Vaccine for inducing antibodies to inhibit thymic stromal lymphopoietin

Abstract

The present disclosure pertains to antigens comprising an epitope of TSLP, fused to a peptide tag capable of forming an isopeptide bond. Additionally, the disclosure encompasses vaccines containing said antigen, their uses, and methods of manufacturing.

Description

[0001] P7422PC00

[0002] Vaccine for Inducing Antibodies to Inhibit Thymic Stromal Lymphopoietin

[0003] Technical field

[0004] The present disclosure relates to antigens comprising an epitope of TSLP and fused to a peptide tag. These antigens are displayed on a particle to be used as a vaccine for the prophylaxis and / or treatment of diseases and disorders associated with TSLP.

[0005] Background

[0006] Over the past few decades, diseases caused by malfunctions of the immune system have become significant challenges in modern medicine. Allergic diseases, particularly atopic or Immunoglobulin E (IgE)-mediated allergies, have reached near-epidemic proportions, affecting approximately 30% of the human population. Common allergic conditions include fur allergies, hay fever, dust mite allergies, insect venom allergies, extrinsic asthma, and various food allergies. Similarly, many domestic animals, such as dogs, cats, and horses, suffer from allergies, with severe atopic dermatitis in dogs posing a particularly difficult problem in veterinary medicine.

[0007] Current therapeutic strategies often involve monoclonal antibody therapy targeting specific cytokines or immune regulators. While these therapies have shown high efficacy in treating chronic diseases, they present challenges such as high costs and poor patient compliance due to the need for frequent administrations of large amounts of recombinant protein, which have to be administered potentially every two to four weeks for the patient's lifetime.

[0008] An alternative approach is active immunization, which harnesses the body's own immune system to produce antibodies against self-antigens involved in disease processes. This method could offer a cost-effective and convenient solution by reducing the amount of recombinant protein required, possibly as much as 10,000 times less than monoclonal antibody treatments, and decreasing the frequency of administrations to one to four times per year. However, inducing autoantibodies against self-antigens poses a significant challenge due to B-cell tolerance mechanisms that prevent the immune system from targeting endogenous proteins. P7422PC00

[0009] Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (I L-7)-like cytokine that binds to a heterodimeric receptor complex composed of the TSLP receptor (TSLPR) and IL- 7 receptor alpha (IL-7Ra). Notably, TSLP exists in two isoforms: the short form (sfTSLP), consisting of amino acid residues 97-159, is constitutively expressed in healthy tissues and exhibits anti-inflammatory properties; while the long form (IfTSLP), consisting of amino acid residues 29-159, is upregulated in diseased tissues and is pro- inflammatory. TSLP is predominantly produced by epithelial cells and mast cells in humans and serves as a critical regulator of T-helper type 2 (TH2)-mediated inflammatory conditions. It stimulates myeloid dendritic cells to promote the differentiation of naive CD4+ T cells into TH2 effector cells, which produce allergypromoting cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL- 13), and tumor necrosis factor-alpha (TNF-a). Elevated levels of TSLP have been associated with atopic dermatitis lesions and severe asthma. Additionally, IfTSLP is closely associated with skin fibrosis, epidermal hyperplasia, angiogenesis, and homeostatic tolerogenic regulation. Experimental and clinical studies have highlighted the role of IfTSLP in various immune-mediated skin diseases, including atopic dermatitis, psoriasis, bullous pemphigoid, systemic sclerosis, chronic spontaneous urticaria, Behget’s disease, vitiligo, rosacea, systemic lupus erythematosus, and alopecia areata.

[0010] To this end, a human monoclonal antibody, Tezepelumab, which blocks TSLP, has been tested for its efficacy and safety in patients with severe, uncontrolled asthma. Administration of Tezepelumab in these patients led to fewer exacerbations and improved lung function, asthma control, and health-related quality of life compared to those who received placebo (Wechsler et al., New England Journal of Medicine, 2021, DOI: 10.1056 / NEJMoa2034975).

[0011] There is a need for a novel vaccination strategy that can modulate excessive TH2- mediated inflammatory conditions by specifically targeting IfTSLP , which is upregulated in disease-affected tissues, while sparing the short form (sfTSLP) that is constitutively expressed in healthy tissue and contributes to epithelial homeostasis. A vaccine specifically inducing antibodies against IfTSLP would provide a safe, cost- effective, and durable therapeutic approach for the treatment or prevention of TSLP- associated inflammatory and allergic conditions in humans and animals. By eliciting anti-TSLP autoantibodies, such a vaccine can attenuate or prevent the pathological P7422PC00 effects arising from excessive IfTSLP expression, thereby alleviating disease symptoms and restoring immune balance.

[0012] Summary

[0013] The present disclosure relates to vaccine / antigen compositions and methods for breaking B-cell tolerance to self-antigens by displaying such antigens in a high-density, repetitive manner on the surface of a particle. Without being bound by theory, it is believed that this particulate presentation mimics pathogen-associated molecular patterns (PAMPs), thereby effectively activating B cells and promoting the production of autoantibodies against the displayed self-antigen. In particular, the disclosure utilizes a split-protein Tag / Catcher capsid virus-like particle (cVLP) system, wherein antigen display is achieved through the formation of a covalent isopeptide bond between genetically fused Tag and Catcher binding partners, enabling stable and efficient antigen conjugation to particle-forming proteins such as cVLPs or nanoparticles.

[0014] To minimize the risk of undesired autoimmunity, including induction of antibodies targeting the short form of thymic stromal lymphopoietin (sfTSLP) expressed in healthy tissue, vaccine antigens have been designed to include epitopes exclusively present in the long form of TSLP (IfTSLP) — such as those within amino acid residues 29-96, which are selectively expressed in diseased tissue. Vaccines based on these epitoperestricted antigens induce antibodies that selectively recognize IfTSLP, thereby avoiding cross-reactivity with sfTSLP or structurally related cytokines. This design strategy also mitigates the theoretical risk of inducing autoreactive cytotoxic T cells.

[0015] In accordance with the present disclosure, computational protein design techniques have been applied to generate de novo TSLP antigens restricted to the disease- associated region of IfTSLP. These designed antigens are displayed on the surface of Tag / Catcher-based cVLPs to enhance their immunogenicity and ability to overcome B- cell tolerance.

[0016] The inventors demonstrate that such de novo TSLP antigens can be designed, expressed, and stably presented on cVLPs, and that the resulting vaccines elicit antibodies capable of binding epitopes overlapping or corresponding to those recognized by the therapeutic monoclonal antibody Tezepelumab. P7422PC00

[0017] Thus, in an aspect, the present disclosure concerns a vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle-forming protein are linked via an isopeptide bond or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0018] In an aspect, the present disclosure concerns a vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0019] In an aspect, the present disclosure concerns a vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP), wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0020] In an aspect, the present disclosure concerns a vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues P7422PC00 present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0021] In an aspect, the present disclosure concerns a vaccine for use in medicine, wherein the vaccine is as described herein.

[0022] In an aspect, the present disclosure concerns a vaccine for use in the prophylaxis and / or treatment of a disease, wherein the vaccine is as described herein, and wherein the disease is an inflammatory disease, a fibrotic disorder, a cancer, a chronic epithelial barrier disorders, a skin disease, and / or an autoimmune disease.

[0023] In an aspect, the present disclosure concerns an antigen as described herein, fused to a (second) peptide tag as described herein.

[0024] In an aspect, the present disclosure concerns a polynucleotide encoding an antigen as described herein fused to a (second) peptide tag as described herein.

[0025] In an aspect, the present disclosure concerns an expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0026] In an aspect, the present disclosure concerns an expression system such as a vector comprising: P7422PC00 i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0027] In an aspect, the present disclosure concerns a cell comprising the polynucleotide as described herein.

[0028] In an aspect, the present disclosure concerns a cell expressing: i. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as described herein; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

[0029] In an aspect, the present disclosure concerns a cell expressing: i. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as described herein; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), preferably as described herein, P7422PC00 wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

[0030] In an aspect, the present disclosure concerns a host cell, wherein the host cell comprises an expression system as described herein.

[0031] In an aspect, the present disclosure concerns a method of administering a vaccine for the prophylaxis and / or treatment of a disease in a subject in need thereof, comprising the step of i. administering at least one composition as described herein to a subject at least once for prophylaxis and / or treatment of a disease as described herein.

[0032] In an aspect, the present disclosure concerns a kit of parts comprising i. a vaccine as described herein, or an expression system as described herein, and ii. optionally, a medical instrument or other means for administering the vaccine, and iii. instructions for use.

[0033] In an aspect, the present disclosure concerns a method of isolating an antigen as described herein, fused to a peptide tag as described herein, wherein the method comprises expressing said antigen fused to a peptide tag in a cell, such as a mammalian cell, or such as microorganism, such as a bacteria, such as E. coli.

[0034] In an aspect, the present disclosure concerns a method of manufacturing the vaccine as described herein, comprising the step of: i. Providing an expression system as described herein; and ii. Recovering the vaccine.

[0035] Description of Figures P7422PC00

[0036] Figure 1: Recombinant Expression of TSLP Antigens in HEK293TT Cells

[0037] A / B) HEK293TT cells were transfected with plasmid DNA encoding TSLP antigens #21-#25. Three days post-transfection, supernatants were collected for the detection of recombinant TSLP proteins by Western blot analysis using an anti-cTag antibody. Each protein sample was analyzed under both reduced and non-reduced conditions (- / + DTT). A positive control for anti-cTag detection was included on each Western blot for reference.

[0038] C) Verification of the Conformational Integrity of the Tezepelumab Neutralizing Epitope. A sandwich ELISA was performed to assess the binding between the commercial monoclonal antibody Tezepelumab and 9 out of 10 recombinant TSLP antigen constructs. ELISA plates were coated with Tezepelumab and incubated with supernatant from HEK293TT cells transfected with plasmid DNA encoding TSLP antigens #21-#30. Bound TSLP antigens were detected using an anti-cTag antibody, confirming their interaction with Tezepelumab.

[0039] Figure 2: Expression Analysis of TSLP Antigens in E. coli Shuffle Cells.

[0040] A-D) TSLP antigens (#21-#30) were expressed in E. coli Shuffle cells. Samples were collected before and after induction of protein expression (- / + IPTG) and analyzed by SDS-PAGE, including both pellet (P) and supernatant (S) fractions following sonication. Black boxes highlight the induction of recombinant TSLP proteins and their presence in the soluble fraction. The data demonstrate that 7 out of 10 constructs were successfully expressed as soluble proteins.

[0041] Figure 3

[0042] A) E. coli produced TSLP antigens bind the commercial antibody Tezepelumab. Seven TSLP antigen constructs (#21 , #23, #26, #27, #28, #29, and #30) expressed as soluble proteins in E. coli were purified using cTag chromatography. Each of these constructs was subsequently designed with both an N-terminal Tag (RumtrunkD9N) and Catcher (Mooncake) to enable conjugation to the surface of VLPs (AP205 gentically fused to the corresponding split protein binding partner). These new designs resulted in 14 new antigen constructs (#35-#48) of which 11 were successfully expressed in E. coli. All the E.coli produced TSLP antigens (both with and without Tag / Catcher fusion) were then tested in duplicate for their binding to the commercial monoclonal antibody Tezepelumab to confirm the integrity of the neutralizing epitope. ELISA plates were P7422PC00 coated with Tezepelumab and incubated with a 3-fold serial dilution of TSLP proteins, starting at 100 pg / mL. Bound TSLP antigens were detected using an anti-cTag antibody. The binding levels are presented as the area under the curve (AUG) with mean ± SD.

[0043] B / C) Coupling of Tag / Catcher TSLP Antigens (B: #35, #37, #38, #39, and #40) and (C: #43-#48) to VLP. TSLP antigens were conjugated to VLPs (AP205) containing the complementary split-protein binding partner (RumtrunkD9N and Mooncake) in a 1 :2 molar ratio of VLP to antigen. The mixtures were incubated overnight at 4°C. To evaluate the stability of the resulting antigen:VLP complexes, aliquots were collected both before and after centrifugation at 20,000g for 2 minutes and analyzed by SDS- PAGE. Isopeptide-conjugated antigen:VLP complexes are indicated by dashed boxes on the gel. The absence of significant loss of these bands after centrifugation demonstrates the stability of the antigen:VLP complexes. If the complexes had aggregated or agglomerated, they would have precipitated during centrifugation, resulting in a diminished signal. Excess unconjugated antigen and VLP are also visible on the SDS-PAGE gel, confirming the presence of unbound components alongside the stable conjugates.

[0044] Figure 4: TSLP vaccine formulations

[0045] Analysis of TSLP-VLP vaccine formulations by density gradient ultracentrifugation. Mixing of Tag / Catcher-TSLP antigens to VLPs (genetically fused to a complimentary binding partner, RumtrunkD9N and Mooncake)) in a ~1 :2 ratio (antigenA / LP) allowed for the conjugation / display of TSLP antigens on the VLPs. The resulting vaccine formulations: AV49 (panel A), AV50 (panel B), AV51 (panel C), AV52 (panel D), AV 53 (panel E), AV54 (panel F) and AV62 (panel G) were purified over an iodixanol density gradient by ultracentrifugation to assess the formation of antigen:VLP complexes. This analysis showed that the conjugated antigen:VLP protein was primarily detected in post-UC fractions 4-7, which contain high-density particulate proteins and thus indicate that presence of antigen-displaying VLP. Some Excess unconjugated TSLP antigen could be detected in the low-density fractions (F12-F13) containing soluble protein. P7422PC00

[0046] Dynamic light-scattering analysis. The post-UC fractions containing the antigen:VLP complexes were subsequently dialysed into PBS + 400mM sucrose and analysed by Dynamic light scattering (DLS) to assess the monodispercity of the antigen:VLP complexes. Except for AV49, DLS analysis of all the vaccine formulations showed the presence of particles of ~50nm in diameter with a polydispersity <15% (panel A2-G2).

[0047] Vaccine stability test and antigen coupling efficiency. A spin stability test for each vaccine demonstrates that the AV50-AV54 and AV62) vaccines is stable in solution (Fig 4H-J). In contrast, the AV49 vaccine demonstrate some instability (Fig. 4H). For all vaccine formulations densitometry analysis estimated the antigen coupling efficiency to be -23-38%, corresponding to approximately 41-68 TSLP antigens per VLP (Fig. 4H- J).

[0048] Figure 5: TSLP-VLP Vaccines Induce TSLP-Specific Antibodies in Mice

[0049] Groups of BALB / c mice (n=4) were immunized using a 2-week prime-boost-boost regimen (total of 3 doses) with seven different TSLP vaccines (A 49-AV54, AV62). Each dose contained 1.5 pg of VLP-displayed TSLP antigen. Vaccines AV49-AV54 were based on TSLP antigen constructs #21, #23, #26, #28, #29, and #30, respectively, while AV62 contained full-length human TSLP. All vaccines were formulated with the extrinsic adjuvant AddaVax. The levels of TSLP-specific antibodies in mouse serum were measured by ELISA and are shown as the area under the curve (AUG) with mean ± SD. Serum samples were collected after a single immunization (1st bleed, Figure 5A) and after two immunizations (Figure 5B). Negative controls included pre-bleed samples and serum from mice immunized with two doses of VLP-only (Mooncake-AP205).

[0050] Figure 6: Vaccine-induced antibodies compete against Tezepelumab for binding to huTSLP

[0051] In a competition ELISA, serum from immunized mice (n=4) were tested in a 2-fold dilution starting from 1 :10 for the ability to compete against Tezepelumab (0.1 g / mL) for binding to huTSLP (0.1 g / well). Data is plotted as percentage competition with mean ± SD, compared to the negative control “cVLP only”. P7422PC00

[0052] Figure 7: Vaccine-induced antibodies demonstrate biological efficacy by inhibition of huTSLP receptor activation in a huTSLP reporter cell line

[0053] The biological activity of the vaccine-induced antibody response was investigated by measuring the ability of IgG purified from serum from immunized mice to inhibit TSLP receptor activation in a HEK293 huTSLP reporter cell line (Invivogen). 10ng / mL huTSLP was incubated with IgG from immunized mice in a 2-fold dilution starting from 1 M, before added to 50.000 cells seeded in a 96-well plate. Cells were incubated overnight at 37°C, 5% CO2. SEAP reporter activity was quantified according to manufacturers protocol and plotted as percentage activity (% activity) with mean + SD, as compared to the negative control (PBS only, “no Ab”). As negative controls, cells were unstimulated (i.e. PBS) or stimulated in the presence of IgG from mice immunized with a non-TSLP cVLP-targeting vaccine (cVLP-AgX). Data show individual inhibitory capacity of immunized mice, in a dose-dependent manner.

[0054] Figure 8: sfTSLP-specific antibodies binding to newly generated TSLP antigens To investigate if the newly genenerated TSLP antigens contain any sfTSLP-specific epitopes, serum from Balb / c mice (n=6) immunized in a 2-week interval prime-boost- boost regimen with a cVLP-sfTSLP(63aa) vaccine was tested for binding to the different antigens (without Catcher). Data show that #21 and #23 have higher binding specificity for sfTSLP-specific Abs, as compared to #26, #28, #29 and #30.

[0055] Figure 9: Expression of double-display antigens and vaccine formulations Antigen designs displaying two different newly generated TSLP antigens on the same Catcher molecule (miniMooncake, engineered Mooncake) were designed based on #26 and #30, with either rigid helical linkers or flexible linkers between the antigens and Catcher molecule. Expression was induced with a final concentration of 1mM IPTG. Samples were taken before (-IPTG) and after (+IPTG) induction, as well as the pellet and supernatant following sonication of the cell pellet. White boxes indicate the induction band. Double display antigen constructs designed with a rigid helical linker between the antigens and the Catcher construct demonstrated best expression and solubility (#54 and #55), as compared to the constructs containing flexible linkers (#56 and #57). #54 and #55 was purified by cTag chromatography. P7422PC00

[0056] Figure 10: cVLP-#54 vaccine formulation

[0057] #54 was mixed with Tag-cVLP in a 1:2 molar excess. The vaccine was incubated overnight at 4°C before excess antigen was removed by density gradient ultracentrifugation (UC) using an Optiprep™ gradient (23, 29, 35%) (A). To assess stability, the vaccine was subjected to a spin stability test. Specifically, a vaccine sample was spun at 16.000g for 2min and an equal volume of pre- and post-spin samples were loaded on an SDS-PAGE gel, to assess potential loss in the post-spin sample due to precipitation or aggregation (B). The antigen coupling efficiency was estimated to 23% by calculating the percentage conjugation (i.e. number of bound antigens divided by the total available binding sites (=180) per cVLP) by densitometric analysis using ImageLab. This corresponds to ~41 Catcher molecules binding per cVLP, displaying ~82 TSLP antigens due to double display. (C) Purified vaccine was quality checked Dynamic Light scattering (DLS) analysis, showing a monodisperse population with a diameter of ~45-50nm, with low percent polydispersity (%Pd<15).

[0058] Figure 11: cVLP-#55 vaccine formulation

[0059] #55 was mixed with Tag-cVLP in a 1:2 molar excess. The vaccine was incubated overnight at 4°C before excess antigen was removed by density gradient ultracentrifugation (UC) using an Optiprep™ gradient (23, 29, 35%) (A). To assess stability, the vaccine was subjected to a spin stability test. Specifically, a vaccine sample was spun at 16.000g for 2min and an equal volume of pre- and post-spin samples were loaded on an SDS-PAGE gel, to assess potential loss in the post-spin sample due to precipitation or aggregation (B). The antigen coupling efficiency was estimated to 23% by calculating the percentage conjugation (i.e. number of bound antigens divided by the total available binding sites (=180) per cVLP) by densitometric analysis using ImageLab. This corresponds to ~41 Catcher molecules binding per cVLP, displaying ~82 TSLP antigens due to double display. (C) Purified vaccine was quality checked Dynamic Light scattering (DLS) analysis, showing a monodisperse population with a diameter of ~46nm, with low percent polydispersity (%Pd<15).

[0060] Figure 12: Immunogenicity of cVLP-Displayed Al-Generated TSLP Antigens Female BALB / c mice (n = 6-7 per group) were immunized following a 2-week prime- boost-boost regimen with 3 pg of TSLP antigen per dose, displayed on a capsid virus-like particle (cVLP) platform. Blood samples were collected 12 days after each immunization. P7422PC00

[0061] Human TSLP (huTSLP)-specific IgG titers were determined in serum obtained after the first and second immunizations (1st and 2nd bleeds, respectively) by ELISA.

[0062] Animals received one of the following vaccine formulations:

[0063] • Single-antigen vaccines: cVLPs displaying individual Al-generated TSLP antigens (cVLP-#26 or cVLP-#30).

[0064] • Dual-display vaccines: miniMooncake (miniMC) constructs co-displaying two Al-generated TSLP variants (cVLP-#54 = #26-miniMC-#30 and cVLP-#55 = #30-miniMC-#26).

[0065] • Heterologous prime-boost regimen: priming with cVLP-#26 followed by boosting with cVLP-#30.

[0066] Detailed description

[0067] Definitions

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied to facilitate the understanding of the present invention.

[0069] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. In addition, as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of”.

[0070] As used in the specification and claims, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B". The term “or” as used herein does not necessarily imply that the elements are mutually exclusive; i.e. “A or B” is intended to include either A alone, B alone, or both A and B, unless it is apparent that A and B are mutually exclusive.

[0071] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Similarly, terms such as “one or more” or “at least one” include both the singular and plural form of the respective feature. P7422PC00

[0072] Particle-forming protein: As used herein, the term “particle-forming protein” refers to proteins that are capable of self-assembling into defined, three-dimensional structures, such as nanoparticles, virus-like particles, viral capsid proteins, or other ordered macromolecular assemblies. These proteins naturally or through engineering possess the inherent ability to organize into particulate forms under suitable conditions without forming disordered aggregates or inclusion bodies. The skilled person will recognize that particle-forming proteins are distinct from proteins that aggregate non-specifically, as they maintain structural fidelity and functional properties within the assembled particle, unlike for instance proteins which aggregate in e.g. inclusion bodies.

[0073] Epitope: As used herein, the term "epitope" refers to the specific part of an antigen recognized by an antibody, B-cell receptor, or T-cell receptor. Epitopes can be classified as either linear or conformational.

[0074] Linear epitope: A linear epitope, also referred to as a sequential epitope, consists of a continuous sequence of amino acids in the primary structure of a protein. These epitopes are recognized based on the specific order of amino acids, and their antigenic properties remain intact even if the protein is denatured, as the sequence remains unaltered.

[0075] Conformational epitope: A conformational epitope, also known as a discontinuous epitope, is composed of amino acids that are not contiguous in the primary sequence of a protein but are brought into proximity by the protein’s three-dimensional folding. These epitopes are typically recognized by antibodies when the protein maintains its native conformation. Denaturation of the protein may disrupt the structure of a conformational epitope, rendering it unrecognizable by the immune system. Further information regarding conformational epitopes might be found, inter alia, at the corresponding Wikipedia page, see e.g. https: / / en.wikipedia.org / wiki / Conformational_epitope.

[0076] The number of amino acids in a conformational epitope can vary, but it typically consists of 5 to 20 amino acids. However, there is no strict number, as conformational epitopes are defined by the spatial arrangement of amino acids rather than a specific sequence. P7422PC00

[0077] Immunogenic fragments thereof: As used herein, the term “immunogenic fragments thereof” refers to portions of a polypeptide that retain immunological relevance. Such fragments include sequences that comprise one or more epitopes capable of eliciting an immune response, such as antibody generation or T-cell activation. The skilled person will understand that these fragments are of sufficient length and structural integrity to preserve the immunogenic properties of the full-length polypeptide, thereby ensuring their functionality in immunological applications.

[0078] Sequence identity: As used herein, the term 'at least 70% sequence identity' encompasses sequences with 71% sequence identity, 72% sequence identity, 73% sequence identity, 74% sequence identity, 75% sequence identity, 76% sequence identity, 77% sequence identity, 78% sequence identity, 79% sequence identity, 80% sequence identity, 81% sequence identity, 82% sequence identity, 83% sequence identity, 84% sequence identity, 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity, and 100% sequence identity.

[0079] The Vaccine of the present disclosure

[0080] Thymic Stromal Lymphopoietin (TSLP) is a cytokine involved in the regulation of immune responses and is particularly significant in conditions such as inflammatory diseases, fibrotic disorders, cancer, chronic epithelial barrier disorders, and / or an autoimmune diseases.

[0081] Thus, in an some aspects, the disclosure provides a protein vaccine or a DNA / RNA vaccine against TSLP.

[0082] Thus, in an aspect, the present disclosure concerns a vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), P7422PC00 wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0083] In an aspect, the present disclosure concerns a vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0084] In some embodiments, the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond.

[0085] In some embodiments, the antigen and particle-forming protein upon expression in a cell are linked via an ester bond.

[0086] In some embodiments the coupled particle is secreted from the cell.

[0087] The epitope, the antigen, the first and the second peptide tag, the particle-forming protein, fusion of the antigen to the particle-forming protein, and further elements of the vaccine are further described herein in the corresponding sections of the disclosure.

[0088] It will be evident to the skilled person that the different sections of the present disclosure are provided to facilitate understanding and clarity. However, the teachings and embodiments described in these sections are not intended to be read in isolation. Rather, they are to be considered in conjunction with one another, and the various features, aspects, and embodiments disclosed herein are intended to be combined in any manner that would be understood as technically feasible and beneficial by the skilled person.

[0089] In some embodiments, Thymic Stromal Lymphopoietin (TSLP) is human TSLP. P7422PC00

[0090] TSLP exists in at least two isoforms in humans, commonly referred to as the long form (IfTSLP) and the short form (sfTSLP).

[0091] The long form of TSLP (herein referred as IfTSLP) is the full-length variant that is predominantly associated with pro-inflammatory activity and immune regulation. It is expressed in various tissues, including epithelial cells, and is upregulated in response to environmental stimuli such as allergens or pathogens. This form of TSLP plays a crucial role in the activation of dendritic cells and the subsequent modulation of T-cell responses, making it a target of interest in immunological research and therapeutic interventions.

[0092] In contrast, the short form of TSLP (herein referred as sfTSLP) is a truncated variant that arises from an alternative promoter and differs in its amino acid sequence from IfTSLP. sfTSLP is generally expressed constitutively in tissues and has been associated with homeostatic functions, including the maintenance of epithelial integrity. Importantly, sfTSLP lacks certain amino acid residues present in IfTSLP that are critical for its interaction with immune cell receptors, thereby rendering it functionally distinct from the long form.

[0093] The differentiation between IfTSLP and sfTSLP is of particular relevance in therapeutic applications, as many immune-modulating effects attributed to TSLP are specific to the long form. Consequently, the aim of the present disclosure is to provide vaccines and other therapeutics designed to specifically recognize and respond to epitopes unique to IfTSLP while avoiding cross-reactivity with sfTSLP. Without being bound by theory, this selective targeting is believed to ensure that pro-inflammatory pathways can be modulated without disrupting the homeostatic roles of sfTSLP.

[0094] Thus, in an aspect, the present disclosure concerns a vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP), P7422PC00 wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0095] In an aspect, the present disclosure concerns a vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0096] In some embodiments, IfTSLP is human IfTSLP.

[0097] Thus, in some embodiments, IfTSLP is human IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0098] Amino acid residues 29 to 96 of SEQ ID NO: 45 are the amino acid residues associated with the diseases described herein, while amino acid residues 97-159 are amino acids that are also present in sfTSLP.

[0099] In some embodiments, sfTSLP is human sfTSLP.

[0100] Thus, in some embodiments, sfTSLP is human sfTSLP as set forth in SEQ ID NO: 46, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0101] In some embodiments, TSLP is canine TSLP, as set forth in SEQ ID NO: 47, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0102] In some embodiments, TSLP is feline TSLP, as set forth in SEQ ID NO: 48, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto. P7422PC00

[0103] In some embodiments, the first and / or second polynucleotide is DNA. In some embodiments, the first and / or second polynucleotide is RNA. In some embodiments, one of the first and / or second polynucleotide is DNA, and the other one of the first and / or second polynucleotide is RNA.

[0104] In some embodiments, the RNA is formulated in a lipid particle formulation.

[0105] In an aspect, the present disclosure is directed to a composition comprising the vaccine described herein. In some embodiments, the compositions may further comprise an acceptable carrier.

[0106] In some embodiments, the composition may further comprise an adjuvant. It will be evident to the skilled person which suitable adjuvant might be used.

[0107] In some embodiments, the composition may further comprise an Addavax adjuvant.

[0108] The Epitope

[0109] As described herein, the present disclosure is directed in some aspects to a vaccine which results in the display on a particle of an antigen comprising an epitope of TSLP.

[0110] In some embodiments, the epitope is comprised in an antigen as described in the section “the antigen” of the present disclosure.

[0111] In some embodiments, the epitope is a linear epitope.

[0112] In some embodiments, the epitope is a conformational epitope. A conformational epitope, also known as a discontinuous epitope, is composed of amino acid residues that are not contiguous in the primary sequence of a protein but are brought into spatial proximity through the three-dimensional folding of the protein. These epitopes are typically recognized by antibodies when the protein maintains its native conformation, as their structural arrangement is critical for immune recognition.

[0113] In some embodiments, the epitope comprises or consists of one or more amino acid residues present in an isoform of TSLP, e.g. IfTSLP, which is associated with a P7422PC00 disease, which amino acid residues are absent in the corresponding variant of TSLP, e.g. sfTSLP, which is not associated with said disease.

[0114] In some embodiments, the epitope comprises or consists of two or more amino acid residues wherein the spatial arrangement of these residues in the antigen displayed by the vaccine is preserved to replicate their configuration in the native three-dimensional structure of TSLP.

[0115] Thus, in some embodiments, the epitope comprises or consists of two or more amino acid residues of TSLP, wherein: i. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the corresponding three-dimensional structure of TSLP.

[0116] In some embodiments, the epitope comprises or consists of two or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, of TSLP, wherein: i. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the three-dimensional structure of TSLP.

[0117] In some embodiments, TSLP is canine TSLP, as set forth in SEQ ID NO: 47, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0118] In some embodiments, TSLP is feline TSLP, as set forth in SEQ ID NO: 48, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0119] In some embodiments, TSLP is human TSLP. In some embodiments, TSLP is human IfTSLP. Thus, in some embodiments, IfTSLP is human IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0120] As described herein, the aim of an aspect of the present disclosure is to provide vaccines and other therapeutics designed to specifically recognize and respond to epitopes unique to IfTSLP while avoiding cross-reactivity with sfTSLP. P7422PC00

[0121] Thus, in some embodiments, the epitope comprises or consists of one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP).

[0122] Thus, the term “an epitope of IfTSLP” as used herein refers to an epitope that is exclusively present in IfTSLP and is absent in sfTSLP.

[0123] It will be evident to the skilled person, that as long as an epitope comprises or consists of at least one residue which is present in IfTSLP and is absent in sfTSLP, said epitope is an epitope of IfTSLP.

[0124] In some embodiments, the epitope comprises or consists of two or more amino acid residues unique to IfTSLP and absent in sfTSLP. Importantly, the spatial arrangement of these residues in the antigen displayed by the vaccine is preserved to replicate their configuration in the native three-dimensional structure of IfTSLP. This conservation of the epitope’s structural integrity ensures that the antigen can effectively mimic the native protein, eliciting an immune response specifically targeting IfTSLP.

[0125] Thus, In some embodiments, the epitope comprises or consists of two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the corresponding three-dimensional structure of IfTSLP.

[0126] In some embodiments, the epitope comprises or consists of two or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the three-dimensional structure of IfTSLP, wherein IfTSLP is as set forth in SEQ ID NO: 45. P7422PC00

[0127] Methods to determine whether two or more amino acid residues maintain the same spatial arrangement are known in the art. Thus, the skilled person may employ any of these methods.

[0128] For example, the skilled person may employ the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure of the antigen and the corresponding two or more amino acid residues within the three- dimensional structure of IfTSLP. If the RMSD is less than, e.g. 2 A, is assessed that said amino acid residues in the antigen maintain the same spatial arrangement as in IfTSLP, such as wherein IfTSLP is IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0129] Root Mean Square Deviation (RMSD) is a widely accepted and routinely used parameter in structural biology for quantifying the similarity between three-dimensional conformations of proteins or of selected amino acid residues thereof. RMSD has long been employed in the fields of protein engineering, homology modelling, structural alignment, and molecular dynamics to evaluate whether two structures maintain the same spatial arrangement. Numerous software packages commonly used in the art (e.g., PyMOL, Chimera, VMD, MOE, and various MD analysis suites) provide built-in RMSD calculation tools, and the parameter is considered a standard descriptor of structural equivalence. Thus, the use of RMSD to assess whether two or more amino acid residues maintain a comparable spatial orientation is conventional and immediately accessible to the skilled person. Further guidance on how to use RSMD can be found for example at the corresponding Wikipedia page: https: / / en.wikipedia.org / wiki / Root_mean_square_deviation_of_atomic_positions.

[0130] Thus, in some embodiments, the epitope comprises or consists of two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure of the antigen and the corresponding two or more amino acid residues within the three-dimensional structure of IfTSLP is less than 2 A, such as less than 1 .9 A, such as less than 1 .8 A, 1.7, 1.6, 1 .5, 1 .4, 1.3, 1.2, 1.2, such as less than 1 A. P7422PC00

[0131] In some embodiments, the conformational epitope comprises or consists of two or more amino acid residues of IfTSLP, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45, and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure the antigen and the corresponding two or more amino acid residues within the three-dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A, wherein IfTSLP is as set forth in SEQ ID NO: 45.

[0132] In some embodiments, the conformational epitope comprises or consists of amino acid residues of IfTSLP, wherein: i. the acid residues of IfTSLP are selected from residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45, and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure the antigen and the corresponding two or more amino acid residues within the three-dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A, wherein IfTSLP is as set forth in SEQ ID NO: 45.

[0133] Thus, in some embodiments, the Root Mean Square Deviation (RMSD) of the amino acid residues within the three-dimensional structure of the antigen and the corresponding amino acid residues within the three-dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A.

[0134] In some embodiments, the conformational epitope comprises or consists of two or more amino acid residues of IfTSLP, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues of IfTSLP. P7422PC00

[0135] The three-dimensional structure of IfTSLP is publicly available and can be retrieved for example by consulting PDB: 5J13 (human TSLP in complex with Tezepelumab), or PDB: 5J11 (human TSLP in complex with the receptor). Alternatively a prediction of the three-dimensional structure of IfTSLP can be used instead.

[0136] It will be evident to the skilled person that where a three-dimensional structure of the antigen displaying the epitope is not available, a prediction of the three-dimensional structure of the antigen might be used instead. Thus, in some embodiments, the structure of the antigen and / or of IfTSLP is a predicted three-dimensional structure. The skilled person knows how to generate a prediction of the three-dimensional structure of a polypeptide given the corresponding sequence. Any known method to generate a three-dimensional structure of a polypeptide might be employed.

[0137] Three-dimensional structures to be used with the present invention may be available through various databases or can be predicted structures. AlphaFold3, referred to herein as AlphaFold, represents a state-of-the-art artificial intelligence (Al) system developed by DeepMind for predicting the three-dimensional (3D) structures of proteins from their amino acid sequences. This system is detailed in the publication: Jumper, J., Evans, R., Pritzel, A. et al. "Highly accurate protein structure prediction with AlphaFold." Nature 596, 583-589 (2021). htps: / / doi.org / 10.1038 / s41586-021-03819-2.

[0138] AlphaFold DB is an online database which hosts over 200 million entries, encompassing the human proteome as well as the proteomes of 47 other key organisms relevant to research and global health. These entries are freely accessible at https: / / alphafold.ebi.ac.uk / . The (predicted) structure of can be retrieved by inputting identifiers such as the protein name, gene name, or UniProt accession number.

[0139] Structures of polypeptides not included in the AlphaFold DB may for example be predicted using the source code available at https: / / github.com / google- deepmind / alphafold, and / or a Colab notebook accessible at https: / / colab.research.google.com / github / deepmind / alphafold / blob / main / notebooks / Alp haFold.ipynb. To generate a three-dimensional structure using the Colab notebook, the amino acid sequence of the antigen can be inserted. P7422PC00

[0140] The skilled person will understand that any similar software to predict the three- dimensional structure of a protein, such as previous or future iterations of AlphaFold, might be equally employed.

[0141] The Antigen

[0142] The vaccines described herein comprise a particle-forming protein comprising a first pepetide tag and an antigen fused to a second peptide tag. The epitopes described herein above are comprised within the antigen. The antigen is described in further detail in the present section.

[0143] In addition, in an aspect, the present disclosure concerns an antigen as described herein, fused to a (second) peptide tag as described herein.

[0144] In some embodiments, said antigen may be displayed by any particle capable of displaying the antigen via an isopeptide bond, or an ester bond, such as those described in the section “the particle-forming protein” of the present disclosure.

[0145] In some embodiments, said antigen may comprise an epitope of TSLP as described in the section “the epitope” of the present disclosure.

[0146] In some embodiments, said antigen is fused to a second peptide, such as those described in the section “the first and second peptide tag” of the present disclosure.

[0147] In an aspect, the present disclosure concerns a polynucleotide encoding an antigen as described herein fused to a (second) peptide tag as described herein. Said polynucleotide may be employed in the process of manufacturing the vaccine as described herein, and / or may be employed as a component of a DNA / RNA vaccine.

[0148] As described herein, in some aspects, the present vaccine is a vaccine against human IfTSLP, such as human IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0149] To minimize the risk of cross-reactivity by the one or more antibodies induced by the vaccine described herein, it is preferable to design the antigen such that the inclusion of amino acid residues shared with the short form of Thymic Stromal Lymphopoietin P7422PC00

[0150] (sfTSLP) is minimized or entirely avoided. Cross-reactivity occurs when antibodies generated against the antigen also recognize and bind to sfTSLP, which may lead to unintended effects due to the functional roles of sfTSLP in homeostasis.

[0151] Thus, in some embodiments, less than 20%, such as less than 15%, 10%, 5% of the amino acid residues of the antigen are amino acid residues which are present both in sfTLSP and IfTSLP, such as none of the amino acid residues of the antigen are amino acid residues which are present both in sfTSLP and IfTSLP.

[0152] In some embodiments, the antigen comprises one or more epitopes of TSLP, wherein each epitope of TSLP comprises one or more amino acid residues present in IfTSLP and absent in sfTSLP.

[0153] In some embodiments, the antigen does not comprise epitopes of sfTSLP. As described in the section “The Epitope” of the present disclosure, an “epitope of sfTSLP” is and epitope consisting of amino acids which are present in sfTSLP, i.e. none of the amino acid residues of said epitope of sfTSLP is present in IfTSLP.

[0154] Thus, in some embodiments, none of the one or more epitopes of the antigen comprise amino acid residues present in sfTLSP. In other words, in some embodiments, the antigen does not comprise epitopes which exclusively consist of amino acids absent in the IfTSLP.

[0155] In some embodiments, the antigen is a scaffold protein, such as a minimal scaffold protein, comprising an epitope of TSLP.

[0156] Suitable scaffolds and minimal scaffold constructs are well known in the art, and methods for their design, optimization, and use in epitope presentation have been extensively described. The skilled person can therefore select an appropriate scaffold without undue burden. Nevertheless, the present application, further provides examples of scaffold which are suitable for carrying out the teachings of the present disclosure. It will be evident that the skilled person following the teachings disclosed in the examples will be able to arrive to other scaffolds, or minimal scaffolds, equally suitable. P7422PC00

[0157] Scaffold proteins are structural frameworks that can display epitopes in a defined spatial arrangement, thereby preserving their conformational integrity and improving their immunogenic potential.

[0158] A scaffold protein, particularly a minimal scaffold protein, provides a compact and stable platform for presenting the epitope of TSLP in a manner that closely mimics its native conformation. This might be of particular relevance for vaccine against IfTSLP, because the scaffold itself does not share the three-dimensional structure of TSLP and, thus, the likelihood of cross-reactivity with the short form of TSLP (sfTSLP) is minimized. This structural distinction may ensure that antibodies elicited by the vaccine specifically target IfTSLP without interfering with sfTSLP’s physiological functions. Scaffold-based antigens can also facilitate efficient production and enhance the structural resilience of the vaccine, contributing to its overall efficacy and safety.

[0159] Nevertheless, it will be evident to the skilled person that a scaffold protein, such as a minimal scaffold protein, might be used to present any epitope of TSLP, such as an epitope of human TSLP, canine TSLP, feline TSLP, or any other TSLP known in the art.

[0160] A “minimal scaffold refers to a structural framework or protein that is engineered or selected to provide a simplified configuration sufficient to stably present an epitope or functional domain. A minimal scaffold maintains the conformational integrity of the epitope while excluding non-essential regions or sequences that are not required for stability, folding, or function.

[0161] An advantage of using a scaffold is that residues which should not be targeted by the antibodies induced by the construct are omitted. This ensures that the resulting immune response focuses on the intended epitope(s), minimizes recognition of irrelevant or undesired regions, and improves both the precision and functional relevance of the antibodies generated.

[0162] In some embodiments, the antigen is a scaffold protein, such as a minimal scaffold protein, comprising an epitope of IfTSLP. The skilled person knows how to design such a scaffold protein in which an epitope of IfTSLP as described herein above has been introduced. P7422PC00

[0163] In some embodiments, the antigen is a scaffold protein, such as a minimal scaffold protein, comprising a conformational epitope of IfTSLP. The skilled person knows how to design such a scaffold protein in which a conformational epitope of IfTSLP as described herein above has been introduced.

[0164] In some embodiments, the antigen is a scaffold protein, such as a minimal scaffold protein, comprising said epitope of IfTSLP, and wherein the amino acid residues of said epitope in the vaccine maintain the same spatial arrangement between each other as in the corresponding three-dimensional structure of IfTSLP, such as how described in the section “The Epitope” of the present disclosure.

[0165] In some embodiments, the antigen is a scaffold protein, such as a minimal scaffold protein, wherein the scaffold protein does not comprise an epitope of sfTSLP.

[0166] In order to obtain IfTSLP epitopes which preferably do not comprise any epitope of sfTSLP, several strategies are available to the skilled person.

[0167] One such strategy entails retaining specific regions or residues only present in IfTSLP, as described herein, and building a scaffold protein around those regions or residues as is known in the art, for instance using RFdiffusion (Watson et al., 2023).

[0168] Another strategy would be to scramble the sfTSLP sequence around the specific regions or residues, for instance using ProteinMPNN (Dauparas et al., 2022).

[0169] Besides the use of the above methods, the skilled person knows that the scaffold may need to meet some structural requirements. In particular, the inclusion of charged residues (such as K, R, D), or of polar and hydrophobic residues (such as F, L) may increase solubility of the antigen and / or recognition by antibodies. G and D residues can be included to increase flexibility of the antigen and / or exposure of the epitope. Should more rigidity be desired, proline or charged residues can be added to one or both termini. It may be advantageous to include a disordered central region, which may favour epitope accessibility, and / or to prevent the inclusion of strong beta sheet motifs to reduce the risk of aggregation. Cysteine residues can be avoided to prevent P7422PC00 formation of disulphide bonds, and may also simplify expression and folding of the antigen. Including aromatic residues may also help boost immunogenicity.

[0170] For all strategies, the skilled person can readily determine whether the obtained epitopes have the desired properties of inducing antibodies targeting IfTSLP, and not sfTLSP, for instance as described herein above.

[0171] The specific regions or residues of interest, of which one or more are retained in the final epitope, independently of the strategy used, will now be further detailed.

[0172] In some embodiments, the scaffold protein comprising the epitope has a length of 100 to 300 amino acids, such as 100 to 200 amino acids, such as 110 to 190 amino acids, such as 120 to 180 amino acids, such as 130 to 170 amino acids, such as 140 to 160 amino acids, such as around 150 amino acids, or such as 100 to 190 amino acids, 100 to 180 amino acids, 100 to 170 amino acids, 100 to 160 amino acids, 100 to 150 amino acids, 100 to 140 amino acids, 100 to 130 amino acids, 100 to 120 amino acids, such as around 110 amino acids, around 120 amino acids, around 130 amino acids, around 140 amino acids, around 150 amino acids, around 160 amino acids, around 170 amino acids. In some embodiments, the scaffold protein comprising the epitope has a length of 200 to 300 amino acids, such as 210 to 290 amino acids, such as 220 to 280 amino acids, such as 230 to 270 amino acids, such as 240 to 260 amino acids, such as around 250 amino acids, or such as 200 to 290 amino acids, 200 to 280 amino acids, 200 to 270 amino acids, 200 to 260 amino acids, 200 to 250 amino acids, 200 to 240 amino acids, 200 to 230 amino acids, 200 to 220 amino acids, such as around 210 amino acids, around 220 amino acids, around 230 amino acids, around 240 amino acids, around 250 amino acids, around 260 amino acids or around 280 amino acids.

[0173] In some embodiments, the antigen does not comprise any amino acid residue of sfTLSP.

[0174] In some embodiments, the antigen does not comprise a polypeptide consisting of sfTLSP or an immunogenic fragment thereof. P7422PC00

[0175] In some embodiments, IfTSLP is human IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0176] The amino acid residues which are unique to the IfTSLP as set forth in SEQ ID NO: 45 are amino acid residues 29 to 96. Thus, in some embodiments, the antigen comprises or consists of a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof.

[0177] While the amino acid residues unique to the IfTSLP are located within residues 29 to 96 of SEQ ID NO: 45, in some embodiments, the antigen may additionally include further amino acid residues of IfTSLP. Without being bound by theory these additional residues may enhance the stability and / or folding of the antigen.

[0178] It will be understood by the skilled person that where additional residues of sfTSLP are included, their three-dimensional spatial arrangement relative to residues 29 to 96 of SEQ ID NO: 45 might be, in some embodiments, preserved. This structural conservation might ensure that the antigen mimics part of the native conformation of IfTSLP, thereby contributing to maintain the epitope’s immunogenicity and specificity.

[0179] In view of the present disclosure, the skilled person will further understand that, in some embodiments, it is possible to introduce further amino acid residues of sfTSLP without introducing in the antigen epitopes of sfTSLP, for example by not introducing a consecutive sequence of amino acid residues present in sfTSLP which is at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7 amino acid residues long.

[0180] In some embodiments, disulfide bonds between Cysteines which are present in IfTSLP are preserved in the antigen displaying an epitope of TSLP. Thus, in some embodiments, the antigen comprises a disulfide bond between Cys34 and Cys110; between Cys90 and Cys137; or between Cys69 and Cys75.

[0181] In some embodiments, the antigen comprises a disulfide bond between Cys34 and Cys110; between Cys90 and Cys137; and between Cys69 and Cys75. P7422PC00

[0182] In some embodiments, the antigen comprises a disulfide bond between Cys34 and Cys110. In some embodiments, the antigen comprises a disulfide bond between between Cys90 and Cys137. In some embodiments, the antigen comprises a disulfide bond between Cys69 and Cys75.

[0183] In some embodiments, the antigen comprises or consists of: a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of Cys110, Cys137, Arg150, or Arg153 of SEQ ID NO: 45.

[0184] In some embodiments, said polypeptide further comprises Cys110 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Cys137 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Arg150 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Arg153 of SEQ ID NO: 45.

[0185] In some embodiments, the antigen comprises: a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of Phe98, Ala99, Leu106, Tyr109, Cys110, Cys137, Leu 138, Arg150, or Arg153 of SEQ ID NO: 45.

[0186] In some embodiments, said polypeptide further comprises Phe98 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Ala99 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Leu106 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Tyr109 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Leu138 of SEQ ID NO: 45.

[0187] In some embodiments, said polypeptide further comprises one or more of: E139; V141; or Q143 as set forth in SEQ ID NO: 45.

[0188] In some embodiments, said polypeptide further comprises Glu139 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Val141 of SEQ ID NO: 45. In some embodiments, said polypeptide further comprises Gln143 of SEQ ID NO: 45. P7422PC00

[0189] In some embodiments, the antigen comprises a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 139 to 153 of SEQ ID NO: 45; such as a polypeptide comprising one or more of amino acid residues 146 to 153 of SEQ ID NO: 45.

[0190] In some embodiments, the antigen further comprises a polypeptide having one or more of amino acid residues Leu144, Gly146, Leu147, Trp148, Arg149, Phe151 , orAsn152 of SEQ ID NO: 45.

[0191] In some embodiments, the antigen comprises: a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of: Phe98, Ala99, Leu106, Tyr109, Cys110, Cys137, Leu138,Glu139, Val141 , Leu144, Gln143, Gly146, Leu147, Trp148, Arg149, Arg150, Phe151, Asn152, or Arg153 of SEQ ID NO: 45.

[0192] In some embodiments, the antigen comprises: a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of Phe98, Ala99, Leu106, Tyr109, Cys110, Cys137, Leu 138, and / or an immunogenic polypeptide, having at least 95% identity to amino acid residues 139 to 153 of SEQ ID NO: 45; such as a polypeptide comprising one or more of amino acid residues 146 to 153 of SEQ ID NO: 45, such as a polypeptide having one or more of amino acid residues Leu144, Gly146, Leu147, Trp148, Arg149, Phe151 , or Asn152 of SEQ ID NO: 45.

[0193] In some embodiments, the antigen does not comprise a polypeptide comprising amino acid residues 97 to 159, such as amino acid residues 110 to 159, 120 to 159, 130 to 159, 140 to 159, 97 to 140, 97 to 130, 97 to 120, 97 to 110 of SEQ I D NO: 45, such as the antigen does not comprising a polypeptide having 96%, 97%, 98% or 99% sequence identity thereto, nor one or more immunogenic fragments thereof. P7422PC00

[0194] In some embodiments, the antigen comprises at least 5, such as at least 10, at least 15, at least 20, 25, 30, 35, 40, 45, 50 amino acid residues which are absent in IfTSLP and / or sfTSLP.

[0195] In some embodiments, the antigen comprises or consists of a polypeptide consisting of: i. SEQ ID NO: 49; ii. SEQ ID NO: 50; iii. SEQ ID NO: 51 ; iv. SEQ ID NO: 52; v. SEQ ID NO: 53; vi. SEQ ID NO: 54; vii. SEQ ID NO: 55; or a polypeptide having at least 95% sequence identity thereto.

[0196] Degenerate sequences encoding the corresponding antigens as encoded by these sequences can also be used as an alternative

[0197] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0198] 49, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0199] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0200] 50, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0201] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0202] 51 , or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0203] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0204] 52, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto. P7422PC00

[0205] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0206] 53, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0207] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0208] 54, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0209] In some embodiments, the antigen comprises a polypeptide as set forth in SEQ ID NO:

[0210] 55, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0211] In some embodiments, the antigen fused to the (second) peptide tag comprises a polypeptide consisting of: i. SEQ ID NO: 59; ii. SEQ ID NO: 60; iii. SEQ ID NO: 61 ; iv. SEQ ID NO: 62; v. SEQ ID NO: 63; vi. SEQ ID NO: 64; vii. SEQ ID NO: 65; viii. SEQ ID NO: 67; ix. SEQ ID NO: 68; x. SEQ ID NO: 69; xi. SEQ ID NO: 70; xii. SEQ ID NO: 71 ; xiii. SEQ ID NO: 72; or a polypeptide having at least 95% sequence identity thereto.

[0212] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 59, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto. P7422PC00

[0213] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 60, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0214] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 61 , or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0215] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 62, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0216] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 63, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0217] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 64, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0218] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 65, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0219] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 65, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0220] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 66, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0221] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 67, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto. P7422PC00

[0222] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 68, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0223] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 69, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0224] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 70, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0225] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 71 , or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0226] In some embodiments, the antigen fused to the (second) peptide tag comprises or consists of a polypeptide as set forth in SEQ ID NO: 72, or a polypeptide having at least 95%, such as at least 96%, 97%, 98% or 99%, sequence identity thereto.

[0227] In some embodiments, the antigen is capable of eliciting an immune reaction in an animal, such as a mammal, such as a Homo sapiens, a dog, cat, cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, and / or a bird, such as a chicken and / or a fish, such as a salmon.

[0228] The skilled person knows how to design antigens which comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a form TSLP associated with disease and absent in the corresponding wild type / asymptomatic form TSLP.

[0229] In some embodiments, the antigen further comprises an additional tag.

[0230] In some embodiments, the antigen further comprises a polyhistidine tag or cTag.

[0231] In some embodiments, the antigen further comprises a polyhistidine tag. P7422PC00

[0232] In some embodiments, the antigen further comprises a cTag.

[0233] In some embodiments, a linker connects the (second) peptide tag and the antigen. Suitable likers are known in the art. A polypeptide of sequence GGS is an example of such suitable linker.

[0234] In some embodiments, the vaccine comprises two or more antigens fused to a second peptide tag as described herein. Said two or more antigens comprise each an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP).

[0235] In some embodiments, the vaccine comprises two or more antigens fused to a second peptide tag as described herein, wherein the first antigen is as set forth in SEQ ID NO: 51 (#26) and the second antigen is as set forth in SEQ ID NO: 55 (#30).

[0236] In some embodiments, the vaccine comprises two or more antigens fused to a second peptide tag as described herein, wherein said two or more antigens, such as the two or more antigens as described herein, are fused to said second peptide via a linker. In some embodiments said linker is a rigid helical linker. In some embodiments, the linker is a flexible linker. Suitable rigid helical linkers and flexible linkers are well known in the art. In some embodiments, one or more antigens are fused to the N terminal of the second peptide tag as described herein and one or more antigens are fused to the C terminal of the second peptide.

[0237] In some embodiments, one or more antigens are fused to the N terminal of the second peptide tag as described herein and one or more antigens are fused to the C terminal of the second peptide, and the second peptide is as set forth in SEQ ID NO: 81.

[0238] Examples of such constructs, where one antigen is fused to the N terminal of the second peptide tag and one antigen is fused to the C terminal of the second peptide, are polypeptides as set forth in SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79 or SEQ ID NO: 80 P7422PC00

[0239] Thus, in some embodiments, the vaccine comprise a polypeptide as set forth in: SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79 or SEQ ID NO: 80.

[0240] In some embodiments, the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 77.

[0241] In some embodiments, the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 78

[0242] In some embodiments, the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 79

[0243] In some embodiments, the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 80

[0244] Antibodies induced by the vaccine

[0245] As described herein, the present disclosure is directed in some aspects to a vaccine which is capable of displaying an antigen comprising an epitope of TSLP on a particle .

[0246] Thus, in some embodiments the vaccine is capable of inducing in a subject one or more antibodies which have affinity for an epitope of TSLP, as described herein such as in the section “The Epitope” of the present disclosure. Thus, in some embodiments, the vaccine is capable of inducing in a subject one or more antibodies specific to TSLP.

[0247] In some embodiments, the vaccine is capable of inducing in a subject one or more antibodies which have affinity for a conformational epitope of IfTSLP as described herein, which conformational epitope is absent in sfTSLP.

[0248] In some embodiments, the vaccine is capable of inducing one or more antibodies which have a Kd (Dissociation Constant) for IfTSLP which is at least 2-fold, 3-fold, 5- fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold lower than the Kd of sfTSLP. P7422PC00

[0249] In some embodiments, the vaccine is not cross-reactive to sfTSLP. In other words, the vaccine does not induce the production of antibodies targeting sfTSLP, or epitopes of sfTSLP which are absent from IfTSLP.

[0250] In some embodiments, the vaccine is capable of inducing in a subject one or more antibodies which have affinity for, and / or are capable of binding to, a conformational epitope of IfTSLP, wherein the conformational epitope comprises one or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, selected from a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0251] The particle-forming protein

[0252] As described herein, the present disclosure is directed in some aspects to a vaccine which results in the display on a particle of an antigen comprising an epitope of TSLP. This is achieved by the binding of an antigen fused to a first peptide tag as described herein to a particle-forming protein as described herein via an isopeptide bond or an ester bond.

[0253] It will be evident to the skilled person that any of the peptide tags described in the section “the first and second peptide tags” of the present disclosure might be fused to any of the particle-forming proteins described herein.

[0254] In some embodiments, the particle-forming protein is fused to a first peptide tag as described in the section “the first and second peptide tag” disclosure.

[0255] In some embodiments, the particle-forming protein forms a particle displaying the antigen as described in the section “the antigen” of the present disclosure.

[0256] As used herein, the term “particle-forming protein” refers to proteins that are capable of self-assembling into defined, three-dimensional structures, such as nanoparticles, virus-like particles, viral capsid proteins, or other ordered macromolecular assemblies. These proteins naturally or through engineering possess the inherent ability to organize into particulate forms under suitable conditions without forming disordered aggregates or inclusion bodies. The skilled person will recognize that particle-forming proteins are distinct from proteins that aggregate non-specifically, as they maintain structural fidelity P7422PC00 and functional properties within the assembled particle, unlike for instance proteins which aggregate in e.g. inclusion bodies.

[0257] Thus, in some embodiments, the particle-forming protein is a protein which is capable of self-assembling into macromolecular assemblies, such as nanoparticles, virus-like particles, or viral capsid proteins. In some embodiments, the particle-forming protein is a protein which is capable of self-assembling into defined macromolecular assemblies. . In some embodiments, the particle-forming protein is a protein which is capable of self-assembling into ordered macromolecular assemblies.

[0258] In some embodiments, the particle-forming protein is: a nanoparticle-forming protein; a virus protein, in particular a virus capsid protein. In some embodiments, the particle is a nanoparticle or a virus-like particle.

[0259] In some embodiments, the particle-forming protein is a nanoparticle-forming protein.

[0260] In some embodiments, the particle-forming protein is a virus protein, such an adenovirus protein, in particular an adenovirus capsid protein.

[0261] In some embodiments, the particle is a virus-like particle.

[0262] In some embodiments, the particle is a capsid virus-like particle (cVLP).

[0263] In some embodiments the particle-forming protein is a protein from a virus, such as an adenovirus. In some embodiments, the particle-forming protein is a protein from a hepatitis virus such as hepatitis B or E, for example a core protein from hepatitis B virus. In some embodiments, the particle-forming protein is a protein from a norovirus such as NoV. In some embodiments, the particle-forming protein is a protein from a papilloma virus such as Human Papilloma Virus (HPV), preferably HPV16 or HPV18, such as HPV L1. In some embodiments, the particle-forming protein a protein from a polyomavirus such as polyomavirus vp1 (PyV). In some embodiments, the particleforming protein is a protein from a calicivirus such as feline calicivirus (FCV), preferably FCV VP1. In some embodiments, the particle-forming protein is a protein from a circovirus such as a porcine circovirus (PCV), preferably PCV2 ORF2. In some embodiments, the particle-forming protein is a protein from a nervous necrosis virus P7422PC00

[0264] (NNV), such as NNV coat protein. In some embodiments, the particle-forming protein is a protein from a parvovirus such as canine parvovirus (CVP), preferably CPV VP2, goose parvovirus (GPV) or porcine parvovirus (PPV), preferably structural proteins from GPV or PPV, or parvovirus B19. In some embodiments, the particle-forming protein is a protein from a protoparvovirus such as an enteritis virus, for example mink enteritis virus (MEV), preferably MEV VP2, or duck plague virus (DPV), preferably a DPV structural protein.

[0265] The protein may be a particle-forming 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 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).

[0266] In some embodiments, the particle-forming is a bacteriophage protein, such as a protein from 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.

[0267] In some embodiments, the particle-forming protein has at least 70% sequence identity, such as 75%, such as 80%, such as 85%, such as 90%, such as 95% such as 99%, such as 100% sequence identity to:

[0268] MsDps2 as set forth in SEQ ID NO: 33;

[0269] AP205 as set forth in SEQ ID NO: 34; ferritin as set forth in SEQ ID NO: 35; i301 as set forth in SEQ ID NO: 36; replicase polyprotein 1a (pp1a); a lumazine synthase as set forth in SEQ ID NO: 38;

[0270] Hbc as set forth in SEQ ID NO: 39; tandemHBc as set forth in SEQ ID NO: 40; the 2-oxo acid dehydrogenase subunit E2 as set forth in SEQ ID NO: 41 ; or P7422PC00 the norovirus capsid protein as set forth in SEQ ID NO: 42.

[0271] In some embodiments, the particle-forming protein has at least 70% sequence identity to MsDps2 as set forth in SEQ ID NO: 33.

[0272] In some embodiments, the particle-forming protein has at least 70% sequence identity to AP205 as set forth in SEQ ID NO: 34.

[0273] In some embodiments, the particle-forming protein has at least 70% sequence identity to ferritin as set forth in SEQ ID NO: 35.

[0274] In some embodiments, the particle-forming protein has at least 70% sequence identity to i301 as set forth in SEQ ID NO: 36.

[0275] In some embodiments, the particle-forming protein has at least 70% sequence identity to replicase polyprotein 1a (pp1a).

[0276] In some embodiments, the particle-forming protein has at least 70% sequence identity to a lumazine synthase as set forth in SEQ ID NO: 38.

[0277] In some embodiments, the particle-forming protein has at least 70% sequence identity to Hbc as set forth in SEQ ID NO: 39.

[0278] In some embodiments, the particle-forming protein has at least 70% sequence identity to tandemHBc as set forth in SEQ ID NO: 40.

[0279] In some embodiments, the particle-forming protein has at least 70% sequence identity to the 2-oxo acid dehydrogenase subunit E2 as set forth in SEQ ID NO: 41.

[0280] In some embodiments, the particle-forming protein has at least 70% sequence identity to the norovirus capsid protein as set forth in SEQ ID NO: 42.

[0281] In some embodiments, the particle-forming protein is a viral capsid protein or a viral envelope protein such as a glycoprotein. P7422PC00

[0282] In some embodiments, the particle-forming protein is a protein of a recombinant adenovirus. In some embodiments, the particle-forming protein is an adenovirus and the first peptide tag is SpyTag. In some embodiments, the particle-forming protein is an adenovirus and the first peptide tag is SpyCatcher.

[0283] In some embodiments, the particle-forming protein attached to the first peptide is as set forth in SEQ ID NO: 73; SEQ ID NO: 74; or a polypeptide having 95% sequence identity thereto.

[0284] In some embodiments, the particle-forming protein attached to the first peptide is as set forth in SEQ ID NO: 73, or a polypeptide having 95% sequence identity thereto.

[0285] In some embodiments the particle-forming protein attached to the first peptide is as set forth in SEQ ID NO: 74 or a polypeptide having 95% sequence identity thereto.

[0286] The first and second peptide tags

[0287] The first and the second peptide tags can bind to one another either by the formation of an isopeptide bond, or via an ester bond. The terms “first peptide tag” and “first peptide” may be used interchangeably herein; similarly, a “second peptide” may refer to a “second peptide tag”.

[0288] Peptide pairs, consisting of a first peptide tag and a second peptide tag which are capable of binding to one another 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 as described in Zakeri et al., 2012, and in Zakeri et al., 2010.

[0289] As described herein, the first peptide is fused to a particle-forming protein, such as the particle-forming protein described in the corresponding section of the present disclosure.

[0290] As described herein, the second peptide is fused to an antigen, such as the antigen described in the corresponding section of the present disclosure.

[0291] The term "peptide tag" as used herein in some embodiments refers to a small peptide fragment which may be designed or derived directly from a particle-forming protein P7422PC00 which naturally forms an intramolecular isopeptide bond. Peptide tags may also be identified by using a known binding partner, for example derived from a particle-forming protein naturally forming an intramolecular isopeptide bond, to screen a peptide library. The candidate peptide tags may thus be from a library, e.g. a peptide library, which can be screened for candidate peptide tags. They may also be designed in silico.

[0292] A peptide pair as understood herein thus in some embodiments consists of two peptide tags which can interact via the spontaneous formation of an isopeptide bond.

[0293] Generally, these are also called a “tag and catcher” system, where the longer of the two peptide tags is termed “catcher” while the shorter of the two peptide tags is termed “tag”. For instance, the SpyTag / SpyCatcher system consists of a first peptide tag (SpyTag) and a second peptide tag (SpyCatcher).

[0294] In some embodiments, the peptide pair as understood herein consists of two peptide tags which can interact via the spontaneous formation of an ester bond. Useful peptide tags able to form such spontaneous ester bonds are further described in Young et al., 2017. An example of an esther-forming split-protein pair is the fragment corresponding to amino acid residues 439-587 of cpe0147 (Uniprot B1 R775) (SEQ ID NO: 32) and the fragment corresponding to amino acid residues 565-587 of cpe0147 (Uniprot B1R775) (SEQ ID NO: 22).

[0295] The “tag” may be between 5-50 amino acids in length e.g. from 10, 20, 30, 40 to 50 amino acids in length and may bind covalently via an isopeptide bond to a binding partner as defined herein. Thus, the “tag” may comprise one reactive residue involved in an isopeptide bond in the isopeptide protein used to design the binding partner (and the binding partner may comprise the other reactive residue involved in that bond), as described herein.

[0296] In some embodiments, the “tag” has a length between 7 and 47 amino acids, such as between 8 and 46 amino acids, such as between 9 and 45 amino acids, such as between 10 and 44 amino acids, such as between 11 and 43 amino acids, such as between 12 and 42 amino acids, such as between 13 and 41 amino acids, such as between 14 and 40 amino acids, such as between 15 and 39 amino acids, such as between 16 and 38 amino acids, such as between 17 and 37 amino acids, such as between 18 and 36 amino acids, such as between 19 and 35 amino acids, such as P7422PC00 between 20 and 34 amino acids, such as between 21 and 33 amino acids, such as between 22 and 32 amino acids, such as between 23 and 31 amino acids, such as between 24 and 30 amino acids, such as between 25 and 29 amino acids, such as between 26 and 28 amino acids, such as 27 amino acids. In some embodiments, the

[0297] “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.

[0298] In some embodiments, the “catcher” is at least 20 amino acids in length. Preferably, the “catcher” has a length of 5 amino acids or more, such as 10 amino acids or more, such as 15 amino acids or more, such as 20 amino acids or more, such as 25 amino acids, such as 30 amino acids, such as 35 amino acids, such as 40 amino acids, such as 45 amino acids, such as 50 amino acids, such as 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 amino acids or more. In preferred embodiments, the “catcher” is at least 20 amino acids in length. In some embodiments, the “catcher” is between 75 to 125 amino acids in length.

[0299] Preferably, the “catcher” has an amino acid sequence which consists of more amino acid residues than the “tag”.

[0300] In some embodiments where an isopeptide bond is formed between the first and the second peptide tags, the first or the second peptide tag 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); 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); Rum7Tag (SEQ ID NO: 12); Rumtrunk D9N (SEQ ID NO: 13); RumTrunkTag (SEQ ID NO: 14); BacTag (SEQ ID NO: 15); Bac2Tag (SEQ ID NO: 16); Bac3Tag (SEQ ID NO: 17); Bac4Tag (SEQ ID NO: 18); Bac5Tag (SEQ ID NO: 19); PsCsTag (SEQ ID NO: 20); Clib9 (SEQ ID NO: 21); Ktag (SEQ ID NO: 76) or a homologue or fragment thereof having at least 70% homology or identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least P7422PC00

[0301] 98%, such as at least 99% identity thereto, with the proviso that said homologue or fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

[0302] In some embodiments where an isopeptide bond is formed between the first and the second peptide tags, the first or the second peptide tag is: SpyCatcher (SEQ ID NO: 23); Spycatcherv2 (SEQ ID NO: 24); Spycatcherv3 (SEQ ID NO: 25); SdyCatcher (SEQ ID NO: 26); SnoopCatcher (SEQ ID NO: 27); MoonCake (SEQ ID NO: 28); Katl (SEQ ID NO: 29); QueenCatcher (SEQ ID NO: 30); PsCsCatcher (SEQ ID NO: 31); MiniMC (SEQ ID NO: 81); or homologues or fragments thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said homologues or fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

[0303] In some embodiments where an isopeptide bond is formed between the first and the second peptide tags, the first or the second peptide tag is: MoonCake (SEQ ID NO: 28); MiniMC (SEQ ID NO: 81); or homologues or fragments thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said homologues or fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N). P7422PC00

[0304] In some embodiments, the isopeptide bond between the first and second peptide tag is an isopeptide bond between: i. SEQ ID NO: 28 (MoonCake) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto; and / or ii. SEQ ID NO: 28 (Katl) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); P7422PC00 l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto; and / or iii. SEQ ID NO: 29 (QueenCatcher) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto and / or iv. SEQ ID NO: 81 (MiniMC) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); P7422PC00 g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto.

[0305] In some embodiments, the isopeptide bond between the first and second peptide tag is an isopeptide bond between: i. SEQ ID NO: 28 (MoonCake) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto; and / or P7422PC00 ii. SEQ ID NO: 81 (MiniMC) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto.

[0306] In some embodiments, the one or more first peptide tags are fused to the N-terminal end, to the C-terminal end of the particle-forming protein and / or inserted in-frame into the coding sequence of the particle-forming protein, optionally by a linker.

[0307] In some embodiments, one or more second peptide tags are fused to the N-terminal end, to the C-terminal end of the antigen and / or inserted in-frame into the coding sequence of the antigen, optionally by a linker.

[0308] In some embodiments, the peptide pair comprises or consists of truncated or modified versions of any of the above, i.e. further engineered peptide pairs, which however retain the ability to form an isopeptide bond.

[0309] A peptide tag may be altered, e.g. mutations or alterations may be introduced in any one or any two of the first or second peptide tag, i.e. in any one or any two of the tag and catcher. The peptide tag, i.e. the first, or the second peptide tag, should be able to P7422PC00 covalently bind to a corresponding binding partner via an isopeptide bond, or via an ester bond, spontaneously. In this respect, each peptide tag preferably comprises one of the reactive amino acid residues involved in the formation of an isopeptide bond in the isopeptide protein. Hence, each peptide tag comprises only one reactive residue from the isopeptide bond and does not comprise both reactive residues involved. Further, if the peptide tag is modified or mutated, the reactive residue in that fragment preferably remains unchanged. This means that when a homologue of a peptide tag is used, the homologue preferably still contains the reactive residue which was originally present in the original peptide tag. In some embodiments, however, the reactive residue in that fragment is also changed if the peptide tag is modified or mutated.

[0310] Preferably, the reactive residue present in the tag is an asparagine or an aspartate residue, which can form an isopeptide bond with the reactive residue of the binding partner or modified binding partner (the catcher), as described above. Thus, one peptide tag contains one reactive residue while the other peptide tag contains the other reactive residue, and thus no single peptide tag contain both reactive residues.

[0311] In some embodiments, both reactive residues are involved in the formation of an isopeptide bond. In some embodiments, the reactive residue of the first peptide tag is different than 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 an aspartate residue. Preferably, the reactive residue present in the “tag” is an asparagine or an aspartate residue. In some embodiments, the reactive residue present in the “tag” is a lysine residue. These residues together may form the isopeptide bond.

[0312] Fusion of the first peptide tag to the protein and fusion of the second peptide tag to the antigen

[0313] Changing the position where the first peptide tag is fused to the protein may allow changing the orientation of the antigen on the particle. This may be performed to enable the best possible display of the most important epitopes of the antigen. The best possible orientation may be different from antigen to antigen.

[0314] Epitopes of specific monoclonal antibodies may be mapped on the antigen structure, whereby it is possible to determine which epitopes are accessible after conjugation of P7422PC00 the antigen to the particle-forming protein. Specifically, one may measure binding between a specific monoclonal antibody and the complex of the antigen bound to the particle forming protein (antigen: particle complex), such as by using ELISA or another affinity-measuring technique (e.g. Attana), and thereby determine the orientation of the antigen. Cryo-electron microscopy may also be used to determine the structure of the entire antigemparticle complex. If the antigen contains a functional binding epitope, binding-assays may be conducted to determine if the epitope is exposed or hidden in the final antigemparticle complex.

[0315] Changing the position where the second peptide tag is fused to the antigen will allow changing the orientation of the antigen on the particle. This may be performed to enable the best possible display of the most important epitopes of the antigen. The best possible orientation may be different from antigen to antigen.

[0316] 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 in the coding sequence of the protein. The fusion protein may comprise a linker between the first peptide tag and the protein.

[0317] 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 in the coding sequence of the antigen. The fusion protein may comprise a linker between the second peptide tag and the antigen.

[0318] Further elements of the vaccine

[0319] In some embodiments, the first polynucleotide further comprises a secretion or excretion signal, whereby the particle forming protein fused to the first peptide tag is secreted or excreted from the endoplasmic reticulum of the cell.

[0320] In some embodiments, the secretion or excretion signal comprises or consists of: MFPFALLYVLSVSFRKIFILQLVGLVLT (SEQ ID NO: 43). P7422PC00

[0321] In some embodiments, the secretion or excretion signal comprises or consists of MGSAALLLWVLLLWVPGSNG (SEQ ID NO: 44).

[0322] It will be evident to the skilled person that the sequences above are merely examples, and that any known secretion or excretion signal known in the art to promote secretion or extraction from the endoplasmic reticulum of the cell might be used. Thus, any signal sequence described herein might substitute by other suitable signal sequence.

[0323] In some embodiments the coupled particle is secreted from the cell.

[0324] Expression systems

[0325] Herein is also provided an expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0326] Herein is also provided an expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0327] The expression system may consist of or comprise a polycistronic RNA construct and / or a DNA construct, from which the transcribed mRNA is polycistronic. Thus, in P7422PC00 some embodiments, the first and the second polynucleotides of the expression system are encoded on the same ribonucleic acid molecule. In some embodiments, the first and the second polynucleotides of the expression system lie within the same open reading frame, whereby only one promoter sequence is needed to transcribe both polynucleotides. In some embodiments, the first and the second polynucleotides of the expression system lie within separate open reading frames and may thus be regulated by separate promoters.

[0328] The first peptide tag, the second peptide tag, the protein and / or the antigen may be as defined herein elsewhere.

[0329] The term “expression system” refers to a genetic construct designed to produce a protein and / or an RNA inside a cell. Thus, the expression system may comprise RNA and / or DNA, which is translated or transcribed to a protein or DNA, respectively, inside the cell.

[0330] The expression system may comprise the sequences necessary for gene expression in the cell. These may include a promoter, a translation initiation sequence such as a ribosomal binding site, a start codon, a termination codon, and a transcription termination sequence. There are differences in the enzymes responsible for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors must comprise elements for expression that are appropriate for the chosen host. For example, prokaryotic expression systems may comprise a Shine-Dalgarno sequence at the translation initiation site for the binding of ribosomes, while eukaryotic expression systems may contain a Kozak consensus sequence.

[0331] 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, whereby cells comprising the expression system may be selected for, or a screenable marker, such as a reporter gene, i.e. a gene that allows for differentiation between cells comprising or not comprising the expression system, whereby cells comprising the expression system may be identified. Examples of such markers include antibiotic resistance genes, auxotrophic markers and genes expressing detectable compounds, such as coloured and / or fluorescent compounds. P7422PC00

[0332] 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.

[0333] 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 each be under the control of a first and / or second promoter, respectively, which may be identical or different. They may also be under the control of a single promoter.

[0334] The first and the second polynucleotides of the expression system may be comprised within the same molecule. The first and the second polynucleotides of the expression system may alternatively be comprised within different molecules, such as within two or more separate molecules.

[0335] The first and / or the 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 also from the cell.

[0336] The present expressions systems can be used for prophylaxis and / or treatment of a wide range of diseases as disclosed herein.

[0337] Cells

[0338] The invention further relates to a cell, such as a host cell, comprising one or more polynucleotides and / or an expression system as disclosed herein. The one or more polynucleotides and / or expression system may have a sequence that is codon- optimised. Codon optimisation methods are known in the art and allow optimised expression in a heterologous host organism or cell.

[0339] Thus, in an aspect, the present disclosure concerns a cell comprising the polynucleotide as described herein. P7422PC00

[0340] In some embodiments the cell comprises the expression system as described herein.

[0341] In an aspect, the present disclosure concerns a cell expressing: i. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as described herein; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

[0342] In an aspect, the present disclosure concerns a cell expressing: i. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as described herein; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), preferably as described herein, wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

[0343] In some embodiments the cell may be selected from a bacterial cell, yeast cell, a fungal cell, a plant, a mammalian cell, and / or an insect cell.

[0344] 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. P7422PC00

[0345] In some embodiments, the cell is a microorganism.

[0346] In an aspect, the present disclosure concerns a host cell, wherein the host cell comprises an expression system as described herein.

[0347] In some embodiments the host cell may be selected from a bacterial cell, yeast cell, a fungal cell, a plant, a mammalian cell, and / or an insect cell.

[0348] In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is a fungal cell. In some embodiments, the host cell is a plant cell. In some embodiments, the host cell is a mammalian cell, such as a human cell. In some embodiments, the host cell is an insect cell.

[0349] Methods for expressing a polypeptide as described herein, such as a first polypeptide and / or a second polypeptide in a cell, such as a host cell, are known in the art. The first or second polypeptide may be heterologously expressed from corresponding polynucleotide sequences cloned into the genome of the cell or they may be comprised within a vector. For example, a first and / or second polynucleotide coding for the first and / or second polypeptide is cloned into the genome, and a first and / or second polynucleotide coding for the first and / or second polypeptide is comprised within a vector transformed or transfected into the cell.

[0350] Expression of a polypeptide, such as the first and second polypeptides in the cell may occur in a transient manner. When the polynucleotide encoding one of the polypeptides is cloned into the genome, an inducible promoter may be cloned as well to control expression of the polypeptides. Such inducible promoters are known in the art.

[0351] Alternatively, genes coding for suppressors of gene silencing may also be cloned into the genome or into a vector transfected within the cell.

[0352] In some embodiments, the cell, such as the host cell, may be selected from 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 P7422PC00

[0353] 293, Nicotiana tabacum cv. Samsun NN or Solanum tuberosum cv. Solara. Thus in some embodiments, the cell is Escherichia coli. In another embodiment, the cell is Spodoptera frugiperda. In some embodiments, the cell is Pichia Pastoris. In some embodiments, the cell is Saccharomyces cerevisiae. In some embodiments, the cell is Hansenula polymorpha. In some embodiments, the cell is Drosophila Schneider 2. In some embodiments, the cell is Lactococcus lactis. In some embodiments, the cell is Chinese hamster ovary (CHO). In some embodiments, the cell is Human Embryonic Kidney 293. In some embodiments, the cell is Trichoplusiani (BTI-TN-5B1-4). In In some embodiments, the cell is Nicotiana tabacum cv. Samsun NN. In some embodiments, the cell is Solanum tuberosum cv. Solara.

[0354] Use of the vaccine in medicine

[0355] In an aspect, the present disclosure concerns a vaccine for use in medicine, wherein the vaccine is as described herein.

[0356] In an aspect, the present disclosure concerns a vaccine for use in the prophylaxis and / or treatment of a disease, wherein the vaccine is as described herein, and wherein the disease is an inflammatory disease, a fibrotic disorder, a cancer, a chronic epithelial barrier disorder, a skin disease, and / or an autoimmune disease. The vaccine may be administered to a subject in need thereof, in particular to a human subject, suffering, suspected of suffering, or at risk of developing the disease.

[0357] It will be evident to the skilled person that, in some embodiments, a specific disease might be categorized in multiple ways. For example a atopic dermatitis, a disease which might benefit from immunization with the vaccine disclosed herein, might be considered an inflammatory disease, a chronic epithelial barrier disorder, and / or a skin disease. Thus, the skilled person will understand that the categories mentioned above are not to be considered as mutually exclusive.

[0358] In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is a fibrotic disorder. In some embodiments, the disease is a cancer. In some embodiments, the disease is a chronic epithelial barrier disorder. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is a skin disease. P7422PC00

[0359] In some embodiments, the skin disease is: atopic dermatitis, psoriasis, bullous pemphigoid, systemic sclerosis, chronic spontaneous urticaria, Behget’s disease, vitiligo, rosacea, systemic lupus erythematosus, and alopecia areata.

[0360] In an aspect, the present disclosure concerns a method of administering a vaccine for the prophylaxis and / or treatment of a disease in a subject in need thereof, comprising the step of i. administering at least one composition comprising the vaccine as described herein to a subject at least once for prophylaxis and / or treatment of a disease as described herein.

[0361] In some embodiments, the composition comprising the vaccine as described herein is boosted by administration in a form or body part different from the previous administration.

[0362] In some embodiments, the vaccine is administered to the area most likely to be the receptacle of a given disease.

[0363] In some embodiments, the subject is an animal, such as a mammal, such as a dog, a cat, a cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, most preferably such as a human being; or a bird, such as a chicken or a fish, such as a salmon.

[0364] In some embodiments, the composition is administered in combination with any other vaccine.

[0365] In some embodiments, the composition forms a part of a vaccine cocktail.

[0366] Kits of parts

[0367] In an aspect, the present disclosure concerns a kit of parts comprising i. a vaccine as described herein, or an expression system as described herein, and ii. optionally, a medical instrument or other means for administering the vaccine, and iii. instructions for use. P7422PC00

[0368] In some embodiments, the kit of parts comprises a second active ingredient.

[0369] Isolation of the antigen and manufacture of the vaccine

[0370] In an aspect, the present disclosure concerns a method of isolating an antigen as described herein, said antigen being fused to a peptide tag as described herein, wherein the method comprises expressing said antigen fused to a peptide tag in a cell, such as the cell described herein, such as a mammalian cell, or such as a microorganism, such as a bacteria, such as E. coli.

[0371] The skilled person will understand that any method known in the art which is suitable for isolating a polypeptide expressed by a cell might be employed to isolate the antigen described herein. This includes for example lysis of the host cell using mechanical, chemical, or enzymatic methods to release the expressed antigen. Depending on the nature of the host cell and expression system, methods such as sonication, bead milling, or the use of detergents or lysozyme may be employed to disrupt the cell membrane while preserving the structural integrity of the antigen.

[0372] Following cell lysis, techniques such as centrifugation or filtration may be used to separate soluble components from cell debris. The antigen fused to a peptide tag can then be isolated using affinity purification techniques, which exploit the specific binding properties of the tag. For instance, if the tag is a polyhistidine tag, immobilized metal affinity chromatography (IMAC) can be used.

[0373] In cases where the antigen forms inclusion bodies or insoluble aggregates, solubilization followed by refolding protocols may be applied to recover the antigen in its functional form. Further purification steps, such as size exclusion chromatography or ion exchange chromatography, may be employed to ensure the antigen meets the required purity standards for downstream applications.

[0374] Alternative methods, including automated purification systems or cell-free expression platforms, may also be utilized depending on the scale and application of the antigen. These methods allow for scalability, reproducibility, and compliance with regulatory standards while ensuring that the antigen retains its intended structure and functionality. P7422PC00

[0375] In an aspect, the present disclosure concerns a method of manufacturing the vaccine as described herein, comprising the step of: i. Providing an expression system as described herein; and ii. Recovering the vaccine.

[0376] The skilled person will understand that any method known in the art which is suitable for manufacturing a vaccine might be employed. This includes for example methods involving recombinant protein expression in host cells, such as bacterial, yeast, insect, or mammalian cells, followed by purification steps to isolate the antigen or antigenic particle. The expression system may be configured to ensure proper folding, post- translational modifications, and assembly of the antigen or antigen-particle complex to maintain immunogenicity and structural fidelity.

[0377] In addition to traditional expression and purification techniques, advanced methodologies such as cell-free expression systems or in vivo assembly of nanoparticles may also be employed. Downstream processes, including chromatographic purification, ultrafiltration, and sterile filtration, may be utilized to ensure the final vaccine product meets required purity and safety standards.

[0378] Furthermore, techniques such as encapsulation, lyophilization, or formulation into nanoparticles or lipid vesicles might be applied to enhance the vaccine’s stability, delivery, or immunogenic efficacy. The method of manufacturing may be tailored to the specific antigen and its intended application, ensuring scalability, reproducibility, and compliance with regulatory requirements for vaccine production.

[0379] Methods for manufacturing a vaccine targeting IL-11

[0380] Also disclosed herein is a method of manufacturing a vaccine targeting IL-11, comprising the steps of: a) Designing an antigen sequence comprising an epitope or mimotope of TSLP, preferably wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP); b) Producing a polynucleotide encoding the antigen or producing a polypeptide comprising or consisting of the antigen; c) Formulating the polynucleotide or the polypeptide as a vaccine. P7422PC00

[0381] All the elements of such vaccines have been described herein above. In some embodiments, the vaccine comprises a particle-forming protein comprising a first peptide tag, and the vaccine comprises the antigen fused to a second peptide tag.

[0382] In some embodiments the antigen comprises a scaffold protein, which can be designed for instance using RFdiffusion, ProteinMPNN or glycan masking strategies, as described herein elsewhere.

[0383] Items

[0384] 2. A vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0385] 3. A vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP, wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0386] 4. A vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP), P7422PC00 wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0387] 5. A vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0388] 6. The vaccine according to any one of the preceding items, wherein the epitope is a conformational epitope of IfTSLP.

[0389] 7. The vaccine according to any one of the preceding items, wherein less than 20%, such as less than 15%, 10%, 5% of the amino acid residues of the antigen are amino acid residues which are present both in sfTLSP and IfTSLP, such as none of the amino acid residues of the antigen are amino acid residues which are present both in sfTSLP and IfTSLP.

[0390] 8. The vaccine according to any one of the preceding items, wherein the antigen comprises one or more epitopes of TSLP, wherein each epitope of TSLP comprises one or more amino acid residues present in IfTSLP and absent in sfTSLP.

[0391] 9. The vaccine according to any one of the preceding items, wherein the antigen does not comprise epitopes of sfTSLP

[0392] 10. The vaccine according to any one of the preceding items, wherein none of the one or more epitopes of TSLP comprise amino acid residues present in sfTLSP. P7422PC00

[0393] 11 . The vaccine according to any one of the preceding items, wherein the antigen does not comprise epitopes which exclusively consist of amino acids absent in the IfTSLP.

[0394] 12. The vaccine according to any one of the preceding items, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising an epitope of IfTSLP.

[0395] 13. The vaccine according to any one of the preceding items, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising a conformational epitope of IfTSLP.

[0396] 14. The vaccine according to any one of the preceding items, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising said epitope of IfTSLP, and wherein the amino acid residues of said epitope in the vaccine maintain the same spatial arrangement between each other as in the corresponding three-dimensional structure of IfTSLP.

[0397] 15. The vaccine according to any one of the preceding items, wherein the antigen does not comprise any amino acid residue of sfTLSP.

[0398] 16. The vaccine according to any one of the preceding items, wherein the antigen does not comprise a polypeptide consisting of sfTLSP or an immunogenic fragment thereof.

[0399] 17. The vaccine according to any one of the preceding items, wherein the epitope comprises two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the corresponding three- dimensional structure of IfTSLP.

[0400] 18. The vaccine according to any one of the preceding items, wherein the epitope comprises two or more amino acid residues of IfTSLP, wherein: P7422PC00 i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure of the antigen and the corresponding two or more amino acid residues within the three- dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A.

[0401] 19. The vaccine according to any one of the preceding items, wherein the vaccine is capable of inducing in a subject one or more antibodies specific to IfTSLP, preferably wherein said antibodies do not bind to sfTSLP.

[0402] 20. The vaccine according to any one of the preceding items, wherein the vaccine is not capable of inducing antibodies in a subject which bind to the corresponding short-form variant of TSLP (sfTSLP).

[0403] 21. The vaccine according to any one of the preceding items, wherein the vaccine is capable of inducing in a subject one or more antibodies which have an affinity for IfTSLP which is at least 2-fold, 3-fold, 5-fold, 10-fold greater than the affinity for sfTSLP.

[0404] 22. The vaccine according to any one of the preceding items, wherein the vaccine is capable of inducing in a subject one or more antibodies which have affinity for a conformational epitope of IfTSLP, which is absent in sfTSLP.

[0405] 23. The vaccine according to any one of the preceding items, wherein the vaccine is capable of inducing one or more antibodies which have a Kd (Dissociation Constant) for IfTSLP which is at least 2-fold, 3-fold, 5-fold, 10-fold lower than sfTSLP.

[0406] 24. The vaccine according to any one of the preceding items, wherein the vaccine is not cross-reactive to sfTSLP. P7422PC00

[0407] 25. The vaccine according to any one of the preceding items, wherein IfTSLP is human IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

[0408] 26. The vaccine according to any one of the preceding items, wherein the antigen comprises a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof.

[0409] 27. The vaccine according to any one of the preceding items, wherein the antigen comprises: a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of: Cys110, Cys137, Arg150, or Arg153 of SEQ ID NO: 45.

[0410] 28. The vaccine according to any one of the preceding items, wherein the antigen comprises: a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of: F98, A99, L106, Try109, Cys110, Cys137, Arg150, or Arg153 of SEQ ID NO: 45.

[0411] 29. The vaccine according to any one of the preceding items, wherein said polypeptide further comprises one or more of: Glu139; Val141 ; or Gln143 as set forth in SEQ ID NO: 45.

[0412] 30. The vaccine according to any one of the preceding items, wherein the antigen comprises a polypeptide having at least 95% identity to amino acid residues 139 to 153 of SEQ ID NO: 45; such as a polypeptide comprising one or more of amino acid residues 146 to 153 of SEQ ID NO: 45.

[0413] 31. The vaccine according to any one of the preceding items, wherein the antigen further comprises a polypeptide having one or more of amino acid residues Leu144, Gly146, Leu147, Trp148; Arg149; Phe151, Asn152 of SEQ ID NO: 45. P7422PC00

[0414] 32. The vaccine according to any one of the preceding items, wherein the antigen does not comprise a polypeptide amino acid residues 97 to 159, such as amino acid residues 110 to 159, 120 to 159, 130 to 159, 140 to 159, 97 to 140, 97 to 130, 97 to 120, 97 to 110 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof.

[0415] 33. The vaccine according to any one of the preceding items, wherein the epitope comprises two or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the three-dimensional structure of IfTSLP, wherein IfTSLP is as set forth in SEQ ID NO: 45.

[0416] 34. The vaccine according to any one of the preceding items, wherein the conformational epitope comprises two or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, of IfTSLP, wherein: i. the two or more amino acid residues are selected from residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45, and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure the antigen and the corresponding two or more amino acid residues within the three- dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A, wherein IfTSLP is as set forth in SEQ ID NO: 45.

[0417] 35. The vaccine according to any one of the preceding items, wherein the vaccine is capable of inducing in a subject one or more antibodies which have affinity for, and / or are capable of binding to, a conformational epitope of IfTSLP, wherein the conformational epitope comprises one or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, selected from a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto. P7422PC00

[0418] 36. The vaccine according to any one of the preceding items, wherein the antigen comprises a polypeptide selected from: i. SEQ ID NO: 49; ii. SEQ ID NO: 50; iii. SEQ ID NO: 51 ; iv. SEQ ID NO: 52; v. SEQ ID NO: 53; vi. SEQ ID NO: 54; vii. SEQ ID NO: 55; or a polypeptide having at least 95% sequence identity thereto.

[0419] 37. The vaccine according to any one of the preceding items, wherein the antigen fused to the (second) peptide comprises a polypeptide consisting of: i. SEQ ID NO: 59; ii. SEQ ID NO: 60; iii. SEQ ID NO: 61 ; iv. SEQ ID NO: 62; v. SEQ ID NO: 63; vi. SEQ ID NO: 64; vii. SEQ ID NO: 65; viii. SEQ ID NO: 67; ix. SEQ ID NO: 68; x. SEQ ID NO: 69; xi. SEQ ID NO: 70; xii. SEQ ID NO: 71 ; xiii. SEQ ID NO: 72; or a polypeptide having at least 95% sequence identity thereto.

[0420] 38. The vaccine according to any one of the preceding items, wherein the vaccine comprises two or more antigens fused to the second peptide tag.

[0421] 39. The vaccine according to any one of the preceding items, wherein the vaccine comprises two or more antigens fused to the second peptide tag, and wherein said two or more antigens comprise each an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present P7422PC00 in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP).

[0422] 40. The vaccine according to any one of the preceding items, wherein the vaccine comprises two or more antigens fused to the second peptide tag, wherein the first antigen is as set forth in SEQ ID NO: 51 (#26) and the second antigen is as set forth in SEQ ID NO: 55 (#30).

[0423] 41 . The vaccine according to any one of the preceding items, wherein the vaccine comprises two or more antigens fused to the second peptide tag, wherein the first antigen is as set forth in SEQ ID NO: 51 (#26) and the second antigen is as set forth in SEQ ID NO: 55 (#30), and the second peptide is as set forth in SEQ ID NO: 81.

[0424] 42. The vaccine according to any one of the preceding items, wherein the vaccine comprises two or more antigens fused to the second peptide tag via a linker.

[0425] 43. The vaccine according to item 41 , wherein said linker is a rigid helical linker.

[0426] 44. The vaccine according to item 41 , wherein the linker is a flexible linker.

[0427] 45. The vaccine according to any one of the preceding items, wherein one or more antigens are fused to the N terminus of the second peptide tag and one or more antigens are fused to the C terminus of the second peptide tag.

[0428] 46. The vaccine according to any one of the preceding items, wherein one or more antigens are fused to the N terminal of the second peptide tag and one or more antigens are fused to the C terminus of the second peptide tag, and the second peptide tag is as set forth in SEQ ID NO: 81.

[0429] 47. The vaccine according to any one of the preceding items, wherein the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79 or SEQ ID NO: 80. P7422PC00

[0430] 48. The vaccine according to any one of the preceding items, wherein the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 77.

[0431] 49. The vaccine according to any one of the preceding items, wherein vaccine comprises a polypeptide as set forth in: SEQ ID NO: 78

[0432] 50. The vaccine according to any one of the preceding items, wherein vaccine comprises a polypeptide as set forth in: SEQ ID NO: 79

[0433] 51. The vaccine according to any one of the preceding items, wherein vaccine comprises a polypeptide as set forth in: SEQ ID NO: 80

[0434] 52. The vaccine according to any one of the preceding items, wherein the particle is a nanoparticle.

[0435] 53. The vaccine according to any one of the preceding items, wherein particle forming protein has at least 70% sequence identity, such as 75%, such as 80%, such as 85%, such as 90%, such as 95% such as 99%, such as 100% sequence identity to:

[0436] MsDps2 as set forth in SEQ ID NO: 33;

[0437] AP205 as set forth in SEQ ID NO: 34; ferritin as set forth in SEQ ID NO: 35; i301 as set forth in SEQ ID NO: 36; replicase polyprotein 1a (pp1a); a lumazine synthase as set forth in SEQ ID NO: 38;

[0438] Hbc as set forth in SEQ ID NO: 39; tandemHBc as set forth in SEQ ID NO: 40; the 2-oxo acid dehydrogenase subunit E2 as set forth in SEQ ID NO: 41 ; or the norovirus capsid protein as set forth in SEQ ID NO: 42.

[0439] 54. The vaccine according to any one of the preceding items, wherein the particleforming protein attached to the first peptide tag is as set forth in SEQ ID NO: 73; SEQ ID NO: 74; or a polypeptide having 95% sequence identity thereto.

[0440] 55. The vaccine according to any one of the preceding items, wherein the particle forming protein is a virus-like particle. P7422PC00

[0441] 56. The vaccine according to any one of the preceding items, wherein the particle forming protein is a viral capsid protein or a viral envelope protein such as a glycoprotein.

[0442] 57. The vaccine according to any one of the preceding items, wherein the first or the second peptide tag 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); 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); Rum7Tag (SEQ ID NO: 12); Rumtrunk D9N (SEQ ID NO: 13); RumTrunkTag (SEQ ID NO: 14); BacTag (SEQ ID NO: 15); Bac2Tag (SEQ ID NO: 16); Bac3Tag (SEQ ID NO: 17); Bac4Tag (SEQ ID NO: 18); Bac5Tag (SEQ ID NO: 19); PsCsTag (SEQ ID NO: 20); Clib9 (SEQ ID NO: 21); Ktag (SEQ ID NO: 76) or a fragment thereof having at least 70% homology or identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

[0443] 58. The vaccine according to any one of the preceding items, wherein the first or the second peptide tag is: SpyCatcher (SEQ ID NO: 23); Spycatcherv2 (SEQ ID NO: 24); Spycatcherv3 (SEQ ID NO: 25); SdyCatcher (SEQ ID NO: 26); SnoopCatcher (SEQ ID NO: 27); MoonCake (SEQ ID NO: 28); Katl (SEQ ID NO: 29);

[0444] QueenCatcher (SEQ ID NO: 30); PsCsCatcher (SEQ ID NO: 31); MiniMC (SEQ ID NO: 81), or fragments thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least P7422PC00

[0445] 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

[0446] 59. The vaccine according to any one of the preceding items wherein the isopeptide bond between the first and second peptide tag is an isopeptide bond between: i. SEQ ID NO: 28 (MoonCake) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto; and / or ii. SEQ ID NO: 29 (Katl) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); P7422PC00 f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto; and / or iii. SEQ ID NO: 30 (QueenCatcher) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% identity thereto and / or P7422PC00 iv. SEQ ID NO: 81 (MiniMC) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% homology or identity thereto.

[0447] 60. The vaccine according to any one items 2, or 4 to 58, wherein the first and / or second polynucleotide is DNA.

[0448] 61. The vaccine according to any one items 2, or 4 to 59, wherein the first and / or second polynucleotide is RNA.

[0449] 62. The vaccine according to item 60, wherein the RNA is formulated in a lipid particle formulation.

[0450] 63. The vaccine according to any one of the preceding items, wherein one or more first peptide tags are fused to the N-terminal end, to the C-terminal end of the particle forming protein and / or inserted in-frame into the coding sequence of the particle forming protein, optionally by a linker. P7422PC00

[0451] 64. The vaccine according to any one of items 2, 4 to 62, wherein one or more second peptide tags are fused to the N-terminal end, to the C-terminal end of the antigen and / or inserted in-frame into the coding sequence of the antigen, optionally by a linker.

[0452] 65. The vaccine according to any one of items 2, 4 to 63, wherein the first polynucleotide further comprises a secretion or excretion signal, whereby the particle forming protein fused to the first peptide tag is secreted or excreted from the endoplasmic reticulum of the cell.

[0453] 66. The vaccine according to item 64, wherein the secretion or excretion signal comprises or consists MFPFALLYVLSVSFRKIFILQLVGLVLT (SEQ ID NO: 43).

[0454] 67. The vaccine according to any one of the preceding items, wherein the antigen further comprises a polyhistidine tag or a cTag.

[0455] 68. The vaccine according to any one of the preceding items, wherein the antigen is capable of eliciting an immune reaction in an animal, such as a mammal, such as a Homo sapiens, a dog, cat, cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, and / or a bird, such as a chicken and / or a fish, such as a salmon.

[0456] 69. A composition comprising a vaccine according to any one of the preceding items.

[0457] 70. The composition according to item 68, wherein the composition further comprises an acceptable carrier.

[0458] 71. The composition according to any one of item 68 to 69, wherein the composition further comprises an adjuvant.

[0459] 72. A vaccine, or a composition, for use in medicine, wherein the vaccine or the composition is according to any one of the preceding items.

[0460] 73. A vaccine, or a composition, for use in the prophylaxis and / or treatment of a disease, wherein the vaccine or the composition is according to any one of the preceding items, and wherein the disease is an inflammatory disease, a fibrotic P7422PC00 disorder, a cancer, a chronic epithelial barrier disorders, a skin disease, and / or an autoimmune disease.

[0461] 74. An antigen as defined in any one of the preceding items, fused to a (second) peptide tag as defined in any one of the preceding items.

[0462] 75. A polynucleotide encoding an antigen as defined in any one of the preceding items fused to a (second) peptide tag as defined in any one of the preceding items.

[0463] 76. A cell comprising the polynucleotide according to item 74.

[0464] 77. The cell according to item 75, wherein the cell is a mammalian cell, an insect cell, or a microorganism.

[0465] 78. The cell according to item 75, wherein the cell is a mammalian cell.

[0466] 79. The cell according to item 75, wherein the cell is a microorganism.

[0467] 80. The cell according to item 75, wherein the cell is a Bacterium.

[0468] 81. The cell according to item 75, wherein the cell is E. Coli.

[0469] 82. An expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0470] 83. An expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and P7422PC00 ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0471] 84. The expression system according to item 81 to 82, wherein the first peptide tag, the second peptide tag, the particle forming protein and / or the antigen are as defined in any one of items 2, or 4 to 67.

[0472] 85. The expression system according to any one of items 81 to 83, wherein the first polynucleotide and the second polynucleotide are comprised within the same nucleic acid molecule, or within two different nucleic acid molecules.

[0473] 86. The expression system according to any one of items 81 to 84, wherein the first polynucleotide and the second polynucleotide are both DNA polynucleotides or RNA polynucleotides.

[0474] 87. The expression system according to any one of items 81 to 85, wherein the first and second polynucleotides are comprised within one vector such as a viral vector or a plasmid, or wherein the first and second polynucleotides are comprised within two vectors such as two viral vectors; two plasmids; or one viral vector and one plasmid.

[0475] 88. The expression system according to item 86, wherein the viral vector is an adenoviral vector, such as a modified adenoviral vector, e.g. the replicationdeficient simian adenovirus vector ChAdOxI , or a modified vaccinia Ankara (MVA) vector.

[0476] 89. The expression system according to any one of items 81 to 87, wherein the expression system comprises or consists of a plasmid. P7422PC00

[0477] 90. The expression system according to any one of items 81 to 88, wherein the expression system comprises or consists of an mRNA.

[0478] 91. A cell expressing: i. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as defined in any one of the preceding items; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

[0479] 92. A cell expressing: i. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as defined in any one of the preceding items; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

[0480] 93. The cell according to any one of items 90 to 91 , comprising the vaccine according to any one of items 2 to 67, the polynucleotide according to item 74, or the expression system according to any one of items 81 to 89.

[0481] 94. A host cell, wherein the host cell comprises an expression system according to any one of items 81 to 89. P7422PC00

[0482] 95. The host cell according to item 93, wherein the host cell is a bacterial cell, a yeast cell, a fungal cell, a plant cell, a mammalian cell, or an insect cell.

[0483] 96. A method of administering a vaccine, or a composition, for the prophylaxis and / or treatment of a disease in a subject in need thereof, comprising the step of i. administering at least one composition comprising a vaccine as defined in any one of items 1 to 67, or a composition according to any of items 68 to 70, to a subject at least once for prophylaxis and / or treatment of a disease as defined in any one of the preceding items.

[0484] 97. The method of administering a vaccine, or a composition for the use according to item 95, wherein the composition is boosted by administration in a form or body part different from the previous administration.

[0485] 98. The method of administering a vaccine for the use according to any one of items 95 to 96, wherein the vaccine is administered to the area most likely to be the receptacle of a given disease.

[0486] 99. The method of administering a vaccine for the use according to any one of items 95 to 97, wherein the subject is an animal, such as a mammal, such as a dog, a cat, a cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, most preferably such as a human being; or a bird, such as a chicken or a fish, such as a salmon.

[0487] 100. The method of administering a vaccine for the use according to any one of items 95 to 98, wherein the composition is administered in combination with any other vaccine.

[0488] 101 . The method of administering a vaccine for the use according to any one of items 95 to 99, wherein the composition forms a part of a vaccine cocktail.

[0489] 102. A kit of parts comprising i. a vaccine as defined in any one of the preceding items, a polynucleotide according to item 74, or an expression system according to any one of items 81 to 87, and P7422PC00 ii. optionally, a medical instrument or other means for administering the vaccine, and iii. instructions for use.

[0490] 103. The kit of parts according to item 101 , comprising a second active ingredient.

[0491] 104. A method of isolating an antigen as defined in any one of the preceding items, fused to a peptide tag as defined in any one of items 1 to 67 wherein the method comprises expressing said antigen fused to a peptide tag in a cell, such as a mammalian cell, or microorganism, such as a bacteria, such as E. coli.

[0492] 105. A method of manufacturing a vaccine according to any one of items 1 to 67, comprising the step of: i. Providing an expression system as defined in any one of items 81 to 89; and ii. Recovering the vaccine.

[0493] Items 2

[0494] 1. A vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP), wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0495] 2. A vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and P7422PC00 ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0496] 3. The vaccine according to any one of the preceding items, wherein the epitope is a conformational epitope of IfTSLP.

[0497] 4. The vaccine according to any one of the preceding items, wherein less than 20%, such as less than 15%, 10%, 5% of the amino acid residues of the antigen are amino acid residues which are present both in sfTLSP and IfTSLP, such as none of the amino acid residues of the antigen are amino acid residues which are present both in sfTSLP and IfTSLP.

[0498] 5. The vaccine according to any one of the preceding items, wherein the antigen does not comprise epitopes of sfTSLP.

[0499] 6. The vaccine according to any one of the preceding items, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising said epitope of IfTSLP, and wherein the amino acid residues of said epitope in the vaccine maintain the same spatial arrangement between each other as in the corresponding three-dimensional structure of IfTSLP.

[0500] 7. The vaccine according to any one of the preceding items, wherein the epitope comprises two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the corresponding three- dimensional structure of IfTSLP. P7422PC00

[0501] 8. The vaccine according to any one of the preceding items, wherein the epitope comprises two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure of the antigen and the corresponding two or more amino acid residues within the three- dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1 .8 A, 1 .7, 1.6, 1.5, 1 .4, 1 .3, 1 .2, 1.2, such as less than 1 A.

[0502] 9. The vaccine according to any one of the preceding items, wherein i. the antigen comprises a polypeptide, preferably an immunogenic polypeptide, having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; and ii. wherein said polypeptide further comprises one or more of Phe98, Ala99, Leu106, Tyr109, Cys110, Cys137, Leu 138, and / or an immunogenic polypeptide, having at least 95% identity to amino acid residues 139 to 153 of SEQ ID NO: 45; such as a polypeptide comprising one or more of amino acid residues 146 to 153 of SEQ ID NO: 45, such as a polypeptide having one or more of amino acid residues Leu144, Gly146, Leu147, Trp148, Arg149, Phe151 , or Asn152 of SEQ ID NO: 45.

[0503] 10. The vaccine according to any one of the preceding items, wherein the particleforming protein has at least 70% sequence identity, such as 75%, such as 80%, such as 85%, such as 90%, such as 95% such as 99%, such as 100% sequence identity to:

[0504] MsDps2 as set forth in SEQ ID NO: 33;

[0505] AP205 as set forth in SEQ ID NO: 34; ferritin as set forth in SEQ ID NO: 35; i301 as set forth in SEQ ID NO: 36; replicase polyprotein 1a (pp1a); a lumazine synthase as set forth in SEQ ID NO: 38; P7422PC00

[0506] Hbc as set forth in SEQ ID NO: 39; tandemHBc as set forth in SEQ ID NO: 40; the 2-oxo acid dehydrogenase subunit E2 as set forth in SEQ ID NO: 41 ; or the norovirus capsid protein as set forth in SEQ ID NO: 42.

[0507] 11. The vaccine according to any one of the preceding items, wherein the first or the second peptide tag 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); 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); Rum7Tag (SEQ ID NO: 12); Rumtrunk D9N (SEQ ID NO: 13); RumTrunkTag (SEQ ID NO: 14); BacTag (SEQ ID NO: 15); Bac2Tag (SEQ ID NO: 16); Bac3Tag (SEQ ID NO: 17); Bac4Tag (SEQ ID NO: 18); Bac5Tag (SEQ ID NO: 19); PsCsTag (SEQ ID NO: 20); Clib9 (SEQ ID NO: 21); Ktag (SEQ ID NO: 76) or a fragment thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

[0508] 12. The vaccine according to any one of the preceding items, wherein the first or the second peptide tag is: SpyCatcher (SEQ ID NO: 23); Spycatcherv2 (SEQ ID NO: 24); Spycatcherv3 (SEQ ID NO: 25); SdyCatcher (SEQ ID NO: 26); SnoopCatcher (SEQ ID NO: 27); MoonCake (SEQ ID NO: 28); Katl (SEQ ID NO: 29);

[0509] QueenCatcher (SEQ ID NO: 30); PsCsCatcher (SEQ ID NO: 31); or fragments thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least P7422PC00

[0510] 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

[0511] 13. A vaccine for use in the prophylaxis and / or treatment of a disease, wherein the vaccine is according to any one of the preceding items, and wherein the disease is an inflammatory disease, a fibrotic disorder, a cancer, a chronic epithelial barrier disorders, a skin disease, and / or an autoimmune disease.

[0512] 14. An antigen as defined in any one of the preceding items, fused to a (second) peptide tag as defined in any one of the preceding items.

[0513] 15. An expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

[0514] Examples

[0515] Example 1: TSLP antigen design

[0516] To enhance the safety of a TSLP vaccine, the investors of the present disclosure designed human TSLP antigens based on the disease-associated region of IfTSLP (amino acids 29-96). Utilizing Al and computational protein design approaches, the inventors successfully grafted this region onto a scaffold protein (see e.g. the sequences in the following examples) . In addition to the core residues (amino acids 29-96), the inventors incorporated additional amino acids: 98, 99, 106, 110 and 137- 153. P7422PC00

[0517] Among these, amino acids 110 and 137 are known to form a disulfide bridge with amino acids 34 and 90, respectively, in the native IfTSLP protein, whereas the remaining amino acids contribute to the integrity of the specific epitope recognized by the monoclonal antibody Tezepelumab. This antibody has demonstrated robust safety and efficacy in treating severe asthma in humans, and was used to test the antigens described in the following examples.

[0518] The inclusion of these additional amino acid residues of sfTSLP was merely intended to create a stable protein, whose structure could be assessed by testing its binding to Tezepelumab, since to be recognized by said antibody it is necessary for the full epitope to be present. As described throughout the whole description, it will also be evident that these additional amino acid residues are not essential for carrying out alternative embodiments described in the description.

[0519] Example 2: 9 out of 10 TSLP antigen designs express in HEK293TT cells

[0520] HEK293tt cells transfected with DNA encoding TSLP antigens #21-#30 (i.e. SEQ ID NO: 49-58, see table below) express and secrete protein (Fig. 1A-B). Supernatants were harvested 3 days post transfection for western blot analysis and protein (- / + DTT) was detected by an anti-cTag. P7422PC00

[0521] A positive control for anti-cTag detection was included on each Western blot (Fig. 1A- B). Data show, that 9 / 10 antigen designs express (#25 is not detected). However, in the unreduced state significant oligomerization is observed for all constructs.

[0522] Example 3: Binding of the commercial monoclonal antibody Tezepelumab verifies the fold and integrity of a neutralizing epitope in HEK-expressed TSLP antigens

[0523] 9 / 10 secreted proteins present in HEK293tt supernatant described in Example 2 bind to the commercial antibody Tezepelumab in a Sandwich ELISA, verifying the conformational integrity of the neutralizing epitope recognized by Tezepelumab. ELISA plates were coated with Tezepelumab and incubated with supernatant from HEK293TT cells transfected with DNA encoding TSLP antigens #21-#30. TSLP antigens bound to Tezepelumab was detected by anti-cTag detection (Fig. 1C).

[0524] Example 4: Expression and Solubility of TSLP Antigen Designs in E. coli Shuffle Cells. Ten TSLP antigen constructs (#21-#30 - SEQ ID NO:49-58 ) were expressed in E. coli Shuffle cells. Samples were collected before and after induction of protein expression (- / + IPTG) and analyzed by SDS-PAGE, including both pellet (P) and supernatant (S) fractions following sonication. The presence of induced protein and its solubility are indicated by black boxes on the gel.

[0525] The data revealed that 7 out of the 10 constructs (#21, #23, #26, #27, #28, #29, and #30) were successfully expressed as soluble proteins (Fig. 2A-D). These soluble constructs were further modified with N-terminal split protein binding Tag (RumtrunkD9N) or Catcher (Mooncake) sequences to enable display on VLPs (AP205 genetically fused to the corresponding split protein binding partner, Mooncake or RumtrunkD9N, respectively), resulting in 14 new antigen constructs (see e.g. Example 5). Of these, 11 were successfully purified as soluble proteins.

[0526] Example 5: Verification of Neutralizing Epitope Integrity in E. coli-Produced TSLP Antigens via Binding to the Commercial Monoclonal Antibody Tezepelumab Seven TSLP antigen constructs (#21 , #23, #26, #27, #28, #29, and #30) that were successfully expressed as soluble proteins in E. coli were purified using cTag chromatography. Each of these constructs was further modified with N-terminal split protein binding Tag and Catcher sequences for VLP display (AP205 genetically fused to the corresponding split protein binding partner, Mooncake or RumtrunkD9N, P7422PC00 respectively), resulting in a total of 14 new antigen constructs (#35-#48), of which 11 were expressed and purified as soluble proteins.

[0527] To verify the integrity of the neutralizing epitope, all 14 antigens and relevant control proteins were tested for binding to the commercial monoclonal antibody Tezepelumab. Duplicate ELISA assays were performed, with Tezepelumab-coated plates incubated with a 3-fold serial dilution of TSLP proteins starting at 100 pg / mL. Bound antigens were detected using an anti-cTag antibody, and the binding levels were quantified as area under the curve (AUG) with mean ± SD (Fig. 3).

[0528] The data demonstrate that all tested proteins bind to Tezepelumab in the sandwich ELISA, confirming the conformational integrity of the neutralizing epitope recognized by Tezepelumab. Additionally, the inclusion of N-terminal Tag or Catcher sequences does not disrupt the folding or epitope accessibility of the TSLP antigens.

[0529] Example 6: E. coli-Produced Tag / Catcher TSLP Antigens Can Be Unidirectionally Displayed on the Surface of VLPs

[0530] To evaluate the unidirectional display of Tag / Catcher (RumtrunkD9N / Mooncake) TSLP antigens described in the previous examples on the surface of VLPs, the antigens were P7422PC00 mixed with VLPs (AP205) containing the complementary split-protein Tag / Catcher binding partner (Mooncake or RumtrunkD9N, see e.g. Example 7) at a 1 :2 molar ratio (VLP:antigen). The mixture was incubated overnight at 4°C to allow conjugation.

[0531] Samples were analyzed by SDS-PAGE both before and after a spin stability test (- / + spin). The results indicate that, for all tested TSLP antigens, stable VLP:antigen (VLP:Ag) complexes were formed, as shown by the absence of protein loss after centrifugation. The stable complexes are highlighted with dashed-line boxes on the gel. Unconjugated VLPs are marked with dotted-line boxes, while excess unbound antigen is visible outside the boxed regions (Fig. 3B-C).

[0532] These findings confirm the successful and stable unidirectional display of TSLP antigens on AP205 VLP surfaces.

[0533] Example 7: Quality Assessment of TSLP-VLP Vaccine Formulations

[0534] Unidirectional display of TSLP antigens on VLPs was achieved by conjugating Tag / Catcher(RumtrunkD9N / Mooncake)-TSLP antigens to VLPs (AP205) with complementary binding partners. The resulting vaccine formulations (AV49-AV54, AV62) were purified using iodixanol density gradient ultracentrifugation. Conjugated TSLP-VLP complexes were predominantly detected in high-density particulate protein fractions (fractions 4-7, Figure 4A.1-G.1).

[0535] Dynamic light scattering (DLS) analysis revealed that most TSLP-VLP vaccine formulations (AV50-AV54, AV62) contained non-aggregated, monodisperse particles with a polydispersity index (PDI) below 15% and an average diameter of ~50 nm (Fig. P7422PC00

[0536] 4B.2-G.2). However, DLS analysis of the AV49 formulation indicated a less homogeneous population, with a PDI of -38% and an average diameter of -190 nm (Fig. 4A.2).

[0537] Spin stability testing demonstrated that most vaccine formulations (AV50-AV54, AV62) remained stable in solution (Fig. 4H-J). In contrast, the AV49 formulation exhibited some instability under the same conditions (Fig. 4H).

[0538] Densitometry analysis estimated the antigen coupling efficiency for all formulations to be -23-38%, corresponding to approximately 41-68 TSLP antigens per VLP (Fig. 4H- J). These results confirm the quality and stability of the TSLP-VLP vaccines, with the exception of AV49, which displayed reduced homogeneity and stability(Fig. 4H).

[0539] Example 8: Induction of Human TSLP (huTSLP)-Specific Antibodies in Mice Groups of BALB / c mice (n=4) were immunized using a 2-week prime-boost-boost regimen (a total of three doses) with seven different TSLP vaccine formulations (AV49- AV54, AV62). Each dose contained 1.5 pg of VLP-displayed TSLP antigen. Vaccines A 49-AV54 were based on TSLP antigen constructs #21 , #23, #26, #28, #29, and #30, while AV62 was based on full-length human TSLP (FL huTSLP). All vaccines were formulated with the extrinsic adjuvant Addavax. TSLP-specific antibody levels in serum were measured and presented as the area under the curve (AUG) with mean ± SD. After a single immunization, all mice seroconverted to varying degrees depending on the vaccine administered (Fig. 5A). In general, the levels of TSLP-specific antibodies induced by AV49-AV54 were lower compared to those induced by the full- length VLP-TSLP vaccine (AV62). Antibody levels were boosted for all vaccine groups following a second immunization (Fig. 5B). As expected, control serum from pre-bleed samples and mice immunized with VLP only (Mooncake-AP205) showed no detectable TSLP-specific antibodies. These results demonstrate that all TSLP vaccines tested can induce specific antibody responses in mice, with antibody levels increasing after booster immunizations.

[0540] Example 9: Vaccine-induced antibodies compete against Tezepelumab for binding to huTSLP

[0541] Tezepelumab, a monoclonal antibody targeting thymic stromal lymphopoietin (TSLP), has demonstrated clinical efficacy in the treatment of severe asthma. It is therefore P7422PC00 relevant to assess whether immunization with the cVLP-huTSLP (full-length) vaccine elicits antibodies recognizing epitopes that overlap with, or are spatially proximal to, those bound by Tezepelumab. Such antibodies are expected to share a similar neutralizing or therapeutic potential.

[0542] To evaluate this, a competition ELISA was performed to determine whether vaccine- induced antibodies could compete with Tezepelumab for binding to human TSLP (huTSLP). Sera from immunized mice (n = 4) were serially diluted two-fold starting at 1 :10 and incubated with huTSLP (0.1 pg / well) in the presence of Tezepelumab (0.1 pg / mL). The degree of inhibition of Tezepelumab binding was quantified and expressed as percentage competition (mean ± SD) relative to the negative control group (cVLP without antigen).

[0543] Conclusion:

[0544] The results (Figure 6) demonstrate that all tested TSLP vaccine constructs induced antibodies capable of competing with Tezepelumab for binding to huTSLP in a dosedependent manner. This finding confirms that the vaccine-induced antibody response includes specificities directed to the same or overlapping functional epitopes as Tezepelumab, thereby supporting the therapeutic potential of the cVLP-huTSLP (FL) (AV62) and related vaccine formulations.

[0545] Example 10: Vaccine-induced antibodies demonstrate biological efficacy by inhibition of huTSLP receptor activation in a huTSLP reporter cell line

[0546] The biological activity of the vaccine-induced antibody response was investigated by measuring the ability of IgG purified from serum from immunized mice to inhibit TSLP receptor activation in a HEK293 huTSLP reporter cell line (Invivogen). 10ng / mL huTSLP was incubated with IgG from immunized mice in a 2-fold dilution starting from 1 pM, before added to 50.000 cells seeded in a 96-well plate. Cells were incubated overnight at 37°C, 5% CO2. SEAP reporter activity was quantified according to manufacturers protocol and plotted as percentage activity (% activity) with mean + SD, as compared to the negative control (PBS only, “no Ab”). As negative controls, cells were unstimulated (i.e. PBS) or stimulated in the presence of IgG from mice immunized with a non-TSLP cVLP-targeting vaccine (cVLP-AgX). Data show individual inhibitory capacity of immunized mice, in a dose-dependent manner. P7422PC00

[0547] Conclusion: This data (Figure 7) demonstrates that all the five newly designed vaccines induce TSLP receptor blocking antibodies, however to different degrees.

[0548] Example 11: sfTSLP-specific antibodies binding to designed TSLP antigens

[0549] To investigate if the newly designed TSLP antigens contain any sfTSLP-specific epitopes, serum from Balb / c mice (n=6) immunized in a 2-week interval prime-boost- boost regimen with a cVLP-sfTSLP(63aa) vaccine was tested for binding to the different antigens (without Catcher). Data show that #21 and #23 have higher binding specificity for sfTSLP-specific Abs, as compared to #26, #28, #29 and #30.

[0550] Conclusion: This data (Figure 8) shows that 4 / 6 designed TSLP antigens are weakly recognized by sfTSLP-specific antibodies, important as a safety aspect as sfTSLP is present in healthy tissue, and supports the original antigen design, using the N-terminal region of IfTSLP for antigen scaffolding, thereby theoretically inducing an antibody response binding IfTSLP present upregulated under inflammatory conditions.

[0551] Example 12: Expression of double-display antigens and vaccine formulations Antigen designs displaying two different newly generated TSLP antigens on the same Catcher molecule (miniMooncake, engineered Mooncake) were designed based on #26 and #30, with either rigid helical linkers or flexible linkers between the antigens and Catcher molecule, i.e.

[0552] #54) #26-miniMC-#30 (rigid helical linkers)

[0553] #55 #30-miniMC-#26 (rigid helical linkers)

[0554] #56 #26-miniMC-#30 (flexible linkers)

[0555] #57 #30-miniMC-#26 (flexible linkers)

[0556] All constructs were designed with a cTag for purification and cloned for expression in E.coli shuffle cells. Expression was induced with a final concentration of 1mM IPTG. Samples were taken before (-IPTG) and after (+IPTG) induction, as well as the pellet and supernatant following sonication of the cell pellet. White boxes indicate the induction band. Double display antigen constructs designed with a rigid helical linker between the antigens and the Catcher construct demonstrated best expression and solubility (#54 and #55), as compared to the constructs containing flexible linkers (#56 and #57). #54 and #55 was purified by cTag chromatography. P7422PC00

[0557] Conclusion: This data (Figure 9) demonstrates successful expression of TSLP double display antigens, with the purpose of focusing the vaccine-induced antibody response towards the TSLP region (shared between the two displayed antigens) and away from the scaffolds (different between the two displayed antigens). Furthermore, data demonstrate that designs with rigid, helical linkers result in better yield and solubility of the antigen designs.

[0558] Example 13: cVLP-#54 vaccine formulation

[0559] #54 was mixed with Tag-cVLP in a 1:2 molar excess. The vaccine was incubated overnight at 4°C before excess antigen was removed by density gradient ultracentrifugation (UC) using an Optiprep™ gradient (23, 29, 35%) (Fig. 10A). To assess stability, the vaccine was subjected to a spin stability test. Specifically, a vaccine sample was spun at 16.000g for 2min and an equal volume of pre- and postspin samples were loaded on an SDS-PAGE gel, to assess potential loss in the postspin sample due to precipitation or aggregation (Fig. 10B). The antigen coupling efficiency was estimated to 23% by calculating the percentage conjugation (i.e. number of bound antigens divided by the total available binding sites (=180) per cVLP) by densitometric analysis using ImageLab. This corresponds to ~41 Catcher molecules binding per cVLP, displaying ~82 TSLP antigens due to double display. Purified vaccine was quality checked Dynamic Light scattering (DLS) analysis, showing a monodisperse population with a diameter of ~45-50nm, with low percent polydispersity (%Pd<15).

[0560] Conclusion: This data (Figure 10) demonstrates successful vaccine formulation for a TSLP double-display antigen. This approach results in the display of ~41 Catcher molecules per VLP, enabling display of the double amount of TSLP antigens.

[0561] Example 14: cVLP-#55 vaccine formulation

[0562] #55 was mixed with Tag-cVLP in a 1:2 molar excess. The vaccine was incubated overnight at 4°C before excess antigen was removed by density gradient ultracentrifugation (UC) using an Optiprep™ gradient (23, 29, 35%) (Fig. A). To assess stability, the vaccine was subjected to a spin stability test. Specifically, a vaccine sample was spun at 16.000g for 2min and an equal volume of pre- and post-spin samples were loaded on an SDS-PAGE gel, to assess potential loss in the post-spin sample due to precipitation or aggregation (Fig. B). The antigen coupling efficiency was P7422PC00 estimated to 23% by calculating the percentage conjugation (i.e. number of bound antigens divided by the total available binding sites (=180) per cVLP) by densitometric analysis using ImageLab. This corresponds to ~41 Catcher molecules binding per cVLP, displaying ~82 TSLP antigens due to double display. Purified vaccine was quality checked Dynamic Light scattering (DLS) analysis, showing a monodisperse population with a diameter of ~46nm, with low percent polydispersity (%Pd<15).

[0563] Conclusion: This data (Figure 11) demonstrates successful vaccine formulation for a TSLP double-display antigen. This approach results in the display of ~41 Catcher molecules per VLP, enabling display of the double amount of TSLP antigens.

[0564] Sequence Overview

[0565] SEQ ID NO: 49 - #21 ELVARETLTVNASNAADIDALLARIDAWAAEAEANPGWVLRVRVAPADFTNCDFEKIK AAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFA RNWLKALQYCSTTITFTIELVRVERREPCNAKERVKQIYGLWRRFNR SEQ ID NO: 50 - #23 SYEDEEWTITNQEELNAALEKVEKLIKEAEKKGIGVSVTIDADFTNCDFEKIKAAYLSTIS KDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEKFALDFLKTLK YCKGIKKIKFSYEKPLSSVKCNSKERIFQIYGLWRRFNR SEQ ID NO: 51 - #26 DTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFTNCDFEKIKAAYLSTISKDLITYM SGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQKTAEVLSEYCGNE KIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWRRFNR SEQ ID NO: 52 - #27 SSAYEESLALQQKHLPELTAIANKINAHDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTE FNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAINTAKVLVEYCGVDKALIDELL NLIDEAGIESHRNPACLEQVSQLQGLWRRFNR SEQ ID NO: 53 - #28 SRLEEDLAAVLADPRWSPEDKALVTEIYQLRIENPMDFTNCDFEKIKAAYLSTISKDLIT YMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQAILKTLKEYCSP EVAEVAEKEHELALKLSDKGDRKKCLEQVSQLQGLWRRFNR SEQ ID NO: 54 - #29’ P7422PC00

[0566] VTPADEAALAANRAAAAALNAALGPLDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEF

[0567] NNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQNIKKTLIEYCPENAAKIEAIYNA

[0568] QLAVPAINGDKKKCLEQVSQLQGLWRRFNR

[0569] SEQ ID NO: 55 - #30

[0570] SAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTV

[0571] SCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQATADVLVEYCGVDKELVDELKALAA

[0572] KIPPSIGSRKACLEQVSQLQGLWRRFNR

[0573] SEQ ID NO: 56 - #22

[0574] DVDAVIAAIRAHAAAGDPATLTRTIENATYEELKRLLALAAELAAQGYRATITVDFTNCD

[0575] FEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAK

[0576] EPFAKNFLKTLEYCGKVNVTVTVERAAPEPQKCNAKERIYQIYQLWRRFNR

[0577] SEQ ID NO: 57 - #24

[0578] EEVETETLTISPADLYDKITAALEEWEKKAEELSKKTGEKYEVQLSLAPVDFTNCDFEKI

[0579] KAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPF

[0580] AKNFIKALAFCPESFTVTITIVKVDPNTKCNSKERVNQIYNLWRRFNR

[0581] SEQ ID NO: 58 - #25

[0582] MKPLEQVKAAIAALEATVKATGKDVTVTINYSSKTVTLNAEEFLELVNAFLALANALSDK

[0583] SVGFSINADFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQS

[0584] LTFNPTAGCASLAKEKFAQDFAKTMIFCKEVKVTSITISTSKSQGPCTSKERIFQIYGLW RRFNR

[0585] SEQ ID NO: 59 - #35 - Mooncake-KL_164_2-cTag (based on #21)

[0586] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0587] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG

[0588] SGSGGSGELVARETLTVNASNAADIDALLARIDAWAAEAEANPGWVLRVRVAPADFT

[0589] NCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCAS

[0590] LAKEPFARNWLKALQYCSTTITFTIELVRVERREPCNAKERVKQIYGLWRRFNRGGSE PEA

[0591] SEQ ID NO: 60 - #36 - RumtrunkD9N-KL_164_2-cTag (based on #21)

[0592] GNPLIVMVNDTTKVKGGSGGSELVARETLTVNASNAADIDALLARIDAWAAEAEANPG

[0593] WVLRVRVAPADFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTE

[0594] IQSLTFNPTAGCASLAKEPFARNWLKALQYCSTTITFTIELVRVERREPCNAKERVKQI

[0595] YGLWRRFNRGGSEPEA

[0596] SEQ ID NO: 61 - #37 - Mooncake-KL_159_13-cTag (based on #23)

[0597] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0598] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG P7422PC00

[0599] SGSGGSGSYEDEEWTITNQEELNAALEKVEKLIKEAEKKGIGVSVTIDADFTNCDFEKI

[0600] KAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEKF

[0601] ALDFLKTLKYCKGIKKIKFSYEKPLSSVKCNSKERIFQIYGLWRRFNRGGSEPEA

[0602] SEQ ID NO: 62 - #38 - RumtrunkD9N-KL_159_13-cTag (based on #23)

[0603] GNPLIVMVNDTTKVKGGSGGSSYEDEEWTITNQEELNAALEKVEKLIKEAEKKGIGVS

[0604] VTIDADFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTF

[0605] NPTAGCASLAKEKFALDFLKTLKYCKGIKKIKFSYEKPLSSVKCNSKERIFQIYGLWRRF

[0606] NRGGSEPEA

[0607] SEQ ID NO: 63 - #39 - Mooncake-KL_helix_158_0 (based on #26)

[0608] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0609] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG

[0610] SGSGGSGDTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFTNCDFEKIKAAYLSTI

[0611] SKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQKTAEVL

[0612] SEYCGNEKIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWRRFNRGGSEPEA

[0613] SEQ ID NO: 64 - #40 - RumtrunkD9N-KL_helix_158_0 (based on #26)

[0614] GNPLIVMVNDTTKVKGGSGGSDTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFT

[0615] NCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCAS

[0616] LAKEPFAQKTAEVLSEYCGNEKIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWR

[0617] RFNRGGSEPEA

[0618] SEQ ID NO: 65 - #41 - Mooncake-KL_helix_152_15 (based on #27)

[0619] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0620] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG

[0621] SGSGGSGSSAYEESLALQQKHLPELTAIANKINAHDFTNCDFEKIKAAYLSTISKDLITY

[0622] MSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAINTAKVLVEYCGV

[0623] DKALIDELLNLIDEAGIESHRNPACLEQVSQLQGLWRRFNRGGSEPEA

[0624] SEQ ID NO: 66 - #42 - RumtrunkD9N-KL_helix_152_15 (based on #27)

[0625] GNPLIVMVNDTTKVKGGSGGSSSAYEESLALQQKHLPELTAIANKINAHDFTNCDFEKI

[0626] KAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPF

[0627] AINTAKVLVEYCGVDKALIDELLNLIDEAGIESHRNPACLEQVSQLQGLWRRFNRGGS EPEA

[0628] SEQ ID NO: 67 - #43 - Mooncake-KL_helix_160_5-cTag (based on #28)

[0629] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0630] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG

[0631] SGSGGSGSRLEEDLAAVLADPRWSPEDKALVTEIYQLRIENPMDFTNCDFEKIKAAYL P7422PC00

[0632] STISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQAILK TLKEYCSPEVAEVAEKEHELALKLSDKGDRKKCLEQVSQLQGLWRRFNRGGSEPEA SEQ ID NO: 68 - #44 - RumtrunkD9N-KL_helix_160_5-cTag (based on #28)

[0633] GNPLIVMVNDTTKVKGGSGGSSRLEEDLAAVLADPRWSPEDKALVTEIYQLRIENPMD FTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGC ASLAKEPFAQAILKTLKEYCSPEVAEVAEKEHELALKLSDKGDRKKCLEQVSQLQGLW RRFNRGGSEPEA

[0634] SEQ ID NO: 69 - #45 - Mooncake-KL_helix_150_7-cTag (based on #29)

[0635] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0636] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG

[0637] SGSGGSGVTPADEAALAANRAAAAALNAALGPLDFTNCDFEKIKAAYLSTISKDLITYM

[0638] SGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQNIKKTLIEYCPENA AKIEAIYNAQLAVPAINGDKKKCLEQVSQLQGLWRRFNRGGSEPEA

[0639] SEQ ID NO: 70 - #46 - RumtrunkD9N-KL_helix_150_7-cTag (based on #29)

[0640] GNPLIVMVNDTTKVKGGSGGSVTPADEAALAANRAAAAALNAALGPLDFTNCDFEKIK

[0641] AAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFA

[0642] QNIKKTLIEYCPENAAKIEAIYNAQLAVPAINGDKKKCLEQVSQLQGLWRRFNRGGSEP EA

[0643] SEQ ID NO: 71 - #47 - Mooncake-KL_helix_146_18-cTag (based on #30)

[0644] MGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQS

[0645] WVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG SGSGGSGSAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAAYLSTISKDLITYMSGTKS TEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQATADVLVEYCGVDKELVD

[0646] ELKALAAKIPPSIGSRKACLEQVSQLQGLWRRFNRGGSEPEA

[0647] SEQ ID NO: 72 - #48 - RumtrunkD9N-KL_helix_146_18-cTag (based on #30)

[0648] GNPLIVMVNDTTKVKGGSGGSSAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAAYLS

[0649] TISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQATAD

[0650] VLVEYCGVDKELVDELKALAAKIPPSIGSRKACLEQVSQLQGLWRRFNRGGSEPEA

[0651] SEQ ID NO: 73 - RumtrunkD9N_AP205_aminoacids

[0652] GNPLIVMVNDTTKVKGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLL

[0653] RQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLA

[0654] TLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA

[0655] SEQ ID NO: 74 - Mooncake_AP205_aminoacids

[0656] FSMGIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDSNGKELAGAMIELRNLSGQTI

[0657] QSWVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDV P7422PC00

[0658] HIGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYV

[0659] SVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFAS

[0660] GNAGLGFLDPTAAIVSSDTTA

[0661] SEQ ID NO: 75 - #2 sign3_6xHls_Mooncake_huTSLP(29-159) AR127A / R130S

[0662] GMKWVTFISLLFLFSSSSRASHHHHHHMGIDTMSGLSGETGQSGNTTIEEDSTTHVKF

[0663] SKRDSNGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGYEL

[0664] AAPITFTVQDNGEVIIQGRLTRGDVHIGGSYDFTNCDFEKIKAAYLSTISKDLITYMSGT

[0665] KSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSET

[0666] QINATQAMKKARKSKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ

[0667] SEQ ID NO: 77 #54 #26-miniMC-#30 (rigid helical linkers) (cTag for purification)

[0668] #26 - aa 1-158

[0669] Linker - aa 159-170 miniMC - aa 171-245

[0670] Linker - aa 246-257

[0671] #30 - aa 258-403

[0672] GGS linker cTag - aa 407-410

[0673] DTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFTNCDFEKIKAAYLSTISKDLITYM

[0674] SGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQKTAEVLSEYCGNE

[0675] KIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWRRFNRAEAAAKEAAAKASGDST

[0676] THVKFSKRDSNGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAP

[0677] EGYELAAPITFTVAEAAAKEAAAKASAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAA

[0678] YLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQA

[0679] TADVLVEYCGVDKELVDELKALAAKIPPSIGSRKACLEQVSQLQGLWRRFNRGGSEP

[0680] EA

[0681] SEQ ID NO: 78 #55 #30-miniMC-#26 (rigid helical linkers)

[0682] #30 - aa 1-146

[0683] Linker - aa 147 - 157 miniMC - aa 158-233

[0684] Linker - aa 234-245

[0685] #26 - aa 246-403

[0686] GGS linker cTag - aa 407-410

[0687] SAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTV

[0688] SCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQATADVLVEYCGVDKELVDELKALAA P7422PC00

[0689] KIPPSIGSRKACLEQVSQLQGLWRRFNRAEAAAKEAAAKASGDSTTHVKFSKRDSNG

[0690] KELAGAM I ELRN LSGQTIQSWVSDGTVKDFYLM PGTYQFVETAAPEGYELAAPITFTV

[0691] AEAAAKEAAAKADTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFTNCDFEKIKAA

[0692] YLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQK

[0693] TAEVLSEYCGNEKIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWRRFNRGGSEP EA

[0694] SEQ ID NO: 79 #56 #26-miniMC-#30 (flexible linkers)

[0695] #26 aa 1-158

[0696] Linker aa 159-166

[0697] MiniMC - aa 167-241

[0698] Linker - aa 242-249

[0699] #30 - aa 250-395

[0700] GGS linker

[0701] Ctag aa 399-402

[0702] DTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFTNCDFEKIKAAYLSTISKDLITYM

[0703] SGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQKTAEVLSEYCGNE

[0704] KIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWRRFNRGSGGSSGGSGDSTTHV

[0705] KFSKRDSNGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGY

[0706] ELAAPITFTVGSGGSSGGSAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAAYLSTISK

[0707] DLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQATADVLVE

[0708] YCGVDKELVDELKALAAKIPPSIGSRKACLEQVSQLQGLWRRFNRGGSEPEA

[0709] SEQ ID NO: 80 #57 #30-miniMC-#26 (flexible linkers)

[0710] #30 aa 1-146

[0711] Linker aa 147-154

[0712] MiniMC - aa 155-229

[0713] Linker - aa 230-2237

[0714] #26 - aa 238-395

[0715] GGS linker

[0716] Ctag aa 399-402

[0717] SAAELAAALAKLKEIAAKLNKHDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTV

[0718] SCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQATADVLVEYCGVDKELVDELKALAA

[0719] KIPPSIGSRKACLEQVSQLQGLWRRFNRGSGGSSGGSGDSTTHVKFSKRDSNGKEL

[0720] AGAM I ELRN LSGQTIQSWVSDGTVKDFYLM PGTYQFVETAAPEGYELAAPITFTVGSG GSSGGDTLAKAKELAAEPLTTPEKVAEFGALMAALSASLDFTNCDFEKIKAAYLSTISK P7422PC00

[0721] DLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEPFAQKTAEVLSE

[0722] YCGNEKIKELYEISLKAADQMAPGSREKCLEQVSQLQGLWRRFNRGGSEPEA

[0723] SEQ ID NO: 81 miniMC

[0724] DSTTHVKFSKRDSNGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVET AAPEGYELAAPITFTV

[0725] References

[0726] Wechsler et al., New England Journal of Medicine, 2021 , DOI: 10.1056 / NEJMoa2034975

Claims

P7422PC00Claims1 . A vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

2. A vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

3. A vaccine comprising: i. a particle-forming protein comprising a first peptide tag, and ii. an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP), wherein the antigen and the particle-forming protein are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

4. A vaccine comprising: i. a first polynucleotide encoding a particle-forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic StromalP7422PC00Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and particle-forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

5. The vaccine according to any one of the preceding claims, wherein the epitope is a conformational epitope of IfTSLP.

6. The vaccine according to any one of the preceding claims, wherein less than 20%, such as less than 15%, 10%, 5% of the amino acid residues of the antigen are amino acid residues which are present both in sfTLSP and IfTSLP, such as none of the amino acid residues of the antigen are amino acid residues which are present both in sfTSLP and IfTSLP.

7. The vaccine according to any one of the preceding claims, wherein the antigen comprises one or more epitopes of TSLP, wherein each epitope of TSLP comprises one or more amino acid residues present in IfTSLP and absent in sfTSLP.

8. The vaccine according to any one of the preceding claims, wherein the antigen does not comprise epitopes of sfTSLP9. The vaccine according to any one of the preceding claims, wherein none of the one or more epitopes of TSLP comprise amino acid residues present in sfTLSP.

10. The vaccine according to any one of the preceding claims, wherein the antigen does not comprise epitopes which exclusively consist of amino acids absent in the IfTSLP.

11. The vaccine according to any one of the preceding claims, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising an epitope of IfTSLP.P7422PC0012. The vaccine according to any one of the preceding claims, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising a conformational epitope of IfTSLP.

13. The vaccine according to any one of the preceding claims, wherein the antigen is a scaffold protein, such as a minimal scaffold protein, comprising said epitope of IfTSLP, and wherein the amino acid residues of said epitope in the vaccine maintain the same spatial arrangement between each other as in the corresponding three-dimensional structure of IfTSLP.

14. The vaccine according to any one of the preceding claims, wherein the antigen does not comprise any amino acid residue of sfTLSP.

15. The vaccine according to any one of the preceding claims, wherein the antigen does not comprise a polypeptide consisting of sfTLSP or an immunogenic fragment thereof.

16. The vaccine according to any one of the preceding claims, wherein the epitope comprises two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the corresponding three- dimensional structure of IfTSLP.

17. The vaccine according to any one of the preceding claims, wherein the epitope comprises two or more amino acid residues of IfTSLP, wherein: i. the two or more amino acid residues are selected from amino acid residues present in IfTSLP and absent in sfTSLP; and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure of the antigen and the corresponding two or more amino acid residues within the three- dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A.P7422PC0018. The vaccine according to any one of the preceding claims, wherein the vaccine is capable of inducing in a subject one or more antibodies specific to IfTSLP, preferably wherein said antibodies do not bind to sfTSLP.

19. The vaccine according to any one of the preceding claims, wherein the vaccine is not capable of inducing antibodies in a subject which bind to the corresponding short-form variant of TSLP (sfTSLP).

20. The vaccine according to any one of the preceding claims, wherein the vaccine is capable of inducing in a subject one or more antibodies which have an affinity for IfTSLP which is at least 2-fold, 3-fold, 5-fold, 10-fold greater than the affinity for sfTSLP.21 . The vaccine according to any one of the preceding claims, wherein the vaccine is capable of inducing in a subject one or more antibodies which have affinity for a conformational epitope of IfTSLP, which is absent in sfTSLP.

22. The vaccine according to any one of the preceding claims, wherein the vaccine is capable of inducing one or more antibodies which have a Kd (Dissociation Constant) for IfTSLP which is at least 2-fold, 3-fold, 5-fold, 10-fold lower than sfTSLP.

23. The vaccine according to any one of the preceding claims, wherein the vaccine is not cross-reactive to sfTSLP.

24. The vaccine according to any one of the preceding claims, wherein IfTSLP is human IfTSLP as set forth in SEQ ID NO: 45, or has at least 95% identity thereto, such as 96%, 97%, 98% or 99% identity thereto.

25. The vaccine according to any one of the preceding claims, wherein the antigen comprises a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof.P7422PC0026. The vaccine according to any one of the preceding claims, wherein the antigen comprises: a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of: Cys110, Cys137, Arg150, or Arg153 of SEQ ID NO: 45.

27. The vaccine according to any one of the preceding claims, wherein the antigen comprises: a polypeptide having at least 95% identity to amino acid residues 29 to96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof; wherein said polypeptide further comprises one or more of: F98, A99, L106, Try109, Cys110, Cys137, Arg150, or Arg153 of SEQ ID NO: 45.

28. The vaccine according to any one of the preceding claims, wherein said polypeptide further comprises one or more of: Glu139; Val141; or Gln143 as set forth in SEQ ID NO: 45.

29. The vaccine according to any one of the preceding claims, wherein the antigen comprises a polypeptide having at least 95% identity to amino acid residues 139 to 153 of SEQ ID NO: 45; such as a polypeptide comprising one or more of amino acid residues 146 to 153 of SEQ ID NO: 45.

30. The vaccine according to any one of the preceding claims, wherein the antigen further comprises a polypeptide having one or more of amino acid residues Leu144, Gly146, Leu147, Trp148; Arg149; Phe151, Asn152 of SEQ ID NO: 45.

31. The vaccine according to any one of the preceding claims, wherein the antigen does not comprise a polypeptide amino acid residues 97 to 159, such as amino acid residues 110 to 159, 120 to 159, 130 to 159, 140 to 159, 97 to 140, 97 to 130,97 to 120, 97 to 110 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto, or one or more immunogenic fragments thereof.

32. The vaccine according to any one of the preceding claims, wherein the epitope comprises two or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, of IfTSLP, wherein:P7422PC00 i. the two or more amino acid residues are selected from amino acid residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45; and ii. the two or more amino acid residues maintain in the vaccine the same spatial arrangement between each other as in the three-dimensional structure of IfTSLP, wherein IfTSLP is as set forth in SEQ ID NO: 45.

33. The vaccine according to any one of the preceding claims, wherein the conformational epitope comprises two or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, of IfTSLP, wherein: i. the two or more amino acid residues are selected from residues 29 to 96 of IfTSLP as set forth in SEQ ID NO: 45, and ii. the Root Mean Square Deviation (RMSD) of the two or more amino acid residues within the three-dimensional structure the antigen and the corresponding two or more amino acid residues within the three- dimensional structure of IfTSLP is less than 2 A, such as less than 1.9 A, such as less than 1.8 A, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.2, such as less than 1 A, wherein IfTSLP is as set forth in SEQ ID NO: 45.

34. The vaccine according to any one of the preceding claims, wherein the vaccine is capable of inducing in a subject one or more antibodies which have affinity for, and / or are capable of binding to, a conformational epitope of IfTSLP, wherein the conformational epitope comprises one or more amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues, selected from a polypeptide having at least 95% identity to amino acid residues 29 to 96 of SEQ ID NO: 45, such as 96%, 97%, 98% or 99% sequence identity thereto.

35. The vaccine according to any one of the preceding claims, wherein the antigen comprises a polypeptide selected from: i. SEQ ID NO: 49; ii. SEQ ID NO: 50; iii. SEQ ID NO: 51 ; iv. SEQ ID NO: 52; v. SEQ ID NO: 53; vi. SEQ ID NO: 54; vii. SEQ ID NO: 55; orP7422PC00 a polypeptide having at least 95% sequence identity thereto.

36. The vaccine according to any one of the preceding claims, wherein the antigen fused to the (second) peptide comprises a polypeptide consisting of: i. SEQ ID NO: 59; ii. SEQ ID NO: 60; iii. SEQ ID NO: 61 ; iv. SEQ ID NO: 62; v. SEQ ID NO: 63; vi. SEQ ID NO: 64; vii. SEQ ID NO: 65; viii. SEQ ID NO: 67; ix. SEQ ID NO: 68; x. SEQ ID NO: 69; xi. SEQ ID NO: 70; xii. SEQ ID NO: 71 ; xiii. SEQ ID NO: 72; or a polypeptide having at least 95% sequence identity thereto.

37. The vaccine according to any one of the preceding claims, wherein the vaccine comprises two or more antigens fused to the second peptide tag.

38. The vaccine according to any one of the preceding claims, wherein the vaccine comprises two or more antigens fused to the second peptide tag, and wherein said two or more antigens comprise each an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in a long form TSLP (IfTSLP) and absent in the corresponding short form TSLP (sfTSLP).

39. The vaccine according to any one of the preceding claims, wherein the vaccine comprises two or more antigens fused to the second peptide tag, wherein the first antigen is as set forth in SEQ ID NO: 51 (#26) and the second antigen is as set forth in SEQ ID NO: 55 (#30).P7422PC0040. The vaccine according to any one of the preceding claims, wherein the vaccine comprises two or more antigens fused to the second peptide tag, wherein the first antigen is as set forth in SEQ ID NO: 51 (#26) and the second antigen is as set forth in SEQ ID NO: 55 (#30), and the second peptide is as set forth in SEQ ID NO: 81.41 . The vaccine according to any one of the preceding claims, wherein the vaccine comprises two or more antigens fused to the second peptide tag, wherein said two or more antigens are fused to said second peptide via a linker.

42. The vaccine according to claim 41 , wherein said linker is a rigid helical linker.

43. The vaccine according to claim 41 , wherein the linker is a flexible linker.

44. The vaccine according to any one of the preceding claims, wherein one or more antigens are fused to the N terminal end of the second peptide tag as described herein and one or more antigens are fused to the C terminal end of the second peptide.

45. The vaccine according to any one of the preceding claims, wherein one or more antigens are fused to the N terminal end of the second peptide tag as described herein and one or more antigens are fused to the C terminal end of the second peptide, and the second peptide is as set forth in SEQ ID NO: 81.

46. The vaccine according to any one of the preceding claims, wherein the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79 or SEQ ID NO: 80.

47. The vaccine according to any one of the preceding claims, wherein the vaccine comprises a polypeptide as set forth in: SEQ ID NO: 77.

48. The vaccine according to any one of the preceding claims, wherein vaccine comprises a polypeptide as set forth in: SEQ ID NO: 78P7422PC0049. The vaccine according to any one of the preceding claims, wherein vaccine comprises a polypeptide as set forth in: SEQ ID NO: 7950. The vaccine according to any one of the preceding claims, wherein vaccine comprises a polypeptide as set forth in: SEQ ID NO: 8051. The vaccine according to any one of the preceding claims, wherein the particle is a nanoparticle.

52. The vaccine according to any one of the preceding claims, wherein particle forming protein has at least 70% sequence identity, such as 75%, such as 80%, such as 85%, such as 90%, such as 95% such as 99%, such as 100% sequence identity to:MsDps2 as set forth in SEQ ID NO: 33;AP205 as set forth in SEQ ID NO: 34; ferritin as set forth in SEQ ID NO: 35; i301 as set forth in SEQ ID NO: 36; replicase polyprotein 1a (pp1a); a lumazine synthase as set forth in SEQ ID NO: 38;Hbc as set forth in SEQ ID NO: 39; tandemHBc as set forth in SEQ ID NO: 40; the 2-oxo acid dehydrogenase subunit E2 as set forth in SEQ ID NO: 41 ; or the norovirus capsid protein as set forth in SEQ ID NO: 42.

53. The vaccine according to any one of the preceding claims, wherein the particleforming protein attached to the first peptide is as set forth in SEQ ID NO: 73; SEQ ID NO: 74; or a polypeptide having 95% sequence identity thereto.

54. The vaccine according to any one of the preceding claims, wherein the particle forming protein is a virus-like particle.

55. The vaccine according to any one of the preceding claims, wherein the particle forming protein is a viral capsid protein or a viral envelope protein such as a glycoprotein.P7422PC0056. The vaccine according to any one of the preceding claims, wherein the first or the second peptide tag 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); 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); Rum7Tag (SEQ ID NO: 12); Rumtrunk D9N (SEQ ID NO: 13); RumTrunkTag (SEQ ID NO: 14); BacTag (SEQ ID NO: 15); Bac2Tag (SEQ ID NO: 16); Bac3Tag (SEQ ID NO: 17); Bac4Tag (SEQ ID NO: 18); Bac5Tag (SEQ ID NO: 19); PsCsTag (SEQ ID NO: 20); Clib9 (SEQ ID NO: 21); Ktag (SEQ ID NO: 76) or a fragment thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).

57. The vaccine according to any one of the preceding claims, wherein the first or the second peptide tag is: SpyCatcher (SEQ ID NO: 23); Spycatcherv2 (SEQ ID NO: 24); Spycatcherv3 (SEQ ID NO: 25); SdyCatcher (SEQ ID NO: 26); SnoopCatcher (SEQ ID NO: 27); MoonCake (SEQ ID NO: 28); Katl (SEQ ID NO: 29);QueenCatcher (SEQ ID NO: 30); PsCsCatcher (SEQ ID NO: 31); MiniMC (SEQ ID NO: 81), or fragments thereof having at least 70% identity thereto, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, with the proviso that said fragments comprise the reactive amino acid residue involved in the formation of the isopeptide bond, or wherein where the reactive amino acid residue is an Aspartic acid (D), the reactive amino acid residue is mutated to an Asparagine (N).P7422PC0058. The vaccine according to any one of the preceding claims wherein the isopeptide bond between the first and second peptide tag is an isopeptide bond between: i. SEQ ID NO: 28 (MoonCake) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% identity thereto; and / or ii. SEQ ID NO: 29 (Katl) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag);P7422PC00 l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% identity thereto; and / or iii. SEQ ID NO: 30 (QueenCatcher) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag); g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag); p) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% identity thereto and / or iv. SEQ ID NO: 81 (MiniMC) and a) SEQ ID NO: 13 (RumTrunkD9NTag); b) SEQ ID NO: 12 (Rum7Tag); c) SEQ ID NO: 8 (Rum3Tag); d) SEQ ID NO: 7 (Rum2Tag); e) SEQ ID NO: 9 (Rum4Tag); f) SEQ ID NO: 10 (Rum5Tag);P7422PC00 g) SEQ ID NO: 11 (Rum6Tag); h) SEQ ID NO: 14 (RumTrunkTag); i) SEQ ID NO: 1 (SpyTag); j) SEQ ID NO: 2 (SdyTag); k) SEQ ID NO: 6 (RumTag); l) SEQ ID NO: 15 (BacTag); m) SEQ ID NO: 16 (Bac2Tag); n) SEQ ID NO: 17 (Bac3Tag); o) SEQ ID NO: 18 (Bac4Tag);P) SEQ ID NO: 19 (Bac5Tag); or q) SEQ ID NO: 21 (Clib9); or variants thereof having at least 70% identity thereto.

59. The vaccine according to any one claims 2, or 4 to 58, wherein the first and / or second polynucleotide is DNA.

60. The vaccine according to any one claims 2, or 4 to 59, wherein the first and / or second polynucleotide is RNA.

61. The vaccine according to claim 60, wherein the RNA is formulated in a lipid particle formulation.

62. The vaccine according to any one of the preceding claims, wherein one or more first peptide tags are fused to the N-terminal end, to the C-terminal end of the particle forming protein and / or inserted in-frame into the coding sequence of the particle forming protein, optionally by a linker.

63. The vaccine according to any one of claims 2, 4 to 62, wherein one or more second peptide tags are fused to the N-terminal end, to the C-terminal end of the antigen and / or inserted in-frame into the coding sequence of the antigen, optionally by a linker.

64. The vaccine according to any one of claims 2, 4 to 63, wherein the first polynucleotide further comprises a secretion or excretion signal, whereby theP7422PC00 particle forming protein fused to the first peptide tag is secreted or excreted from the endoplasmic reticulum of the cell.

65. The vaccine according to claim 64, wherein the secretion or excretion signal comprises or consists MFPFALLYVLSVSFRKIFILQLVGLVLT (SEQ ID NO: 43).

66. The vaccine according to any one of the preceding claims, wherein the antigen further comprises a polyhistidine tag or a cTag.

67. The vaccine according to any one of the preceding claims, wherein the antigen is capable of eliciting an immune reaction in an animal, such as a mammal, such as a Homo sapiens, a dog, cat, cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, and / or a bird, such as a chicken and / or a fish, such as a salmon.

68. A composition comprising a vaccine according to any one of the preceding claims.

69. The composition according to claim 68, wherein the composition further comprises an acceptable carrier.

70. The composition according to any one of claim 68 to 69, wherein the composition further comprises an adjuvant.

71. A vaccine, or a composition, for use in medicine, wherein the vaccine or the composition is according to any one of the preceding claims.

72. A vaccine, or a composition, for use in the prophylaxis and / or treatment of a disease, wherein the vaccine or the composition is according to any one of the preceding claims, and wherein the disease is an inflammatory disease, a fibrotic disorder, a cancer, a chronic epithelial barrier disorders, a skin disease, and / or an autoimmune disease.

73. An antigen as defined in any one of the preceding claims, fused to a (second) peptide tag as defined in any one of the preceding claims.P7422PC0074. A polynucleotide encoding an antigen as defined in any one of the preceding claims fused to a (second) peptide tag as defined in any one of the preceding claims.

75. A cell comprising the polynucleotide according to claim 74.

76. The cell according to claim 75, wherein the cell is a mammalian cell, an insect cell, or a microorganism.

77. The cell according to claim 75, wherein the cell is a mammalian cell.

78. The cell according to claim 75, wherein the cell is a microorganism.

79. The cell according to claim 75, wherein the cell is a Bacterium.

80. The cell according to claim 75, wherein the cell is E. Coli.81 . An expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

82. An expression system such as a vector comprising: i. a first polynucleotide encoding a particle forming protein comprising a first peptide tag; and ii. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP),P7422PC00 wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first and second peptide tag, and wherein i - ii form a particle displaying said antigen.

83. The expression system according to claim 81 to 82, wherein the first peptide tag, the second peptide tag, the particle forming protein and / or the antigen are as defined in any one of claims 2, or 4 to 67.

84. The expression system according to any one of claims 81 to 83, wherein the first polynucleotide and the second polynucleotide are comprised within the same nucleic acid molecule, or within two different nucleic acid molecules.

85. The expression system according to any one of claims 81 to 84, wherein the first polynucleotide and the second polynucleotide are both DNA polynucleotides or RNA polynucleotides.

86. The expression system according to any one of claims 81 to 85, wherein the first and second polynucleotides are comprised within one vector such as a viral vector or a plasmid, or wherein the first and second polynucleotides are comprised within two vectors such as two viral vectors; two plasmids; or one viral vector and one plasmid.

87. The expression system according to claim 86, wherein the viral vector is an adenoviral vector, such as a modified adenoviral vector, e.g. the replicationdeficient simian adenovirus vector ChAdOxI, or a modified vaccinia Ankara (MVA) vector.

88. The expression system according to any one of claims 81 to 87, wherein the expression system comprises or consists of a plasmid.

89. The expression system according to any one of claims 81 to 88, wherein the expression system comprises or consists of an mRNA.

90. A cell expressing:P7422PC00 iii. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as defined in any one of the preceding claims; and iv. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

91. A cell expressing: iii. a first polynucleotide encoding a particle forming protein fused to a first peptide tag, preferably as defined in any one of the preceding claims; and iv. a second polynucleotide encoding an antigen fused to a second peptide tag, wherein the antigen comprises an epitope of Thymic Stromal Lymphopoietin (TSLP), wherein said epitope comprises one or more amino acid residues present in the long-form variant of TSLP (IfTSLP) and absent in the corresponding short-form variant of TSLP (sfTSLP), wherein the antigen and the particle forming protein upon expression in a cell are linked via an isopeptide bond, or an ester bond, between the first peptide tag and the second peptide tag, whereby i - ii form a particle forming protein particle displaying said antigen.

92. The cell according to claim 90 to 91 , comprising the vaccine according to any one of claims 2 to 67, the polynucleotide according to claim 74, or the expression system according to any one of claims 81 to 89.

93. A host cell, wherein the host cell comprises an expression system according to any one of claims 81 to 89.

94. The host cell according to claim 93, wherein the host cell is a bacterial cell, a yeast cell, a fungal cell, a plant cell, a mammalian cell, or an insect cell.

95. A method of administering a vaccine, or a composition, for the prophylaxis and / or treatment of a disease in a subject in need thereof, comprising the step ofP7422PC00 i. administering at least one composition comprising a vaccine as defined in any one of claims 1 to 67, or a composition according to any of claims 68 to 70, to a subject at least once for prophylaxis and / or treatment of a disease as defined in any one of the preceding claims.

96. The method of administering a vaccine, or a composition for the use according to claim 95, wherein the composition is boosted by administration in a form or body part different from the previous administration.

97. The method of administering a vaccine for the use according to any one of claims 95 to 96, wherein the vaccine is administered to the area most likely to be the receptacle of a given disease.

98. The method of administering a vaccine for the use according to any one of claims 95 to 97, wherein the subject is an animal, such as a mammal, such as a dog, a cat, a cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, most preferably such as a human being; or a bird, such as a chicken or a fish, such as a salmon.

99. The method of administering a vaccine for the use according to any one of claims 95 to 98, wherein the composition is administered in combination with any other vaccine.

100. The method of administering a vaccine for the use according to any one of claims 95 to 99, wherein the composition forms a part of a vaccine cocktail.

101. A kit of parts comprising i. a vaccine as defined in any one of the preceding claims, a polynucleotide according to claim 74, or an expression system according to any one of claims 81 to 87, and ii. optionally, a medical instrument or other means for administering the vaccine, and iii. instructions for use.

102. The kit of parts according to claim 101 , comprising a second active ingredient.P7422PC00103. A method of isolating an antigen as defined in any one of the preceding claims, fused to a peptide tag as defined in any one of claims 1 to 67 wherein the method comprises expressing said antigen fused to a peptide tag in a cell, such as a mammalian cell, or microorganism, such as a bacteria, such as E. coli.

104. A method of manufacturing a vaccine according to any one of claims 1 to 67, comprising the step of: i. Providing an expression system as defined in any one of claims 81 to89; and ii. Recovering the vaccine.

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