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

Abstract

The present invention relates to compositions comprising a modified cucumber mosaic virus (CMV) virus-like particle (VLP), and in particular, to the VLP of the modified CMV comprising a chimeric CMV polypeptide to which is attached a nerve growth factor (NGF) antigen, said chimeric CMV polypeptide comprising a segment of contiguous negatively charged amino acid selected from aspartic acid or glutamic acid; and pharmaceutical compositions thereof, preferably acting as a vaccine platform for generating an immune response, in particular an antibody response, directed against the NGF antigen linked to the VLP of the modified CMV.

Description

Technical Field

[0001] The present invention relates to a veterinary composition comprising modified cucumber mosaic virus (CMV) virus-like particles (VLPs), and in particular, to a modified CMV VLP linked to a nerve growth factor (NGF) antigen, wherein the modified CMV VLP comprises a chimeric CMV polypeptide containing a stretch of continuous negatively charged amino acids, wherein the negatively charged amino acids are selected from aspartic acid or glutamic acid; and a pharmaceutical composition thereof, wherein the composition preferably serves as a vaccine platform for generating an immune response, especially an antibody response, directed against the NGF antigen linked to the modified CMV VLP.

[0002] Related technologies

[0003] Virus-like particles (VLPs) have become an established and accepted vaccine technology, particularly as immunological vehicles 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).

[0004] Recently, a vaccine platform has been described that is based on cucumber mosaic virus (CMV, Bromoviridae, Cucumovirus) virus-like particles (CMV VLPs) and utilizes chemical linker coupling technology to present different antigens, including self-antigens, such as cytokines on its surface, and elicits effective neutralizing antibody responses. These soluble and stable CMV VLPs serve as an excellent platform because of their inherent properties, such as repeated presentation of target antigens to B cell receptors, nanoscale size, and geometry. And activate innate immunity through TLR activation and provision of T cell help (WO2016 / 062720; Zeltins A et al., Vaccines 2 (2017) 30; Bachmann MF et al., Frontiers in Microbiology Vol. 9, Paper 2522, October 2018; von Loga IS et al., Ann. Rheum Dis 2019, 78: 672-675; WO2021 / 260131).

[0005] Although the development process of these universal vaccines based on VLP has made progress, there are still challenges and requirements to be considered, especially the challenges and requirements in the final clinical trial test, product registration, market launch and commercial supply demand. Here, it is particularly important to mention the product characteristics of controllability, such as stability, shelf life, solubility, manufacturability, including scalability, safety, efficacy, bioavailability and other pharmacological properties, and the product characteristics are the key elements of chemical treatment, manufacturing and control (CMC) process, which are low-cost and high-efficiency. Provide these products in sufficient quantities for such final clinical trial tests, product registration, market launch and commercial supply needs are necessary (Pham NG, Int J Pharm, 2020, 585: 119523). Specifically, it is related to the stability of these VLP platforms and vaccines based on VLP (even under various conditions necessary for effective CMC processes). Another unexpected event and problem that adversely affects product characteristics is the aggregation of biological agents and vaccines, respectively (Roberts CJ, Current Opinion in Biotechnology, 2014, 30: 211-217). Although aggregated vaccines may still be able to elicit an immune response (provided their native structure is maintained), and even though they may therefore still be suitable for some laboratory studies, they are not acceptable for GMP products made for clinical studies and the market.

[0006] Therefore, despite the progress made in the development of these VLP-based universal vaccines, there is still a need to develop modified VLP systems that can be adapted to address the challenges that may arise and meet the requirements for final product registration and market launch.

[0007] Nerve growth factor (NGF) was found to be a key factor in the development and maintenance of sensory and sympathetic neurons in the developing nervous system. It acts as a soluble signaling protein that mediates its activity by binding to two different cell surface receptors (NGFR): the high affinity NGF-specific tropomyosin receptor kinase A (TrkA) and the low affinity p75 neurotrophin receptor (p75NTR). The amino acid sequences of canine or feline nerve growth factors and corresponding heterologous homologs from other animal species have been identified and are known to those skilled in the art. Summary of the invention

[0008] We surprisingly found that the composition of the present invention comprising the modified CMV VLP linked to the NGF antigen is not only highly immunogenic and induces high titers of neutralizing antibodies against the NGF antigen in vitro, but also the CMV VLP-NGF conjugate of the present invention maintains its stability and structural integrity. This is particularly surprising because the inclusion of additional negative charges in the protein forming the VLP, such as the insertion of multiple consecutive segments of negatively charged amino acids selected from glutamic acid and aspartic acid according to the present invention, may have a deleterious effect on the formation of the VLP. In contrast, the specific insertion of these consecutive segments of negatively charged amino acids not only improves the stability of the resulting modified CMV VLP under conditions of high temperature and higher ion concentration (compared to the CMV VLP of the prior art), but also after the NGF antigen is linked, the CMV VLP-NGF conjugate of the present invention does not form aggregates in solution and remains stable, whereas the CMV VLP of the prior art forms large aggregates and precipitates after such a link. Such aggregation and aggregate formation of the conjugated CMV VLPs is highly undesirable for drug development and product registration, and the composition of the present invention is very beneficial in substantially reducing or avoiding such undesirable aggregation. In addition, the stability improvement caused by the surface charge modification of the CMV VLPs in higher salt solutions is additionally beneficial to its handleability and ion exchange chromatography (especially anion exchange chromatography) purification or even essential, thereby further advantageously allowing the manufacture of the composition of the present invention to be easily expanded.

[0009] Thus, in a first aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0010] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0011] (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; and

[0012] (ii) a polypeptide comprising, 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, wherein the polypeptide is inserted between any amino acid residues corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39,

[0013] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0014] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0015] Other aspects and embodiments of the present invention will become apparent as the specification proceeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Description of the pET-CMVB2-Ntt-E8* plasmid map with single-cutting restriction enzyme sites.

[0017] Figure 2A : SDS-PAGE gel analysis of VLP purification derived from expression of CMV-Ntt830-E8*. M-protein size marker PageRuler (Thermo Fisher Scientific, #26620); S-soluble protein in cell extract of E. coli C2566 / pET-CMVB2-Ntt-E8*; P-insoluble protein in cell extract; 1-insoluble protein after sucrose gradient (bottom of tube); 2-6-sucrose gradient fractions (60% at the bottom of the tube to 0% at the top). The asterisks (*) in the figure indicate the relative position of the corresponding CMV-Ntt830-E8* chimeric CMV polypeptide in the SDS / PAGE gel.

[0018] Figure 2B : Electron microscopy images of purified CMV-Ntt830-E8* VLPs. The horizontal scale bar corresponds to 500 nm.

[0019] Figure 3 : Description of the pET-CMVB2-Ntt-E4 plasmid map with single-cutting restriction enzyme sites.

[0020] Figure 4 : Description of the pET-CMVB2-Ntt-E8 plasmid map with single-cutting restriction enzyme sites.

[0021] Figure 5 : Description of the pET-CMVB2-Ntt-E12 plasmid map with single-cutting restriction enzyme sites.

[0022] Figure 6: SDS-PAGE (left) and agarose gel (right) analysis of VLP purification derived from expression of CMV-Ntt830-E4. M1 - protein size marker PageRuler (Thermo Fisher Scientific, #26620); M2 - DNA size marker (Thermo Fisher Scientific, #SM0311); T - total protein in E. coli C2566 cells after 18 hours of incubation at 20°C; S - soluble protein in cell extracts after cell disruption and before sucrose gradient (20-60%); P - insoluble protein; 1-6 - sucrose gradient fractions (60% at the bottom of the tube to 0% at the top). The asterisks (*) in the figure indicate the relative position of the corresponding CMV-Ntt830-E4 chimeric CMV polypeptide in the SDS / PAGE gel and the relative position of the typical VLP signal in the agarose gel.

[0023] Figure 7 : SDS-PAGE (left) and agarose gel (right) analysis of VLP purification derived from expression of CMV-Ntt830-E8. M1 - protein size marker PageRuler (Thermo Fisher Scientific, #26620); M2 - DNA size marker (Thermo Fisher Scientific, #SM0311); T - total protein in E. coli C2566 cells after 18 hours of incubation at 20°C; S - soluble protein in cell extracts after cell disruption and before sucrose gradient (20-60%); P - insoluble protein; 1-6 - sucrose gradient fractions (60% at the bottom of the tube to 0% at the top). The asterisks (*) in the figure indicate the relative position of the corresponding CMV-Ntt830-E8 chimeric CMV polypeptide in the SDS / PAGE gel and the relative position of the typical VLP signal in the agarose gel.

[0024] Figure 8: SDS-PAGE (left) and agarose gel (right) analysis 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); T - total protein in E. coli C2566 cells after 18 hours of incubation at 20°C; S - soluble protein in cell extracts after cell disruption and before sucrose gradient (20-60%); P - insoluble protein; 1-6 - sucrose gradient fractions (60% at the bottom of the tube to 0% at the top). The asterisks (*) in the figure indicate the relative position of the corresponding CMV-Ntt830-E12 chimeric CMV polypeptide in the SDS / PAGE gel. No clear and distinct bands corresponding to intact VLPs were observed in the agarose gel.

[0025] Fig. 9 : Electron microscopy images of purified CMV-Ntt830-E4 VLPs. The horizontal scale bar corresponds to 200 nm.

[0026] Fig.10 : Electron microscopy images of purified CMV-Ntt830-E8 VLPs. The horizontal scale bar corresponds to 200 nm.

[0027] Fig.11 : Comparison of thermal stability of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP. Using DNA melting temperature determination program and real-time PCR system, structural changes of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP were monitored in the presence of Sypro-Orange dye. Curve 1 is CMV-Ntt830-E4 VLP, curve 2 is CMV-Ntt830 VLP and curve 3 is buffer control (5mM sodium phosphate, 2mM EDTA, pH 7.5). The corresponding 57°C and 51°C melting points are indicated by arrows.

[0028] Fig.12: Stability of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP in solution in the presence of different concentrations of NaCl. 0.5 mg / ml CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP samples were incubated in 5 mM sodium phosphate, 2 mM EDTA (pH 7.5) in the presence of different concentrations of NaCl (the NaCl molar concentration in each sample is indicated at the bottom of the gel) at room temperature for up to 2 hours. The samples were analyzed by native agarose gel electrophoresis and ethidium bromide staining. Figures A and B show NAGE analysis of CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP samples, respectively. M shows the lane loaded with GeneRuler 1 kb DNA Ladder (SM0311, TFS). The black arrows indicate the position of the loading wells in the gel and the position of the VLPs in each well and gel. The presence of CMV-Ntt830 VLPs in the sample wells after electrophoresis (Panel A) is due to the formation of VLP aggregates that are too large to enter the gel. Unaggregated intact VLPs migrate into the gel.

[0029] Fig.13 : Analysis of CMV-Ntt830 VLPs undergoing anion exchange chromatography. 5 ml of 5 mM sodium borate buffer pH 9.0 containing 1 mg / ml CVMtt-VLPs was loaded onto a 1.0 ml Macro-PrepDEAE Bio-Rad anion exchange cartridge equilibrated with 5 mM sodium borate buffer and eluted stepwise in the presence of 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 by nanodrop 260 nm (for protein concentration) and native agarose gel electrophoresis. Figure A shows the NaCl concentration and 260 nm absorbance plotted for each fraction (1-25). Figure B shows NAGE analysis of the main fraction containing the highest protein concentration (ethidium bromide staining). M shows the lane loaded with GeneRuler 1 kb DNA Ladder (SM0311, TFS). The black arrows indicate the positions of the loading wells in the gel and the positions of the VLPs in each well and the gel. The presence of CMV-Ntt830 VLPs in the loading wells after electrophoresis is due to the formation of VLP aggregates that are too large to enter the gel. Unaggregated intact VLPs migrate into the gel.

[0030] Fig.14: Analysis of CMV-Ntt830-E4 VLPs subjected to anion exchange chromatography. The biomass of E. coli cells expressing CMV-Ntt830-E4 VLPs was resuspended in 50 mM citrate, 5 mM borate buffer pH 9.0, and the cells were lysed using a microfluidizer LM-20. The soluble fraction was clarified by centrifugation and loaded onto 60 ml Fracto-DEAE (XK 26 / 20). An elution buffer comprising 50 mM citrate, 5 mM borate and 1 M NaCl was applied in a continuous gradient to elute the bound VLPs. Figure A shows protein elution and NaCl concentration gradient measured by A260nm (mAU) and conductivity (mS / cm), respectively. The X-axis shows elution volume and fraction number (4-11). The fractions collected from the Fracto-DEAE column were analyzed by NAGE (Figure B) and SDS-PAGE (Figure C). In Figure B, M indicates the lane loaded with GeneRuler 1kb DNALadder (SM0311, TFS), L is the E. coli lysate sample before loading on Fracto DEAE, FT is the flow-through collected from 0 to 150 ml and 4-10 represents the fraction number collected during elution. The black arrows indicate from top to bottom the position of the loading well, the position of the intact CMV-Ntt830-E4 VLP in the gel and the position of the contaminating nucleic acid from the clarified bacterial lysate, respectively. In Figure C, FT is the flow-through collected from 0 to 150 ml and 4-10 represents the fraction number. The black arrow shows the position of the CMV-Ntt830-E4 coat protein stained with Coomassie blue.

[0031] Fig.15A : Purification and purity of recombinant canine mature NGF. SDS-PAGE analysis of the NGF purification process. M-marker, whose band molecular weight is shown in kDa; A-total cell lysate after expression; B-combined elution fractions containing pro-NGF after refolding and partial purification; C-mature NGF after trypsin digestion and final purification. Arrows indicate pro-NGF in lanes A and B and mature NGF in lane C.

[0032] Fig. 15B : PC12 cells were cultured for 5 days with recombinant human mature NGF (R&D systems) produced by mouse myeloma cells (black squares) or with canine mature NGF produced by E. coli as described herein (grey circles). Cells were cultured in the presence of 100, 50, 25, 12.5 and 6.25 ng / ml recombinant NGF and the percentage of cells with well-defined neurites was determined.

[0033] Fig.16A : SDS-PAGE analysis of recombinant mature canine NGF (cNGF) of SEQ ID NO: 31 coupled with CMV-Ntt830 and CMV-Ntt830-E8* VLP.

[0034] M-PageRuler TM Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific, #26620) protein size marker; 1-corresponding purified CMV-Ntt830 and CMV-Ntt830-E8* VLP; 2-CMV VLP derivatized with 5x SMPH and SMPH removed; 3-CMV VLP coupled with an equimolar amount 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.

[0035] Fig. 16B : SDS-PAGE analysis of recombinant mature canine NGF (cNGF) of SEQ ID NO: 31 coupled with CMV-Ntt830-E4 and CMV-Ntt830-E8 VLPs.

[0036] M-PageRuler TM Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific, #26620) protein size marker; 1-corresponding purified CMV-Ntt830-E4 and CMV-Ntt830-E8 VLPs; 2-CMV VLPs after derivatization with 5x SMPH and removal of SMPH; 3-CMV VLPs coupled with an equimolar amount of cNGF; 4-mixed samples 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 bands.

[0037] Fig. 16C : Dynamic light scattering analysis of cNGF-CMV-Ntt830 VLPs. EM analysis was not possible due to vaccine precipitation.

[0038] Fig.16D : Dynamic light scattering analysis of cNGF-CMV-Ntt830-E4 VLPs comprising the cNGF antigen of SEQ ID NO:31.

[0039] Fig.16E : Dynamic light scattering analysis of cNGF-CMV-Ntt830-E4 VLPs containing the cNGF antigen of SEQ ID NO:33

[0040] Fig.16F : Dynamic light scattering analysis of cNGF-CMV-Ntt830-E8*VLPs.

[0041] Figure 16G : Electron microscopy of cNGF-CMV-Ntt830-E4 VLPs.

[0042] Fig.16H : Electron microscopy of cNGF-CMV-Ntt830-E8* VLPs.

[0043] Fig.17A : Evaluation of anti-NGF IgG antibodies in the sera of mice immunized with cNGF-CMV-Ntt830-E8*VLPs. Anti-NGF IgG titers were measured by ELISA in mice immunized twice (day 0 and day 14, indicated by arrows) with 15 μg of cNGF-CMV-Ntt830-E8*VLPs in the presence or absence of Quil A adjuvant (closed and open circles, respectively).

[0044] Fig. 17B : To test the neutralizing IgG antibodies produced in mice, PC12 cells were cultured for 5 days in the presence of 12.5 ng / ml human mature NGF (or in the absence of it as a negative control), in the presence of the indicated concentrations of anti-human NGF polyclonal antibodies (obtained from BioTechne) or purified IgG from naive mice (ms pIgG NAIVE), or purified IgG from mice immunized with the indicated concentrations of cNGF-CMV-Ntt830-E8*VLPs (pooled sera from study days 21, 28, and 35, ms pIgG NGF vacc). Data points represent sample copies.

[0045] Fig.18A : Evaluation of anti-NGF IgG antibodies in the sera of dogs immunized with cNGF-CMV-Ntt830-E8* VLPs. Anti-NGF IgG titers in Group 1 dogs that received the vaccine but not the adjuvant. Arrows indicate vaccine injections on days 0, 21, and 42.

[0046] Fig.18B : Evaluation of anti-NGF IgG antibodies in the sera of dogs immunized with cNGF-CMV-Ntt830-E8* VLPs. The arrows indicate the injections of vaccines on days 0, 21 and 42.

[0047] Fig.18C : Evaluation of anti-CMV IgG titers of sera from dogs immunized with cNGF-CMV-Ntt830-E8* VLPs. Anti-CMV IgG titers of Group 1 dogs that received the vaccine but not the adjuvant. Arrows indicate vaccine injections on days 0, 21, and 42.

[0048] Fig.18D : Evaluation of anti-CMV IgG titers in sera from dogs immunized with cNGF-CMV-Ntt830-E8* VLPs. Arrows indicate the administration of vaccines on days 0, 21 and 42.

[0049] Fig.18E : Evaluation of anti-NGF IgG antibodies in sera of dogs immunized with cNGF-CMV-Ntt830-E4 VLPs in the absence of adjuvant. Five dogs were given cNGF-CMV-Ntt830-E4 VLPs on days 0 and 21. NGF-specific antibodies were evaluated by ELISA in sera collected on days 0, 21, 42, 71, and 91.

[0050] Fig.18F : Evaluation of anti-NGF IgG antibodies in the sera of dogs immunized with cNGF-CMV-Ntt830-E4 VLPs in the presence of aluminum hydroxide. Five dogs were given cNGF-CMV-Ntt830-E4 VLPs with aluminum hydroxide on days 0 and 21. NGF-specific antibodies were measured by ELISA on days 0, 21, 42, 71, and 91.

[0051] Fig.19A: Vaccination with cNGF-CMV-Ntt830-E8*VLP induces dogs to produce NGF neutralizing antibodies. On days 0, 21 and 42, dogs (3 dogs / group) were immunized with 250μg cNGF-CMV-Ntt830-E8*VLP in the presence or absence of adjuvant QuilA. Serum was collected and tested for the presence of neutralizing antibodies using a TF-1-based NGF bioactivity assay. A titration curve for one dog is shown to determine the neutralizing ability and 50% neutralization titer (NT50) of canine serum. 5ng / mL of human mature NGF was pre-incubated with increasing concentrations of IgG, which was purified from serum collected on designated dates after the first administration of the vaccine. The NT50 value, i.e., the IgG concentration that causes 50% inhibition of cell proliferation, was determined using the sigmoid curve fitting model of 4PL.

[0052] Fig.19B : Vaccination with cNGF-CMV-Ntt830-E8*VLP induces dogs to produce NGF neutralizing antibodies. On days 0, 21 and 42, 250 μg of cNGF-CMV-Ntt830-E8*VLP was immunized against dogs (3 dogs / group) in the presence or absence of adjuvant QuilA. Serum was collected and the presence of neutralizing antibodies was tested using a TF-1-based NGF bioactivity assay. Total IgG was purified from canine serum. The ability of 20 μg / mL purified total IgG to neutralize 5 ng / mL human mature NGF was assessed using bioassays. The bars represent group means and standard deviations and the symbols represent individual dogs (mean values ​​of duplicate analyses). Two-way ANOVA and Tukey's multiple comparisons test were performed using GraphPad Prism to compare group means. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0053] Fig.19C: Vaccination with cNGF-CMV-Ntt830-E8*VLP induces dogs to produce mature NGF neutralizing antibodies. On days 0, 21 and 42, dogs (3 dogs / group) were immunized with 250 μg cNGF-CMV-Ntt830-E8*VLP in the presence or absence of the adjuvant QuilA. Serum was collected and the presence of neutralizing antibodies was tested using a TF-1-based NGF bioactivity assay. The NT50 value was plotted against the OD50 value of the anti-NGF IgG serum titer. Total IgG purified from serum with a higher concentration of NGF-specific antibodies is more potent in inhibiting NGF-mediated TF-1 cell proliferation than total IgG purified from canine serum with a lower anti-NGF titer. Symbols represent individual dogs and sampling time points. Different symbols are assigned to different dogs. Closed symbols represent animals vaccinated in the presence of adjuvant, while open symbols represent animals vaccinated in the absence of adjuvant.

[0054] Fig.19D : Vaccination with cNGF-CMV-Ntt830-E4 VLPs induces NGF neutralizing antibodies in dogs. On days 0 and 21, cNGF-CMV-Ntt830-E4 VLPs were administered with aluminum hydroxide to 5 dogs. Serum was collected on day 42 and tested for the presence of neutralizing antibodies using a TF-1-based NGF bioactivity assay. Bars represent group means and standard deviations and symbols represent individual dogs. Dashed lines indicate the detection limit of the assay. DETAILED DESCRIPTION OF THE INVENTION

[0056] 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 the invention belongs. The embodiments, preferred embodiments and / or very preferred embodiments described and disclosed herein should apply to all aspects and other embodiments, preferred embodiments and / or very preferred embodiments, whether or not specifically mentioned again or whether or not repetition thereof is avoided for brevity. As used herein, the article "a / an" refers to one or more than one (i.e., at least one) grammatical object of the article. Unless the context clearly indicates otherwise, the term "or" as used herein should be understood to mean "and / or".

[0057] Virus-like particles (VLP): As used herein, the term "virus-like particles (VLP)" refers to non-replicative or non-infectious particles, preferably non-replicative and non-infectious virus particles, or refers to non-replicative or non-infectious structures, preferably non-replicative and non-infectious structures similar to virus particles, preferably viral capsids. 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 the host cell. The virus-like particles according to the present invention are non-replicative and non-infectious due to the lack of all or part of the viral genome or genome function. The virus-like particles according to the present invention may contain nucleic acids different from their genome. Recombinantly produced virus-like particles typically contain host cell-derived RNA. Typical and preferred embodiments of the virus-like particles according to the present invention are virus capsids composed of the polypeptides of the present invention. Virus-like particles are typically macromolecular assemblies composed of viral coat proteins, which typically contain 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 virus-like capsid structure with an inherent repetitive organization.One characteristic of virus-like particles is their high degree of order and the repetitive arrangement of their subunits.

[0058] Modified CMV virus-like particles (VLPs): The term "modified CMV virus-like particles" refers to virus-like particles comprising at least one chimeric CMV polypeptide as defined and described herein. Typically and preferably, the modified CMV VLP is similar to the structure of the CMV capsid. The modified CMV VLP is non-replicative and / or non-infectious, and at least lacks one or more genes encoding the CMV replication machinery, and typically also lacks one or more genes encoding one or more proteins responsible for viral attachment to the host or entry into the host. This definition also includes modified virus-like particles, in which the aforementioned one or more genes are still present, but inactive. Preferably, the 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, the modified CMV VLP is a macromolecular assembly composed of a CMV polypeptide modified according to the present invention, and typically and preferably, each VLP comprises 180 such protein subunits and chimeric polypeptides, respectively. Therefore, in a preferred embodiment, the modified cucumber mosaic virus (CMV) virus-like particle (VLP) comprises 180 chimeric CMV polypeptides.

[0059] Polypeptide: As used herein, the term "polypeptide" refers to a polymer composed of amino acid monomers linearly linked by amide bonds (also called peptide bonds). It refers to a chain of amino acid molecules, not to a specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides and proteins are included in the definition of polypeptide. As used herein, typically and preferably, the term "polypeptide" shall also refer to a polypeptide as defined above, and encompasses modifications, such as post-translational modifications, including but not limited to glycosylation. In a preferred embodiment, as used herein, the term "polypeptide" shall refer to a polypeptide as defined above and does not encompass modifications, such as post-translational modifications, such as glycosylation. In particular, the peptide having biological activity may then even undergo the modifications, such as the glycosylation, in vivo, for example by bacteria.

[0060] Cucumber mosaic virus (CMV) polypeptide, 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 the coat protein of cucumber mosaic virus (CMV); or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence of CMV shows at least 90%, preferably at least 91%, 92%, 93% or 94%, more preferably at least 95% sequence identity with the coat protein, yet more preferably at least 98% and further more preferably at least 99% sequence identity. Typically and preferably, the CMV polypeptide is capable of forming CMV virus-like particles by self-assembly after expression.

[0061] Coat protein (CP) of cucumber mosaic virus (CMV): As used herein, the term "coat protein (CP) of cucumber mosaic virus (CMV)" refers to the coat protein of cucumber mosaic virus present in nature. Due to the extremely wide host range of cucumber mosaic virus, many different strains and isolates of CMV are known. The coat protein sequences of the strains and isolates have been determined and are known to those skilled in the art. The sequence of the coat protein (CP) of CMV is described in and can be retrieved in known databases such as Genbank, www.dpvweb.net, or www.ncbi.nlm.nih.gov / protein / . Specific examples of the CP of CMV are described in WO 2016 / 062720, page 12, line 8 to page 13, line 25, the contents of which are expressly incorporated herein by reference. Very preferred examples and embodiments of CMV coat protein are provided in SEQ ID NO:39. Therefore, preferably, as used herein, the term "coat protein of cucumber mosaic virus (CMV)" refers to the amino acid sequence of CMV coat protein, wherein the 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%, yet more preferably at least 90%, yet more preferably at least 91%, 92%, 93% or 94%, yet more preferably at least 95%, still more preferably at least 96% or 97%, still more preferably at least 98% and still more preferably at least 99% sequence identity with SEQ ID NO:39.

[0062] It is noteworthy that these virus strains and isolates have highly similar coat protein sequences in different protein domains (including the N-terminus of the coat protein). Specifically, 98.1% of all CMV isolates that were fully sequenced shared more than 85% sequence identity in the first 28 amino acids of their coat protein sequences, and 79.5% of all CMV isolates that were fully sequenced still shared more than 90% sequence identity in the first 28 amino acids of their coat protein sequences.

[0063] Modified CMV polypeptide: As used herein, the term "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 forming CMV virus-like particles by self-assembly after expression. Preferably, the modified CMV polypeptide is a recombinantly modified CMV polypeptide and is capable of forming CMV virus-like particles by self-assembly after expression in Escherichia coli.

[0064] Chimeric CMV polypeptide: As used herein, the term "chimeric CMV polypeptide" refers to a polypeptide as defined herein and according to the present invention, and comprises a CMV polypeptide, preferably consisting of a CMV polypeptide, wherein the CMV polypeptide is modified as defined and described herein to comprise a polypeptide containing, preferably consisting of, a continuous stretch of negatively charged amino acids, and optionally further comprises a helper T cell epitope, wherein the continuous amino acids are independently selected from aspartic acid or glutamic acid, and all components are as defined and described herein. Typically and preferably, the chimeric CMV polypeptide is capable of forming modified CMV virus-like particles by self-assembly after expression. Therefore, in a preferred embodiment, the chimeric CMV polypeptide is capable of forming modified CMV virus-like particles by self-assembly, typically and preferably by self-assembly after expression. Preferably, the chimeric CMV polypeptide is a recombinantly modified CMV polypeptide and is capable of forming CMV virus-like particles by self-assembly after expression in Escherichia 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 wherein 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.

[0065] N-terminal region of CMV polypeptide: As used herein, the term "N-terminal region of CMV polypeptide" refers to the N-terminus of the CMV polypeptide, and specifically, refers to the N-terminus of the CMV coat protein, or the N-terminal region of the CMV polypeptide or the coat protein of CMV, but if the CMV polypeptide or the coat protein comprises an N-terminal methionine residue, the N-terminal region starts at the second amino acid of the N-terminus of the CMV polypeptide or the coat protein of CMV. Preferably, in the case where the CMV polypeptide or the coat protein comprises an N-terminal methionine residue, from a practical point of view, the start codon encoding methionine will usually be deleted and added to the N-terminus of the helper T cell epitope. More preferably, one, two or three other amino acids, preferably one amino acid, may be optionally inserted between the methionine and the Th cell epitope for cloning purposes.

[0066] Recombinant polypeptide: In the context of the present invention, the term "recombinant" when used in the context of a polypeptide refers to a polypeptide obtained by a method comprising at least one step of recombinant DNA technology. Typically and preferably, the recombinant polypeptide is produced in a prokaryotic expression system. It is obvious to the skilled person that a recombinantly produced polypeptide expressed in a prokaryotic expression system (such as E. coli) may contain an N-terminal methionine residue. During maturation of the recombinant polypeptide, the N-terminal methionine residue typically causes the recombinant polypeptide to be cleaved in the expression host. However, the cleavage of the N-terminal methionine may not be complete. Therefore, a preparation of a recombinant polypeptide may contain a mixture of otherwise identical polypeptides having an N-terminal methionine residue and not having an N-terminal methionine residue. Typically and preferably, a recombinant polypeptide preparation contains less than 10%, more preferably less than 5%, and still more preferably less than 1% of a recombinant polypeptide having an N-terminal methionine residue.

[0067] 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 method comprising at least one step of recombinant DNA technology.

[0068] Mutated amino acid sequence: The term "mutated amino acid sequence" refers to an amino acid sequence obtained by introducing a set of defined mutations into an amino acid sequence to be mutated. In the context of the present invention, the amino acid sequence to be mutated is typically and preferably an amino acid sequence of a CMV coat protein. Thus, the mutated amino acid sequence differs from the amino acid sequence of the CMV coat protein by at least one amino acid residue, wherein the mutated amino acid sequence shows at least 90% sequence identity with the amino acid sequence to be mutated. Typically and preferably, the mutated amino acid sequence shows at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence to be mutated. Preferably, the mutated amino acid sequence differs from the sequence to be mutated by at most 11, 10, 9, 8, 7, 6, 4, 3, 2 or 1 amino acid residues, wherein more preferably, the difference is selected from insertion, deletion and amino acid exchange. Preferably, the mutated amino acid sequence differs from the amino acid sequence of the CMV coat protein in at least one amino acid, wherein preferably, the difference is an amino acid exchange.

[0069] The terms "corresponding", "correspond", "corresponds", "corresponding ...

[0070] Sequence identity: The sequence identity of two given amino acid sequences is determined based on the alignment of the two sequences. Algorithms for measuring sequence identity are available to the skilled person. 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 / ), and preferably by the "BLAST" program provided by the NCBI homepage http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using the preset settings provided therein. Typical and preferred standard settings are: expectation threshold: 10; word length: 3; maximum matches within the query range: 0; matrix: BLOSUM62; gap cost: existence 11, extension 1; composition adjustment: conditional composition scoring matrix adjustment.

[0071] Amino acid exchange: The term amino acid exchange refers to the exchange of a specified amino acid residue in an amino acid sequence with any other amino acid residue having a different chemical structure, preferably with another protein amino acid residue. Therefore, compared to the insertion or deletion of amino acids, amino acid exchange does not change the total number of amino acids in the amino acid sequence.

[0072] As used herein, the term "isoelectric point", abbreviated as pI, refers to the pH at which a molecule carries no net charge or is electrically neutral on a statistical average. Specifically, the term "isoelectric point" is used herein to refer to the pH at which an antigen used in the present invention and composed of amino acids carries no net charge or is electrically neutral on a statistical average. At pH below its pI, such antigens carry a net positive charge; at pH above its pI, they carry a net negative charge. Typically and preferably, when referring to a pI value, and in particular, when referring to the pI value of an antigen of the present invention and an antigen within the present disclosure, the pI value is determined by entering the primary amino acid sequence of the particular protein and antigen, respectively, into the ExPASy Compute pI / MW tool described by Gasteiger et al. (Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, MR, Appel, RD, and Bairoch, A., Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005)). Thus, the ExPASy Compute pI / MW tool referred to herein refers to the tool described by Gasteiger et al. The tool calculates the theoretical isoelectric point pI and Mw for a specified Swiss-Prot / TrEMBL entry or a user-entered amino acid sequence. Protein pI was calculated using amino acid pK values ​​as described by Bjellqvist et al., which were defined by examining the migration of polypeptides in an immobilized pH gradient gel environment containing 9.2 M and 9.8 M urea between pH 4.5 and 7.3 at either 15°C or 25°C (Bjellqvist, B. et al., 1993, Electrophoresis 14: 1023-1031; Bjellqvist, B. et al., 1994, Electrophoresis 15: 529-539).

[0073] Epitope: The term epitope refers to a continuous or discontinuous portion of a polypeptide or antigen that can be specifically bound by an antibody or T-cell receptor in the context of an MHC molecule. In the case of antibodies, specific binding excludes nonspecific binding but does not necessarily exclude cross-reactivity. An epitope typically contains 5 to 20 amino acids in a spatial configuration that is unique to the antigenic site.

[0074] T helper (Th) cell epitope: As used herein, the interchangeable terms "helper T cell epitope or Th cell epitope" refer to an epitope that can be recognized by helper Th cells. Typically and preferably, as used herein, the term "Th cell epitope" refers to a Th cell epitope that can bind to at least one, preferably more than one, class II MHC molecule. The simplest way to determine whether a peptide sequence is a Th cell epitope is to measure the ability of the peptide to bind to individual class II MHC molecules. This can be measured based on the ability of the peptide to compete with known Th cell epitope peptides for binding to class II MHC molecules. Representative selections of HLA-DR molecules are described, for example, in Alexander J et al., Immunity (1994) 1: 751-761. The affinity of a Th cell epitope for a class II MHC molecule should be at least 10 -5M. A representative set of class II MHC molecules present in different individuals is shown in Panina-Bordignon P et al., Eur J Immunol (1989) 19: 2237-2242. Therefore, as used herein, the term "Th cell epitope" preferably refers to a Th cell epitope that produces a measurable T cell response after immunization and boosting. In addition, and yet more preferably, as used herein, the term "Th cell epitope" preferably refers to a Th cell epitope that is capable of binding with an affinity of at least 500 nM to at least one, preferably at least two and even more preferably at least three DR alleles selected from the group consisting of DR1, DR2w2b, DR3, DR4w4, DR4w14, DR5, DR7, DR52a, DRw53, DR2w2a; and preferably selected from the group consisting of DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a (as described in Alexander J et al., Immunity (1994) 1:751-761 and references cited therein); a preferred binding assay for assessing said affinity is the binding assay described in Sette A et al., J Immunol (1989) 142:35-40. In an even more preferred manner, as used herein, the term "Th cell epitope" refers to a Th cell epitope capable of binding with an affinity of at least 500 nM to at least one, preferably at least two and even more preferably at least three DR alleles selected from the group consisting of DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a (as described in Alexander J et al., Immunity (1994) 1:751-761 and references cited therein); a preferred binding assay for assessing said affinity is the binding assay described by Sette A et al., J Immunol (1989) 142:35-40. Th cell epitopes have been described and are known to those skilled in the art, such as 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.

[0075] Amino acid linker: As used herein, the term "amino acid linker" refers to a linker consisting entirely of amino acid residues. The amino acid residues of the amino acid linker are composed of naturally occurring amino acids or non-natural amino acids, all L-forms or all D-forms or mixtures thereof as known in the art. The amino acid residues of the amino acid linker are preferably naturally occurring amino acids, all L-forms or all D-forms or mixtures thereof. In a preferred embodiment, the amino acid linker consists of naturally occurring α amino acids (all in their L-configuration).

[0076] G linker: As used herein, the term "G linker" refers to an amino acid linker consisting of glycine amino acid residues only. The G linker according to the present invention comprises at least two glycine residues and at most ten glycine residues.

[0077] GS linker: As used herein, the term "GS linker" refers to an amino acid linker consisting only of glycine and serine amino acid residues. The GS linker according to the present invention comprises at least one glycine and at least one serine residue. Typically and preferably, the GS linker has a length of up to 30 amino acids.

[0078] 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.

[0079] As used herein, the term "amino acid" refers to an organic compound containing the functional groups amine (-NH2) and carboxylic acid (-COOH) and zwitterions thereof and typically and preferably containing side chains unique to each amino acid. The term "amino acid" typically and preferably includes naturally occurring amino acids, such as protein amino acids (produced by RNA translation), non-protein amino acids (produced by other metabolic mechanisms, such as post-translational modifications), standard or regular amino acids (directly encoded by codons of the genetic code), and non-standard or non-regular 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" also includes chemically synthesized non-natural amino acids; amino acids such as alpha-(α-), beta-(β-), gamma-(γ-) and delta-(δ-) and mixtures thereof in any ratio; and, if applicable, such as alpha-(α-) amino acids, any isomeric forms of amino acids, i.e., their D-stereoisomers and L-stereoisomers (alternatively described according to the (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 a preferred embodiment, the term amino acid refers to an alpha amino acid, preferably to a naturally occurring alpha amino acid, more preferably to a naturally occurring alpha amino acid in the L-configuration.

[0080] Association: As used herein, the term "association" or "association" refers to all possible ways by which two molecules can be joined together, preferably chemical interactions. 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, for example, on covalent bonds, such as esters, ethers, phosphates, carbon-phosphorus bonds, carbon-sulfur bonds, such as thioethers, or imide bonds.

[0081] First attachment site: As used herein, the phrase "first attachment site" refers to an element that is naturally present in a virus-like particle or artificially added to a virus-like particle and can be connected to a second attachment site. The first attachment 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 ions, phenylmethylsulfonyl fluoride, or a chemically reactive group, such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, a histidyl group or a combination thereof. A preferred embodiment of a chemically reactive group as the first attachment site is an amino group of an amino acid residue, preferably an amino group of a side chain of a lysine residue. The first attachment site is typically located on the surface, and is preferably located on the outer surface of the VLP. A plurality of first attachment sites are present on the surface, preferably on the outer surface of the VLP, typically in a repetitive configuration. In a preferred embodiment, the first attachment site is associated with the VLP via at least one covalent bond, preferably via at least one peptide bond. In another preferred embodiment, the first attachment site is naturally present in the VLP. Alternatively, in a preferred embodiment, the first attachment site is artificially added to the VLP. In a very preferred embodiment, the first attachment site is the amino group of a lysine residue of the amino acid sequence of the VLP polypeptide.

[0082] Second attachment site: As used herein, the phrase "second attachment site" refers to an element that is naturally present in an antigen or artificially added to an antigen and can be connected to the first attachment site. The second attachment site of an antigen is preferably a protein, polypeptide, peptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound, such as biotin, fluorescein, retinol, digoxin, metal ion, phenylmethylsulfonyl fluoride, or a chemically reactive group, such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidyl group, a histidyl group or a combination thereof. A preferred embodiment of a chemically reactive group as the second attachment site is a sulfhydryl group, preferably a sulfhydryl group in a cysteine ​​residue. Therefore, the term "antigen having at least one second attachment site" refers to a construct comprising an antigen and at least one second attachment site. However, particularly for a second attachment site that is not naturally present in an antigen, such a construct typically and preferably further comprises a "linker". In another preferred embodiment, the second attachment site is associated with the antigen via at least one covalent bond, preferably via at least one peptide bond. In another embodiment, the second attachment site is naturally present in the antigen. In another more preferred embodiment, the second attachment site is artificially added to the antigen via a linker, wherein the linker comprises or alternatively consists of cysteine.Preferably, the linker is fused to the antigen via a peptide bond.

[0083] Linked: As used herein, the term "linked / linkage" refers to all possible ways by which at least one first linking site and at least one second linking site are joined together, preferably chemical interactions. 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, phosphates, carbon-phosphorus bonds, carbon-sulfur bonds, such as thioethers, or imide bonds. In certain preferred embodiments, the first linking site and the second linking site are linked via at least one covalent bond, preferably via at least one non-peptide bond and even more preferably via a complete non-peptide covalent bond. However, as used herein, the term "linked" should not only refer to the direct connection of at least one first linking site to at least one second linking site, but also alternatively and preferably refer to the indirect connection of at least one first linking site to at least one second linking site via an intermediate molecule and here typically and preferably by using at least one, preferably one heterobifunctional cross-linking agent. In other embodiments, the first attachment site is linked to the second attachment site via at least one covalent bond, preferably via at least one peptide bond, and even more preferably entirely via peptide bonds.

[0084] Linker: As used herein, a "linker" associates the second attachment site with the antigen or already comprises or consists of the second attachment site. Preferably, as used herein, a "linker" already comprises the second attachment site, typically and preferably as an amino acid residue, preferably as a cysteine ​​residue. Preferably, the linker is a linker containing at least one amino acid residue, or even more preferably a linker consisting entirely of amino acid residues. The amino acid residues of the linker are preferably composed of naturally occurring amino acids or non-natural amino acids, all L-type or all D-type or mixtures thereof known in the art. A more preferred embodiment of the linker according to the present invention is a molecule comprising a sulfhydryl or cysteine ​​residue and therefore, such molecules are also encompassed within the present invention. Other linkers suitable for use in the present invention are molecules comprising C1-6 alkyl-, cycloalkyl (such as cyclopentyl or cyclohexyl), cycloalkenyl, aryl or heteroaryl moieties. In addition, linkers preferably comprising C1-C6 alkyl-, cycloalkyl-(C5, C6), aryl- or heteroaryl-moieties and other amino acids can also be used as linkers of the present invention and should be encompassed within the scope of the present invention. The linker is preferably associated with the antigen via at least one covalent bond, more preferably via at least one peptide bond.

[0085] Antigen: As used herein, the term "antigen" refers to a molecule that can be bound by an antibody or T cell receptor (TCR) (if presented by an MHC molecule). Antigens are also capable of being recognized by the immune system and / or capable of inducing a humoral immune response and / or a cellular immune response that causes activation of B lymphocytes and / or T lymphocytes. An antigen may have one or more epitopes (e.g., a B epitope and a T epitope). As used herein, an antigen may also be a mixture of several individual antigens.

[0086] Ordered and repetitive antigen arrays: As used herein, the term "ordered and repetitive antigen arrays" refers to a repetitive pattern of antigens, typically and preferably characterized by a high-order uniformity of the spatial arrangement of the antigens relative to the modified CMV VLPs. In one embodiment of the invention, the repetitive pattern may be a geometric pattern. Certain embodiments of the invention (such as antigens linked to modified CMV VLPs) are typical and preferred examples of suitable ordered and repetitive antigen arrays, and in addition, they have a strictly repetitive antigen sub-order, preferably 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 further more preferably 1.6 to 7 nanometers.

[0087] Coupling efficiency: The coupling efficiency of virus-like particles and specific antigens is determined by SDS-PAGE of the coupling reaction. The intensity of the Coomassie brilliant blue staining band corresponding to the components of the coupling reaction is determined by densitometry and used to calculate the coupling efficiency. The coupling efficiency is defined as the ratio of the amount of VLP polypeptides (i) coupled to the antigen relative to the total amount of (ii) VLP polypeptides. 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 more preferably at least 35% or at least 40%. The coupling efficiency can also be expressed by the total number of antigens connected to the modified CMV VLP. The coupling efficiency can depend on the nature of the antigen, and the total number of antigens connected 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.

[0088] Nerve Growth Factor (NGF): As used herein and when referring to the antigen in the composition of the present 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 a corresponding heterologous homolog from any other species, preferably from a non-human animal, or to 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 a corresponding heterologous homolog from any other species, preferably from a non-human animal. 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 comprises, preferably consists of, a polypeptide of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, 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 92%, more preferably at least 95% and even more preferably at least 98% sequence identity with any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58. The NGF antigen typically and preferably comprises biological activity, preferably in a cell proliferation assay. In addition, the NGF antigen, when administered to an animal in the form of a composition of the present invention, is typically and preferably capable of inducing the animal to produce anti-NGF antibodies, wherein the anti-NGF antibodies are preferably capable of neutralizing the biological activity of NGF in an in vitro assay, as described herein (see Example 6). As used herein and when referring to the NGF antigen, the term "biological activity" refers to the activity of the NGF antigen in a cell proliferation assay, wherein preferably, the cell proliferation assay is based on the NGF-dependent human erythroleukemia TF-1 cell line, wherein still more preferably, the cell proliferation assay is performed under conditions substantially as described in Example 6 herein.

[0089] Adjuvant: As used herein, the term "adjuvant" refers to a stimulator of the immune response and / or a substance that typically allows the production of a depot in the host, which, when combined with the composition, vaccine or pharmaceutical composition of the present invention, can achieve a stronger immune response. Different types of adjuvants with different mechanisms of action have been described and are capable of enhancing antigen-specific antibody responses (Pulendran B et al., 2021, Nature Reviews Drug Discovery 20: 454-475). Typical and preferred adjuvants are mineral salts (e.g. aluminum hydroxide, aluminum phosphate), microcrystalline tyrosine, emulsions, microparticles, saponins (QuilA), cytokines, immunopotentiators, microbial components / products, liposomes, complexes and mucosal adjuvants, which are known and described, for example, in Adjuvant Compendium NIAID and VAC (nih.gov) or Aguilar et al. (Aguilar JC et al., 2007, Vaccine 25:3752-3762), Gerdts (Gerdts V, 2015, Berliner und Münchener Wochenschrift 128:456-463) and Pasquale et al. (Pasquale et al., 2015, Vaccines 3:320-343). As used herein, the term "adjuvant" may also include a mixture of adjuvants. Virus-like particles are sometimes described as adjuvants. However, as used in the context of this application, the term "adjuvant" refers to an adjuvant that is not a modified virus-like particle of the present invention. Instead, "adjuvant" refers to another different component in the composition, vaccine or pharmaceutical composition of the present invention.

[0090] Immunostimulatory substances: As used herein, the term "immunostimulatory substances" refers to substances that can induce and / or enhance immune responses. As used herein, immunostimulatory substances include, but are not limited to, substances that activate toll-like receptors and substances that induce cytokine secretion. Toll-like receptor activating substances include, but are not limited to, immunostimulatory nucleic acids, peptidoglycans, lipopolysaccharides, lipoteichoic acid, imidazoquinoline compounds, flagellin, and immunostimulatory organic substances, such as paclitaxel.

[0091] 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, wherein both ribonucleic acids and deoxyribonucleic acids can be double-stranded or single-stranded. Preferred ISS-NAs are deoxyribonucleic acids, wherein more preferably, the deoxyribonucleic acids are single-stranded. Preferably, the immunostimulatory nucleic acid contains at least one CpG motif comprising an unmethylated C. Very preferred immunostimulatory nucleic acids contain at least one CpG motif, wherein the at least one CpG motif comprises or preferably consists of at least one, preferably one, CG dinucleotide, wherein C is unmethylated. Preferably, but 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. In the context of the present invention, particularly preferred are ISS-NAs capable of stimulating dendritic cells to produce IFN-α. Immunostimulatory nucleic acids suitable for the purposes of the present invention are described, for example, in WO 2007 / 068747 A1.

[0092] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a nucleic acid sequence comprising 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 and are 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: (a) single-stranded and double-stranded DNA; (b) DNA, which is a mixture of single-stranded and double-stranded regions; (c) single-stranded and double-stranded RNA; (d) RNA, which is a mixture of single-stranded and double-stranded regions; and (e) hybrid molecules comprising DNA and RNA, which 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) alkyl phosphorothioates, (f) 5-nitroindole deoxyribofuranoyl, (g) 5-methyldeoxycytosine and (h) 5,6-dihydro-5,6-dihydroxydeoxythymidine. Phosphorothioate nucleotides are protected from degradation in cells or organisms and are therefore preferred nucleotide modifications. Unmodified oligonucleotides consisting entirely of phosphodiester-bound nucleotides are typically more active than modified nucleotides and are therefore generally preferred in the context of the present invention. Most preferred are oligonucleotides consisting entirely of phosphodiester-bound oligonucleotides, wherein more preferably, the oligonucleotide is single-stranded. More preferred are oligonucleotides capable of stimulating cells, preferably dendritic cells, to produce IFN-α. Very preferred oligonucleotides capable of stimulating cells to produce IFN-α are selected from CpG type A and CpG type C. More preferred are RNA molecules without Cap.

[0093] CpG motif: As used herein, the term "CpG motif" refers to a pattern of nucleotides including an unmethylated central CpG, i.e., an unmethylated CpG dinucleotide in which C is unmethylated; the central CpG is surrounded by at least one base (preferably one or two nucleotides) flanking the central CpG (flanking 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 located at its 5' and 3' ends. Without being bound by theory, the bases on either side of the CpG confer a significant portion of the activity of the CpG oligonucleotide.

[0094] Oligonucleotides containing unmethylated CpG: As used herein, the term "oligonucleotides containing unmethylated CpG" or "CpG" refers to oligonucleotides containing at least one CpG motif, preferably oligodeoxynucleotides. Therefore, CpG contains at least one unmethylated cytosine, guanine dinucleotide. Preferred CpG stimulates / activates (e.g., produces mitogenic effect) or induces or increases the expression of cytokines by vertebrate bone marrow-derived cells. For example, CpG can be used to activate B cells, NK cells and antigen-presenting cells, such as dendritic cells, monocytes and macrophages. Preferably, CpG refers to an oligodeoxynucleotide containing an unmethylated cytosine and a guanosine after its 3', preferably a single-stranded oligodeoxynucleotide, wherein the unmethylated cytosine is linked to the guanosine by a phosphate bond, wherein preferably, the phosphate bond is a phosphodiester bond or a phosphorothioate bond, and wherein more preferably, the phosphate bond is a phosphodiester bond. CpG may include nucleotide analogs, such as analogs containing thiophosphate bonds, and may be double-stranded or single-stranded. In general, double-stranded molecules are more stable in vivo, while single-stranded molecules have enhanced immunological activity. Preferably, as used herein, CpG is an oligonucleotide having a length of at least about ten nucleotides and comprising at least one CpG motif, wherein more preferably, the length of the CpG is 10 to 60, more preferably 15 to 50, still more preferably 20 to 40, still more preferably about 30 and most preferably exactly 30 nucleotides. CpG may be composed of methylated nucleotides and / or unmethylated nucleotides, wherein the at least one CpG motif comprises at least one CG dinucleotide, wherein C is unmethylated. CpG may also comprise methylated and unmethylated sequence segments, wherein the at least one CpG motif comprises at least one CG dinucleotide, wherein C is unmethylated. Very preferably, CpG refers to a single-stranded oligodeoxynucleotide containing an unmethylated cytosine and a guanosine 3' thereafter, wherein the unmethylated cytosine is linked to the guanosine by a phosphodiester bond. CpG may include nucleotide analogs, such as analogs containing a phosphorothioate bond, and may be double-stranded or single-stranded. In general, phosphodiester CpG is a type A CpG as shown below, while phosphorothioate stabilized CpG is a type B CpG. In the context of the present invention, a preferred CpG oligonucleotide is a type A CpG.

[0095] A-type CpG: As used herein, the term "A-type CpG" or "D-type CpG" refers to an oligodeoxynucleotide (ODN) comprising at least one CpG motif. A-type CpG preferentially stimulates the activation of T cells and the maturation of dendritic cells and is capable of stimulating IFN-α production. In A-type CpG, the nucleotides of at least one CpG motif are linked by at least one phosphodiester bond. A-type CpG comprises at least one phosphodiester-bonded CpG motif, which may be flanked at its 5' end and / or preferably at its 3' end by thiophosphate-bonded nucleotides. Preferably, the CpG motif, and preferably here, the CG dinucleotide comprising at least one, preferably two nucleotides and its immediately flanking region, are composed of phosphodiester nucleotides. Preferred A-type CpGs are composed entirely of phosphodiester (PO)-bonded nucleotides. Typically and preferably, the poly G motif comprises or alternatively consists of at least one guanosine (G's), preferably at least three, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 G's, most preferably at least 10 G's. Preferably, the A-type CpG of the present invention comprises or alternatively consists of a palindromic sequence.

[0096] Packaging: As used herein, the term "packaging" refers to the state of a polyanionic macromolecule or an immunostimulatory substance relative to a core particle and a VLP, respectively. As used herein, the term "packaging" includes covalent binding, such as chemical coupling; or non-covalent binding, such as ionic interactions, hydrophobic interactions, hydrogen bonds, etc. The term also includes the enclosure or partial enclosure of polyanionic macromolecules. Therefore, polyanionic macromolecules or immunostimulatory substances can be enclosed by VLPs in the absence of actual binding, especially in the absence of covalent binding. In a preferred embodiment, at least one polyanionic macromolecule or immunostimulatory substance is packaged inside the VLP, most preferably in a non-covalent manner. In the case where the immunostimulatory substance is a nucleic acid (preferably DNA), the term packaging means that the nucleic acid is inaccessible to nuclease hydrolysis, preferably inaccessible to deoxyribonuclease hydrolysis (such as deoxyribonuclease I or omnipotent nuclease), wherein preferably, the accessibility is analyzed as described in Examples 11-17 of WO2003 / 024481A2.

[0097] Effective amount: As used herein, the term "effective amount" refers to an amount necessary to achieve a desired biological effect or an amount sufficient to achieve 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 amounts can be routinely determined by those skilled in the art. The effective amount may vary depending on the specific composition administered and the size of the individual. A person of ordinary skill can determine the effective amount of a specific composition of the present invention empirically without undue experimentation. Preferably, the term "effective amount" refers to an amount that (i) treats or prevents a specific disease or condition described herein; (ii) alleviates, improves or eliminates one or more symptoms of a specific disease or condition described herein; or (iii) prevents or delays the onset of one or more symptoms of a specific disease or condition described herein.

[0098] Animal: As used herein and as an individual of the composition of the present invention to which the VLPs of the modified CMV are administered, the term "animal" refers to a non-human animal, 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), or primates. Preferably, the individual is a non-human mammal (such as a dog, cat, horse, sheep, cattle, or pig). In a preferred embodiment, the individual is a non-human mammal selected from a dog, cat, horse, sheep, cattle, or pig.

[0099] Veterinary composition: As used herein, the term "veterinary composition" refers to a composition suitable for use in non-human animals.

[0100] Treatment: As used herein, the term "treatment / treat / treated / treating" refers to prevention and / or treatment. In one embodiment, the term "treatment / treat / treated / treating" refers to therapeutic treatment. In another embodiment, the term "treatment / treat / treated / treating" refers to preventive and therapeutic treatment. Preferably, the beneficial or desired clinical results of the treatment include, but are not limited to, relief of symptoms, reduction in the extent of the disease or condition, stabilization of the disease or condition state (i.e., no worsening), delay or slowing of the progression of the disease or condition, improvement or alleviation of the disease or condition state.

[0101] In a first aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0102] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0103] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39; and

[0104] (ii) a polypeptide comprising, 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, wherein the polypeptide is inserted between any amino acid residues corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39,

[0105] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0106] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0107] Therefore, in another aspect, the present invention provides a composition comprising

[0108] (a) A modified CMV VLP comprising at least one first attachment site;

[0109] (b) at least one nerve growth factor (NGF) antigen, wherein the antigen comprises at least one second binding site;

[0110] Wherein the VLP of the modified CMV comprises at least one chimeric CMV polypeptide, and wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the following polypeptide:

[0111] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39; and

[0112] (ii) a polypeptide comprising, 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 corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39; and

[0113] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0114] In a preferred embodiment, the chimeric CMV polypeptide further comprises a helper T cell epitope, wherein preferably, the helper T cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein more preferably, the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 39, and wherein even more preferably, the helper T cell epitope is derived from tetanus toxin or is a PADRE sequence, wherein very preferably, the Th cell epitope comprises, even more preferably consists of, the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In another very preferred embodiment, the CMV polypeptide is a CMV coat protein 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 with SEQ ID NO: 39.

[0115] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0116] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0117] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39; and

[0118] (ii) a polypeptide comprising, 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 corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39, and

[0119] (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; and

[0120] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0121] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0122] In another very preferred embodiment, the stretch of consecutive negatively charged amino acids comprises, preferably consists of, SEQ ID NO:1 or SEQ ID NO:2.

[0123] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0124] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0125] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 39, wherein preferably, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 95% sequence identity with SEQ ID NO: 39; and

[0126] (ii) a polypeptide comprising, 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 corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39, and wherein the stretch of consecutive negatively charged amino acids comprises, preferably consists of, SEQ ID NO: 1 or SEQ ID NO: 2;

[0127] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0128] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0129] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0130] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0131] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 39, wherein preferably, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 95% sequence identity with SEQ ID NO: 39;

[0132] (ii) a polypeptide comprising, 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 corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39, and wherein the stretch of consecutive negatively charged amino acids comprises, preferably consists of, SEQ ID NO: 1 or SEQ ID NO: 2; and

[0133] (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; and

[0134] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0135] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0136] 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 wherein the first amino acid linker and the second amino acid linker are independently selected from: (a.) a polyglycine linker (G linker) having an amino acid sequence (Gly) with a length of n=2-10 n (b.) a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably, the GS linker has (GS) r (G s S) t (GS) uAn amino acid sequence of wherein r=0 or 1, s=1-5, t=1-5 and u=0 or 1; 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 wherein the first amino acid linker and the second amino acid linker are independently selected from a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein the GS linker has (GS) r (G s S) t (GS) u In another very preferred embodiment, the first amino acid linker comprises, preferably consists of, SEQ ID NO: 8. In another very preferred embodiment, the second amino acid linker comprises, preferably consists of, SEQ ID NO: 4 or SEQ ID NO: 9. In another very preferred embodiment, the polypeptide comprises, preferably consists of, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In another very preferred embodiment, the polypeptide consists of, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In another very preferred embodiment, the polypeptide comprises, preferably consists of, a stretch of consecutive negatively charged amino acids, inserted between the amino acid residues corresponding to the amino acid residues at position 84 and position 85 of SEQ ID NO: 39 of the CMV polypeptide.

[0137] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0138] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0139] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 39, wherein preferably, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 95% sequence identity with SEQ ID NO: 39; and

[0140] (ii) a polypeptide comprising, 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 of the CMV polypeptide corresponding to any amino acid residue between position 75 and position 85 of SEQ ID NO:39, and wherein the polypeptide comprises, preferably consists of SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and wherein preferably the polypeptide is inserted between the amino acid residues of the CMV polypeptide corresponding to the amino acid residues at position 84 and position 85 of SEQ ID NO:39; and

[0141] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0142] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0143] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0144] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0145] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 39, wherein preferably, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 95% sequence identity with SEQ ID NO: 39;

[0146] (ii) a polypeptide comprising, 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 of the CMV polypeptide corresponding to any amino acid residue between position 75 and position 85 of SEQ ID NO:39, and wherein the polypeptide comprises, preferably consists of SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and wherein preferably the polypeptide is inserted between the amino acid residues of the CMV polypeptide corresponding to the amino acid residues at position 84 and position 85 of SEQ ID NO:39;

[0147] (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; and

[0148] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0149] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0150] In another very preferred embodiment, the CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein 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.

[0151] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0152] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0153] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39;

[0154] (ii) a polypeptide comprising 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 corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39; and

[0155] wherein the CMV polypeptide comprises, 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

[0156] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0157] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0158] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0159] (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of:

[0160] (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39;

[0161] (ii) a polypeptide comprising 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 corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO: 39;

[0162] (iii) a helper T cell epitope, wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide; and

[0163] wherein the CMV polypeptide comprises, 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

[0164] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0165] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0166] The embodiments, preferred embodiments and very preferred embodiments described and disclosed herein shall apply to all aspects and other embodiments, preferred embodiments and / or very preferred embodiments, regardless of whether they are specifically mentioned again or whether repetition thereof is avoided for the sake of brevity.

[0167] In a preferred embodiment, the CMV polypeptide comprises, preferably consists of, an amino acid sequence of a CMV coat protein or a mutated amino acid sequence, wherein the mutated amino acid sequence shows at least 90%, preferably at least 91%, 92%, 93, 94% or 95%, more preferably at least 96%, 97% or 98% and again more preferably at least 99% sequence identity with the CMV coat protein; wherein preferably, the mutated amino acid sequence differs from the amino acid sequence to be mutated in at least one and at most 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues, and wherein more preferably, the differences are selected from (i) insertions, (ii) deletions, (iii) amino acid exchanges, and (iv) any combination of (i) to (iii).

[0168] In a preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 92% sequence identity with 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 with 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 with 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.

[0169] In a preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 92% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 93% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 39. In another preferred embodiment, the CMV polypeptide consists of a CMV coat protein or an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 39.

[0170] 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 with SEQ ID NO:39. In a very preferred embodiment, the CMV polypeptide is a CMV coat protein or an amino acid sequence having at least 90%, preferably 95% sequence identity with SEQ ID NO:39. In a preferred embodiment, the CMV polypeptide is a CMV coat protein having 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, wherein the CMV coat protein comprises SEQID NO:39. In a preferred embodiment, the CMV polypeptide comprises a CMV coat protein, wherein the CMV coat protein consists of SEQID NO:39. In a preferred embodiment, the CMV polypeptide consists of a CMV coat protein, wherein the CMV coat protein consists of SEQ ID NO:39.

[0171] 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 with 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 with 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 with 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 with 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 with 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 with 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 99% sequence identity with SEQ ID NO: 40.

[0172] In a preferred embodiment, the CMV polypeptide comprises or preferably consists of: (i) an amino acid sequence of a CMV coat protein, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO: 39; or (ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 39; and wherein the amino acid sequence as defined in (i) or (ii) comprises SEQ ID NO: 40 or an amino acid sequence region, wherein the amino acid sequence region has at least 90% sequence identity with SEQ ID NO: 40. In a preferred embodiment, the CMV polypeptide comprises or preferably consists of: (i) an amino acid sequence of a CMV coat protein, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO: 39; or (ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 39; and wherein the amino acid sequence as defined in (i) or (ii) comprises SEQ ID NO: 40 or an amino acid sequence region, wherein the amino acid sequence region has at least 95% sequence identity with SEQ ID NO: 40. In a preferred embodiment, the CMV polypeptide comprises or preferably consists of: (i) an amino acid sequence of a CMV coat protein, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO:39; or (ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO:39; and wherein the amino acid sequence as defined in (i) or (ii) comprises SEQ ID NO:40.

[0173] In a preferred embodiment, the number of amino acid substitutions in the N-terminal region is equal to or lower than the number of amino acids consisting of the helper T cell epitope. In a preferred embodiment, the N-terminal substitution region of the CMV polypeptide consists of 5 to 15 consecutive amino acids. In a preferred embodiment, the N-terminal substitution region of the CMV polypeptide consists of 9 to 14 consecutive amino acids. In a preferred embodiment, the N-terminal substitution 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 up to 20 amino acids.

[0174] In a preferred embodiment of the present invention, the Th cell epitope is selected from TT 830-843 (SEQ ID NO: 41), PADRE (SEQ ID NO: 42), HA307-319 (SEQ ID NO: 43), HBVnc 50-69 (SEQ ID NO: 44), CS378-398 (SEQ ID NO: 45), MT 17-31 (SEQ ID NO: 46) and TT 947-967 (SEQ ID NO: 47). In a preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin or a 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 a PADRE sequence. In a preferred embodiment, the Th cell epitope comprises the amino acid sequence SEQ ID NO: 41 or SEQ ID NO: 42. In a very preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:42. In a very 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 very preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO:42. In a very preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO:42.

[0175] In a preferred embodiment, the CMV polypeptide comprises or preferably consists of the amino acid sequence of the CMV coat protein, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO:39 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:39; and wherein the amino acid sequence comprises SEQ ID NO:40, and wherein the helper T cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein the N-terminal substituted region of the CMV polypeptide consists of 11 to 13 consecutive amino acids, preferably consists of 11 consecutive amino acids, and wherein 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, preferably consists of the amino acid sequence SEQ ID NO:5, wherein the polypeptide is inserted between any amino acid residues of the CMV polypeptide corresponding to any amino acid residue between position 75 and position 85 of SEQ ID NO:39. In another preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between any amino acid residues corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide of SEQ ID NO:39.

[0176] In a preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 and less than 12 amino acids. In a preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 10 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 9 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 8 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 9 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 8 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4, 5, 6, 7 or 8 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 or 8 amino acids. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 amino acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids has a length of 4 amino acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids has a length of 5 amino acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids has a length of 6 amino acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids has a length of 7 amino acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids has a length of 8 amino acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids has a length of 9 amino acids.

[0177] In another preferred embodiment, the stretch of consecutive negatively charged amino acids is independently selected from aspartic acid or glutamic acid, wherein the aspartic acid or the glutamic acid is independently selected from its L-configuration or its D-configuration in each case. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one aspartic acid in L-configuration or D-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one aspartic acid in L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one aspartic acid in D-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one glutamic acid in L-configuration or D-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one glutamic acid in L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one glutamic acid in D-configuration.

[0178] In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one aspartic acid in L-configuration and at least one glutamic acid in L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids consists of aspartic acid and glutamic acid both in L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids consists of aspartic acid or glutamic acid both in L-configuration.

[0179] In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least one aspartic acid or at least one glutamic acid. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least two aspartic acids or at least two glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least three aspartic acids or at least three glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four aspartic acids or at least four glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four aspartic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least five glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least six glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least seven glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least eight glutamic acids. In another preferred embodiment, the stretch of consecutive negatively charged amino acids consists only of aspartic acid. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids consists only of glutamic acid.

[0180] In another preferred embodiment, the stretch of consecutive 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 L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least two aspartic acids or at least two glutamic acids, wherein at least two aspartic acids or at least two glutamic acids are in L-configuration. In another preferred embodiment, the stretch of consecutive 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 L-configuration. In another preferred embodiment, the stretch of consecutive 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 L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four aspartic acids, wherein the at least four aspartic acids are in L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least four glutamic acids, wherein the at least four glutamic acids are in L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least five glutamic acids, wherein the at least five glutamic acids are in the L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least six glutamic acids, wherein the at least six glutamic acids are in the L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least seven glutamic acids, wherein the at least seven glutamic acids are in the L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids comprises at least eight glutamic acids, wherein the at least eight glutamic acids are in the L-configuration. In another preferred embodiment, the stretch of consecutive negatively charged amino acids consists only of aspartic acids, wherein the aspartic acids are in the L-configuration. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids consists only of glutamic acids, wherein the glutamic acids are in the L-configuration.

[0181] In a preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 10 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In a preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 9 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 9 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4, 5, 6, 7 or 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4, 5, 6, 7 or 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 or 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 5 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 6 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 7 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 9 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid.

[0182] In a preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 10 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 9 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 to 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 9 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4, 5, 6, 7 or 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 to 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4, 5, 6, 7 or 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 or 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 3 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 4 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 5 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 6 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration.In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 7 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 8 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration. In another preferred embodiment, the stretch of continuous negatively charged amino acids has a length of 9 amino acids, wherein the stretch of continuous negatively charged amino acids consists only of glutamic acid, wherein the glutamic acid is in the L-configuration.

[0183] In another very preferred embodiment, the stretch of consecutive negatively charged amino acids comprises SEQ ID NO: 1 or SEQ ID NO: 2. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids consists of SEQ ID NO: 1 or SEQ ID NO: 2. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids comprises SEQ ID NO: 1. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids consists of SEQ ID NO: 1. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids comprises SEQ ID NO: 2. In another very preferred embodiment, the stretch of consecutive negatively charged amino acids consists of SEQ ID NO: 2.

[0184] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker, wherein the first amino acid linker is located 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, wherein the first amino acid linker is located 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, wherein the first amino acid linker is located 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 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.

[0185] In a preferred embodiment, the first amino acid linker has a length of at most 30 amino acids. In a preferred embodiment, the first amino acid linker has a length of at most 20, 19, 18, 17 or 16 amino acids. In a preferred embodiment, the first amino acid linker has a length of at most 15 amino acids. In a preferred embodiment, the first amino acid linker has at most 14 amino acids. In a preferred embodiment, the first amino acid linker has at most 13 amino acids. In a preferred embodiment, the first amino acid linker has at most 12 amino acids. In a preferred embodiment, the first amino acid linker has at most 11 amino acids. In a preferred embodiment, the first amino acid linker has at most 10 amino acids. In a preferred embodiment, the first amino acid linker has at most 9 amino acids. In a preferred embodiment, the first amino acid linker has at most 8 amino acids. In a preferred embodiment, the first amino acid linker has at most 7 amino acids. In a preferred embodiment, the first amino acid linker has at most 6 amino acids. In a preferred embodiment, the first amino acid linker has at most 5 amino acids. In a preferred embodiment, the first amino acid linker has at most 4 amino acids. In a preferred embodiment, the first amino acid linker has at most 3 amino acids. In a preferred embodiment, the first amino acid linker has at most 2 amino acids. In a preferred embodiment, the first amino acid linker consists of one amino acid. In a preferred embodiment, the second amino acid linker has a length of at most 30 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 20, 19, 18, 17 or 16 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 15 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 14 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 13 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 12 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 11 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 10 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 9 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 8 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 7 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 6 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 5 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 4 amino acids. In a preferred embodiment, the second amino acid linker has a length of at most 3 amino acids.In a preferred embodiment, the second amino acid linker has a length of at most 2 amino acids.In a preferred embodiment, the second amino acid linker consists of one amino acid.

[0186] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a first amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the stretch of consecutive negatively charged amino acids, and wherein the first amino acid linker is selected from: (a.) a polyglycine linker (Gly) with a length of n=2-10 n (b.) a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably, the GS linker has (GS) r (G s S) t (GS) u An amino acid sequence of , wherein r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; 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.

[0187] In a preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids further comprises a second amino acid linker, wherein the second amino acid linker is located at the N-terminus or C-terminus of the stretch of consecutive negatively charged amino acids, and wherein the second amino acid linker is selected from: (a.) a polyglycine linker (Gly) with a length of n=2-10; n (b.) a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably, the GS linker has (GS) r (G s S) t (GS) u An amino acid sequence of wherein r=0 or 1, s=1-5, t=1-5 and u=0 or 1; 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 wherein the first and second amino acid linkers are independently selected from: (a.) a polyglycine linker (G linker) having an amino acid sequence (Gly) with a length of n=2-10 n(b.) a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably, the GS linker has (GS) r (G s S) t (GS) u An amino acid sequence of , wherein r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; 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.

[0188] 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 wherein the first and second amino acid linkers are independently selected from a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein the GS linker has (GS) r (G s S) t (GS) u An amino acid sequence of , wherein r=0 or 1, s=1-5, t=1-5 and u=0 or 1; and 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.

[0189] In a preferred embodiment, the first amino acid linker is a polyglycine linker (Gly) with a length of n=2-10. n In a preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker) comprising 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) comprising at least one glycine and at least one serine, and wherein the first amino acid linker has Gly-Ser at its N-terminus. In another preferred embodiment, the GS linker has (GS) r (G s S) t (GS) u An amino acid sequence of wherein r=0 or 1, s=1-5, t=1-5 and u=0 or 1. In another preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker) having (GS) r (G s S) t (GS) uIn another preferred embodiment, the GS linker has a length of at most 15, 14, 13, 12, 11, preferably 10, 9, 8, 7 amino acids, and more preferably at most 6 amino acids. In another preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker), and the GS linker has the amino acid sequence SEQ ID NO: 8. In another preferred embodiment, the first amino acid linker has the amino acid sequence SEQ ID NO: 8. In a preferred embodiment, the first 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.

[0190] In a preferred embodiment, the second amino acid linker is a polyglycine linker (Gly) with a length of n=2-10. 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 wherein the second amino acid linker has Gly-Ser at its N-terminus. In another preferred embodiment, the second amino acid linker is a glycine-serine linker (GS linker) having (GS) r (G s S) t (GS) u In another preferred embodiment, the GS linker has a length of at most 15, 14, 13, 12, 11, preferably 10, 9, 8, 7 amino acids, and more preferably at most 6 amino acids. In another preferred embodiment, the second amino acid linker is a glycine-serine linker (GS linker), and the GS linker has the amino acid sequence SEQ ID NO: 9.

[0191] 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 an amino acid linker (GS* linker) comprising at least one Gly, at least one Ser and at least Cys, and the second amino acid linker has Gly-Ser at its N-terminus. In another preferred embodiment, the second amino acid linker (GS* linker) has a length of at most 15, 14, 13, 12, 11, preferably 10, 9 amino acids, and more preferably a length of at most 7 or 6 amino acids. In another preferred embodiment, the second amino acid linker is an amino acid linker (GS* linker), and the GS* linker has the amino acid sequence SEQ ID NO:4.

[0192] In a preferred embodiment, the first and second amino acid linkers are independently polyglycine linkers (Gly) with a length of n=2-10. n In a preferred embodiment, the first and the second amino acid linkers are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine. In a preferred embodiment, the first and the second amino acid linkers are independently amino acid linkers (GS* linkers) comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys and Cys, and wherein the second amino acid linker has Gly-Ser at its N-terminus. In another preferred embodiment, the GS linker has (GS) r (G s S) t (GS) u An amino acid sequence of wherein r=0 or 1, s=1-5, t=1-5 and u=0 or 1. In another preferred embodiment, the first and second amino acid linkers are independently glycine-serine linkers (GS linkers) having (GS) r (G s S) t (GS) u An amino acid sequence wherein r=0 or 1, s=2, 3 or 4, t=1, 2 or 3, and u=0 or 1.

[0193] In another preferred embodiment, the first amino acid linker and / or the second amino acid linker comprises, preferably consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 9. In another very preferred embodiment, the first amino acid linker comprises, preferably consists of SEQ ID NO: 8. In another very preferred embodiment, the second amino acid linker comprises, preferably consists of SEQ ID NO: 4 or SEQ ID NO: 9. In another very preferred embodiment, the second amino acid linker comprises, preferably consists of SEQ ID NO: 4. In another very preferred embodiment, the second amino acid linker comprises, preferably consists of SEQ ID NO: 9. In another very 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 another very 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. In another very 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:9.

[0194] In a preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of consecutive negatively charged amino acids has a length of at most 30 amino acids. In a preferred embodiment, the polypeptide has a length of at most 25, 24, 23, 22 or 21 amino acids. In a preferred embodiment, the polypeptide has a length of at most 20 amino acids. In a preferred embodiment, the polypeptide has a length of at most 19 amino acids. In a preferred embodiment, the polypeptide has a length of at most 18 amino acids. In a preferred embodiment, the polypeptide has a length of at most 17 amino acids. In a preferred embodiment, the polypeptide has a length of at most 16 amino acids. In a preferred embodiment, the polypeptide has a length of at most 15 amino acids. In a preferred embodiment, the polypeptide has a length of at most 14 amino acids. In a preferred embodiment, the polypeptide has a length of at most 13 amino acids. In a preferred embodiment, the polypeptide has a length of at most 12 amino acids. In a preferred embodiment, the polypeptide has a length of at most 11 amino acids. In a preferred embodiment, the polypeptide has a length of at most 10 amino acids. In a preferred embodiment, the polypeptide has a length of at most 9 amino acids. In a preferred embodiment, the polypeptide has a length of at most 8 amino acids. In a preferred embodiment, the polypeptide has a length of at most 7 amino acids. In a preferred embodiment, the polypeptide has a length of at most 6 amino acids. In a preferred embodiment, the polypeptide has a length of at most 5 amino acids. In a preferred embodiment, the polypeptide has a length of at most 4 amino acids. In another very preferred embodiment, the polypeptide consists of this stretch of consecutive negatively charged amino acids.

[0195] In another very preferred embodiment, the polypeptide comprises SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51. In another very preferred embodiment, the polypeptide consists of SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51. In another very preferred embodiment, the polypeptide comprises SEQ ID NO:49. In another very preferred embodiment, the polypeptide comprises SEQ ID NO:50. In another very preferred embodiment, the polypeptide comprises SEQ ID NO:51. In another very preferred embodiment, the polypeptide consists of SEQ ID NO:49. In another very preferred embodiment, the polypeptide consists of SEQ ID NO:50. In another very preferred embodiment, the polypeptide consists of SEQ ID NO:51.

[0196] In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between any amino acid residues corresponding to any amino acid residue between position 75 and position 85 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 75 and position 76 of the CMV polypeptide. In another very preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 76 and position 77 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 77 and position 78 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 78 and position 79 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 79 and position 80 of SEQ ID NO:39 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 80 and position 81 of SEQ ID NO:39 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 75 and position 81 of SEQ ID NO:82 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 82 and position 83 of SEQ ID NO:39 of the CMV polypeptide. In another preferred embodiment, the polypeptide comprising, preferably consisting of a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 83 and position 84 of SEQ ID NO:39 of the CMV polypeptide. In another very preferred embodiment, the polypeptide comprising, preferably consisting of, a stretch of consecutive negatively charged amino acids is inserted between the amino acid residues corresponding to the amino acid residues at position 84 and position 85 of SEQ ID NO:39.

[0197] In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between the amino acid residues at positions 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between the amino acid residues at positions 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between the amino acid residues at positions 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein 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. In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at positions 86 and 87 of SEQ ID NO:48. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at position 86 and position 87 of SEQ ID NO:48.

[0198] In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between the amino acid residues at positions 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between the amino acid residues at positions 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between the amino acid residues at positions 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein 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. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at positions 86 and 87 of SEQ ID NO:48. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at position 86 and position 87 of SEQ ID NO:48.

[0199] In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 and 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 first and second amino acid linkers, wherein the first and the second amino acid linkers are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine 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* linkers), wherein the first and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0200] In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 and 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 first and second amino acid linkers, wherein the first and the second amino acid linkers are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine 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* linkers), wherein the first and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0201] In a very 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 very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a very 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 very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0202] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0203] (a) a modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12;

[0204] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0205] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one non-peptide covalent bond.

[0206] In an embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a very 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 very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12. In a very preferred embodiment, the VLP of the modified CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In a very preferred embodiment, the modified CMV VLP comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the modified CMV VLP comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the modified CMV VLP comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 12.

[0207] The VLPs of the modified CMV of the present invention can be expressed in a prokaryotic or eukaryotic expression system. Preferred systems are Escherichia coli, yeast, insect cells and mammalian cell lines. Very preferably, the VLPs of the modified CMV are obtained by expressing a chimeric CMV polypeptide in Escherichia coli, and wherein preferably, the expression is carried out at a temperature between 10°C and 25°C, preferably at a temperature of 20°C. As indicated above, the recombinantly produced polypeptide may include an N-terminal methionine residue. In one embodiment, the chimeric CMV polypeptide therefore includes an N-terminal methionine residue. However, typically and preferably, the chimeric CMV polypeptide is cut at the N-terminal methionine residue.

[0208] In another preferred embodiment, the VLP of this modified CMV further comprises at least one immunostimulating substance. In a very preferred embodiment, this immunostimulating substance is encapsulated in the modified VLP of the present invention. In another preferred embodiment, immunostimulating substance is mixed with modified VLP of the present invention. It is well known in the art and especially disclosed in WO2003 / 024481 to be suitable for immunostimulating substances of the present invention.

[0209] In another embodiment of the present invention, the immunostimulatory substance consists of DNA or RNA of non-eukaryotic origin. In another preferred embodiment, the immunostimulatory substance is selected from: (a) immunostimulatory nucleic acid; (b) peptidoglycan; (c) lipopolysaccharide; (d) lipoteichoic acid; (e) imidazoquinoline compound; (f) flagellin; (g) lipoprotein; and (h) any mixture of at least one substance in (a) to (g). In another preferred embodiment, the immunostimulatory substance is an immunostimulatory nucleic acid, wherein the immunostimulatory nucleic acid is selected from: (a) ribonucleic acid; (b) deoxyribonucleic acid; (c) chimeric nucleic acid; and (d) any mixture of (a), (b) and / or (c). In another preferred embodiment, the immunostimulatory nucleic acid is a ribonucleic acid, and wherein the ribonucleic acid is a bacterial RNA. In another preferred embodiment, the immunostimulatory nucleic acid is poly (IC) or a derivative thereof. In another preferred embodiment, the immunostimulatory nucleic acid is a deoxyribonucleic acid, wherein the deoxyribonucleic acid is an oligonucleotide containing unmethylated CpG.

[0210] In a very preferred embodiment, the immunostimulatory substance is an unmethylated CpG-containing oligonucleotide. In another preferred embodiment, the unmethylated CpG-containing oligonucleotide is an A-type CpG. In another preferred embodiment, the A-type CpG comprises a palindromic sequence. In another preferred embodiment, the palindromic sequence is flanked by guanosine entities at its 5' end and 3' end. In another preferred embodiment, the palindromic sequence is flanked by at least 3 and at most 15 guanosine entities at its 5' end, and wherein the palindromic sequence is flanked by at least 3 and at most 15 guanosine entities at its 3' end.

[0211] In another preferred embodiment, the immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, and wherein preferably, the unmethylated CpG-containing oligonucleotide comprises a palindromic sequence, and wherein more preferably, the CpG motif of the unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, and wherein again more preferably, the palindromic sequence is SEQ ID NO: 52. In another 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 entirely of phosphodiester-bonded nucleotides.

[0212] In another aspect, the present invention provides a composition comprising (a) a modified CMV VLP as defined herein, wherein the modified CMV VLP comprises at least one first attachment site; and (b) at least one NGF antigen, wherein the antigen comprises at least one second attachment site; wherein (a) and (b) are connected via the at least one first and the at least one second attachment site, typically and preferably via at least one covalent non-peptide bond. Methods for connecting the modified VLP to the antigen via the first and the second attachment sites are described in, for example, WO2002 / 056905, WO2004 / 084940, and WO2016 / 062720.

[0213] Therefore, in another aspect, the present invention provides a composition comprising (a) a modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site; and (b) at least one NGF antigen, wherein the antigen comprises at least one second attachment site; wherein (a) and (b) are linked via the at least one first attachment site and the at least one second attachment site, typically and preferably via at least one covalent non-peptide bond, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of: (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO:39; and (ii) a polypeptide comprising, 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 of the CMV polypeptide corresponding to any amino acid residue between position 75 and position 85 of SEQID NO:39.

[0214] In a very preferred embodiment, the at least one first connection site does not constitute or is not part of a polypeptide comprising the stretch of continuous negatively charged amino acids. In a very preferred embodiment, all of the first connection sites do not constitute or are not part of a polypeptide comprising the stretch of continuous negatively charged amino acids. In a very preferred embodiment, the at least one first connection site does not constitute or is not part of a polypeptide comprising the stretch of continuous negatively charged amino acids. In a very preferred embodiment, all of the first connection sites do not constitute or are not part of a continuous negatively charged amino acids. In a very preferred embodiment, the first connection site is connected to the second connection site only via one or more covalent bonds. In a very preferred embodiment, the at least one antigen is connected to the VLP of the modified CMV only via one or more covalent bonds. In a very preferred embodiment, all of the antigens are connected to the VLP of the modified CMV only via one or more covalent bonds.

[0215] In another preferred embodiment, the first attachment site is linked to the second attachment site via at least one covalent non-peptide bond. In another preferred embodiment, all of the first attachment sites are linked to the second attachment sites via at least one covalent non-peptide bond. In another very preferred embodiment, the first attachment site is an amino group, preferably an amino group of lysine. In another very preferred embodiment, all of the first attachment sites are amino groups, preferably amino groups of lysine.

[0216] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0217] (a) a modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12;

[0218] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0219] Wherein (a) and (b) are linked via the at least one first linking site and the at least one second linking site, via at least one covalent non-peptide bond; and wherein the at least one first linking site does not constitute or is not part of a polypeptide comprising the stretch of continuous negatively charged amino acids; and wherein preferably, the first linking site is an amino group, preferably an amino group of lysine, and wherein more preferably, the second linking site is a thiol group, preferably a thiol group of a cysteine ​​residue or a thiol group that has been chemically linked to the NGF antigen.

[0220] In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a very 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 very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0221] The connection between the modified virus-like particles and the antigen by a disulfide bond is typically unstable, especially for molecules containing a sulfhydryl moiety, and in addition, the stability in serum is lower than, for example, a thioether connection (Martin FJ. and Papahadjopoulos D. (1982) J. Biol. Chem. 257: 286-288). Therefore, in another very preferred embodiment of the present invention, the association or connection of the VLP of the modified CMV with at least one antigen does not contain a disulfide bond. Here, more preferably, at least one second connection site contains or is preferably a sulfhydryl group. Preferably, all of the second connection sites contain or are preferably sulfhydryl groups. In another preferred embodiment, the at least one first connection site is not or does not contain a sulfhydryl group. In another preferred embodiment, all of the first connection sites are not or do not contain a sulfhydryl group. In a preferred embodiment, the at least one first connection site is not or does not contain a sulfhydryl group of cysteine. In a preferred embodiment, all of the first connection sites are not or do not contain a sulfhydryl group of cysteine. In another very preferred embodiment, the second connection site is a sulfhydryl group, preferably a sulfhydryl group of cysteine. In another very preferred embodiment, all of said second attachment sites are sulfhydryl groups, preferably sulfhydryl groups of cysteine.

[0222] In a very preferred embodiment, at least one first attachment site is an amino group, preferably an amino group of a lysine residue, and at least one second attachment site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue or a sulfhydryl group that has been chemically linked to an antigen. In a very preferred embodiment, all of the first attachment sites are amino groups, preferably amino groups of lysine residues, and the second attachment sites are sulfhydryl groups, preferably sulfhydryl groups of cysteine ​​residues or a sulfhydryl group that has been chemically linked to an antigen. In another preferred embodiment, only one of the second attachment sites associates with the first attachment site via at least one non-peptide covalent bond, so that the combination of the antigen and the modified CMV VLP is achieved in a single and uniform type, wherein the only second attachment site that associates with the first attachment site is a sulfhydryl group, and wherein the antigen and the modified CMV VLP interact via the association to form an ordered and repeated antigen array.

[0223] In a preferred embodiment of the present invention, the antigen is connected to the VLP of the modified CMV by chemical cross-linking, typically and preferably using a heterobifunctional cross-linking agent. Therefore, in a preferred embodiment, the NGF antigen is connected to the VLP of the modified CMV via the at least one first connection site and the at least one second connection site, via at least one covalent non-peptide bond, by chemical cross-linking, typically and preferably by a heterobifunctional cross-linking agent. In a preferred embodiment, the heterobifunctional cross-linking agent contains a functional group that can react with the preferred first connection site of the VLP of the modified CMV (preferably with an amino group, more preferably with an amino group of a lysine residue), and can react with the preferred second connection site (i.e., a thiol group, preferably a thiol group of a cysteine ​​residue inherent to the antigen, or a thiol group artificially added to the antigen) and optionally also another functional group for reduction reaction. Several heterobifunctional cross-linking agents are known in the art. These cross-linking agents include the preferred cross-linking agent 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 cross-linking agents available from, for example, Pierce Chemical Company and having a functional group reactive toward amino groups and a functional group reactive toward sulfhydryl groups. After reacting with amino groups and achieving thioether linkage with sulfhydryl groups, the above cross-linking agents all cause amide bond formation. In a very preferred embodiment, the heterobifunctional cross-linking agent is SMPH. Therefore, in a preferred embodiment, the NGF antigen is linked to the VLP of the modified CMV by chemical cross-linking, typically and preferably by a heterobifunctional cross-linking agent, via the at least one first linking site and the at least one second linking site, via at least one covalent non-peptide bond, and wherein the heterobifunctional cross-linking agent is SMPH. Another class of cross-linking agents suitable for practicing the present invention is characterized by the introduction of a disulfide bond between the antigen and the modified VLP after coupling. Preferred cross-linking agents belonging to this class include, for example, SPDP and sulfo-LC-SPDP (Pierce).

[0224] Therefore, in another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0225] (a) a modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12;

[0226] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0227] Wherein (a) and (b) are linked via a heterobifunctional cross-linker, via the at least one first attachment site and the at least one second attachment site, via at least one covalent non-peptide bond, wherein the heterobifunctional cross-linker is SMPH, and wherein the at least one first attachment site does not constitute or is not part of a polypeptide comprising the stretch of continuous negatively charged amino acids, and wherein the at least one first attachment site is an amino group, preferably an amino group of lysine, and wherein the at least one second attachment site is a thiol group, preferably a thiol group of a cysteine ​​residue or a thiol group that has been chemically linked to an NGF antigen.

[0228] In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a very 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 very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0229] Using a heterobifunctional cross-linking agent to connect the antigen to the VLP of modified CMV allows the antigen to be connected to the VLP of modified CMV in a directional manner. Other methods for connecting the antigen to the VLP of modified CMV include using carbodiimide EDC and NHS to cross-link the antigen to the VLP of modified CMV. The antigen may also be first thiolated via the reaction with SATA, SATP or iminothiolane. After the antigen removes the protecting group when necessary, it can then be coupled with the VLP of modified CMV as follows. After separating the excess thiolation agent, the antigen is reacted with the VLP of modified CMV, and the VLP of the modified CMV is activated in advance by a heterobifunctional cross-linking agent comprising a cysteine ​​reactive part and therefore presents at least one or several functional groups reactive to cysteine ​​residues, which can react with the thiolated antigen, such as described above. A small amount of reducing agent is optionally included in the reaction mixture. In other methods, antigens are linked to the VLPs of modified CMV using homobifunctional cross-linkers such as glutaraldehyde, DSG, BM[PEO]4, BS3 (Pierce), or other known homobifunctional cross-linkers in which the functional groups are reactive toward the amino or carboxyl groups of the modified VLPs.

[0230] In a very preferred embodiment of the present invention, the antigen is linked to a lysine residue of the VLP of a modified CMV via a cysteine ​​residue added to the N-terminus or C-terminus of the antigen or a native cysteine ​​residue within the antigen. In a preferred embodiment, the composition of the present invention further comprises a linker, wherein the linker associates the antigen with the second attachment site, and wherein preferably, the linker comprises or alternatively consists of the second attachment site.

[0231] Engineering the second attachment site to the antigen is achieved by the association of a linker, which typically and preferably contains at least one amino acid suitable for use as the second attachment site, according to the disclosure of the present invention. Therefore, in a preferred embodiment of the present invention, the linker is associated with the antigen by at least one covalent bond, preferably by at least one, preferably one, peptide bond. Preferably, the linker comprises or consists of the second attachment site. In another preferred embodiment, the linker comprises a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue. In another preferred embodiment, the linker comprises or is a cysteine ​​residue. In another preferred embodiment of the present invention, the linker consists of amino acids, wherein more preferably, the linker consists of up to 15 amino acids. In another preferred embodiment of the present invention, such amino acid linkers contain 1 to 10 amino acids.

[0232] In yet another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0233] (a) a modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10;

[0234] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and

[0235] Wherein (a) and (b) are linked by a heterobifunctional cross-linker, via the at least one first linking site and the at least one second linking site, via at least one covalent non-peptide bond, wherein the heterobifunctional cross-linker is SMPH, and wherein the at least one first linking site does not constitute or is not part of a polypeptide comprising the stretch of continuous negatively charged amino acids, and wherein the at least one first linking site is an amino group, preferably an amino group of lysine, and wherein the at least one second linking site is a thiol group, preferably a thiol group that has been chemically linked to the NGF antigen.

[0236] In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the VLP of the modified CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the VLP of the modified CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10.

[0237] In another preferred embodiment, the antigen is nerve growth factor (NGF), which is selected from human NGF, canine NGF (cNGF), feline NGF (fNGF), horse NGF (eNGF), bovine NGF (bNGF) and porcine NGF (pNGF), preferably selected from canine NGF (cNGF) or feline NGF (fNGF), and 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 one 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 with any one 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 another preferred embodiment, the NGF antigen comprises a polyhistidine tag of at least two consecutive and at most 12 consecutive histidine residues, which tag is preferably located at the C-terminus or N-terminus of the NGF antigen. In another 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, which tag is preferably located at the C-terminus or N-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 4, 6, 8 or 10 consecutive histidine residues, which tag is preferably located at the C-terminus or N-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 4 consecutive histidine residues, which tag is preferably located at the C-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 4 consecutive histidine residues, which tag is preferably located at the N-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 6 consecutive histidine residues consisting of SEQ ID NO: 34, which tag is preferably located at the C-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 6 consecutive histidine residues consisting of SEQ ID NO: 34, and the tag is preferably located at the N-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 8 consecutive histidine residues, and the tag is preferably located at the C-terminus of the NGF antigen.In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 8 consecutive histidine residues, which is preferably located at the N-terminus of the NGF antigen. In another preferred embodiment, the NGF antigen comprises a polyhistidine tag of 10 consecutive histidine residues, which is preferably located at the C-terminus or N-terminus of the NGF antigen.

[0238] In an alternative embodiment, the antigen is human NGF. In another 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% sequence identity and even more preferably at least 98% amino acid sequence identity to SEQ ID NO:54. In another embodiment, the antigen comprises SEQ ID NO:54. In another embodiment, the antigen consists of SEQ ID NO:54.

[0239] In another very preferred embodiment, the antigen is canine NGF. In a very 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% sequence identity and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In another preferred embodiment, the antigen comprises SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In another preferred embodiment, the antigen consists of SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In another very 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% sequence identity and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 30. In another very preferred embodiment, the antigen comprises SEQ ID NO: 30. In another very preferred embodiment, the antigen consists of SEQ ID NO: 30. In another 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 92%, more preferably at least 95% sequence identity and even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 31. In another very preferred embodiment, the antigen comprises SEQ ID NO: 31. In another very preferred embodiment, the antigen consists of SEQ ID NO: 31. In another 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 92%, more preferably at least 95% sequence identity and even more preferably at least 98% amino acid sequence identity to SEQ ID NO: 33. In another very preferred embodiment, the antigen comprises SEQ ID NO: 33. In another very preferred embodiment, the antigen consists of SEQ ID NO: 33.

[0240] In another very 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% sequence identity and even more preferably at least 98% amino acid sequence identity with SEQ ID NO:30, and wherein the NGF antigen further comprises a polyhistidine tag consisting of at least two 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, which tag 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 another very 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% sequence identity and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 30, and wherein 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 tag 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 another very 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% sequence identity and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 30, and wherein the NGF antigen further comprises a polyhistidine tag of 6 consecutive histidine residues consisting of SEQ ID NO: 34, which tag 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 another very 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% sequence identity and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 30, and wherein the NGF antigen further comprises a polyhistidine tag consisting of SEQ ID NO: 34, 6 consecutive histidine residues located at the N-terminus of the NGF antigen. In another very preferred embodiment, the antigen comprises SEQ ID NO: 30, and wherein the NGF antigen further comprises a polyhistidine tag of at least two consecutive and at most 12 consecutive histidine residues, preferably 4, 6, 8 or 10 consecutive histidine residues, more preferably 6 consecutive histidine residues, which tag consists of SEQ ID NO: 34 and 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 another very preferred embodiment, the antigen comprises SEQ ID NO:30, and wherein the NGF antigen further comprises a polyhistidine tag of 6 consecutive histidine residues, which tag consists of SEQ ID NO:34 and 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 another very preferred embodiment, the antigen comprises SEQ ID NO:30, and wherein the NGF antigen further comprises a polyhistidine tag of 6 consecutive histidine residues, which tag consists of SEQ ID NO:34 and is located at the N-terminus of the NGF antigen. In another very preferred embodiment, the antigen consists of SEQ ID NO:30.

[0241] In another very preferred embodiment, the antigen is feline NGF. In another very 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 92%, at least 93% or at least 94%, more preferably at least 95%, at least 96% or at least 97% sequence identity and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO:55. In another very preferred embodiment, the antigen comprises SEQ ID NO:55. In another very preferred embodiment, the antigen consists of SEQ ID NO:55.

[0242] In another preferred embodiment, the antigen is horse NGF. In another very 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 92%, at least 93% or at least 94%, more preferably at least 95%, at least 96% or at least 97% sequence identity and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO:56. In another very preferred embodiment, the antigen comprises SEQ ID NO:56. In another very preferred embodiment, the antigen consists of SEQ ID NO:56.

[0243] In another preferred embodiment, the antigen is bovine NGF. In another very 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 92%, at least 93% or at least 94%, more preferably at least 95%, at least 96% or at least 97% sequence identity and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO:57. In another very preferred embodiment, the antigen comprises SEQ ID NO:57. In another very preferred embodiment, the antigen consists of SEQ ID NO:57.

[0244] In another very preferred embodiment, the antigen is porcine NGF. In another very 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 92%, at least 93% or at least 94%, more preferably at least 95%, at least 96% or at least 97% sequence identity and even more preferably at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 58. In another very preferred embodiment, the antigen comprises SEQ ID NO: 58. In another very preferred embodiment, the antigen consists of SEQ ID NO: 58.

[0245] Without being bound, we believe that for antigens with higher isoelectric points and thus for antigens that will have an overall positive charge under conditions for binding, it is particularly possible to reduce and avoid unwanted aggregation and aggregate formation of VLPs of conjugated CMV. Therefore, in a preferred embodiment, the NGF antigen has an isoelectric point higher than 6.5. In a preferred embodiment, the NGF antigen has an isoelectric point higher than 6.5 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point greater than 6.5 as determined by the ExPASy Compute pI / MW tool described by Gasteiger et al. (Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, MR, Appel, RD and Bairoch, A., Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (edd): The Proteomics Protocols Handbook, Humana Press (2005)). 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, as determined by the ExPASy Compute pI / MW tool described by Gasteiger et al. (Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, MR, Appel, RD and 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 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 pI / 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 pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.0. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or 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 equal to or greater than 7.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.0 and less than 13.0, preferably less than 12.5 and more preferably less than 12.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.1, 7.2, 7.3 or 7.4. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.1, 7.2, 7.3 or 7.4 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 equal to or greater than 7.1, 7.2, 7.3 or 7.4, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.1, 7.2, 7.3 or 7.4 and less than 13.0, preferably less than 12.5 and more preferably less than 12.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.5. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 7.5 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 7.5, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 7.5 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 7.6, 7.7, 7.8 or 7.9. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 7.6, 7.7, 7.8 or 7.9 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0.In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.6, 7.7, 7.8 or 7.9, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 7.6, 7.7, 7.8 or 7.9 and less than 13.0, preferably less than 12.5 and more preferably less than 12.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 8.0. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 8.0 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 equal to or greater than 8.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 8.0 and less than 13.0, preferably less than 12.5 and more preferably less than 12.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 8.1, 8.2, 8.3 or 8.4. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or greater than 8.1, 8.2, 8.3 or 8.4 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 equal to or greater than 8.1, 8.2, 8.3 or 8.4, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 8.1, 8.2, 8.3 or 8.4 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 8.5. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 8.5 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 8.5, as determined by the ExPASy Compute pI / MW tool. In a preferred embodiment, the NGF antigen has an isoelectric point equal to or higher than 8.5 and lower than 13.0, preferably lower than 12.5 and more preferably lower than 12.0, as determined by the ExPASy Compute pI / MW tool.

[0246] In a very preferred embodiment, the polypeptide comprising the stretch of consecutive negatively charged amino acids comprises SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In another very preferred embodiment, the polypeptide consists of SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In another very preferred embodiment, the polypeptide comprises SEQ ID NO: 49. In another very preferred embodiment, the polypeptide comprises SEQ ID NO: 50. In another very preferred embodiment, the polypeptide comprises SEQ ID NO: 51. In another very preferred embodiment, the polypeptide consists of SEQ ID NO: 49. In another very preferred embodiment, the polypeptide consists of SEQ ID NO: 50. In another very preferred embodiment, the polypeptide consists of SEQ ID NO: 51.

[0247] In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between the amino acid residues at positions 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between the amino acid residues at positions 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between the amino acid residues at positions 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein 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. In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at positions 86 and 87 of SEQ ID NO:48. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at position 86 and position 87 of SEQ ID NO:48.

[0248] In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein the polypeptide comprising the stretch of consecutive negatively charged amino acids is inserted between the amino acid residues at positions 88 (Ser) and 89 (Thr) of SEQ ID NO:5, between the amino acid residues at positions 84 (Ser) and 85 (Thr) of SEQ ID NO:39, or between the amino acid residues at positions 86 (Ser) and 87 (Thr) of SEQ ID NO:48. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:39 or SEQ ID NO:48, wherein 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. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at positions 86 and 87 of SEQ ID NO:48. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is inserted between the amino acid residues at positions 88 and 89 of SEQ ID NO:5. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:39, wherein the polypeptide is inserted between the amino acid residues at positions 84 and 85 of SEQ ID NO:39. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO:48, wherein the polypeptide is inserted between the amino acid residues at position 86 and position 87 of SEQ ID NO:48.

[0249] In a very preferred embodiment, the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 and 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 first and second amino acid linkers, wherein the first and the second amino acid linkers are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine 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* linkers), wherein the first and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0250] In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 and 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 first and second amino acid linkers, wherein the first and the second amino acid linkers are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine 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* linkers), wherein the first and / or the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0251] In a very 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 very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In a very 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 very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0252] In a very preferred embodiment, the modified CMV VLP 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 very preferred embodiment, the modified CMV VLP comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the modified CMV VLP comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the modified CMV VLP comprises 180 copies of the chimeric CMV polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In a very preferred embodiment, the modified CMV VLP 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 very preferred embodiment, the modified CMV VLP comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the modified CMV VLP comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 11. In a very preferred embodiment, the modified CMV VLP comprises 180 copies of the chimeric CMV polypeptide consisting of the amino acid sequence of SEQ ID NO: 12.

[0253] In another very preferred embodiment, the antigen is canine NGF. In a very 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% sequence identity and even more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO:30 or SEQ ID NO:31 or SEQ ID NO:33.

[0254] In yet another aspect, the present invention provides a composition, preferably a veterinary composition, comprising

[0255] (a) a modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10;

[0256] (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and wherein 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% sequence identity and even more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO:30 or SEQ ID NO:31 or SEQ ID NO:33; and

[0257] Wherein (a) and (b) are linked by a heterobifunctional cross-linker, via the at least one first linking site and the at least one second linking site, via at least one covalent non-peptide bond, wherein the heterobifunctional cross-linker is SMPH, and wherein the at least one first linking site does not constitute or is not part of a polypeptide comprising the stretch of continuous negatively charged amino acids, and wherein the at least one first linking site is an amino group, preferably an amino group of lysine, and wherein the at least one second linking site is a thiol group, preferably a thiol group that has been chemically linked to the NGF antigen.

[0258] In a very preferred embodiment, the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the VLP of the modified CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10. In a very preferred embodiment, the VLP of the modified CMV comprises 180 identical chimeric CMV polypeptides, wherein the chimeric CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 10.

[0259] In another preferred embodiment, the antigen comprises SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In another preferred embodiment, the antigen consists of SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33. In another very 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% sequence identity and even more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO: 30. In another very preferred embodiment, the antigen comprises SEQ ID NO: 30. In another very preferred embodiment, the antigen consists of SEQ ID NO: 30. In another 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% sequence identity and 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 another very preferred embodiment, the antigen comprises SEQ ID NO:31. In another very preferred embodiment, the antigen consists of SEQ ID NO:31. In another very preferred embodiment, the antigen consists of SEQ ID NO:33. In another 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% sequence identity and 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 another very preferred embodiment, the antigen comprises SEQ ID NO:33. In another very preferred embodiment, the antigen consists of SEQ ID NO:33.In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, 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% sequence identity and still more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 33, and preferably, all of the first attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids. In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence SEQ ID NO: 10, 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% sequence identity and still more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO: 30, and preferably, all of the first attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids. In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence SEQ ID NO:11, 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% sequence identity and still more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO:30, and preferably, all of the first attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids.In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence SEQ ID NO: 10, 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% sequence identity and still 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 attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids. In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence SEQ ID NO: 11, 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% sequence identity and still 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 attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids. In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence SEQ ID NO: 12, 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% sequence identity and still more preferably at least 96%, 97% or at least 98% or at least 99% amino acid sequence identity with SEQ ID NO: 30, and preferably, all of the first attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids. In a very preferred embodiment, the VLP of the modified CMV comprises at least one copy, preferably 180 copies, of the chimeric CMV polypeptide comprising the amino acid sequence SEQ ID NO: 12, 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% sequence identity and still 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 attachment sites do not constitute or are not part of a polypeptide comprising the stretch of consecutive negatively charged amino acids.

[0260] 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. Very preferably, the modified CMV VLPs or the CMV VLPs are obtained by expressing the chimeric CMV polypeptide in E. coli, and wherein preferably, the expression is achieved at a temperature between 10°C and 35°C.

[0261] Thus, in another aspect, the present invention provides a composition comprising (a) a modified cucumber mosaic virus (CMV) virus-like particle (VLP) comprising at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of: (i) a CMV polypeptide, wherein the CMV polypeptide comprises 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, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, wherein the polypeptide is inserted into the CMV polypeptide corresponding to SEQ ID NO: between any amino acid residues of any amino acid residues between position 75 and position 85 of NO:39; and (b) at least one antigen, wherein the antigen comprises at least one second attachment site, and wherein the antigen is nerve growth factor (NGF); and wherein (a) and (b) are connected via the at least one first attachment site and the at least one second attachment site, via at least one covalent non-peptide bond, and wherein the VLP of the modified CMV is obtained by expressing the chimeric CMV polypeptide in Escherichia coli, and wherein preferably, the expression is achieved at a temperature between 10°C and 35°C.

[0262] In another aspect, the present invention provides a method for producing a composition of the present invention, comprising purifying a modified cucumber mosaic virus (CMV) virus-like particle (VLP) from a recombinant bacterial host expressing the modified CMV VLP, wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of: (i) a CMV polypeptide, wherein the CMV polypeptide comprises 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, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, wherein the polypeptide is inserted into the CMV polypeptide corresponding to SEQ ID NO: NO:39, between any amino acid residue between position 75 and position 85; and wherein the method comprises the following steps: (a) lysing the bacterial host; (b) clarifying the lysate obtained by the lysis; (c) purifying the VLP of the modified CMV from the clarified lysate by anion exchange chromatography (AEX); wherein the steps are performed in a given order.

[0263] In a preferred embodiment, the composition comprises an adjuvant. Typical and preferred adjuvants are mineral salts (e.g., aluminum hydroxide, aluminum phosphate), microcrystalline tyrosine, emulsions, microparticles, saponins (Quil A), cytokines, immunopotentiators, microbial components / products, liposomes, complexes, and mucosal adjuvants, which are known and described in, for example, Adjuvant Compendium NIAID and VAC (nih.gov) or Aguilar et al. (Aguilar JC et al., 2007, Vaccine 25:3752-3762), Gerdts (Gerdts V, 2015, Berliner und Münchener Wochenschrift 128:456-463) and Pasquale et al. (Pasquale et al., 2015, Vaccines 3:320-343). In a preferred embodiment, the composition comprises an adjuvant, wherein the adjuvant is aluminum hydroxide. In another preferred embodiment, the composition lacks an adjuvant.

[0264] In another aspect, the present invention provides a vaccine comprising or alternatively consisting of: a composition of the present invention comprising the VLP of the modified CMV and at least one NGF antigen as described herein. Contemplated are vaccines 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, wherein the adjuvant is aluminum hydroxide. In another preferred embodiment, the vaccine lacks an adjuvant. In a preferred embodiment, the vaccine comprises an effective amount of the composition of the present invention.

[0265] In another 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 herein; 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 take a form containing salts, buffers, adjuvants, or other substances required 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.

[0266] Another aspect of the present invention is a method of immunization, which comprises administering to an animal a composition of the present invention as described herein, a vaccine of the present invention as described herein, or a pharmaceutical composition as described herein, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog. In a preferred embodiment, the method comprises administering to an animal a composition of the present invention as described herein, a vaccine of the present invention as described herein, or a pharmaceutical composition as described herein, wherein the animal is a dog or a cat, preferably, wherein the animal is a dog. In a preferred embodiment, the method comprises administering to the animal an effective amount of a composition of the present invention, the vaccine, or the pharmaceutical composition, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog.

[0267] In another aspect, the present invention provides a modified CMV VLP as described herein, a composition of the present invention as described herein, a vaccine of the present invention as described herein, or a pharmaceutical composition as described herein, for use in a method for immunizing an animal or a human, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog, wherein the method comprises administering to the animal an effective amount of the modified CMV VLP, the composition of the present invention, the vaccine, or the pharmaceutical composition, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog.

[0268] Another aspect of the present invention is a method for inducing an animal to produce neutralizing antibodies against NGF, which comprises administering a composition of the present invention as described herein, a vaccine of the present invention as described herein, or a pharmaceutical composition as described herein to the animal, wherein preferably, 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 present invention as described herein, a vaccine of the present invention as described herein, or a pharmaceutical composition as described herein to an animal, 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, the vaccine, or the pharmaceutical composition of the present invention to the animal, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog.

[0269] In another aspect, the present invention provides a modified CMV VLP as described herein, a composition of the present invention as described herein, a vaccine of the present invention as described herein, or a pharmaceutical composition as described herein, for use in a method for immunizing an animal, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog, wherein the method comprises administering an effective amount of the modified CMV VLP, the composition of the present invention, the vaccine, or the pharmaceutical composition to the animal, wherein preferably, the animal is a dog or a cat, more preferably, wherein the animal is a dog. Example

[0270] Example 1: Construction and production of surface charge-modified CMV VLPs

[0271] Different chimeric CMV polypeptides according to the invention are prepared and subsequently expressed to produce the modified CMV VLPs of the invention.

[0272] To this end, chimeric CMV polypeptides were prepared, in particular, different polypeptides comprising consecutive negatively charged amino acids, i.e., polypeptides consisting of 4, 8 or 12 glutamic acid residues ("E4" - SEQ ID NO: 1; "E8" - SEQ ID NO: 2; "E12" - SEQ ID NO: 3), so that the glutamic acid residues were inserted between the 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 comprises a helper T cell epitope derived from tetanus toxoid TT830 (SEQ ID NO: 6). The corresponding nucleic acid sequence encoding the modified CMV polypeptide CMV-Ntt830 (SEQ ID NO: 7) was prepared as described in Example 3 of WO2016 / 062720A1.

[0273] The prepared chimeric CMV polypeptide further comprises linkers flanking the introduced E4, E8 and E12 polypeptides at both ends. In detail, the prepared chimeric CMV polypeptide comprises a GGS linker or a GGGS linker (SEQ ID NO: 8) directly located at the N-terminus of the introduced E4, E8 and E12 polypeptides, and a GGGSGS linker (SEQ ID NO: 9) or a CGGGSGS linker (SEQ ID NO: 4) directly located at the C-terminus of the introduced E4, E8 and E12 polypeptides.

[0274] The resulting amino acid sequences of the prepared chimeric CMV polypeptides were named "CMV-Ntt830-E4", "CMV-Ntt830-E8", "CMV-Ntt830-E8*" and "CMV-Ntt830-E12" and had the following amino acid sequences:

[0275] "CMV-Ntt830-E4": SEQ ID NO:10;

[0276] "CMV-Ntt830-E8": SEQ ID NO:11;

[0277] "CMV-Ntt830-E8*": SEQ ID NO:12;

[0278] "CMV-Ntt830-E12": SEQ ID NO:13.

[0279] The corresponding nucleotide sequence of the preferred chimeric CMV polypeptide is as follows:

[0280] "CMV-Ntt830-E4": SEQ ID NO:14;

[0281] "CMV-Ntt830-E8": SEQ ID NO:15;

[0282] "CMV-Ntt830-E8*": SEQ ID NO:16;

[0283] "CMV-Ntt830-E12": SEQ ID NO:17.

[0284] First, a chimeric CMV polypeptide CMV-Ntt830-E8* was prepared. Here and in the first step, the E8 coding sequence including the flanking linkers was incorporated into the modified CMV using PCR mutagenesis. A PCR fragment encoding the E8 sequence including the flanking linkers and the 3' end fragment of the modified CMV was amplified using two-step PCR using the following oligonucleotides:

[0285] Forward: E8*-1F (SEQ ID NO: 18)

[0286] Forward: E8*-2F (SEQ ID NO: 19)

[0287] Reverse: CMcpR (SEQ ID NO: 20).

[0288] Therefore, the PCR reaction was performed using E8*-1F / CMcpR oligonucleotides and pET-CMV-Ntt830 plasmid as templates. Template pET-CMV-Ntt830 was prepared as described in Example 3 of WO2016 / 062720A1. After a second PCR using oligonucleotides E8*-2F / CMcpR and the PCR product obtained from the first PCR, the target PCR product was obtained. The resulting PCR product was cloned into the auxiliary vector pTZ57 (InsTAclone PCR Cloning Kit, Fermentas#K1214). The plasmid containing the PCR product was amplified in Escherichia coli XL1-Blue cells, and the plasmid DNA was purified and sequenced using the BigDye cycle sequencing kit and ABI Prism 3100 Gene Analyzer (Applied Biosystems). As a result, the auxiliary plasmid pTZ-CMV-E8* was obtained without PCR errors.

[0289] As a next step, the BamHI / HindIII fragment of pTZ-CMV-E8* was cloned back into the pET-CMV-Ntt830B helper vector using the same restriction sites to obtain the expression vector pET-CMVB2-Ntt-E8C ( Figure 1 ).

[0290] The auxiliary vector pET-CMV-Ntt830B is used to introduce a DNA sequence encoding a polypeptide comprising a continuous stretch of negatively charged amino acids into the corresponding CMV DNA sequence of CMV-Ntt830, and a sequence containing a BamHI site is introduced at the corresponding position for subsequent cloning. The nucleic acid sequence encoding CMV-Ntt830 was prepared as described in Example 3 of WO2016 / 062720A1 and corresponds to SEQ ID NO: 14 of WO2016 / 062720A1.

[0291] The BamHI site was introduced by two-step PCR mutagenesis using the oligonucleotides listed below and previously constructed pET-CMV-Ntt830 as template. As indicated, the template pET-CMV-Ntt830 was prepared as described in Example 3 of WO2016 / 062720A1.

[0292] 1st PCR: Forward-pET-90 primer (annealed to pET28a+) (SEQ ID NO: 21)

[0293] Reverse - RGSYrev (SEQ ID NO:22)

[0294] 2nd PCR forward - RGSYdir (SEQ ID NO: 23)

[0295] Reverse-CMV-AgeR (SEQ ID NO: 24)

[0296] After both PCR products were purified, the next PCR was performed to join the PCR fragments (5 cycles without primers, followed by 25 cycles with primers pET-90 and CMV-AgeR).

[0297] After amplifying the gene, the resulting PCR product was directly cloned into the pTZ57R / T vector (InsTAclone PCR Cloning Kit, Fermentas #K1214). Escherichia coli XL1-Blue cells were used as hosts for cloning and plasmid amplification.

[0298] To avoid RT-PCR errors, several pTZ57 plasmid clones containing the CMV-Ntt830 gene were sequenced using the BigDye cycle sequencing kit and the ABI Prism 3100 Gene Analyzer (Applied Biosystems). After sequencing, the pTZ-plasmid clones without sequence errors were cut using NcoI and AgeI enzymes, and the clones contained the CMV-Ntt830B gene via the introduced BamHI site. The fragments were then cloned into the NcoI / AgeI sites of pET-CMV-Ntt830 to obtain the auxiliary vector pET-CMV-Ntt830B.

[0299] 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:

[0300] 1) 3 g of biomass was suspended in 20 ml of 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, 5 mM mercaptoethanol, pH 9.0, and the suspension was treated with ultrasound (Hielscher sonicator UP200S, 16 min, amplitude 70%, cycle 0.5);

[0301] 2) Centrifuge the lysate at 11000 rpm for 20 minutes at +4°C;

[0302] 3) In a 35 ml tube, prepare a sucrose gradient (20-60%) in a buffer containing 50 mM sodium citrate, 5 mM sodium borate, 2 mM EDTA, 0.5% TX-100;

[0303] 4) Overlay 5 ml of VLP sample on the sucrose gradient;

[0304] 5) Centrifuge for 6 hours using a SW32 rotor Beckman (25,000 rpm at +18°C);

[0305] 6) Divide the contents of each gradient tube into 6 ml fractions and combine the corresponding fractions;

[0306] 7) Analyze the gradient fractions on SDS.

[0307] After sucrose gradient purification, SDS-PAGE analysis of VLPs revealed homogeneous CMV-Ntt830-E8* coat protein monomers ( Figure 2A ) and electron microscopy imaging showed intact VLPs ( Figure 2B ).

[0308] Chimeric CMV polypeptides CMV-Ntt830-E4, CMV-Ntt830-E8 and CMV-Ntt830-E12 were prepared accordingly and as follows. The first step was to incorporate the polyglutamic acid coding sequence including the flanking linkers into the modified CMV using PCR mutagenesis. The PCR fragment encoding the polyglutamic acid sequence including the flanking linkers and the 3' end fragment of the modified CMV was amplified by PCR using the following pairs of oligonucleotides and plasmid pET-CMVB2-Ntt-E8* as templates:

[0309] 1) Forward: E4-F (SEQ ID NO: 25)

[0310] Reverse: CMcpR (SEQ ID NO: 20);

[0311] 2) Forward: E8-F (SEQ ID NO: 26)

[0312] Reverse: CMcpR (SEQ ID NO: 20);

[0313] 3) Forward: E12-F (SEQ ID NO: 27)

[0314] Reverse: CMcpR (SEQ ID NO: 20).

[0315] The obtained PCR product was cloned into the auxiliary vector pTZ57 (InsTAclone PCR Cloning Kit, Fermentas #K1214). The plasmid containing the PCR product was amplified in E. coli XL1-Blue cells, and the plasmid DNA was purified and sequenced using the BigDye cycle sequencing kit and the ABI Prism 3100 Gene Analyzer (Applied Biosystems). Thus, the auxiliary plasmids pTZ-CMV-E4, pTZ-CMV-E8 and pTZ-CMV-E12 were obtained without PCR errors.

[0316] Subsequently, the 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, the expression vector pET-CMVB2-Ntt-E4 ( Figure 3 )、pET-CMVB2-Ntt-E8( Figure 4 ) and pET-CMVB2-Ntt-E12( Figure 5 The expression vector was 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. After sucrose gradient purification, SDS-PAGE analysis of the VLPs showed that all three polyglutamic acid constructs obtained nearly homogeneous CMV coat protein monomers ( Figure 6 , Figure 7 , Figure 8 However, agarose gel analysis showed that only CMV-Ntt830-E4 and CMV-Ntt830-E8 formed complete particles, while CMV-E12 did not form complete particles ( Figure 6 , Figure 7 , Figure 8 ). Electron microscopy imaging showed that CMV-Ntt830-E4 and CMV-Ntt830-E8 formed complete VLPs ( Fig. 9 , Fig.10 ).

[0317] Example 2: Improved stability of the surface charge-modified CMV VLPs of the present invention compared to the prior art CMV VLPs

[0318] Thermal stability

[0319] The enhanced thermal stability of the surface charge-modified CMV VLPs of the present invention was demonstrated by measuring the denaturation of prior art CMV-Ntt830 VLPs (prepared as described in Examples 3 and 4 of WO2016 / 062720A1) and the CMV-Ntt830-E4 VLPs of the present invention as the temperature increased and determining the corresponding melting temperatures.

[0320] For this purpose, a thermal shift assay involving temperature-induced denaturation and fluorescent dyes was used. Orange (Sigma, Saint Louis, USA). This dye is naturally quenched in solution, but as the VLPs denature at elevated temperatures, Orange interacts with exposed hydrophobic amino acids and the core and emits a fluorescent signal that is measured by fluorimetry. The melting peak curve and melting temperature are determined from the resulting melting curve (fluorescent signal versus temperature). A solution containing 0.5 mg / ml of sucrose density gradient purified (as described in Example 1 above) CMV-Ntt830 VLPs or CMV-Ntt830-E4 VLPs in 5 mM sodium phosphate, 2 mM EDTA pH 7.5 was analyzed using the real-time PCR system MJ Mini (Bio-Rad, Hercules, USA) using the DNA melting temperature determination program. The data were analyzed using Opticon monitoring software and the melting curves were processed under a four-parameter smoothing setting. Fig.11 The melting peak curves of purified CMV-Ntt830 VLP and CMV-Ntt830-E4 VLP are shown.

[0321] The corresponding melting temperatures were estimated to be 51° C. and 57° C., demonstrating that the thermal stability of the surface charge-modified CMV VLPs according to the present invention is enhanced compared to the prior art CMV-Ntt830 VLPs.

[0322] Ionic Strength / Salt Stability

[0323] Ionic strength plays an important role in capsid stability. Salt in solution interacts with charged residues on the coat protein and VLP surface, affecting the water shell and detrimental to hydrophobic exposure, and thus affecting overall VLP stability.

[0324] The relative stability of CMV-Ntt830 VLPs and CMV-Ntt830-E4 VLPs to NaCl was tested by incubating purified VLPs (0.5 mg / ml in 5 mM sodium phosphate, 2 mM EDTA pH 7.5) at room temperature at various NaCl concentrations. After 2 hours in the presence of 20 mM NaCl, CMV-Ntt830 VLPs became relatively unstable and formed large aggregates that were visible to the eye and visualized by native gel electrophoresis ( Fig.12 In contrast, CMV-Ntt830-E4 VLPs showed no evidence of aggregate formation even at NaCl concentrations as high as 0.4 M ( Fig.12 ).

[0325] The improved stability in higher salt solutions caused by the surface charge modification of the modified CMV VLPs of the present invention plays an important role in the processability of ion exchange chromatography as described in Example 3.

[0326] Example 3: Compared with the prior art CMV VLP, the purification potential of the surface charge modified CMV VLP of the present invention is improved

[0327] Sucrose gradient / buffered ultracentrifugation purification steps used in laboratory-scale CMV VLP manufacturing methods as described in the prior art (such as in Examples 2 to 4 of WO2016 / 062720A1) and for preparing modified CMV VLPs of the present invention as described in Example 1 above provide CMV VLPs of suitable yield and purity for subsequent conjugation, vaccine manufacturing and preclinical evaluation. However, this method cannot be used simply and cost-effectively to produce vaccines for commercial purposes.

[0328] Ion exchange chromatography (IEX) is typically easily scaled up and used in downstream processes for commercial production of biologics. It is based on reversible ionic interactions between charged molecules / macromolecules in solution and fixed oppositely charged chromatography resins. An example is anion exchange chromatography (AEX), where the stationary phase (resin) is a positively and negatively charged molecule, such as a bound protein. The interaction of the resin with the sample can be controlled by applying a counter ion (such as Cl - ) to destroy. IEX is often used in a bind / elute mode to provide rapid capture, high-resolution purification, and concentration of the desired sample. It can be used in the initial stages (e.g., after lysate clarification), in the middle stages, or at the end of the downstream process.

[0329] In order for IEX to effectively bind and elute CMV VLPs, CMV VLPs must be stable in the ionic environment encountered during the binding and elution stages. The charges on both the ion exchange resin and the elution salt contribute to the ionic environment.

[0330] Prior art CMV-Ntt830 VLPs as well as modified CMV VLPs of the present invention (such as CMV-Ntt830-E4, CMV-Ntt830-E8 and CMV-Ntt830-E8*) carry a net negative charge at about pH 9 and below, as indicated by their migration toward the positively charged electrode in NAGE. Therefore, anion exchange chromatography (AIX) is a technique that is expected to have worked for both CMV VLP particles.

[0331] However, this is not the case, because CMV-Ntt830 VLPs as described above in Example 2 are relatively unstable in solution in the presence of up to 20 mM NaCl and form aggregates that precipitate. In contrast, the modified CMV VLPs of the present invention, such as CMV-Ntt830-E4 VLPs, do not form aggregates at NaCl concentrations up to 0.4 M ( Fig.12 , Panel B). The improved stability in higher salt solutions resulting from surface charge modification of VLPs is essential for their tractability by ion exchange chromatography.

[0332] Improved anion exchange chromatography (AEX) purification

[0333] To test the processability of prior art CMV-Ntt830 VLPs by anion exchange chromatography (AEX), sucrose gradient purified VLPs were prepared as described in Examples 2 to 4 of WO2016 / 062720A1. 5 ml of CMV-Ntt830 VLPs (1 mg / ml) were buffer exchanged in 5 mM sodium borate pH 9 and loaded onto a 1.0 ml Macro-PrepDEAE 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 measured at 260 nm using a Nanodrop spectrophotometer for measuring protein, and native agarose gel electrophoresis (NAGE) was performed.

[0334] The resulting chromatograms plotted against protein elution and NaCl concentration for the corresponding fractions ( Fig.13, Panel A) shows that CMV-Ntt830 VLPs do not elute as a single peak, which is unique to AIX. Instead, CMV-Ntt830 VLPs elute in a broad, nonspecific manner during loading (at 0 M NaCl) and subsequent elution steps (at a range of NaCl concentrations, primarily 0.2 to 0.8 M). Crucially, after elution from the column, the fractions containing the VLPs are turbid and contain a significant proportion of VLP aggregates, as indicated by the presence of ethidium bromide-stained VLPs in the loading wells after NAGE ( Fig.13 , Panel B). The tendency of CMV-Ntt830 VLPs to aggregate and elute in a non-discrete manner precludes the ready use of this approach for scaled-up manufacturing.

[0335] In contrast, non-aggregated CMV-Ntt830-E4 VLPs can be easily purified from crude lysates using AEX. Clarified lysate prepared from E. coli expressing CMV-Ntt830-E4 VLPs (as described in Example 1) was loaded in 50 mM citrate, 5 mM borate buffer pH 9.0 onto 60 ml Fracto-DEAE (Merck) in an XK 26 / 20 column equilibrated with the same buffer and eluted using the same buffer by applying a 0 to 1.0 M continuous NaCl gradient. The eluate was monitored at A260 nm to measure protein and conductivity to monitor salt concentration. Clarified lysate, flow-through, and fractions were collected and subjected to NAGE and SDS-PAGE.

[0336] The obtained chromatograms, SDS-PAGE and NAGE analysis ( Fig.14 ) showed that CMV-Ntt830-E4 VLPs were not present in the flow-through and were completely bound to Fracto-DEAE. VLPs were subsequently eluted in a relatively narrow concentration range of 0.2 to 0.5 M NaCl. In addition, there was no evidence of VLP aggregates in the loading wells of the native agarose gel. Coomassie brilliant blue stained SDS-polyacrylamide gels showed that high purity VLP coat proteins were obtained from crude bacterial lysates.

[0337] Example 4: Cloning, expression and purification of recombinant mature NGF

[0338] Cloning of recombinant NGF

[0339] A cDNA construct (SEQ ID NO:28) was resynthesized and cloned into a pBHA vector (BIONEER), which consisted of a cat full-length NGF propeptide sequence, a canine mature NGF sequence, and a C-terminal glycine-cysteine-glycine motif. The canine NGF sequence was codon optimized. The resulting amino acid sequence of the cat full-length NGF propeptide is provided in SEQ ID NO:29 of the canine mature NGF sequence comprising SEQ ID NO:30. The canine mature NGF amino acid sequence connected to the C-terminal glycine-cysteine-glycine motif is provided in SEQ ID NO:31.

[0340] Similarly, a cDNA construct (SEQ ID NO: 32) was resynthesized and cloned into a pBHA vector (BIONEER) consisting of a cat full-length NGF propeptide sequence, a canine mature NGF sequence, a C-terminal glycine-cysteine-glycine motif, and a his tag. The included his tag did not achieve any effect for purification, but its presence increased the refolding efficiency in downstream processes. The resulting amino acid sequence is provided in SEQ ID NO: 33, which includes the canine mature NGF sequence of SEQ ID NO: 30 and a His6 tag (SEQ ID NO: 34).

[0341] The construct was subcloned into the expression vector by PCR. Briefly, NGF-pBHA plasmid was used as a template, where NGF forward primer (SEQ ID NO: 35) and NGF reverse primer (SEQ ID NO: 36) contained XbaI and HindIII sites, respectively.

[0342] The NGFPCR product was subjected to 1% agarose gel electrophoresis in TAE buffer and then the NGF fragment was extracted using the 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 in 1x FastDigest buffer at +37°C according to the manufacturer's protocol. The pET42a plasmid (Novagen) was digested in the same manner. The NGF and vector digested DNA fragments were analyzed by agarose gel electrophoresis and extracted as described above. The NGF fragment was ligated in the pET42a vector overnight at room temperature using T4 ligase according to the manufacturer's protocol.

[0343] The NGF-pET42a construct was transformed into chemically competent E. coli DH5α cells by heat shock method. The cells were suspended in 1 ml LB medium and incubated at +37°C with shaking for 1 hour and inoculated on LB agar containing 60 μg / ml kanamycin and incubated overnight at 37°C. Single colonies were inoculated in LB medium containing 30 μg / ml kanamycin and incubated overnight at +37°C with shaking. DNA was extracted from single clone cultures using GeneJet plasmid small scale purification kit (ThermoFisher Scientific) according to the manufacturer's protocol.

[0344] The correct sequence of the NGF constructs of SEQ ID NO: 28 and SEQ ID NO: 32 was confirmed by Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0345] Expression and purification of recombinant canine mature NGF

[0346] The NGF-pET42a plasmid was transformed into chemically competent E. coli BL21-DE3 (Sigma-Aldrich) cells. The cells were suspended in 1 ml LB medium and incubated at +37°C with shaking for 1 hour. The cells were plated on LB agar containing 60 μg / ml kanamycin and incubated overnight at 37°C. Several colonies of BL21-DE3 cells transformed with NGF-pET42 were inoculated in LB medium containing 30 μg / ml kanamycin and incubated overnight at 37°C and then added to 2x TY medium containing 30 μg / ml kanamycin and cultured at 37°C with shaking until an OD of 0.7 units was reached. 540nm Recombinant protein expression was induced by adding IPTG to a final concentration of 1 mM and the cells were cultured for another 4 hours at 37°C with shaking. The biomass was collected by centrifugation at 5000 g for 15 minutes, frozen and stored at -70°C.

[0347] 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 the cells were lysed by sonication using a UP200S (Hielscher) ultrasonic device. The sonicated product was centrifuged at 15 557 g for 40 minutes. The supernatant was discarded and the lysis buffer was added to the pellet, which was resuspended by sonication. The suspension was centrifuged at 15 557 g for 15 minutes and the supernatant was discarded again. This washing step was repeated three more times. Finally, the pellet was washed with 50% lysis buffer and 3.5 M urea. After resuspension and centrifugation, the pellet was dissolved with 8 M guanidine hydrochloride and 0.1 M dithiothreitol. The suspension was homogenized by sonication for 10 minutes and then centrifuged at 15 557 g for 25 minutes. The supernatant (containing dissolved denatured NGF) was collected and filtered using a 45 μm filter, then added dropwise to refolding buffer (0.75M L-arginine, 0.1M Tris, 1mM EDTA, 5mM reduced glutathione and 0.5mM oxidized glutathione pH 9.5) at 7°C with constant stirring until the final concentration was 5ml NGF solution / 100ml refolding buffer. After overnight incubation, the refolding solution was centrifuged at 10 000g for 10 minutes and the supernatant was collected and incubated at +7°C for one week. The solution was diluted three times with deionized water, warmed to room temperature and the pH was adjusted to 6.8 with . The solution was then centrifuged at 7 000g for 10 minutes at room temperature to remove the precipitate and loaded on a 5ml Capto S cation exchange column previously equilibrated with 50mM sodium phosphate buffer (pH 6.5). The protein was then eluted via a gradient of 0-1 M NaCl in 50 mM sodium phosphate buffer (pH 6.5). The eluted fractions were analyzed using SDS-PAGE and the fractions containing proNGF were pooled and concentrated to 2-3 mg / ml via ultrafiltration. The refolded proNGF was digested in-house for 4 hours at a 30:1 volume ratio with TrypZean (Sigma-Aldrich, catalog number T3449) trypsin solution. The reaction was terminated by adding PMSF to a final concentration of 1 mM and then loaded onto a Superdex 200 10 / 300 GL size exclusion column equilibrated with 0.5 M NaCl and 30 mM phosphate (pH 6.8). Fractions were collected and analyzed using SDS-PAGE ( Fig.15A ; The resulting amino acid sequence of the cDNA construct of SEQ ID NO:28 and the feline full-length NGF propeptide of SEQ ID NO:29 is shown) and the fractions containing mature NGF were mixed and concentrated to a concentration of 2 mg / ml by ultrafiltration.

[0348] The authenticity of canine recombinant mature NGF was confirmed using a bioassay, which showed that canine mature NGF had similar neurite-inducing activity as mouse mature NGF (commercially produced by R&D systems). Fig. 15B : The resulting amino acid sequence of the cDNA construct of SEQ ID NO:28 and the feline full-length NGF propeptide of SEQ ID NO:29 is shown); known functions of properly folded and biologically active mature NGF.

[0349] Example 5. Coupling of canine recombinant mature NGF and modified CMV VLP

[0350] Various NGF antigens containing canine mature NGF (SEQ ID NO: 30) were covalently linked to various modified CMV VLPs prepared as described above. The connection was achieved according to the method described in Schmitz N et al., J Exp Med (2009) 206: 1941-1955. In brief, 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 NHS esters that react with lysine on the surface of VLPs. The amount of SMPH added was approximately 5 times the molar excess over one VLP coat protein monomer. The cross-linker that did not react with the VLPs 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).

[0351] In detail, and for the coupling of the cNGF antigen with SEQ ID NO: 33 to CMV-Ntt830-E4 VLPs: Using a BCA protein assay kit (TFS, catalog number 23225) with a protein concentration of 7.43 mg / ml, a solution of CMV-Ntt830-E4 VLPs in 5mM NaHPO4 pH 7.5, 2mM EDTA pH 8.0 was diluted to a working concentration of 1.5 mg / ml in a 50 ml tube (Sarstedt, sterile, catalog number 62.559.001) with a sample volume of 3x44 ml, thus the total volume for derivatization was 132 ml. A 50 mM (19 mg / ml) SMPH solution in DMSO was prepared just before use.

[0352] To derivatize CMV-Ntt830-E4 VLPs with SMPH, 264 μl of 50 mM SMPH in DMSO was added to each of the three previously prepared tubes containing 44 ml of CMV-Ntt830-E4 VLPs. The mixture was vortexed for 5 seconds and incubated for 1 hour at room temperature. To remove excess SMPH, the mixture was further centrifuged at 3214 g for 7 minutes in an Eppendorf 5810R centrifuge on an Amicon-Ultra-15 100K unit (Merck-Millipore, catalog number UFC910024). By three more centrifugation runs, the buffer was exchanged with 5 mM NaHPO4 pH 7.5, 2 mM EDTA using the same parameters. After the last centrifugation run, the total volume was adjusted to 132 ml (same as before derivatization). The 260 nm UV absorption was measured and the concentration of the derivatized CMV-Ntt830-E4 VLPs was estimated to be 1.5 mg / ml.

[0353] Briefly, cNGF antigen is then added to VLPs at a molar ratio of about 0.5:1 to 1:1 (relative to the corresponding chimeric CMV polypeptide monomer, relative to the surface charge modified CMV VLPs previously derivatized with SMPH) while shaking at room temperature, typically for 3 hours. The engineered free cysteine ​​of the cNGF antigen reacts with the maleimide in the cross-linker SMPH bound to the VLPs to form a stable covalent linkage.

[0354] In detail, and for the coupling of cNGF antigen with SEQ ID NO: 33 with CMV-Ntt830-E4 VLPs: The coupling reaction was carried out in six 50 ml tubes (Sarstedt, sterile, catalog number 62.559.001). In each tube, 22 ml of derivatized CMV-Ntt830-E4 VLPs (1.5 mg / ml, 60 μM, relative to CMV monomer) were mixed with 3.82 ml of buffer-exchanged cNGF 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 with DSG Titertek (Flow Laboratories) at room temperature by tumbling rotation. Uncoupled cNGF was removed by gel filtration on a Superdex 200 column (running buffer 20 mM NaHPO4 pH 7.5, 2 mM EDTA). 10 ml of a solution containing cNGF-CMV-Ntt830-E4 VLPs was loaded onto a HiLoad 26 / 600 Superdex 200 preparative column equilibrated with 20 mM NaHPO4 pH 7.5, 2 mM EDTA. The fractions containing cNGF-CMV-Ntt830-E4 VLPs were mixed and filtered through a 0.2 μm filter (Sarstedt, catalog number 83.1826.001). The sample volume collected after gel filtration was 280 ml. The sample was further concentrated to 230 ml by 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 NaHPO4 pH 7.5, 2 mM EDTA buffer. The 260 nm UV absorbance was measured (A230 = 6.628 and A260 = 3.722).

[0355] In order to demonstrate the covalent conjugation of cNGF antigen to VLP, the coupling reaction was analyzed by SDS-PAGE. After chemical coupling of cNGF to CMV-Ntt830, CMV-Ntt830-E4, CMV-Ntt830-E8 and CMV-Ntt830-E8* VLP, a significant conjugation band was observed ( Fig.16A and Fig. 16B However, cNGF-CMV-Ntt VLPs formed large aggregates (1400-1700 nm) ( Fig. 16C ) and precipitated rapidly and completely from solution.

[0356] 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. Fig.16D , Fig.16E and Fig.16F ) and electron microscopy ( Figure 16G and Fig.16H ) analysis showed that the modified VLP conjugates did not aggregate and were stable in solution.

[0357] Example 6: Induction of neutralizing antibodies by immunization with various conjugates of the present invention coupled with canine mature NGF and modified CMV VLP

[0358] Immunization of mice

[0359] Balb / c mice were divided into two groups (n=4 / group). The first group was immunized with 150 μl canine mature NGF-CMV-Ntt830-E8*VLPs twice, 14 days apart, and the VLPs were formulated to 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*VLPs formulated to 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). Blood was obtained before each immunization and on days 21, 28, 35 and 42 after the first vaccination. Serum was prepared by centrifuging the blood samples in serum tubes at 10,000 x g for 10 minutes. The serum was stored at about -20°C until analysis.

[0360] Vaccination of dogs

[0361] Six male beagles (obtained from Marshall US) aged 22 to 26 months at the time of first administration were randomly divided into 2 groups (n=3 / group). The first group was immunized three times with 1.0 ml of cNGF-CMV-Ntt830-E8*VLPs prepared to a concentration of 250 μg / ml in sodium phosphate buffer pH 7.5. The second group was similarly immunized with cNGF-CMV-Ntt830-E8*VLPs prepared to a concentration of 250 μg / ml in sodium phosphate buffer pH 7.5 and 100 μg / ml Adjuvant (vivoGen vac-quil) treatment. 24 hours before the first (day 0), second (day 21) and third (day 42) immunization, blood samples were drawn from each animal using a disposable needle and syringe. Blood was also drawn on days 63, 84 and 105. 6 ml blood samples were collected in inert tubes and set aside at ambient temperature. After clot formation, the tubes were centrifuged and the serum was collected in inert tubes and stored at about -20°C until IgG purification and / or analysis was performed.

[0362] In another study, 10 adult beagles aged 9 months at the time of inclusion were divided into 2 groups. Immunization used cNGF-CMV-Ntt830-E4 VLPs containing the cNGF antigen of SEQ ID NO: 33. Therefore, the first group of 5 dogs was treated with 250 μg of cNGF-CMV-Ntt830-E4 VLPs per dose formulated with 1.7 mg of aluminum hydroxide, while the second group of 5 dogs was treated with 250 μg of cNGF-CMV-Ntt830-E4 VLPs per dose without aluminum hydroxide. Dogs were administered subcutaneously at two times on day 0 and day 21 of the study. Serum samples were also collected on days 42, 71, and 91 throughout the study.

[0363] Measurement of NGF and CMV-VLP-specific IgG antibodies

[0364] For mice and dogs immunized with cNGF-CMV-Ntt830-E8*VLPs, anti-NGF- and CMV-Ntt830-E8*-VLP-specific IgG antibodies were measured in the serum by ELISA. For dogs immunized with cNGF-CMV-Ntt830-E4VLPs, anti-NGF-specific IgG antibodies were measured in the serum by ELISA.

[0365] Maxisorp ELISA plates were coated with canine recombinant mature NGF protein or CMV-Ntt830-E8*-VLPs at a concentration of 1 μg / ml in 0.1 M sodium carbonate buffer pH 9.6 at 4°C overnight as detailed for immunization with cNGF-CMV-Ntt830-E8*VLPs. The plates were washed and SuperBlock was added for 2 hours at room temperature. TM(PBS) blocking buffer (ThermoFisher / Life Technologies Europe) and then washed again. Serum samples were pre-diluted 1:9 or 1:100 in PBS containing 2% BSA, 0.05% Tween 20, transferred to ELISA plates and diluted ten times 3-fold. After incubation for 2 hours at room temperature and washing, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG with Fcγ fragment specificity (Jackson ImmunoResearch Europe Ltd.) or HRP-labeled rabbit anti-canine IgG (H+L)-HRP (Jackson ImmunoResearch Europe Ltd.) was added, which were diluted 1:2000 or 1:2500 in PBS containing 2% BSA, 0.05% Tween-20 (PBS pH 7.4 (1x) Gibco). After incubation and washing, the samples were diluted 1:2000 or 1:2500 in PBS containing 2% BSA, 0.05% Tween-20 (PBS pH 7.4 (1x) Gibco). TM Colorimetric development was performed using the TMB substrate kit (Thermo Fisher / Life Technologies Europe). The enzyme 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 describes the reciprocal of the dilution that reaches half the maximum OD value.

[0366] Neutralization assay in PC12 cells.

[0367] The biological activity of recombinantly produced canine mature NGF was determined and the neutralizing capacity of antibodies induced by immunization of mice was evaluated using an in vitro assay that measures mature NGF-mediated neurite outgrowth in rat adrenal pheochromocytoma cell culture (PC-12). 5×10 4PC-12 cells were seeded in duplicate in 24-well tissue culture plates coated with type I collagen (Thermo Fisher / Life Technologies Europe) (10 μg / ml) in an assay medium containing RPMI 1640 (Sigma-Aldrich, Switzerland), 2 mM L-glutamine (Gibco), 2.4 g / L HEPES (AppliChem Germany GmbH), 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); and incubated overnight at 37° C., 5% CO 2 . The next day, the medium in the wells was replaced with assay medium (RPMI 1640, 1x A / A, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.5% FBS) containing mouse mature NGF (R&D, 1156-NG-100), human mature NGF (R&D, 256-GF-100 / CF) or recombinantly produced canine mature NGF at a final concentration of 12.5 ng / ml, as well as human mature NGF polyclonal antibody (R&D AF-256-NA), human mature NGF monoclonal antibody (R&D MAB256-500) or IgG purified from vaccinated mice. NGF was omitted from the negative control wells (starvation medium alone) and the antibody was omitted from the positive control wells (starvation medium containing 12.5 ng / ml NGF). After 5 days, the cells were stained with 0.05% w / v crystal violet solution and examined by microscopy. Bright field images of several fields were captured on an inverted microscope Leica DM IL LED (Leica Microsystems (UK) Ltd.), HIPLAN I 20x objective, using Q-Capture Pro 7 software. Cells with and without neurite outgrowth (defined as extension of cell body width) were counted and the proportion of neurite-positive cells was determined for each treatment.

[0368] Neutralization assay of TF-1 cells

[0369] The neutralizing capacity of sera from dogs immunized with cNGF-CMV-Ntt830-E8* VLPs and cNGF-CMV-Ntt830-E4 VLPs was determined using a bioactivity assay that involves measuring the proliferation of the TF-1 erythroblastoma cell line (American Type Culture Collection, ATCC, Manassas, VA).

[0370] For immunization with cNGF-CMV-Ntt830-E8* VLPs, TF-1 cells were collected, washed three times with PBS (PBS pH 7.4 (1×) Gibco) and incubated at 10 μg / ml in starvation medium (RPMI 1640 medium (ATCC modified) supplemented with heat-inactivated 10% FBS, 1× A / A 5 Culture the cells at a density of 10 cells per well overnight. 4 TF-1 cells were seeded in a total of 100 μl assay medium (RPMI without phenol red, containing 10% FBS, 2 mM GlutaMax (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, 25 μg / mL amphotericin B) per well of a flat-bottom 96-well plate.

[0371] To test the in vitro neutralizing activity of antibodies generated by immunization with cNGF-CMV-Ntt830-E8* VLPs, sera from immunized dogs were collected and purified using Invitrogen Dynabeads for mouse IgG purification. TM Protein G (Thermo Fisher / Life Technologies Europe) and Pierce Protein A magnetic beads for dogs (Thermo Fisher / Life Technologies Europe) were used to purify total IgG according to the manufacturer's instructions. The ability of purified total IgG to neutralize NGF bioactivity was tested by incubating a constant concentration of 5 ng / ml human mature NGF (R&D, 256-GF-100 / CF) with increasing concentrations of purified canine total IgG (625-20000 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 1 / 4-ounce canines that had been starved overnight. 4TF-1 cells and quantified cell proliferation during the last 24 hours of a total incubation time of 72 hours using a BrdU-based cell proliferation ELISA (Roche). The manufacturer's instructions were followed and color development was stopped using 5% sulfuric acid. The absorbance at 450 nm was measured using a reference wavelength of 690 nm.

[0372] The percentage of proliferation corresponding to each IgG dilution was calculated relative to the proliferation measured by serum-purified IgG collected at baseline before infection (day 0). Data are expressed as percentage proliferation versus IgG concentration. GraphPad Prism (version Windows 8.0.0, GraphPad Software, San Diego, California USA, www.graphpad.com) was used to fit the sigmoidal 4PL curve to determine the IgG concentration required for 50% inhibition of proliferation (50% neutralization titer NT50).

[0373] After immunization with cNGF-CMV-Ntt830-E4 VLPs, NGF neutralizing antibodies were determined in dogs as follows: TF-1 cells were collected and washed three times with PBS and then incubated at 2×10 5 The cells were resuspended at a cell density of 10 cells in starvation medium (RPMI without phenol red (Sigma), which contained 10% HI-FBS, 2mM GlutaMax (Gibco), 10mM HEPES (Sigma), 1mM sodium pyruvate (Sigma), 4500mg / L glucose (Gibco), 1500mg / L sodium bicarbonate, 100U / mL penicillin, 100μg / mL streptomycin, 25μg / mL amphotericin B (100x anti-Gibco)). Serum samples were heat inactivated at 56°C for 30 minutes, then diluted 1:25 in starvation medium (4 times the final concentration of 1:100) and 2-fold serial dilutions were performed. hNGF was diluted to 20ng / mL (4 times the final concentration of 5ng / mL) and 25μL was added to wells containing 25μL pre-diluted serum or 25μL starvation medium (positive control wells). Instead of hNGF, 50μL starvation medium was added to the negative control wells. The hNGF-serum / antibody mixture was incubated at room temperature for 1 hour. Serum-starved TF-1 cells were collected and plated at 1 × 10 cells per well in a flat-bottom 96-well plate. 4Add 50 μL of cell suspension at a cell density of 10 cells. The final sample volume per plate is 100 μL per well. The cell culture plates are incubated in a 5% CO2 cell incubator at +37°C for approximately 68 hours. Cell viability is quantified by Promega CellTiter 96Aqueous OneSolution Cell Proliferation Assay (Promega). Add 20 μL of CellTiter per well. AqueousOne Solution reagent. The culture plates were incubated in a humidified 5% CO2 incubator at +37°C for 7 hours. The absorbance at 490nm was recorded with 700nm as the reference wavelength. To determine the IC50 value, a setting curve was generated by plotting the OD values ​​of the serum samples against the dilution factor using GraphPad prism software (GraphPad Prism 8 and 9 for Windows, GraphPad Software, San Diego, California USA). The IC50 value was determined using a 4-PL regression curve fitting model, corresponding to the dilution factor of the half-maximum OD value. The serum titer of the sample at different time points was defined and depicted as the IC50 value of the curve fit.

[0374] result

[0375] Mouse experiments

[0376] For mice immunized with cNGF-CMV-Ntt830-E8*VLP and Quil A, cNGF-specific IgG antibodies were detected in sera collected from day 14 ( Fig.17A ). The antibody titer in the serum was measured to increase further on day 21 (7 days after the second injection on day 14). The high titer was maintained until the experiment was terminated on day 42. After two immunizations with cNGF-CMV-Ntt830-E8*VLP vaccine in the absence of adjuvant, NGF-specific IgG antibodies were detected in sera isolated from day 21. The co-administration of Quil A adjuvant has the effect of enhancing immunity and enhances the specific antibody response by about 10 times.

[0377] To test whether the anti-NGF IgG antibodies induced by immunization with cNGF-CMV-Ntt830-E8*VLPs are neutralizing, they were tested in a PC12-based bioassay in which NGF acts as a neurotrophic factor inducing differentiation and neurite outgrowth. IgG was purified from pooled sera collected before immunization (naive ms pIgG) and from pooled sera collected on days 21, 28, and 35 after cNGF-CMV-Ntt830-E8*VLP / QuilA boost immunization (ms pIgG NGFvacc). Fig. 17B It was shown that IgG purified from immune sera neutralized NGF, whereas IgG from naive mice did not neutralize NGF.

[0378] Dog Experiment

[0379] For animals that received cNGF-CMV-Ntt830-E8* VLPs but no adjuvant, the number of Fig.18A ) detected anti-NGF IgG titers were observed in serum collected from day 42 (week 3 after the second vaccine administration). The NGF-specific IgG titers in serum were highest on day 42 (week 3 after the second vaccine administration). After the third injection on day 42, the titers of all animals remained constantly high until day 63 and then gradually decreased. The magnitude of anti-CMV IgG titers was similar to those measured for canine mature NGF, but there were slight differences in the reaction kinetics ( Fig.18C ). Anti-CMV IgG antibodies were somewhat delayed and only clearly detectable from day 42 after the second immunization, with peak titers measured in sera on day 63 after the third immunization, followed by a decline in titers.

[0380] For animals immunized with cNGF-CMV-Ntt830-E8* VLPs in combination with adjuvant Quil A, anti-NGF IgG antibodies were first detected in the serum on day 21 after a single administration of the vaccine on day 0 ( Fig.18B The second and third doses of vaccine increased the titers in two of the three animals, with peak titers measured in sera collected on day 63. The third animal reached its peak titer on day 42, suggesting that the third dose of vaccine may not increase the antibody response. The kinetics and magnitude of anti-CMV IgG antibody titers were similar to those measured for canine mature NGF ( Fig.18D ).

[0381] For animals that received cNGF-CMV-Ntt830-E8* VLPs but no adjuvant, 4 of 5 study animals had positive results on day 21 ( Fig.18E ) Detectable anti-NGF IgG titers were observed in serum collected on day 42. The highest titer was observed on day 21 after the second dose was administered on day 42.

[0382] For animals immunized with cNGF-CMV-Ntt830-E4 VLPs in combination with aluminum hydroxide, anti-NGF IgG antibodies were detected in all animals at week 3 after a single vaccine administration on day 0 ( Fig.18F ). The second dose of vaccine increased the mean group titer.

[0383] The neutralizing capacity of anti-NGF IgG antibodies induced in response to vaccination with cNGF-CMV-Ntt830-E8*VLPs was analyzed using a bioassay based on NGF-mediated proliferation of TF-1 cells. IgG antibodies purified from immunized dogs inhibited the proliferation induced by mature NGF in a concentration-dependent manner, whereas IgG antibodies purified from pre-immune sera of the same animals did not ( Fig.19A Vaccination with cNGF-CMV-Ntt830-E8*VLP induced high neutralization titers, and co-administration of the vaccine with Quil A adjuvant further increased the neutralization titer ( Fig.19B This observation reflects the anti-NGF ELISA IgG titers in these dogs. A clear correlation was observed between anti-NGF titers and neutralization capacity ( Fig.19C IgG purified from sera with high vaccine-specific titers had increased potency with respect to inhibition of NGF-mediated TF-1 cell proliferation.

[0384] Vaccination with cNGF-CMV-Ntt830-E4 VLPs induces neutralizing anti-NGF antibody titers. On day 42, high levels of neutralizing anti-NGF antibodies ( Fig.19D ).

[0385] These results show that the conjugate of canine mature NGF coupled to the modified VLPs comprising chimeric CMV polypeptides according to the present invention can overcome immune tolerance to endogenous target antigens and induce NGF-specific IgG antibodies in dogs (target species). In addition, these antibodies can effectively neutralize canine mature NGF activity in vitro.

Claims

1. A composition, preferably a veterinary composition, comprising (a) A modified CMV VLP, wherein the modified CMV VLP comprises at least one first attachment site, and wherein the modified CMV VLP comprises at least one chimeric CMV polypeptide, wherein the at least one chimeric CMV polypeptide comprises, preferably consists of, the following polypeptide (i) a CMV polypeptide, wherein the CMV polypeptide comprises 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, wherein the negatively charged amino acids are independently selected from aspartic acid or glutamic acid, wherein the polypeptide is inserted between any amino acid residues of the CMV polypeptide corresponding to any amino acid residue between position 75 and position 85 of SEQ ID NO: 39, (b) at least one antigen, wherein the antigen comprises at least one second binding site, and wherein the antigen is nerve growth factor (NGF); and Wherein (a) and (b) are linked via at least one non-peptide covalent bond through the at least one first linking site and the at least one second linking site.

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 the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO:

39.

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

42.

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

39.

5. A composition as claimed in any one of the preceding claims, wherein the CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO:5, wherein 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.

6. A CMV composition as claimed in any one of the preceding claims, wherein the stretch of consecutive negatively charged amino acids has a length of 3 to 10 amino acids.

7. A CMV composition as claimed in any preceding claim, wherein the stretch of consecutive negatively charged amino acids consists only of glutamic acid.

8. The CMV composition of any one of the preceding claims, wherein 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 wherein the first amino acid linker and the second amino acid linker are independently selected from: (a.) A polyglycine linker (G linker) having an amino acid sequence (Gly) of length n=2-10 n ; (b.) A glycine-serine linker (GS linker), which comprises at least one glycine and at least one serine, wherein preferably, the GS linker has (GS) r (G s S) t (GS) u wherein r=0 or 1, s=1-5, t=1-5 and u=0 or 1; 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.

9. The composition according to any one of the preceding claims, 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, preferably consists of, the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

11. A composition as claimed in any one of the preceding claims, wherein the at least one first attachment site is not comprised in the polypeptide comprising the stretch of consecutive negatively charged amino acids, or is not a part of the polypeptide comprising the stretch of consecutive negatively charged amino acids.

12. A composition as claimed in any preceding claim, wherein the first attachment site is an amino group, preferably an amino group of a lysine residue, and wherein the at least one second attachment site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine ​​residue.

13. A composition as described in any of the preceding claims, wherein the antigen is selected from canine NGF (cNGF), feline NGF (fNGF), horse NGF (eNGF), bovine NGF (bNGF) and porcine NGF (pNGF), wherein preferably, the antigen is canine NGF (cNGF) or feline NGF (fNGF), and wherein more preferably, the antigen is canine NGF (cNGF).

14. A composition according to any one of the preceding claims, wherein the antigen comprises or preferably consists of: An amino acid sequence selected from any one 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 with any one 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. A composition according to any one of the preceding claims, wherein the antigen comprises or preferably consists of: An amino acid sequence selected from any one 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 with any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:

55.

16. A composition as claimed in any preceding claim for use in a method of inducing neutralising antibodies against NGF in an animal.

17. A 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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