

[0379] Suitable autoimmune diseases include: asthma, psoriasis, MS, rheumatoid arthritis, reactive arthritis, lupus, inflammatory bowel syndrome / disease, type 1 diabetes, Guillain-Barre syndrome, demyelinating polyneuropathy, Graves’ disease, Hashimo’s thyroiditis, Myasthenia gravis, vasculitis, pernicious anemia, ulcerative colitis, antiphospholipid syndrome, Kawasaki disease, alopecia, vitiligo, scleroderma, Sjogren’s syndrome, crohn’s disease, coeliac disease, Addison’s disease, narcolepsy.

[0380] Suitable cardiovascular diseases include: angina, heart attack, heart failure, coronary heart disease, stroke, transient ischemic attack, peripheral arterial disease, aortic disease, atherosclerosis, hypertension, cerebrovascular disease, renal artery stenosis, aneurysm, cardiomyopathy, pulmonary heart disease, arrythmia, dysrhythmia, endocarditis, cardiomegaly, myocarditis, valvular heart disease, congenital heart disease, rheumatic heart disease.

[0381] Suitable metabolic diseases include: hypercholesterolemia, hypertriglyceridemia, diabetes, hyperlipidemia, hyperbilirubinemia, hypercalcemia.

[0382] Suitable inflammatory diseases may include any of the above infections or autoimmune diseases. Suitable inflammatory diseases may include include: arthritis, asthma, tuberculosis, periodontis, chronic ulcers, sinusitis, hepatitis, glomerulonephritis, inflammatory bowel syndrome / disease, preperfusion injury, transplant rejection, sickle cell disease, allergies, cardiovascular disease, psoriasis, cytokine-mediated pruritus, COPD, diabetes, bronchitis, Crohn’s disease, atherosclerosis, dermatitis, arteritis, lupus.

[0383] Suitable neurological diseases include: Alzheimer’s, ataxia, ALS, Bells palsy, brain tumours, aneurysms, epilepsy, Guillain-Barre syndrome, hydrocephalus, Meningitis, MS, muscular dystrophy, neurocutaneous syndromes, Parkinson’s, migraines, encephalitis, myasthenia gravis, dementia, seizures, spinal muscular atrophy, motor neuron disease, scoliosis, neuropathy, chronic fatigue syndrome, cerebal palsy.

[0384] Suitable rheumatological degenerative diseases include: rheumatoid arthritis, psoriasis arthritis, spondylarthropathy, osteoarthritis, lupus, systemic sclerosis.

[0385] Suitable addictions include: alcohol, nicotine, caffeine, amphetamines, opioids, sedatives, hypnotics, anxiolytics, cocaine, cannabinoids, hallucinogenics, phenycylcidine.

[0386] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a disease or condition which involves, or has an associate symptom of, itching. In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a disease or condition which involves, or is mediated by, IL31 i.e. an IL31 mediated disease.

[0387] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a disease or condition, suitably an immune-mediated disease or condition, suitably selected from: dermatological conditions, allergic conditions, and onco- haematological conditions.

[0388] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a disease or condition which involves itching selected from: dermatological conditions, allergic conditions, and onco-haematological conditions.

[0389] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a disease or condition which involves or is mediated by IL31 selected from: dermatological conditions, allergic conditions, and onco-haematological conditions.

[0390] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a dermatological disease or condition. In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a dermatological disease or condition which involves itching. In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a dermatological disease or condition which involves or is mediated by IL31.

[0391] Suitably the VLP or an immunogenic composition thereof of the invention comprises IL31 which is an inflammatory cytokine, and which acts to increase the symptom of itching, especially chronic itching. Therefore, use of the VLP or an immunogenic composition thereof of the invention as a treatment stimulates the formation of anti-IL-31 antibodies in the subject, these antibodies then bind IL31 and reduce its physiological effects in the subject including a reduction in itching. Furthermore, the use of the VLP described herein allows breaking of B cell tolerance to endogenous IL-31 proteins so that neutralizing antibodies can be efficiently generated to a higher level in the subject.

[0392] In one embodiment therefore, the VLP or the immunogenic composition are for use in the prevention or treatment of a disease or condition selected from: dermatological conditions, allergic conditions, and onco-haematological conditions by reducing itching.

[0393] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of a dermatological disease or condition by reducing itching. Suitable dermatological conditions include: Atopic dermatitis, acne, hives, rosacea, epidermolysis bullosa, ichthyosis, scleroderma, vitiligo, hidradenitis suppurativa, shingles, pemphigus, alopecia, contact dermatitis, eczema, psoriasis, prurigo nodularis, senile pruritus, renal pruritus, hepatic pruritus, and amyloidosis.

[0394] Suitable allergic conditions include: eczema, urticaria, contact dermatitis, hay fever, asthma, hives, rhinitis, arthritis, drug allergies, house dust allergy, animal allergies, plant allergies, insect allergies, and food allergies.

[0395] Suitable onco-haematological conditions include: Aplastic anaemia, Autoimmune Haemolytic anaemia, sickle cell anaemia, Sideroblastic Amentia, Antiphospholipid Antibody Syndrome, Arteritis, Basal Cell Carcinoma, Bladder Cancer, Bony and Spinal Metastasis, Breast Cancer, Chemotherapy-Induced Peripheral Neuropathies, Chemotherapy-Related Mucositis, Colon Cancer, Cryoglobulinemia, Cutaneous T-Cell Lymphoma, Mycosis Fungoides, Deep Vein Thrombophlebitis, Disseminated Intravascular Coagulation, Ductal Carcinoma In Situ, Endometrial Cancer and Uterine Sarcoma, Factor V Leiden, Gilbert Syndrome, Hamartoma, Head and Neck Cancers, Hemochromatosis, Hemophilia, Heparin-Induced Thrombocytopenia, Hepatoma (Hepatocellular Carcinoma), Hodgkin Lymphoma, Hypercalcemia Associated with Malignancy, Hypereosinophilic Syndrome, Hypersplenism, Inferior Vena Cava Syndrome, Laryngeal Cancer, Leukemia, Acute Lymphoblastic (ALL) in Adults, Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, lung cancer, Lymphoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Multiple Myeloma, Myelodysplastic Syndromes (MDS), Myeloproliferative Neoplasms, Neutropenia, Oral Cavity Neoplasms, Oropharyngeal Cancer, Ovarian Cancer, Paget Disease of the Breast, Pancoast Tumor, Pancreatic Cancer, Polycythemia Vera, Porphyria, Prostate Cancer, Protein C Deficiency Protein S Deficiency, Prothrombin 20210 (Mutation), Radiation Sickness, Rectal Cancer , Renal Cell Carcinoma, Rhabdomyosarcoma, Salivary Gland Tumors, Squamous Cell Carcinoma, Cutaneous, Superior Vena Cava Syndrome, Thalassemia, Thrombophilia and Hypercoagulable States, Thrombotic Thrombocytopenic Purpura, Thymus Cancer, Transfusion Reaction, Hemolytic, Venous Insufficiency Ulcers, Vitamin B12 Deficiency, Von Hippel-Lindau Syndrome, von Willebrand Disease, and Wiskott- Aldrich Syndrome.

[0396] In one embodiment, the VLP or the immunogenic composition are for use in the prevention or treatment of dermatitis, optionally atopic dermatitis, senile pruritus, or allergic dermatitis, or prurigo nodularis.

[0397] Suitably the invention further provides a method of generating IL31 antibodies, the method comprising using a VLP according to the first aspect, or an immunogenic composition according to the ninth aspect of the invention in the immunisation of a subject, to generate anti-l L31 antibodies and recovering said antibodies, optionally further formulating the recovered antibodies.

[0398] Suitably such a method may allow the generation of large amounts of anti-l L31 antibodies in a safe and effective manner. Suitably such a method may be performed in animals, sutiably therefore the subject may be an animal as defined below, suitably a mouse or rabbit.

[0399] Suitably the step of immunisation may comprise administration of the VLP according to the first aspect, or an immunogenic composition according to the ninth aspect to the subject as defined below.

[0400] Suitably recovering the antibodies may comprise extracting blood from the subject, and suitably isolating the antibodies from the serum thereof. Alternatively, recovering the antibodies may comprise fusing antibody-secreting spleen cells from immunized subjects with immortal myeloma cells to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant. Suitably recovering the antibodies in this embodiment comprises isolation of the antibody from culture supernatant of the hybridoma cell line.

[0401] Suitably the method may comprise a step of screening the subject serum (or hybridoma) for antibody titre and isotype.

[0402] Suitably the method may comprise a step of purification of the antibodies. Suitably by any means known in the art such as immunoprecipitation, or affinity binding to a resin, which may be contacted with the serum or supernatant under suitable conditions to allow the antibodies to bind to the resin. Suitably the antibodies may then be eluted.

[0403] Suitably the recovered anti-l L31 antibodies may themselves be used in therapy. Suitably for use in the treatment or prevention of a disease in a subject in need thereof. Suitably the recovered IL31 antibodies may therefore be formulated for use as a medicament, suitably into a pharmaceutical composition for use as a medicament.

[0404] Suitably, an effective amount for administration to the subject is an effective amount to prevent or treat the disease. Suitable effective amounts can be readily determined by the skilled medical practitioner.

[0405] Suitably a dose comprises an effective amount. A suitable dose of the VLP may comprise: 10- 100 micrograms, suitably 10-80 micrograms, suitably 20-60 micrograms, suitably 20-40 micrograms.

[0406] Suitably the VLP or immunogenic composition may be administered by any route. Suitably the

[0407] VLP or immunogenic composition may be administered enterally or parenterally. Suitably the VLP or immunogenic composition may be administered orally, rectally, vaginally, sublingually, by injection, transdermally, or by inhalation.

[0408] In one embodiment, the VLP or immunogenic composition may be administered by injection, suitably by subcutaneous or by intramuscular injection.

[0409] In one embodiment, the VLP or immunogenic composition may be administered by inhalation, suitably by nasal inhalation.

[0410] Subject

[0411] The present invention relates to the prevention and / or treatment of a disease in a subject by using the VLP or immunogenic composition thereof.

[0412] Suitably the subject may be human or animal. Suitably therefore the prevention and / or treatment of disease may be in the veterinary field. Suitable animals may include any mammal such as dogs, cats, cows, horses, pigs, mice, rats, monkeys etc.

[0413] In one embodiment, the subject is a horse or a dog. Suitably horses having or which have been diagnosed with sweet itch, or dogs having or which have been diagnosed with dermatitis, suitably atopic dermatitis.

[0414] Suitably the subject may be adult or child. Suitably the subject may be male or female. In one embodiment, the subject is an adult human. Suitably the subject may be any age. Suitably the subject is a human adult of old or elder age. Suitably a human adult aged over 50 years, suitably over 55 years, suitably over 60 years, suitably over 65 years, suitably over 70 years of age.

[0415] Suitably the subject may have been diagnosed with a disease. Suitably any diseases listed above. Alternatively, the subject may be suspected of having a disease. Suitably the subject may display one or more symptoms of a disease. Suitably any diseases listed above.

[0416] Alternatively, the subject may be at risk of contracting a disease. Suitably the subject may have one or more risk factors associated with a disease. Suitable risk factors may include: weight, smoking, alcohol or substance addiction, age, sex, race, inheritance for example. Suitable risk factors may further include a genetic predisposition to a disease, for example by expression of particular gene, or by the presence of a particular mutation in a gene.

[0417] In one embodiment, subjects that have been diagnosed with a disease or who have one or more symptoms of a disease are provided with the VLP or immunogenic composition for treatment of the disease. Suitably the subject may have already received or is being treated for a disease. Suitably the subject may be intractable to current treatments or standard of care for said disease. Suitably therefore the subject may be intractable i.e., non-responsive to standard treatments for any diseases listed herein, such as anti-itch creams or anti-histamine medications. Suitably therefore the subject may have an intractable itch, suitably which is non-responsive to antiitch creams or anti-histamine.

[0418] In one embodiment, the subject is a human adult of over 50 years in age having any of the above listed conditions. Suitably having an intractable itch.

[0419] In one embodiment, subjects that are at risk of developing a disease are provided with the VLP or immunogenic composition for prevention of the disease.

[0420] Other Uses

[0421] The present invention further relates to use of the VLP in research and in the diagnosis of diseases.

[0422] Suitably the VLP of the first aspect may be used in research. Suitably the VLP may be used as a detection tool. Suitably the VLP may be used as a label. Suitably in such embodiments, the VLP comprises a functional molecule which is a fluorescent molecule. Suitably the VLP may comprise IL31 polypeptides functional molecules and a further functional molecule which is a fluorescent molecule.

[0423] Suitably the VLP may comprise a further functional molecule which is an antigen binding molecule such as an antibody, and a further functional molecule which is a fluorescent molecule. Suitably the antigen binding molecule may specifically bind a cell surface receptor. Suitable cell surface receptors are discussed elsewhere herein, however suitably the cell surface receptor is specific to a cell type. Suitably therefore the VLP is capable of binding to, and labelling, specific cell types.

[0424] Suitably the VLP may be used as a carrier. Suitably in such embodiments, the VLP may comprise a cargo. Suitably the cargo may be contained within the VLP, suitably within the VLP shell. Suitably the cargo may be a therapeutic molecule. Suitably therefore the VLP may not in itself be a therapeutic, but may be a carrier of a therapeutic molecule. Suitable therapeutic molecules may include oligonucleotides, small molecules, peptides, for example. In one embodiment, the therapeutic molecule may comprise an antisense oligonucleotide which may act to repress expression of a particular nucleic acid. In another embodiment, the therapeutic molecule may comprise a cytotoxic chemical which may act to trigger cell death. Suitably, in such embodiments, the VLP is targeted to a particular site, for example to a particular cell or cell type where the therapeutic molecule is required. Suitably this is achieved by the VLP comprising a further functional molecule which is an antigen binding molecule such as an antibody. Suitably the antigen binding molecule may specifically bind to a cell surface receptor. Sutiably to a cell surface receptor specific to the target cell. Suitably binding to the cell surface receptor may stimulate uptake of the VLP into the cell. Suitably therefore, the VLP is capable of binding to specific cell types and delivering cargo thereto.

[0425] In a further aspect of the invention, there is provided a carrier VLP comprising the features of the first aspect, and in addition a cargo, wherein the cargo is contained within the VLP shell. Suitably the cargo is a therapeutic molecule.

[0426] Suitably the VLP of the first aspect may also be used in diagnosis.

[0427] Suitably the VLP comprises a further functional molecule which is an antigen binding molecule, such as an antibody. Suitably the antibody specifically binds an antigen derived from a disease causing agent as discussed hereinabove. Suitably from an infectious agent such as a virus, bacterium, fungus, protozoan, or archaeon.

[0428] Suitably, therefore, the VLP is capable of binding to a disease causing agent and allowing detection thereof.

[0429] Suitably therefore the VLP of the invention may be used in a method of diagnosing a disease. Suitably there is provided a method of diagnosing a disease in a subject comprising:

[0430] (a) Providing a VLP according to the first aspect of the invention, wherein the further functional molecule is an antigen binding molecule directed towards an antigen derived from a disease causing agent;

[0431] (b) Mixing the VLP with a suitable sample from the subject;

[0432] (c) Detecting whether the VLP precipitates;

[0433] (d) Diagnosing the presence of a disease if the VLP precipitates.

[0434] Suitably, given that the further functional molecule is an antigen binding molecule, the VLP further comprises a third binding protein. The third binding protein is described elsewhere herein. Suitably the antigen binding protein is indirectly attached to the second binding protein via a third binding protein.

[0435] Suitably detection is via precipitation of the VLP bound to the disease causing agent. Suitably detecting precipitation may comprise visual confirmation, or testing with a spectrometer.

[0436] Suitably if no precipitation occurs, the disease is not present. Suitably, the VLP may also comprise a further functional molecule which is a fluorescent molecule. Suitably such a further functional molecule may be attached to a chemical modification of the first binding protein. In such embodiments, suitably the detection step may comprise detecting the presence of fluorescence in the sample. Suitably the detection step may comprise detecting the presence of fluorescent precipitation in the sample. Suitably diagnosing the presence of a disease if fluorescent precipitation occurs.

[0437] Advantageously, the use of fluorescence allows more sensitive detection of the precipitation in a sample.

[0438] A suitable sample from a subject may be a blood sample, saliva sample, serum sample, sputum sample, sperm sample, mucus sample, CSF sample. Suitably the sample is a fluid sample.

[0439] Suitably the method of diagnosis may further comprise a step of incubating the sample with the VLP. Suitably for a period of time sufficient to allow the VLP to bind to any antigens in the sample and precipitate. Suitably for at least 1 minute, suitably up to 30 minutes, suitably up to 25 minutes, suitably up to 20 minutes, suitably up to 15minutes.

[0440] Suitable diseases which may be detected by the method may be any of those listed herein above.

[0441] Suitably the method of diagnosis may further comprise a step of treatment of the subject if a disease is diagnosed. Suitably treatment of the subject may comprise administering an effective amount of any known treatment for the relevant disease to the subject.

[0442] BRIEF DESCRIPTION OF THE DRAWINGS

[0443] Figure 1. Expression of murine and human IL31 (mlL31 and hl L31), cloned downstream of a ColE7 domain, in E. coli. (A) Schematic arrangement of expression cassettes placed within a pET Duet-derived plasmid backbone used for recombinant expression in E. coli. The illustrated example is mlL31. (B) Tertiary structure of mlL31 (dark) and hlL31 (light), generated by Alfaphold2 and overlaid using iCn3D. (C) SDS-PAGE of mlL31 (left) and hl L31 (right), cloned downstream of the identical folding chaperone domain ColE7, after induction in E. coli. Addition of IPTG induces the helper enzymes DsbC and Erv1 P required for disulfide bond formation (white arrows, marked by *). Addition of anhydrotetracycline (aTc) induces transcription of the IL31 containing cassette. The gels shown display cytosolic fractions obtained after 2h cultivation of cells in LB media. Figure 2. N-terminal peptide boundaries of mature IL31. Data show the strength of prediction of signal-peptide (SP) boundaries, performed SignalP 6.0, which has been shown to be highly accurate artificial intelligence-based SP predictor of signal peptide type and length (Teufel et al. 2022). Light-, medium-, and dark-grey lines indicate the probability of N-terminal-, centralhydrophobic, and c-terminal signal peptide sequence, respectively. The light grey dashed line represents the likelihood of mature peptide sequence. The vertical dashed line denotes the position where maximal likelihood of C-terminal part of signal peptide drops which identifies the most upstream possible start of the mature sequence. The bottom sequence in both panels shows the amino acid sequence. The sequence of N, H, C and O characters depicts computationally predicted identity of each position as part of N-terminal (N), hydrophobic (H), or C-terminal signal peptide (C), as well as mature peptide (‘O’).

[0444] Figure 3. Expression of modified hl L31 constructs harbouring N- and C-terminal deletions in E. coli. (A). Multi-sequence alignment of mature primate IL31 sequences (SEQ ID NOs: 69- 73), showing the site of sequence deletions in DU73416 and DU73424 (dashed boxes), as well as conserved locations of helices A-D (horizontal bars), and receptor-interface-contacting residues (vertical shaded boxes). (B) Expression and purification via IMAC of DU71416 (left) and DU71424 constructs harbouring modified human IL31 (hlL31). Shown are SDS-PAGE analyses of cytosolic fractions subjected to chromatography on Ni-agarose with flow through, wash fractions containing 80 mM imidazole, and elution fractions (E) containing 250 mM imidazole. White arrows denote the target protein.

[0445] Figure 4. Design of modified linkers regulating domain flexibility in hlL31 constructs. (A) Schematic indicating additional amino acids incorporated into the GS2 linker in DU71416 connecting the TEV-restriction site and the mature hlL31 peptide, yielding the constructs DU71462 (part of which is shown in SEQ ID NO: 74) and DU71463 (part of which is shown in SEQ ID NO: 75), respectively. (B) SDS-PAGE analysis of both clones of cytosolic E. coli fractions (exemplary protein sections labelled as SEQ ID NO: 76 and 77) subjected to IMAC chromatography, as in Fig. 3. White arrows denote the target protein.

[0446] Figure 5. Immunogenicity of the IL31 sequence in 71462. Virus-Like-Particles presenting either murine IL31 or hlL31 were manufactured and injected into female C57B / 6j mice subcutaneously on two occasions. One week after the second dose, mouse serum was assayed for the presence of specific antibodies by ELISA where either murine or human IL31 was immobilized to the test plate, respectively. Data shown represent detected titres against dilution for all mice, as well as median for each group. No IgG was detected in pre-immune serum (not shown). Figure 6. Modification of linker length to not only allow high level expression but also additional ability to purify the ColE7-IL31 fusion protein not only on its own, but after it has been linked to the VLP surface. (A) Three linker designs: the top sequence (SEQ ID NO: 68), contained in plasmid DU73849, is identical in structure to the linker in DU73928; the middle design (SEQ ID NO: 4), contained in DU73854 (SEQ ID NO: 57), exhibits omission of the C-terminal and TEV-site regions, resulting in eight amino acids (i.e. just the GS1 linker); the bottom design (SEQ ID NO: 51) (contained in DU73355, SEQ ID NO: 58), is shortened to three amino acids.

[0447] (B) SDS-PAGE of the epitope protein in plasmid DU73355, showing poor expression levels.

[0448] (C) IMAC of cytosols expressing either epitopes from DU73849 or DU74854 were mixed with cytosol expressing VLP scaffold from DU67867, followed by IMAC chromatography as described above. The arrows indicate epitope proteins (white, light grey) and VLP scaffold (dark grey) respectively.

[0449] Certain embodiments of the invention will now be described with reference to the following examples:

[0450] EXAMPLES

[0451] Materials & Methods used in all Examples

[0452] Cloning

[0453] Cloning was outsourced and completed as paid-for service by the cloning group of the MRC Protein Phosphorylation Unit / Reagents and Services School of Life Sciences, University of Dundee. Cloning was performed using standard custom-designed template PCR-mediated recombineering. All sequences were verified by sequencing.

[0454] Structural Imaging

[0455] Structural models were generated using AlphaFold2 using MMseqs2 via the ColabFold v1 .5.5 access portal. Analysis and annotation was performed using icn3d software in online mode as available through the National Center for Biotechnology Information (NCBI) at

[0456] Protein expression

[0457] Expression plasmids listed in the Results section were transfected into a proprietary strain termed P912, this strain is derived from BL21 / DE3 but harbouring chromosomal integration of the disulfate transferase DsbC and the disulfide isomerase Erv1 P, respectively, both under the control of an arabinose inducible promoter. E,coli BL21 / DE3 is available from for example: NewEngland BioLabs GmbH, C2527I, LOT: 10129891 , and could be used without the additional enzymes to produce the VLPs described herein. The additional enzymes increase folding efficiency of the proteins therefore, optionally, such an E.coli strain may be modified to comprise nucleic acids encoding the enzymes DsbC and Erv1 P under the control of an inducible promoter, by known molecular biology techniques such as transformation with an expression construct or vector encoding such proteins. One such available system for the expression of the enzymes DsbC and Erv1 P is the ‘CyDisCo’ system, which may be transfected into E.coli as described in: Gqciarz et al. Microb Cell Fact (2017) 16:108, such a strain may be used in a similar manner to produce the VLPs herein.

[0458] Upon addition of 0.1 % (w / v) of IPTG, these enzymes are expressed in the cytosol of E.coli and afford the formation of intramolecular disulfide bridges in the recombinant protein of interest.

[0459] Transformed strains were inoculated in typically 40 ml of LB broth from glycerol stocks and incubated in shaker flasks overnight at 30C. The following morning, they were diluted into 400 ml LB and adjusted to 37C. Upon reaching of cellular density defined by OD595 = 0.8-1.0 temperature was adjusted to 16C. For induction of the epitope protein (ColE7-linker-l L31 ), 0.1 % IPTG was added and, 15min later, anhydrotetracycline (aTc) was added to final 40 ng / ml to achieve induction of target recombinant protein of interest. For induction of VLP scaffold protein, IPTG was added to 0.3 mM. Both epitope and VLP were either induced separately or in combination as indicated in Fig. 1c. Induction was carried out for 2.5-3h unless otherwise indicated.

[0460] Protein purification by IMAC

[0461] Biomass was harvested by sedimenting cells and resuspending in lysis buffer consisting of 25 mM Tris / HCI, pH 7.4, 300 mM NaCI, 10 U / ml Benzonase, followed by disruption of cells using an Emulsiflex high pressure homogenizer (three passes). Lysates were incubated for 1h at 4°C to allow for DNA digestion, followed by clarification by centrifugation at 5,000 rpm for 10 min. Clarified lysates were serially filtered through 0.44 and 0.22 pm filters to obtain cytosol fractions. Cytosol was adjusted to 10 mM imidazole and passed over Ni-NTA agarose (from either Qiagen or Cube Biotech GmbH) using a Perkin Elmer micro HPLC pump at 1 ml / min at room temperature in 1ml column volume, followed by washing with 80 mM imidazole in the same buffer and elution at 250 mM imidazole.

[0462] VLP manufacture

[0463] VLP scaffolds were obtained by transfecting plasmid DU67867 (SEQ ID NO: 26), based on plasmid pET-Duet 1 (Novagen), exactly as described above This plasmid contained an inframe fusion of the Hepatitis B capsid (HBc) protein (SEQ ID NO: 35) and Im7 (SEQ ID NO: 36). Specifically, the amino acid sequence of the HBc protein was optimised to account for the insertion of a binding protein as follows: the negatively charged amino acids E77 and D78 were deleted to reduce the net-negative charge in the Major Immunodominant Region, the C- terminal sequence which binds RNA in native virus was removed following residue V149, and a positive-net charge sequence was added on the C-terminus to stabilize the VLPs via inserting six histidine residues downstream of V149, which are not exposed to the protein surface. The in-frame fusion was arranged by extending Im7 (SEQ ID NO: 36) on either side with a secondary linker consisting of GGGGSGGGGS (SEQ ID NO: 13) and extended on the N-terminal end with a sequence encoding M1-Leu76 of Hepatitis B core antigen, and on the C-terminal end with a sequence encoding Pro79-V149 of Hepatitis B core antigen. The HBc- Im7 fusion protein is provided in SEQ ID NO: 3 (DNA sequence in SEQ ID NO: 16).

[0464] Expression of VLP protein was induced by addition of 0.3 mM IPTG at 16°C for 16h. Cytosolic fractions were prepared as described above, except for lowering the NaCI concentration to 100mM in the lysis buffer. VLP scaffolds were purified by sequential chromatography using CaptoCore 700 on an AKTA FPLC system, followed by anion exchange chromatography using Nuvia HP agarose in a linear gradient from 0.1-1M NaCI in the presence of 200mM arginine. Identity and purity of VLP scaffolds eluting at approx. 600mM NaCI was verified by SDS PAGE. VLP’s decorated with desired cytokine epitopes were obtained by mixing purified VLP scaffolds (HBc-lm7) with IMAC-purified epitopes (ColE7-linker-IL31) having various different linker structures made as explained below in example 1 by adding volumes for both the VLP- scaffold and the epitope-containing fractions, respectively, to yield an approx. 1 :1 stoichiometric ratio, based on SDS PAGE analysis, followed by polishing and rebuffering on Sephacryl 300 HR in a 50 ml column to obtain purified VLPs formulated in PBS containing 0.05% polysorbate 80. These were stored at -20°C until in vivo application.

[0465] In vivo immunogenicity

[0466] VLPs were made to 5% (v / v) alum (Alum Hydrogel) and injected subcutaneously in the interscapular dorsal midline at approx. 10 pg in a 100 pl volume into female C57BI / 6j mice aged 8-10 weeks. Booster injections were given 14 days later. Antibody titres were determined from either tail-vein sampled peripheral blood or blood ascertained at terminal cardiac puncture, using standard ELISA with recombinant murine IL31 immobilized at 1 pg / ml in carbonate buffer ph9.0, blocking with PBS / 2% BSA, and using anti-mouse IgG-HRP at 1 :20,000 for detection. Example 1

[0467] The expression of recombinant Interleukin 31 (I L31) was achieved in E. coli by cloning IL31 fused in-frame to the C-terminus of a domain called Colicin-E7 (ColE7, amino acid sequence in SEQ ID NO: 1 , DNA sequence in SEQ ID NO: 14), which served as a chaperone for folding, while at the same time also allowing subsequent placement of the translated protein onto the surface of a Virus like Particle. For purification purposes, a short peptide tag (amino acid sequence in SEQ ID NO: 2, DNA sequence in SEQ ID NO: 15) allowing immobilized-metal affinity chromatography (IMAC as explained above) was placed on the N-terminus. The arrangement is shown in Fig. 1A. The open reading frame for the catalytic domain of Colicin E7 (GenBank accession Genbank: KJ470776.1), starting with E444, was modified to harbour the mutations: R538A, E542A (Ku, Nucleic Acids Research, 2002), His569A (Ko, Structure, 1999), ensuring complete catalytic inactivity with retained Im7-binding capacity. In addition to the functional protein domains (purification tag, ColE7, I L31 ), the figure also shows the linkers connecting the ColE7 and an intervening protease site allowing TEV1 -mediated cleavage (ENLYFQG, SEQ ID NO: 5; DNA sequence in SEQ ID NO: 18), as well as the linker connecting the TEV1 site and IL31. The linkers are flexible, composed of glycine and serine, structurally disordered, and termed GS1 (GGGSSGSG, SEQ ID NO: 4; DNA sequence in SEQ ID NO: 17) and GS2 (SGGGSG, SEQ ID NO: 6; DNA sequence in SEQ ID NO: 19), respectively. Expression of the recombinant cassette is driven by a tetA / tetR promoter, which triggers transcription of target sequence after addition of anhydrotetracycline (aTc). The cassette as such is placed within a plasmid derived from a pET-Duet backbone. For the murine IL31 sequence (mlL31 , amino acid sequence in SEQ ID NO: 10, DNA sequence in SEQ ID NO: 23), the resulting plasmid is termed DU73849 (SEQ ID NO: 27), for the human IL31 sequence (hl L31 , amino acid sequence in SEQ ID NO: 9, DNA sequence in SEQ ID NO: 22) the resulting sequence is termed DU73928 (SEQ ID NO: 28), respectively.

[0468] Although the primary protein sequences of hlL31 and mlL31 , respectively, are only 32% identical, superposition of the Alphafold2-generated tertiary structures yielded a root mean square deviation of only 1.8Å, confirming highly conserved domain fold, as shown in Fig. 1 B. Both structures exhibit an entirely preserved 4-helix domain fold, with a similar codon usage and a single conserved intramolecular disulfide bond. The expression level of recombinant proteins in E. coli is heavily influenced by the promoter used, as well as the initial translation sequence and the first translated domain which stabilizes protein folding. Therefore, cloning mlL31 and hl L31 sequences into the same cassette downstream of ColE7 was expected to yield similar magnitudes of expression. By contrast, the experimental evidence obtained showed a dramatic difference, where ColE7- mlL31 was highly expressed, while ColE7-hlL31 was only weakly detectable (Fig. 1C). Both proteins were expressed in an E. coli strain which was genetically modified to harbour enzymes driving expression of disulfide transferase proteins (marked by white arrows in Fig. 1C). These allow the intact folding of proteins with intact disulfide bonds in E. coli, which otherwise would be sorted into misfolded inclusion bodies.

[0469] This unexpected result led to review of the N-terminal starting amino acids in both hl L31 and mlL31. As shown in Fig. 2, murine IL31 harbours a clearly defined signal peptide with 90% likelihood of accurate signal peptide boundary localisation (Fig. 2 top, dashed light grey line). The boundary (vertical arrow in Fig. 2 top) was incorporated into the sequence used in clone 73849. By contrast, the precise localisation of the signal peptide boundary in human IL31 can be predicted with less precision (denoted by the broader vertical arrow in the bottom graph of Fig. 2). The site chosen as start of mature hl L31 for clone DU73928 is denoted by white arrow. It is uncertain if the physiological signal peptide boundaries have any function at all for a recombinant protein positioned downstream of a heterologous chaperone domain when expressed in E. coli, as is the case here.

[0470] Given the unexpectedly low expression, two alternative constructs of hl L31 were tested, where either three additional amino acids at the N-terminus where removed (yielding clone DU71416, SEQ ID NO: 29, containing the hlL31 variant of the amino acid SEQ ID NO: 11 and of DNA SEQ ID NO: 24) or, in addition to this deletion, also the three amino acids at the C-terminus were removed, which is disordered (yielding clone DU71424, SEQ ID NO: 30, containing hl L31 variant of amino acid SEQ ID NO: 12 and of DNA SEQ ID NO: 25). These are shown in Fig. 3A. All modifications leave intact the helical domain structure, as well as the receptor binding interfaces conserved among primate IL31 sequences.

[0471] Expression of both clones in E. coli cytosol was improved compared to the parent clone DU73928, but was still not as high as murine IL31 (Fig. 3B). However, purification of both construct by immobilised metal ion chromatography (IMAC as above) showed poor binding to Ni-agarose, suggesting steric occlusion of the purification handle needed to produce pure protein.

[0472] Based on the above results, further modifications were engineered to increase domain flexibility by elongating linker GS2 upstream of the IL31 domain. This is shown in Fig. 4A. The linker GS2 contained in clone DU71416 was extended by either three additional amino acids (arriving at GS2 linker SEQ ID NO: 7, DNA sequence in SEQ ID NO: 20), yielding clone DU71462 (SEQ ID NO: 31), or by seven additional amino acids (arriving at GS2 linker SEQ ID NO: 8, DNA sequence in SEQ ID NO: 21), yielding clone DU71463 (SEQ ID NO: 32), respectively. Both of these further modifications yielded comparable cytosolic expression to the parent clone DU71416 but with significantly improved binding to Ni-agarose in IMAC chromatography as above (Fig. 4B).

[0473] Example 2

[0474] Based on these results, construct DU71462 was incorporated into a Virus-Like Particle (VLP), manufactured, and assessed for its ability to generate antibodies specific for human IL31 in mice. As shown in Fig. 5, this construct yielded high hlL31-specific antibody titres, confirming that the folding of the protein is indistinguishable from native wild type IL31 in terms of immune recognition.

[0475] Example 3

[0476] Finally, further work was performed to explore if and to which degree the length of the linker has an effect of downstream purification strategy for the VLP. This work was carried out with plasmid constructs harbouring mouse IL31 using the same techniques as described in example 1 for assembly of the constructs. It was found that, upon shortening the linker between ColicinE7 and IL31 to just the GS1 linker (SEQ ID NO: 4), the resultant fusion protein expressed in E.coli cytosol could be directly purified via immobilized metal affinity chromatography (IMAC), when it had been pre-attached to the VLP scaffold (Fig. 6). It was further found that there is an optimal length of linker, such that a linker that was only 3 amino acids (GSG, SEQ ID NO: 51) resulted in poor expression levels. However, shortening the linker from 21 to eight amino acids resulted in this beneficial effect of direct purification.

[0477] The most likely reason for the successful IMAC-mediated purification is that, with the shortened linker between ColE7and IL31 , the affinity purification tag is far removed from the VLP surface, as depicted in Fig. 6c, therefore allowing increased access to the immobilized metal ions in the IMAC stationary phase.

[0478] A summary of the expression constructs is provided in the table below.

[0479] REFERENCES

[0480] Bachmann MF, Zeltins A, Kalnins G, Balke I, Fischer N, Rostaher A, Tars K, Favrot C., Vaccination against I L-31 for the treatment of atopic dermatitis in dogs. Journal of Allergy and Clinical Immunology, 2018. 142(1):279-81. Olomski F, Fettelschoss V, Jonsdottir S, Birkmann K, Thoms F, Marti E, Bachmann MF, Kundig TM, Fettelschoss-Gabriel A., Interleukin 31 in insect bite hypersensitivity — Alleviating clinical symptoms by active vaccination against itch. Allergy, 2020. 75(4):862-71.

[0481] Teufel, F., et al., SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol, 2022. 40(7): 1023-1025. SEQUENCES

[0482] Amino acid sequences

[0483] SEQ ID NO: 1 (Colicin-E7 i.e. Col7 used in VLP)

[0484] SEQ ID NO: 2 (purification tag)

[0485] SEQ ID NO: 3 (HBc-lm7 fusion in DU67867)

[0486] ( secondary linkers underlined, lm7 in italics , His-tag in bold )

[0487] SEQ ID NO: 4 (GS1 linker)

[0488] SEQ ID NO: 5 (TEV site)

[0489] SEQ ID NO: 6 (GS2 linker used in DU73928, DU71416, DU73849 and DU71424)

[0490] SEQ ID NO: 7 (GS2 linker used in DU71462)

[0491] SEQ ID NO: 8 (GS2 linker [GS2v2] used in DU71463)

[0492] SEQ ID NO: 9 (mature human IL31, hlL31, in DU73928) SEQ ID NO: 10 (mature murine IL31, mlL31, in DU73849)

[0493] SEQ ID NO: 11 (mature human IL31, hlL31, AN-RLL, in DU71416, DU71462, DU71463)

[0494] SEQ ID NO: 12 (mature human IL31, hlL31, AN-RLL, AC- A AT in DU71424)

[0495] SEQ ID NO: 13 (secondary linker surrounding Im7 in plasmid DU67867)

[0496] SEQ ID NO: 68 (full primary linker in DU73928, DU71424, DU71416, DU73849)

[0497] SEQ ID NO: 33 (full primary linker in DU71462)

[0498] SEQ ID NO: 34 (full primary linker in DU71463)

[0499] SEQ ID NO: 35 (Hepatitis B capsid used in VLP)

[0500] ( note 'X' indicates position of binding protein insertion, for example of lm7 insertion, as shown above in SEQ ID NO : 3 )

[0501] SEQ ID NO: 36 (Im7 used in VLP) SEQ ID NO: 37 (Barstar used in VLP)

[0502] SEQ ID NO: 38 (Barnase used in VLP)

[0503] SEQ ID NO: 39 (wild type Barstar, ref seq WP_007408543)

[0504] SEQ ID NO: 40 (wild type Barnase, ref seq WP_223204413)

[0505] SEQ ID NO: 41 (wild type Colicin E7, ref seq WP_024258710)

[0506] SEQ ID NO: 42 (wild type Hepatitis B capsid protein, ref seq YP_009173868)

[0507] [ immunodominant region underlined]

[0508] SEQ ID NO: 43 (Colicin E7-linker[21aa]-mouse IL31, in DU73849)

[0509] [ italics = purification tag, underline = linker]

[0510] SEQ ID NO: 44 (Colicin E7-linker[21aa]-human IL31, in DU73928)

[0511] [ italics = purification tag, underline = linker]

[0512] SEQ ID NO: 45 (Colicin E7-linker[21aa]-human IL31 AN-RLL, in DU71416)

[0513] [ italics = purification tag, underline = linker]

[0514] SEQ ID NO: 46 (Colicin E7-linker[21aa]-human IL31 AN-RLL AC- A AT, in DU71424)

[0515] [ italics = purification tag, underline = linker]

[0516] SEQ ID NO: 47 (Colicin E7-linker[24aa]-human IL31 AN-RLL, in DU71462)

[0517] [ italics = purification tag, underline = linker] SEQ ID NO: 48 (Colicin E7-linker[28aa]-human IL31 AN-RLL, in DU71463)

[0518] [ italics = purification tag, underline = linker]

[0519] SEQ ID NO: 49 (Colicin E7-linker[8aa]-murine IL31 in DU73854)

[0520] [ italics = purification tag, underline = linker]

[0521] SEQ ID NO: 50 (Colicin E7-linker[3aa]~ murine IL31 in DU73355)

[0522] [ italics = purification tag, underline = linker]

[0523] SEQ ID NO: 51 (shortened GS1 linker, used in DU73355)

[0524] SEQ ID NO: 52 (TEV cleavage site / sequence)

[0525] SEQ ID NO: 53 (HRV3C cleavage site / sequence)

[0526] SEQ ID NO: 54 (Factor Xa cleavage site / sequence) SEQ ID NO: 55 (Thrombin cleavage site / sequence)

[0527] SEQ ID NO: 56 (Enterokinase cleavage site / sequence)

[0528] SEQ ID NO: 78 (wild type Im7 protein, accession # Q03708.2)

[0529] Nucleotide sequences

[0530] SEQ ID NO: 14 (Colicin-E7 i.e. Col7 used in VLP)

[0531] SEQ ID NO: 15 (purification tag)

[0532] SEQ ID NO: 16 (HBc-lm7 fusion in DU67867) SEQ ID NO: 17 (GS1 linker)

[0533] SEQ ID NO: 18 (TEV site used in all constructs)

[0534] SEQ ID NO: 19 (GS2 linker used in DU73928, DU71416, DU73849 and DU71424)

[0535] SEQ ID NO: 20 (GS2 linker used in DU71462)

[0536] SEQ ID NO: 21 (GS2 linker used in DU71463)

[0537] SEQ ID NO: 22 (mature human IL31, hlL31, in DU73928)

[0538] SEQ ID NO: 23 (mature murine IL31, mlL31, in DU73849)

[0539] SEQ ID NO: 24 (mature human IL31, hlL31, AN-RLL, in DU71416, DU71462, DU71463)

[0540] SEQ ID NO: 25 (mature human IL31, hlL31, AN-RLL, AC- A AT in DU71424)

[0541] SEQ ID NO: 26 (DU67867)

[0542]

[0543] SEQ ID NO: 27 (DU73849)

[0544]

[0545] SEQ ID NO: 28 (DU73928)

[0546]

[0547] SEQ ID NO: 29 (DU71416)

[0548] SEQ ID NO: 30 (DU71424)

[0549]

[0550] SEQ ID NO: 31 (DU71462)

[0551]

[0552] SEQ ID NO: 32 (DU71463)

[0553]

[0554] SEQ ID NO: 57 (DU73854)

[0555] 

[0556]

[0557] SEQ ID NO: 58 (DU73355) SEQ ID NO: 59 (Colicin E7-linker[21aa]-human IL31, in DU73849)

[0558] SEQ ID NO: 60 (Colicin E7-linker[21aa]-human IL31, in DU73928)

[0559] SEQ ID NO: 61 (Colicin E7-linker[21aa]-human IL31 AN-RLL, in DU71416)

[0560]

[0561] SEQ ID NO: 62 (Colicin E7-linker[21aa]-human IL31 AN-RLL AC- A AT, in DU71424)

[0562] SEQ ID NO: 63 (Colicin E7-linker[24aa]-human IL31 AN-RLL, in DU71462)

[0563] SEQ ID NO: 64 (Colicin E7-linker[28aa]-human IL31 AN-RLL, in DU71463)

[0564] SEQ ID NO: 65 (Colicin E7-linker[8aa]-murine IL31 in DU73854) SEQ ID NO: 66 (Colicin E7-linker[3aa]~ murine IL31 in DU73355) SEQ ID NO: 67 (T7 promoter)

Claims

CLAIMS1. A virus-like particle (VLP) comprising:One or more viral capsid protein,One or more pairs of binding proteins, each pair of binding proteins comprising a first binding protein and a second binding protein, wherein the pair of binding proteins comprises a bacterial toxin and its inhibitor,One or more functional molecules, wherein at least one of the functional molecules is an IL31 polypeptide, or a functional fragment thereof, wherein each viral capsid protein is attached to a first binding protein, wherein each functional molecule is attached to a second binding protein via a linker, and wherein the first and second binding proteins are capable of binding to each other.

2. A VLP according to claim 1 , wherein the one or more viral capsid proteins are homodimers.

3. A VLP according to claim 1 or 2, wherein each linker comprises at least 8 amino acid residues, preferably between 8 and 30 amino acid residues.

4. A VLP according to any preceding claim, wherein the linker comprises at least one region that is rich in glycine and serine residues, preferably wherein a majority of the residues in the first region are glycine and serine, more preferably wherein the or each region consists of glycine and serine residues.

5. A VLP according to claim 4, wherein the linker comprises a first and a second region that are rich in glycine and serine residues, preferably wherein a majority of the residues in the first and second region are glycine and serine, more preferably wherein the first and second region consist of glycine and serine residues.

6. A VLP according to claims 4 or 5, wherein the linker further comprises a third region, preferably wherein the third region comprises a cleavage site, more preferably a protease cleavage site, even more preferably a TEV protease cleavage site having a sequence according to SEQ ID NO: 52.

7. A VLP according to any of claims 4-6, wherein the linker comprises the following structure from N-to-C terminus: the first region, the third region, and the second region.

8. A VLP according to any of claims 4-7, wherein the or each region that is rich in glycine and serine residues comprises a sequence selected from: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

9. A VLP according to claims 7 or 8, wherein the linker comprises a sequence with 70% or greater identity to SEQ ID NO: 4, SEQ ID NO: 33, or SEQ ID NO: 34.

10. A VLP according to any preceding claim, wherein the IL31 polypeptide or a functional fragment thereof is human IL31 or a functional fragment thereof, preferably wherein the IL31 polypeptide comprises a sequence 70% or greater identity to SEQ ID NO: 9 or 11 or 12.

11. A VLP according to any preceding claim, wherein the first binding protein is the bacterial toxin inhibitor, and the second binding protein is the bacterial toxin, optionally wherein the bacterial toxin inhibitor is selected from Im7, Im8, Im9, Im2, and Barstar, and / or optionally wherein the bacterial toxin is a bacterial nuclease, preferably the bacterial nuclease is selected from: ColE7, C0IE8, ColE9, ColE2, and Barnase.

12. A VLP according to any preceding claim, wherein the bacterial toxin and its inhibitor are ColE7 and Im7, preferably wherein Im7 comprises a sequence with 70% or greater identity to SEQ ID NO: 36 and / or wherein ColE7 comprises a sequence with 70% or greater identity to SEQ ID NO: 1.

13. A VLP according to any preceding claim, wherein the viral capsid protein is a Hepatitis B capsid protein, preferably a Hepatitis B capsid protein of 70% or greater identity to SEQ ID NO: 42.

14. A VLP according to claim 13, wherein the first binding protein is inserted into the major immunodominant region of the Hepatitis B capsid protein wherein the major immunodominant region is located between amino acids 75 to 81 of SEQ ID NO: 42, preferably the first binding protein is inserted between amino acid residues 76 and 79 of the major immunodominant region of the Hepatitis B capsid protein according to SEQ ID NO: 42, preferably wherein the Hepatitis B capsid protein comprises a sequence according to SEQ ID NO: 35.

15. A VLP according to any preceding claim, further comprising a secondary linker, preferably wherein each viral capsid protein is attached to a first binding protein via a secondary linker,more preferably wherein the secondary linker comprises a sequence according to SEQ I D NO: 13.

16. A VLP according to any preceding claim, wherein each viral capsid protein attached to a first binding protein comprises a sequence having at least 70% identity to SEQ ID NO: 3.

17. A VLP according to any preceding claim, wherein each functional molecule is an IL31 polypeptide or a functional fragment thereof.

18. A functional fusion protein comprising an IL31 polypeptide or a functional fragment thereof fused via a linker to a binding protein wherein the binding protein is a bacterial toxin.

19. A functional fusion protein according to claim 18, wherein the IL31 polypeptide, the linker, and the binding protein are defined according to any of claims 1-17.

20. A functional fusion protein according to claim 18 or 19 comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 43, 44, 45, 46 ,47, 48, 49, or 50, preferably wherein the functional fusion protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 47, 48 or 49.

21. A functional fusion protein according to any of claims 18-20, wherein the protein further comprises an affinity purification tag.

22. One or more nucleic acids encoding the functional fusion protein according to any of claims 18-21 , preferably wherein the nucleic acid comprises a sequence having at least 70% identity to SEQ ID NO: 59-66, more preferably wherein the nucleic acid comprises a sequence having at least 70% identity to SEQ ID NO: 63, 64 or 65.

23. One or more vectors comprising the one or more nucleic acids of claim 22, preferably wherein the vector comprises a sequence having at least 70% identity to SEQ ID NO: 27-32, 57 or 58, more preferably wherein the vector comprises a sequence having at least 70% identity to SEQ I D NO: 31 , 32 or 57.

24. A host cell comprising the one or more nucleic acids of claim 22 or the one or more vectors of claim 23.

25. A host cell comprising one or more vectors, the one or more vectors comprising a first nucleic acid encoding a viral capsid protein attached to a first binding protein, and a second nucleic acid encoding an I L31 polypeptide or a functional fragment thereof attached via a linker to a second binding protein; wherein the first and second binding proteins are capable of binding to each other.

26. A host cell according to claim 25, wherein the first nucleic acid is selected from a sequence having at least 70% identity to SEQ ID NO: 16, and wherein the second nucleic acid is selected from a sequence having at least 70% identity to SEQ ID NO: 59-66, preferably wherein the second nucleic acid is selected from a sequence having at least 70% identity to SEQ ID NO: 63, 64, or 65.

27. A process of producing a virus-like particle (VLP) in a single host cell comprising:(a) Providing a host cell comprising one or more vectors, wherein the one or more vectors comprise:(i) a first nucleic acid encoding a viral capsid protein attached to a first binding protein; and(ii) a second nucleic acid encoding an IL31 polypeptide or a functional fragment thereof attached via a linker to a second binding protein;(b) Culturing the host cell under conditions suitable to express proteins from the first and second nucleic acids;(c) Forming VLPs from the proteins; and(d) Optionally further comprising a step of recovering the virus-like particles (VLPs), and optionally a step of formulating the VLPs28. A process of producing a virus-like particle (VLP), comprising:(a) Providing a first host cell comprising one or more vectors, wherein the one or more vectors comprise a first nucleic acid encoding a viral capsid protein attached to a first binding protein;(b) Providing at least one further host cell comprising one or more vectors, wherein the one or more vectors comprise a second nucleic acid encoding an IL31 polypeptide or a functional fragment thereof attached via a linker to a second binding protein;(c) Culturing the host cells under conditions suitable to express proteins from the first and second nucleic acids;(d) Recovering the proteins;(e) Mixing the proteins to form virus-like particles under conditions allowing binding of the proteins; and(f) Optionally further comprising a step of recovering the virus-like particles (VLPs), and optionally a step of formulating the VLPs29. An immunogenic composition comprising a VLP according to any of claims 1-17.

30. A VLP according to of any of claims 1-17, or an immunogenic composition according to claim 29, for use as a medicament, preferably wherein the medicament is a vaccine.

31. A VLP according to of any of claims 1-17, or an immunogenic composition according to claim 29, for use in the prevention and / or treatment of an immune-mediated condition or disease, preferably for use in the prevention and / or treatment of a condition selected from: dermatological conditions, allergic conditions, and onco-haematological conditions, more preferably for use in the prevention and / or treatment of a dermatological condition involving itching, still more preferably for use in the prevention and / or treatment of dermatitis.

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