Veterinary compositions of modified virus-like particles of CMV and NGF antigens

Abstract

The present invention relates to compositions comprising modified virus-like particles (VLPs) of Cucumber Mosaic Virus (CMV), particularly modified VLPs of CMV comprising a chimeric CMV polypeptide comprising a stretch of consecutive negatively charged amino acids selected from aspartic acid or glutamic acid to which a nerve growth factor (NGF) antigen is bound, and pharmaceutical compositions thereof, which preferably function as a vaccine platform for generating an immune response, particularly an antibody response, against the NGF antigen bound to the modified CMV VLP.

Description

[Technical Field]

[0001] The present invention relates to veterinary compositions comprising modified virus-like particles (VLPs) of Cucumber Mosaic Virus (CMV), in particular modified VLPs of CMV comprising a chimeric CMV polypeptide comprising a stretch of consecutive negatively charged amino acids selected from aspartic acid or glutamic acid to which a nerve growth factor (NGF) antigen is attached, and pharmaceutical compositions thereof, which preferably function as a vaccine platform for generating an immune response, in particular an antibody response, against the NGF antigen attached to the modified CMV VLP. [Background technology]

[0002] Virus-like particles (VLPs) have become an established and accepted vaccine technology, especially as immunological carriers for inducing strong immune responses against conjugated antigens (Zeltins A, Mol Biotechnol (2013) 53:92-107; Jennings GT and Bachmann MF, Annu Rev Pharmacol Toxicol (2009) 49:303-26, Jennings GT and Bachmann MF, Biol Chem (2008) 389:521-536).

[0003] Recently, a vaccine platform based on cucumber mosaic virus (CMV, family Bromoviridae, genus Cucumovirus) virus-like particles (CMV VLPs) has been described using chemical linker coupling technology to present various antigens, including autoantigens such as cytokines, on their surface and induce effective neutralizing antibody responses. These soluble and stable CMV VLPs serve as an outstanding platform due to their unique properties, such as repeated presentation of target antigens to B cell receptors, nanoscale dimensions and geometry, and activation of innate immunity through TLR activation and T cell helper responses (WO 2016 / 062720; Zeltins A et al. Vaccines 2 (2017) 30; Bachmann MF et al. Frontiers in Microbiology Vol. 9, Article 2522, October 2018; von Loga IS et al. Ann. Rheum Dis 2019, 78:672-675; WO 2021 / 260131).

[0004] Despite the progress made in developing these multifunctional VLP-based vaccines, challenges and requirements remain, particularly for potential clinical trials, product registration, market launch, and commercial supply. Control of product characteristics, such as stability, shelf life, solubility, manufacturability (including scalability), safety, efficacy, bioavailability, and other pharmacological properties, is particularly important. These are key elements of the chemistry, manufacturing, and control (CMC) process required to cost-effectively deliver these products in sufficient quantities for potential clinical trials, product registration, market launch, and commercial supply (Pham NG, Int J Pharm, 2020, 585:119-523). The stability of these VLP platforms and VLP-based vaccines, even under the various conditions required for an effective CMC process, is particularly important. Another undesirable event and challenge that can adversely affect product performance is aggregation, both for biopharmaceuticals and vaccines, respectively (Roberts CJ, Current Opinion in Biotechnology, 2014, 30:211-217). Aggregated vaccines, if their native structure is maintained, can still induce an immune response, but while still suitable for some laboratory-scale testing, they are not acceptable for clinical studies and commercially manufactured GMP products.

[0005] Thus, despite the progress made in developing these multifunctional VLP-based vaccines, there remains a need for the development of modified VLP systems adapted to address potential challenges and meet potential product registration and market launch requirements.

[0006] Nerve growth factor (NGF) was discovered as a key factor for the development and maintenance of sensory and sympathetic neurons in the developing nervous system. It functions as a soluble signaling protein that mediates its activity by binding to two distinct cell surface receptors (NGF-Rs): the high-affinity NGF-specific tropomyosin receptor kinase A (TrkA) and the low-affinity p75 neurotrophin receptor (p75NTR). The amino acid sequences of canine and feline nerve growth factor and their corresponding orthologs from other animal species have been identified and are known to those skilled in the art. Summary of the Invention

[0007] The present inventors have surprisingly found that compositions of the present invention comprising modified CMV VLPs to which an NGF antigen is bound are not only highly immunogenic and induce high titers of neutralizing antibodies against the NGF antigen in vitro, but also that the CMV VLP-NGF conjugates of the present invention retain their stability and structural integrity. This was particularly surprising because the inclusion of additional negative charges within VLP-forming proteins, such as the inserted stretch of consecutive negatively charged amino acids selected from glutamic acid and aspartic acid described in the present invention, can adversely affect VLP formation. In contrast, under conditions of high temperature and high ionic strength, the specific insertion of these consecutive stretches of negatively charged amino acids not only further stabilizes the resulting modified CMV VLPs compared to prior art CMV VLPs, but also that the CMV VLP-NGF conjugates of the present invention do not form aggregates and remain stable in solution upon binding to an NGF antigen, whereas prior art CMV VLPs form large aggregates and precipitate upon binding. Such aggregation and the formation of aggregated, conjugated CMV VLPs are highly undesirable for drug development and product registration, and the substantial reduction or avoidance of such undesirable aggregation by the compositions of the present invention is highly beneficial. Furthermore, the improved stability in high salt solutions resulting from surface charge modifications to CMV VLPs is further beneficial or even important for their processability and purification by ion exchange chromatography, particularly anion exchange chromatography, and advantageously allows for easier scalable manufacture of the compositions of the present invention.

[0008] Therefore, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one first binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; A modified VLP of CMV comprising, preferably consisting of, (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); a composition, preferably a veterinary composition, comprising: (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0009] Further aspects and embodiments of the present invention will become apparent as this description continues. [Brief explanation of the drawings]

[0010] [Figure 1] Illustration of the pET-CMVB2-Ntt-E8* plasmid map with single-cutting restriction enzyme sites. [Figure 2A]SDS-PAGE gel analysis of purified VLPs derived from expression of CMV-Ntt830-E8*. M - protein size marker PageRuler (Thermo Fisher Scientific, #26620). S - soluble protein in the cell extract of E. coli C2566 / pET-CMVB2-Ntt-E8*. P - insoluble protein in the cell extract. 1 - insoluble protein after sucrose gradient (bottom of tube). 2 - 6 - sucrose gradient fractions (from 60% at the bottom to 0% at the top). Asterisks (*) indicate the relative position of the corresponding CMV-Ntt830-E8* chimeric CMV polypeptide in the SDS / PAGE gel. [Figure 2B] Electron microscopy image of purified CMV-Ntt830-E8* VLPs. Horizontal bars correspond to 500 nm. [Figure 3] Illustration of the pET-CMVB2-Ntt-E4 plasmid map with single-cutting restriction enzyme sites. [Figure 4] Illustration of the pET-CMVB2-Ntt-E8 plasmid map with single-cutting restriction enzyme sites. [Figure 5] Illustration of the pET-CMVB2-Ntt-E12 plasmid map with single-cutting restriction enzyme sites. [Figure 6] SDS-PAGE gel analysis (left) and agarose gel analysis (right) of purified VLPs derived from expression of CMV-Ntt830-E4. M1 - protein size marker PageRuler (Thermo Fisher Scientific, #26620), M2 - DNA size marker (Thermo Fisher Scientific, #SM0311), total protein in E. coli C2566 cells after 18 h of incubation at -20°C, S - soluble protein in cell extract after cell division prior to the sucrose gradient (20-60%), P - insoluble protein, and 1-6-sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisks (*) indicate the relative positions of the corresponding CMV-Ntt830-E4 chimeric CMV polypeptides in the SDS / PAGE gel and typical VLP signals in the agarose gel. [Figure 7] SDS-PAGE gel analysis (left) and agarose gel analysis (right) of purified VLPs derived from expression of CMV-Ntt830-E8. M1 - protein size marker PageRuler (Thermo Fisher Scientific, #26620), M2 - DNA size marker (Thermo Fisher Scientific, #SM0311), total protein in E. coli C2566 cells after 18 h of incubation at -20°C, S - soluble protein in cell extract after cell division prior to the sucrose gradient (20-60%), P - insoluble protein, and 1-6-sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisks (*) in the figure indicate the relative position of the corresponding CMV-Ntt830-E8* chimeric CMV polypeptide in the SDS / PAGE gel and the typical VLP signal in the agarose gel. [Figure 8] SDS-PAGE gel analysis (left) and agarose gel analysis (right) of purified VLPs derived from expression of CMV-Ntt830-E12. M1 - protein size marker PageRuler (Thermo Fisher Scientific, #26620), M2 - DNA size marker (Thermo Fisher Scientific, #SM0311), total protein in E. coli C2566 cells after 18 h of incubation at -20°C, S - soluble protein in cell extract after cell division prior to the sucrose gradient (20-60%), P - insoluble protein, and 1-6-sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisks (*) in the figure indicate the relative position of the corresponding CMV-Ntt830-E12 chimeric CMV polypeptide in the SDS / PAGE gel. No clear, distinct band corresponding to intact VLPs was observed in the agarose gel. [Figure 9] Electron microscopy image of purified CMV-Ntt830-E4 VLPs. Horizontal bars correspond to 200 nm. [Figure 10] Electron microscopy image of purified CMV-Ntt830-E8 VLPs. Horizontal bars correspond to 200 nm. [Figure 11] Comparison of the thermal stability of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP. Structural changes in CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP were monitored in the presence of Sypro-Orange dye using a DNA melting temperature determination program and a real-time PCR system. Curve 1 is for CMV-Ntt830-E4 VLP, curve 2 is for CMV-Ntt830 VLP, and curve 3 is for the buffer control (5 mM Na phosphate, 2 mM EDTA, pH 7.5). The melting temperatures of 57°C and 51°C, respectively, are indicated by arrows. [Figure 12] Stability of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP in solution in the presence of different NaCl concentrations. Samples of 0.5 mg / mL CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP were incubated for up to 2 hours at room temperature in 5 mM Na phosphate, 2 mM EDTA (pH 7.5) with different NaCl concentrations (the molar concentration of NaCl in each sample is indicated at the bottom of the gel). Samples were analyzed by native agarose gel electrophoresis and ethidium bromide staining. Panels A and B show NAGE analysis of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP samples, respectively. M indicates the lane loaded with GeneRuler 1 kb DNA ladder (SM0311, TFS). Black arrows indicate the location of the loaded gel within the well and the VLPs within the well and gel. The presence of CMV-Ntt830 VLPs in the loaded wells after electrophoresis (panel A) is due to the formation of VLP aggregates that are too large to enter the gel. Intact, unaggregated VLPs migrated inside the gel. [Figure 13]Analysis of CMV-Ntt830 VLPs subjected to anion exchange chromatography. Five mL of 1 mg / mL CVMtt-VLPs in 5 mM sodium borate buffer (pH 9.0) was loaded onto a 1.0 mL Macro-Prep DEAE Bio-Rad anion exchange cartridge equilibrated with 5 mM sodium borate buffer and eluted stepwise with increasing concentrations of NaCl (0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1.0, and 2.0 M). Fractions were collected and analyzed for protein concentration and native agarose gel electrophoresis at 260 nm using a Nanodrop™. Panel A shows the 260 nm absorbance values ​​plotted against NaCl concentration and the respective fractions (1–25). Panel B shows NAGE analysis (ethidium bromide staining) of the major fraction containing the highest protein concentration. M indicates the lane loaded with GeneRuler 1 kb DNA ladder (SM0311, TFS). Black arrows indicate the position of the loaded gel within the well and the position of the VLPs within the well and gel. The presence of CMV-Ntt830 VLPs in the loaded well after electrophoresis is due to the formation of VLP aggregates that are too large to enter the gel. Intact, unaggregated VLPs migrated to the inside of the gel. [Figure 14]Analysis of CMV-Ntt830-E4 VLPs subjected to anion exchange chromatography. Biomass of Escherichia coli (E. coli) cells expressing CMV-Ntt830-E4 VLPs was resuspended in 50 mM citrate, 5 mM borate buffer (pH 9.0), and cells were lysed using a microfluidizer LM-20. The soluble fraction was clarified by centrifugation and loaded onto a 60 mL Fracto-DEAE (XK 26 / 20) column. Bound VLPs were eluted by applying an elution buffer containing 50 mM citrate, 5 mM borate, and 1 M NaCl in a continuous gradient. Panel A shows the protein elution and NaCl gradient measured by A260 nm (mAU) and conductivity (mS / cm), respectively. The x-axis indicates the elution volume and fraction number (4–11). Fractions collected from the Fracto-DEAE column were analyzed by NAGE (panel B) and SDS-PAGE (panel C). In panel B, M indicates the lane loaded with GeneRuler 1 kb DNA ladder (SM0311, TFS), L indicates a sample of E. coli lysate before loading onto the Fracto-DEAE column, FT indicates the flow-through collected from 0 to 150 mL, and 4 to 10 indicate the fraction numbers collected during elution. Black arrows from top to bottom indicate the location of the loaded well, the location of the intact CMV-Ntt830-E4 VLP inside the gel, and contaminating nucleic acids from the clarified bacterial lysate, respectively. In panel C, FT indicates the flow-through collected from 0 to 150 mL, and 4 to 10 indicate the fraction numbers. Black arrows indicate the location of the Coomassie blue-stained CMV-Ntt830-E4 coat protein. [Figure 15A] Purification and authenticity of recombinant canine mature NGF. SDS-PAGE analysis of the NGS purification process. A - total cell lysate after expression, B - pooled fractions containing proNGF after refolding and partial purification, C - mature NGF after trypsin digestion and final purification, with M-marker and molecular weights of bands indicated in kDa. Arrows indicate proNGF in lanes A and B, and mature NGF in lane C. [Figure 15B]PC12 wells were grown for 5 days with recombinant human mature NGF (black squares) produced in mouse myeloma cells (R&D Systems) or canine mature NGF (gray circles) produced in E. coli as described herein. Cells were grown in the presence of 100, 50, 25, 12.5, and 6.25 ng / mL of recombinant NGF, and the percentage of cells with defined neurite outgrowth was determined. [Figure 16A] SDS-PAGE analysis of the coupling of recombinant mature canine NGF (cNGF) of SEQ ID NO: 31 with CMV-Ntt830 and CMV-Ntt830-E8* VLPs. M-PageRuler™ Plus Prestained Protein Ladder, 10-250 kDa (Thermo Fisher Scientific, #26620) protein size markers; 1—corresponding purified CMV-Ntt830 and CMV-Ntt830-E8* VLPs; 2—CMV VLP derivatized with 5x SMPH and SMPH removal; 3—CMV VLP coupled with a molar equivalent of cNGF; 4—mixed sample of CMV-Ntt830-E8* and cNGF without SMPH derivatization; 5—purified cNGF. Asterisks indicate the location of the observable CMV VLP-NGF conjugate band. [Figure 16B] SDS-PAGE analysis of the coupling of recombinant mature canine NGF (cNGF) of SEQ ID NO: 31 to CMV-Ntt830-E4 and CMV-Ntt830-E8 VLPs. M-PageRuler™ Plus Prestained Protein Ladder, 10-250 kDa (Thermo Fisher Scientific, #26620) protein size markers; 1—corresponding purified CMV-Ntt830-E4 and CMV-Ntt830-E8 VLPs; 2—CMV VLP derivatization with 5x SMPH and removal of SMPH; 3—CMV VLP coupled with a molar equivalent of cNGF; 4—mixture of CMV-Ntt830-E4 or CMV-Ntt830-E8 and cNGF without SMPH derivatization; 5—purified cNGF. Asterisks indicate the location of the observable CMV VLP-cNGF conjugate band. [Figure 16C]Dynamic light scattering analysis of cNGF-CMV-Ntt830 VLPs. EM analysis was not possible due to vaccine precipitation. [Figure 16D] Dynamic light scattering analysis of cNGF-CMV-Ntt830-E4 VLPs containing the cNGF antigen of SEQ ID NO: 31. [Figure 16E] Dynamic light scattering analysis of cNGF-CMV-Ntt830-E4 VLPs containing the cNGF antigen of SEQ ID NO: 33. [Figure 16F] Dynamic light scattering analysis of cNGF-CMV-Ntt830-E8*VLPs. [Figure 16G] Electron microscopy of cNGF-CMV-Ntt830-E4 VLPs. [Figure 16H] Electron microscopy of cNGF-CMV-Ntt830-E8*VLPs. [Figure 17A] Evaluation of anti-NGF IgG antibodies from the sera of mice immunized with cNGF-CMV-Ntt830-E8*VLP. Anti-NGF IgG titers were measured by ELISA in mice immunized twice (days 0 and 14 indicated by arrows) with 15 μg of cNGF-CMV-Ntt830-E8*VLP with or without Quil A adjuvant (filled and open circles, respectively). [Figure 17B] To test for neutralizing IgG antibodies generated in mice, PC12 cells were grown for 5 days in the presence of 12.5 ng / mL human mature NGF (or none as a negative control) in the presence of either anti-human NGF polyclonal antibody (BioTechne) or purified IgG from naive mice (ms pIgG NAIVE) or mice immunized with cNGF-CMV-Ntt830-E8*VLP (pooled serum from study days 21, 28, and 35, ms pIgG NGF Vaccine) at the indicated concentrations. Data points represent sample replicates. [Figure 18A]Evaluation of anti-NGF IgG antibodies from the serum of dogs immunized with cNGF-CMV-Ntt830-E8*VLP. Anti-NGF IgG titers from dogs in Group 1 that received the vaccine without adjuvant. Arrows indicate injections of vaccine administered on days 0, 21, and 42. [Figure 18B] Evaluation of anti-NGF IgG antibodies from the serum of dogs immunized with cNGF-CMV-Ntt830-E8*VLP. Anti-NGF IgG titers from three dogs in Group 2 that received a vaccine containing the adjuvant QuilA®. Arrows indicate injections of vaccine administered on days 0, 21, and 42. [Figure 18C] Evaluation of anti-CMV IgG titers from sera of dogs immunized with cNGF-CMV-Ntt830-E8*VLP. Anti-CMV IgG titers from dogs in Group 1 that received the vaccine without adjuvant. Arrows indicate injections of vaccine administered on days 0, 21, and 42. [Figure 18D] Evaluation of anti-CMV IgG antibody titers from sera of dogs immunized with cNGF-CMV-Ntt830-E8*VLP. Anti-CMV IgG titers from dogs in Group 2 that received a vaccine containing the adjuvant QuilA®. Arrows indicate vaccine injections administered on days 0, 21, and 42. [Figure 18E] Assessment of anti-NGF IgG antibodies from sera of dogs immunized with adjuvant-free cNGF-CMV-Ntt830-E4 VLP. Five dogs were administered cNGF-CMV-Ntt830-E4 VLP on days 0 and 21. NGF-specific antibodies were assessed by ELISA in sera collected on days 0, 21, 42, 71, and 91. [Figure 18F] Evaluation of anti-NGF IgG antibodies from the serum of dogs immunized with cNGF-CMV-Ntt830-E4 VLP in the presence of aluminum hydroxide. Five dogs were administered cNGF-CMV-Ntt830-E4 VLP with aluminum hydroxide on days 0 and 21. NGF-specific antibodies were determined by ELISA on days 0, 21, 42, 71, and 91. [Figure 19A] Vaccination with cNGF-CMV-Ntt830-E8*VLP induces NGF-neutralizing antibodies in dogs. Dogs (3 dogs per group) were immunized with 250 μg of cNGF-CMV-Ntt830-E8*VLP in the presence or absence of the adjuvant QuilA on days 0, 21, and 42. Serum was collected and tested for the presence of neutralizing antibodies using a TF-1-based NGF bioactivity assay. Representation of a titration curve from one dog to determine the neutralizing capacity of canine serum and the 50% neutralization titer (NT50). 5 ng / mL human mature NGF was preincubated with increasing concentrations of IgG purified from serum collected on the indicated days after the first vaccination. NT50 values, i.e., the IgG concentration resulting in 50% inhibition of cell proliferation, were determined using a 4PL sigmoidal curve fit model. [Figure 19B] Vaccination with cNGF-CMV-Ntt830-E8*VLP induces NGF-neutralizing antibodies in dogs. Dogs (3 dogs per group) were immunized with 250 μg of cNGF-CMV-Ntt830-E8*VLP in the presence or absence of the adjuvant QuilA on days 0, 21, and 42. Serum was collected and tested for the presence of neutralizing antibodies using a TF-1-based NGF bioactivity assay. Total IgG was purified from dog serum. The ability of 20 μg / mL of purified total IgG to neutralize 5 ng of human mature NGF / mL was assessed using a bioassay. Bars represent mean group values ​​with standard deviations, and symbols represent individual dogs (means of duplicate determinations). Two-way ANOVA with Tukey's multiple comparison test was performed to compare group means using GraphPad Prism. *p<0.05, **<0.01, ***p<0.001, ****p<0.0001. [Figure 19C]Vaccination with cNGF-CMV-Ntt830-E8*VLP induces mature NGF-neutralizing antibodies in dogs. Dogs (3 dogs per group) were immunized with 250 μg of cNGF-CMV-Ntt830-E8*VLP in the presence or absence of the adjuvant QuilA on days 0, 21, and 42. Serum was collected and tested for the presence of neutralizing antibodies using a TF-1-based NGF bioactivity assay. NT50 values ​​were plotted against OD50 values ​​of anti-NGF IgG serum titers. Total IgG purified from serum with higher concentrations of NGF-specific antibodies was more potent, inhibiting NGF-mediated TF-1 cell proliferation than total IgG purified from serum of dogs with lower anti-NGF titers. Symbols represent individual dogs and sampling time points. Different symbols were assigned to different dogs. Filled symbols represent animals vaccinated in the presence of adjuvant, while open symbols represent animals vaccinated without adjuvant. [Figure 19D] Vaccination with cNGF-CMV-Ntt830-E4 VLP induces NGF-neutralizing antibodies in dogs. cNGF-CMV-Ntt830-E4 VLP containing aluminum hydroxide was administered to five dogs on days 0 and 21. Serum was collected on day 42 and tested for the presence of neutralizing antibodies using a TF-1-based NGF bioactivity assay. Bars represent mean group values ​​with standard deviations, and symbols represent individual dogs. The dotted line indicates the detection limit of the assay. DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The embodiments, preferred embodiments, and highly preferred embodiments described and disclosed herein should apply to all aspects and other embodiments, preferred embodiments, and highly preferred embodiments, whether specifically recited or their repetition is avoided for the sake of brevity. The articles "a" and "an," as used herein, refer to one or more than one (i.e., at least one) of the grammatical object of the article. As used herein, the term "or" should be understood to mean "and / or" unless the context clearly dictates otherwise.

[0012] Virus-like particle (VLP): As used herein, the term "virus-like particle (VLP)" refers to a non-replicative or non-infectious, preferably non-replicative and non-infectious, virus particle, or a non-replicative or non-infectious, preferably non-replicative and non-infectious structure resembling a virus particle, preferably a viral capsid. As used herein, the term "non-replicative" refers to the inability to replicate the genome contained in the VLP. As used herein, the term "non-infectious" refers to the inability to enter a host cell. Virus-like particles according to the present invention are non-replicative and non-infectious because they lack all or part of the viral genome or genome function. Virus-like particles according to the present invention may contain nucleic acids distinct from their genome. Recombinantly produced virus-like particles typically contain host cell-derived RNA. A typical and preferred embodiment of a virus-like particle according to the present invention is a viral capsid composed of a polypeptide of the present invention. Virus-like particles are typically macromolecular assemblies composed of viral coat proteins, typically containing 60, 120, 180, 240, 300, 360, or more than 360 protein subunits per virus-like particle. Typically and preferably, the interaction of these subunits results in the formation of a viral capsid or viral capsid-like structure with a unique repetitive organization. One characteristic of virus-like particles is the highly ordered repetitive arrangement of their subunits.

[0013] Modified CMV virus-like particle (VLP): The term "modified CMV virus-like particle" refers to a virus-like particle comprising at least one chimeric CMV polypeptide as defined herein and described herein. Typically and preferably, modified CMV VLPs resemble the structure of a CMV capsid. Modified CMV VLPs are non-replicative and / or non-infectious and lack at least one gene or genes encoding the CMV replication machinery, and typically also lack one or more genes encoding one or more proteins involved in viral binding to or entry into a host. This definition also includes modified virus-like particles in which one or more of the above genes are still present but inactive. Preferably, non-replicative and / or non-infectious modified virus-like particles are obtained by recombinant gene technology and typically and preferably do not contain a viral genome. Preferably, modified CMV VLPs are macromolecular assemblies composed of CMV polypeptides according to the present invention, typically and preferably comprising 180 such protein subunits and chimeric polypeptides per VLP. Thus, in a preferred embodiment, the modified virus-like particle (VLP) of Cucumber Mosaic Virus (CMV) comprises 180 chimeric CMV polypeptides.

[0014] Polypeptide: As used herein, the term "polypeptide" refers to a polymer composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). It refers to a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, and proteins are included within the definition of polypeptide. As used herein, the term "polypeptide" also typically and preferably refers to a polypeptide as defined above and includes modifications such as post-translational modifications, including but not limited to glycosylation. In a preferred embodiment, as used herein, the term "polypeptide" refers to a polypeptide as defined above and does not include modifications such as post-translational modifications, including glycosylation. In particular, for such biologically active peptides, such modifications, such as glycosylation, can occur subsequently in vivo, for example, by bacteria.

[0015] Cucumber mosaic virus (CMV) polypeptide, i.e., CMV polypeptide: As used herein, the term "Cucumber mosaic virus (CMV) polypeptide" refers to a polypeptide comprising, or preferably consisting of, (i) the amino acid sequence of a coat protein of Cucumber mosaic virus (CMV), or (ii) a variant amino acid sequence, wherein the variant amino acid sequence and the amino acid sequence of the coat protein of CMV exhibit at least 90%, preferably at least 91%, 92%, 93%, or 94%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% sequence identity. Typically and preferably, the CMV polypeptide is capable of self-assembly upon expression to form CMV virus-like particles.

[0016] Cucumber mosaic virus (CMV) coat protein (CP): As used herein, the term "Cucumber mosaic virus (CMV) coat protein (CP)" refers to the coat protein of naturally occurring Cucumber mosaic virus. Due to the extremely broad host range of Cucumber mosaic virus, many different strains and isolates of CMV are known. The sequences of the coat proteins of these strains and isolates have been determined and are known to those skilled in the art. The sequence of the CMV coat protein (CP) is listed and searchable in known databases, such as Genbank, www.dpvweb.net, or www.ncbi.nlm.nih.gov / protein / . An exemplary CMV CP is listed on page 12, line 8 to page 13, line 25 of WO 2016 / 062720, the disclosures of which are expressly incorporated herein by reference. A highly preferred example and embodiment of a CMV coat protein is provided in SEQ ID NO: 39. Therefore, preferably, as used herein, "coat protein of Cucumber Mosaic Virus (CMV)" refers to the amino acid sequence of the coat protein of CMV, which amino acid sequence comprises, or preferably consists of, SEQ ID NO: 39, or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 91%, 92%, 93% or 94%, even more preferably at least 95%, even more preferably at least 96% or 97%, even more preferably at least 98%, and even more preferably at least 99% sequence identity to SEQ ID NO: 39.

[0017] It is noteworthy that these strains and isolates have highly similar coat protein sequences in different protein domains, including the N-terminus of the coat protein. In particular, 98.1% of all fully sequenced CMV isolates share greater than 85% sequence identity within the first 28 amino acids of their coat protein sequence, and still 79.5% of all fully sequenced CMV isolates share greater than 90% sequence identity within the first 28 amino acids of their coat protein sequence.

[0018] Modified CMV polypeptide: As used herein, "modified CMV polypeptide" refers to a CMV polypeptide comprising, or preferably consisting of, a CMV polypeptide and a helper T cell epitope. Typically, the modified CMV polypeptide is capable of self-assembly to form CMV virus-like particles upon expression. Preferably, the modified CMV polypeptide is a recombinant modified CMV polypeptide that is capable of self-assembly to form CMV virus-like particles upon expression in Escherichia coli (E. coli).

[0019] Chimeric CMV polypeptide: As used herein, "chimeric CMV polypeptide" refers to a polypeptide as defined herein and in accordance with the present invention, comprising, and preferably consisting of, a CMV polypeptide, the CMV polypeptide being modified as defined and described herein, including a polypeptide comprising, and preferably consisting of, a stretch of contiguous negatively charged amino acids independently selected from aspartic acid or glutamic acid, and optionally further comprising a helper T cell epitope, all components as defined and described herein. Typically and preferably, the chimeric CMV polypeptide is capable of self-assembly upon expression to form modified virus-like particles of CMV. Thus, in preferred embodiments, the chimeric CMV polypeptide is capable of self-assembly, typically and preferably upon expression, to form modified virus-like particles of CMV. Preferably, the chimeric CMV polypeptide is a recombinant modified CMV polypeptide that is capable of self-assembly upon expression to form CMV virus-like particles in Escherichia coli (E. coli). Typically and preferably, the helper T cell epitope replaces the N-terminal region of the CMV polypeptide, and the substituted N-terminal region of the CMV polypeptide consists of 5 to 15 consecutive amino acids. Preferably, the helper T cell epitope replaces the N-terminal region of the CMV polypeptide, and the substituted N-terminal region of the CMV polypeptide consists of 5 to 15 consecutive amino acids, preferably 9 to 14, 9 to 13, or 10 to 13 consecutive amino acids, more preferably 11 to 13 consecutive amino acids, and most preferably 11, 12, or 13 consecutive amino acids.

[0020] N-terminal region of a CMV polypeptide: As used herein, the term "N-terminal region of a CMV polypeptide" refers to either the N-terminus of the CMV polypeptide, particularly the N-terminus of a CMV coat protein, or the N-terminal region of the CMV polypeptide or coat protein, beginning with the second amino acid at the N-terminus of the CMV polypeptide or coat protein if the CMV polypeptide or coat protein contains an N-terminal methionine residue. Preferably, if the CMV polypeptide or coat protein contains an N-terminal methionine residue, from a practical standpoint, the start codon encoding the methionine is usually deleted and a T helper (Th) cell epitope is added to the N-terminus. More preferably, one, two, or three additional amino acids, preferably one amino acid, can be optionally inserted between the start methionine and the Th cell epitope for cloning purposes.

[0021] Recombinant polypeptide: In the context of the present invention, the term "recombinant polypeptide," when used in the context of a polypeptide, refers to a polypeptide obtained by a process including at least one step of recombinant DNA technology. Typically and preferably, recombinant polypeptides are produced in prokaryotic expression systems. It will be apparent to those skilled in the art that recombinantly produced polypeptides expressed in prokaryotic expression systems, such as E. coli, may contain an N-terminal methionine residue. The N-terminal methionine residue is typically cleaved from the recombinant polypeptide in the expression host during maturation of the recombinant polypeptide. However, cleavage of the N-terminal methionine may be incomplete. Thus, a preparation of recombinant polypeptides may contain a mixture of otherwise identical polypeptides, both with and without an N-terminal methionine residue. Typically and preferably, a preparation of recombinant polypeptides contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant polypeptides with an N-terminal methionine residue.

[0022] Recombinant modified virus-like particle: In the context of the present invention, the term "recombinant modified virus-like particle" refers to a modified virus-like particle (VLP) obtained by a process comprising at least one step of recombinant DNA technology.

[0023] Mutant amino acid sequence: The term "mutant amino acid sequence" refers to an amino acid sequence obtained by introducing a defined set of mutations into a mutated amino acid sequence. In the context of the present invention, the mutated amino acid sequence is typically and preferably the amino acid sequence of a CMV coat protein. Thus, the mutated amino acid sequence differs from the amino acid sequence of a CMV coat protein by at least one amino acid residue, and the mutated amino acid sequence and the mutated amino acid sequence exhibit at least 90% sequence identity. Typically and preferably, the mutated amino acid sequence and the mutated amino acid sequence exhibit at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Preferably, the mutated amino acid sequence and the mutated sequence differ by at most 11, 10, 9, 8, 7, 6, 4, 3, 2, or 1 amino acid residue, and more preferably, the difference is selected from insertions, deletions, and amino acid exchanges. Preferably, the variant amino acid sequence differs from the amino acid sequence of the coat protein of CMV by at least one amino acid, and preferably the difference is an amino acid exchange.

[0024] The terms "corresponding," "corresponding," or "corresponds," as used herein to describe the relationship of specific positions of one or more amino acid residues in a polypeptide and an amino acid sequence, respectively, refer to the position of one or more amino acid residues in an amino acid sequence that correspond to one or more given and specific amino acid residues in another amino acid sequence, which can be identified in a sequence alignment, typically and preferably using the BLASTP algorithm, most preferably using standard settings. Typical and preferred standard settings are: expectation threshold: 10; word size: 3; maximum matches within query: 0; matrix: BLOSUM62; gap cost: presence 11, extension 1; composition adjustment: conditional composition score matrix adjustment.

[0025] Sequence identity: The sequence identity of two given amino acid sequences is determined based on the alignment of both sequences. Algorithms for determining sequence identity are available to those skilled in the art. Preferably, the sequence identity of two amino acid sequences is determined using publicly available computer homology programs such as the "BLAST" program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) or "CLUSTALW" (http: / / www.genome.jp / tools / clustalw / ), preferably by the "BLAST" program provided on the NCBI homepage at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using the default settings provided therein. Typical and preferred standard settings are: expectation threshold: 10; word size: 3; maximum match within query range: 0; matrix: BLOSUM62; gap cost: existence 11, extension 1; composition adjustment: conditional composition score matrix adjustment.

[0026] Amino acid exchange: The term amino acid exchange refers to the replacement of a given amino acid residue in an amino acid sequence with any other amino acid residue having a different chemical structure, preferably with another proteinogenic amino acid residue. Thus, in contrast to an amino acid insertion or deletion, an amino acid exchange does not change the total number of amino acids in the amino acid sequence.

[0027] As used herein and abbreviated as pI, the term "isoelectric point" refers to the pH at which a molecule has no net charge or is electrically neutral on statistical average. In particular, the term "isoelectric point" is used herein to refer to the pH at which antigens composed of amino acids have no net charge or are electrically neutral on statistical average. At a pH below the pI, such antigens have a net positive charge, and at a pH above the pI, they have a net negative charge. Typically and preferably, when referring to pI values, and in particular pI values ​​of antigens of the present invention and within this disclosure, the pI values ​​are determined by inputting the primary amino acid sequence for the particular protein and antigen, respectively, into the ExPASyCompute pI / MW tool described by Gasteiger et al. (Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, MR, Appel, RD, & Bairoch, A., Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). Accordingly, when referred to herein, the ExPASyCompute pI / MW tool is the tool described by Gasteiger et al. This tool calculates the theoretical isoelectric point (pI) and Mw of a specific Swiss-Prot / TrEMBL entry or a user-entered amino acid sequence. The pI of a protein is calculated using the amino acid pK values ​​described by Bjellqvist et al. (Bjellqvist, B. et al., 1993, Electrophoresis 14:1023-1031; Bjellqvist, B. et al., 1994, Electrophoresis 15:529-539), which are defined by examining polypeptide migration between pH 4.5 and 7.3 in immobilized pH gradient gels with 9.2 M and 9.8 M urea at 15°C or 25°C.

[0028] Epitope: The term "epitope" refers to a continuous or discontinuous portion of a polypeptide or antigen, which, within the context of an MHC molecule, is capable of being specifically bound by an antibody or T-cell receptor. With respect to antibodies, specific binding excludes nonspecific binding, but does not necessarily exclude cross-reactivity. An epitope typically comprises 5 to 20 amino acids in a spatial conformation unique to the antigenic site.

[0029] Helper T (Th) cell epitope: As used herein, the term "helper T cell epitope or Th cell epitope, as used interchangeably," refers to an epitope capable of being recognized by helper Th cells. Typically and preferably, as used herein, the term "Th cell epitope" refers to a Th cell epitope capable of binding to at least one, preferably two or more MHC class II molecules. The simplest way to determine whether a peptide sequence is a Th cell epitope is to measure the peptide's ability to bind to individual MHC class II molecules. This can be measured by the peptide's ability to compete with the binding of known Th cell epitope peptides to MHC class II molecules. Representative selection of HLA-DR molecules is described, for example, in Alexander J, et al., Immunity (1994) 1:751-761. The affinity of a Th cell epitope for an MHC class II molecule should be at least 10 -5M. A representative collection of MHC class II molecules present in different individuals is shown in Panina-Bordignon P, et al., Eur J Immunol (1989) 19:2237-2242. Consequently, as used herein, the term "Th cell epitope" preferably refers to a Th cell epitope that generates a measurable T cell response upon immunization and boosting. Additionally and even more preferably, the term "Th cell epitope", as used herein, refers to a Th cell epitope capable of binding to at least one, preferably at least two, and even more preferably at least three DR alleles selected from DR1, DR2w2b, DR3, DR4w4, DR4w14, DR5, DR7, DR52a, DRw53, DR2w2a, preferably selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a, with an affinity of at least 500 nM (as described in Alexander J et al., Immunity (1994) 1:751-761 and references cited therein), and a preferred binding assay for assessing such affinity is that described in Sette A et al., J Immunol (1989) 142:35-40. In an even more preferred manner, the term "Th cell epitope", as used herein, refers to a Th cell epitope capable of binding to at least one, preferably at least two, and even more preferably at least three DR alleles selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a, preferably with an affinity of at least 500 nM (as described in Alexander J, et al., Immunity (1994) 1:751-761 and references cited therein), and a preferred binding assay for assessing said affinity is that described in Sette A, et al., J Immunol (1989) 142:35-40.Th cell epitopes have been described by Alexander J, et al., Immunity (1994) 1:751-761, Panina-Bordignon P, et al., Eur J Immunol (1989) 19:2237-2242, Calvo-Calle JM et al., J Immunol (1997) 159:1362-1373, and Valmori D, et al., J Immunol (1992) 149:717-721, and are known to those skilled in the art.

[0030] Amino acid linker: As used herein, the term "amino acid linker" refers to a linker consisting solely of amino acid residues. The amino acid residues of an amino acid linker are composed of naturally occurring amino acids or non-natural amino acids known in the art, all L or all D, or mixtures thereof. The amino acid residues of an amino acid linker are preferably naturally occurring amino acids, all L or all D, or mixtures thereof. In a preferred embodiment, the amino acid linker is composed of naturally occurring alpha amino acids, all of which are in the L configuration.

[0031] G-linker: As used herein, the term "G-linker" refers to an amino acid linker consisting of only glycine amino acid residues. A G-linker according to the present invention contains at least two glycine residues and up to 10 glycine residues.

[0032] GS-linker: As used herein, the term "GS-linker" refers to an amino acid linker consisting of only glycine and serine amino acid residues. A GS-linker according to the present invention contains at least one glycine residue and at least one serine residue. Typically and preferably, the GS-linker has a length of up to 30 amino acids.

[0033] GS*-linker: As used herein, the term "GS*-linker" refers to an amino acid linker comprising at least one glycine, at least one serine, and at least one amino acid residue selected from Thr, Ala, Lys, and Cys. Typically and preferably, the GS*-linker has a length of up to 30 amino acids.

[0034] As used herein, the term "amino acid" refers to an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH) and their zwitterions, typically and preferably with a side chain specific to each amino acid. The term "amino acid" typically and preferably includes naturally occurring amino acids, such as proteinogenic amino acids (produced by RNA translation), non-proteinogenic amino acids (produced by other metabolic mechanisms, e.g., post-translational modifications), standard or canonical amino acids (directly encoded by codons in the genetic code), and non-standard or non-canonical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include non-eukaryotic and eukaryotic amino acids. As used herein, the term "amino acid" includes chemically synthesized and unnatural amino acids; amino acids such as alpha (α-), beta (β-), gamma (γ-), and delta- (δ-), and mixtures thereof in any ratio, and, as applicable to alpha-(α-) amino acids, also includes any isomer of an amino acid, i.e., D- and L-stereoisomers (alternatively addressed by (R) and (S) nomenclature), and mixtures thereof in any ratio, including a 1:1 racemic ratio. The terms "D-stereoisomer," "L-stereoisomer," "D-amino acid," or "L-amino acid" refer to the chiral alpha carbon of an amino acid. In preferred embodiments, the term amino acid refers to an alpha amino acid, preferably a naturally occurring alpha amino acid, more preferably an alpha amino acid that occurs naturally in its L-configuration.

[0035] Interacted: As used herein, the term "interacted" or "interaction" refers to all possible ways, preferably chemical interactions, by which two molecules are bound together. Chemical interactions include covalent and non-covalent interactions. Typical examples of non-covalent interactions are ionic interactions, hydrophobic interactions, or hydrogen bonds, while covalent interactions are based on covalent bonds such as esters, ethers, phosphoesters, carbon-phosphorus bonds, carbon-sulfur bonds such as thioethers, or imide bonds, for example.

[0036] Binding site, first: As used herein, the phrase "first binding site" refers to a naturally occurring or artificially added element on a virus-like particle, and a second binding site to which the first binding site can be attached. The first binding site is preferably a protein, polypeptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound such as biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride, or a chemically reactive group such as an amino group, a carboxy group, a sulfhydryl group, a hydroxy group, a guanidinyl group, a histidinyl group, or a combination thereof. A preferred embodiment of the chemically reactive group that is the first binding site is an amino group of an amino acid residue, preferably the amino group of the side chain of a lysine residue. The first binding site is typically located on the surface, preferably the outer surface of the VLP. Multiple first binding sites are present on the surface, preferably the outer surface of the VLP, typically in a repetitive configuration. In a preferred embodiment, the first binding site interacts with the VLP via at least one covalent bond, preferably via at least one peptide bond. In a more preferred embodiment, the first binding site is naturally present with the VLP. Alternatively, in a preferred embodiment, the first binding site is artificially added to the VLP. In a highly preferred embodiment, the first binding site is the amino group of a lysine residue in the amino acid sequence of the VLP polypeptide.

[0037] Binding site, second: As used herein, the phrase "second binding site" refers to a naturally occurring or artificially added element with an antigen to which the antigen and the first binding site can be bound. The second binding site of an antigen is preferably a protein, polypeptide, peptide, amino acid, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound such as biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride, or a chemically reactive group such as an amino group, a carboxy group, a sulfhydryl group, a hydroxy group, a guanidinyl group, a histidinyl group, or a combination thereof. A preferred embodiment of the chemically reactive group that is the second binding site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue. Thus, the term "antigen having at least one second binding site" refers to a construct comprising an antigen and at least one second binding site. However, particularly for second binding sites that are not naturally occurring in the antigen, such constructs typically and preferably further comprise a "linker." In another preferred embodiment, the second binding site interacts with the antigen via at least one covalent bond, preferably via at least one peptide bond. In a further embodiment, the second binding site is naturally present in the antigen. In a further preferred embodiment, the second binding site is artificially added to the antigen via a linker, which comprises or alternatively consists of a cysteine. Preferably, the linker is fused to the antigen by a peptide bond.

[0038] Bonded: As used herein, the term "bonded" or "bond" refers to all possible ways, preferably chemical interactions, by which at least one first binding moiety and at least one second binding moiety are bound together. Chemical interactions include covalent and non-covalent interactions. Typical examples of non-covalent interactions are ionic interactions, hydrophobic interactions, or hydrogen bonds, while covalent interactions are based on covalent bonds such as, for example, esters, ethers, phosphoesters, carbon-phosphorus bonds, carbon-sulfur bonds such as thioethers, or imide bonds. In certain preferred embodiments, the first binding moiety and the second binding moiety are bound by at least one covalent bond, preferably at least one non-peptide bond, and even more preferably only non-peptide covalent bonds. However, as used herein, the term "linked" can refer not only to the direct linkage between at least one first binding site and at least one second binding site, but also, alternatively and preferably, to the indirect linkage between at least one first binding site and at least one second binding site via one or more intermediate molecules, typically and preferably herein by using at least one, preferably one, heterobifunctional cross-linker. In other preferred embodiments, the first binding site and the second binding site are linked by at least one covalent bond, preferably at least one peptide bond, and even more preferably only peptide bonds.

[0039] Linker: As used herein, a "linker" either allows a second binding site to interact with an antigen, or already contains or consists of a second binding site. Preferably, as used herein, a "linker" already contains a second binding site, typically and preferably as a single amino acid residue, preferably as a cysteine ​​residue. Preferred linkers are those containing at least one amino acid residue, and even more preferred are linkers consisting solely of amino acid residues. The amino acid residues of the linker are preferably composed of naturally occurring or unnatural amino acids known in the art, all L or all D, or mixtures thereof. Further preferred embodiments of linkers according to the present invention are molecules containing a sulfhydryl group or a cysteine ​​residue, and therefore such molecules are also encompassed within the scope of the present invention. Additional linkers useful in the present invention are molecules containing a C1-6 alkyl moiety, a cycloalkyl moiety such as cyclopentyl or cyclohexyl, a cycloalkenyl moiety, an aryl moiety, or a heteroaryl moiety. Additionally, linkers preferably comprising a C1-C6 alkyl moiety, a cycloalkyl (C5, C6) moiety, an aryl moiety, or a heteroaryl moiety, as well as one or more additional amino acids, can also be used as linkers for the present invention and are within the scope of the present invention. The interaction between the antigen and the linker is preferably via at least one covalent bond, more preferably via at least one peptide bond.

[0040] Antigen: As used herein, the term "antigen" refers to a molecule that, when presented by an MHC molecule, can be bound by an antibody or a T cell receptor (TCR). An antigen can further be recognized by the immune system and / or induce a humoral and / or cellular immune response that results in the activation of B and / or T lymphocytes. An antigen can have one or more epitopes (B epitopes and T epitopes). As used herein, an antigen can also be a mixture of several individual antigens.

[0041] Specific and repetitive antigen array: As used herein, the term "specific and repetitive antigen array" typically and preferably refers to a repeating pattern of antigens characterized by a high degree of uniformity in the spatial arrangement of antigens on a modified CMV VLP. In one embodiment of the present invention, the repeating pattern may be a geometric pattern. Certain embodiments of the present invention, such as antigens bound to a modified CMV VLP, are typical and preferred examples of suitable specific and repetitive antigen arrays. In addition, they preferably have a strict repeating paracrystalline order of antigens with a spacing of 1 to 30 nanometers, preferably 2 to 15 nanometers, even more preferably 2 to 10 nanometers, even more preferably 2 to 8 nanometers, and even more preferably 1.6 to 7 nanometers.

[0042] Coupling efficiency: The coupling efficiency of virus-like particles to specific antigens is determined by SDS-PAGE of the coupling reaction. The intensity of the Coomassie blue-stained bands corresponding to the components of the coupling reaction is determined by densitometry and used to calculate the coupling efficiency. Coupling efficiency is defined as the ratio of the amount of VLP polypeptide (i) coupled to the antigen to the total amount of VLP polypeptide (ii). Typically and preferably, the coupling efficiency is at least 5%, 10%, preferably at least 15%, more preferably at least 20%, 25%, or at least 30%, and even more preferably at least 35% or at least 40%. Coupling deficiency can also be expressed by the total number of antigens conjugated to the modified CMV VLP. Coupling deficiency may depend on the nature of the antigen, and the total number of antigens conjugated to the modified CMV VLP is typically and preferably at least 5, at least 7, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, and at least 50 antigens.

[0043] Nerve Growth Factor (NGF): As used herein, and when referring to an antigen of the compositions of the invention, the term "nerve growth factor (NGF)" refers to a polypeptide comprising, preferably consisting of, the amino acid sequence of canine or feline nerve growth factor or the corresponding orthologue from other species, preferably non-human animals, or a polypeptide having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% sequence identity with the amino acid sequence of canine or feline nerve growth factor or the corresponding orthologue from other species, preferably non-human animals. The term "NGF antigen" is used interchangeably herein. Preferred NGF antigens from various animal species are canine NGF (cNGF), feline NGF (fNGF), equine NGF (eNGF), bovine NGF (bNGF), and porcine NGF (pNGF), preferably canine NGF (cNGF) or feline NGF (fNGF), and the NGF antigen preferably comprises, or preferably consists of, a polypeptide of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% sequence identity to any of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58. NGF antigens typically and preferably comprise biological activity, preferably in a cell proliferation assay. In addition, when administered to an animal in the form of a composition of the present invention, the NGF antigen is typically and preferably capable of inducing anti-NGF antibodies in the animal, and the anti-NGF antibodies are capable of neutralizing the biological activity of NGF in an in vitro assay, preferably as described herein (see Example 6). As used herein, and when referring to an NGF antigen, the term "biological activity" refers to the activity of the NGF antigen in a cell proliferation assay, preferably the cell proliferation assay is based on the NGF-dependent human erythroleukemia TF-1 cell line, and even more preferably the cell proliferation assay is performed under conditions essentially as described in Example 6 herein.

[0044] Adjuvant: As used herein, the term "adjuvant" refers to an immune response stimulator and / or substance that is typically capable of creating a depot in the host that can provide a more enhanced immune response when combined with the composition, vaccine, or pharmaceutical composition of the present invention, respectively. Various types of adjuvants with different mechanisms of action have been described that can enhance antigen-specific antibody responses (Pulendran B et al., 2021, Nature Reviews Drug Discovery 20:454-475). Typical and preferred adjuvants are inorganic salts (e.g., aluminum hydroxide, aluminum phosphate), microcrystalline tyrosine, emulsions, microparticles, saponins (Quil A), cytokines, immunopotentiators, bacterial components / products, liposomes, complexes, and mucosal adjuvants that are known and are similarly described, for example, in the Adjuvant Compendium NIAID and VAC (nih.gov) or in Aguilar et al. (Aguilar JC et al., 2007, Vaccine 25:3752-3762), Gerdts (Gerdts V, 2015, Berliner und Munchener Tierarztliche Wochenschrift 128:456-463), and Pasquale et al. (Pasquale et al. 2015, Vaccines 3:320-343). (Pasquale et al. 2015, Vaccines 3:320-343). As used herein, the term "adjuvant" also includes a mixture of adjuvants. Virus-like particles are sometimes described as adjuvants. However, as used within the context of this application, the term "adjuvant" refers to an adjuvant that is not a modified virus-like particle of the present invention. Rather, "adjuvant" relates to an additional, separate component of a composition, vaccine, or pharmaceutical composition of the present invention.

[0045] Immunostimulatory substance: As used herein, the term "immunostimulatory substance" refers to a substance that can induce and / or enhance an immune response. As used herein, immunostimulatory substances include, but are not limited to, Toll-like receptor activators and substances that induce cytokine secretion. Toll-like receptor activators include, but are not limited to, immunostimulatory nucleic acids, peptidoglycans, lipopolysaccharides, lipoteichoic acid, imidazoquinoline compounds, flagellin, lipoproteins, and immunostimulatory organic substances such as taxol.

[0046] Immunostimulatory nucleic acid (ISS-NA): As used herein, the term immunostimulatory nucleic acid refers to a nucleic acid capable of inducing and / or enhancing an immune response. Immunostimulatory nucleic acids include ribonucleic acids and, in particular, deoxyribonucleic acids, and both ribonucleic acids and deoxyribonucleic acids can be either double-stranded or single-stranded. Preferred ISS-NAs are deoxyribonucleic acids, and more preferably, the deoxyribonucleic acids are single-stranded. Preferably, the immunostimulatory nucleic acid contains at least one CpG motif containing an unmethylated C. Highly preferred immunostimulatory nucleic acids contain at least one CpG motif, which comprises or preferably consists of at least one, preferably one, CG dinucleotide, and the C is unmethylated. Preferably, although not necessarily, the CG dinucleotide is part of a palindromic sequence. The term immunostimulatory nucleic acid also refers to a nucleic acid containing a modified base, preferably 4-bromo-cytosine. ISS-NAs capable of stimulating IFN-alpha production in dendritic cells are particularly preferred in the context of the present invention. Immunostimulatory nucleic acids useful for the purposes of the present invention are described, for example, in WO 2007 / 068747 A1.

[0047] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a nucleic acid sequence containing two or more nucleotides, preferably about 6 to about 200 nucleotides, more preferably 20 to about 100 nucleotides, and most preferably 20 to 40 nucleotides. Oligonucleotides are polyribonucleotides or polydeoxyribonucleotides, preferably selected from (a) unmodified RNA or DNA and (b) modified RNA or DNA. Modifications may include backbone or nucleotide analogs. Oligonucleotides are preferably selected from the group consisting of (a) single-stranded and double-stranded DNA, (b) DNA that is a mixture of single-stranded and double-stranded regions, (c) single-stranded and double-stranded RNA, (d) RNA that is a mixture of single-stranded and double-stranded regions, and (e) hybrid molecules containing DNA and RNA that are single-stranded, or more preferably double-stranded, or a mixture of single-stranded and double-stranded regions. Preferred nucleotide modifications / analogs are selected from the group consisting of (a) peptide nucleic acids, (b) inosine, (c) tritylated bases, (d) phosphorothioates, (e) alkylphosphothioates, (f) 5-nitroindole deoxyribofurylanosyl, (g) 5-methyldeoxycytosine, and (h) 5,6-dihydro-5,6-dihydroxydeoxythymidine. Phosphorothioated nucleotides are protected against degradation in cells or organisms and are therefore preferred nucleotide modifications. Unmodified oligonucleotides consisting only of phosphodiester-linked nucleotides are typically more active than modified nucleotides and are therefore generally preferred in the context of the present invention. Oligonucleotides consisting only of phosphodiester-linked oligonucleotides are most preferred, and more preferably, the oligonucleotides are single-stranded. More preferred are oligonucleotides capable of stimulating IFN-alpha production in cells, preferably dendritic cells. Highly preferred oligonucleotides capable of stimulating IFN-alpha production in cells are selected from A-type CpG and C-type CpG. Cap-free RNA molecules are even more preferred.

[0048] CpG motif: As used herein, the term "CpG motif" refers to a pattern of nucleotides comprising an unmethylated central CpG, a so-called unmethylated CpG dinucleotide, in which the C is unmethylated and is surrounded by at least one base, preferably one or two nucleotides adjacent to (the 3' and 5' sides of) the central CpG. Typically and preferably, as used herein, a CpG motif comprises, or alternatively consists of, an unmethylated CpG dinucleotide and two nucleotides at its 5' and 3' ends. Without being bound by theory, the bases adjacent to the CpG are responsible for a significant portion of the activity of a CpG oligonucleotide.

[0049] Unmethylated CpG-containing oligonucleotide: As used herein, the term "unmethylated CpG-containing oligonucleotide" or "CpG" refers to an oligonucleotide, preferably an oligodeoxynucleotide, containing at least one CpG motif. Thus, CpG contains at least one unmethylated cytosine, guanine dinucleotide. Preferred CpGs stimulate / activate vertebrate bone marrow-derived cells, e.g., by having a mitogenic effect on such cells or by inducing or increasing cytokine expression by such cells. For example, CpGs may be useful in activating antigen-presenting cells, such as B cells, NK cells and dendritic cells, monocytes, and macrophages. Preferably, CpG refers to an oligodeoxynucleotide, preferably a single-stranded oligodeoxynucleotide, containing an unmethylated cytosine followed by a guanosine at its 3' end, wherein the unmethylated cytosine and the guanosine are linked by a phosphate bond, preferably a phosphodiester or phosphorothioate bond, and more preferably a phosphodiester bond. CpGs can include nucleotide analogs, such as analogs containing phosphorothioester bonds, and can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immunoreactivity. Preferably, as used herein, CpGs are oligonucleotides at least about 10 nucleotides in length and containing at least one CpG motif; more preferably, the CpGs are 10-60, more preferably 15-50, even more preferably 20-40, even more preferably about 30, and most preferably exactly 30 nucleotides in length. CpGs can be composed of methylated and / or unmethylated nucleotides, wherein the at least one CpG motif contains at least one CG dinucleotide, with the C being unmethylated. CpGs can also include methylated and unmethylated sequence stretches, wherein the at least one CpG motif contains at least one CG dinucleotide, with the C being unmethylated.Highly preferably, CpG refers to a single-stranded oligodeoxynucleotide containing an unmethylated cytosine followed by a guanosine at the 3' end, and the unmethylated cytosine and the guanosine are linked by a phosphodiester bond. CpG can include nucleotide analogs, such as analogs containing phosphorothioester bonds, and can be double-stranded or single-stranded. Generally, phosphodiester CpG is A-type CpG as shown below, while phosphothioester-stabilized CpG is B-type CpG. In the context of the present invention, the preferred CpG oligonucleotide is A-type CpG.

[0050] A-type CpG: As used herein, the term "A-type CpG" or "D-type CpG" refers to an oligodeoxynucleotide (ODN) containing at least one CpG motif. A-type CpGs can preferentially stimulate T cell activation and dendritic cell maturation and stimulate IFN-alpha production. In A-type CpGs, the nucleotides of at least one CpG motif are linked by at least one phosphodiester bond. A-type CpGs contain at least one phosphodiester-linked CpG motif, which may be flanked at its 5'-end and / or preferably at its 3'-end by phosphorothioate-linked nucleotides. Preferably, the CpG motif, here preferably a CG dinucleotide and its immediately adjacent region, including at least one, preferably two nucleotides, are composed of phosphodiester nucleotides. Preferred A-type CpGs consist exclusively of phosphodiester (PO)-linked nucleotides. Typically and preferably, the poly-G motif comprises or alternatively consists of at least 1, preferably at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 G (guanosine), most preferably at least 10 G. Preferably, the A-type CpG of the present invention comprises or alternatively consists of a palindromic sequence.

[0051] Packaged: As used herein, the term "packaged" refers to the state of a polyanionic macromolecule or immunostimulatory substance relative to a core particle and a VLP, respectively. As used herein, the term "packaged" includes binding, which may be covalent, e.g., by chemical coupling, or non-covalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc. The term also includes encapsulation or partial encapsulation of a polyanionic macromolecule. Thus, a polyanionic macromolecule or immunostimulatory substance can be encapsulated by a VLP without the presence of an actual binding, particularly a covalent binding. In a preferred embodiment, at least one polyanionic macromolecule or immunostimulatory substance is packaged within a VLP, most preferably in a non-covalent manner. When the immunostimulatory substance is a nucleic acid, preferably DNA, the term "packaged" means that the nucleic acid is inaccessible to nuclease hydrolysis, preferably inaccessible to DNAse hydrolysis (e.g., DNase I or benzonase), preferably as assayed as described in Examples 11-17 of WO 2003 / 024481 A2.

[0052] Effective amount: As used herein, the term "effective amount" refers to an amount necessary or sufficient to realize a desired biological effect. An effective amount of a composition, or alternatively a pharmaceutical composition, is an amount that achieves this selected result, and such an amount can be routinely determined by one of ordinary skill in the art. The effective amount may vary depending on the particular composition administered and the size of the subject. One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation. Preferably, the term "effective amount" refers to an amount that (i) treats or prevents a particular disease or disorder, (ii) attenuates, ameliorate, or eliminates one or more symptoms of a particular disease or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease or disorder described herein.

[0053] Animal: As used herein, the term "animal," a subject in need of administration of a composition of the invention comprising a modified CMV VLP, refers to non-human animals, including vertebrates, mammals, rodents (e.g., guinea pigs, hamsters), canines (e.g., dogs), felines (e.g., cats), porcines (e.g., pigs), equines (e.g., horses), and primates. Preferably, the subject is a non-human mammal (e.g., dog, cat, horse, sheep, cow, or pig, etc.). In a preferred embodiment, the subject is a non-human mammal selected from a dog, cat, horse, sheep, cow, or pig.

[0054] Veterinary composition: As used herein, the term "veterinary composition" refers to a composition suitable for use in non-human animals.

[0055] Treatment: As used herein, the terms "treatment," "treat," "treated," or "treating" refer to prophylaxis and / or therapy. In one embodiment, the terms "treatment," "treat," "treated," or "treating" refer to therapeutic treatment. In another embodiment, the terms "treatment," "treat," "treated," or "treating" refer to prophylactic treatment. Preferably, beneficial or desired clinical results of the treatment include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease or disorder, stabilization (i.e., not worsening) of the disease or disorder, delay or slowing of the progression of the disease or disorder, or improvement or palliation of the disease or disorder state.

[0056] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one first binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; a modified VLP of CMV comprising, preferably consisting of,

[0057] (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); Including, The present invention provides a composition, preferably a veterinary composition, wherein (a) and (b) are linked by said at least one first binding moiety and said at least one second binding moiety via at least one covalent non-peptide bond.

[0058] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV comprising at least one first binding site; (b) at least one nerve growth factor (NGF) antigen, wherein the antigen comprises at least one second binding site; The modified VLP of CMV comprises at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, the polypeptide comprising an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0059] In a preferred embodiment, the chimeric CMV polypeptide further comprises a helper T cell epitope, preferably the helper T cell epitope replaces the N-terminal region of the CMV polypeptide, more preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 39, even more preferably the helper T cell epitope is derived from tetanus toxin or is the PADRE sequence, and most preferably the Th cell epitope comprises, and even more preferably consists of, the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In an even more highly preferred embodiment, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 92%, more preferably at least 95%, and even more preferably at least 98% sequence identity to SEQ ID NO: 39.

[0060] Therefore, in another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; A modified VLP of CMV comprising, preferably consisting of, (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); a composition, preferably a veterinary composition, comprising (a) and (b) link the at least one first binding site and the at least one second binding site via at least one covalent non-peptide bond.

[0061] In a further highly preferred embodiment, the stretch of contiguous negatively charged amino acids comprises, and preferably consists of, SEQ ID NO:1 or SEQ ID NO:2.

[0062] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39, preferably the CMV polypeptide is a coat protein of CMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO:39, and wherein the stretch of consecutive negatively charged amino acids comprises, and preferably consists of, SEQ ID NO:1 or SEQ ID NO:2; and (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); (a) and (b) link the at least one first binding site and the at least one second binding site via at least one covalent non-peptide bond.

[0063] Therefore, in another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39, preferably the CMV polypeptide is a coat protein of CMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO:39, and wherein the stretch of consecutive negatively charged amino acids comprises, and preferably consists of, SEQ ID NO:1 or SEQ ID NO:2; and (iii) a chimeric CMV polypeptide comprising, and preferably consisting of, a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0064] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is arranged at the N-terminus of the stretch of consecutive negatively charged amino acids, and the second amino acid linker is arranged at the C-terminus of the stretch of consecutive negatively charged amino acids, and the first amino acid linker and the second amino acid linker are each selected from the group consisting of: (a) an amino acid sequence (Gly) having a length of n=2 to 10; n (b.) a polyglycine linker (G-linker) having at least one glycine and at least one serine; (b.) a glycine-serine linker (GS-linker) having r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS) r (G s S) t (GS) uand (c.) an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys. In a preferred embodiment, the polypeptide comprising the stretch of contiguous negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is positioned at the N-terminus of the stretch of contiguous negatively charged amino acids and the second amino acid linker is positioned at the C-terminus of the stretch of contiguous negatively charged amino acids, and the first amino acid linker and the second amino acid linker are glycine-serine linkers (GS-linkers) comprising at least one glycine and at least one serine, wherein the GS linker has r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS). r (G s S) t (GS) u or an amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, and Cys (GS*-linker), having the amino acid sequence:

[0065] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39, preferably the CMV polypeptide is a coat protein of CMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO:39, and wherein the polypeptide comprises, and preferably consists of, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51, and preferably wherein the polypeptide is inserted between amino acid residues in the CMV polypeptide corresponding to amino acid residues 84 and 85 of SEQ ID NO:39; A modified VLP of CMV comprising, preferably consisting of, (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); a composition, preferably a veterinary composition, comprising (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0066] Therefore, in another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39, preferably the CMV polypeptide is a coat protein of CMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO:39, and wherein the polypeptide comprises, and preferably consists of, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51, and preferably wherein the polypeptide is inserted between amino acid residues in the CMV polypeptide corresponding to amino acid residues 84 and 85 of SEQ ID NO:39; (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; A modified VLP of CMV comprising, preferably consisting of, (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); a composition, preferably a veterinary composition, comprising (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0067] In even more highly preferred embodiments, the CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48.

[0068] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, the CMV polypeptide comprising an amino acid sequence having at least 75% sequence identity to the coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising a stretch of consecutive negatively charged amino acids, the negatively charged amino acids being independently selected from aspartic acid or glutamic acid, the polypeptide being inserted between any amino acid residues in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; A modified VLP of CMV comprising, preferably consisting of, wherein the CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48; (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); a composition, preferably a veterinary composition, comprising (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0069] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, the modified VLP of CMV comprising at least one binding site, the modified VLP of CMV comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising a stretch of consecutive negatively charged amino acids, the negatively charged amino acids being independently selected from aspartic acid or glutamic acid, the polypeptide being inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; and preferably consisting of wherein the CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5, between the amino acid residues at positions 84 and 85 of SEQ ID NO:39, or between the amino acid residues at positions 86 and 87 of SEQ ID NO:48; and (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF); a composition, preferably a veterinary composition, comprising (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0070] The embodiments, preferred embodiments and highly preferred embodiments described and disclosed herein should apply to all aspects and other embodiments, preferred embodiments and highly preferred embodiments, whether specifically mentioned again or whether their repetition is avoided for the sake of brevity.

[0071] In a preferred embodiment, the CMV polypeptide comprises, and preferably consists of, the amino acid sequence of a CMV coat protein or a mutated amino acid sequence, wherein the mutated amino acid sequence and the CMV coat protein share at least 90%, preferably at least 91%, 92%, 93, 94% or 95%, more preferably at least 96%, 97% or 98%, and even more preferably at least 99% sequence identity; preferably, the mutated amino acid sequence and the mutated amino acid sequence differ by a minimum of 1 and a maximum of 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues; more preferably, these differences are selected from (i) insertions, (ii) deletions, (iii) amino acid exchanges, and (iv) any combination of (i)-(iii).

[0072] In a preferred embodiment, the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39.

[0073] In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 39.

[0074] In a preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of the coat protein of CMV or an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 39.

[0075] In a preferred embodiment, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 75%, preferably 85%, sequence identity to SEQ ID NO: 39. In a preferred embodiment, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO: 39. In a preferred embodiment, the CMV coat protein comprises SEQ ID NO: 39. In a preferred embodiment, the CMV coat protein consists of SEQ ID NO: 39. In a preferred embodiment, the CMV polypeptide comprises a CMV coat protein. In a preferred embodiment, the CMV polypeptide consists of a CMV coat protein. In a preferred embodiment, the CMV polypeptide comprises a CMV coat protein, and the CMV coat protein comprises SEQ ID NO: 39. In a preferred embodiment, the CMV polypeptide comprises a CMV coat protein, and the CMV coat protein consists of SEQ ID NO: 39. In a preferred embodiment, the CMV polypeptide consists of a coat protein of CMV, and the coat protein of CMV consists of SEQ ID NO:39.

[0076] In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40 or an amino acid sequence region, wherein the amino acid sequence region has at least 75% sequence identity to SEQ ID NO:40. In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40 or an amino acid sequence region, wherein the amino acid sequence region has at least 80% sequence identity to SEQ ID NO:40. In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40 or an amino acid sequence region, wherein the amino acid sequence region has at least 85% sequence identity to SEQ ID NO:40. In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40 or an amino acid sequence region, wherein the amino acid sequence region has at least 90% sequence identity to SEQ ID NO:40. In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40 or an amino acid sequence region, wherein the amino acid sequence region has at least 95% sequence identity to SEQ ID NO:40. In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40 or an amino acid sequence region, wherein the amino acid sequence region has at least 98% sequence identity to SEQ ID NO:40. In a preferred embodiment, the CMV polypeptide comprises SEQ ID NO:40, or an amino acid sequence region, which has at least 99% sequence identity to SEQ ID NO:40.

[0077] In preferred embodiments, the CMV polypeptide comprises, or preferably consists of, (i) an amino acid sequence of a coat protein of CMV, the amino acid sequence comprising, or preferably consisting of, SEQ ID NO: 39, or (ii) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39, wherein the amino acid sequence defined in (i) or (ii) comprises SEQ ID NO: 40 or an amino acid sequence region having at least 90% sequence identity to SEQ ID NO: 40. In preferred embodiments, the CMV polypeptide comprises, or preferably consists of, (i) an amino acid sequence of a coat protein of CMV, the amino acid sequence comprising, or preferably consisting of SEQ ID NO: 39, or (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 39, wherein the amino acid sequence defined in (i) or (ii) comprises SEQ ID NO: 40 or an amino acid sequence region having at least 95% sequence identity to SEQ ID NO: 40. In a preferred embodiment, the CMV polypeptide comprises (i) an amino acid sequence of a coat protein of CMV, wherein the amino acid sequence comprises, or preferably consists of, an amino acid sequence of SEQ ID NO: 39, or (ii) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39, wherein the amino acid sequence defined in (i) or (ii) comprises, or preferably consists of, SEQ ID NO: 40.

[0078] In a preferred embodiment, the number of amino acids substituted in the N-terminal region is equal to or less than the number of amino acids constituting the helper T cell epitope. In a preferred embodiment, the substituted N-terminal region of the CMV polypeptide consists of 5 to 15 consecutive amino acids. In a preferred embodiment, the substituted N-terminal region of the CMV polypeptide consists of 9 to 14 consecutive amino acids. In a preferred embodiment, the substituted N-terminal region of the CMV polypeptide consists of 11 to 13 consecutive amino acids. In a preferred embodiment, the N-terminal region of the CMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO: 39. In a preferred embodiment, the N-terminal region of the CMV polypeptide comprises amino acids 2 to 12 of SEQ ID NO: 39. In a preferred embodiment, the N-terminal region of the CMV polypeptide consists of amino acids 2 to 12 of SEQ ID NO: 39. In a preferred embodiment, the helper T cell epitope consists of a maximum of 20 amino acids.

[0079] In a preferred embodiment of the present invention, the Th cell epitope is selected from TT830-843 (SEQ ID NO: 41), PADRE (SEQ ID NO: 42), HA307-319 (SEQ ID NO: 43), HBVnc50-69 (SEQ ID NO: 44), CS378-398 (SEQ ID NO: 45), MT17-31 (SEQ ID NO: 46), and TT947-967 (SEQ ID NO: 47). In a preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin or is the PADRE sequence. In a preferred embodiment, the helper T cell epitope is derived from a human vaccine. In a preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin. In a preferred embodiment, the Th cell epitope is the PADRE sequence. In a preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In a highly preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In a highly preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 41. In a preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 41. In a highly preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 42. In a highly preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 42.

[0080] In a preferred embodiment, the CMV polypeptide comprises, or preferably consists of, the amino acid sequence of a CMV coat protein, the amino acid sequence comprising, or preferably consisting of, SEQ ID NO: 39 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 39, the amino acid sequence comprising SEQ ID NO: 40, and the helper T cell epitope replaces the N-terminal region of the CMV polypeptide, the substituted N-terminal region of the CMV polypeptide consisting of 11 to 13 contiguous amino acids, preferably 11 contiguous amino acids, and more preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO: 39. In a preferred embodiment, the chimeric CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 5, and the polypeptide is inserted between any amino acid residues of the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39. In a preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between any amino acid residues of the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO:39.

[0081] In a preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 and less than 12 amino acids. In a preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 10 amino acids. In a more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 9 amino acids. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 8 amino acids. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 9 amino acids. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 8 amino acids. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4, 5, 6, 7, or 8 amino acids. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 or 8 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 5 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 6 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 7 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 8 amino acids. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 9 amino acids.

[0082] In a further preferred embodiment, the stretch of contiguous negatively charged amino acids are independently selected from aspartic acid or glutamic acid, wherein the aspartic acid or the glutamic acid, in each instance, is independently selected from its L-configuration or its D-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one aspartic acid in the L-configuration or the D-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one aspartic acid in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one aspartic acid in the D-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one glutamic acid in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one glutamic acid in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one glutamic acid in the D-configuration.

[0083] In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one aspartic acid in the L-configuration and at least one glutamic acid in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids consists entirely of aspartic acids and glutamic acids in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids consists entirely of aspartic acids or glutamic acids in the L-configuration.

[0084] In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one aspartic acid or at least one glutamic acid. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least two aspartic acids or at least two glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least three aspartic acids or at least three glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four aspartic acids or at least four glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four aspartic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least five glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least six glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least 7 glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least 8 glutamic acids. In a further preferred embodiment, the stretch of consecutive negatively charged amino acids consists exclusively of aspartic acids. In a further highly preferred embodiment, the stretch of consecutive negatively charged amino acids consists exclusively of glutamic acids.

[0085] In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least one aspartic acid or at least one glutamic acid, wherein the at least one aspartic acid or the at least one glutamic acid is in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least two aspartic acids or at least two glutamic acids, wherein the at least two aspartic acids or the at least two glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least three aspartic acids or at least three glutamic acids, wherein the at least three aspartic acids or the at least three glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least four aspartic acids or at least four glutamic acids, wherein the at least four aspartic acids or the at least four glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least four aspartic acids, wherein the at least four aspartic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least four glutamic acids, wherein the at least four glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least five glutamic acids, wherein the at least five glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least six glutamic acids, wherein the at least six glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least seven glutamic acids, wherein the at least seven glutamic acids are in the L-configuration. In a further preferred embodiment, the stretch of contiguous negatively charged amino acids comprises at least eight glutamic acids, wherein the at least eight glutamic acids are in the L-configuration. In a more preferred embodiment, the stretch of contiguous negatively charged amino acids consists solely of aspartic acid, wherein the aspartic acid is in the L-configuration.In a more highly preferred embodiment, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, wherein the glutamic acid is in the L-configuration.

[0086] In a preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 10 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 9 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 8 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a still more preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 9 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 8 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4, 5, 6, 7, or 8 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 8 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4, 5, 6, 7, or 8 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 or 8 amino acids, and the stretch of contiguous negatively charged amino acids consists solely of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 amino acids and the stretch consists exclusively of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 amino acids and the stretch consists exclusively of glutamic acid.In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 5 amino acids and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 6 amino acids and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 7 amino acids and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 8 amino acids and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 9 amino acids and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid.

[0087] In a preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 10 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 9 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 to 8 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 9 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 8 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4, 5, 6, 7, or 8 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 to 8 amino acids, the stretch of contiguous negatively charged amino acids consists solely of glutamic acid, and the glutamic acid is in the L-configuration. In even more preferred embodiments, the stretch of contiguous negatively charged amino acids is 4, 5, 6, 7, or 8 amino acids in length, and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acids, which are in the L-configuration. In even more preferred embodiments, the stretch of contiguous negatively charged amino acids is 4 or 8 amino acids in length, and the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acids, which are in the L-configuration.In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 3 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 4 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 5 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 6 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 7 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 8 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration. In a further preferred embodiment, the length of the stretch of contiguous negatively charged amino acids is 9 amino acids, the stretch of contiguous negatively charged amino acids consists exclusively of glutamic acid, the glutamic acid being in the L-configuration.

[0088] In an even more highly preferred embodiment, the stretch of contiguous negatively charged amino acids comprises SEQ ID NO: 1 or SEQ ID NO: 2. In an even more highly preferred embodiment, the stretch of contiguous negatively charged amino acids consists of SEQ ID NO: 1 or SEQ ID NO: 2. In an even more highly preferred embodiment, the stretch of contiguous negatively charged amino acids comprises SEQ ID NO: 1. In an even more highly preferred embodiment, the stretch of contiguous negatively charged amino acids consists of SEQ ID NO: 1. In an even more highly preferred embodiment, the stretch of contiguous negatively charged amino acids comprises SEQ ID NO: 2. In an even more highly preferred embodiment, the stretch of contiguous negatively charged amino acids consists of SEQ ID NO: 2.

[0089] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker, which is disposed at the N-terminus or C-terminus of the stretch of consecutive negatively charged amino acids. In a preferred embodiment, the polypeptide further comprises a first amino acid linker, which is disposed at the N-terminus of the stretch of consecutive negatively charged amino acids. In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker, which is disposed at the C-terminus of the stretch of consecutive negatively charged amino acids. In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a second amino acid linker. In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is positioned at the N-terminus of the stretch of consecutive negatively charged amino acids and the second amino acid linker is positioned at the C-terminus of the stretch of consecutive negatively charged amino acids.

[0090] In a preferred embodiment, the first amino acid linker is up to 30 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 20, 19, 18, 17, or 16 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 15 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 14 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 13 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 12 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 11 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 10 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 9 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 8 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 7 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 6 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 5 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 4 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 3 amino acids in length. In a preferred embodiment, the first amino acid linker is up to 2 amino acids in length. In a preferred embodiment, the first amino acid linker consists of 1 amino acid. In a preferred embodiment, the second amino acid linker is up to 30 amino acids in length. In a preferred embodiment, the second amino acid linker is up to 20, 19, 18, 17, or 16 amino acids in length. In a preferred embodiment, the second amino acid linker is up to 15 amino acids in length. In a preferred embodiment, the second amino acid linker is up to 14 amino acids in length. In a preferred embodiment, the second amino acid linker is up to 13 amino acids in length. In a preferred embodiment, the second amino acid linker is up to 12 amino acids in length. In a preferred embodiment, the second amino acid linker is up to 11 amino acids in length.In a preferred embodiment, the second amino acid linker is at most 10 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 9 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 8 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 7 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 6 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 5 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 4 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 3 amino acids in length. In a preferred embodiment, the second amino acid linker is at most 2 amino acids in length. In a preferred embodiment, the second amino acid linker consists of 1 amino acid.

[0091] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker, the first amino acid linker being located at the N-terminus or C-terminus of the stretch of consecutive negatively charged amino acids, and the first amino acid linker being (a) a polyglycine linker (Gly) having a length of n=2 to 10. n (b) a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, preferably the GS linker has r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS) r (G s S) t (GS) u and (c) an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys and Cys.

[0092] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a second amino acid linker, the second amino acid linker being located at the N-terminus or C-terminus of the stretch of consecutive negatively charged amino acids, and the second amino acid linker being (a) a polyglycine linker (Gly) having a length of n=2 to 10. n (b) a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, preferably the GS linker has r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS) r (G s S) t (GS) u and (c) an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys. In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is located at the N-terminus of the stretch of consecutive negatively charged amino acids and the second amino acid linker is located at the C-terminus of the stretch of consecutive negatively charged amino acids, and the first amino acid linker and the second amino acid linker are selected from the group consisting of: (a.) an amino acid sequence (Gly) having a length of n=2 to 10 n (b.) a polyglycine linker (G-linker) having at least one glycine and at least one serine; (b.) a glycine-serine linker (GS-linker) having r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS) r (G s S) t (GS) uand (c.) an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys and Cys.

[0093] In a preferred embodiment, the polypeptide comprising the stretch of contiguous negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is disposed at the N-terminus of the stretch of contiguous negatively charged amino acids, and the second amino acid linker is disposed at the C-terminus of the stretch of contiguous negatively charged amino acids, and the first amino acid linker and the second amino acid linker are glycine-serine linkers (GS-linkers) comprising at least one glycine and at least one serine, and the GS linker has r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS). r (G s S) t (GS) u or an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys and Cys, having the amino acid sequence:

[0094] In a preferred embodiment, the first amino acid linker is a polyglycine linker (Gly) having a length of n=2 to 10. n In a preferred embodiment, the first amino acid linker is a glycine-serine linker (GS-linker) containing at least one glycine and at least one serine. In a preferred embodiment, the first amino acid linker is a glycine-serine linker (GS-linker) containing at least one glycine and at least one serine, and the first amino acid linker has Gly-Ser at the N-terminus. In a further preferred embodiment, the GS linker is such that r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS).r (G s S) t (GS) u In a more preferred embodiment, the first amino acid linker is a glycine-serine linker (GS-linker), wherein r=0 or 1, s=3 or 4, t=1, 2 or 3, and u=0 or 1 (GS) r (G s S) t (GS) u In a further preferred embodiment, the GS-linker has a length of up to 15, 14, 13, 12, or 11 amino acids, preferably 10, 9, 8, or 7 amino acids, and even more preferably a length of up to 6 amino acids. In a further preferred embodiment, the first amino acid linker is a glycine-serine linker (GS-linker), and the GS linker has the amino acid sequence of SEQ ID NO: 8. In a further preferred embodiment, the first amino acid linker has the amino acid sequence of SEQ ID NO: 8. In a preferred embodiment, the first amino acid linker is a glycine-serine linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys.

[0095] In a preferred embodiment, the second amino acid linker is a polyglycine linker (Gly) having a length of n=2 to 10. n In a preferred embodiment, the second amino acid linker is a glycine-serine linker (GS-linker) consisting of at least one glycine and at least one serine. In a preferred embodiment, the second amino acid linker is a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, and the second amino acid linker has Gly-Ser at the N-terminus. In a further preferred embodiment, the second amino acid linker is a glycine-serine linker (GS-linker), and the GS linker has r=0 or 1, s=3 or 4, t=1, 2 or 3, and u=0 or 1 (GS). r (G s S)t (GS) u In a further preferred embodiment, the GS-linker is up to 15, 14, 13, 12, or 11 amino acids in length, preferably 10, 9, 8, or 7 amino acids, and more preferably up to 6 amino acids in length. In a further preferred embodiment, the second amino acid linker is a glycine-serine linker (GS-linker), and the GS linker has the amino acid sequence of SEQ ID NO:9.

[0096] In a preferred embodiment, the second amino acid linker is an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys. In a preferred embodiment, the second amino acid linker is an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least Cys. In a preferred embodiment, the second amino acid linker is a glycine-serine linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least Cys, and the second amino acid linker has a Gly-Ser at the N-terminus. In a further preferred embodiment, the length of the second amino acid linker (GS*-linker) is at most 15, 14, 13, 12, or 11 amino acids, preferably 10 or 9 amino acids, and more preferably at most 7 or 6 amino acids. In a further preferred embodiment, the second amino acid linker is an amino acid linker (GS*-linker), wherein the GS* linker has the amino acid sequence of SEQ ID NO:4.

[0097] In a preferred embodiment, the first amino acid linker and the second amino acid linker are independently a polyglycine linker (Gly) having a length of n=2 to 10. nIn a preferred embodiment, the first amino acid linker and the second amino acid linker are independently a glycine-serine linker (GS-linker) containing at least one glycine and at least one serine. In a preferred embodiment, the first amino acid linker and the second amino acid linker are independently an amino acid linker (GS*-linker) containing at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys, and the second amino acid linker has Gly-Ser at the N-terminus. In a further preferred embodiment, the GS linker is such that r=0 or 1, s=1 to 5, t=1 to 5, and u=0 or 1 (GS). r (G s S) t (GS) u In a further preferred embodiment, the first amino acid linker and the second amino acid linker are independently a glycine-serine linker (GS-linker), wherein r=0 or 1, s=2, 3 or 4, t=1, 2 or 3, and u=0 or 1 (GS) r (G s S) t (GS) u It has the amino acid sequence:

[0098] In a further preferred embodiment, the first amino acid linker and / or the second amino acid linker comprise, preferably consist of, an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:9. In a further highly preferred embodiment, the first amino acid linker comprises, preferably consists of SEQ ID NO:8. In a further highly preferred embodiment, the second amino acid linker comprises, preferably consists of SEQ ID NO:4 or SEQ ID NO:9. In a further highly preferred embodiment, the second amino acid linker comprises, preferably consists of SEQ ID NO:4. In a further highly preferred embodiment, the second amino acid linker comprises, preferably consists of SEQ ID NO:9. In a further highly preferred embodiment, the first amino acid linker comprises, preferably consists of SEQ ID NO:8, and the second amino acid linker comprises, preferably consists of SEQ ID NO:4 or SEQ ID NO:9. In a further highly preferred embodiment, the first amino acid linker comprises, preferably consists of SEQ ID NO:8, and the second amino acid linker comprises, preferably consists of SEQ ID NO:4 or SEQ ID NO:9. In a further highly preferred embodiment, the first amino acid linker comprises, and preferably consists of, SEQ ID NO:8, and the second amino acid linker comprises, and preferably consists of, SEQ ID NO:9.

[0099] In a preferred embodiment, the polypeptide comprising, and preferably consisting of, a stretch of contiguous negatively charged amino acids is at most 30 amino acids in length. In a preferred embodiment, the polypeptide is at most 25, 24, 23, 22, or 21 amino acids in length. In a preferred embodiment, the polypeptide is at most 20 amino acids in length. In a preferred embodiment, the polypeptide is at most 19 amino acids in length. In a preferred embodiment, the polypeptide is at most 18 amino acids in length. In a preferred embodiment, the polypeptide is at most 17 amino acids in length. In a preferred embodiment, the polypeptide is at most 16 amino acids in length. In a preferred embodiment, the polypeptide is at most 15 amino acids in length. In a preferred embodiment, the polypeptide is at most 14 amino acids in length. In a preferred embodiment, the polypeptide is at most 13 amino acids in length. In a preferred embodiment, the polypeptide is at most 12 amino acids in length. In a preferred embodiment, the polypeptide is at most 11 amino acids in length. In a preferred embodiment, the polypeptide is at most 10 amino acids in length. In a preferred embodiment, the polypeptide is at most 9 amino acids in length. In a preferred embodiment, the polypeptide is at most 8 amino acids in length. In a preferred embodiment, the polypeptide is at most 7 amino acids in length. In a preferred embodiment, the polypeptide is at most 6 amino acids in length. In a preferred embodiment, the polypeptide is at most 5 amino acids in length. In a preferred embodiment, the polypeptide is at most 4 amino acids in length. In a further preferred embodiment, the polypeptide consists of said stretch of contiguous negatively charged amino acids.

[0100] In an even more highly preferred embodiment, the polypeptide comprises SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51. In an even more highly preferred embodiment, the polypeptide consists of SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51. In an even more highly preferred embodiment, the polypeptide comprises SEQ ID NO:49. In an even more highly preferred embodiment, the polypeptide comprises SEQ ID NO:50. In an even more highly preferred embodiment, the polypeptide comprises SEQ ID NO:51. In an even more highly preferred embodiment, the polypeptide consists of SEQ ID NO:49. In an even more highly preferred embodiment, the polypeptide consists of SEQ ID NO:50. In an even more highly preferred embodiment, the polypeptide consists of SEQ ID NO:51.

[0101] In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between any amino acid residues of the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 75 and 76 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 76 and 77 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 77 and 78 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 78 and 79 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 79 and 80 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 80 and 81 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 75 and 81 of SEQ ID NO: 39. In a further preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of contiguous negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 82 and 83 of SEQ ID NO: 39.In a further preferred embodiment, the polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 83 and 84 of SEQ ID NO: 39. In an even more highly preferred embodiment, the polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids is inserted between amino acid residues of the CMV polypeptide corresponding to amino acid residues 84 and 85 of SEQ ID NO: 39.

[0102] In highly preferred embodiments, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between amino acid residues 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between amino acid residues 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In highly preferred embodiments, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48. In highly preferred embodiments, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5. In a highly preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48.

[0103] In highly preferred embodiments, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between amino acid residues 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between amino acid residues 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In highly preferred embodiments, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48.

[0104] In a highly preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between amino acid residue 88 (Ser) and amino acid residue 89 (Thr) of SEQ ID NO: 5, and the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker and the second amino acid linker are independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, or an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys, and the first amino acid linker and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0105] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between amino acid residue 88 (Ser) and amino acid residue 89 (Thr) of SEQ ID NO: 5, and the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker and the second amino acid linker are independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, or an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys, and the first amino acid linker and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0106] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0107] Therefore, in another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a modified VLP of CMV, wherein the modified VLP of CMV comprises at least one first binding site, and the modified VLP of CMV comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); (a) and (b) are linked by the at least one first binding moiety and the at least one second binding moiety via at least one covalent non-peptide bond.

[0108] In an embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides comprise, and preferably consist of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides comprise, and preferably consist of, the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides comprise, and preferably consist of, the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides comprise, and preferably consist of, the amino acid sequence of SEQ ID NO: 12.

[0109] The modified CMV VLPs of the present invention may be expressed in prokaryotic or eukaryotic expression systems. Preferred systems are E. coli, yeast, insect cells, and mammalian cell lines. Highly preferred modified CMV VLPs are obtained by expression of the chimeric CMV polypeptide in E. coli, preferably at a temperature of 10°C to 25°C, preferably 20°C. As noted above, recombinantly produced polypeptides may include an N-terminal methionine residue. Thus, in one embodiment, the chimeric CMV polypeptide includes an N-terminal methionine residue. However, typically and preferably, the N-terminal methionine residue is cleaved from the chimeric CMV polypeptide.

[0110] In a further preferred embodiment, the modified VLP of the CMV further comprises at least one immunostimulatory agent. In a highly preferred embodiment, the immunostimulatory agent is encapsulated in the modified VLP of the invention. In another preferred embodiment, the immunostimulatory agent is mixed with the modified VLP of the invention. Immunostimulatory agents useful in the present invention are generally known in the art and are disclosed, inter alia, in WO 2003 / 024481.

[0111] In another embodiment of the present invention, the immunostimulatory substance comprises DNA or RNA of non-eukaryotic origin. In a further preferred embodiment, the immunostimulatory substance is selected from the group consisting of (a) immunostimulatory nucleic acids, (b) peptidoglycans, (c) lipopolysaccharides, (d) lipoteichoic acid, (e) imidazoquinoline compounds, (f) flagellins, (g) lipoproteins, and (h) any mixture of at least one of the substances (a) to (g). In a further preferred embodiment, the immunostimulatory substance is an immunostimulatory nucleic acid, which is selected from the group consisting of (a) ribonucleic acids, (b) deoxyribonucleic acids, (c) chimeric nucleic acids, and (d) any mixture of (a), (b), and / or (c). In a further preferred embodiment, the immunostimulatory nucleic acid is a ribonucleic acid, which is bacterial-derived RNA. In a further preferred embodiment, the immunostimulatory nucleic acid is poly(IC) or a derivative thereof. In a further preferred embodiment, the immunostimulatory nucleic acid is a deoxyribonucleic acid, and the deoxyribonucleic acid is an unmethylated CpG-containing oligonucleotide.

[0112] In a highly preferred embodiment, the immunostimulatory agent is an unmethylated CpG-containing oligonucleotide. In a further preferred embodiment, the unmethylated CpG-containing oligonucleotide is an A-type CpG. In a further preferred embodiment, the A-type CpG is a palindromic sequence. In a further preferred embodiment, the palindromic sequence is flanked at its 5'-end and at its 3'-end by a guanosine backbone. In a further preferred embodiment, the palindromic sequence is flanked at its 5'-end by at least three and at most 15 guanosine backbone, and the palindromic sequence is flanked at its 3'-end by at least three and at most 15 guanosine backbone.

[0113] In another preferred embodiment, the immunostimulatory agent is an unmethylated CpG-containing oligonucleotide, preferably wherein the unmethylated CpG-containing oligonucleotide comprises a palindromic sequence, more preferably wherein the CpG motif of the unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, and even more preferably wherein the palindromic sequence is SEQ ID NO: 52. In a further preferred embodiment, the immunostimulatory nucleic acid is an unmethylated CpG-containing oligonucleotide consisting of SEQ ID NO: 53, wherein the unmethylated CpG-containing oligonucleotide consists solely of phosphodiester-linked nucleotides.

[0114] In a further aspect, the present invention provides a composition comprising: (a) a modified VLP of CMV as defined herein, wherein the modified VLP of CMV comprises at least one first binding site; and (b) at least one NGF antigen, wherein the antigen comprises at least one second binding site, wherein (a) and (b) are linked by the at least one first binding site and the at least one second binding site, typically and preferably via at least one covalent non-peptide bond. Methods for linking the modified VLP to the antigen by the first binding site and the second binding site are described, for example, in WO2002 / 056905, WO2004 / 084940, and WO2016 / 062720.

[0115] Thus, in a further aspect, the present invention provides a composition comprising: (a) a modified VLP of CMV, wherein the modified VLP of CMV comprises at least one binding site; and (b) at least one NGF antigen, wherein the antigen comprises at least one second binding site, wherein (a) and (b) typically and preferably link the at least one first binding site and the at least one second binding site via at least one covalent non-peptide bond; and wherein the modified VLP of CMV comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide is linked to the at least one NGF antigen. The polypeptide comprises, preferably consists of, (i) a CMV polypeptide, the polypeptide comprising a CMV coat protein or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39, and (ii) a polypeptide comprising, preferably consisting of, a stretch of consecutive negatively charged amino acids, the negatively charged amino acids being independently selected from aspartic acid or glutamic acid, the polypeptide being inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39.

[0116] In a highly preferred embodiment, the at least one first binding site does not consist of, or is not a part of, a portion of a polypeptide comprising the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, all of the at least one first binding site does not consist of, or is not a part of, a portion of a polypeptide comprising the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, the at least one first binding site does not consist of, or is not a part of, a portion of the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, all of the first binding sites do not consist of, or are not a part of, a portion of the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, the first binding site and the second binding site are linked only by one or more covalent bonds. In a highly preferred embodiment, the at least one antigen is linked to the modified VLP of CMV only by one or more covalent bonds. In a highly preferred embodiment, all of the antigens are linked to the modified VLP of CMV only by one or more covalent bonds.

[0117] In a further preferred embodiment, the first binding site is linked to the second binding site via at least one covalent non-peptide bond. In a further preferred embodiment, all of the first binding sites are linked to the second binding site via at least one covalent non-peptide bond. In a further highly preferred embodiment, the first binding sites are amino groups, preferably lysine amino groups. In a further highly preferred embodiment, all of the first binding sites are amino groups, preferably lysine amino groups.

[0118] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, wherein the modified VLP of CMV comprises at least one first binding site, and the modified VLP of CMV comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); (a) and (b) link the at least one first binding site and the at least one second binding site via at least one covalent non-peptide bond by a heterobifunctional crosslinker, preferably the at least one first binding site is not composed of or is not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids, preferably the first binding site is an amino group, here preferably the amino group of a lysine, more preferably the second binding site is a sulfhydryl group, preferably the sulfhydryl group of a cysteine ​​residue or a sulfhydryl group chemically bound to an NGF antigen.

[0119] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0120] The bond between the modified virus-like particle and the antigen via a disulfide bond is typically unstable, especially for molecules containing sulfhydryl moieties, and is also less stable in serum than, for example, a thioether bond (Martin FJ. and Papahadjopoulos D. (1982) J. Biol. Chem. 257:286-288). Therefore, in a further highly preferred embodiment of the present invention, the interaction or binding between the modified CMV VLP and the at least one antigen does not involve a disulfide bond. Accordingly, it is further preferred that at least one second binding site comprises, or preferably is, a sulfhydryl group. Preferably, all of the second binding sites comprise, or preferably are, sulfhydryl groups. In a further preferred embodiment, the at least one first binding site is not or does not contain a sulfhydryl group. In a further preferred embodiment, all of the first binding sites are not or do not contain a sulfhydryl group. In preferred embodiments, the at least one first binding site is not or does not contain a cysteine ​​sulfhydryl group. In even more preferred embodiments, all of the first binding sites are not or do not contain a cysteine ​​sulfhydryl group. In even more highly preferred embodiments, the second binding site is a sulfhydryl group, preferably a cysteine ​​sulfhydryl group. In even even more highly preferred embodiments, all of the second binding sites are sulfhydryl groups, preferably cysteine ​​sulfhydryl groups.

[0121] In a highly preferred embodiment, at least one first binding site is an amino group, preferably an amino group of a lysine residue, and at least one second binding site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue or a sulfhydryl group chemically bound to an antigen. In a highly preferred embodiment, all of the first binding sites are amino groups, preferably amino groups of lysine residues, and all of the second binding sites are sulfhydryl groups, preferably a sulfhydryl group of a cysteine ​​residue or a sulfhydryl group chemically bound to an antigen. In a further preferred embodiment, only one of the second binding sites interacts with the first binding site through at least one non-peptide covalent bond to provide a single, uniform type of binding of the antigen to the modified VLP of CMV, and the only second binding site that interacts with the first binding site is a sulfhydryl group, and the antigen and the modified VLP of CMV interact through this interaction to form an ordered, repetitive antigen array.

[0122] In one preferred embodiment of the present invention, the antigen is conjugated to the modified CMV VLP by chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker. Thus, in a preferred embodiment, the NGF antigen is conjugated to the modified CMV VLP by chemical cross-linking, typically and preferably by at least one covalent non-peptide bond, via the heterobifunctional cross-linker via the at least one first binding site and the at least one second binding site. In a preferred embodiment, the heterobifunctional cross-linker contains a functional group capable of reacting with a preferred first binding site, preferably an amino group, more preferably the amino group of one or more lysine residues of the modified CMV VLP, and a functional group capable of reacting with a preferred second binding site, i.e., a sulfhydryl group, preferably the sulfhydryl group of one or more cysteines inherent in or artificially added to the antigen, and optionally further available for reaction by reduction. Several heterobifunctional cross-linkers are known in the art. These include the preferred crosslinker succinimidyl-6-(b-maleimidopropionamide)hexanoate (SMPH) (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, Sulfo-KMUS SVSB, SIA, and other crosslinkers available, for example, from Pierce Chemical Company, that have one functional group reactive toward amino groups and one functional group reactive toward sulfhydryl groups. All of the above crosslinkers result in the formation of an amide bond after reaction with an amino group and a thioether bond with a sulfhydryl group. In a highly preferred embodiment, the heterobifunctional crosslinker is SMPH.Therefore, in a preferred embodiment, the NGF antigen is chemically cross-linked to the modified CMV VLP via at least one first binding site and at least one second binding site, typically and preferably via at least one covalent non-peptide bond, using a heterobifunctional cross-linker, where the heterobifunctional cross-linker is SMPH. Another class of cross-linkers suitable for the practice of the present invention is characterized by the introduction of a disulfide bond between the antigen and the modified VLP upon coupling. Preferred cross-linkers in this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).

[0123] Therefore, in a further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, wherein the modified VLP of CMV comprises at least one first binding site, and the modified VLP of CMV comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); (a) and (b) are linked by at least one covalent non-peptide bond via a heterobifunctional crosslinker through the at least one first binding site and the at least one second binding site, preferably the heterobifunctional crosslinker is SMPH, the at least one first binding site does not consist of or is not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids, the at least one first binding site is an amino group, thereby preferably an amino group of a lysine, and the at least one second binding site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue or a sulfhydryl group chemically bound to an NGF antigen.

[0124] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0125] Conjugating an antigen to a modified CMV VLP using a heterobifunctional crosslinker allows the antigen to be conjugated to the modified CMV VLP in an oriented manner. Other methods for conjugating an antigen to a modified CMV VLP include crosslinking the antigen to the modified CMV VLP using the carbodiimide EDC and NHS. The antigen may also be first thiolated, for example, via reaction with SATA, SATP, or iminothiolane. After optional deprotection, the antigen may be coupled to the modified CMV VLP as follows: After separating excess thiolation reagent, the antigen is reacted with a modified CMV VLP preactivated with a heterobifunctional crosslinker containing a cysteine-reactive moiety. Thus, as described above, the thiolated antigen presents at least one functional group reactive to a cysteine ​​residue that can react with the antigen. Optionally, a small amount of a reducing agent is included in the reaction mixture. In a further method, the antigen is conjugated to the modified CMV VLP using a homobifunctional crosslinker such as glutaraldehyde, DSG, BM[PEO]4, BS3 (Pierce), or other known homobifunctional crosslinkers that have functional groups reactive to the amine or carboxy groups of the modified VLP.

[0126] In highly preferred embodiments of the invention, the antigen is linked to a lysine residue of the modified VLP of CMV by a cysteine ​​residue added to either the N- or C-terminus, or by a naturally occurring cysteine ​​residue within the antigen. In preferred embodiments, the composition of the invention further comprises a linker that allows the antigen to interact with the second binding site, and preferably the linker comprises or alternatively consists of the second binding site.

[0127] Engineering a second binding site to the antigen is typically and preferably achieved by interaction of a linker containing at least one amino acid suitable as a second binding site according to the present disclosure. Thus, in a preferred embodiment of the present invention, the linker interacts with the antigen via at least one covalent bond, preferably at least one, preferably one peptide bond. Preferably, the linker comprises, or alternatively consists of, the second binding site. In a further preferred embodiment, the linker comprises a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue. In a preferred embodiment, the linker comprises or is a cysteine ​​residue. In a further preferred embodiment of the present invention, the linker consists of amino acids, more preferably the linker consists of up to 15 amino acids. In a further preferred embodiment of the present invention, such amino acid linkers contain 1 to 10 amino acids.

[0128] In a still further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, wherein the modified VLP of CMV comprises at least one first binding site, and the modified VLP of CMV comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10; (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); (a) and (b) link the at least one first binding site and the at least one second binding site via at least one covalent non-peptide bond by a heterobifunctional crosslinker, the preferred heterobifunctional crosslinker being SMPH, the at least one first binding site not consisting of or not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids, the at least one first binding site being an amino group, preferably an amino group of a lysine, and the at least one second binding site being a sulfhydryl group, preferably a sulfhydryl group chemically bound to an NGF antigen.

[0129] In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides comprise, and preferably consist of, the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides consist of the amino acid sequence of SEQ ID NO: 10.

[0130] In another preferred embodiment, the antigen is a nerve growth factor (NGF) selected from human NGF (hNGF), canine NGF (cNGF), feline (fNGF), equine NGF (eNGF), bovine NGF (bNGF) and porcine NGF (pNGF), preferably a nerve growth factor (NGF) selected from canine NGF (cNGF) or feline NGF (fNGF), more preferably the antigen is canine NGF (cNGF). In a preferred embodiment, the antigen comprises, or preferably consists of, an amino acid sequence selected from any of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, or an amino acid sequence having at least 90% or at least 91%, preferably at least 92%, at least 93%, or at least 94%, more preferably at least 95%, at least 96%, or at least 97%, even more preferably at least 98% or at least 99% sequence identity to any of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of at least two consecutive and up to 12 consecutive histidine residues, preferably located at the C-terminus or the N-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive histidine residues, preferably located at the C-terminus or N-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of 4, 6, 8, or 10 consecutive histidine residues, preferably located at the C-terminus or N-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of 4 consecutive histidine residues, preferably located at the C-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of 4 consecutive histidine residues, preferably located at the N-terminus of the NGF antigen.In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of six consecutive histidine residues consisting of SEQ ID NO: 34, preferably located at the C-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of six consecutive histidine residues consisting of SEQ ID NO: 34, preferably located at the N-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of eight consecutive histidine residues, preferably located at the C-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of eight consecutive histidine residues, preferably located at the N-terminus of the NGF antigen. In a further preferred embodiment, the NGF antigen comprises a polyhistidine tag of ten consecutive histidine residues, preferably located at the C-terminus or N-terminus of the NGF antigen.

[0131] In an alternative embodiment, the antigen is human NGF. In a further embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 54, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 54. In a further embodiment, the antigen comprises SEQ ID NO: 54. Furthermore, the antigen consists of SEQ ID NO: 54.

[0132] In an even more highly preferred embodiment, the antigen is canine NGF. In an even more highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity to SEQ ID NO:30, or SEQ ID NO:31, or SEQ ID NO:33. In an even more preferred embodiment, the antigen comprises SEQ ID NO:30, or SEQ ID NO:31, or SEQ ID NO:33. In an even more highly preferred embodiment, the antigen comprises, or preferably consists of SEQ ID NO:30, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity to SEQ ID NO:30. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO:30. In an even more highly preferred embodiment, the antigen consists of SEQ ID NO:30. In a further preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 31, or an amino acid sequence having sequence identity with SEQ ID NO: 31 of at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity. In a further highly preferred embodiment, the antigen comprises SEQ ID NO: 31. In a further highly preferred embodiment, the antigen consists of SEQ ID NO: 31. In a further highly preferred embodiment, the antigen comprises, or preferably consists of SEQ ID NO: 33, or an amino acid sequence having sequence identity with SEQ ID NO: 33 of at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity. In a further highly preferred embodiment, the antigen comprises SEQ ID NO: 33. In a further highly preferred embodiment, the antigen consists of SEQ ID NO: 33.

[0133] In a further highly preferred embodiment, the antigen comprises or consists of SEQ ID NO: 30, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 30, and the NGF antigen further comprises a polyhistidine tag of at least 2 consecutive and up to 12 consecutive histidine residues, preferably 4, 6, 8 or 10 consecutive histidine residues, more preferably 6 consecutive histidine residues consisting of SEQ ID NO: 34, preferably located at the C-terminus or N-terminus of the NGF antigen, more preferably located at the C-terminus of the NGF antigen. In a further highly preferred embodiment, the antigen comprises or consists of SEQ ID NO: 30, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 30, and the NGF antigen further comprises a polyhistidine tag of 4, 6, 8 or 10 consecutive histidine residues, preferably 6 consecutive histidine residues consisting of SEQ ID NO: 34, which is preferably located at the C-terminus or N-terminus of the NGF antigen, more preferably at the C-terminus of the NGF antigen. In an even more highly preferred embodiment, the antigen comprises or consists of SEQ ID NO: 30, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 30, wherein the NGF antigen further comprises a polyhistidine tag of six consecutive histidine residues consisting of SEQ ID NO: 34, preferably located at the C-terminus or N-terminus of the NGF antigen, and even more preferably located at the C-terminus of the NGF antigen. In an even more highly preferred embodiment, the antigen comprises or consists of SEQ ID NO: 30, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 30, wherein the NGF antigen further comprises a polyhistidine tag of six consecutive histidine residues consisting of SEQ ID NO: 34, located at the N-terminus of the NGF antigen.In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 30, and the NGF antigen further comprises a polyhistidine tag of at least 2 consecutive and up to 12 consecutive histidine residues, preferably 4, 6, 8, or 10 consecutive histidine residues, and more preferably 6 consecutive histidine residues of SEQ ID NO: 34, preferably located at the C-terminus or N-terminus of the NGF antigen, and more preferably located at the C-terminus of the NGF antigen. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 30, and the NGF antigen further comprises a polyhistidine tag of 6 consecutive histidine residues of SEQ ID NO: 34, preferably located at the C-terminus or N-terminus of the NGF antigen, and more preferably located at the C-terminus of the NGF antigen. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 30, and the NGF antigen further comprises a polyhistidine tag of 6 consecutive histidine residues of SEQ ID NO: 34, preferably located at the N-terminus of the NGF antigen. In a further highly preferred embodiment, the antigen consists of SEQ ID NO:30.

[0134] In an even more highly preferred embodiment, the antigen is feline NGF. In an even more highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 55, or an amino acid sequence having at least 90%, or at least 91%, preferably at least 92%, at least 93%, or at least 94%, more preferably at least 95%, at least 96%, at least 97%, and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 55. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 55. In an even more highly preferred embodiment, the antigen consists of SEQ ID NO: 55.

[0135] In a further preferred embodiment, the antigen is equine NGF. In an even more highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 56, or an amino acid sequence having at least 90% or at least 91%, preferably at least 92%, at least 93%, or at least 94%, more preferably at least 95%, at least 96%, or at least 97%, and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 56. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 56. In an even more highly preferred embodiment, the antigen consists of SEQ ID NO: 56.

[0136] In a further preferred embodiment, the antigen is bovine NGF. In an even more highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 57, or an amino acid sequence having at least 90%, or at least 91%, preferably at least 92%, at least 93%, or at least 94%, more preferably at least 95%, at least 96%, at least 97%, and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 57. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 57. In an even more highly preferred embodiment, the antigen consists of SEQ ID NO: 57.

[0137] In an even more highly preferred embodiment, the antigen is porcine NGF. In an even more highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 58, or an amino acid sequence having at least 90% or at least 91%, preferably at least 92%, at least 93%, or at least 94%, more preferably at least 95%, at least 96%, or at least 97%, and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 58. In an even more highly preferred embodiment, the antigen comprises SEQ ID NO: 58. In an even more highly preferred embodiment, the antigen consists of SEQ ID NO: 58.

[0138] Without being bound, the inventors believe that undesired aggregation and the formation of aggregated conjugated CMV VLPs can be particularly reduced and avoided for antigens with higher isoelectric points, and therefore, for antigens with an overall positive charge under the conditions used for conjugation. Thus, in a preferred embodiment, the NGF antigen has an isoelectric point greater than 6.5. In a preferred embodiment, the NGF antigen has an isoelectric point greater than 6.5 and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of greater than 6.5 as determined by the ExPASy Compute pl / MW tool as described in Gasteiger et al. (Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, MR, Appel, RD, & Bairoch, A., Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). In a preferred embodiment, the NGF antigen has an isoelectric point of greater than 6.5 as determined by the ExPASy Compute pl / MW tool as described in Gasteiger et al. (Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, MR, Appel, RD, & Bairoch, A., Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). It has an isoelectric point greater than 6.5 and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool as described in the Protocols Handbook, Humana Press (2005). In preferred embodiments, the NGF antigen has an isoelectric point greater than 6.6, 6.7, 6.8, or 6.9.In a preferred embodiment, the NGF antigen has an isoelectric point greater than 6.6, 6.7, 6.8, or 6.9 and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point greater than 6.6, 6.7, 6.8, or 6.9, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point greater than 6.6, 6.7, 6.8, or 6.9 and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.0 or greater. In a preferred embodiment, the NGF antigen has an isoelectric point greater than 7.0 and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.0 or greater, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.0 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.1, 7.2, 7.3, or 7.4 or greater. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.1, 7.2, 7.3, or 7.4 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.1, 7.2, 7.3, or 7.4 or greater, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.1, 7.2, 7.3 or 7.4 or more and less than 13.0, preferably less than 12.5, more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.5 or more.In a preferred embodiment, the NGF antigen has an isoelectric point of 7.5 or more and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.5 or more, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the antigen has an isoelectric point of 7.5 or more and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.6, 7.7, 7.8, or 7.9 or more. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.6, 7.7, 7.8, or 7.9 or more and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.6, 7.7, 7.8, or 7.9 or greater, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 7.6, 7.7, 7.8, or 7.9 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.0 or greater. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.0 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.0 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.0 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.1, 8.2, 8.3, or 8.4 or greater.In a preferred embodiment, the NGF antigen has an isoelectric point of 8.1, 8.2, 8.3, or 8.4 or more and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.1, 8.2, 8.3, or 8.4 or more, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.1, 8.2, 8.3, or 8.4 or more and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.5 or more. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.5 or more and less than 13.0, preferably less than 12.5, and more preferably less than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.5 or greater as determined by the ExPASy Compute pl / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point of 8.5 or greater and less than 13.0, preferably less than 12.5, and more preferably less than 12.0, as determined by the ExPASy Compute pl / MW tool.

[0139] In highly preferred embodiments, the polypeptide comprising the stretch of contiguous negatively charged amino acids comprises SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51. In even more highly preferred embodiments, the polypeptide consists of SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51. In even more highly preferred embodiments, the polypeptide comprises SEQ ID NO:49. In even more highly preferred embodiments, the polypeptide comprises SEQ ID NO:50. In even more highly preferred embodiments, the polypeptide comprises SEQ ID NO:51. In even more highly preferred embodiments, the polypeptide consists of SEQ ID NO:49. In even more highly preferred embodiments, the polypeptide consists of SEQ ID NO:50. In even more highly preferred embodiments, the polypeptide consists of SEQ ID NO:51.

[0140] In highly preferred embodiments, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between amino acid residues 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between amino acid residues 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In highly preferred embodiments, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48. In highly preferred embodiments, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5. In a highly preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48.

[0141] In highly preferred embodiments, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between amino acid residues 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between amino acid residues 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In highly preferred embodiments, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39, or SEQ ID NO:48, and the polypeptide comprising the stretch of contiguous negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:5, between amino acid residues 84 and 85 of SEQ ID NO:39, or between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, and the polypeptide is inserted between amino acid residues 88 and 89 of SEQ ID NO:5. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, and the polypeptide is inserted between amino acid residues 84 and 85 of SEQ ID NO:39. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, and the polypeptide is inserted between amino acid residues 86 and 87 of SEQ ID NO:48.

[0142] In a highly preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between amino acid residue 88 (Ser) and amino acid residue 89 (Thr) of SEQ ID NO: 5, and the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker and the second amino acid linker are independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, or an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys, and the first amino acid linker and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0143] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between amino acid residue 88 (Ser) and amino acid residue 89 (Thr) of SEQ ID NO: 5, and the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker and the second amino acid linker are independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, or an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys, and the first amino acid linker and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0144] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0145] In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 11. In a highly preferred embodiment, the modified VLP of CMV comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 12.

[0146] In a further highly preferred embodiment, the antigen is canine NGF. In a further highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94%, or at least 95%, and even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 30, or SEQ ID NO: 31, or SEQ ID NO: 33.

[0147] Thus, in a still further aspect, the present invention provides a method for producing a composition comprising: (a) a modified VLP of CMV, wherein the modified VLP of CMV comprises at least one first binding site, and the modified VLP of CMV comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10; (b) providing a composition, preferably a veterinary composition, comprising at least one antigen, said antigen comprising at least one second binding site, said antigen being nerve growth factor (NGF), said antigen comprising, or preferably consisting of, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO:30, or SEQ ID NO:31, or SEQ ID NO:33; (a) and (b) are preferably linked by at least one covalent non-peptide bond via a heterobifunctional crosslinker through the at least one first binding site and the at least one second binding site, the preferred heterobifunctional crosslinker being SMPH, the at least one first binding site not consisting of or being part of a polypeptide comprising the stretch of consecutive negatively charged amino acids, the at least one first binding site being an amino group, thereby preferably an amino group of a lysine, and the at least one second binding site being a sulfhydryl group, preferably a sulfhydryl group chemically bound to an NGF antigen.

[0148] In a highly preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides comprise, and preferably consist of, the amino acid sequence of SEQ ID NO: 10. In a highly preferred embodiment, the modified VLP of CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptides consist of the amino acid sequence of SEQ ID NO: 10.

[0149] In a further preferred embodiment, the antigen comprises SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In a further preferred embodiment, the antigen consists of SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In a further highly preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 30, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 30. In a further highly preferred embodiment, the antigen comprises SEQ ID NO: 30. In a further highly preferred embodiment, the antigen consists of SEQ ID NO: 30. In a further preferred embodiment, the antigen comprises, or preferably consists of, SEQ ID NO: 31, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 31. In a further highly preferred embodiment, the antigen comprises SEQ ID NO: 31. In a further highly preferred embodiment, the antigen consists of SEQ ID NO: 31. In a further highly preferred embodiment, the antigen consists of SEQ ID NO: 33. In a further preferred embodiment, the antigen comprises, or preferably consists of SEQ ID NO: 33, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 33. In a further highly preferred embodiment, the antigen comprises SEQ ID NO: 33. In a further highly preferred embodiment, the antigen consists of SEQ ID NO:33.In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and the antigen comprises, or preferably consists of, SEQ ID NO:30, or SEQ ID NO:31, or SEQ ID NO:33, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94%, or at least 95%, even more preferably at least 96%, 97%, or at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:30 or SEQ ID NO:31 or SEQ ID NO:33, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 10, and the antigen comprises, or preferably consists of, SEQ ID NO: 30 or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 30, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids.In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and the antigen comprises, or preferably consists of, SEQ ID NO: 30 or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 30, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 10, and the antigen comprises, or preferably consists of, SEQ ID NO: 31 or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 31, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and the antigen comprises, or preferably consists of, SEQ ID NO: 31, or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 31, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids.In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 12, and the antigen comprises, or preferably consists of, SEQ ID NO: 30 or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 30, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids. In a highly preferred embodiment, the modified VLP of CMV comprises at least one, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 12, and the antigen comprises, or preferably consists of, SEQ ID NO: 31 or an amino acid sequence having at least 90%, preferably at least 91% or 92%, more preferably at least 93%, 94% or at least 95%, even more preferably at least 96%, 97%, or at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 31, and preferably all of the first binding site is not contained in or is not part of the polypeptide comprising the stretch of contiguous negatively charged amino acids.

[0150] The modified VLPs of the present invention can be prepared in prokaryotic or eukaryotic expression systems. Preferred systems are E. coli, yeast, insect cells, and mammalian cell lines. Highly preferred are the modified CMV VLPs or the CMV VLPs obtained by expression of the chimeric CMV polypeptide in E. coli, preferably at a temperature between 10°C and 35°C.

[0151] Accordingly, in another aspect, the present invention provides (a) a modified virus-like particle (VLP) of Cucumber Mosaic Virus (CMV), comprising at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, the CMV polypeptide comprising a coat protein of CMV or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39; and (ii) a polypeptide comprising, and preferably consisting of, a stretch of contiguous negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and the polypeptide corresponds to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39. and (b) at least one antigen, the antigen comprising at least one second binding site, the antigen being nerve growth factor (NGF), wherein (a) and (b) are linked by at least one covalent non-peptide bond between the at least one first binding site and the at least one second binding site, and the modified VLP of CMV is obtained by expression of the chimeric CMV polypeptide in Escherichia coli (E. coli), preferably wherein the expression is effected at a temperature between 10°C and 35°C.

[0152] In another aspect, the invention provides a process for producing a composition of the invention, comprising purifying the modified virus-like particle (VLP) of Cucumber Mosaic Virus (CMV) from a recombinant bacterial host expressing the modified VLP of CMV, wherein the modified VLP of CMV comprises at least one chimeric CMV polypeptide, the at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, the CMV polypeptide comprising an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; and (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residues in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39, the process comprising: (a) lysing the bacterial host; (b) clarifying the lysate obtained by the lysis; and (c) purifying the modified CMV VLP from the clarified lysate by anion exchange chromatography (AEX), the steps being performed in the given order.

[0153] In a preferred embodiment, the composition comprises an adjuvant. Typical and preferred adjuvants are inorganic salts (e.g., aluminum hydroxide, aluminum phosphate), microcrystalline tyrosine, emulsions, microparticles, saponin (Quil A), cytokines, immunopotentiators, bacterial components / products, liposomes, complexes, and mucosal adjuvants known in the art, such as those described in the Adjuvant Compendium NIAID and VAC (nih.gov) or in Aguilar et al. (Aguilar JC et al., 2007, Vaccine 25:3752-3762), Gerdts (Gerdts V, 2015, Berliner und Munchener Tierarztliche Wochenschrift 128:456-463) and Pasquale et al. (Pasquale et al., 2015, Vaccines 3:320-343). (Pasquale et al. 2015, Vaccines 3:320-343). In a preferred embodiment, the composition comprises an adjuvant, and the adjuvant is aluminum hydroxide. In another preferred embodiment, the composition lacks an adjuvant.

[0154] In a further aspect, the present invention provides a vaccine, preferably a veterinary vaccine comprising, or alternatively consisting of, a composition of the present invention comprising the modified VLP of CMV as described herein and at least one NGF antigen. Vaccines are encompassed in which the composition of the present invention comprises any one of the technical features disclosed herein, alone or in any possible combination. In a preferred embodiment, the vaccine further comprises an adjuvant. In a preferred embodiment, the vaccine comprises an adjuvant, and the adjuvant is aluminum hydroxide. In a further preferred embodiment, the vaccine lacks an adjuvant. In a preferred embodiment, the vaccine comprises an effective amount of a composition of the present invention.

[0155] In a further aspect, the present invention relates to a pharmaceutical composition comprising (a) a composition of the present invention as described herein or a vaccine of the present invention as described in the present invention, and (b) a pharmaceutically acceptable carrier, diluent and / or excipient. The diluent includes sterile aqueous (e.g., saline) or non-aqueous solutions and suspensions. The pharmaceutical composition of the present invention may be in a form containing salts, buffers, adjuvants, or other substances desirable to improve the efficacy of the conjugate. In a preferred embodiment, the pharmaceutical composition comprises an effective amount of the vaccine of the present invention. In a preferred embodiment, the pharmaceutical composition comprises an adjuvant.

[0156] A further aspect of the present invention is a method of immunization comprising administering a composition of the invention as described herein, a vaccine of the invention as described herein, or a pharmaceutical composition as described herein to an animal or a human, preferably wherein the animal is a dog or a cat, more preferably wherein the animal is a dog. In a preferred embodiment, the method comprises administering a composition of the invention as described herein, a vaccine of the invention as described herein, or a pharmaceutical composition as described herein to an animal or a human, wherein the animal is a dog or a cat, preferably wherein the animal is a dog. In a preferred embodiment, the method comprises administering an effective amount of the composition of the invention, the vaccine, or the pharmaceutical composition to the animal or the human, preferably wherein the animal is a dog or a cat, more preferably wherein the animal is a dog.

[0157] In a further aspect, the present invention provides a modified VLP of CMV as described herein, a composition of the invention as described herein, a vaccine of the invention as described herein, or a pharmaceutical composition as described herein for use in a method of immunizing an animal, preferably wherein the animal is a dog or cat, more preferably wherein the animal is a dog, the method comprising administering to the animal or the human an effective amount of the modified VLP of CMV, the composition of the invention, the vaccine, or the pharmaceutical composition, preferably wherein the animal is a dog or cat, more preferably wherein the animal is a dog.

[0158] A further aspect of the present invention is a method for inducing neutralizing antibodies against NGF in an animal, comprising administering to the animal or human a composition of the invention as described herein, a vaccine of the invention as described herein, or a pharmaceutical composition as described herein, preferably wherein the animal is a dog or cat, more preferably wherein the animal is a dog. In a preferred embodiment, the method comprises administering to the animal or human a composition of the invention as described herein, a vaccine of the invention as described herein, or a pharmaceutical composition as described herein, wherein the animal is a dog or cat, preferably wherein the animal is a dog. In a preferred embodiment, the method comprises administering to the animal or human an effective amount of the composition, vaccine, or pharmaceutical composition of the invention, preferably wherein the animal is a dog or cat, more preferably wherein the animal is a dog.

[0159] In a further aspect, the present invention provides a modified VLP of CMV as described herein, a composition of the invention as described herein, a vaccine of the invention as described herein, or a pharmaceutical composition as described herein for use in a method of immunizing an animal, preferably wherein the animal is a dog or cat, more preferably wherein the animal is a dog, the method comprising administering to the animal or the human an effective amount of the modified VLP of CMV, the composition of the invention, the vaccine, or the pharmaceutical composition, preferably wherein the animal is a dog or cat, more preferably wherein the animal is a dog.

[0160] example Example 1 Construction and production of surface charge-modified CMV VLPs Different chimeric CMV polypeptides according to the invention were prepared and subsequently expressed to yield modified CMV VLPs of the invention.

[0161] For this purpose, chimeric CMV polypeptides were prepared, including different polypeptides of consecutive negatively charged amino acids, i.e., polypeptides consisting of either 4, 8, or 12 glutamic acid residues ("E4" - SEQ ID NO: 1, "E8" - SEQ ID NO: 2, and "E12" - SEQ ID NO: 3), in which the glutamic acid residues were inserted between amino acid residues Ser(88) and Tyr(89) of the modified CMV polypeptide CMV-Ntt830 (SEQ ID NO: 5). The modified CMV polypeptide CMV-Ntt830 contains a helper T cell epitope derived from tetanus toxoid TT830 (SEQ ID NO: 6). The corresponding nucleic acid sequence (SEQ ID NO: 7) encoding the modified CMV polypeptide CMV-Ntt830 was prepared as described in Example 3 of WO 2016 / 062720 A1.

[0162] The prepared chimeric CMV polypeptides further comprise linkers flanking the incorporated E4, E8, and E12 polypeptides at both ends, specifically comprising either a GGS-linker or a GGGS-linker (SEQ ID NO:8) directly at the N-terminus of the incorporated E4, E8, and E12 polypeptides, and either a GGGSGS-linker (SEQ ID NO:9) or a CGGGSGS-linker (SEQ ID NO:4) directly at the C-terminus of the incorporated E4, E8, and E12 polypeptides.

[0163] The resulting amino acid sequences of the prepared chimeric CMV polypeptides are designated "CMV-Ntt830-E4," "CMV-Ntt830-E8," "CMV-Ntt830-E8*," and "CMV-Ntt830-E12," and are referred to below: "CMV-Ntt830-E4": SEQ ID NO: 10, "CMV-Ntt830-E8": SEQ ID NO: 11, "CMV-Ntt830-E8*": SEQ ID NO: 12, "CMV-Ntt830-E12": has an amino acid sequence as shown in SEQ ID NO:13.

[0164] The corresponding nucleotide sequence of the preferred chimeric CMV polypeptide is as follows: "CMV-Ntt830-E4": SEQ ID NO: 14, "CMV-Ntt830-E8": SEQ ID NO: 15, "CMV-Ntt830-E8*": SEQ ID NO: 16, "CMV-Ntt830-E12": as set forth in SEQ ID NO: 17.

[0165] First, a chimeric CMV polypeptide, CMV-Ntt830-E8*, was prepared, where in the first step PCR mutagenesis was used to induce the integration of the coding sequence of E8, including the flanking linker, into the modified CMV. The PCR fragment encoding the E8 sequence, including the flanking linker, and the 3'-end fragment of the modified CMV were ligated using the following oligonucleotides: Forward: E8*-1F (SEQ ID NO: 18) Forward: E8*-2F (SEQ ID NO: 19) Reverse: Amplified by two-step PCR using CMcpR (SEQ ID NO: 20).

[0166] Therefore, a PCR reaction was performed using the E8*-1F / CMcpR oligonucleotide and the pET-CMV-Ntt830 plasmid as a template. The template, pET-CMV-Ntt830, was prepared as described in Example 3 of WO 2016 / 062720 A1. The target PCR product was obtained after a second PCR using the oligonucleotide E8*-2F / CMcpR and the PCR product from the first PCR. The resulting PCR product was cloned into the helper vector pTZ57 (InsTAclone PCR Cloning Kit, Fermentas #K1214). The PCR product-containing plasmid was amplified in E. coli XL1-Blue cells, and the plasmid DNA was purified and sequenced using a BigDye cycle sequencing kit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). As a result, the helper plasmid pTZ-CMV-E8* was obtained without PCR errors.

[0167] As a next step, the BamHRI / HindIII fragment of pTZ-CMV-E8* was cloned back into the pET-CMV-Ntt830B helper vector using the same restriction sites, resulting in the expression vector pET-CMVB2-Ntt-E8C (Fig. 1 ).

[0168] The helper vector pET-CMV-Ntt830B was used to incorporate a polypeptide containing a stretch of consecutive negatively charged amino acids encoding a DNA sequence in the corresponding CMV DNA sequence of CMV-Ntt830, and a BamHI site-containing sequence was incorporated into the corresponding position for subsequent cloning. The CMV-Ntt830 encoding nucleic acid sequence was prepared as described in Example 3 of WO 2016 / 062720A1 and corresponds to SEQ ID NO: 14 of WO 2016 / 062720A1.

[0169] A BamHI site was incorporated by two-step PCR mutagenesis using the oligonucleotides listed below and the previously constructed pET-CMV-Ntt830 as template. As indicated, the template pET-CMV-Ntt830 was prepared as described in Example 3 of WO 2016 / 062720 A1. 1st PCR: Forward - pET-90 primer (anneals to pET28a+) (SEQ ID NO: 21) Reverse - RGSYrev (SEQ ID NO: 22) Second PCR forward - RGSYdir (SEQ ID NO: 23) Reverse-CMV-AgeR (SEQ ID NO: 24)

[0170] After purification of both PCR products, a subsequent PCR was performed to join the PCR fragments (5 cycles without primers, then 25 cycles using primers pET-90 and CMV-AgeR).

[0171] After gene amplification, the resulting PCR product was directly cloned into the pTZ57R vector (InsTAclone PCR Cloning Kit, Fermentas #K1214). E. coli XL1-Blue cells were used as a host for cloning and plasmid amplification.

[0172] To avoid RT-PCR errors, several pTZ57 plasmid clones containing the CMV-Ntt830 gene were sequenced using a BigDye cycle sequencing kit and an ABI Prism3100 Genetic analyzer (Applied Biosystems). After sequencing, pTZ-plasmid clones containing the CMV-Ntt830B gene with an integrated BamHI site were digested with Ncol and Agel enzymes. The fragment was then subcloned into the Ncol / Agel site of pET-CMV-Ntt830 to obtain the helper vector pET-CMV-Ntt830B.

[0173] CMV-Ntt830-E8* VLPs were produced in E. coli C2566 cells (New England Biolabs, USA). VLPs were produced using E. coli cell culture, biomass processing and purification methods as follows: 1) 3 g of biomass is suspended in 20 mL of 50 mM Na-citrate, 5 mM Na-borate, 5 mM EDTA, 5 mM mercaptoethanol (pH 9.0) and the suspension is treated with ultrasound (Hielscher ultrasonicator UP200S, 16 min, amplitude 70%, cycle 0.5); 2) Centrifuge the lysate at 11000 rpm for 20 minutes at +4°C; 3) Prepare a sucrose gradient (20-60%) in a 35 mL tube in a buffer containing 50 mM Na-citrate, 5 mM Na-borate, 2 mM EDTA, and 0.5% TX-100. 4) Layering 5 mL of the VLP sample onto a sucrose gradient; 5) Centrifuge for 6 hours using an SW32 rotor (Beckman) (25,000 rpm, +18°C). 6) Divide the contents of each gradient tube into 6 mL fractions and pool the corresponding fractions; 7) Gradient fractions were prepared as follows: analyzed with SDS.

[0174] SDS-PAGE analysis of the sucrose gradient purification demonstrated homogeneous CMV-Ntt830-E8* coat protein monomers (Figure 2A), and electron microscopy showed intact VLPs (Figure 2B).

[0175] The chimeric CMV polypeptides CMV-Ntt830-E4, CMV-Ntt830-E8, and CMV-Ntt830-E12 were prepared accordingly and as follows: The first step was the incorporation of the coding sequence of the flanking linker-containing polyglutamate into the modified CMV using PCR mutagenesis. The PCR fragment encoding the flanking linker-containing polyglutamate sequence and the 3'-end fragment of the modified CMV were amplified by PCR using the following oligonucleotide pair and the plasmid pET-CMVB2-Ntt-E8* as a template: 1) Forward: E4-F (SEQ ID NO: 25) Reverse: CMcpR (SEQ ID NO: 20), 2) Forward: E8-F (SEQ ID NO: 26) Reverse: CMcpR (SEQ ID NO: 20), 3) Forward: E12-F (SEQ ID NO: 27) Reverse: Amplified by two-step PCR using CMcpR (SEQ ID NO: 20).

[0176] The resulting PCR product was cloned into the helper vector pTZ57 (InsTAclone PCR Cloning Kit, Fermentas #K1214). The PCR product-containing plasmid was amplified in E. coli XL1-Blue cells, and the plasmid DNA was purified and sequenced using a BigDye cycle sequencing kit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Thus, PCR error-free helper plasmids pTZ-CMV-E4, pTZ-CMV-E8, and pTZ-CMV-E12 were obtained.

[0177] Next, BamHI / HindIII-digested fragments of pTZ-CMV-E4, pTZ-CMV-E8, and pTZ-CMV-E12 were cloned back into pET-CMV-Ntt830B (see above) using the same restriction sites. Thus, expression vectors pET-CMVB2-Ntt-E4 (Figure 3), pET-CMVB2-Ntt-E8 (Figure 4), and pET-CMVB2-Ntt-E12 (Figure 5) were obtained. The expression vectors were transformed into E. coli C2566 cells (New England Biolabs, USA). VLPs were produced using the E. coli cell culture, biomass processing, and purification methods described above for CMV-Ntt830-E8* VLPs. SDS-PAGE analysis of VLPs after sucrose gradient purification demonstrated that nearly homogeneous CMV coat protein monomers were obtained for all three polyglutamate constructs (Figures 6, 7, and 8). However, agarose gel analysis indicated that integrated particles were formed only with CMV-Ntt830-E4 and CMV-Ntt830-E8, but not with CMV-E12 (Figures 6, 7, and 8). Electron microscopy demonstrated that CMV-Ntt830-E4 and CMV-Ntt830-E8 formed intact VLPs (Figures 9 and 10).

[0178] Example 2 Improved stability of the surface charge-modified CMV VLPs of the present invention compared to prior art CMV VLPs thermal stability The increased thermal stability of the surface charge-modified CMV VLPs of the invention was demonstrated by measuring the denaturation of prior art CMV-Ntt830 VLPs prepared as described in Examples 3 and 4 of WO 2016 / 062720 A1, and the denaturation of CMV-Ntt830-E4 VLPs of the invention as a function of increasing temperature and determining their respective melting temperatures.

[0179] A thermal shift assay involving heat-induced denaturation and the fluorescent dye SYPRO® Orange (Sigma, Saint Louis, USA) was used for this purpose. The dye is naturally quenched in solution, but as the VLPs unfold with increasing temperature, SYPRO® Orange interacts with exposed hydrophobic amino acids and the core, emitting a fluorescent signal, which is measured by fluorometry. From the resulting melting curves (fluorescence signal vs. temperature), the melting peak curves and melting points were determined. Solutions containing 0.5 mg / mL of sucrose density gradient-purified CMV-Ntt830 VLP or CMV-Ntt830-E4 VLP (as described in Example 1 above) in 5 mM Na phosphate, 2 mM EDTA, pH 7.5 were assayed using a real-time PCR system MJ Mini (Bio-Rad, Hercules, USA) using a DNA melting point determination program. Data were analyzed using Option Monitor software, and melting curves were processed with a smooth setting of 4. FIG. 11 shows the melting peak curves for purified CMV-Ntt830 VLPs and CMV-Ntt830-E4 VLPs.

[0180] The respective melting points were estimated to be 51° C. and 57° C., demonstrating the increased thermal stability of the surface charge-modified CMV VLPs according to the present invention compared to the prior art CMV-Ntt830 VLPs.

[0181] Ionic strength / salt stability Ionic strength is important for capsid stability: salt in solution interacts with the coat protein and charged residues on the VLP surface, affecting the water shell and distancing the hydrophobic exposure, thereby affecting overall VLP stability.

[0182] The relative stability of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP to NaCl was tested by incubating purified VLP (0.5 mg / mL in 5 mM Na phosphate, 2 mM EDTA, pH 7.5) at room temperature with various NaCl concentrations. After 2 hours in the presence of 20 mM NaCl, CMV-Ntt830 VLP was relatively unstable, forming a significant proportion of aggregates that were visible to the eye and demonstrable by native gel electrophoresis (Figure 12). In contrast, there was no evidence of aggregate formation for CMV-Ntt830-E4 VLP, even at NaCl concentrations up to 0.4 M (Figure 12).

[0183] The improved stability in high salt solutions resulting from the surface charge modification of the modified CMV VLPs of the present invention is important for their processability by ion exchange chromatography as described in Example 3.

[0184] Example 3 Improved purification ability of the surface charge-modified CMV VLPs of the present invention compared to prior art CMV VLPs The sucrose gradient / cushion ultracentrifugation purification step of preparations of modified CMV VLPs of the invention, as used in laboratory-scale CMV VLP production processes as described in the prior art, such as Examples 2-4 of WO 2016 / 062720 A1, and as described in Example 1 above, provides CMV VLPs in yields and purities suitable for subsequent conjugation, vaccine production, and preclinical evaluation, although this method cannot be used simply and cost-effectively to produce a commercial vaccine.

[0185] Ion exchange chromatography (IEX) is typically easily scalable and is used downstream in commercial biopharmaceutical manufacturing. It is based on reversible ionic interactions between charged molecules / macromolecules in solution and an immobilized, oppositely charged chromatography resin. One example is anion exchange chromatography (AEX), where the stationary phase (resin) is positively charged and negatively charged molecules such as proteins are bound to it. The interaction between the resin and the sample is via Cl - The IEX can be disrupted by the application of counterions such as HCl, ...

[0186] For CMV VLPs to be effectively bound and eluted by IEX, the CMV VLPs must be stable to the ionic environment encountered during the binding and elution steps. The charge of the ion exchange resin and the elution salts contribute to the ionic environment.

[0187] Prior art CMV-Ntt830 VLPs and modified CMV VLPs of the invention, such as CMV-Ntt830-E4, CMV-Ntt830-E8, and CMV-Ntt830-E8*, have a net negative charge at a pH of approximately 9 or less, as demonstrated by their migration toward a positively charged electrode in NAGE. Therefore, anion exchange chromatography (AIX) is a technique that is expected to work for both CMV VLP particles.

[0188] However, this is not the case for the CMV-Ntt830 VLP described above in Example 2, which is relatively unstable and forms aggregates that precipitate already in solutions with 20 mM NaCl. In contrast, modified CMV VLPs of the invention, such as the CMV-Ntt830-E4 VLP, do not form aggregates at NaCl concentrations up to 0.4 M (Figure 12, panel B). The improved stability in high salt solutions resulting from surface charge modifications of the VLP is essential for its processability by ion exchange chromatography.

[0189] Enhanced purification by anion exchange chromatography (AEX) To test the processability of prior art CMV-Ntt830 VLPs using anion exchange chromatography (AEX), sucrose gradient-purified VLPs were prepared as described in Examples 2-4 of WO 2016 / 062720 A1. Five milliliters of CMV-Ntt830 VLPs (1 mg / mL) were buffer-exchanged into 5 mM sodium borate (pH 9) and loaded onto a 1.0 mL Macro-Prep DEAE Bio-Rad anion Exchange cartridge equilibrated with the same buffer. After the loading step, the NaCl concentration in the elution buffer was increased stepwise (0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1.0, and 2.0 M). Fractions were collected and subjected to native agarose gel electrophoresis (NAGE) at 260 nm using a Nanodrop spectrophotometer to measure protein.

[0190] The resulting chromatograms of protein elution and NaCl concentration plotted against the corresponding fractions (Figure 13, panel A) show that CMV-Ntt830 VLPs did not elute as a single peak, as is typical for AIX. Instead, CMV-Ntt830 VLPs eluted in a broad, nonspecific manner during loading (at 0 M NaCl) and subsequent elution steps, primarily spanning a range of NaCl concentrations from 0.2 to 0.8 M. Critically, VLP-containing fractions after elution from the column were opaque and contained a significant proportion of aggregated VLPs, as demonstrated by the presence of ethidium bromide-stained VLPs in the loading wells after NAGE (Figure 13, panel B). The tendency of CMV-Ntt830 VLPs to aggregate and elute in a nondispersed manner precludes the immediate use of this methodology for scale-up manufacturing.

[0191] In contrast, non-aggregated CMV-Ntt830-E4 VLPs were easily purified from crude lysates using AEX. A sorted lysate prepared from Escherichia coli (E. coli) expressing CMV-Ntt830-E4 VLPs (as described in Example 1) in 50 mM citrate, 5 mM borate buffer (pH 9.0) was loaded onto a 60 mL Fracto-DEAE (Merck) in an XK26 / 20 column equilibrated with the same buffer and eluted by applying a continuous NaCl gradient from 0 to 1.0 M in the same buffer. The eluate was monitored at A260 nm to measure protein, and conductivity was measured to monitor salt concentration. The clarified lysate, flow-through, and fractions were collected and subjected to NAGE and SDS-PAGE.

[0192] The resulting chromatogram, SDS-PAGE, and NAGE analyses (Figure 14) show that CMV-Ntt830-E4 VLPs were not present in the flow-through and were completely bound to Fracto-DEAE. The VLPs were subsequently eluted over a relatively narrow range of 0.2–0.5 M NaCl. Additionally, there was no evidence of aggregated VLPs in the loading wells of the native agarose gel. A Coomassie Blue-stained SDS-polyacrylamide gel demonstrated that highly pure VLP coat protein was obtained from the crude bacterial lysate.

[0193] Example 4 Cloning, expression and purification of recombinant mature NGF Cloning of recombinant NGF A cDNA construct (SEQ ID NO: 28) consisting of the full-length feline NGF propeptide sequence, the canine mature NGF sequence, and the C-terminal glycine-cysteine-glycine motif was synthesized de novo and cloned into the pBHA vector (BIONEER Company). The canine NGF sequence was codon-optimized. The resulting amino acid sequence of the full-length feline NGF propeptide is provided in SEQ ID NO: 29, which includes the canine mature NGF sequence of SEQ ID NO: 30. The amino acid sequence of canine mature NGF with the C-terminal glycine-cysteine-glycine motif attached is provided in SEQ ID NO: 31.

[0194] Similarly, a cDNA construct (SEQ ID NO: 32) consisting of the full-length feline NGF propeptide sequence, the canine mature NGF sequence, and a C-terminal glycine-cysteine-glycine motif and a His tag was synthesized de novo and cloned into the pBHA vector (BIONEER Company). The included His tag does not play any role in purification, but its presence increases the refolding efficiency in downstream processes. The resulting amino acid sequence is provided in SEQ ID NO: 33, which contains the canine mature NGF sequence of SEQ ID NO: 30, and a His6 tag (SEQ ID NO: 34).

[0195] The construct was subcloned into an expression vector by PCR. Briefly, the NGF-pBHA plasmid was used as a template with the NGF forward primer (SEQ ID NO: 35) and the NGF reverse primer (SEQ ID NO: 36), which contain XbaI and HindIII sites, respectively.

[0196] The NGF PCR product was subjected to 1% agarose gel electrophoresis in TAE buffer, and the NGF fragment was then extracted using a GeneJet DNA Elution Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. The NGF fragment was digested with FastDigest XbaI and HindIII (Thermo Fisher Scientific) restriction enzymes for 30 minutes at +37°C in 1x FastDigest buffer according to the manufacturer's protocol. The pET42 plasmid (Novagen) was digested in the same manner. NGF and vector-digested DNA fragments were analyzed by agarose gel electrophoresis and extracted as described above. The NGF fragment was ligated with the pET42 vector using T4 ligase overnight at room temperature according to the manufacturer's protocol.

[0197] The NGF-pET42a construct was transformed into chemically competent E. coli DH5α cells by heat shock. Cells were suspended in 1 mL of LB medium, incubated at +37°C for 1 hour with shaking, and plated onto LB agar containing 60 μg / mL kanamycin and incubated at 37°C overnight. Individual colonies were plated onto LB medium containing 30 μg / mL kanamycin and incubated at +37°C with shaking overnight. DNA was extracted from individual clonal cultures using a GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0198] The correct sequences of the NGF constructs of SEQ ID NO:28 and SEQ ID NO:32 were confirmed by Sanger sequencing using a BigDye Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0199] Expression and purification of recombinant canine mature NGF The NGF-pET42a plasmid was transformed into chemically competent E. coli BL21-DE3 (Sigma-Aldrich) cells. The cells were suspended in 1 mL of LB medium and incubated at 37°C for 1 hour with shaking. The cells were plated onto LB agar containing 60 μg / mL kanamycin and incubated at 37°C overnight. Several colonies of NGF-pET42-transformed BL21-DE3 cells were plated onto LB medium containing 30 μg / mL kanamycin, incubated at 37°C overnight, and then added to 2x TY medium containing 30 μg / mL kanamycin to obtain an OD of 0.7 units. 540nm The cells were grown at 37°C with shaking until a final concentration of 1 mM was reached. Recombinant protein expression was induced by adding IPTG to a final concentration of 1 mM, and the cells were grown for an additional 4 hours with shaking at 37°C. The biomass was harvested by centrifugation at 5000g for 15 minutes, frozen, and stored at -70°C.

[0200] The biomass was suspended in lysis buffer (40 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM PMSF, 1 mM DTT, and 1% Triton X-100) and lysed by sonication using a UP200S (Hielscher) ultrasonic device. The resulting sonicate was centrifuged at 15,557 g for 40 min. The supernatant was discarded, and lysis buffer was added to the sonicated, resuspended pellet. The suspension was centrifuged at 15,557 g for 15 min, and the supernatant was again discarded. This washing step was repeated three more times. The pellet was finally washed with 50% lysis buffer and 3.5 M urea. After resuspension and centrifugation, the pellet was solubilized with 8 M guanidine hydrochloride and 0.1 M dithiothreitol. The suspension was homogenized by sonication for 10 min and then centrifuged at 15,557 g for 25 min. The supernatant (containing solubilized denatured NGF) was collected and filtered using a 45 μm filter. Then, under constant stirring, refolding buffer (0.75 M L-arginine, 0.1 M Tris, 1 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione, pH 9.5) was added dropwise at 7°C to a final concentration of 5 mL of NGF solution per 100 mL of refolding buffer. After overnight incubation, the refolding solution was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected and incubated at +7°C for 1 week. The solution was diluted 3-fold with deionized water, warmed to room temperature, and the pH was adjusted to 6.8 with acetic acid. The solution was then centrifuged at 7,000 g for 10 minutes at room temperature to remove the precipitate and loaded onto a 5 mL Capto S cation exchange column pre-equilibrated with 50 mM sodium phosphate buffer (pH 6.5). The protein was then eluted with a gradient of 0 to 1 M NaCl in 50 mM sodium phosphate buffer (pH 6.5). The eluted fractions were analyzed by SDS-PAGE, and the proNGF-containing fractions were pooled and concentrated to 2 to 3 mg / mL using ultrafiltration. The reconstituted proNGF was digested with TrypZean (Sigma-Aldrich, catalog no. T3449) trypsin solution at a volume ratio of 30:1 for 4 hours at room temperature.The reaction was stopped by adding PMSF to a final concentration of 1 mM and then loaded onto a Superdex200 10 / 300GL size exclusion column equilibrated with 0.5 M NaCl and 30 mM phosphate (pH 6.8).

[0201] Fractions were collected and analyzed by SDS-PAGE (shown in Figure 15A for the cDNA construct of SEQ ID NO: 28 and the resulting amino acid sequence of the full-length feline NGF propeptide of SEQ ID NO: 29), and fractions containing mature NGF were pooled and concentrated by ultrafiltration to a concentration of 2 mg / mL.

[0202] The authenticity of the recombinant canine mature NGF was confirmed using bioassays in which canine mature NGF and mouse mature NGF (commercially produced by R&D systems) were similarly active in induced neurites (shown in Figure 15B for the cDNA construct of SEQ ID NO: 28 and the resulting amino acid sequence of the full-length feline NGF propeptide of SEQ ID NO: 29), demonstrating the known function of properly folded, biologically active mature NGF.

[0203] Example 5 Coupling of recombinant canine mature NGF to modified CMV VLPs Various NGF antigens, including canine mature NGF (SEQ ID NO: 30), were covalently conjugated to various modified CMV VLPs prepared as described above. Conjugation was carried out according to the method described in Schmitz N, et al., J Exp Med (2009) 206: 1941-1955).

[0204] Briefly, purified CMV-Ntt830, CMV-Ntt830-E4, CMV-Ntt830-E8, or CMV-Ntt830-E8* VLPs were diluted to 1.5 mg / mL and reacted with the heterobifunctional chemical crosslinker succinimidyl-6-(b-maleimidopropionamide)hexanoate (SMPH) for 1 hour at room temperature (RT). SMPH contains an NHS ester that reacts with lysines on the surface of VLPs. The amount of SMPH added was approximately 5-fold molar excess over one VLP coat protein monomer. Unreacted crosslinker was removed by centrifugation using an Amicon-Ultra-0.5, 100K centrifugal filter (Merck-Millipore, #UFC910024). The SMPH-derivatized VLPs were then washed three times with 5 mM Na2HPO4, 2 mM EDTA (pH 7.5).

[0205] Specifically, the coupling of a cNGF antigen having SEQ ID NO: 33 to CMV-Ntt830-E4 VLP was described: A solution of CMV-Ntt830-E4 VLP in 5 mM NaHPO (pH 7.5), 2 mM EDTA, with a protein concentration of 7.43 mg / mL from the BCA Protein Assay Kit (TFS, catalog no. 23225), was diluted to a working concentration of 1.5 mg / mL with 5 mM NaHPO (pH 7.5), 2 mM EDTA (pH 8.0) in a 50 mL tube (Sarstedt, sterile, catalog no. 62.559.001) with a sample volume of 3 times 44 mL. The total volume for derivatization was therefore 132 mL. A 50 mM (19 mg / mL) SMPH solution in DMSO was prepared directly before use.

[0206] For derivatization of CMV-Ntt830-E4 VLP with SMPH, 264 μL of 50 mM SMPH solution in DMSO was added to each of three previously prepared tubes containing 44 mL of CMV-Ntt830-E4 VLP. The mixture was vortexed for 5 seconds and incubated at RT for 1 hour. To remove excess SMPH, the mixture was centrifuged in an Amicon-Ultra-15 100K unit (Merck-Millipore, UFC910024) at 3214 g for 7 minutes in an Eppendorf 5810R centrifuge. The buffer was exchanged for 5 mM NaHPO (pH 7.5), 2 mM EDTA by centrifuging three more times with the same parameters. After the final centrifugation, the total volume was adjusted to 132 mL (same as before derivatization). UV absorption at 260 nm was measured and the concentration of derivatized CMV-Ntt830-E4 VLPs was estimated at 1.5 mg / mL.

[0207] Briefly, the cNGF antigen was then added to the surface-charge-modified CMV VLPs pre-derivatized with SMPH at a molar ratio of approximately 0.5:1 to 1:1 based on the respective chimeric CMV polypeptide monomers, typically over a period of 3 hours at room temperature with shaking. The engineered free cysteine ​​of the cNGF antigen reacted with the maleimide of the cross-linker SMPH bound to the VLPs to form a stable covalent bond.

[0208] Specifically, coupling of a cNGF antigen bearing SEQ ID NO: 33 to CMV-Ntt830-E4 VLP was described. The coupling reaction was carried out in six 50 mL tubes (Sarstedt, sterile, catalog no. 62.559.001). In each tube, 22 mL of derivatized CMV-Ntt830-E4 VLP (1.5 mg / mL, 60 μM relative to CMV monomer) was mixed with 3.82 mL of buffer-exchanged cNGF of SEQ ID NO: 33 (2.33 mg / mL, 172.6 μM). This resulted in a molar ratio of CMV monomer:NGF monomer = 1:0.5. The reaction mixture was incubated at RT by rotation using a DSG Titertek (Flow Laboratories). Uncoupled cNGF was removed by gel filtration on a Superdex 200 column (running buffer 20 mM NaHPO4 (pH 7.5), 2 mM EDTA). A 10 mL solution containing cNGF-CMV-Ntt830-E4 VLPs was loaded onto a HiLoad 26 / 600 Superdex 200prep-grade column equilibrated with 20 mM NaHPO4 (pH 7.5), 2 mM EDTA. Fractions containing cNGF-CMV-Ntt830-E4 VLPs were pooled and filtered through a 0.2 μm filter (Sarstedt, catalog number 83.1826.001). The collected sample volume after gel filtration was 280 mL. The sample was further concentrated to 230 mL using an Amicon-Ultra-15,100K (Merck-Millipore, catalog number UFC910024) and filtered through a 0.2 μm filter (Sarstedt, catalog number 83.1826.001). The concentration was measured by Qubit and the final concentration was adjusted to 0.7 mg / mL with sterile 20 mM NaHPO (pH 7.5), 2 mM EDTA buffer. UV absorbance at 260 nm was measured (A = 6.628 and A = 3.722).

[0209] To demonstrate covalent conjugation of the cNGF antigen to the VLPs, the coupling reactions were analyzed by SDS-PAGE. Prominent conjugation bands were observed after chemical coupling of CMV-Ntt830, CMV-Ntt830-E4, CMV-Ntt830-E8, and CMV-Ntt830-E8* VLPs with cNGF (Figures 16A and 16B). However, cNGF-CMV-Ntt VLPs formed large aggregates (1400–1700 nm) (Figure 16C) that rapidly and completely precipitated from solution.

[0210] In contrast, after covalent conjugation of cNGF to CMV-Ntt830-E4, CMV-Ntt830-E8, and CMV-Ntt830-E8* VLPs, the resulting modified VLP conjugates remained soluble and did not precipitate from solution. Analysis by dynamic light scattering (DLS) (Figures 16D, 16E, and 16F) and electron microscopy (Figures 16G and 16H) indicated that the modified VLP conjugates did not aggregate and were stable in solution.

[0211] Example 6 Induction of neutralizing antibodies by immunization with various conjugates of the present invention of canine mature NGF coupled to modified CMV VLP Immunization of mice Balb / c mice were assigned to two groups (n = 4 per group). The first group was immunized twice, 14 days apart, with 150 μL of canine mature NGF-CMV-Ntt830-E8* VLP formulated at a concentration of 100 μg / mL in 20 mM NaP, 2 mM EDTA (pH 7.5). The second group was similarly treated with canine mature NGF-CMV-Ntt830-E8* VLP formulated at a concentration of 100 μg / mL in 20 mM NaP, 2 mM EDTA (pH 7.5) and 100 μg / mL Quil-A adjuvant (InvivoGen vac-quil). Before each immunization, blood samples were similarly collected on days 21, 28, 35, and 42 after the first vaccination. Serum was prepared by spinning blood samples in serum tubes at 10,000 x g for 10 minutes and stored at approximately -20°C until assayed.

[0212] Immunization of dogs Six male beagles (obtained from Marshall US), aged 22–26 months at the time of first dosing, were randomly assigned to two groups (n=3 per group). The first group was immunized three times with 1.0 mL of cNGF-CMV-Ntt830-E8*VLP formulated at a concentration of 250 μg / mL in sodium phosphate buffer (pH 7.5). The second group was similarly treated with cNGF-CMV-Ntt830-E8*VLP formulated at a concentration of 250 μg / mL in sodium phosphate buffer (pH 7.5) and 100 μg / mL Quil-A® adjuvant (InvivoGen vac-quil). Blood samples were collected from the jugular vein of each animal using a single-use needle and syringe 24 hours before the first immunization (day 0), second immunization (day 21), and third immunization (day 42). Blood was also collected on days 63, 84, and 105. 6 mL blood samples were collected in inert tubes and allowed to sit at ambient temperature. After clotting, the tubes were centrifuged and serum was collected in inert tubes and stored at approximately -20°C until IgG purification and / or assay.

[0213] In a further study, 10 adult beagle dogs, over 9 months of age at enrollment, were assigned to two groups. For immunization, cNGF-CMV-Ntt830-E4 VLPs containing the cNGF antigen of SEQ ID NO: 33 were used. Thus, five dogs in the first group were treated with 250 μg of cNGF-CMV-Ntt830-E4 VLPs / dose formulated with 1.7 mg of aluminum hydroxide, while five dogs in the second group were treated with 250 μg of cNGF-CMV-Ntt830-E4 VLPs / dose without aluminum hydroxide. The dogs were subcutaneously administered twice, on study days 0 and 21. Serum samples were also collected throughout the study on days 42, 71, and 91.

[0214] Measurement of NGF- and CMV-VLP-specific IgG antibodies Serum anti-NGF and CMV-Ntt830-E8*VLP-specific IgG antibodies were measured by ELISA for mice and dogs immunized with cNGF-CMV-Ntt830-E8*VLP.Serum anti-NGF-specific IgG antibodies were measured by ELISA for dogs immunized with cNGF-CMV-Ntt830-E4 VLP.

[0215] As detailed for immunization with cNGF-CMV-Ntt830-E8*VLP, Maxisorp ELISA plates were coated overnight at 4°C with recombinant canine mature NGF protein or CMV-Ntt830-E8*-VLP in 0.1 M Na-carbonate buffer (pH 9.6) at a concentration of 1 μg / mL. Plates were washed, and SuperBlock™ (PBS) blocking buffer (Thermo Fisher / Life Technologies Europe) was added for 2 h at RT and then washed again. Serum samples were prediluted 1:9 or 1:100 in 2% BSA in PBS containing 0.05% Tween 20 and transferred to ELISA plates for ten 3-fold serial dilutions. After 2 hours of incubation at room temperature, the plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, Fc gamma fragment specific (Jackson ImmunoResearch Europe Ltd.) or HRP-conjugated rabbit anti-dog IgG (H+L)-HRP (Jackson ImmunoResearch Europe Ltd.), diluted 1:2000 or 1:2500, respectively, in 2% BSA in PBS containing 0.05% Tween-20 (PBS, pH 7.4 (1x), Gibco), was added. After incubation and washing, the Pierce™ TMB Substrate Kit (Thermo Fisher / Life Technologies Europe) was used for colorimetric development. The enzymatic reaction was stopped by adding 5% H2SO4, and the absorbance at 450 nm was measured spectrophotometrically using an ELISA reader (Tecan Spark 10). The OD50 titer represents the reciprocal of the dilution that reached half the maximum OD value.

[0216] Neutralization assay PC12 cells An in vitro assay measuring mature NGF-mediated neurite outgrowth in rat adrenal pheochromocytoma cell cultures (PC-12) was used to determine the biological activity of recombinantly produced canine mature NGF and to assess the neutralizing capacity of antibodies induced by immunization of mice. 24-well tissue culture plates coated with type I collagen (Thermo Fisher / Life Technologies Europe) (10 μg / mL) were cultured at 5 × 10 cells / well with assay medium containing RPMI 1640 (Sigma-Aldrich Switzerland), 2 mM L-glutamine (Gibco), 2.4 g / L HEPES (AppliChem GmbH Germany), 2.5 g / L glucose (Sigma-Aldrich Switzerland), and further supplemented with 10% heat-inactivated fetal bovine serum (FBS Premium, PAN Biotech, Germany), 10% horse serum (kindly provided by Evax, Switzerland), 1x antibiotic-antimycotic (A / A) (Gibco, Thermo Fisher / Life Technologies Europe), and 1 mM sodium pyruvate (Sigma-Aldrich, Switzerland). 4PC-12 cells were seeded in duplicate at 1000p per well and incubated overnight at 37°C, 5% CO. The next day, the well medium was replaced with assay medium (RPMI 1640, 1x A / ATCC, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.5% FBS) containing recombinant canine mature NGF at a final concentration of 12.5 ng / mL, including mouse (R&D, 1156-NG-100), human mature NGF (R&D, 256-GF-100 / CF), human mature NGF polyclonal antibody (R&D AF-256-NA), human mature NGF monoclonal antibody (R&D MAB256-500), or purified IgG. NGF was omitted from negative control wells (starvation medium only), and antibody was omitted from positive control wells (12.5 ng / mL NGF in starvation medium). After 5 days, cells were stained with 0.05% w / v crystal violet solution and examined microscopically. Bright-field images from several fields were captured using Q-Capture Pro 7 software on an inverted microscope Leica DM IL LED (Leica Microsystems (UK) Ltd) with a HI PLAN I 20x objective. Cells with and without neurite outgrowth (defined as the width of the extending cell body) were counted to determine the percentage of neurite-positive cells for each treatment.

[0217] Neutralization assay TF-1 cells The neutralizing capacity of sera from dogs immunized with cNGF-CMV-Ntt830-E8* VLP and cNGF-CMV-Ntt830-E4 VLP was determined using a bioactivity assay involving measuring proliferation of the TF-1 erythroblastoma cell line (American Type Culture Collection (ATCC), Manassas, VA).

[0218] For immunization with cNGF-CMV-Ntt830-E8*VLP, TF-1 cells were harvested, washed three times with PBS (PBS (pH 7.4) (1x) Gibco), and 10 5The cells were cultured overnight in starvation medium (RPMI 1640 medium (ATCC modified) supplemented with 10% heat-inactivated FBS and 1x AxA) at a cell density of 10 cells / mL. 4 TF-1 cells were seeded per well of a 96-well flat-bottom plate in a total of 100 μL of assay medium (phenol red-free RPMI containing 10% FBS, 2 mM GlutaMax, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, 1500 mg / L sodium bicarbonate, 100 U / mL penicillin, 100 μg / mL streptomycin, and 25 μg / mL amphotericin B).

[0219] To test the in vitro neutralizing activity of antibodies generated by immunization with cNGF-CMV-Ntt830-E8* VLP, serum from immunized dogs was collected, and total IgG was purified using Invitrogen Dynabeads™ Protein G (Thermo Fisher / Life Technologies Europe) for mouse IgG purification and Pierce Protein A magnetic beads (Thermo Fisher / Life Technologies Europe) for dog IgG purification according to the manufacturer's instructions. The ability of purified total IgG to neutralize NGF bioactivity was tested by incubating a fixed concentration of 5 ng / mL of human mature NGF (R&D, 256-GF-100 / CF) with increasing concentrations of purified canine total IgG (625-20,000 ng / mL), human mature NGF polyclonal antibody (R&D AF-256-NA), or human mature NGF monoclonal antibody (R&D MAB256-500) at room temperature for 1 hour. The NGF-antibody solution was then added to 10 4 TF-1 cells were added and cell proliferation was quantified over the final 24 hours of a 72-hour incubation period using a BrdU-based cell proliferation ELISA (Roche). Color development was stopped using 5% sulfuric acid according to the manufacturer's instructions. Absorbance was measured at 450 nm with a reference wavelength of 690 nm.

[0220] The percent growth for each IgG dilution was calculated relative to the growth measured for IgG purified from serum collected at baseline before infection (day 0). Data were expressed as percent growth versus IgG concentration. GraphPad Prism (version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com) was used to fit a sigmoidal 4PL curve to determine the IgG concentration required to obtain 50% inhibition of growth (50% neutralization titer NT50).

[0221] NGF neutralizing antibodies in dogs after immunization with cNGF-CMV-Ntt830-E4 VLP were assayed as follows: TF-1 cells were harvested and cultured at 2 × 10 in starvation medium (phenol red-free RPMI (Sigma) containing 10% HI-FBS, 2 mM GlutaMax (Gibco), 10 mM HEPES (Sigma), 1 mM sodium pyruvate (Sigma), 4500 mg / L glucose (Gibco), 1500 mg / L sodium bicarbonate, 100 U / mL penicillin, 100 μg / mL streptomycin, 25 μg / mL amphotericin B (100x anti-Gibco)). 5 The cells were washed three times with PBS before being resuspended at a cell density of 1 x 10 cells / mL. Serum samples were heat-inactivated at 56°C for 30 minutes and then diluted 1:25 (4x final concentration of 1:100) in starvation medium, followed by two-fold serial dilutions. hNGF was diluted to 20 ng / mL (4x final concentration of 5 ng / mL) and 25 μL was added to wells containing 25 μL of prediluted serum or 25 μL of starvation medium (positive control wells). Instead of hNGF, 50 μL of starvation medium was added to negative control wells. The hNGF-serum / antibody mixture was incubated for 1 hour at room temperature. Serum-starved TF-1 cells were harvested and 50 μL of the cell suspension was added to 1 x 10 cells in a flat-bottom 96-well plate. 4Cells were added at a cell density of 100 cells / well. The final sample volume per plate was 100 μL / well. The cell culture plates were incubated in a cell culture incubator at 37°C with 5% CO2 for approximately 68 hours. Cell viability was quantified using the Promega CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). 20 μL of CellTiter 96® Aqueous One Solution Reagent was added per well. The plates were incubated at 37°C in a humidified 5% CO2 incubator for 7 hours. Absorbance was measured at 490 nm with a reference wavelength of 700 nm. To determine IC50 values, titer curves were generated by plotting OD values ​​against the dilution factor of the serum samples using GraphPad prism software (versions 8 and 9 for Windows, GraphPad Software, San Diego, California, USA). The 4-PL regression curve fit model was used to determine the IC50 value, the dilution factor corresponding to half the maximum OD value. The serum titers of the samples at different time points were defined and presented as IC50 values ​​of the curve fit.

[0222] result Mouse experiments For mice immunized with cNGF-CMV-Ntt830-E8*VLP together with Quil A, cNGF-specific IgG antibodies were detected in serum collected from day 14 onward (FIG. 17A). A further increase in antibody titers was measured in serum on day 21, 7 days after the second injection was administered on day 14. Titers remained high until the end of the experiment on day 42. After two immunizations with the adjuvant-free cNGF-CMV-Ntt830-E8*VLP vaccine, NGF-specific IgG antibodies were detected in serum isolated from day 21 onward. Coadministration of Quil A adjuvant had an immune-enhancing effect, enhancing the specific antibody response by approximately 10-fold.

[0223] To test whether the anti-NGF IgG antibodies induced by immunization with cNGF-CMV-Ntt830-E8*VLP were neutralizing, we tested them in a PC12-based bioassay, in which NGF acts as a neurotrophic factor to induce differentiation and neurite outgrowth. IgG was purified from pooled sera collected before immunization (ms pIgG NAIVE) and on days 21, 28, and 35 after the cNGF-CMV-Ntt830-E8*VLP / QuilA boost (ms pIgG NGF vaccine). Figure 17B shows that IgG purified from immune sera neutralized NGF, but not IgG from naive mice.

[0224] Dog experiments In animals receiving cNGF-CMV-Ntt830-E8*VLP in the absence of adjuvant, detectable anti-NGF IgG titers were observed in serum collected 21 days after a single dose of vaccine (Figure 18A). NGF-specific IgG titers were highest in serum collected on day 42, 3 weeks after the second dose of vaccine. After the third injection on day 42, titers remained consistently high until day 63 and then gradually declined in all animals. The magnitude of anti-CMV IgG titers was similar to that measured against canine mature NGF, but the kinetics of the response were slightly different (Figure 18C). Anti-CMV IgG antibodies were somewhat delayed, with only clear detectable titers from day 42 onward after the second immunization, and peak titers were measured in serum on day 63 after the third immunization, after titers had declined.

[0225] For animals immunized with cNGF-CMV-Ntt830-E8*VLP in combination with the adjuvant Quil A, anti-NGF IgG antibodies were first detected in serum on day 21 after a single dose of vaccine on day 0 (Figure 18B). The second and third doses of vaccine increased titers in two of the three animals. Peak titers were measured in serum collected on day 63. The third animal achieved a peak titer on day 42, suggesting that the third dose of vaccine may not have increased the antibody response. The kinetics and magnitude of anti-CMV IgG antibody titers were similar to those measured against canine mature NGF (Figure 18D).

[0226] In animals receiving cNGF-CMV-Ntt830-E8*VLP in the absence of adjuvant, detectable anti-NGF IgG titers were observed in 4 of 5 test animals in serum collected 21 days after a single dose of vaccine (FIG. 18E). The highest titers were observed 21 days after the second dose on day 42.

[0227] For animals immunized with cNGF-CMV-Ntt830-E4 VLPs in combination with aluminum hydroxide, anti-NGF IgG antibodies were detected in all animals 3 weeks after a single dose of vaccine on day 0 (FIG. 18F). A second dose of vaccine increased the mean group titers.

[0228] The neutralizing ability of anti-NGF IgG antibodies induced in response to vaccination with cNGF-CMV-Ntt830-E8*VLP was analyzed using a bioassay based on NGF-mediated proliferation of TF-1 cells. IgG antibodies purified from immunized dogs inhibited mature NGF-induced proliferation in a concentration-dependent manner, whereas IgG antibodies purified from preimmune serum of the same animals failed to do so (Fig. 19A). Vaccination with cNGF-CMV-Ntt830-E8*VLP induced high neutralizing titers that could be further increased by coadministration of a vaccine containing Quil A adjuvant (Fig. 19B). This observation mirrored the anti-NGF ELISA IgG titers in these dogs. A clear correlation between anti-NGF titers and neutralizing ability was observed (Fig. 19C). IgG purified from serum with high vaccine-specific titers enhanced potency in inhibiting NGF-mediated proliferation of TF-1 cells.

[0229] Vaccination with cNGF-CMV-Ntt830-E4 VLPs induced neutralizing anti-NGF antibody titers. High levels of neutralizing anti-NGF antibodies were observed on day 42 in sera collected from dogs immunized twice with cNGF-CMV-Ntt830-E4 in the presence of aluminum hydroxide (Fig. 19D).

[0230] These results demonstrate that the conjugate of canine mature NGF coupled to a modified VLP containing a chimeric CMV polypeptide according to the present invention can overcome immune tolerance to the endogenous target antigen and induce NGF-specific IgG antibodies in the target species, dogs. In addition, these antibodies were able to effectively neutralize canine mature NGF in vitro.

Claims

1. 1. A composition, preferably a veterinary composition, comprising: (a) a modified VLP of CMV, said modified VLP of CMV comprising at least one first binding site, said modified VLP of CMV comprising at least one chimeric CMV polypeptide, said at least one chimeric CMV polypeptide comprising: (i) a CMV polypeptide, wherein the CMV polypeptide comprises an amino acid sequence having at least 75% sequence identity to a coat protein of CMV or SEQ ID NO: 39; (ii) a polypeptide comprising, and preferably consisting of, a stretch of consecutive negatively charged amino acids, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, and wherein the polypeptide is inserted between any amino acid residue in the CMV polypeptide corresponding to any amino acid residue between positions 75 and 85 of SEQ ID NO: 39; A modified VLP of CMV comprising, and preferably consisting of, (b) at least one antigen, wherein the antigen comprises at least one second binding site, and the antigen is nerve growth factor (NGF); Including, A composition, wherein (a) and (b) are linked by said at least one first binding site and said at least one second binding site via at least one covalent non-peptide bond.

2. 2. The composition of claim 1, wherein the chimeric CMV polypeptide further comprises a helper T cell epitope, wherein the helper T cell epitope replaces an N-terminal region of the CMV polypeptide, and wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:

39.

3. 3. The composition of claim 2, wherein the helper T cell epitope is derived from tetanus toxin or is a PADRE sequence, and preferably the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO:

42.

4. A composition of CMV according to any one of claims 1 to 3, wherein the CMV polypeptide is a coat protein of CMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO:

39.

5. 5. The composition of claim 1, wherein the CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between amino acid residues 88 and 89 of SEQ ID NO:

5.

6. 6. The composition of CMV of any one of claims 1 to 5, wherein the stretch of consecutive negatively charged amino acids has a length of 3 to 10 amino acids.

7. 7. The composition of claim 1, wherein the stretch of consecutive negatively charged amino acids consists solely of glutamic acid.

8. The polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is disposed at the N-terminus of the stretch of consecutive negatively charged amino acids and the second amino acid linker is disposed at the C-terminus of the stretch of consecutive negatively charged amino acids, and the first amino acid linker and the second amino acid linker are (a.) an amino acid sequence (Gly) of length n=2 to 10; ｎ a polyglycine linker (G-linker) having the formula: (b.) a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, preferably wherein the GS linker has r=0 or 1, s=1-5, t=1-5, and u=0 or 1 (GS); ｒ (G ｓ S) ｔ (GS) ｕ a glycine-serine linker having the amino acid sequence (c.) an amino acid linker (GS*-linker) comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, and Cys; 8. The composition of CMV of any one of claims 1 to 7, independently selected from the group consisting of:

9. The composition according to any one of claims 1 to 8, wherein the polypeptide comprises, preferably consists of, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO:

51.

10. The composition of claim 1 , wherein the chimeric CMV polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

11. 11. The composition of any one of claims 1 to 10, wherein the at least one first binding site is not contained in or part of a polypeptide comprising the stretch of consecutive negatively charged amino acids.

12. 12. The composition of any one of claims 1 to 11, wherein the first binding site is an amino group, preferably an amino group of a lysine residue, and the at least one second binding site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue.

13. The composition according to any one of claims 1 to 12, wherein the antigen is selected from canine NGF (cNGF), feline NGF (fNGF), equine NGF (eNGF), bovine NGF (bNGF) and porcine NGF (pNGF), preferably the antigen is canine NGF (cNGF) or feline NGF (fNGF), more preferably the antigen is canine NGF (cNGF).

14. 14. The composition of any one of claims 1 to 13, wherein the antigen comprises, or preferably consists of, an amino acid sequence selected from any of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity to any of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:

58.

15. 15. The composition of any one of claims 1 to 14, wherein the antigen comprises, or preferably consists of, an amino acid sequence selected from any of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 55, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity to any of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO:

55.

16. A composition according to any one of claims 1 to 15 for use in a method for inducing neutralising antibodies against NGF in an animal.

17. 17. The composition for use according to claim 16, wherein the animal is a dog.

18. The composition for use according to claim 16, wherein the animal is a cat.

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