Nucleic Acid Vaccines

Abstract

The present invention relates to nanoparticle-based modular compositions based on nucleic acids, such as DNA and RNA, that are particularly useful for the prevention and / or treatment of diseases and disorders.

Description

[Technical field]

[0001] The present invention relates to nanoparticle-based modular compositions based on nucleic acids, such as DNA and RNA, that are particularly useful for the prevention and / or treatment of diseases and disorders. [Background technology]

[0002] Vaccines remain the most effective means to prevent and control the spread of infectious diseases. Live attenuated vaccines are highly immunogenic and induce long-lived antibody responses even after a single vaccination. In contrast, modern subunit vaccines (i.e., based on soluble protein antigens) show high safety but are poorly immunogenic and fail to induce similar long-lasting antibody responses in humans. Thus, it is very difficult for simple subunit vaccines to induce strong and sustained immune responses against pathogens and disease-associated antigens.

[0003] However, the licensed human papillomavirus (HPV) vaccines (Cervarix®, Gardasil®, and Gardasil 9®) are an important exception, as they have immunogenicity comparable to live attenuated vaccines and can induce very strong and durable antibody responses in humans even with a single dose (Schiller et al., 2018, Schiller et al., 2012, De Vincenzo et al., 2014). Importantly, the vaccine is formed by the self-assembly of HPV major capsid protein into virus-like particles (VLPs), which are thought to be responsible for the high efficacy of the vaccine.

[0004] Indeed, many studies have established a strong causal relationship between the high immunogenicity of VLPs and their structural similarity to native viruses, and several strategies have been pursued to utilize VLPs as scaffolds for the presentation of heterologous antigens, including self-antigens. These studies collectively demonstrate that multivalent, repeated antigen display can indeed significantly increase the immunogenicity of antigens and can even induce long-lasting immunity.

[0005] We previously described the development of a VLP-based modular vaccine platform using a split protein (tag / catcher) conjugation technology to attach antigens to the surface of the VLP in a unidirectional, multivalent and iterative manner (WO 2016 / 112921). To date, this technology has become a very powerful and versatile vaccine platform.

[0006] In contrast to protein-based vaccines, DNA and / or RNA-based vaccines have a major advantage due to their simple and low-cost production, where the steps of recombinant expression and purification of the vaccine antigens are omitted. Indeed, batches of clinical vaccines can usually be generated already as soon as the antigen-encoding sequences become available. A further advantage is that the manufacturing process is highly scalable and cell-free. Furthermore, the production of several different vaccines in one facility requires only minimal adaptation of the manufacturing process to the specific vaccine formulation. Finally, these vaccines allow the in vivo expression of complex proteins that are difficult or impossible to produce recombinantly with current expression systems.

[0007] However, so far DNA vaccines have been shown to induce relatively weak immune responses to antigens when tested in humans and non-human primates, limiting their commercial application.

[0008] Therefore, there is an urgent need for vaccines that combine the high immunogenicity of multivalent particulate vaccines with the manufacturing advantages associated with nucleic acid-based vaccines. Summary of the Invention

[0009] The present invention provides a nucleic acid-based vaccine, which when delivered to eukaryotic cells during vaccination, is translated into self-assembled nanoparticles that display vaccine antigens in a unidirectional, iterative and multivalent manner by utilizing a split protein tag / catcher conjugation system.

[0010] Repetitive multivalent antigen display enhances the immunogenicity of vaccine antigens and allows the induction of strong antigen-specific immune responses after vaccination. At the same time, nucleic acid (DNA and / or mRNA)-based vaccine technologies have significant manufacturing advantages, since they allow the elimination of upstream and downstream steps associated with recombinant production of vaccine antigens. Furthermore, delivery of vaccine antigens as nucleic acid sequences allows for in vivo translation of the encoded proteins, which may otherwise be difficult or impossible to produce recombinantly.

[0011] In this specification, i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; A composition is provided in which the antigen and the protein, when expressed in a cell, are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii display the antigen.

[0012] Also provided herein are the compositions disclosed herein for use in the prevention and / or treatment of a disease in a subject in need thereof.

[0013] Also provided herein are methods for preventing or treating a disease in a subject in need of the composition.

[0014] In this specification, i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; An expression system is also provided in which, when the first and second polynucleotides are expressed in a cell, the antigen and the protein are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle displaying the antigen, i-ii.

[0015] In this specification, i. a first polynucleotide encoding a protein fused to a first peptide tag, said first polynucleotide preferably as defined in any one of the preceding claims; ii. A cell expressing a second polynucleotide encoding an antigen fused to a second peptide tag, preferably said second polynucleotide as defined in any one of the preceding claims, A cell is also provided in which, when expressed in the cell, the antigen and the protein are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii display the antigen.

[0016] Also provided herein is a host cell comprising the expression system disclosed herein.

[0017] Provided herein is a method for administering a composition for use in the prevention and / or treatment of a disease in a subject in need thereof, comprising the steps of: i. obtaining at least one of the compositions disclosed herein; ii. administering said composition at least once to a subject for the prevention and / or treatment of a disease as defined herein.

[0018] In this specification, i. a composition or expression system as defined herein; ii. optionally, a medical device or other means for administering the composition; and iii. A kit of parts including instructions for use is also provided. [Brief description of the drawings]

[0019] [Figure 1] Confocal laser scanning microscopy (CLSM) of C2C12 mouse cells 72 hours after co-transfection with SpyCatcher (SpyC)-eGFP and Spytagged (SpytT) HBcore. The pictures show a clear particulate enhanced green fluorescent protein (eGFP) fluorescent signal with a perinuclear distribution (all panels). [Diagram 2] Confocal laser scanning microscopy (CLSM) of C2C12 mouse cells 72 hours after co-transfection with SpyCatcher (SpyC)-eGFP and Spytagged (SpytT) AP205. The pictures show a homogenous green smear of eGFP fluorescence throughout the cells (all panels). Scale bar indicates 50 μm. [Diagram 3] Western blot (WB) probed with polyclonal anti-e-GFP antibody for detection of eGFP in pre-ultracentrifugation (UC input) and post-ultracentrifugation (UC) fractions (3-21 and 31) of sonicated C2C12 cells co-transfected with Spytagged (SpytT) AP205 coat protein and SpyCatcher (SpyC)-eGFP. The theoretical size of eGFP is 41 kDa, but conjugation of eGFP and AP205 results in 59 kDa. A band of approximately 59 kDa is seen in eGFP weekly and more intensely in the UC input sample, but not in the post-UC fraction sample. Thus, there is no indication that eGFP-bound AP205 particles have been formed. [Figure 4]Western blot (WB) probed with polyclonal anti-e-GFP antibody for detection of eGFP in pre-ultracentrifugation (UC input) and post-ultracentrifugation (UC) fractions (3-21 and 31) of sonicated C2C12 cells co-transfected with Spytagged (SpytC) HBcore antigen and SpyCatcher (SpyC)-eGFP. The theoretical size of eGFP is 41 kDa, but the conjugation of eGFP and HBcore antigen is 64 kDa. A band of approximately 64 kDa was seen in the UC input sample and in the post-UC fraction, indicating the formation of particulate structures from the conjugated SpyTHBc and SpyC eGFP proteins. [Diagram 5] Vector map of pVAX1 (V26020, thermoFisher). CMV promoter: bases 137-724, T7 promoter / priming site: bases 664-683, multiple cloning site: bases 696-811, bovine growth hormone (BGH) reverse priming site: bases 823-840, BGH polyadenylation signal: bases 829-1053, kanamycin resistance gene: bases 1226-2020, pUC origin: bases 2320-2993. [Figure 6A] Expression of particle-forming subunit protein from plasmid DNA. Western blot (WB) image of particle-forming subunit after transfection of HEK cells with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunit. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies coupled to horseradish peroxidase (HRP) were used for detection of particle-forming subunit. Sample: sign3-SpyC-i301-ctag. Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 35.7 kDa. The picture shows bands of expected size in cell and supernatant samples of all constructs, thus demonstrating the expression and secretion of particle-forming subunit protein in HEK cells after plasmid DNA transfection. [Figure 6B]Expression of particle-forming subunit protein from plasmid DNA. Western blot (WB) image of particle-forming subunit after transfection of HEK cells with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunit. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to horseradish peroxidase (HRP) were used for detection of particle-forming subunit. Sample: sign8-tandemHBc-SpyC. Primary antibody: aHBc (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 52.3 kDa. The picture shows bands of expected size in cell and supernatant samples of all constructs, thus demonstrating the expression and secretion of particle-forming subunit protein in HEK cells after plasmid DNA transfection. [Figure 6C] Expression of particle-forming subunit protein from plasmid DNA. Western blot (WB) image of particle-forming subunit after transfection of HEK cells with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunit. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to horseradish peroxidase (HRP) were used for detection of particle-forming subunit. Sample: sign8-tandemHBc-SpyC. Primary antibody: aHBc (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 23.12 kDa. The picture shows bands of expected size in cell and supernatant samples of all constructs, thus demonstrating the expression and secretion of particle-forming subunit protein in HEK cells after plasmid DNA transfection. [Figure 6D]Expression of particle-forming subunit protein from plasmid DNA. Western blot (WB) image of particle-forming subunit after transfection of HEK cells with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunit. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to horseradish peroxidase (HRP) were used for detection of particle-forming subunit. Sample: sign9-Ferritin-SpyC. Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 32.6 kDa. The picture shows bands of expected size in cell and supernatant samples of all constructs, thus demonstrating the expression and secretion of particle-forming subunit protein in HEK cells after plasmid DNA transfection. [Figure 7A] Expression of soluble proteins from plasmid DNA. Western blot image of soluble proteins after plasmid DNA transfection in HEK cells. HEK cells were transfected with plasmid DNA encoding soluble proteins. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to HRP were used for detection of soluble proteins. Sample: sign8-SpyT-eGFP. Primary antibody: aGFP-HRP. Expected size: 30.8kDa. The picture shows bands of expected size in cells and supernatants of all constructs, thus demonstrating the expression and secretion of particle-forming subunit proteins in HEK cells after plasmid DNA transfection. [Figure 7B]Expression of soluble proteins from plasmid DNA. Western blot image of soluble proteins after plasmid DNA transfection in HEK cells. HEK cells were transfected with plasmid DNA encoding soluble proteins. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to HRP were used for detection of soluble proteins. Sample: sign8-SpyT-eGFP. Primary antibody: aGFP-HRP. Expected size: 29 kDa. The picture shows bands of expected size in cells and supernatants of all constructs, thus demonstrating the expression and secretion of particle-forming subunit proteins in HEK cells after plasmid DNA transfection. [Figure 7C] Expression of soluble proteins from plasmid DNA. Western blot image of soluble proteins after plasmid DNA transfection in HEK cells. HEK cells were transfected with plasmid DNA encoding soluble proteins. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to HRP were used for detection of soluble proteins. Sample: sign8-SpyC-His. Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 15.4 kDa. The picture shows bands of expected size in cells and supernatants of all constructs, thus demonstrating the expression and secretion of particle-forming subunit proteins in HEK cells after plasmid DNA transfection. [Figure 7D]Expression of soluble proteins from plasmid DNA. Western blot image of soluble proteins after plasmid DNA transfection in HEK cells. HEK cells were transfected with plasmid DNA encoding soluble proteins. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to HRP were used for detection of soluble proteins. Sample: sign7-Pfs25-SpyT-Ctag. Primary antibody: aCtag-biotin. Secondary antibody: strep-HRP. Expected size: 24.3kDa. The picture shows bands of expected size in cells and supernatants of all constructs, thus demonstrating the expression and secretion of particle-forming subunit proteins in HEK cells after plasmid DNA transfection. [Figure 7E] Expression of soluble proteins from plasmid DNA. Western blot image of soluble proteins after plasmid DNA transfection in HEK cells. HEK cells were transfected with plasmid DNA encoding soluble proteins. Cells and supernatants were harvested 6 days after transfection and run on WB. Relevant antibodies conjugated to HRP were used for detection of soluble proteins. Sample: sign8-SpyT-eGFP. Primary antibody: aGFP-HRP. Expected size: 41.7kDa. The picture shows bands of expected size in cells and supernatants of all constructs, thus demonstrating the expression and secretion of particle-forming subunit proteins in HEK cells after plasmid DNA transfection. [Figure 8A]Validation of conjugation of eGFP to different particle-forming proteins. Western blot (WB) images of eGFP bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding eGFP + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used to detect GFP bound to particle-forming subunits. Samples: sign8-SpyT-eGFP and sign9-SpyC-ferritin. Primary antibody: aGFP-HRP. Expected size: 62 kDa. The pictures show bands of expected size of eGFP bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that eGFP and particles with corresponding tags / catchers can be bound in vitro after co-transfection into HEK cells. [Figure 8B] Validation of conjugation of eGFP to different particle-forming proteins. Western blot (WB) images of eGFP bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding eGFP + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used to detect GFP bound to particle-forming subunits. Samples: sign8-SpyC-eGFP and sign8-Hbc-SpyT. Primary antibody: aHBc (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 64.8 kDa. The pictures show bands of expected size of eGFP bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that eGFP and particles with corresponding tags / catchers can be bound in vitro after co-transfection into HEK cells. [Figure 8C] Validation of conjugation of eGFP to different particle-forming proteins. Western blot (WB) images of eGFP bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding eGFP + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used to detect GFP bound to particle-forming subunits. Samples: sign8-tandemHBc-SpyC and sign8-SpyT-eGFP. Primary antibody: aGFP-HRP. Expected size: 83.1 kDa. The pictures show bands of expected size of eGFP bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This indicates that eGFP and particles with corresponding tags / catchers can be bound in vitro after co-transfection into HEK cells. [Figure 8D]Validation of conjugation of eGFP to different particle-forming proteins. Western blot (WB) images of eGFP bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding eGFP + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used for detection of GFP bound to particle-forming subunits. Samples: sign3-SpyC-i301-ctag and sign8-SpyT-eGFP. Primary antibody: aGFP-HRP. Expected size: 66.5 kDa. The pictures show bands of expected size of eGFP bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that eGFP and particles with corresponding tags / catchers can be bound in vitro after co-transfection into HEK cells. [Figure 8E] Validation of conjugation of eGFP to different particle-forming proteins. Western blot (WB) images of eGFP bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding eGFP + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used to detect GFP bound to particle-forming subunits. Samples: sign9-SpyT-E2 and sign8-SpyC-eGFP. Primary antibody: aGFP-HRP. Expected size: 72.45 kDa. The pictures show bands of expected size of eGFP bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This indicates that eGFP and particles with corresponding tags / catchers can be bound in vitro after co-transfection into HEK cells. [Figure 8F] Validation of conjugation of eGFP to different particle-forming proteins. Western blot (WB) images of eGFP bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding eGFP + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used to detect GFP bound to particle-forming subunits. Samples: sign9-SpyT-LS and sign8-SpyC-eGFP. Primary antibody: aGFP-HRP. Expected size: 61.7 kDa. The pictures show bands of expected size of eGFP bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This indicates that eGFP and particles with corresponding tags / catchers can be bound in vitro after co-transfection into HEK cells. [Figure 9A] Validation of SpyC conjugation to different particle-forming proteins. Western blot (WB) images of SpyC bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding SpyC and plasmid DNA encoding particle-forming proteins + corresponding tags. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used for detection of SpyC bound to particle-forming subunits. Samples: sign8-SpyC-His and sign9-SpyT-E2. Primary antibody: aHis-HRP. Expected size: 46.15 kDa. The pictures show bands of the expected size of SpyC bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that SpyC and particles with the corresponding tags / catchers can be conjugated in vitro after co-transfection in HEK cells. [Figure 9B]Validation of SpyC conjugation to different particle-forming proteins. Western blot (WB) images of SpyC bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding SpyC and plasmid DNA encoding particle-forming proteins + corresponding tags. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used for detection of SpyC bound to particle-forming subunits. Samples: sign8-SpyC-His and sign9-SpyT-E2. Primary antibody: aHis-HRP. Expected size: 35.4 kDa. The pictures show bands of the expected size of SpyC bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that SpyC and particles with the corresponding tags / catchers can be conjugated in vitro after co-transfection in HEK cells. [Figure 9C] Validation of SpyC conjugation to different particle-forming proteins. Western blot (WB) images of SpyC bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding SpyC and plasmid DNA encoding particle-forming proteins + corresponding tags. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used for detection of SpyC bound to particle-forming subunits. Samples: sign8-SpyC-His and sign8-Hbc-SpyT. Primary antibody: aHis-HRP. Expected size: 38.5 kDa. The pictures show bands of the expected size of SpyC bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that SpyC and particles with the corresponding tags / catchers can be conjugated in vitro after co-transfection in HEK cells. [Figure 9D]Validation of SpyC conjugation to different particle-forming proteins. Western blot (WB) images of SpyC bound to particle-forming proteins after co-transfection of plasmid DNA into HEK cells. HEK cells were co-transfected with plasmid DNA encoding SpyC and plasmid DNA encoding particle-forming proteins + corresponding tags. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used for detection of SpyC bound to particle-forming subunits. Samples: sign8-Norovirus-SpyT and sign8-SpyC-His. Primary antibody: aHis-HRP. Expected size: 78.16 kDa. The pictures show bands of the expected size of SpyC bound to different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that SpyC and particles with the corresponding tags / catchers can be conjugated in vitro after co-transfection in HEK cells. [Figure 10]Validation of Pfs25 conjugation to different particle-forming proteins. Western blot (WB) images of Pfs25 bound to particle-forming proteins after co-transfection of HEK cells with plasmid DNA. HEK cells were co-transfected with plasmid DNA encoding Pfs25-SpyT and plasmid DNA encoding particle-forming protein + corresponding catcher. Cells and supernatants were harvested 6 days after co-transfection. Relevant antibodies conjugated to HRP were used to detect Pfs25 bound to particle-forming subunits. A. Samples: sign9-SpyC-Ferritin and sign7-Pfs25-SpyT-Ctag. Primary antibody: aCtag-biotin. Secondary antibody: strep-HRP. Expected size: 56.9a. B. Samples: sign3-SpyC-i301-Ctag and sign7-Pfs25-SpyT-Ctag. Primary antibody: aCtag-biotin. Secondary antibody: strep-HRP. Expected size: 60 kDa. The pictures show bands of the expected size for Pfs25 bound to the different particle-forming subunit proteins in cells and supernatants of all constructs. This shows that particles carrying Pfs25 and the corresponding tags / catchers can be bound in vitro after co-transfection in HEK cells. [Figure 11A]Validation of nanoparticle formation by ultracentrifugation (UC) and WB. Western blot (WB) image of UC fraction from the supernatant of transfected HEK cells. HEK cells were transfected with plasmid DNA encoding particle-forming subunit proteins. The supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect the particle-forming subunits. If any particles were formed, a band of the corresponding size would be expected in fractions 3-8. Sample: sign3-SpyC-i301-Ctag. Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 36kDa. The picture shows particle formation with all constructs and it can be seen that a band of the expected size is present in the relevant fractions (3-8). This therefore indicates that after transfection of HEK cells with plasmid DNA, not only was there expression and secretion of the particle subunits, but also particle formation in vitro. [Figure 11B]Validation of nanoparticle formation by ultracentrifugation (UC) and WB. Western blot (WB) image of UC fraction from the supernatant of transfected HEK cells. HEK cells were transfected with plasmid DNA encoding particle-forming subunit proteins. The supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect the particle-forming subunits. If any particles were formed, a band of the corresponding size would be expected in fractions 3-8. Sample: sign8-tandemHBc-SpyCatcher. Primary antibody: aHBc (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 52kDa. The picture shows particle formation with all constructs and it can be seen that a band of the expected size is present in the relevant fractions (3-8). This therefore indicates that after transfection of HEK cells with plasmid DNA, not only was there expression and secretion of the particle subunits, but also particle formation in vitro. [Figure 11C]Validation of nanoparticle formation by ultracentrifugation (UC) and WB. Western blot (WB) image of UC fraction from the supernatant of transfected HEK cells. HEK cells were transfected with plasmid DNA encoding particle-forming subunit proteins. The supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect the particle-forming subunits. If any particles were formed, a band of the corresponding size would be expected in fractions 3-8. Sample: sign9-Ferritin-SpyC. Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 32.6kDa. The picture shows particle formation with all constructs and it can be seen that a band of the expected size is present in the relevant fractions (3-8). This therefore indicates that after transfection of HEK cells with plasmid DNA, not only was there expression and secretion of the particle subunits, but also particle formation in vitro. [Figure 12A]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign8-SpyT-eGFP+sign9-SpyC-Ferritin. Primary antibody: aGFP-HRP. Expected size 62kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 12B]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign8-SpyC-His+sign9-SpyT-E2. Primary antibody: aHis-HRP. Expected size 46.15kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 12C]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign8-SpyC-His+sign9-LS-SpyT. Primary antibody: aHis-HRP. Expected size 35.4kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 12D]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign9-SpyC-Ferritin+sign7-Pfs25-SpyT. Primary antibody: aCtag-biotin. Secondary antibody: strep-HRP. Expected size 56.9kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 12E]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign3-SpyC-i301+sign7-Pfs25-SpyT. Primary antibody: aCtag-biotin. Secondary antibody: strep-HRP. Expected size 60kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 12F]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign9-SpyT-E2+sign8-SpyC-eGFP. Primary antibody: aGFP-HRP. Expected size 72.45kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 12G]Validation of nanoparticle conjugate formation by UC and Western Blot (WB). WB image of UC fraction from the supernatant of transfected HEK cells. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Supernatant was harvested 6 days after transfection and loaded onto an optirep density gradient. The gradient was centrifuged for 3 hours 30 minutes at 47800RPM and 16oC and fractionated into 12 fractions. Each fraction was run with WB where the relevant antibody conjugated to HRP was used to detect antigen bound to particle-forming subunits. If any particles were formed, bands of the corresponding size are expected in fractions 3-8. Sample: sign9-SpyT-E2+sign8-SpyC-eGFP. Primary antibody: aGFP-HRP. Expected size 61.7kDa. The photographs show bound particle formation for all constructs, with bands of the expected size in the relevant fractions (3-8), thus demonstrating not only expression and secretion of particle subunits and soluble antigens, but also the formation of antigen-bound particles in vitro after co-transfection of plasmid DNA into HEK cells. [Figure 13A]Visualization of particle formation by transmission electron microscopy (TEM). Electron microscopy images of UC purified supernatant from HEK cells transfected with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunits or co-transfected with plasmid DNA encoding soluble antigens + tags / catchers and plasmid DNA encoding particle-forming proteins + corresponding tags / catchers. Supernatants were harvested 6 days after transfection and loaded onto optirep density gradients. Gradients were centrifuged for 3 hours 30 minutes at 47800 RPM, 16oC and fractionated into 12 fractions. Fractions containing particles were pooled and dialyzed into 1xPBS for TEM imaging. Sample: sign9-Ferritin-SpyC. Expected size: 12nm. The picture shows particles of expected size formed in the SN of transfected or co-transfected cells. Thus, we can further confirm that particles and conjugated particles can be formed in the supernatant of transfected or co-transfected cells in vitro from plasmid DNA. [Figure 13B]Visualization of particle formation by transmission electron microscopy (TEM). Electron microscopy images of UC purified supernatants from HEK cells transfected with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunits or co-transfected with plasmid DNA encoding soluble antigens + tag / catcher and plasmid DNA encoding particle-forming proteins + corresponding tag / catcher. Supernatants were harvested 6 days after transfection and loaded onto optirep density gradients. Gradients were centrifuged for 3 hours 30 minutes at 47800RPM, 16oC and fractionated into 12 fractions. Fractions containing particles were pooled and dialyzed into 1xPBS for TEM imaging. Sample: sign8-SpyC-His+sign9-LS-SpyT. Expected size: 12nm. The picture shows particles of the expected size formed in the SN of transfected or co-transfected cells. Thus, the inventors are further able to confirm that particles and conjugated particles can be formed in the supernatants of cells transfected or co-transfected in vitro with plasmid DNA. [Figure 13C]Visualization of particle formation by transmission electron microscopy (TEM). Electron microscopy images of UC purified supernatants from HEK cells transfected with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunits or co-transfected with plasmid DNA encoding soluble antigens + tags / catchers and plasmid DNA encoding particle-forming proteins + corresponding tags / catchers. Supernatants were harvested 6 days after transfection and loaded onto optirep density gradients. Gradients were centrifuged for 3 hours 30 minutes at 47800RPM, 16oC and fractionated into 12 fractions. Fractions containing particles were pooled and dialyzed into 1xPBS for TEM imaging. Sample: sign9-SpyT-E2+sign8-SpyC-His. Expected size: 12nm. The picture shows particles of the expected size formed in the SN of transfected or co-transfected cells. Thus, the inventors are further able to confirm that particles and conjugated particles can be formed in the supernatants of cells transfected or co-transfected in vitro with plasmid DNA. [Figure 13D]Visualization of particle formation by transmission electron microscopy (TEM). Electron microscopy images of UC purified supernatants from HEK cells transfected with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunits or co-transfected with plasmid DNA encoding soluble antigens + tag / catcher and plasmid DNA encoding particle-forming proteins + corresponding tag / catcher. Supernatants were harvested 6 days after transfection and loaded onto optirep density gradients. Gradients were centrifuged for 3 hours 30 minutes at 47800RPM, 16oC and fractionated into 12 fractions. Fractions containing particles were pooled and dialyzed into 1xPBS for TEM imaging. Sample: sign8-SpyT-eGFP+sign9-SpyC-Ferritin. Expected size: 12nm. The picture shows particles of the expected size formed in the SN of transfected or co-transfected cells. Thus, the inventors are further able to confirm that particles and conjugated particles can be formed in the supernatants of cells transfected or co-transfected in vitro with plasmid DNA. [Figure 13E]Visualization of particle formation by transmission electron microscopy (TEM). Electron microscopy images of UC purified supernatants from HEK cells transfected with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunits or co-transfected with plasmid DNA encoding soluble antigens + tag / catcher and plasmid DNA encoding particle-forming proteins + corresponding tag / catcher. Supernatants were harvested 6 days after transfection and loaded onto optirep density gradients. Gradients were centrifuged for 3 hours 30 minutes at 47800RPM, 16oC and fractionated into 12 fractions. Fractions containing particles were pooled and dialyzed into 1xPBS for TEM imaging. Sample: sign3-SpyC-i301-Ctag+sign7-Pfs25-SpyT-Ctag. Expected size: 25nm. The picture shows particles of the expected size formed in the SN of transfected or co-transfected cells. Thus, the inventors are further able to confirm that particles and conjugated particles can be formed in the supernatants of cells transfected or co-transfected in vitro with plasmid DNA. [Figure 13F]Visualization of particle formation by transmission electron microscopy (TEM). Electron microscopy images of UC purified supernatants from HEK cells transfected with plasmid DNA. HEK cells were transfected with plasmid DNA encoding particle-forming subunits or co-transfected with plasmid DNA encoding soluble antigens + tag / catcher and plasmid DNA encoding particle-forming proteins + corresponding tag / catcher. Supernatants were harvested 6 days after transfection and loaded onto optirep density gradients. Gradients were centrifuged for 3 hours 30 minutes at 47800RPM, 16oC and fractionated into 12 fractions. Fractions containing particles were pooled and dialyzed into 1xPBS for TEM imaging. Sample: sign9-SpyC-Ferritin+sign7-Pfs25-SpyT-Ctag. Expected size: 12nm. The picture shows particles of the expected size formed in the SN of transfected or co-transfected cells. Thus, the inventors are further able to confirm that particles and conjugated particles can be formed in the supernatants of cells transfected or co-transfected in vitro with plasmid DNA. [Figure 14A]Verification that conjugation of antigen to nanoparticle-forming protein occurred intracellularly. Western blot (WB) images of soluble antigen bound to particle-forming protein after co-transfection of HEK cells with plasmid DNA. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. At harvest, cells were resuspended in 1xSDS+DTT to prevent further binding. Cell samples and supernatants in 1xSDS+DTT were run in WB. Relevant antibodies conjugated to HRP were used to detect soluble antigen bound to particle-forming subunits. Therefore, if any binding was seen in WB, it must have occurred intracellularly before harvest. Samples: sign8-SpyC-His and sign9-LS-SpyT. Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 35.4 kDa. The picture shows that the particles and soluble antigens can bind not only in the supernatant but also inside the cells. Indeed, a band of the expected binding size was visualized in cells harvested with SDS+DTT, indicating that binding occurred inside the cells during in vitro culture. [Figure 14B]Verification that conjugation of antigen to nanoparticle-forming protein occurred intracellularly. Western blot (WB) images of soluble antigen bound to particle-forming protein after co-transfection of HEK cells with plasmid DNA. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. At harvest, cells were resuspended in 1xSDS+DTT to prevent further binding. Cell samples and supernatants in 1xSDS+DTT were run in WB. Relevant antibodies conjugated to HRP were used to detect soluble antigen bound to particle-forming subunits. Therefore, if any binding was seen in WB, it must have occurred intracellularly before harvest. Sample: sign8-SpyT-eGFP+sign9-SpyC-Ferritin (B1822+B1772). Primary antibody: aSpyC (mouse serum). Secondary antibody: anti-mouse HRP. Expected size: 63.4 kDa. The picture shows that the particles and soluble antigens can bind not only in the supernatant but also inside the cells. Indeed, a band of the expected binding size was visualized in cells harvested with SDS+DTT, indicating that binding occurred inside the cells during in vitro culture. [Figure 14C]Verification that conjugation of antigen to nanoparticle-forming protein occurred intracellularly. Western blot (WB) images of soluble antigen bound to particle-forming protein after co-transfection of HEK cells with plasmid DNA. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. At harvest, cells were resuspended in 1xSDS+DTT to prevent further binding. Cell samples and supernatants in 1xSDS+DTT were run in WB. Relevant antibodies conjugated to HRP were used to detect soluble antigen bound to particle-forming subunits. Therefore, if any binding was seen in WB, it must have occurred intracellularly before harvest. Samples: sign3-SpyC-i301-ctag (B1774) and sign8-SpyT-eGFP (B1822). Primary antibody: aHis-HRP. Expected size: 66.5 kDa. The picture shows that the particles and soluble antigen can bind not only in the supernatant but also inside the cells. Indeed, a band of the expected binding size was visualized in cells harvested with SDS+DTT, indicating that binding occurred inside the cells during in vitro culture. [Figure 14D]Verification that conjugation of antigen to nanoparticle-forming protein occurred intracellularly. Western blot (WB) images of soluble antigen bound to particle-forming protein after co-transfection of HEK cells with plasmid DNA. HEK cells were co-transfected with plasmid DNA encoding soluble antigen + tag / catcher and with plasmid DNA encoding particle-forming protein + corresponding tag / catcher. Cells and supernatants were harvested 6 days after co-transfection. At harvest, cells were resuspended in 1xSDS+DTT to prevent further binding. Cell samples and supernatants in 1xSDS+DTT were run in WB. Relevant antibodies conjugated to HRP were used to detect soluble antigen bound to particle-forming subunits. Therefore, if any binding was seen in WB, it must have occurred intracellularly before harvest. Samples: sign8-eGFP-SpyC (B2009) and sign9-LS-SpyT (B1929) lanes 1 (cells), 2 (SN) compared with sign8-eGFP (B2033) and sign9-LS-SpyT (B1929) lanes 3 (cells), 4 (SN). Primary antibody: aGFP-HRP. Expected: B2009+B1929 should be able to bind, but B2033+B1929 should not be able to bind (because B2033 does not have a catcher). The photo shows that particles and soluble antigens can bind not only in the supernatant but also inside the cells. Indeed, a band of the expected binding size was visualized in cells harvested with SDS+DTT, indicating that binding occurred inside the cells in in vitro culture. [Figure 15A]Validation of immunogenicity of plasmid DNA vaccines encoding particle-forming subunits. ELISA titers of total IgG in mice after plasmid DNA immunization. Plasmid DNA encoding particles and / or soluble antigens were used for vaccination of mice. Balb / c mice were immunized with 30ug LS-SpyT (B1929) and 30ug SpyC (B1928) (N=6), or 30ug SpyC (B1928) (N=4), or 30ug E2-SpyT (B1930) and 30ug SpyC (B1928). DNA was formulated in PBS and injected into the right thigh muscle. Mice were immunized on day 0 and week 5, and blood was taken at weeks 3 and 4 after prime and boost. Serum was separated from blood and ELISA was performed for detection of anti-SpyC IgG. For this purpose, 96-well plates (Nunc MaxiSorp) were coated with SpyC in PBS. IgG against SpyC was detected with HRP-conjugated goat anti-mouse IgG (Life technologies, A16072). Plates were developed with TMB X-tra substrate (Kem-En-Tec, 4800A) and absorbance was measured at 450 nM. A. ELISA titers against SpyC 3 and 4 weeks after first immunization in Balb / c mice ELISA titers show that mice receiving DNA encoding particles and DNA encoding SpyC have higher IgG titers against SpyC after the first immunization compared to mice receiving only DNA encoding SpyC. Moreover, this trend is even higher after booster immunization. This is true for both groups of mice receiving SpyC in combination with either LS or E2 particles. [Figure 15B]Validation of immunogenicity of plasmid DNA vaccines encoding particle-forming subunits. ELISA titers of total IgG in mice after plasmid DNA immunization. Plasmid DNA encoding particles and / or soluble antigens were used for vaccination of mice. Balb / c mice were immunized with 30ug LS-SpyT (B1929) and 30ug SpyC (B1928) (N=6), or 30ug SpyC (B1928) (N=4), or 30ug E2-SpyT (B1930) and 30ug SpyC (B1928). DNA was formulated in PBS and injected into the right thigh muscle. Mice were immunized on day 0 and week 5, and blood was taken at weeks 3 and 4 after prime and boost. Serum was separated from blood and ELISA was performed for detection of anti-SpyC IgG. For this purpose, 96-well plates (Nunc MaxiSorp) were coated with SpyC in PBS. IgG against SpyC was detected with HRP-conjugated goat anti-mouse IgG (Life technologies, A16072). Plates were developed with TMB X-tra substrate (Kem-En-Tec, 4800A) and absorbance was measured at 450 nM. B. ELISA titers against SpyC 3 and 4 weeks after booster immunization in Balb / c mice ELISA titers show that mice receiving DNA encoding particles and DNA encoding SpyC have higher IgG titers against SpyC after the first immunization compared to mice receiving only DNA encoding SpyC. Moreover, this trend becomes even higher after the booster immunization. This is true for both groups of mice receiving SpyC in combination with either LS or E2 particles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure provides a nucleic acid-based vaccine, which upon delivery to eukaryotic cells during vaccination, is translated into self-assembled nanoparticles that display vaccine antigens in a unidirectional, iterative and multivalent manner by utilizing a split protein tag / catcher conjugation system.

[0021] Repetitive multivalent antigen display enhances the immunogenicity of vaccine antigens and allows the induction of strong antigen-specific immune responses after vaccination. At the same time, nucleic acid (DNA and / or mRNA)-based vaccine technologies have significant manufacturing advantages, since they allow the omission of upstream and downstream steps associated with recombinant production of vaccine antigens. Furthermore, delivery of vaccine antigens as nucleic acid sequences allows for in vivo translation of the encoded proteins, which may otherwise be difficult or impossible to produce recombinantly.

[0022] The inventive solution represents a novel approach to create a versatile nucleic acid-based vaccine delivery platform capable of efficiently displaying antigenic epitopes and inducing long-term protective immunity.

[0023] definition The term "isopeptide bond" as used herein refers to an amide bond between a carboxyl group and an amino group, at least one of which is not believed to originate from the protein backbone or to be part of the protein backbone. Isopeptide bonds can form within a single protein, or occur between two peptides or between a peptide and a protein. Thus, isopeptides can form intramolecularly within a single protein, or intermolecularly, i.e., between two peptide / protein molecules. Usually, isopeptide bonds can occur intramolecularly between two reactive amino acids (lysine and asparagine or aspartic acid). For this process to occur, the two reactive amino acids must be in close proximity in a hydrophobic environment, often containing aromatic residues. Finally, the autocatalytic process can be facilitated by catalytic aspartate or glutamic acid residues that are not themselves involved in the isopeptide bond.

[0024] In the case of intermolecular isopeptide bonds, the bonds typically occur between lysine residues and asparagine, aspartic acid, glutamine, or glutamic acid residues, or the terminal carboxyl group of a protein or peptide chain, or between the alpha amino terminus of a protein or peptide chain and asparagine, aspartic acid, glutamine, or glutamic acid. Each residue of the pair involved in an isopeptide bond is referred to herein as a reactive residue. Thus, isopeptide bonds can be formed between lysine and asparagine residues, or between lysine and aspartic acid residues. In particular, isopeptide bonds can occur between the side chain amine of lysine and the carboxamide group of asparagine.

[0025] The term "open reading frame" as used herein refers to a nucleotide sequence that includes 1) a translation initiation codon, 2) one or more codons encoding one or more gene products of interest, preferably one or more proteins, and 3) a translation stop codon, in a 5' to 3' direction, whereby 1), 2) and 3) are operably linked in frame, whereby an open reading frame is thus composed of a multiple of three nucleotides (triplets).

[0026] The term "sequence variant" refers to a polypeptide and / or polynucleotide sequence having at least 70%, such as 75%, for example 80%, such as 85%, for example 90%, such as 95%, for example 96%, such as 97%, for example 98%, such as 99%, for example 99.5%, such as 100% sequence identity to said polypeptide and / or polynucleotide sequence.

[0027] The term "antigenic variant" refers to a variant of a full length polypeptide or a fragment of said polypeptide, the fragment comprising an epitope recognised by cytotoxic T cells, helper T cells and / or B cells of the host. The fragment may be more immunogenic and thus induce a stronger and / or longer lasting immune response than the original polypeptide from which it is derived. Preferably, the immunogenic portion of the antigenic variant comprises at least 30%, preferably at least 50%, particularly at least 75% and especially at least 90% (e.g. 95% or 98%) of the amino acid sequence of the reference sequence. The immunogenic portion preferably comprises all of the epitope region of the reference sequence. The immunogenicity of the antigenic variant may be verified by any method known in the art, such as the method described in Wadhwa et al., 2015.

[0028] As described herein, a first peptide tag and a second peptide tag (or binding partner) refer to a first and a second peptide tag that are bound to each other via an isopeptide bond, preferably a spontaneous isopeptide bond. Preferably, the first peptide tag comprises one of the reactive residues involved in the isopeptide bond and the second peptide tag comprises the other reactive residue involved in the isopeptide bond.

[0029] The term "spontaneous" as used herein refers to bonds, particularly acute peptide bonds, that can be formed within a protein or between peptides or proteins (e.g., between a first peptide tag and a second peptide tag) in the absence of other agents (e.g., enzyme catalysts) and / or chemical modification of the protein or peptide, e.g., without native chemical ligation or chemical conjugation. Spontaneous isopeptide bonds can thus be formed naturally, in the absence of enzymes or other exogenous substances or without chemical modification. However, particularly spontaneous isopeptide or covalent bonds may require the presence of a glutamic or aspartic acid residue in one of the peptides / proteins involved in the conjugation to allow the formation of the bond.

[0030] The term "virus-like particle" or "VLP" refers to one or more recombinantly expressed viral proteins, such as viral capsid proteins, that spontaneously assemble into polymeric particulate structures that mimic the morphology of the viral coat but lack infectious genetic material.

[0031] The term "particle" as used herein refers to a virus-like particle or nanoparticle capable of displaying an antigen on its surface, as described herein. The surface may be an internal surface, i.e., the surface facing the interior of the particle, or an external surface, i.e., the surface facing the surroundings of the particle.

[0032] The term "self-assembly" refers to the process in which a system of existing components adopts a more organized structure under certain conditions through interactions between the components themselves. In this context, self-assembly refers to the inherent ability of proteins such as viral proteins, e.g. capsid proteins, and / or phage proteins, to self-assemble into particles, especially virus-like particles in the absence of other viral proteins, when exposed to certain conditions. "Self-assembly" does not exclude the possibility that cellular proteins, e.g. chaperones, may be involved in the process of intracellular VLP or nanoparticle assembly. The self-assembly process may be sensitive and fragile and may be influenced by factors such as, but not limited to, the choice of expression host, the choice of expression conditions, and the maturation conditions of the virus-like particle. The viral capsid protein may form a VLP alone or in combination with several viral capsid proteins, optionally all identical.

[0033] The term "consistent orientation" as used herein refers to the orientation of the antigen and its spatial orientation on the surface of the particle disclosed herein, i.e., the inner or outer surface of the particle, preferably at least the outer surface. As disclosed herein, when an antigen fused to a second peptide tag is linked to a protein containing a second peptide tag, one molecule of the antigen can only be linked to a single particle-forming protein at a unique site on both the antigen and the protein, creating a uniform and / or consistent presentation of the antigen with a consistent orientation. In contrast, for example, a streptavidin homotetramer may crosslink several proteins on the outer surface of a biotinylated VLP, creating an irregular and inconsistent orientation of the antigen. Furthermore, it is very difficult to use streptavidin as a bridging molecule for conjugating, for example, a biotinylated antigen to a biotinylated VLP, because multiple biotin binding sites allow crosslinking and aggregation of the biotinylated VLP.

[0034] The term "equally spaced" as used herein refers to the antigens of the present invention forming a pattern on the surface of a VLP or nanoparticle. Such a pattern may be a symmetrical, circular, and / or bouquet-like pattern of antigens.

[0035] The term "treatment" refers to the amelioration of a health problem. Treatment may also be protective and / or preventative, or may reduce the risk of developing a disease and / or infection. Treatment may also cure or ameliorate a disease and / or infection.

[0036] The term "prophylactic" refers to reducing the risk of developing a disease and / or infection. Prophylactic can also refer to preventing the development of a disease and / or infection.

[0037] The term "loop" refers to a secondary structure in a polypeptide in which the polypeptide chain reverses its general direction, sometimes called a turn.

[0038] The term "vaccine cocktail" refers to a mixture of antigens administered together. Vaccine cocktails can be administered as a single dose or as several doses administered over a period of time. The time interval can be, but is not limited to, administration within the same year, month, week, day, hour and / or minute. Concurrent vaccination and vaccine cocktail can be used interchangeably.

[0039] The term "autoantigen" refers to an endogenous antigen produced within a previously normal cell as a result of abnormal cellular metabolism.

[0040] composition In this specification, i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; A composition is provided in which the antigen and the protein, when expressed in a cell, are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii display the antigen.

[0041] Said compositions are useful for the prevention and / or treatment of a disease or disorder as described herein below.

[0042] Administration of the composition comprising the first and second polynucleotides to a subject may induce a stronger immune response in the subject compared to administration of a composition comprising a polypeptide encoded by the first and second polynucleotides, wherein the first and second polypeptides are linked via an isopeptide bond or an ester bond between the first and second peptide tags prior to administration to the subject.

[0043] The first and second peptide tags are selected from peptides that have an inherent ability to form isopeptide or ester bonds, thereby linking them to each other. The first polynucleotide encodes a protein that has the ability to form particles, such as nanoparticles or virus-like particles (VLPs), preferably spontaneously. Thus, when expressed in a cell, the first polynucleotide results in the formation of a particle formed by the protein fused to the first peptide tag, while the second polynucleotide results in the expression of a fusion protein that includes or consists of an antigen fused to the second peptide tag. The assembled particles may closely resemble viruses or other pathogenic organisms that are recognized by the immune system, but are non-infectious because they do not contain pathogenic genetic material. The spontaneous formation of isopeptide or ester bonds between the first and second peptide tags results in the formation of particles that display the antigen on their surface, preferably the outer surface. Importantly, in addition to consistent antigen orientation, such nanoparticles and VLPs unexpectedly possess highly beneficial antigen display properties, including consistent antigen orientation, high density, and regular spacing. Thus, the resulting nanoparticles and VLPs are capable of inducing strong humoral responses and overcoming B cell tolerance. The successful formation of such nanoparticles or VLPs can be assessed by relevant methods known to those skilled in the art, such as those disclosed in Examples 2 and 3 of the present disclosure. Such nanoparticles or VLPs surprisingly allow for antigen display with consistent antigen orientation, high density, and regular spacing for both large and small antigens, with improved efficiency of antigen binding compared to chemical conjugation methods.

[0044] The first and second polynucleotides may be DNA or RNA, and preferably, the first polynucleotide and the second polynucleotide may both be DNA or both be RNA.

[0045] The mRNA transcribed from the RNA construct and / or DNA construct may be polycistronic. In some embodiments, the first and second polynucleotides are encoded on the same ribonucleic acid molecule. In some embodiments, the first and second polynucleotides are in the same open reading frame, such that only one promoter sequence is required to transcribe both polynucleotides. In some embodiments, the first and second polynucleotides are in separate open reading frames, and therefore may be controlled by separate promoters.

[0046] protein In a preferred embodiment, the protein is a particle-forming protein. For example, the protein can be a viral capsid protein, such as a glycoprotein, or a viral envelope protein. In some embodiments, the protein is derived from a mammalian virus, such as a human virus.

[0047] In some embodiments, the protein is a protein from a hepatitis virus, such as hepatitis B or E, e.g., core protein from hepatitis B virus. In some embodiments, the protein is a protein from a norovirus, such as NoV. In some embodiments, the protein is a protein from a human papillomavirus (HPV), preferably a papillomavirus, such as HPV16 or HPV18, such as HPV L1. In some embodiments, the protein is a protein from a polyomavirus, such as polyomavirus vp1 (PyV). In some embodiments, the protein is a protein from a calicivirus, such as feline calicivirus (FCV), preferably FCV VP1. In some embodiments, the protein is a protein from a circovirus, such as porcine circovirus (PCV), preferably PCV2 ORF2. In some embodiments, the protein is a protein from a nervous necrosis virus (NNV), such as NNV coat protein. In some embodiments, the protein is a protein from a parvovirus, such as canine parvovirus (CVP), preferably CPV VP2, goose parvovirus (GPV) or porcine parvovirus (PPV), preferably a structural protein from GPV or PPV, or parvovirus B19. In some embodiments, the protein is a protein from a protoparvovirus, such as an enteritis virus, e.g. mink enteritis virus (MEV), preferably MEV VP2, or duck plague virus (DPV), preferably a DPV structural protein.

[0048] The protein may be a protein from a plant virus, such as a cowpea virus, a tobacco virus, a tomato virus, a cucumber virus, or a potato virus. In some embodiments, the plant virus is a mosaic virus, preferably a cowpea mosaic virus (CPMV). In some embodiments, the plant virus is a tobacco mosaic virus (TMV). In some embodiments, the plant virus is a tomato spotted wilt virus (TSWV). In some embodiments, the plant virus is a tomato yellow leaf curl virus (TYLCV). In some embodiments, the plant virus is a cucumber mosaic virus (CMV). In some embodiments, the plant virus is a potato virus Y (PVY).

[0049] In some embodiments, the protein is a bacteriophage protein, such as Salmonella virus P22, MS2, QBeta, PRR1, PP7, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage M11, bacteriophage MX1, bacteriophage NL95, bacteriophage f2, or Cb5. Additional relevant bacteriophage proteins are described in Lieknina et al., 2019.

[0050] In some embodiments, the protein is a protein from a virus common to mammals, particularly humans. Without being bound by theory, such viruses may be more suitable for expression in mammalian cells, particularly human cells, than viruses commonly found in organisms other than mammals. Thus, in some embodiments, the virus is a hepatitis virus, such as hepatitis B or E virus. In some embodiments, the virus is a norovirus. In some embodiments, the virus is a papillomavirus, such as a human papillomavirus (HPV), preferably HPV16 or HPV18. In some embodiments, the virus is a polyomavirus. In some embodiments, the virus is a parvovirus.

[0051] In some embodiments, the protein is a particle-forming protein such as ferritin, i301, replicase polyprotein 1a (pp1a) or lumazine synthase. Other particle-forming proteins are known in the art and may be used.

[0052] First and second peptide tags Peptide pairs consisting of a first peptide tag and a second peptide tag that can bind to each other via the (spontaneous) formation of an isopeptide bond are known in the art or can be designed or obtained by methods known in the art, in particular the methods described in Zakeri et al., 2012 and Zakeri et al., 2010.

[0053] The term "peptide tag" as used herein generally refers to a small peptide fragment that can be directly designed or derived from a protein that naturally forms intramolecular isopeptide bonds. Peptide tags can also be identified by using known binding partners, for example, from proteins that naturally form intramolecular isopeptide bonds, to screen peptide libraries. Thus, candidate peptide tags can be from libraries, for example, peptide libraries, from which candidate peptide tags can be screened. Candidate peptide tags can also be designed in silico.

[0054] Thus, a peptide pair as understood herein consists of two peptide tags that can interact through the spontaneous formation of isopeptide bonds or through ester bonds. In general, these are also called "tag and catcher" systems, where the longer of the two peptide tags is called "catcher" and the shorter of the two peptide tags is called "tag". For example, the SpyTag / SpyCatcher system consists of a first peptide tag (SpyTag) and a second peptide tag (SpyCatcher).

[0055] In some embodiments, a peptide pair as understood herein consists of two peptide tags that can interact via the spontaneous formation of an ester bond. Useful peptide tags that can form such spontaneous ester bonds are further described in Young et al., 2017.

[0056] A "tag" is 5-50 amino acids in length, e.g., 10, 20, 30, 40-50 amino acids in length, and can be covalently attached via an isopeptide bond to a binding partner as defined herein. Thus, a "tag" can include one reactive residue involved in an isopeptide bond in an isopeptide protein used to design a binding partner as described herein (the binding partner can include other reactive residues involved in the bond).

[0057] In some embodiments, the "tag" has a length of between 7 and 47 amino acids, such as between 8 and 46 amino acids, for example between 9 and 45 amino acids, such as between 10 and 44 amino acids, for example between 11 and 43 amino acids, such as between 12 and 42 amino acids, for example between 13 and 41 amino acids, such as between 14 and 40 amino acids, for example between 15 and 39 amino acids, such as between 16 and 38 amino acids, for example between 17 and 37 amino acids, such as between 18 and 36 amino acids, for example between 19 and 35 amino acids, such as between 20 and 34 amino acids, for example between 21 and 33 amino acids, such as between 22 and 32 amino acids, for example between 23 and 31 amino acids, such as between 24 and 30 amino acids, for example between 25 and 29 amino acids, such as between 26 and 28 amino acids, for example 27 amino acids. In some embodiments, the "tag" has a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 amino acids.

[0058] In some embodiments, the "catcher" is at least 20 amino acids in length. Preferably, the "catcher" has a length of 5 or more amino acids, such as 10 or more amino acids, such as 15 or more amino acids, such as 20 or more amino acids, such as 25 or more amino acids, such as 30 or more amino acids, such as 35 or more amino acids, such as 40 or more amino acids, such as 45 or more amino acids, such as 50 or more amino acids, such as 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 or more amino acids. In some embodiments, the "catcher" is at least 20 amino acids in length. In some embodiments, the "catcher" is between 75 and 125 amino acids in length.

[0059] Preferably, the "catcher" has an amino acid sequence consisting of more amino acid residues than the "tag".

[0060] In some embodiments, one of the first and second peptide tags is selected from the group consisting of SpyTag (SEQ ID NO:1), SdyTag (SEQ ID NO:2), SnoopTag (SEQ ID NO:3), PhoTag (SEQ ID NO:4), EntTag (SEQ ID NO:5), KTag, BacTag (SEQ ID NO:15), Bac2Tag (SEQ ID NO:16), Bac3Tag (SEQ ID NO:17), Bac4Tag (SEQ ID NO:18), RumTrunkTag (SEQ ID NO:13 or SEQ ID NO:14), Rum7Tag (SEQ ID NO:12), RumTag (SEQ ID NO:6), Rum2Tag (SEQ ID NO:7), Rum3Tag (SEQ ID NO:8), Rum4Tag (SEQ ID NO:9), Rum5Tag (SEQ ID NO:10), Rum6Tag (SEQ ID NO:11) and Bac5Tag (SEQ ID NO:19). The nucleic acid sequences encoding the tags are as follows: SpyTag (SEQ ID NO:35), SdyTag (SEQ ID NO:36), SnoopTag (SEQ ID NO:37), PhoTag (SEQ ID NO:38), EntTag (SEQ ID NO:39), KTag, BacTag (SEQ ID NO:49), Bac2Tag (SEQ ID NO:50), Bac3Tag (SEQ ID NO:51), Bac4Tag (SEQ ID NO:52), RumTrunkTag (SEQ ID NO:47 or SEQ ID NO:48), Rum7Tag (SEQ ID NO:46), RumTag (SEQ ID NO:40), Rum2Tag (SEQ ID NO:41), Rum3Tag (SEQ ID NO:42), Rum4Tag (SEQ ID NO:43), Rum5Tag (SEQ ID NO:44), Rum6Tag (SEQ ID NO:45) and Bac5Tag (SEQ ID NO:46).

[0061] In some embodiments, the other of the first and second peptide tags is selected from the group consisting of SpyCatcher (SEQ ID NO:55), SdyCatcher (SEQ ID NO:56), SnoopCatcher (SEQ ID NO:57) and an ester-forming split-protein pair. Examples of ester-forming split-protein pairs are the fragment corresponding to amino acid residues 439-587 of cpe0147 (Uniprot B1R775) (SEQ ID NO:34, DNA sequence: SEQ ID NO:68) and the fragment corresponding to amino acid residues 565-587 of cpe0147 (Uniprot B1R775) (SEQ ID NO:20, DNA sequence: SEQ ID NO:54). Other first or second peptide tags are presented in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, with corresponding DNA sequences as SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0062] In some embodiments, the peptide pair comprises or consists of a SpyTag and a SpyCatcher. In some embodiments, the peptide pair comprises or consists of a SpyTag and a SdyCatcher. In some embodiments, the peptide pair comprises or consists of a SnoopTag and a SnoopCatcher.

[0063] In some embodiments, the peptide pairs comprise or consist of truncated or modified versions of any of the above, i.e., peptide pairs that retain the ability to form isopeptide bonds but that have been further engineered.

[0064] The peptide pair may be modified, for example, by introducing mutations or alterations into any one or two of the first or second peptide tags, i.e., into any one or two of the tag and catcher. The peptide tag, i.e., the first or second peptide tag, should be able to spontaneously covalently bind to the corresponding binding partner via an isopeptide bond or via an ester bond. In this respect, each peptide tag preferably comprises one of the reactive amino acid residues involved in the formation of an isopeptide bond in an isopeptide protein. Thus, each peptide tag comprises only one reactive residue from an isopeptide bond, and does not comprise both reactive residues involved. Furthermore, when a peptide tag is modified or mutated, the reactive residues in its fragments preferably remain unchanged. This means that when a homologue of a peptide tag is used, the homologue preferably still comprises the reactive residues originally present in the original peptide tag. However, in some embodiments, when a peptide tag is modified or mutated, the reactive residues in its fragments also change.

[0065] Preferably, the reactive residue present in the tag is an asparagine or aspartic acid residue, which can form an isopeptide bond with a reactive residue of the binding partner or modified binding partner (catcher), as described above. Thus, one peptide tag contains one reactive residue and the other peptide tag contains the other reactive residue, such that no single peptide tag contains both reactive residues.

[0066] In some embodiments, both reactive residues participate in the formation of an isopeptide bond. In some embodiments, the reactive residue of the first peptide tag is different from the reactive residue of the second peptide tag. Preferably, the reactive residue present in the "catcher" is a lysine residue. In some embodiments, the reactive residue present in the "catcher" is an asparagine or aspartic acid residue. Preferably, the reactive residue present in the "tag" is an asparagine or aspartic acid residue. In some embodiments, the reactive residue present in the "tag" is a lysine residue. These residues can be combined to form an isopeptide bond.

[0067] Without being bound by theory, a third residue may be involved in the formation of an isopeptide bond. Although not directly participating in the bond, this third residue may mediate the formation of the bond. Typically, the third residue is a glutamic acid residue. The modified binding partner preferably comprises this third residue. In other words, the peptide tag, i.e., either the first, second or third peptide tag, preferably does not comprise this third residue that is present instead in the modified binding partner.

[0068] Thus, a peptide pair may comprise or consist of truncated or modified versions of any of the aforementioned peptide tags, so long as the peptide pair retains the ability to form an isopeptide bond.

[0069] Thus, in some embodiments, one of the first and second peptide tags is SpyTag (SEQ ID NO:1), SdyTag (SEQ ID NO:2), SnoopTag (SEQ ID NO:3), PhoTag (SEQ ID NO:4), EntTag (SEQ ID NO:5), KTag, BacTag (SEQ ID NO:15), Bac2Tag (SEQ ID NO:16), Bac3Tag (SEQ ID NO:17), Bac4Tag (SEQ ID NO:18), RumTrunkTag (SEQ ID NO:13 or SEQ ID NO:14), Rum7Tag (SEQ ID NO:12), RumTag (SEQ ID NO:6), Rum2Tag (SEQ ID NO:7), Rum3Tag (SEQ ID NO:8), Rum4Tag (SEQ ID NO:9), R um5Tag (SEQ ID NO:10), Rum6Tag (SEQ ID NO:11), Bac5Tag (SEQ ID NO:19) and homologues thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99% homology thereto.Similarly, the nucleic acid sequence of one of the first and second peptide tags may be SpyTag (SEQ ID NO: 35), SdyTag (SEQ ID NO: 36), SnoopTag (SEQ ID NO: 37), PhoTag (SEQ ID NO: 38), EntTag (SEQ ID NO: 39), KTag, BacTag (SEQ ID NO: 49), Bac2Tag (SEQ ID NO: 50), Bac3Tag (SEQ ID NO: 51), Bac4Tag (SEQ ID NO: 52), RumTrunkTag (SEQ ID NO: 47 or SEQ ID NO: 48), Rum7Tag (SEQ ID NO: 46), RumTag (SEQ ID NO: 40), Rum2Tag (SEQ ID NO: 41), Rum3Tag (SEQ ID NO: 42), Rum4Tag (SEQ ID NO: 43), Rum5Tag (SEQ ID NO: 44), Rum6Tag (SEQ ID NO: 45), Rum7Tag (SEQ ID NO: 46), RumTag (SEQ ID NO: 47), Rum8Tag (SEQ ID NO: 48), Rum9Tag (SEQ ID NO: 49), Rum10Tag (SEQ ID NO: 50), Rum11Tag (SEQ ID NO: 51), Rum12Tag (SEQ ID NO: 52), Rum13Tag (SEQ ID NO: 53), Rum14Tag (SEQ ID NO: 54), Rum15Tag (SEQ ID NO: 55), Rum16Tag (SEQ ID NO: 56), Rum17Tag (SEQ ID NO: 57), Rum18Tag (SEQ ID NO: 58), Rum19Tag (SEQ ID NO: 59), Rum20Tag (SEQ ID NO: Tag (SEQ ID NO: 44), Rum6Tag (SEQ ID NO: 45), Bac5Tag (SEQ ID NO: 46) and variants thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0070] In some embodiments, the other of the first and second peptide tags is selected from the group consisting of SpyCatcher (SEQ ID NO: 21), SdyCatcher (SEQ ID NO: 22), SnoopCatcher (SEQ ID NO: 23), an ester-forming split-protein pair (a fragment corresponding to amino acid residues 439 to 587 of cpe0147 (Uniprot B1R775) (SEQ ID NO: 34, DNA sequence: SEQ ID NO: 68) and cpe0147 (Uniprot B1R775) (such as a fragment corresponding to amino acid residues 565-587 of SEQ ID NO:20, DNA sequence: SEQ ID NO:54), SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, and homologues thereof having at least 60% homology thereto, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% homology thereto. Similarly, the other nucleic acid sequence of the first and second peptide tags is selected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 and variants thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99% sequence identity thereto.

[0071] In some embodiments the peptide pair is SpyTag (SEQ ID NO: 1) or a homologue thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto, and It comprises or consists of SpyCatcher (SEQ ID NO: 21) or a homologue thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto.Similarly, the nucleic acid sequence of the peptide pair may be SpyTag (SEQ ID NO: 35) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto, and SpyC. atcher (SEQ ID NO: 55) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0072] In some embodiments the peptide pair is SpyTag (SEQ ID NO: 2) or a homologue thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto, and and SdyCatcher (SEQ ID NO: 22) or a homologue thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto.Similarly, the nucleic acid sequence of the peptide pair may be SpyTag (SEQ ID NO: 36) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto, and SpyC. atcher (SEQ ID NO: 56) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0073] In some embodiments the peptide pair is SnoopTag (SEQ ID NO:3) or a homologue thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto, and and SnoopCatcher (SEQ ID NO: 23) or a homologue thereof having at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto.Similarly, the nucleic acid sequence of the peptide pair may be SnoopTag (SEQ ID NO: 37) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto, and SnoopTag (SEQ ID NO: 37) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% sequence identity thereto. It may comprise or consist of pCatcher (SEQ ID NO: 57) or a variant thereof having at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0074] Fusion of a first peptide tag to a protein and a second peptide tag to an antigen By changing the position where the first peptide tag is fused to the protein, the orientation of the antigen on the particle can be changed. This can be done to allow optimal display of the most important epitopes of the antigen. The optimal orientation may vary from antigen to antigen.

[0075] The epitopes of a particular monoclonal antibody may be mapped onto the antigen structure, which allows one to determine which epitopes are accessible after conjugation of the antigen to the particle. Specifically, the orientation of the antigen may be determined by measuring the binding between a particular monoclonal antibody and a complex of the antigen bound to the particle (antigen:particle complex), such as by using ELISA or another affinity measurement technique (e.g., Attana). Cryo-electron microscopy may be used to determine the structure of the entire antigen:particle complex. If the antigen contains a functional binding epitope, a binding assay may be performed to determine whether the epitope is exposed or hidden in the final antigen:particle complex.

[0076] By changing the position where the second peptide tag is fused to the antigen, the orientation of the antigen on the particle can be changed. This can be done to allow optimal display of the most important epitopes of the antigen. The optimal orientation may vary from antigen to antigen.

[0077] In some embodiments, the first peptide tag is fused to the N-terminus of the protein. In other embodiments, the first peptide tag is fused to the C-terminus of the protein. In other embodiments, the first polynucleotide is inserted in frame into the coding sequence of the protein. The fusion protein may include a linker between the first peptide tag and the protein.

[0078] Similarly, in some embodiments, the second peptide tag is fused to the N-terminus of the antigen. In other embodiments, the second peptide tag is fused to the C-terminus of the antigen. In other embodiments, the second polynucleotide is inserted in frame into the coding sequence of the protein. The fusion protein may include a linker between the second peptide tag and the antigen.

[0079] Disease and medical indications The present invention is a novel, versatile and easy-to-use approach to conjugate various antigens to nanoparticles or VLPs directly in the cells where the nanoparticles or VLPs are expressed, i.e. in vivo. Depending on the antigen, the composition can be used to prevent and / or treat a wide range of diseases. Diseases that the present invention can be used to prevent and / or treat include, but are not limited to, cancer, cardiovascular diseases, allergic diseases, chronic diseases, neurological diseases, and / or infectious diseases. Antigens are typically peptides, polypeptides or proteins or fragments thereof, i.e. they comprise or consist of amino acid sequences.

[0080] In some embodiments, at least one cancer disease associated antigen is linked to a protein, such as a particle-forming protein described herein, via an interaction between a first peptide tag and a second peptide tag. In further embodiments, the composition may be used for the prevention and / or treatment of cancer and / or cancer with which the antigen is associated.

[0081] In some embodiments, at least one cardiovascular disease associated antigen is linked to a protein, such as a particle-forming protein described herein, via an interaction between a first peptide tag and a second peptide tag. In further embodiments, the composition may be used in the prevention and / or treatment of cardiovascular disease and / or the cardiovascular disease with which the antigen is associated.

[0082] In some embodiments, at least one antigen associated with an allergic disease is linked to a protein, such as a particle-forming protein described herein, via an interaction between the first and second peptide tags. In further embodiments, the composition can be used for the prevention and / or treatment of an allergic disease and / or an allergic disease associated with the antigen.

[0083] In some embodiments, at least one infectious disease associated antigen is linked to a protein, such as a particle-forming protein described herein, via an interaction between a first peptide tag and a second peptide tag, hi further embodiments, the composition can be used for the prevention and / or treatment of an infectious disease and / or an infectious disease with which the antigen is associated.

[0084] In some embodiments, at least one chronic disease associated antigen is linked to a protein, such as a particle-forming protein described herein, via an interaction between a first peptide tag and a second peptide tag, hi further embodiments, the composition can be used for the prevention and / or treatment of a chronic disease and / or a chronic disease with which the antigen is associated.

[0085] In some embodiments, at least one antigen associated with a neurological disorder is linked to a protein, such as a particle-forming protein described herein, via an interaction between a first peptide tag and a second peptide tag. In further embodiments, the composition can be used for the prevention and / or treatment of a neurological disorder and / or a neurological disorder with which the antigen is associated.

[0086] In some embodiments, an antigen associated with at least one viral disease is linked to a protein, such as a particle-forming protein described herein, via an interaction between a first peptide tag and a second peptide tag. In further embodiments, the composition can be used for the prevention and / or treatment of a chronic disease associated with the antigen. In some embodiments, the viral disease is caused by a coronavirus, such as SARS-CoV-2, malaria, tuberculosis, HIV, or influenza.

[0087] A non-exhaustive list of antigens that may be used in the present invention is outlined in Tables 1 and 2. Table 1 further provides examples of specific diseases with which the antigens are associated, as well as examples of patient groups that may be in need of prevention and / or treatment with the present compositions.

[0088] Relevant antigens include hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, neuraminidase, human melanoma protein gp100, human melanoma protein melan-A / MART1, HIV envelope protein, M2e, VAR2CSA, ICAM1, CSP, dengue virus NS1, dengue virus envelope protein, chikungunya virus envelope protein, tyrosinase, HCV E2, NA17-Ant, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1, EGRFvIII, endoglin, ANGPTL-3, CSPG4, CTLA-4, HER2, IgE, IL-1 beta, IL-5, IL-13, IL-17, IL-22, IL-31, IL-33, TSLP, NGF and (IHNV) G protein, lymphotoxins such as lymphotoxin alpha or beta, lymphotoxin receptor, receptor activator of nuclear factor kB ligand, vascular endothelial growth factor VEGF, VEGF receptor, IL-23 p19, ghrelin, CCL21, CXCL12, SDF-1, M-CSF, MCP-1, endoglin, GnRH, TRH, eotaxin, bradykinin, BLC, TNF-α, amyloid beta peptide A, angiotensin, gastrin, progastrin, CETP, CCR5, C5a, CXCR4, Des-Arg-bradykinin, GnRH peptide, angiotensin peptide or TNF peptide.

[0089] In some embodiments, the antigen is hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, neuraminidase, human melanoma protein gp100, human melanoma protein melan-A / MART1, HIV envelope protein, M2e, VAR2CSA, ICAM1, CSP, dengue virus NS1, dengue virus envelope protein, chikungunya virus envelope protein, tyrosinase, HCV E2, NA17-Ant, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1, EGRFvIII, endoglin, ANGPTL-3, CSPG4, CTLA-4, HER2, IgE, IL-1 beta, IL-5, IL-13, IL-17, IL-22, IL-31, IL-33, TSLP, NGF and (IHNV) G protein, lymphotoxins such as lymphotoxin alpha or beta, lymphotoxin receptor, receptor activator of nuclear factor kB ligand, vascular endothelial growth factor VEGF, VEGF receptor, IL-23 An antigenic fragment or variant of p19, ghrelin, CCL21, CXCL12, SDF-1, M-CSF, MCP-1, endoglin, GnRH, TRH, eotaxin, bradykinin, BLC, TNF-α, amyloid beta peptide A, angiotensin, gastrin, progastrin, CETP, CCR5, C5a, CXCR4, Des-Arg-bradykinin, GnRH peptide, angiotensin peptide or TNF peptide, which may be better at inducing a stronger immune response than the corresponding antigen. Thus, in some embodiments the protein sequence of the antigenic fragment or antigenic variant is a homologue of the corresponding antigen and has at least 60% homology thereto, such as at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% homology thereto. In some embodiments the antigenic fragment or antigenic variant is encoded by a polypeptide. The polypeptide may consist of or comprise a nucleic acid sequence variant of the corresponding native antigen, wherein the nucleic acid sequence variant has at least 60% sequence identity thereto, such as at least 65%, such as at least 70%, for example at least 60%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0090] Desirably, the cells are capable, upon transfection and expression of the antigen and particle-forming protein, each fused to a tag as described herein, of forming self-assembled particles displaying the antigen linked to the particle-forming protein, which may be assessed by relevant methods known to those skilled in the art, such as those disclosed in Examples 2 and 3 of the present disclosure.

[0091] The compositions of the invention may also be used against other diseases and / or may use other antigens than those listed herein.

[0092] In one embodiment of the invention, the medical indication is selected from the group consisting of cardiovascular disease, immune inflammatory disease, chronic disease, neurological disease, infectious disease, and cancer. In a particular embodiment, the medical indication is an immune inflammatory disease. In another particular embodiment, the medical indication is a cardiovascular disease. In another embodiment, the medical indication is a chronic disease. In another embodiment, the medical indication is a neurological disease. In another embodiment, the medical indication is an infectious disease. In another embodiment, the medical indication is cancer.

[0093] In another embodiment, the antigen is a polypeptide, peptide and / or antigenic fragment of a polypeptide associated with an abnormal physiological response, such as cardiovascular disease and / or an allergic response / disease. In a particular embodiment, the abnormal physiological response is cancer.

[0094] In a further embodiment, the antigen is a protein, peptide and / or antigenic fragment associated with a medical indication disclosed herein.

[0095] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0096] [Table 2-1] [Table 2-2]

[0097] Cancer and related antigens In 2012, more than 14 million adults worldwide were diagnosed with cancer and more than 8 million died from the disease. Therefore, there is a need for efficient cancer treatments.

[0098] One of the hallmarks of cancer cells is the abnormal expression levels of genes and proteins. One example of a cancer-associated gene is HER2, which is overexpressed in 20% of all breast cancers and is associated with high metastatic potential and decreased patient survival. Cancer cells express cancer-associated antigens in a manner that immunologically distinguishes them from normal cells, but most cancer-associated antigens are only weakly immunogenic because most cancer-associated antigens are "self" proteins that are generally tolerated by the host. The present compositions can be used to express in cells of a subject particles that display antigens that can activate the immune system in response to antigens, such as cancer-associated antigens, to overcome immune tolerance to such antigens. Different cancers are characterized by having different cancer-associated antigens. Survivin is believed to be overexpressed in most cancer cells and may also be used in the present invention. Thus, the present invention may be used to treat / prevent most types of cancer that overexpress tumor-associated antigens.

[0099] The invention thereby provides compositions capable of activating the immune system to react against, for example, cancer-associated antigens and overcome immune tolerance to such antigens, hi further embodiments, the compositions may be used for the prevention and / or treatment of cancers associated with the antigen.

[0100] In another embodiment, the present invention is used to treat / prevent any type of cancer that overexpresses the antigen. The type of cancer for which the present invention can be used is determined by the choice of antigen.

[0101] Oncoviruses are known to cause cancer. Thus, in one embodiment, the vaccine of the invention comprises an oncovirus-associated antigen linked to a protein, preferably a particle-forming protein, via an isopeptide bond or an ester bond.

[0102] In a further embodiment, the composition may be used for the prevention and / or treatment of cancer with which the antigen is associated.

[0103] In one embodiment, the antigen is selected from the group consisting of adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary (CUP), Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, adult acute lymphocytic, leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung The antigen fragment of a protein or peptide or polypeptide associated with a cancer selected from the group consisting of carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, pediatric non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, adult soft tissue carcinosarcoma, skin cancer, basal and squamous cell skin cancer, melanoma skin cancer, Merkel cell skin cancer, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, primary macroglobulinemia, and Wilms' tumor.

[0104] In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, and uterine serous cancer.

[0105] These Her2 / Neu (ERBB2) and / or survivin or antigen fragments thereof are linked to the VLPs or nanoparticles described herein to form VLPs or nanoparticles that can react against cells with high expression of Her2 / Neu (ERBB2) and / or survivin, for example, and activate the immune system to overcome immune tolerance. In one embodiment, Her2 / Neu (ERBB2) and / or survivin can be used for the prevention and / or treatment of the cancer diseases and / or other cancer diseases disclosed herein. Using similar reasoning, other cancer disease associated antigens may be used for any cancer disease. Such antigens may be selected from the group consisting of interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein Melan-A / MART1, tyrosinase, NA17-Ant, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1, and (IHNV) G protein.

[0106] In one embodiment, the antigen of the present disclosure is Her2 / Neu (ERBB2) and / or survivin or an antigenic fragment thereof, which antigen is associated with and directed to at least one of the types of cancer disclosed herein. In one embodiment, the antigen of the present disclosure is interleukin-17, hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, human melanoma protein gp100, human melanoma protein melan-A / MART1, tyrosinase, NA17-Ant, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1, and (IHNV) G protein or an antigenic fragment thereof, which antigen is associated with and directed to at least one of the types of cancer disclosed herein.

[0107] Cardiovascular Disease and Related Antigens An estimated 17.3 million people died from cardiovascular disease in 2008, which represents 30% of the total deaths worldwide. Addressing risk factors such as tobacco use, unhealthy diet and obesity, physical inactivity, hypertension, diabetes, elevated lipids, etc., is important for the prevention of cardiovascular disease. However, the need for preventive pharmaceutical measures is becoming increasingly important. The present invention may be used to treat / prevent most types of cardiovascular disease. The type of cardiovascular disease for which the present invention may be used is determined by the choice of antigen.

[0108] In one embodiment of the invention, the antigen is an antigenic fragment of a protein or peptide or polypeptide associated with a disease selected from the group including lipid disorders such as hyperlipidemia, Type I, II, III, IV, or V hyperlipidemia, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatosis, cholesterol acetyltransferase deficiency, arteriosclerotic conditions (e.g., atherosclerosis), coronary artery disease, cardiovascular disease.

[0109] In one embodiment of the invention, the antigen is an antigenic fragment of a protein or peptide or polypeptide associated with a cardiovascular disease. In a further embodiment, the cardiovascular disease is selected from the group consisting of dyslipidemia, atherosclerosis, and hypercholesterolemia.

[0110] One example of a polypeptide related to cardiovascular disease is PCSK9, which acts on cholesterol homeostasis. Inhibition of PCSK9 has medical significance as it can lower plasma and / or serum low-density lipoprotein cholesterol (LDL-C) levels. Reducing LDL-C reduces the risk of heart attacks, etc.

[0111] Linking a PCSK9 antigen to a VLP or nanoparticle forms a PCSK9-VLP / nanoparticle-based vaccine that can activate the immune system to produce antibodies that either bind PCSK9 and remove it from the bloodstream or block PCSK9 from binding to the LDL receptor. In one embodiment, the composition can be used for the prevention and / or treatment of the cardiovascular diseases disclosed herein and / or other cardiovascular diseases. Using similar reasoning, other cardiovascular disease-related antigens may be used for any cardiovascular disease.

[0112] In a preferred embodiment, the antigen comprises PSCK9 or an antigenic fragment thereof, and the antigen is associated with and directed to at least one of the cardiovascular diseases disclosed herein and / or other cardiovascular diseases.

[0113] Another example of a polypeptide related to cardiovascular disease is ANGPTL3, which acts on cholesterol homeostasis. Inhibition of ANGPTL3 has medical significance as it can reduce plasma and / or serum low-density lipoprotein cholesterol (LDL-C) levels. Reducing LDL-C reduces the risk of heart attacks, etc.

[0114] Linking ANGPTL3 antigen to VLP or nanoparticles forms an ANGPTL3-VLP / nanoparticle-based vaccine that can activate the immune system to produce antibodies that either bind ANGPTL3 and remove it from the bloodstream or block ANGPTL3 from binding to the LDL receptor, thereby lowering LDL-C levels and the risk of heart attack. In one embodiment, the composition can be used for the prevention and / or treatment of the cardiovascular diseases disclosed herein and / or other cardiovascular diseases. Using similar reasoning, other cardiovascular disease-related antigens can be used for any cardiovascular disease.

[0115] In a preferred embodiment, the antigen comprises ANGPTL3 or an antigenic fragment thereof, and the antigen is associated with and directed to at least one of the cardiovascular diseases disclosed herein and / or other cardiovascular diseases.

[0116] Immunoinflammatory diseases and associated antigens The prevalence of immune inflammatory diseases worldwide has risen dramatically in both developed and developing countries. According to statistics from the World Health Organization, it is estimated that hundreds of millions of subjects worldwide suffer from allergic rhinitis and 300 million people suffer from asthma, which significantly affects the quality of life of individuals and negatively impacts the socio-economic well-being of societies.

[0117] Interleukin 5 (IL-5) has been shown to play a key role in eosinophilic inflammation in various types of allergy, including severe eosinophilic asthma. Eosinophils are regulated by IL-5 with respect to their recruitment, activation, growth, differentiation, and survival, which has identified this cytokine as a prime target for therapeutic intervention.

[0118] Linking an IL-5 antigen or a fragment thereof to the particle-forming protein of the invention results in the formation of an IL-5-VLP / nanoparticle-based vaccine that can activate the immune system to respond to IL-5. Thus, the IL-5-based composition described in the present invention can be used to treat / prevent eosinophilic asthma or other immunoinflammatory diseases. Other immunoinflammatory disease-associated antigens (e.g., IgE or interleukin 17 or IL-17) can be used by the present invention using similar reasoning. Thus, the IL-17-based composition described in the present invention can be used to treat / prevent eosinophilic asthma or other immunoinflammatory diseases. The type of asthma or allergy or other immunoinflammatory disease for which the invention can be used is determined by the choice of antigen. In one embodiment, the antigen is an antigenic fragment of one or more asthma or immunoinflammatory disease-associated proteins or peptides or polypeptides disclosed herein. In a preferred embodiment, the asthma or immunoinflammatory disease is selected from the group consisting of eosinophilic asthma, allergy, nasal polyps, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome, and Churg-Strauss syndrome.

[0119] In a preferred embodiment, the antigen comprises IL-5, IL-17, or an antigenic fragment thereof, and the antigen is associated with and directed to at least one of the asthma or allergic diseases and / or other immunoinflammatory diseases disclosed herein.

[0120] Infectious diseases and related antigens Tuberculosis and malaria are two major infectious diseases. In 2012, an estimated 207 million cases of malaria occurred, resulting in over 500,000 deaths. Also, in 2012, an estimated 8.6 million people developed tuberculosis, and 1.3 million people died from the disease. Current treatments are inadequate, and some have led to drug resistance. Thus, new and efficient drugs are needed to treat / prevent tuberculosis and malaria. Malaria or tuberculosis-related antigens or fragments thereof are linked to the VLPs or nanoparticles of the invention to form a VLP or nanoparticle-based vaccine that can activate the immune system to respond, for example, to malaria or tuberculosis. Using similar reasoning, the present invention can be used to treat / prevent most infectious diseases. The type of infectious disease for which the present invention can be used is determined by the choice of antigen.

[0121] The year 2020 has been marked by the SARS-CoV-2 / COVID-19 pandemic, which has infected millions of people worldwide and therefore has attracted considerable attention to this infectious disease. Influenza is another infectious disease of interest.

[0122] In one embodiment, the antigen fused to the second peptide tag of the invention is an antigenic fragment of a protein or peptide or polypeptide associated with an infectious disease such as tuberculosis and / or malaria.

[0123] In one embodiment, an antigen from Plasmodium falciparum is fused to a second peptide tag for use in the treatment / prevention of malaria.

[0124] In a further embodiment, an antigen from M. tuberculosis is fused to a second peptide tag for use in the treatment / prevention of tuberculosis.

[0125] In further embodiments, the antigen is selected from the group consisting of Ag85A from Mycobacterium tuberculosis, PfRH5 from Plasmodium falciparum, VAR2CSA (domains, ID1-ID2a) from Plasmodium falciparum, CIDR1a domain, PfEMP1 from Plasmodium falciparum, GLURP from Plasmodium falciparum, MSP3 from Plasmodium falciparum, Pfs25 from Plasmodium falciparum, CSP from Plasmodium falciparum, PfSEA-1 from Plasmodium falciparum, or an antigenic fragment of a disclosed antigen. In another embodiment, the antigen comprises a fusion construct between MSP3 and GLURP (GMZ2) from Plasmodium falciparum.

[0126] In a further embodiment, the antigen is a hemagglutinin (HA) antigen from influenza virus or an antigenic fragment thereof.

[0127] In another embodiment, the antigen of the present invention comprises a protein, or an antigenic fragment thereof, from a pathogenic organism that causes an infectious disease.

[0128] In one embodiment, the antigen is a protein, peptide, and / or antigenic fragment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Thus, in some embodiments, the antigen is a protein, peptide, and / or antigenic fragment of SARS-CoV-2 envelope protein. In some embodiments, the antigen is a protein, peptide, and / or antigenic fragment of SARS-CoV-2 spike protein. In some embodiments, the antigen is a protein, peptide, and / or antigenic fragment of SARS-CoV-2 nucleocapsid protein. In some embodiments, the antigen is a protein, peptide, and / or antigenic fragment of SARS-CoV-2 envelope protein. In certain embodiments, the antigen is amino acids 319-591 of SARS-CoV-2 spike protein RBD (GenBank Accession Number: QIA20044.1). The antigen may be fused to a catcher or tag and may further include a C-tag purification tag.

[0129] Thus, in some embodiments, the compositions described herein include i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to a second peptide tag; The composition, wherein the antigen and the protein, when expressed in a cell, are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii display the SARS-CoV-2 antigen.

[0130] In some embodiments, the compositions described herein comprise: i. a first polynucleotide encoding a protein fused to SpyCatcher; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to a SpyTag.

[0131] In some embodiments, the compositions described herein comprise: i. a first polynucleotide encoding a protein fused to SpyCatcher; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to an SdyTag.

[0132] In some embodiments, the compositions described herein comprise: i. a first polynucleotide encoding a protein fused to SnoopCatcher; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to a SpyTag.

[0133] In some embodiments, the compositions described herein comprise: i. a first polynucleotide encoding a protein fused to a SpyTag; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to SpyCatcher.

[0134] In some embodiments, the compositions described herein comprise: i. a first polynucleotide encoding a protein fused to a SpyTag; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to SdyCatcher.

[0135] In some embodiments, the compositions described herein comprise: i. a first polynucleotide encoding a protein fused to a SnoopTag; ii. a second polynucleotide encoding a SARS-CoV-2 antigen fused to SnoopCatcher.

[0136] In one embodiment, the antigen is an influenza virus protein, peptide and / or antigenic fragment.

[0137] Antigens known to be difficult to express in heterologous systems Some antigens are difficult to express in heterologous expression systems (e.g., mammalian antigens produced in E. coli). Many antigens also exist as multiprotein complexes, further complicating production and formulation. Protein degradation or aggregation during production and conjugation can also cause problems with particle display, especially VLP display. This can lead to antigen insolubility, making in vitro production and use of VLP technology as a vaccine very complicated or impossible.

[0138] These antigens may therefore benefit from being produced by DNA / mRNA in vivo and directly conjugated to VLPs in vivo. This avoids the need to first produce and purify protein / protein complexes, as well as prolonged storage in potentially suboptimal buffers. Below are some examples of a few types of antigens that may benefit from such technology:

[0139] Interleukins: In the context of allergy and asthma, the levels of interleukins are involved in the severity of the disease. Particle technology, especially VLP technology, allows the breakthrough of immune tolerance and controls the levels of interleukins, thus alleviating the symptoms of some diseases. However, many interleukins are difficult to produce (especially in E. coli, but also in various other expression systems) and are often insoluble. Of these, IL-13, IL-31 and IL-17A could benefit from current mRNA / DNA technology.

[0140] Thus, in some embodiments, the antigen is IL-13. In some embodiments, the antigen is IL-31. In some embodiments, the antigen is IL-17.

[0141] Similarly, PCSK9 is involved in cholesterol levels, so research has focused on creating a PCSK9 vaccine. In the SARS-CoV-2 pandemic, the mutation rate of the virus is so high that new vaccines may be needed to protect against the various variants. However, both of these antigens need to be produced in eukaryotic cells, which makes production lines costly and time-consuming. Furthermore, PCSK9 has proven very difficult to stably bind to particles, including VLPs, and development has been significantly delayed due to issues with protein aggregation and degradation, requiring new PCSK9 designs to be successful. This antigen may therefore benefit from mRNA / DNA delivery technologies to reduce production time and costs.

[0142] Thus, in some embodiments, the antigen is PCSK9.

[0143] Finally, HIV trimers and influenza stem trimers are also very large proteins, making them difficult proteins to bind to particles such as VLPs, and artificially designed stem trimers have inherent stability issues. It is also known from the literature that many HIV variant sequences have proven impossible to incorporate into stem-only HIV vaccine designs due to instability issues (Zhang et al., 2021). In in vivo systems where these antigens are delivered as DNA / mRNA (different binding efficiencies and pressures while eliminating the need for prolonged processing of unstable antigens before binding to particles), DNA / mRNA particle technology could be a solution.

[0144] Thus, in some embodiments, the antigen is an HIV trimer, hi some embodiments, the antigen is an influenza virus trimer.

[0145] Expression system In this specification, i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; An expression system is also provided in which, when the first and second polynucleotides are expressed in a cell, the antigen and the protein are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle displaying the antigen, i-ii.

[0146] The expression system may consist of or include a polycistronic RNA and / or DNA construct, from which the mRNA transcribed is polycistronic. Thus, in some embodiments, the first and second polynucleotides of the expression system are encoded on the same ribonucleic acid molecule. In some embodiments, the first and second polynucleotides of the expression system are in the same open reading frame, thereby requiring only one promoter sequence to transcribe both polynucleotides. In some embodiments, the first and second polynucleotides of the expression system are in separate open reading frames, and therefore may be controlled by separate promoters.

[0147] The first peptide tag, the second peptide tag, the protein and / or the antigen may be as defined elsewhere herein.

[0148] The term "expression system" refers to a genetic construct designed to produce protein and / or RNA within a cell. Thus, an expression system can include RNA and / or DNA that is translated or transcribed into protein or DNA, respectively, within a cell.

[0149] An expression system may contain sequences necessary for gene expression in a cell. These may include a promoter, a translation initiation sequence such as a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Because there are differences in the enzymes involved in protein synthesis between prokaryotes and eukaryotes, an expression vector must contain expression elements appropriate for the selected host. For example, a prokaryotic expression system may contain a Shine-Dalgarno sequence at the translation initiation site for ribosome binding, while a eukaryotic expression system may contain a Kozak consensus sequence.

[0150] The expression system may additionally comprise a marker, such as a selectable marker, i.e. a gene that confers a trait suitable for artificial selection, so that cells containing the expression system can be selected, or a screenable marker, such as a reporter gene, i.e. a gene that allows for the distinction between cells containing the expression system and cells that do not, so that cells containing the expression system can be identified. Examples of such markers include antibiotic resistance genes, auxotrophic markers, and genes that express detectable compounds, such as colored and / or fluorescent compounds.

[0151] In some embodiments, the first polynucleotide and the second polynucleotide are both DNA polynucleotides. In some embodiments, the first polynucleotide and the second polynucleotide are both RNA polynucleotides. In some embodiments, the first polynucleotide or the second polynucleotide is a DNA polynucleotide and the other is an RNA polynucleotide.

[0152] The first polynucleotide and / or the second polynucleotide may be under the control of a promoter, such as an inducible promoter or a constitutive promoter. The first and / or the second polynucleotide may be under the control of a first and / or a second promoter, respectively, which may be the same or different, or they may be under the control of a single promoter.

[0153] The first and second polynucleotides of the expression system may be contained within the same molecule, or alternatively, the first and second polynucleotides of the expression system may be contained within different molecules, such as within two or more separate molecules.

[0154] The first and / or second polynucleotide may further comprise a secretion or excretion signal to obtain a fusion protein comprising such a signal, whereby the protein fused to the first peptide tag and / or the antigen fused to the second peptide tag is secreted or excreted from the endoplasmic reticulum and, optionally, from the cell.

[0155] The expression system of the present invention may be used for the prevention and / or treatment of a wide range of diseases as disclosed herein above.

[0156] Cells and host cells The present invention further relates to a cell, such as a host cell, comprising the polynucleotide and / or expression system disclosed herein. The polynucleotide and / or expression system may have a codon-optimized sequence. Codon optimization methods are known in the art and allow for optimized expression in heterologous host organisms or cells. In one embodiment, the cell may be selected from the group comprising bacterial, yeast, fungal, plant, mammalian and / or insect cells.

[0157] Methods for expressing a first and / or second polypeptide in a cell, such as a host cell, are well known in the art. The first or second polypeptide may be heterologously expressed from a corresponding polynucleotide sequence cloned into the genome of the cell, or the first or second polypeptide may be contained within a vector. For example, the first and / or second polynucleotides encoding the first and / or second polypeptides are cloned into the genome and the first and / or second polynucleotides encoding the first and / or second polypeptides are contained within a vector that is transformed or transfected into the cell.

[0158] The expression of the first and second polypeptides in the cell can occur in a transient manner.When the polynucleotide encoding one of the polypeptides is cloned into the genome, an inducible promoter can also be cloned to control the expression of the polypeptide.Such inducible promoters are well known in the art.Alternatively, a gene encoding a suppressor of gene silencing can also be cloned into the genome or into a vector transfected into the cell.

[0159] Therefore, further herein: i. a first polynucleotide encoding a protein fused to a first peptide tag, said first polynucleotide preferably as defined in any one of the preceding claims; ii. A cell expressing a second polynucleotide encoding an antigen fused to a second peptide tag, preferably said second polynucleotide as defined in any one of the preceding claims, The antigen and the protein, when expressed in the cell, are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby providing a cell in which i-ii form a particle displaying the antigen.

[0160] In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a mammalian cell, such as a human cell. In some embodiments, the cell is an insect cell.

[0161] In certain embodiments, the cell, such as a host cell, may be selected from the group including Escherichia coli, Spodoptera frugiperda (sf9), Trichoplusia ni (BTI-TN-5B1-4), Pichia Pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Drosophila Schneider 2 (S2), Lactococcus lactis, Chinese hamster ovary (CHO), human embryonic kidney 293, Nicotiana tabacum cv. Samsun NN, and Solanum tuberosum cv. Solara. Thus, in one embodiment, the cell is Escherichia coli. In another embodiment, the cell is Spodoptera frugiperda. In another embodiment, the cell is Pichia Pastoris. In another embodiment, the cell is Saccharomyces cerevisiae. In another embodiment, the cell is Hansenula polymorpha. In another embodiment, the cell is Drosophila Schneider 2. In another embodiment, the cell is Lactococcus lactis. In another embodiment, the cell is Chinese hamster ovary (CHO). In another embodiment, the cell is human embryonic kidney 293. In another embodiment, the cell is Trichoplusia ni (BTI-TN-5B1-4). In another embodiment, the cell is Nicotiana tabacum cv. Samsun NN. In another embodiment, the cell is Solanum tuberosum cv. Solara.

[0162] In some embodiments, a cell, such as a host cell, i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; The antigen and the protein, when expressed in a cell, are linked via an isopeptide bond or an ester bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii display the antigen, and the cell is selected from the group including bacterial, yeast, fungal, plant, mammalian and / or insect cells.

[0163] The present cells, such as host cells, can be used in the prevention and / or treatment of a wide range of diseases, as disclosed herein above. EXAMPLES

[0164] Materials and Methods for Examples 1-2 and 3 The gene sequences of Spytagged Hepatitis B core antigen, Spytagged Acinetobacter bacteriophage AP205 major coat protein and SpyCatcher fused enhanced green fluorescent protein (eGFP) were codon optimized and synthesized by Geneart.

[0165] array: >pVax1_ SpyTag-AP205 AHIVMVDAYKPTKGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA* (DNA sequence of SEQ ID NO: 85, SEQ ID NO: 88) >pVAX1_HBc-SpyTag MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHVSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKLRQILWFHISCLTFGRETVLEYLVSFGVWIRTPTAYRPPNAPILSTLPETTVVGGGGGSPGGGTPSPGGGGSQSPGGGGSQSGESQCGSAHIVMVDAYKPTK* (DNA sequence of SEQ ID NO: 86, SEQ ID NO: 89) >pVAX1-SPYCEGFP GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGSGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK* (DNA sequence of SEQ ID NO: 87, SEQ ID NO: 90) Modular combinatorial INFUSION shuffling and cloning of the constructs into a plasmid vector suitable for DNA immunization (pVAX1 - see Figure 5) was performed.

[0166] Mouse-derived C2C12 myoblast progenitor cells were transfected (reverse transfection, 2x24 well plates using Lipofectamine 2000 reagent) with: A) SPYCEGFP (n=3) B) SPYCEGFP and SpyTAP205 (n=3) C) SPYCEGFP and HBcSpyT (n=3)

[0167] Example 2 - Formation of distinct intracellular particles in transfected mammalian cells Co-transfection of eukaryotic cells with eGFP (genetically fused to SpyCatcher at the N-terminus) and Hepatitis B core antigen (genetically fused to SpyTag at the C-terminus) resulted in clear expression of both components, which could then bind to each other (through the interaction of SpyTag and SpyCatcher) to form a particulate complex displaying eGFP. Specifically, confocal laser scanning microscopy (CLSM) of mouse cells harvested 72 hours after co-transfection with SpyCatcher-eGFP and Spytagged HBcore, respectively, revealed a clear perinuclear distribution of eGFP fluorescent signal, indicating the specificity of eGFP (see Figure 1).

[0168] In contrast, transfection with SpyCatcher-eGFP and Spytagged AP205 coat protein (previously shown to spontaneously form VLPs upon expression in E. coli ) resulted in a diffuse / dimmed eGFP fluorescent signal throughout the cells (see Figure 2 ).

[0169] Overall, these results indicate that both SpyCatcher eGFP and HBcSpyT proteins were successfully expressed and that eGFP (via spyTag / SpyCatcher) was able to bind to the particulate structures formed by the HBcSpyT capsid protein.

[0170] Example 3 - Co-expression and assembly of microparticle complexes to encoded proteins in mammalian cells To further investigate whether Spytagged coat proteins (AP205 and HBcore) can be expressed to form virus-like particles, and whether SpyCatcher-eGFP protein can also bind to such particles (via spyTag / SpyCatcher), we harvested co-transfected C2C12 cells (48 hours after transfection), then sonicated for 45 seconds (three times, 30%) and fractionated by ultracentrifugation (UC) using iodixanol ultracentrifugal density gradient columns. Post-UC fractions (including controls) were run on SDS-PAGE, transferred to nitrocellulose membranes, and finally probed with a polyclonal HRP-conjugated anti-GFP antibody.

[0171] This experiment demonstrated that co-transfection of SpyCatcher eGFP and SpyTagged AP205 may have led to expression and conjugation of the two proteins, as indicated by the appearance of a protein band in the UC input lane ( / pellet lane) with a size approximately the theoretical size of the conjugate (i.e., 59 kDA), but no protein band of that size was seen in any of the UC fractions (F3-F21) that may have contained particulate proteins (Figure 3).

[0172] However, similar analysis of C2C12 cells cotransfected with SpyT-HBc and SpyCatcher-eGFP shows a clear protein band of approximately 64 kDa (i.e., the theoretical size of the SpyTHBc:SpyCatcher-eGFP conjugate) in both the UC input sample and the post-UC fractions (F3-21, F31, and F32), including the high-density fractions expected to contain the microparticle protein complexes (Figure 4). These results further demonstrate that cotransfection of Spytagged HBcore antigen and SpyCatcher-eGFP can be simultaneously expressed in mammalian cells and assembled into microparticle complexes.

[0173] DNA sequences used in Examples 4-5 to 10 The following nomenclature is used throughout Examples 5 through 10 to refer to DNA sequences that encode the indicated amino acid sequence numbers. [Table 3]

[0174] Example 5 - Expression of soluble antigens and particle-forming proteins from plasmid DNA method The DNA was cloned into the pVAX1 vector (V26020, thermoFisher) and cloned into E. coli (One shot Top10, Invitrogen, C404006). The vector was purified using a midiprep kit.

[0175] HEK293-Freestyle cells were transfected or co-transfected using FreeStyle™ MAX Reagent (16447100, Life Technologies) at 37.5ug in 30mL culture medium.

[0176] After 6 days of incubation, cells and supernatants were harvested. o The supernatant was frozen at -20°C and designated as the sample supernatant (SN). The cells were pelleted by centrifugation at 1200 RPM for 5 minutes and resuspended in PBS with Complete™ Mini, EDTA-free protease inhibitor cocktail (Sigma, 11836170001). The cells were sonicated 3 times at 30%, 45 seconds, and centrifuged at 20.000g for 10 minutes at 4°C. The supernatant was frozen at -20°C and designated as the sample "cells".

[0177] Both cells and SNs were run on a denaturing SDS+DTT gel (15 uL loaded), then the SDS gel was transferred to a nitrocellulose membrane and blocked overnight in 5% milk in TBS-T. Proteins on the membrane were detected with primary antibodies (specified for each gel) and secondary antibodies (specified for each gel) as required. Membranes were washed three times between each antibody and before developing in TBS-T. Membranes were developed with ECL substrate according to the manufacturer's instructions.

[0178] Results and Conclusions After DNA transfection in mammalian cells in vitro, expression and secretion (due to the secretion signal) of particles and soluble antigens could be detected by WB of cells and SN from transfected cells. Western blots show the appearance of bands at the expected sizes of particles or soluble antigens.

[0179] This is shown below. · Sign3-SpyC-i301-ctag ·Sign8-tandemHBc-SpyC ·Sign8-HBc-SpyT ·Sign9-Ferritin-SpyC ·Sign8-SpyT-eGFP ·Sign8-SpyT-eGFP ·Sign8-SpyC-His · Sign7-Pfs25-SpyT-Ctag ·Sign8-SpyT-eGFP

[0180] As can be seen in Figures 6 and 7, bands of the expected size were observed in cell and supernatant samples for all constructs, indicating expression and secretion of the cellular particle-forming subunit proteins and soluble proteins in human (HEK) cells following plasmid DNA transfection.

[0181] Example 6 - Particles expressed from plasmid DNA and protein binding method HEK293-Freestyle cells were co-transfected using FreeStyle™ MAX Reagent (16447100, Life Technologies) at 37.5ug in 30mL culture medium.

[0182] Cells were co-transfected with a vector encoding the particle and a vector encoding a protein carrying a tag-catcher compatible for binding.

[0183] After 6 days of incubation, cells and supernatant were harvested. The supernatant was frozen at -20°C and designated as sample Supernatant (SN). Cells were pelleted by centrifugation at 1200 RPM for 5 minutes and resuspended in PBS with Complete™ Mini, EDTA-free protease inhibitor cocktail (Sigma, 11836170001). Cells were sonicated 3 times at 30%, 45 seconds, and centrifuged at 20 000g for 10 minutes at 4°C. The supernatant was frozen at -20°C and designated as sample "Cells".

[0184] Both cells and SNs were run on a denaturing SDS+DTT gel (15 uL loaded), then the SDS gel was transferred to a nitrocellulose membrane and blocked overnight in 5% milk in TBS-T. Proteins on the membrane were detected with primary antibodies (specified for each gel) and secondary antibodies (specified for each gel) as required. Membranes were washed three times between each antibody and before developing in TBS-T. Membranes were developed with ECL substrate according to the manufacturer's instructions.

[0185] Results and Conclusions After in vitro DNA cotransfection of mammalian (HEK293) cells, conjugation mediated by isopeptide bonds of individually expressed antigen and particle-forming protein could be verified by WB by detection of bands of the expected size of the conjugated antigen and particle-forming protein.

[0186] This is shown below. Sign8-SpyT-eGFP and sign9-SpyC-Ferritin Sign8-SpyC-eGFP and sign8-Hbc-SpyT Sign8-tandemHBc-SpyC and sign8-SpyT-eGFP Sign3-SpyC-i301-ctag and sign8-SpyT-eGFP Sign9-SpyT-E2 and sign8-SpyC-eGFP Sign9-SpyT-LS and sign8-SpyC-eGFP Sign8-SpyC-His and sign9-SpyT-E2 Sign8-SpyC-His and sign9-LS-SpyT Sign8-SpyC-His and sign8-Hbc-SpyT Sign8-SpyC-Ferritin and sign7-Pfs25-SpyT Sign3-SpyC-i301-Ctag and sign7-Pfs25-SpyT Sign8-Norovirus-SpyT and sign8-SpyC-His

[0187] As seen in Figures 8, 9, and 10, bands of the expected sizes for eGFP, SpyCatcher, and Pfs25 (model antigen) bound to different particle-forming subunit proteins were observed in cells and supernatants for all constructs, indicating that antigens and particles with corresponding tags / catchers can be bound in vitro after co-transfection into human (HEK) cells.

[0188] Example 7 - Validation of nanoparticle formation by ultracentrifugation and Western blotting method After transfection and harvesting of HEK cells, the supernatant was loaded onto an Optiprep step gradient (23, 29 and 35%) followed by centrifugation for 3 h 30 min at 47800 g and 16° C. The gradient was then titrated into fractions (F1-F12), each fraction containing approximately 250 uL.

[0189] If particles were formed, they would be expected to be found in the intermediate fractions (F3-F8).

[0190] All fractions were run on a denaturing SDS+DTT gel (15uL loaded), then the SDS gel was transferred to a nitrocellulose membrane and blocked overnight in TBS-T 5% milk. Proteins on the membrane were detected with primary antibodies (specified for each gel) and secondary antibodies (specified for each gel) as required. Membranes were washed three times between each antibody and before developing in TBS-T. Membranes were developed with ECL substrate according to the manufacturer's instructions.

[0191] Results and Conclusions After DNA co-transfection or transfection in mammalian cells in vitro, we were able to detect the formation and possible secretion of particles. Particle formation can be expected if the protein is found within the 3rd to 8th fractions from the density gradient. This was the case in the following cases: ·Sign3-SpyC-i301-Ctag ·Sign8-tandemHBc-SpyCatcher ·Sign9-Ferritin-SpyC ·Sign8-SpyT-eGFP+sign9-SpyC-Ferritin ·Sign8-SpyC-His+sign9-SpyT-E2 ·Sign8-SpyC-His+sign9-LS-SpyT ·Sign9-SpyC-Ferritin+sign7-Pfs25-SpyT ·Sign3-SpyC-i301+sign7-Pfs25-SpyT ·Sign9-SpyT-E2+sign8-SpyC-eGFP ·Sign9-LS-SpyT+sign8-SpyC-eGFP

[0192] As can be seen in Figures 11 and 12, particle formation and antigen-bound particle formation were observed for all depicted constructs, as bands of expected size were present in the relevant fractions (fractions 3-8). This therefore indicates that there was not only expression and secretion of particle subunits and soluble antigen in vivo, but also formation of particles and antigen-bound particles in vitro after transfection of plasmid DNA into human (HEK) cells.

[0193] Example 8 - Visualization of particle formation by transmission electron microscopy After transfection of HEK cells, harvesting, and purification by ultracentrifugation, samples were run under transmission electron microscopy (TEM).

[0194] After cotransfection or transfection of DNA into mammalian cells in vitro, the formation of secreted particles was visualized by TEM.

[0195] This is shown below. ·Sign9-Ferritin-SpyC ·Sign8-SpyC-His+sign9-LS-SpyT ·Sign9-SpyT-E2+sign8-SpyC-His ·Sign8-SpyT-eGFP+sign9-SpyC-Ferritin ·Sign3-SpyC-i301-Ctag+sign7-Pfs25-SpyT-Ctag ·Sign9-SpyC-Ferritin+sign7-Pfs25-SpyT-Ctag

[0196] As can be seen from Figure 13, particles of the expected size were formed in the supernatants of transfected or co-transfected cells, thus further confirming that particles and conjugated particles can be formed in the supernatants of cells transfected or co-transfected in vitro from plasmid DNA.

[0197] Example 9 - Verification of intracellular conjugation of antigens to nanoparticle-forming proteins method The DNA was cloned into the pVAX1 (V26020, thermoFisher) vector and cloned into E. coli (One shot Top10, Invitrogen, C404006). The vector was purified using a midiprep kit.

[0198] HEK293-Freestyle cells were transfected or co-transfected using FreeStyle™ MAX Reagent (16447100, Life Technologies) at 37.5ug in 30mL culture medium.

[0199] After 6 days of incubation, cells and supernatant were harvested. The supernatant was frozen at -20°C and designated as "supernatant (SN)". Cells were pelleted by centrifugation at 1200 RPM for 5 minutes, resuspended in 1x SDS+DTT, frozen at -20°C and designated as "cells".

[0200] Both cells and SNs were run on a denaturing SDS+DTT gel (15uL loaded), then the SDS gel was transferred to a nitrocellulose membrane and blocked overnight in TBS-T 5% milk. Proteins on the membrane were detected with primary antibodies (specified for each gel) and secondary antibodies (specified for each gel) as required. Membranes were washed three times between each antibody and before developing in TBS-T. Membranes were developed with ECL substrate according to the manufacturer's instructions.

[0201] Results and Conclusions This technique allows visualization of binding between particles and soluble antigens inside cells. We previously showed that some particles can bind some antigens, but it was not clear whether this was happening after secretion or already inside the cell. This experiment shows that binding is happening inside the cell, as we observe binding bands from cell substrates harvested with SDS+DTT (these compounds inhibit binding after harvest).

[0202] This is shown below. Sign8-SpyC-His and sign9-LS-SpyT ·Sign8-SpyT-eGFP+sign9-SpyC-Ferritin Sign3-SpyC-i301-ctag and sign8-SpyT-eGFP Sign8-eGFP-SpyC and sign9-LS-SpyT

[0203] As can be seen in Figure 14, particles and soluble antigens could be bound not only in the supernatant but also inside the cells, and indeed a band of the expected binding size was visible in cells harvested with SDS+DTT, indicating that binding occurred inside the cells during in vitro culture.

[0204] Example 10 - Immunization of mice with plasmid DNA method DNA encoding particles and / or soluble antigens was used for vaccination of mice. Balb / c mice were immunized with 30 μg LS-SpyT and 30 μg SpyC (N=6), or 30 μg SpyC (N=4), or 30 μg E2-SpyT and 30 μg SpyC (N=6). DNA was formulated in PBS and injected into the right thigh muscle.

[0205] Mice were immunized on day 0 and week 5, and blood was taken at weeks 3 and 4 after the prime and boost. Serum was isolated from the blood and ELISA was performed for the detection of anti-SpyC IgG. For this purpose, 96-well plates (Nunc MaxiSorp) were coated with 0.1 μg / well SpyC in PBS overnight at 4° C. Plates were blocked for 1 h at room temperature (RT) using 0.5% skim milk in PBS. Mouse sera were diluted 1:50 or 1:10 in blocking buffer and added to the plates in 2-fold dilutions followed by incubation at room temperature for 1 h. Plates were washed 3 times with PBS between steps. To measure total serum IgG, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Life technologies, A16072) was diluted 1:1000 in blocking buffer followed by incubation at room temperature for 1 h. Plates were developed with TMB X-tra substrate (Kem-En-Tec, 4800A) and absorbance was measured at 450 nm. Data were collected on a BioSan HiPo MPP-96 microplate reader and analyzed using GraphPad Prism (version 8.4.3, San Diego, USA).

[0206] Results and Conclusions After DNA immunization of mice, mice receiving DNA encoding particles and DNA encoding SpyC showed higher IgG titers against SpyC only after the first immunization compared to mice receiving only DNA encoding SpyC. Furthermore, this trend became even higher after booster immunizations. This was true for both groups of mice receiving SpyC in combination with either LS or E2 particles.

[0207] This is shown in Figure 15, where mice administered DNA encoding particles and DNA encoding SpyC have higher IgG titers to SpyC after the first immunization compared to mice administered only DNA encoding SpyC, as shown by ELISA.

[0208] Example 11 - Expression of soluble antigens and particle-forming proteins from mRNA HEK293-Freestyle cells will be transfected or co-transfected using FreeStyle™ MAX Reagent (16447100, Life Technologies) with 37.5 μg per 30 mL culture medium.

[0209] After 6 days of incubation, the cells and supernatant will be harvested. The supernatant will be stored at -20 o The supernatant will be frozen at -20°C and designated as sample "supernatant (SN)". The cells will be pelleted by centrifugation at 1200RPM for 5 minutes and resuspended in PBS with Complete™ Mini, EDTA-free protease inhibitor cocktail (Sigma, 11836170001). The cells will be sonicated 3 times at 30%, 45 seconds, and centrifuged at 20000g for 10 minutes at 4°C. The supernatant will be frozen at -20°C and designated as sample "cells".

[0210] Both cells and SNs will be run on a denaturing SDS+DTT gel (15 μL loaded). The SDS gel will be transferred to a nitrocellulose membrane and blocked overnight with 5% milk in TBS-T. Proteins on the membrane will be detected with primary and, if necessary, secondary antibodies. The membrane will be washed three times between each antibody and before developing in TBS-T. The membrane will be developed with ECL substrate according to the manufacturer's instructions.

[0211] After transfection of the mRNA in mammalian cells in vitro, expression and (due to the secretion signal) of the particles and soluble antigens will be detected by Western blotting as described in Example 5.

[0212] Bands of the expected size in cells and supernatants for all constructs (particle-forming subunit proteins or soluble protein antigens) indicate expression and secretion of soluble protein in human (HEK) cells after mRNA transfection.

[0213] Example 12 - Validation of conjugation of soluble antigens to different particle-forming proteins expressed from mRNA HEK293-Freestyle cells will be co-transfected with 37.5 μg (18.75 μg of each RNA) in 30 mL culture medium using FreeStyle™ MAX Reagent (16447100, Life Technologies).

[0214] Cells will be co-transfected with a vector encoding the particle and a vector encoding a protein with a tag-catcher suitable for binding.

[0215] After 6 days of incubation, cells and supernatant will be harvested. The supernatant will be frozen at -20oC and will be referred to as sample Supernatant (SN). Cells will be pelleted by spinning down at 1200RPM for 5 minutes and resuspended in PBS with Complete™ Mini, EDTA-free protease inhibitor cocktail (Sigma, 11836170001). Cells will be sonicated 3 times for 45 seconds at 30% and centrifuged at 20000g for 10 minutes at 4°C. The supernatant will be frozen at -20°C and will be referred to as sample "Cells".

[0216] Both cells and SNs will be run on a denaturing SDS+DTT gel (15 μL loaded). The SDS gel will be transferred to a nitrocellulose membrane and blocked overnight with 5% milk in TBS-T. Proteins on the membrane will be detected with primary and, if necessary, secondary antibodies. The membrane will be washed three times between each antibody and before developing in TBS-T. The membrane will be developed with ECL substrate according to the manufacturer's instructions.

[0217] After co-transfection of mRNA in mammalian cells in vitro, binding of the particles to the expressed, secreted and soluble antigen will be detected by WB by detecting bands of the expected size of the particles and antigen, similar to that described in Example 6.

[0218] Example 13 - Verification of nanoparticle formation when expressed from transfected mRNA by ultracentrifugation and Western blotting After transfection and harvesting of Hek cells with mRNA, the supernatant will be loaded onto an Optiprep step gradient (23, 29 and 35%) followed by centrifugation for 3 hours and 30 minutes at 47800g and 16°C. The gradient will then be titrated into fractions (F1-F12) and each fraction will contain approximately 250uL.

[0219] If particles are formed, they are expected to be found in the intermediate fractions (F3-F8).

[0220] All fractions will be run on a denaturing SDS+DTT gel (15 μL loaded). The SDS gel will be transferred to a nitrocellulose membrane and blocked overnight with 5% milk in TBS-T. Proteins on the membrane will be detected with primary and, if necessary, secondary antibodies. The membrane will be washed three times between each antibody and before developing with TBS-T. The membrane will be developed with ECL substrate according to the manufacturer's instructions.

[0221] After co-transfection or transfection of mRNA in mammalian cells in vitro, particle formation and possible secretion will be detected similarly to that described in Example 7. Particle formation can be expected if the protein is found within the 3rd to 8th fractions from the density gradient.

[0222] Example 14 - Visualization of particle formation by transmission electron microscopy As described in Example 8, secreted and assembled particles expressed from the mRNA constructs in HEK cells will be detected by transmission electron microscopy.

[0223] Example 15 - Validation of intracellular conjugation of antigens to nanoparticle-forming proteins when expressed from mRNA HEK293-Freestyle cells will be transfected or co-transfected with 37.5 μg of RNA per 30 mL culture using FreeStyle™ MAX Reagent (16447100, Life Technologies).

[0224] After 6 days of incubation, the cells and supernatant will be harvested. The supernatant will be frozen at -20°C and will be designated as sample Supernatant (SN). The cells will be pelleted by centrifugation at 1200 RPM for 5 minutes, resuspended in 1xSDS+DTT, frozen at -20°C and will be designated as sample "Cells".

[0225] Both cells and SNs will be run on a denaturing SDS+DTT gel (15 μL loaded). The SDS gel will be transferred to a nitrocellulose membrane and blocked overnight with 5% milk in TBS-T. Proteins on the membrane will be detected with primary and, if necessary, secondary antibodies. The membrane will be washed three times between each antibody and before developing in TBS-T. The membrane will be developed with ECL substrate according to the manufacturer's instructions.

[0226] This allows visualization of binding between the particles and soluble antigen inside the cells, similar to that shown in Example 9. This would indicate that binding occurs intracellularly when the construct is expressed from mRNA.

[0227] Example 16 - mRNA Immunization in Mice The mRNA encoding the particles and / or soluble antigens (particles and antigens, respectively, fused to separate peptide tags) will be used for vaccination in mice, similar to that described in Example 10.

[0228] Balb / c mice will be immunized with either a combination of mRNA encoding the particles and mRNA encoding the soluble antigen, or with mRNA encoding the soluble antigen alone. Mice will be immunized on days 0 and 5, and blood will be taken at weeks 3 and 4 after the prime and boost.

[0229] Serum will be separated from the blood and an ELISA will be performed for the detection of antigen-specific IgG. For this purpose, 96-well plates (Nunc MaxiSorp) will be coated with 0.1 μg / well SpyC in PBS overnight at 4° C. The plates will be blocked for 1 h at room temperature (RT) using 0.5% skim milk in PBS. Mouse sera will be diluted 1:50 in blocking buffer and added to the plates in 2-fold dilutions followed by incubation for 1 h at RT. The plates will be washed 3 times with PBS between steps. To measure total serum IgG, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Life technologies, A16072) will be diluted 1:1000 in blocking buffer followed by incubation for 1 h at RT. The plates will be developed with TMB X-tra substrate (Kem-En-Tec, 4800A) and the absorbance will be measured at 450 nM. Data will be collected on a BioSan HiPo MPP-96 microplate reader and analyzed using GraphPad Prism (version 8.4.3, San Diego, USA).

[0230] Following mRNA immunization in mice, mice administered RNA encoding particles and soluble antigen (thus forming a combined antigen-particle complex) have higher IgG titers against the soluble antigen compared to mice administered RNA encoding the soluble antigen alone. Similar to the results shown in Example 10, this trend is expected to be even higher after booster immunizations. References

[0231] De Vincenzo, R.;Conte, C.;Ricci, C.;Scambia, G.;Capelli, G. Long-term efficacy and safety of human papillomavirus vaccination. Int. J. Womens. Health2014,6, 999-1010. Ilva Lieknina, Gints Kalnins, Inara Akopjana, Janis Bogans, Mihails Sisovs, Juris Jansons, Janis Rumnieks, Kaspars Tars: Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data;J Nanobiotechnology. 2019;17: 61. Schiller, J.;Lowy, D. Explanations for the high potency of HPV prophylactic vaccines. Vaccine2018,36, 4768-4773. Schiller, J.T.;Castellsague, X.;Garland, S.M. A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine2012,30, F123-F138. Wadhwa M, Knezevic I, Kang HN, Thorpe R. Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility. Biologicals. 2015;43(5):298-306. doi:10.1016 / j.biologicals.2015.06.004 Young PG, Yosaatmadja Y, Harris PW, Leung IK, Baker EN, Squire CJ. Harnessing ester bond chemistry for protein ligation. Chem Commun (Camb). 2017;53(9):1502-1505. doi:10.1039 / c6cc09899a Zakeri, B. et al. J. Am. Chem. Soc.,2010,132(13), pp 4526-4527 Zakeri, B. et al. Proceedings of the National Academy of Sciences 109(12), E690-E697. 2012. Zhang, P., Narayanan, E., Liu, Q.et al. A multicladeenv-gagVLP mRNA vaccine elicits tier-2 HIV-1-neutralizing antibodies and reduces the risk of heterologous SHIV infection in macaques. Nat Med(2021). https: / / doi.org / 10.1038 / s41591-021-01574-5

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[0232] item 1. A composition for use in the prevention and / or treatment of a disease, comprising: i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; The composition, wherein the antigen and the protein, when expressed in a cell, are linked via an isopeptide bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii display the antigen.

[0233] 2. The composition for use according to item 1, wherein the protein is a particle-forming protein such as a viral capsid protein, such as a glycoprotein, or a viral envelope protein.

[0234] 3. The protein is selected from the group consisting of a protein from a hepatitis virus, such as hepatitis B or E, for example a core protein from hepatitis B virus, a protein from a norovirus, such as NoV, a protein from a papillomavirus, such as a human papillomavirus (HPV), preferably HPV16 or HPV18, such as HPV L1, a protein from a polyomavirus, such as polyomavirus vp1 (PyV), a protein from a calicivirus, such as feline calicivirus (FCV), preferably FCV VP1, a protein from a circovirus, such as porcine circovirus (PCV), preferably PCV2 ORF2, a protein from a nervous necrosis virus (NNV), such as the NNV coat protein, a protein from a parvovirus, such as canine parvovirus (CVP), preferably CPV VP2, goose parvovirus (GPV) or porcine parvovirus (PPV), preferably GPV or PPV, or a structural protein from parvovirus B19, an enteritis virus, for example a mink enteritis virus (MEV), preferably MEV. 3. The composition for use according to item 2, wherein the protein is a protein from a protoparvovirus, such as VP2, or Duck Plague Virus (DPV), preferably a DPV structural protein.

[0235] 4. The composition for use according to any one of the preceding items, wherein the protein is a bacteriophage protein, such as a protein from Salmonella virus P22, from MS2, QBeta, PRR1, PP7, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage M11, bacteriophage MX1, bacteriophage NL95, bacteriophage f2 or Cb5.

[0236] 5. The composition, wherein the first and / or second peptide tag is derived from the cleavage of a protein containing an intramolecular isopeptide bond, or is identified from a library by using known binding partners, or is designed in silico to obtain complementary binding partners each containing reactive amino acids involved in isopeptide bond formation, preferably the first or second peptide tag is selected from the group consisting of SpyTag (SEQ ID NO: 1), SdyTag (SEQ ID NO: 2), SnoopTag (SEQ ID NO: 3), PhoTag (SEQ ID NO: 4), EntTag (SEQ ID NO: 5), KTag, a 13. The composition for use according to any one of the preceding claims, wherein the tag is selected from the group consisting of BacTag (SEQ ID NO: 15), Bac2Tag (SEQ ID NO: 16), Bac3Tag (SEQ ID NO: 17), Bac4Tag (SEQ ID NO: 18), RumTrunkTag (SEQ ID NO: 13 or SEQ ID NO: 14), Rum7Tag (SEQ ID NO: 12), RumTag (SEQ ID NO: 6), Rum2Tag (SEQ ID NO: 7), Rum3Tag (SEQ ID NO: 8), Rum4Tag (SEQ ID NO: 9), Rum5Tag (SEQ ID NO: 10), Rum6Tag (SEQ ID NO: 11) and Bac5Tag (SEQ ID NO: 19); and / or preferably said first or said second peptide tag comprises a tag selected from the group consisting of SpyCatcher (SEQ ID NO: 21), SdyCatcher (SEQ ID NO: 22), SnoopCatcher (SEQ ID NO: 23), and an ester-forming split protein pair.

[0237] 6. The composition for use according to any one of the preceding items, wherein the disease is a cancer, a lipid disorder, a cardiovascular disease, an immunoinflammatory disease, a chronic disease, a neurological disease, an allergic reaction / disease and / or an infectious disease such as an infectious disease selected from the group consisting of malaria, tuberculosis, and a disease caused by a virus such as coronavirus, e.g. SARS-CoV-2, malaria, tuberculosis, HIV, HCV, dengue, chikungunya, yellow fever, HBV or influenza.

[0238] 7. The composition for use according to any one of the preceding items, wherein the antigen is a protein, peptide and / or antigenic fragment of the group consisting of cancer-specific polypeptides, cardiovascular disease associated polypeptides, asthma associated polypeptides, polypeptides, nasal polyp associated polypeptides, atopic dermatitis associated polypeptides, eosinophilic esophagitis associated polypeptides, hypereosinophilic syndrome associated polypeptides, Churg-Strauss syndrome associated polypeptides and polypeptides associated with pathogenic organisms, preferably wherein the antigen is a protein, peptide and / or antigenic fragment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0239] 8. The antigen is hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, neuraminidase, human melanoma protein gp100, human melanoma protein Melan-A / MART1, HIV envelope protein, M2e, VAR2CSA, ICAM1, CSP, dengue virus NS1, dengue virus envelope protein, chikungunya virus envelope protein, tyrosinase, HCV E2, NA17-Ant, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1, EGRFvIII, endoglin, ANGPTL-3, CSPG4, CTLA-4, HER2, IgE, IL-1beta, IL-5, IL-13, IL-17, IL-22, IL-31, IL-33, TSLP, NGF, (IHNV)G protein, lymphotoxins such as lymphotoxin alpha or beta, lymphotoxin receptor, receptor activator of nuclear factor kB ligand, vascular endothelial growth factor VEGF, VEGF receptor, IL-23 5. The composition according to any one of the preceding claims, wherein the antigen is selected from the group consisting of SARS-CoV-2 envelope protein, SARS-CoV-2 spike protein, SARS-CoV-2 nucleocapsid protein, and SARS-CoV-2 envelope protein.

[0240] 9. A composition according to any one of the preceding items.

[0241] 10. i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag, wherein, when said first and said second polynucleotides are expressed in a cell, said antigen and said protein are linked via an isopeptide bond between said first peptide tag and said second peptide tag, thereby forming a particle displaying said antigen, and optionally said first peptide tag, said second peptide tag, said protein and / or said antigen are as defined in any one of items 1 to 8.

[0242] 11. i. a first polynucleotide encoding a protein fused to a first peptide tag, said first polynucleotide being preferably as defined in any one of the preceding items; ii. a second polynucleotide encoding the antigen fused to a second peptide tag, preferably as defined in any one of the preceding paragraphs, wherein the second polynucleotide is a polypeptide of the present invention; wherein said antigen and said protein, when expressed in said cell, are linked via an isopeptide bond between said first peptide tag and said second peptide tag, thereby forming a particle in which i-ii displays said antigen, and optionally said cell comprises the expression system according to item 10.

[0243] 12. A host cell comprising the expression system described in item 10.

[0244] 13. The expression system according to item 10 and / or the cell or host cell according to any one of items 11-12 for use in a method for inducing an immune response in a subject, the method comprising administering the composition, the polynucleotide or the expression system to the subject at least once.

[0245] 14. A method of administering a composition for use in the prevention and / or treatment of a disease in a subject in need thereof, comprising: i. obtaining at least one composition as defined in any one of items 1 to 9; ii. administering said composition at least once to a subject for the prevention and / or treatment of a disease as defined in any one of the preceding paragraphs.

[0246] 15. i. a composition according to any one of items 1 to 9 or an expression system according to item 10; ii. optionally, a medical device or other means for administering the composition; and iii. A kit of parts including instructions for use.

Claims

1. A composition for use in the prevention and / or treatment of a disease, comprising: i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag; the antigen and the protein, when expressed in a cell, are linked via an isopeptide bond between the first peptide tag and the second peptide tag, thereby forming a particle displaying the antigen, i-ii.

2. The composition for use according to claim 1 , wherein the particle is a virus-like particle and / or the particle is a nanoparticle.

3. A composition for use according to any one of claims 1 to 2, wherein the protein is a particle-forming protein.

4. The composition for use according to any one of claims 1 to 3, wherein the protein is a protein from a plant virus.

5. A composition for use according to any one of claims 1 to 4, wherein the protein is a bacteriophage protein and / or the protein is a particle-forming protein.

6. The first or second peptide tag is SpyTag described in SEQ ID NO: 1, SdyTag described in SEQ ID NO: 2, SnoopTag described in SEQ ID NO: 3, PhoTag described in SEQ ID NO: 4, EntTag described in SEQ ID NO: 5, BacTag described in SEQ ID NO: 15, Bac2Tag described in SEQ ID NO: 16, Bac3Tag described in SEQ ID NO: 17, Bac4Tag described in SEQ ID NO: 18, RumTrunk D9N described in SEQ ID NO: 13 or SEQ ID NO:

14. The composition for use according to any one of claims 1 to 5, comprising a tag selected from the group consisting of: a Rum7Tag as set forth in SEQ ID NO: 12, a RumTag as set forth in SEQ ID NO: 6, a Rum2Tag as set forth in SEQ ID NO: 7, a Rum3Tag as set forth in SEQ ID NO: 8, a Rum4Tag as set forth in SEQ ID NO: 9, a Rum5Tag as set forth in SEQ ID NO: 10, a Rum6Tag as set forth in SEQ ID NO: 11, a Bac5Tag as set forth in SEQ ID NO: 19, a SpyCatcher as set forth in SEQ ID NO: 21, a SdyCatcher as set forth in SEQ ID NO: 22, and a SnoopCatcher as set forth in SEQ ID NO:

23.

7. one or more of said first peptide tags are fused to the N-terminus, C-terminus of said protein and / or inserted in frame into the coding sequence of said protein, optionally with a linker; and / or The one or more second peptide tags are fused to the N-terminus, C-terminus of the antigen and / or inserted in frame into the coding sequence of the antigen, optionally with a linker. A composition for use according to any one of claims 1 to 6.

8. the disease is cancer, dyslipidemia, cardiovascular disease, immunoinflammatory disease, chronic disease, neurological disease, infectious disease and / or allergic reactions / diseases, and / or the antigen is a polypeptide, peptide, and / or an antigenic fragment of a polypeptide associated with or specific for an abnormal physiological response; and / or The composition is used to treat an infectious disease, and the antigen is a protein or peptide, or a fragment thereof, specific to the pathogenic organism causing the infectious disease. A composition for use according to any one of claims 1 to 7.

9. The antigens include hemagglutinin, GD2, EGF-R, CEA, CD52, CD21, neuraminidase, human melanoma protein gp100, human melanoma protein melan-A / MART1, HIV envelope protein, M2e, VAR2CSA, ICAM1, CSP, dengue virus NS1, dengue virus envelope protein, chikungunya virus envelope protein, tyrosinase, HCV E2, NA17-Ant, MAGE-3, HPV 16 E7, HPV L2, PD1, PD-L1, CTLA-4, p53, hCG, Fel d1, EGRFvIII, endoglin, ANGPTL-3, CSPG4, CTLA-4, HER2, IgE, IL-1 beta, IL-5, IL-13, IL-17, IL-22, IL-31, IL-33, TSLP, NGF, (IHNV) G protein, lymphotoxin, receptor activator of nuclear factor kB ligand, vascular endothelial growth factor VEGF, VEGF receptor, IL-23 p19, ghrelin, CCL21, CXCL12, SDF-1, M-CSF, MCP-1, endoglin, GnRH, TRH, eotaxin, bradykinin, BLC, TNF-α, amyloid-β peptide A, angiotensin, gastrin, progastrin, CETP, CCR5, C5a, CXCR4, Des-Arg-bradykinin, GnRH peptide, angiotensin peptide, TNF peptide, IL-13, IL-31, IL- 17A, PCSK9, HIV trimer, and influenza virus trimer, eGFP as set forth in SEQ ID NO:94, Pfs25 as set forth in SEQ ID NO:96, and His purification tag as set forth in SEQ ID NO:95, a protein, peptide and / or antigenic fragment of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), or an antigenic fragment or antigenic variant thereof.

10. i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag, The antigen and the protein, when expressed in a cell, are linked via an isopeptide bond between the first peptide tag and the second peptide tag, thereby forming a particle in which i-ii displays the antigen, the protein being selected from the group consisting of i301 as set forth in SEQ ID NO:72 or a homolog thereof having at least 90% sequence identity thereto, Hbc as set forth in SEQ ID NO:92 or a homolog thereof having at least 90% sequence identity thereto, tandemHBc as set forth in SEQ ID NO:93 or a homolog thereof having at least 90% sequence identity thereto. the composition being selected from the group consisting of a human ferritin as set forth in SEQ ID NO:69, or a homologue thereof having at least 90% sequence identity thereto, a 2-oxoacid dehydrogenase subunit E2 as set forth in SEQ ID NO:97, or a homologue thereof having at least 90% sequence identity thereto, a lumazine synthase as set forth in SEQ ID NO:70, or a homologue thereof having at least 90% sequence identity thereto, and a norovirus capsid protein as set forth in SEQ ID NO:98, or a homologue thereof having at least 90% sequence identity thereto.

11. The composition of claim 10, wherein the first polynucleotide, the protein, the first peptide tag, the second polynucleotide, the antigen, and the second peptide tag are as defined in any one of claims 1 to 9.

12. i. a first polynucleotide encoding a protein fused to a first peptide tag; ii. a second polynucleotide encoding the antigen fused to a second peptide tag, when said first and said second polynucleotides are expressed in a cell, said antigen and said protein are linked via an isopeptide bond between said first peptide tag and said second peptide tag, thereby forming a particle i-ii displaying said antigen; i. the first polynucleotide further comprises a secretion or export signal, such that the protein fused to the first peptide tag is secreted or exported from the endoplasmic reticulum of the cell; and / or ii. The above expression system, wherein the second polynucleotide further comprises a secretion or export signal, such that the antigen fused to the second peptide tag is secreted or exported from the endoplasmic reticulum of the cell.

13. 13. The expression system of claim 12, wherein the first peptide tag, the second peptide tag, the protein and / or the antigen are as defined in any one of claims 1 to 9.

14. i. a) a first polynucleotide encoding a protein fused to a first peptide tag; b) a second polynucleotide encoding the antigen fused to a second peptide tag, said composition, wherein when said first and said second polynucleotides are expressed in a cell, said antigen and said protein are linked via an isopeptide bond or an ester bond between said first peptide tag and said second peptide tag, thereby forming a particle or expression system according to any one of claims 12 to 13, wherein i-ii display said antigen; ii. optionally, a medical device or other means for administering the composition; and iii. a kit of parts, including instructions for use.

15. 15. The kit of parts according to claim 14, wherein the first polynucleotide, the protein, the first peptide tag, the second polynucleotide, the antigen and / or the second peptide tag are as defined in any one of claims 1 to 9.

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