mRNA vaccines containing IL-4 and / or IL-13 RNA and uses thereof

Abstract

The present invention relates to an mRNA vaccine comprising at least one RNA molecule encoding at least one cytokine (preferably IL-4, IL-13, or a fragment thereof) and at least one T cell epitope for treating or preventing disorders associated with aberrant IL-4 and / or IL-13 expression or activity, in particular asthma, atopic dermatitis and allergic disorders.

Description

[Technical field]

[0001] The present invention relates to mRNA vaccines and their uses for treating disorders associated with aberrant IL-4 and / or IL-13 expression or activity, in particular asthma, atopic dermatitis and allergic disorders. [Background technology]

[0002] Allergic disorders are complex diseases resulting from the interaction of multiple genetic and environmental factors. The increase in allergies observed in the past decades is mainly explained by the environmental changes that have occurred during the same period. Among all allergies, allergic asthma, allergic rhinitis, and food allergies are major public health problems. It is estimated that half of the world's population will suffer from allergic diseases by 2050. Thus, the increase in allergic diseases has become a major health problem worldwide, resulting in a significant socio-economic burden for which there is still no effective long-term treatment.

[0003] The pathogenesis of allergic disorders results from exposure of the immune system to allergens. Such exposure is believed to be responsible for the breakdown of tolerance, resulting in a type 2 immune response characterized by the production of type 2 helper T cell (Th2) cytokines such as interleukin 4 (IL-4) and interleukin 13 (IL-13), high levels of immunoglobulin E (IgE) antibodies, and the infiltration and expansion of immune cells in inflamed tissues. Mast cells, basophils, and eosinophils are particularly involved in the release of cytoplasmic granules containing preformed inflammatory mediators such as histamine.

[0004] IL-4 and IL-13 cytokines therefore play important roles in the pathogenesis of allergic disorders. Both cytokines have long been associated with the pathogenesis of allergic disorders and are therapeutically important cytokines based on their biological functions. IL-4 and IL-13 exhibit similar structures and share one receptor subunit (IL-4Rα). However, despite their many similarities, IL-4 and IL-13 are thought to play non-redundant functions in allergy.

[0005] IL-4 is a pleiotropic cytokine involved in the development of allergy (Gour N. & Wills-Karp M., 2015), and elevated levels of IL-4 have been observed in the serum and bronchoalveolar lavage fluid of asthmatic patients. IL-4 is thought to act specifically in the early stages of allergy development. The key role of IL-4 lies in its multiple effects in driving allergy, such as induction of IgE production, upregulation of IgE receptor expression, and differentiation of naive type 0 helper T cells (Th0) into Th2 lymphocytes.

[0006] In contrast, IL-13 is more deeply involved in the effector and later stages of allergic responses (Gour N. & Wills-Karp M., 2015). IL-13 has been shown to be sufficient to induce the major symptoms of allergic disease, including, but not limited to, airway hyperresponsiveness, mucus production, airway smooth muscle changes, and subepithelial fibrosis.

[0007] Thus, IL-4 and IL-13 are promising therapeutic targets for the treatment of allergy, and there is a clear need to improve current strategies to block these molecules, especially to achieve long-term therapeutic effects.

[0008] In recent years, new therapeutic methods have been developed to treat or prevent allergy. These therapeutic methods based on passive immunity specifically target the pathogenic factors involved in allergy. For example, the use of recombinant antibodies against IL-4 and IL-13 or their receptors has been described in the art. However, the use of recombinant antibodies is limited by the high cost, the need to perform repeated injections, and the potential risk of the appearance of anti-drug antibodies (ADA) or other side effects.

[0009] RNA vaccines emerged as a new therapeutic perspective in the 1990s. Great advantages may be associated with the use of RNA as a vaccine, compared to other types of vaccines (e.g. attenuated virus and DNA-based vaccines). Indeed, the use of RNA vaccines may be safer, since there is no risk of mutagenicity or infection. Furthermore, the immunogenicity of RNA can be regulated before in vivo delivery. Higher efficiency of the vaccination process may also be obtained by in vitro modification of the RNA, thereby improving its stability and translatability. Finally, RNA vaccines can be produced very quickly and at low cost.

[0010] However, the use of RNA as a vaccine requires overcoming many challenges, particularly RNA instability, high natural immunogenicity, and in vivo delivery of RNA.

[0011] In the present invention, the applicant provides an RNA vaccine that can be used to induce the production of antibodies against self-proteins (i.e., IL-4 and IL-13), while conventional mRNA vaccines induce the production of antibodies against non-self-proteins by the vaccinated subject. In particular, the RNA vaccine disclosed in the present invention efficiently induces the production of antibodies against self-proteins in the recipient subject without substantial toxicity.

[0012] The applicant therefore provides herein a novel RNA vaccine comprising an RNA sequence encoding a cytokine selected from IL-4 and IL-13 (or a fragment thereof) and a T cell epitope. This novel mRNA vaccine is of particular interest for treating inflammatory disorders such as asthma, atopic dermatitis, and allergic disorders. Summary of the Invention

[0013] The present invention relates to a composition comprising at least one RNA molecule, the at least one RNA molecule comprising: at least one cytokine, or at least one fragment or epitope thereof, preferably wherein the at least one cytokine is interleukin-4 (IL-4) and / or interleukin-13 (IL-13), - at least one T cell epitope, optionally at least one spacer, The present invention encodes at least one amino acid sequence comprising:

[0014] In one embodiment, the at least one cytokine is IL-4.

[0015] In one embodiment, the IL-4 fragment is selected from the group consisting of SEQ ID NOs: 7-10, 13-16, 94-97 and 109-112.

[0016] In one embodiment, the at least one cytokine is IL-13.

[0017] In one embodiment, the IL-13 fragment comprises or is selected from the group consisting of SEQ ID NOs: 25-28, 35-38, 119-122 and 129-132.

[0018] In one embodiment, at least one RNA molecule encodes IL-4, or at least one fragment or epitope thereof, and IL-13, or at least one fragment or epitope thereof.

[0019] In one embodiment, the at least one T cell epitope is 197 , a combination of diphtheria and tetanus epitopes (TpD), an epitope of the tetanus toxin (TT), a universal CD4 polyepitope, variants and fragments thereof.

[0020] In one embodiment, at least one spacer is selected from the group consisting of PMGLP, a cathepsin cleavage site, an amino acid doublet, GP, GPGPG, GGSGGGGSGG, (GGGGS) n (wherein n ranges from 1 to 4), LG, ASG, KG, and RR.

[0021] In one embodiment, at least one RNA molecule is preferably encapsulated in a nanoparticle (eg, a lipid nanoparticle), in a liposome, or in a virus-like particle.

[0022] The present invention further relates to a pharmaceutical composition comprising the composition described herein and at least one pharma- ceutically acceptable excipient.

[0023] Another object of the invention is a vaccine composition comprising the composition described above and optionally at least one adjuvant.

[0024] Another object of the present invention is a composition as described herein for use as a medicament.

[0025] The present invention further relates to a composition, pharmaceutical composition or vaccine composition as described herein for use in treating an inflammatory disorder, preferably the disorder is associated with aberrant IL-4 and / or IL-13 expression or activity.

[0026] In one embodiment, the inflammatory disorder is asthma (either allergic or non-allergic), allergic conditions (e.g., food allergies, venom allergies, animal allergies, drug allergies, hyper-IgE syndrome, allergic rhinitis, allergic conjunctivitis, and allergic enterogastritis, etc.), atopic disorders (e.g., atopic dermatitis, urticaria (including chronic idiopathic urticaria and chronic spontaneous urticaria), eczema, etc.), bullous pemphigoid, respiratory disorders (e.g., allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), etc.), nasal polyposis, and other conditions involving airway inflammation (e.g., eosinophilia, fibrosis, and excess mucus production, e.g., cystic fibrosis and pulmonary fibrosis, systemic sclerosis, and the like). and / or subcutaneous inflammatory disease (SSc); inflammatory and / or autoimmune disorders or conditions, gastrointestinal disorders or conditions (e.g., inflammatory bowel disease (IBD) and eosinophilic esophagitis (EE), as well as eosinophil-mediated gastrointestinal disease, ulcerative colitis, and Crohn's disease); systemic lupus erythematosus, liver disorders or conditions (e.g., cirrhosis, and hepatocellular carcinoma), scleroderma; fibrotic diseases or disorders (e.g., fibrosis of the liver (e.g., fibrosis caused by hepatitis B and / or C virus), scleroderma; solid tumors or cancers, such as leukemia (e.g., B-cell chronic lymphocytic leukemia), glioblastoma, lymphoma (e.g., Hodgkin's lymphoma), and mastocytosis.In one embodiment, the inflammatory disorder is asthma (either allergic or non-allergic), allergic conditions (such as food allergies, venom allergies, animal allergies, drug allergies, anaphylaxis, hyper-IgE syndrome, allergic rhinitis, allergic conjunctivitis, and allergic enterogastritis), atopic disorders (such as atopic dermatitis, urticaria (chronic idiopathic urticaria and chronic spontaneous urticaria, eczema, and the like), bullous pemphigoid, respiratory disorders (such as allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD)), nasal polyposis, and other conditions involving airway inflammation (such as eosinophilia, fibrosis, and excess mucus production, e.g., cystic fibrosis and pulmonary fibrosis, systemic and / or inflammatory and / or autoimmune disorders or conditions, gastrointestinal disorders or conditions (such as inflammatory bowel disease (IBD) and eosinophilic esophagitis (EE), as well as eosinophil-mediated gastrointestinal diseases, ulcerative colitis, and Crohn's disease); systemic lupus erythematosus, liver disorders or conditions (such as cirrhosis, and hepatocellular carcinoma), scleroderma; fibrotic diseases or disorders (such as fibrosis of the liver (such as fibrosis caused by hepatitis B and / or C viruses), scleroderma; solid tumors or cancers, such as leukemia (such as B-cell chronic lymphocytic leukemia), glioblastoma, lymphoma (such as Hodgkin's lymphoma), and mastocytosis.

[0027] In one embodiment, the inflammatory disorder is selected from the group including or consisting of asthma (e.g., allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergies, nasal polyps, and eosinophilic esophagitis, preferably said inflammatory disorder is allergy, asthma, or atopic dermatitis.

[0028] definition In the present invention, the following terms have the following meanings:

[0029] As used herein, the term "about" when referring to a measurable value, such as an amount, temporal duration, and the like, is meant to encompass variations of ±20%, in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, since such variations are appropriate for performing the disclosed methods.

[0030] As used herein, an "adjuvant" is a substance that enhances the immunogenicity of the compositions of the invention. Adjuvants are often given to enhance the immune response and are well known to those skilled in the art.

[0031] As used herein, the term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using the T cell receptor (TCR). APCs process antigens and present them to T cells.

[0032] As used herein, the term "derived from" refers to a relationship between a first molecule and a second molecule. It generally refers to a structural similarity between the first and second molecules, and does not imply or include any process or source limitations on the origin of the first molecule from the second molecule.

[0033] As used herein, the term "epitope" refers to a specific arrangement of amino acids located on a protein to which an antibody or MHC molecule or any binding fragment thereof binds. Epitopes are composed of chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be linear (or contiguous) or conformational, i.e., involving two or more sequences of amino acids in different regions of a protein that may not necessarily be contiguous.

[0034] In particular, as used herein, the term "T cell epitope" refers to an epitope that, when presented by an MHC type II molecule, can be recognized and bound by a TCR, particularly as expressed by a CD4+ T cell (e.g., a Th2 cell). In one embodiment, the T cell expressing the TCR is a helper T cell (e.g., a Th2 cell), and binding of the T cell epitope to the TCR induces activation of the T cell, thereby resulting in the production of proinflammatory molecules by said T cell.

[0035] As used herein, the term "B cell epitope" refers to an epitope that can be recognized and bound by the B cell receptor (BCR) expressed by B cells when present as a soluble molecule in the B cell microenvironment, or can be cross-linked via the BCR when presented on the surface of various cell types. In one embodiment, the binding of a B cell epitope to the BCR induces the activation of a B cell, thereby resulting in its activation and production of specific antibodies by said B cell.

[0036] As used herein, the term "immune response" refers to the reaction that occurs in an organism, particularly in response to a foreign element.It can therefore refer to the action of, for example, lymphocytes (such as B cells and T cells, including CD4+, CD8+, Th1 and Th2 cells), antigen-presenting cells (such as professional antigen-presenting cells, such as dendritic cells), natural killer cells, myeloid cells (such as macrophages, eosinophils, mast cells, basophils, and granulocytes), and macromolecules produced by the above cells or liver (including, but not limited to, antibodies, cytokines, and complements).As used herein, the term "immune response" therefore includes T cell-mediated immune response and / or B cell-mediated immune response.

[0037] As used herein, an antibody that "inhibits the biological activity" or "neutralizes the biological activity" of at least one cytokine selected from IL-4, IL-13, or a mixture thereof is intended to refer to an antibody that inhibits the activity of the cytokine by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more compared to the level of activity of the cytokine in the absence of the antibody. Examples of functional assays that can be used to assess the activity of a cytokine are well known in the art.

[0038] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that does not cause adverse reactions, allergic reactions or other untoward reactions when administered to animals, particularly mammals, preferably humans. Excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, etc. Pharmaceutically acceptable excipients can therefore refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials, or any type of formulation auxiliary. For human administration, preparations must meet the sterility, pyrogenicity, general safety and purity standards required by regulatory authorities such as FDA or EMA.

[0039] As used herein, the term "subject" is intended to include organisms in which an immune response can be elicited (e.g., mammals, particularly humans, primates, dogs, cats, horses, sheep, etc.). In one embodiment, the subject is a human. In one embodiment, the subject may be a "patient", a warm-blooded animal, preferably a human, that is awaiting or receiving medical treatment, or has been or is the subject of medical treatment, or is being monitored for the development of a targeted disease or condition, such as an inflammatory disorder. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is suffering from, preferably diagnosed with, an inflammatory disorder. In one embodiment, the subject is at risk of developing an inflammatory disorder. Examples of risk factors include, but are not limited to, a genetic predisposition, or a family history of an inflammatory disorder.

[0040] The term "therapeutically effective amount" as used herein refers to an amount of the composition described herein that is effective to achieve a particular biological result. Thus, the term "therapeutically effective amount" refers to a level or amount of a composition that is intended to (1) delay or prevent the onset of the targeted disease or condition, (2) slow down or stop the progression, worsening, or decay of one or more symptoms of the targeted disease or condition, (3) bring about an improvement in the symptoms of the targeted disease or condition, (4) reduce the severity or incidence of the targeted disease or condition, or (5) cure the targeted disease or condition, without causing significant negative or harmful side effects to the target. A therapeutically effective amount may be administered prior to the onset of the targeted disease or condition for prophylactic or preventative action. Alternatively or additionally, a therapeutically effective amount may be administered after the onset of the targeted disease or condition for therapeutic action.

[0041] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and preventive or preventative measures, the purpose of which is to prevent or slow down (alleviate) the targeted disease or condition. Those in need of treatment include those who already have the condition, those who are prone to have the condition, or those who need to prevent the condition. After receiving a therapeutic amount of the composition described herein, if the subject shows observable and / or measurable improvement in one or more of the following: reduction in the number of pathogenic cells; reduction in the proportion of total cells that are pathogenic; some degree of relief of one or more of the symptoms associated with a particular condition; reduction in morbidity and mortality; and / or improvement in quality of life issues. The above parameters for evaluating the success of treatment and improvement of a condition can be easily measured by routine procedures familiar to physicians. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention relates to at least one RNA molecule, or a composition comprising at least one RNA molecule, wherein the at least one RNA molecule encodes (i) the amino acid sequence of at least one cytokine or cytokine fragment, (ii) the amino acid sequence of at least one T cell epitope, and optionally (iii) the amino acid sequence of at least one spacer.

[0043] In one embodiment, at least one RNA molecule encodes a single protein construct (preferably a single-chain protein construct) comprising in one amino acid chain: (i) the amino acid sequence of at least one cytokine or cytokine fragment, (ii) the amino acid sequence of at least one T cell epitope, and optionally (iii) the amino acid sequence of at least one spacer.

[0044] In one embodiment, at least one RNA molecule encodes a single protein construct (preferably a single single chain protein construct) comprising in one amino acid chain, from N-terminus to C-terminus, (i) the amino acid sequence of at least one cytokine or cytokine fragment, optionally (ii) the amino acid sequence of at least one spacer, and (iii) the amino acid sequence of at least one T cell epitope. In one embodiment, the single protein construct further comprises, preferably at the N-terminus, a signal peptide.

[0045] In one embodiment, the RNA molecule of the invention comprises, from 5' to 3', (i) a sequence encoding at least one cytokine or cytokine fragment, optionally (ii) a sequence encoding at least one spacer, and (iii) a sequence encoding at least one T cell epitope. In one embodiment, the RNA molecule further comprises, preferably at the 5', a sequence encoding a signal peptide.

[0046] In one embodiment, the at least one T cell epitope is not a T cell epitope derived from the at least one cytokine. More preferably, the at least one T cell epitope is derived from a different organism than the at least one cytokine.

[0047] In one embodiment, at least one cytokine or cytokine fragment is derived from the same organism as the subject to be treated. For example, if the RNA molecule is intended to be used to treat humans, at least one cytokine or fragment thereof is human (either natural or recombinant).

[0048] Thus, in one embodiment, at least one RNA molecule may be described as comprising two domains: a first domain encoding at least one cytokine or a fragment thereof (wherein the at least one cytokine is preferably derived from the same organism as the subject being treated - this domain may therefore be defined as a "self domain"), and a second domain comprising at least one T cell epitope (wherein the at least one T cell epitope is preferably derived from another organism than the subject being treated - this domain may therefore be defined as a "non-self domain").

[0049] In one embodiment, at least one RNA molecule encodes at least one epitope of said at least one cytokine or a fragment thereof.In one embodiment, said at least one epitope is a B cell epitope.In another embodiment, said at least one epitope is a T cell epitope.

[0050] In one embodiment, at least one RNA molecule encodes a cytokine or a fragment thereof with reduced cytokine activity. Without wishing to be bound by any theory, reduced cytokine activity may be particularly relevant, since induction of expression of cytokines with natural activity may induce toxicity. Indeed, in one embodiment, the present invention is aimed at treating diseases associated with abnormal IL-4 and / or IL-13 activity, and administering RNA molecules results in increased expression of functional IL-4 and / or IL-13, which may cause severe side effects.

[0051] In one embodiment, the residual cytokine activity of the cytokine or fragment thereof encoded by at least one RNA molecule of the present invention is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than the cytokine activity measured for the cytokine from which it is derived. Assays that can be used to measure cytokine activity are well known in the art.

[0052] In one embodiment, the cytokine is IL-4 or IL-13, and the residual cytokine activity of the cytokine or fragment thereof encoded by at least one RNA molecule of the present invention can be measured under the conditions of Test A. Test A is an in vitro test based on measuring the proliferation activity induced by serial dilutions of the supernatant (preferably, the supernatant is collected 24 hours after transfection) of cells (e.g., HEK293 cells) transfected with at least one RNA molecule of the present invention, for example, after about 48 hours of incubation, on CTLL-2 cells. Methods for measuring cell proliferation are well known in the art and include, but are not limited to, MTS / PMS assay (including the addition of substrate and reading the optical density at 490 nm after 4 hours of incubation). An example of Test A is shown in Example 2.

[0053] In one embodiment, the residual cytokine activity of a cytokine or fragment thereof encoded by at least one RNA molecule of the present invention, when measured under the conditions of Test A, is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than the cytokine activity measured for the cytokine from which it is derived, at the same dilution.

[0054] Without wishing to be bound by any theory, the applicant suggests that at least one RNA molecule is produced in vitro and delivered in vivo to target cells (such as antigen presenting cells (APCs), in particular dendritic cells), which then results in the production in these cells of at least one protein or peptide. The peptide(s) or protein(s) are then secreted by the cells or degraded by the proteasome in the extracellular compartment and processed into the endoplasmic reticulum to generate epitopes, which are then presented on the cell membrane by MHC class I and class II molecules.

[0055] Presentation of IL-4 and / or IL-13 epitopes (from the RNA construct of the present invention) via MHC class II molecules can result in activation of CD4+ T cells, which then activate B lymphocytes, thereby resulting in the production of antibodies specific to said epitope(s). Furthermore, IL-4 and / or IL-13 epitopes can also be presented by MHC class I molecules and can induce activation of cytotoxic antigen-specific CD8+ T cells, which can then lyse cells expressing the same antigen on their cell surface. Furthermore, presentation of T cell epitopes of the second domain of the RNA molecule of the present invention by MHC molecules can increase the immune response induced after presentation of IL-4 and / or IL-13 epitopes.

[0056] In one embodiment, the at least one RNA molecule is an mRNA molecule, preferably a non-replicating mRNA molecule encoding at least one cytokine or fragment thereof, at least one T cell epitope, and optionally at least one spacer.

[0057] In one embodiment, at least one cytokine or fragment thereof, at least one T cell epitope, and optionally at least one spacer are encoded by different mRNA molecules. In one embodiment, at least one cytokine or fragment thereof, at least one T cell epitope, and optionally at least one spacer are encoded by one mRNA molecule. In one embodiment, at least one cytokine or fragment thereof, at least one T cell epitope, and optionally at least one spacer are comprised in a single protein construct, preferably a single single chain protein construct.

[0058] In one embodiment, the at least one cytokine is IL-4.

[0059] In one embodiment, the IL-4 is derived from a mammalian IL-4.

[0060] In one embodiment, the IL-4 is a variant of a mammalian IL-4, wherein said variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to the mammalian IL-4 from which it is derived.

[0061] As used herein, the terms "identity" or "identical," when used in the context of two or more nucleic acid sequences or two or more polypeptide sequences, refer to the degree of sequence relatedness between the nucleic acid sequences or polypeptides, as determined by the number of matches between strings of two or more nucleic acid residues or amino acid residues, respectively. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) specified by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related nucleic acid sequences or polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two sequences include the GCG program package, ClustalO (Sievers F., et al 2011), the GCG program package, such as GAP (Devereux et al., Nucl. Acid. Res.\2,387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410 (1990)). The BLASTX program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known SmithWaterman algorithm may also be used to determine identity.

[0062] In one embodiment, at least one cytokine is a fragment of IL-4, such as a fragment of IL-4 that comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or 125 amino acids (preferably contiguous amino acids) of the IL-4 molecule from which they are derived.

[0063] In one embodiment, the fragment of IL-4 may comprise or consist of an epitope. In one embodiment, the fragment of IL-4 comprises or consists of at least one epitope of IL-4. Examples of epitopes of IL-4 are shown below.

[0064] In one embodiment, the IL-4 is full-length IL-4.

[0065] In one embodiment of the invention, the IL-4 is human IL-4. Human IL-4 has the sequence SEQ ID NO: 1 (UniProt ID: P05112-1).

[0066] SEQ ID NO: 1HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS

[0067] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding human IL-4. An example of an RNA sequence encoding human IL-4 comprises or consists of SEQ ID NO:2 or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:2.

[0068] SEQ ID NO:2 CACAAGUGCGACAUCACCCUGCAGGAGAUCAUCAAGACCCUGAACAGCCUGACCGAGCAGAAGACCCUGUGCACCGAGCUGACCGUGACCGACAUCUUCGCCGCCAGCAAGAACACCACCGAGAAGGAGACCUUCUGCCGCGCCGCCACCGUGCUGCGCCAGUUCUACAGCCACCACGAGAAGGACACCCGCU GCCUGGGCGCCACCGCCCAGCAGUUCCACCGCCACAAGCAGCUGAUCCGCUUCCUGAAGCGCCUGGACCGCAACCUGUGGGGCUGCCGGCCUGAACAGCUGCCCCGUGAAGGAGGCCAACCAGAGCACCCUGGAGAACUUCCUGGAGCGCCUGAAGACCAUCAUGCGCGAGAAGUACAGCAAGUGCAGCAGC

[0069] In one embodiment, at least one IL-4 cytokine fragment is a peptide comprising or consisting of at least one epitope of human IL-4.

[0070] Examples of epitopes of human IL-4 include, but are not limited to, the following: LQEIIKTLNSLTEQKTLCTELT (SEQ ID NO: 3), KETFCRAATVLRQFY (SEQ ID NO: 4), AQQFHRHKQLIRFLKRLDRNLWGLAG (SEQ ID NO: 5) and LENFERLKTIMREKYSKC (SEQ ID NO: 6).

[0071] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of human IL-4, e.g., SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0072] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising at least one epitope of human IL-4. In one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of human IL-4, thereby conferring a cyclic conformation to said peptide.

[0073] Examples of cyclic peptides containing at least one epitope of human IL-4 include, but are not limited to, CLQEIIKTLNSLTEQKTLCTELTC (SEQ ID NO: 7), CKETFCRAATVLRQFYC (SEQ ID NO: 8), CAQQFHRHKQLIRFLKRLDRNLWGLAGC (SEQ ID NO: 9), and CLENFLERLKTIMREKYSKC (SEQ ID NO: 10).

[0074] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of human IL-4, e.g., SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0075] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding human IL-4 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g., the signal peptide of the native human IL-4 protein.

[0076] In one embodiment, the signal peptide is the signal peptide of human IL-4, preferably comprising or consisting of the sequence of SEQ ID NO: 103 (MGLTSQLLPPLFFLLACAGNFVHG), and may be encoded by the RNA sequence SEQ ID NO: 104 (AUGGGCCUGACCAGCCAGCUGCUGCCCCCCCUGUUCUUCCUGCUGGCCUGCGCCGGCAACUUCGUGCACGGC).

[0077] Other examples of signal peptides that can be used in the present invention include, but are not limited to, peptides having the following sequences: -MKWVTFISLLFLFSSAYS (SEQ ID NO: 88, derived from serum albumin preproprotein) -MQLLSCIALILALV (SEQ ID NO: 89, derived from human IL-2) -MGVKVLFALICIAVAEA (SEQ ID NO: 90, derived from Gaussia luciferase): - MAFLWLLSCWALLGTTFG (SEQ ID NO: 91, from human chymotrypsinogen); -MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 92) and - MNLLLILTFVAAAVA (SEQ ID NO: 93, from human trypsinogen-2).

[0078] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs:78, 84, 117, 127, 137, 140 and 142.

[0079] In one embodiment of the invention, the IL-4 is mouse IL-4. Mouse IL-4 has SEQ ID NO: 11 (UniProt ID: P07750-1).

[0080] SEQ ID NO:11 HIHGCDKNHLREIIGILNEVTGEGTPCTEMDVPNVLTATKNTTESELVCRASKVLRIFYLKHGKTPCLKKNSSVLMELQRLFRAFRCLDSSISCTMNESKSTSLKDFLESLKSIMQMDYS

[0081] In one embodiment, the RNA sequence encoding at least one cytokine comprises a sequence encoding murine IL-4. An example of an RNA sequence encoding murine IL-4 comprises or consists of SEQ ID NO: 12, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO: 12.

[0082] SEQ ID NO:12 CACAUCCAGGCUGCGACAAGAACCACCUGAGGGAGAUCAUCGGCAUCCUGAACGAGGUGACCGGCGAGGGCACCCCCUGCACCGAGAUGGACGUGCCCAACGUGCUGACCGCCACCAAGAACACCACCGAGAGCGAGCUGGUGUGCAGGGCCAGCAAGGUGCUGAGGAUCUUCUACCUG AAGCACGGCAAGACCCCCUGCCUGAAGAAGAACAGCAGCGUGCUGAUGGAGCUGCAGAGGCUGUUCAGGGCCUUCAGGUGCCUGGACAGCAGCAUCAGCUGCACCAUGAACGAGAGCAAGAGCACCAGCCUGAAGGACUUCCUGGAGAGCCUGAAGAGCAUCAUGCAGAUGGACUACAGC

[0083] In one embodiment, the at least one IL-4 cytokine fragment is a peptide comprising or consisting of at least one epitope of murine IL-4.

[0084] Examples of epitopes of mouse IL-4 include, but are not limited to, LREIIGILNEVTGEGTPCTEMD (SEQ ID NO: 13), SELVCRASKVLRIFY (SEQ ID NO: 14), SSVLMELQRLFRAFRCLDS (SEQ ID NO: 15), and LKDFLESLKSIMQMDYS (SEQ ID NO: 16).

[0085] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of mouse IL-4, e.g., SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0086] In one embodiment, one or more cysteine ​​residue(s) are added to the terminus of a peptide comprising or consisting of at least one epitope of mouse IL-4. In one embodiment, one cysteine ​​residue is added to the terminus of a peptide comprising or consisting of at least one epitope of mouse IL-4, thereby conferring a cyclic conformation to the peptide.

[0087] Examples of cyclic peptides containing at least one epitope of mouse IL-4 include, but are not limited to, CLREIGINLEVTGEGTPCTEMDC (SEQ ID NO: 17), CSELVCRASKVLRIFYC (SEQ ID NO: 18), CSSVLMELQRLFRAFRCLDSC (SEQ ID NO: 19) and CLKDFLESLKSIMQMDYSC (SEQ ID NO: 20).

[0088] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of mouse IL-4, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0089] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding mouse IL-4 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g. the signal peptide of the native mouse IL-4 protein.

[0090] In one embodiment, the signal peptide is the signal peptide of mouse IL-4, preferably comprising or consisting of SEQ ID NO: 140 (MGLNPQLVVILLFFLECTRS), which can be encoded by the RNA sequence of SEQ ID NO: 141 (AUGGGCCUGAACCCCCAGCUGGUGGUGAUCCUGCUGUUCUUCC UGGAGUGCACCCGCAGC).

[0091] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 78, 84, 103, 117, 127, 137, and 142.

[0092] In one embodiment of the invention, the IL-4 is canine IL-4. Canine IL-4 has SEQ ID NO: 21 (UniProt ID: O77762-1).

[0093] SEQ ID NO:21 HNFNITIKEIIKMLNILTARNDSCMELTVKDVFTAPKNTSDKEIFCRAATVLRQIYTHNCSNRYLRGLYRNLSSMANKTCSMNEIKKSTLKDFLERLKVIMQKKYYRH

[0094] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding canine IL-4. An example of an RNA sequence encoding canine IL-4 comprises or consists of SEQ ID NO:22, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:22.

[0095] SEQ ID NO:22 CACAACUUCAACAUCACCAUCAAGGAGAUCAUCAAGAUGCUGAACAUCCUGACCGCCCGCAACGACAGCUGCAUGGAGCUGACCGUGAAGGACGUGUUCACCGCCCCCAAGAACACCAGCGACAAGGAGAUCUUCUGCCGCGCCGCCACCGUGCUGCGCCAG AUCUACACCCACAACUGCAGCAACCGCUACCUGCGCGGCCUGUACCGCAACCUGAGCAGCAUGGCCAACAAGACCUGCAGCAUGAACGAGAUCAAGAAGAGCACCCUGAAGGACUUCCUGGAGCGCCUGAAGGUGAUCAUGCAGAAGAAGUACUACCGCCAC

[0096] In one embodiment, the at least one IL-4 cytokine fragment is a peptide comprising or consisting of at least one epitope of canine IL-4.

[0097] Examples of epitopes of canine IL-4 include, but are not limited to, IKEIIKMLNILTARNDSCMELT (SEQ ID NO: 94), KEIFCRAATVLRQIY (SEQ ID NO: 95), RYLRGLYRNLSSMAN (SEQ ID NO: 96), and LKDFLERLKVIMQKKY (SEQ ID NO: 97).

[0098] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of canine IL-4, such as SEQ ID NOs: 94-97.

[0099] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising or consisting of at least one epitope of canine IL-4, hi one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of canine IL-4, thereby conferring a cyclic conformation to the peptide.

[0100] Examples of cyclic peptides containing at least one epitope of canine IL-4 include, but are not limited to, CIKEIIKMLNILTARNDSCMELTC (SEQ ID NO: 105), CKEIFCRAATVLRQIYC (SEQ ID NO: 106), CRYLRGLYRNLSSMANC (SEQ ID NO: 107), and CLKDFLERLKVIMQKKYC (SEQ ID NO: 108).

[0101] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of canine IL-4, for example SEQ ID NOs: 105-108.

[0102] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding canine IL-4 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g., the signal peptide of the native canine IL-4 protein.

[0103] In one embodiment, the signal peptide is the signal peptide of canine IL-4, preferably comprising or consisting of the sequence MGLTSQLIPTLVCLLALTSTFVHG (SEQ ID NO: 78), which can be encoded by the RNA sequence AUGGGCCUGACCAGCCAGCUGAUCCCCACCCUGGUGUGC CUGCUGGCCCUGACCAGCACCUUCGUGCACGGC (SEQ ID NO: 79).

[0104] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 84, 103, 117, 127, 137, 140, and 142.

[0105] In one embodiment of the invention, the IL-4 is equine IL-4. Equine IL-4 has SEQ ID NO: 99 (UniProt ID: P42202).

[0106] SEQ ID NO:99 CKYDITLQEIIKTLNLTDGKGKNSCMELTVADAFGPKNTDGKEICRAAKVLQQYKRHDRSLIKECLSGLDRNLKGMANGTCCTVNEAKKSTLKDFLERLKTIMKEKYSKCS

[0107] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding equine IL-4. An exemplary RNA sequence encoding equine IL-4 comprises or consists of SEQ ID NO: 100, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO: 100.

[0108] SEQ ID NO:100 UGCAAGUACGACAUCACCCUGCAGGAGAUCAUCAAGACCCUGAACCUGACCGACGGCAAGGGCAAGAACAGCUGCAUGGAGCUGACCGUGGCCGACGCCUUCGGCCCCAAGAACACCGACGGCAAGGAGAUCUGCCGCGCCGCCAAGGUGCUGCAGCAGUACAAGC GCCACGACCGCAGCCUGAUCAAGGAGUGCCUGAGCGGCCUGGACCGCAACCUGAAGGGCAUGGCCAACGGCACCUGCUGCACCGUGAACGAGGCCAAGAAGAGCACCCUGAAGGACUUCCUGGAGCGCCUGAAGACCAUCAUGAAGGAGAAGUACAGCAAGUGCAGC

[0109] In one embodiment, the at least one IL-4 cytokine fragment is a peptide comprising or consisting of at least one epitope of equine IL-4.

[0110] Examples of epitopes of equine IL-4 include, but are not limited to, LQEIIKTLNLTDGKGKNSCMELT (SEQ ID NO: 109), KEICRAAKVLQQYK (SEQ ID NO: 110), RSLIKECLSGLDRNLKGMAN (SEQ ID NO: 111), and LKDFLERLKTIMKEKYSKC (SEQ ID NO: 112).

[0111] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of equine IL-4, for example SEQ ID NOs: 109-112.

[0112] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising or consisting of at least one epitope of equine IL-4, hi one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of equine IL-4, thereby conferring a cyclic conformation to the peptide.

[0113] Examples of cyclic peptides that contain at least one epitope of equine IL-4 include, but are not limited to, CLQEIIKTLNLTDGKGKNSCMELTC (SEQ ID NO:113), CKEICRAAKVLQQYKC (SEQ ID NO:114), CRSLIKECLSGLDRNLKGMANC (SEQ ID NO:115), and CLKDFLERLKTIMKEKYSKC (SEQ ID NO:116).

[0114] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of equine IL-4, for example SEQ ID NOs: 113-116.

[0115] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding equine IL-4 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g., the signal peptide of the native equine IL-4 protein.

[0116] In one embodiment, the signal peptide is the signal peptide of equine IL-4, preferably comprising or consisting of SEQ ID NO: 84 (MGLTYQLLPALVCLLACTSFIQG), which can be encoded by the RNA sequence of SEQ ID NO: 85 (AUGGGCCUGACCUACCAGCUGCUGCCCGCCCUGGUGUGCCUG CUGGCCUGCACCAGCUUCAUCCAGGGC).

[0117] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 78, 103, 117, 127, 137, 140, and 142.

[0118] In one embodiment, the at least one cytokine is IL-13.

[0119] In one embodiment, the IL-13 is derived from a mammalian IL-13.

[0120] In one embodiment, the IL-13 is a variant of a mammalian IL-13, wherein the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to the mammalian IL-13 from which it is derived.

[0121] In another embodiment, at least one cytokine is a fragment of IL-13, such as a fragment of IL-13 that comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 amino acids (preferably contiguous amino acids) of the IL-13 molecule from which they are derived.

[0122] In one embodiment, the fragment may consist of or include an epitope. In one embodiment, the fragment of IL-13 comprises or consists of at least one epitope of IL-13. Examples of epitopes of IL-13 are listed below.

[0123] In one embodiment, the IL-13 is full-length IL-13.

[0124] In one embodiment of the invention, the IL-13 is human IL-13. Human IL-13 has SEQ ID NO: 23 (UniProtID: P35225-1).

[0125] SEQ ID NO:23 LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

[0126] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding a human IL-13 protein. An example of an RNA sequence encoding human IL-13 comprises or consists of SEQ ID NO:24, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:24.

[0127] SEQ ID NO:24 CUGACCUGCCUGGGGCGGCUUCGCCAGCCCCGGCCCCGUGCCCCCCAGCACCGCCCUGCGCGAGCUGAUCGAGGAGCUGGUGAACAUCACCCAGAACCAGAAGGCCCCUGUGCAACGGCAGCAUGGUGGAGCAUCAACCUGACCGCCGGCAUGUACUGCGCCGCCCUGGAGAGCCUGAUC AACGUGAGCGGCUGCAGGCCAUCGAGAAGACCCAGCGCAUGCUGAGCGGCUUCUGCCCCCACAAGGUGAGCGCCGGCCAGUUCAGCAGCCUGCACGUGCGCGACACCAAGAUCGAGGUGGCCCAGUUCGUGAAGGACCUGCUGCUGCACCUGAAGAAGCUGUUCCGCGAGGGCCGCUUCAAC

[0128] In one embodiment, at least one IL-13 cytokine fragment is a peptide comprising or consisting of at least one epitope of human IL-13.

[0129] Examples of epitopes of human IL-13 include, but are not limited to, LRELIEELVNITQNQKAPLCNG (SEQ ID NO: 25), NGSMVWSINLTAGMYCA (SEQ ID NO: 26), AGMYCAALESLINVSGCSAIEK (SEQ ID NO: 27), and VAQFVKDLLLHLKKLFREGRFN (SEQ ID NO: 28).

[0130] Thus, according to one embodiment, at least one RNA molecule comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of human IL-13, e.g., SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

[0131] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising or consisting of at least one epitope of human IL-13. In one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of human IL-4, thereby conferring a cyclic conformation to the peptide.

[0132] Examples of cyclic peptides that contain at least one epitope of human IL-13 include, but are not limited to, the following: CLRELIEELVNITQNQKAPLCNGC (SEQ ID NO: 29), CNGSMVWSINLTAGMYCAC (SEQ ID NO: 30), CAGMYCAALESLINVSGCSAIEKC (SEQ ID NO: 31), and CVAQFVKDLLLHLKKLFREGRFNC (SEQ ID NO: 32).

[0133] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of human IL-13, e.g., SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.

[0134] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding human IL-13 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g. the signal peptide of the native human IL-13 protein.

[0135] In one embodiment, the signal peptide is the signal peptide of human IL-13, preferably comprising or consisting of the sequence of SEQ ID NO: 117 (MHPLLNPLLLALGLMALLLTTVIA), and may be encoded by the RNA sequence SEQ ID NO: 118 (AUGCACCCCCUGCUGAACCCCCUGCUGCUGGCCCUGGGCCUGAUGGCCCUGCUGCUGACCACCGUGAUCGCC).

[0136] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 78, 84, 103, 127, 137, 140, and 142.

[0137] In one embodiment of the invention, the IL-13 is mouse IL-13. Mouse IL-13 has SEQ ID NO: 33 (UniProt ID: P20109-1).

[0138] SEQ ID NO:33 APGPVPRSVSLPLTLKELIEELSNITQDQTPLCNGSMVWSVDLAAGGFCVALDSLTNISNCNAIYRTQRILHGLCNRKAPTTVSSLPDTKIEVAHFITKLLSYTKQLFRHGPF

[0139] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding murine IL-13. An example of an RNA sequence encoding murine IL-13 comprises or consists of SEQ ID NO:34, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:34.

[0140] SEQ ID NO:34 GCCCCCGGCCCCGUGCCAGGAGCUGAGCCUGCCCCUGACCCUGAAGGAGCUGAUCGAGGAGCUGAGCAACAUCACCCAGGACCAGACCCCCCUGUGCAACGGCAGCAUGUGUGGAGCGUGGACCUGGCCGCCGGCGGCUUCUGCGUGGCCCUGGACAGCCUGACCA ACAUCAGCAACUGCAACGCCAUCUACAGGACCCAGAGGAUCCUGCACGGCCUGUGCAACAGGAAGGCCCCCACCACCGUGAGCAGCCUGCCCGACACCAAGAUCGAGGUGGCCCACUUCAUCACCAAGCUGCUGAGCUACACCAAGCAGCUGUUCAGGCACGGCCCCUUC

[0141] In one embodiment, the at least one IL-13 cytokine fragment is a peptide comprising or consisting of at least one epitope of mouse IL-13.

[0142] Examples of epitopes of mouse IL-13 include LKELIEELSNITQDQTPLCNG (SEQ ID NO: 35), NGSMVWSVDLAAGGFCV (SEQ ID NO: 36), AGGFCVALDSLTNISNCNAIYR (SEQ ID NO: 37) and VAHFITKLLSYTKQLFRHGPF (SEQ ID NO: 38).

[0143] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of mouse IL-13, such as SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.

[0144] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising at least one epitope of mouse IL-13. In one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of mouse IL-13, thereby conferring a cyclic conformation to the peptide.

[0145] Examples of cyclic peptides containing at least one epitope of mouse IL-13 include, but are not limited to, CLKELIEELSNITQDQTPLCNGC (SEQ ID NO: 39), CNGSMVWSVDLAAGGFCVC (SEQ ID NO: 40), CAGGFCVALDSLTNISNCNAIYRC (SEQ ID NO: 41), and CVAHFITKLLSYTKQLFRHGPFC (SEQ ID NO: 42).

[0146] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of mouse IL-13, e.g., SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.

[0147] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding mouse IL-13 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g. the signal peptide of the native mouse IL-13 protein.

[0148] In one embodiment, the signal peptide is the signal peptide of mouse IL-13, preferably comprising or consisting of the sequence of SEQ ID NO: 142 (MALWVTAVLALACLGGLA), which can be encoded by the RNA sequence SEQ ID NO: 143 (AUGGCCCUGUGGGUGACCGCCGUGCUGGCCCUGGCCUGGGCGGCCUGGCC).

[0149] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 78, 84, 103, 117, 127, 137, and 140.

[0150] In one embodiment of the invention, the IL-13 is canine IL-13. Canine IL-13 has SEQ ID NO: 43 (UniProt ID: Q9N0W9-1).

[0151] SEQ ID NO:43 SPSPVTPSPTLKELIEELVNITQNQASLCNGSMVWSVNLTAGMYCAALESLINVSDCSAIQRTQRMLKALCSQKPAAGQISSERSRDTKIEVIQLVKNLLTYVRGVYRHGNFR

[0152] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding canine IL-13. An example of an RNA sequence encoding canine IL-13 comprises or consists of SEQ ID NO:44, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:44.

[0153] SEQ ID NO:44 AGCCCCAGCCCCGUGACCCCAGCCCCACCCUGAAGGAGCUGAUCGAGGAGCUGGUGAACAUCACCCAGAACCAGGCCAGCUGUGCAACGGCAGCAUGUGUGGAGCGUGAACCUGACCGCCGGCAUGUACUGCGCCGCCCUGGAGAGCCUGAUCAACGUGAGCGACU GCAGCGCCAUCCAGCGCACCCAGCGCAUGCUGAAGGCCCUGUGCAGCCAGAAGCCCGCCGCCGGCCAGAUCAGCAGCGAGCGCAGCCGCGACACCAAGAUCGAGGUGAUCCAGCUGGUGAAGAACCUGCUGACCUACGUGCGCGGCGUGUACCGCCACGGCAACUUCCGC

[0154] In one embodiment, the at least one IL-13 cytokine fragment is a peptide comprising or consisting of at least one epitope of canine IL-13.

[0155] Examples of epitopes of canine IL-13 include, but are not limited to, LKELIEELVNITQNQASLCNG (SEQ ID NO: 119), NGSMVWSVNLTAGMYCA (SEQ ID NO: 120), AGMYCAALESLINVSDCSAIQR (SEQ ID NO: 121), and VIQLVKNLLTYVRGVYRHGNF (SEQ ID NO: 122).

[0156] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of canine IL-13, such as SEQ ID NOs: 119-122.

[0157] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising or consisting of at least one epitope of canine IL-13, hi one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of canine IL-13, thereby conferring a cyclic conformation to the peptide.

[0158] Examples of cyclic peptides containing at least one epitope of canine IL-13 include, but are not limited to, CLKELIEELVNITQNQASLCNGC (SEQ ID NO: 123), CNGSMVWSVNLTAGMYCAC (SEQ ID NO: 124), CAGMYCAALESLINVSDCSAIQRC (SEQ ID NO: 125), and CVIQLVKNLLTYVRGVYRHGNFC (SEQ ID NO: 126).

[0159] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of canine IL-13, such as SEQ ID NOs: 123-126.

[0160] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding canine IL-13 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g., the signal peptide of the native canine IL-13 protein.

[0161] In one embodiment, the signal peptide is the signal peptide of canine IL-13, preferably comprising or consisting of the sequence MALWLTVVIALTCLGGLA (SEQ ID NO: 127), which can be encoded by the RNA sequence AUGGCCCUGUGGCUGACCGUGGUGAUCGCCCUGACCUGCCUGGGCGGCCUGGCC (SEQ ID NO: 128).

[0162] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 78, 84, 103, 117, 137, 140, and 142.

[0163] In one embodiment of the invention, the IL-13 is equine IL-13. Equine IL-13 has SEQ ID NO: 101 (UniProtID: B6C802).

[0164] SEQ ID NO:101 APLPSSMALKELIKELVNITQNQAPLCNGSMVWSVNLTADTYCRALESLSNVSTCSAIQNTRKMLTKLCPHQLSAGQVSSERARDTKIEVIVLVKDLLKNLRKIFHGGKHVDA

[0165] In one embodiment, the RNA sequence encoding at least one cytokine comprises or consists of a sequence encoding equine IL-13. An exemplary RNA sequence encoding equine IL-13 comprises or consists of SEQ ID NO: 102, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO: 102.

[0166] SEQ ID NO:102 GCCCCCCUGCCCAGCAGCAUGGCCCUGAAGGAGCUGAUCAAGGAGCUGGUGAACAUCACCCAGAACCAGGCCCCCUGUGCAACGGCAGCAUGGUGGAGCGUGAACCUGACCGCCGACACCUACUGCCGCGCCCUGGAGAGCCUGAGCAACGUGAGCACCUGCAGCG CCAUCCAGAACACCCGCAAGAUGCUGACCAAGCUGUGCCCCCACCAGCUGAGCGCCGGCCAGGUGAGCAGCGAGCGCGCCCGCGACACCAAGAUCGAGGUGAUCGUGCUGGUGAAGGACCUGCUGAAGAACCUGCGCAAGAUCUUCCACGGCGGCAAGCACGUGGACGCC

[0167] In one embodiment, at least one IL-13 cytokine fragment is a peptide comprising or consisting of at least one epitope of equine IL-13.

[0168] Examples of epitopes of equine IL-13 include, but are not limited to, LKELIKELVNITQNQAPLCNG (SEQ ID NO: 129), NGSMVWSVNLTADTYCRA (SEQ ID NO: 130), ADTYCRALESLSNVSTCSAIQN (SEQ ID NO: 131), and VIVLVKDLLKNLRKIFHGGK (SEQ ID NO: 132).

[0169] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a peptide comprising or consisting of at least one epitope of equine IL-13, such as SEQ ID NOs: 129-132.

[0170] In one embodiment, one or more cysteine ​​residue(s) are added to the termini of a peptide comprising or consisting of at least one epitope of equine IL-13, hi one embodiment, one cysteine ​​residue is added to each terminus of a peptide comprising or consisting of at least one epitope of equine IL-13, thereby conferring a cyclic conformation to the peptide.

[0171] Examples of cyclic peptides that contain at least one epitope of equine IL-13 include, but are not limited to, CLKELIKELVNITQNQAPLCNGC (SEQ ID NO: 133), CNGSMVWSVNLTADTYCRAC (SEQ ID NO: 134), CADTYCRALESLSNVSTCSAIQNC (SEQ ID NO: 135), and CVIVLVKDLLKNLRKIFHGGKC (SEQ ID NO: 136).

[0172] Thus, according to one embodiment, at least one RNA molecule of the invention comprises one or more RNA sequences encoding a cyclic peptide comprising or consisting of at least one epitope of equine IL-13, such as SEQ ID NOs: 133-136.

[0173] In one embodiment, at least one RNA sequence, in particular at least one RNA sequence encoding equine IL-13 or at least one fragment thereof, further comprises an RNA sequence encoding a signal peptide, e.g., the signal peptide of the native equine IL-13 protein.

[0174] In one embodiment, the signal peptide is the signal peptide of equine IL-13, preferably comprising or consisting of the sequence of SEQ ID NO: 137 (MALWLTAVIALACLGGLASP), which can be encoded by the RNA sequence of SEQ ID NO: 138 (AUGGCCCUGUGGCUGACCGCCGUGAUCGCCCUGGCCUGCCU GGGCGGCCUGGCCAGCCCC).

[0175] Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 88 to 93. Other examples of signal peptides that can be used in the present invention include, but are not limited to, SEQ ID NOs: 78, 84, 103, 117, 127, 140, and 142.

[0176] In one embodiment, at least one RNA molecule of the invention encodes (i) at least one amino acid sequence comprising or consisting of IL-4, at least one variant of IL-4, at least one fragment of IL-4, or at least one epitope of IL-4 (particularly one of the above peptides), and (ii) at least one amino acid sequence comprising or consisting of IL-13, at least one variant of IL-13, at least one fragment of IL-13, or at least one epitope of IL-13 (particularly one of the above peptides).

[0177] According to the present invention, at least one RNA molecule encodes at least one T cell epitope. In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence that comprises one T cell epitope. In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence that comprises at least 2, 3, 4, 5, or 10 T cell epitopes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more T cell epitopes), which may be the same or different and may be linked together by at least one spacer. Examples of spacers are listed herein.

[0178] In one embodiment, at least one T cell epitope is a dominant T cell epitope, i.e., an epitope that is non-HLA restricted, hi one embodiment, at least one T cell epitope is a Th2 epitope.

[0179] In one embodiment, the at least one T cell epitope is comprised in a peptide or protein, and the at least one RNA molecule encodes at least one peptide or protein comprising at least one T cell epitope. In one embodiment, the at least one peptide or protein comprising at least one T cell epitope is distinct from a cytokine or a fragment thereof.

[0180] Examples of T cell epitopes, or peptides or proteins that contain at least one T cell epitope, include, but are not limited to, CRMPs. 197 , variants and fragments thereof (e.g., CRM 197(299-312) , CRM 197(425-440) , CRM 197(300-450) ), a combination of diphtheria and tetanus epitopes (TpD), an epitope of tetanus toxin (TT), and the universal polyepitope CD4 (eg, N10, N19).

[0181] In one embodiment, at least one RNA molecule comprises a CRM 197 or a variant or fragment thereof. 197 is a non-toxic mutant of diphtheria toxin having the sequence of SEQ ID NO: 48, which has no toxic activity due to a single base substitution (glycine to glutamic acid at position 52).

[0182] SEQ ID NO:48 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0183] In one embodiment, at least one RNA molecule comprises a CRM 197 In one embodiment, the CRM comprises an RNA sequence encoding a variant of SEQ ID NO: 48, wherein the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO: 48. 197 This variant contains a glycine to glutamic acid mutation at position 52 (SEQ ID NO: 48) and is therefore non-toxic.

[0184] In one embodiment, at least one RNA molecule comprises a full-length CRMP 197 The RNA sequence encoding the

[0185] In one embodiment, at least one RNA molecule comprises a CRM 197RNA sequences encoding fragments of the CRM, e.g., fragments comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids (preferably contiguous amino acids) from SEQ ID NO:48. 197 Specific examples of fragments of are listed herein.

[0186] In one embodiment, the CRM 197 The RNA sequence encoding comprises or consists of SEQ ID NO:49, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:49.

[0187] SEQ ID NO:49

[0188] In one embodiment, at least one RNA molecule comprises or consists of a CRM from amino acid 299 to amino acid 312 of SEQ ID NO: 48. 197 The RNA sequence encoding the fragment of CRM consisting of amino acid 299 to amino acid 312 of SEQ ID NO: 48 is included. 197 A fragment of CRM 197(299-312) and consists of the sequence of SEQ ID NO: 50 (KTTAALSILPGIGS).

[0189] In one embodiment, an RNA molecule of the invention comprises an RNA sequence encoding a variant of SEQ ID NO:50, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:50.

[0190] In one embodiment, at least one RNA molecule comprises a CRM comprising or consisting of at least about 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids (preferably consecutive amino acids) of SEQ ID NO:50. 197(299-312) The present invention includes an RNA sequence encoding a fragment of the above.

[0191] In one embodiment, the RNA sequence encoding at least one T cell epitope, or a peptide or protein comprising at least one T cell epitope, is 197(299-312) The CRM comprises or consists of a sequence encoding the CRM. 197(299-312) An example of an RNA sequence encoding the ribozyme comprises or consists of SEQ ID NO:51, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:51 (AAGACCACCGCCGCCCUGAGCAUCCUGCCCGGCAUCGGCAGC).

[0192] In one embodiment, at least one RNA molecule comprises or consists of a CRM comprising amino acids 425 to 440 of SEQ ID NO: 48. 197The fragment of CRM197 consisting of amino acid 425 to amino acid 440 of SEQ ID NO: 48 contains an RNA sequence encoding a fragment of CRM 197(425-440) and consists of the sequence of SEQ ID NO: 52 (TPLPIAGVLLPTIPGK).

[0193] In one embodiment, an RNA molecule of the invention comprises an RNA sequence encoding a variant of SEQ ID NO:52, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:52.

[0194] In one embodiment, at least one RNA molecule comprises a CRM comprising or consisting of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids (preferably consecutive amino acids) of SEQ ID NO:52. 197(425-440) The present invention includes an RNA sequence encoding a fragment of the above.

[0195] In one embodiment, the RNA sequence encoding at least one T cell epitope, or a peptide or protein comprising at least one T cell epitope, is 197(425-440) The CRM comprises or consists of a sequence encoding the CRM. 197(425-440) An example of an RNA sequence encoding the above comprises or consists of SEQ ID NO:53, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:53 (ACCCCCCUGCCCAUCGCCGGCGUGCUGCUGCCCACCAUCCCCGGCAAG).

[0196] In one embodiment, at least one RNA molecule comprises or consists of a CRM comprising amino acids 300 to 450 of SEQ ID NO:48. 197 The RNA sequence encoding the fragment of the CRM consisting of amino acids 300 to 450 of SEQ ID NO: 48 is included. 197 A fragment of CRM 197(300-450) and consists of the sequence set forth in SEQ ID NO:46.

[0197] SEQ ID NO:46 TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHI

[0198] In one embodiment, an RNA molecule of the invention comprises an RNA sequence encoding a variant of SEQ ID NO:46, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:46.

[0199] In one embodiment, at least one RNA molecule comprises a CRM comprising or consisting of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 or 145 amino acids (preferably consecutive amino acids) of SEQ ID NO:46. 197(300-450) The present invention includes an RNA sequence encoding a fragment of the above.

[0200] In one embodiment, the RNA sequence encoding at least one T cell epitope, or a peptide or protein comprising at least one T cell epitope, is 197(300-450) The CRM comprises or consists of a sequence encoding the CRM. 197(300-450) An example of an RNA sequence encoding the above comprises or consists of SEQ ID NO:47, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:47.

[0201] SEQ ID NO:47 ACCGCCGCCCUGAGCAUCCUGCCCGGCAUCGGCAGCGUGAUGGGCAUCGCCGACGGCGCCGUGCACCACAACACCGAGGAGAUCGUGGCCCAGAGCAUCGCCCUGAGCAGCCUGAUGGUGGCCCAGGCCAUCCCCCUGGUGGGCGAGCUGGUGGACAUCGGCUUCGCCGCCUACAACUUCGUGGAGAGCAUCAUCAACCUGUUCCAGGUGGUGCACAACAGCUAC AACAGGCCCGCCUACAGCCCCGGCCACAAGACCCAGCCCUUCCUGCACGACGGCUACGCCGUGAGCUGGAACACCGUGGAGGACAGCAUCAUCAGGACCGGCUUCCAGGGCGAGAGCGGCCACGACAUCAAGAUCACCGCCGAGAACACCCCCCUGCCCAUCGCCGGCGUGCUGCUGCCCACCAUCCCCGGCAAGCUGGACGUGAACAAGAGCAAGACCCACAUC

[0202] In one embodiment, at least one RNA molecule comprises an RNA sequence encoding TpD, which contains both diphtheria and tetanus epitopes and optionally a cathepsin cleavage site.

[0203] In one embodiment, the TpD comprises or consists of the sequence of SEQ ID NO: 54 (ILMQYIKANSKFIGIPMGLPQSIALSSLMVAQ).

[0204] In one embodiment, at least one RNA molecule comprises an RNA sequence encoding a variant of SEQ ID NO:54, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:54.

[0205] In one embodiment, at least one RNA molecule comprises an RNA sequence encoding a full-length TpD. An example of an RNA sequence encoding a full-length TpD is an RNA sequence comprising or consisting of SEQ ID NO:55, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:55.

[0206] SEQ ID NO:55 AUCCUGAUGCAGUACAUCAGGCCAACAGCAAGUUCAUCGGCAUCCCCAUGGGCCUGCCCCAGAGCAUCGCCCUGAGCAGCCUGAUGGUGGCCCAG

[0207] In one embodiment, at least one RNA molecule comprises an RNA sequence encoding a fragment of TpD, such as a fragment comprising at least about 5, 10, 15, 20, 25 or 30 amino acids (preferably consecutive amino acids) from SEQ ID NO:54.

[0208] In one embodiment, at least one RNA molecule of the invention comprises an RNA sequence encoding a "p2-spacer-p30" construct. P2-spacer-p30 is a peptide that comprises two epitopes of tetanus toxin (TT), namely peptide P2 and peptide P30, and at least one spacer.

[0209] In one embodiment, the spacer comprises 1 to 20 amino acids. In one embodiment, the spacer consists of 2 amino acids. In one embodiment, the spacer consists of RR.

[0210] In one embodiment, p2-spacer-p30 comprises or consists of a peptide "p2TT" consisting of the sequence of SEQ ID NO: 63, a peptide "p30TT" consisting of the sequence of SEQ ID NO: 62, and optionally a spacer (e.g., RR, etc.).

[0211] In one embodiment, p2-spacer-p30 comprises or consists of the construct p2-RR-p30, i.e. the construct comprises or consists of the peptides p2TT and p30TT and spacer RR, having or comprising the sequence of SEQ ID NO: 56 or a variant thereof, preferably having at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO: 56. p2-RR-p30 construct.

[0212] SEQ ID NO:56 QYIKANSKFIGITERRFNNFTVSFWLRVPKVSASHLEQY

[0213] In one embodiment, the at least one T cell epitope, or a peptide or protein or fragment thereof comprising at least one T cell epitope, is full length p2-RR-p30.

[0214] In one embodiment, the at least one T cell epitope, or a peptide or protein or fragment thereof comprising at least one T cell epitope, is a fragment of p2-RR-p30 comprising at least about 5, 10, 15, 20, 25, 30, 32, 35 or 37 amino acids (preferably consecutive amino acids) from SEQ ID NO:56.

[0215] In one embodiment, the RNA molecule of the present invention comprises an RNA sequence encoding p2-RR-p30. An example of an RNA sequence encoding p2-RR-p30 is SEQ ID NO:57, or a sequence comprising or consisting of a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:57.

[0216] SEQ ID NO:57 CAGUACAUCAAGGCCAACAGCAAGUUCAUCGGCAUCACCGAGAGGAGGUUCAACAACUUCACCGUGAGCUUCUGGCUGAGGGUGCCCAAGGUGAGCGCCAGCCACCUGGAGCAGUAC

[0217] In one embodiment, at least one RNA molecule of the invention comprises an RNA sequence encoding one or more universal CD4 polyepitopes. In one embodiment, at least one RNA molecule of the invention comprises an RNA sequence encoding at least two universal CD4 polyepitopes, optionally linked by a spacer(s). Examples of universal CD4 polyepitopes include, but are not limited to, peptides having the sequences of SEQ ID NOs: 58-67 (Table 1). Examples of spacers are provided herein.

[0218] Table 1: Amino acid sequences of the universal CD4 polyepitope [Table 1]

[0219] In one embodiment, at least one T cell epitope is a variant of a sequence selected from SEQ ID NOs: 58-67, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NOs: 58-67.

[0220] In one embodiment, the at least one T cell epitope is a full-length universal CD4 protein polyepitope disclosed in Table 1.

[0221] In one embodiment, at least one T cell epitope is a fragment of a universal CD4 polyepitope comprising at least about 5, 6, 7, 8, 9, 10, 11, or 12 amino acids (preferably consecutive amino acids) of SEQ ID NOs: 58-60, 63, and 65-67, or a fragment of a universal CD4 polyepitope comprising at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids (preferably consecutive amino acids) of SEQ ID NOs: 61-62 and 64.

[0222] In one embodiment, the RNA sequence encoding at least one T cell epitope comprises or consists of a sequence encoding at least two universal CD4 polyepitopes, optionally linked by a spacer(s). Examples of RNA sequences encoding universal CD4 polyepitopes include, but are not limited to, RNA sequences comprising or consisting of the sequences of SEQ ID NOs: 68-77 (Table 2).

[0223] Table 2: Nucleotide sequence of the universal CD4 polyepitope [Table 2]

[0224] In one embodiment, an RNA molecule of the invention comprises an RNA sequence encoding an amino acid sequence comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 universal CD4 polyepitopes of Table 1, optionally linked by spacers.

[0225] In one embodiment, at least one RNA molecule of the invention comprises an RNA sequence encoding at least two identical or different universal CD4 polyepitopes.

[0226] In one embodiment, the RNA molecule of the invention comprises an RNA sequence encoding a recombinant peptide or protein that comprises or consists of at least one T cell epitope, such as N10, N19, which comprise or consist of 10 or 19 human CD4+ T cell epitopes (respectively) derived from an antigen from a pathogen, including but not limited to, TT and proteins from Plasmodium falciparum, influenza virus and Hepatitis B virus (listed in Table 1).

[0227] In one embodiment, at least one RNA molecule of the invention comprises an RNA sequence encoding ten universal CD4 epitopes (herein referred to as N10) as described herein, optionally the universal CD4 polyepitope being linked by spacers, examples of which are listed herein.

[0228] A non-limiting example of an N10 construct is the peptide of SEQ ID NO:80.

[0229] SEQ ID NO:80 VSIDKFRIFCKANPKKGLKFIIKRYTPNNEIDSKGIREDNNITLKLDRCNNKGEKKIAKMEKASSVFNVVNKGFNNFTVSFWLRVPKVSASHLEKGQYIKANSKFIGITEKGPHHTALRQAILCWGELMTLAKGPKYVKQNTLKLATKGFFLLTRILTIPQSLDKGYSGPLKAEIAQRLEDV

[0230] In one embodiment, the RNA molecule comprises an RNA sequence encoding a variant of SEQ ID NO:80, wherein said variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:80.

[0231] In one embodiment, the RNA molecule comprises an RNA sequence encoding the full-length N10 construct. In another embodiment, the RNA molecule comprises an RNA sequence encoding a fragment of N10, such as a fragment comprising at least about 10, 15, 20, 25, 50, 75, 100, 125, 150, or 175 amino acids (preferably consecutive amino acids) from SEQ ID NO:80.

[0232] In one embodiment, the RNA sequence encoding at least one T cell epitope comprises or consists of a sequence encoding N10. An example of an RNA sequence encoding N10 is a sequence comprising or consisting of SEQ ID NO:81, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:81.

[0233] SEQ ID NO:81 GUGAGCAUCGACAAGUUCAGGAUCUUCUGCAAGGCCAACCCCAAGAAGGGCCUGAAGUUCAUCAUCAAGAGGUACACCCCCAACAACGAGAUCGACAGCAAGGGCAUCAGGGAGGACAACAACAUCACCCUGAAGC UGGACAGGUGCAACAACAAGGGCGAAAAGAAGAUCGCCAAGAUGGAGAAGGCCAGCAGCGUGUUCAACGUGGUGAACAAGGGCUUCAACAACUUCACCGUGAGCUUCUGGCUGAGGGUGCCCAAGGUGAGCGCCAGC CACCUGGAGAAGGGCCAGUACAUCAAGGCCAACAGCAAGUUCAUCGGCAUCACCGAAAAGGGCCCCCACCACACCGCCCUGAGGCAGGCCAUCCUGUGCUGGGGCGAGCUGAUGACCCUGGCCAAGGGCCCCAAGU ACGUGAAGCAGAACACCCUGAAGCUGGCCACCAAGGGCUUCUUCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGACAAGGGCUACAGCGGCCCCCUGAAGGCCGAGAUCGCCCAGAGGCUGGAGGACGUG

[0234] In one embodiment, at least one RNA molecule of the invention comprises an RNA sequence encoding the 19 universal CD4 epitopes described herein (referred to herein as N19), optionally the universal CD4 polyepitopes being linked by spacers, examples of which are listed herein.

[0235] A non-limiting example of an N19 construct is the peptide of SEQ ID NO:82.

[0236] SEQ ID NO:82 VSIDKFRIFCKANPKKGLKFIIKRYTPNNEIDSKGIREDNNITLKLDRCNNKGEKKIAKMEKASSVFNVVNKGFNNFTVSFWLRVPKVSASHLEKGQYIKANSKFIGITEKGPHHTALRQAILCWGELMTLAKGPKYVKQNTLKLATKGFFLLTRILTIPQSLDKGYSGPLKAEI AQRLEDVKGVSIDKFRIFCKANPKKGLKFIIKRYTPNNEIDSKGIREDNNITLKLDRCNNKGEKKIAKMEKASSVFNVVNKGFNNFTVSFWLRVPKVSASHLEKGQYIKANSKFIGITEKGPHHTALRQAILCWGELMTLAKGPKYVKQNTLKLATKGFFLLTRILTIPQSLDKG

[0237] In one embodiment, the RNA molecule comprises an RNA sequence encoding a variant of SEQ ID NO:82, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:82.

[0238] In one embodiment, the RNA molecule comprises an RNA sequence encoding a full-length N19 construct. In another embodiment, the RNA molecule comprises an RNA sequence encoding a fragment of N19, such as a fragment comprising at least about 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, or 330 amino acids (preferably consecutive amino acids) from SEQ ID NO:82.

[0239] In one embodiment, the RNA sequence encoding at least one T cell epitope comprises or consists of a sequence encoding N19. An example of an RNA sequence encoding N19 is a sequence comprising or consisting of SEQ ID NO:83, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO:83.

[0240] SEQ ID NO:83

[0241] In one embodiment, at least one RNA molecule of the present invention comprises at least one RNA sequence encoding a spacer. In one embodiment, the cytokine is linked to at least one T cell epitope by a spacer. In one embodiment, the cytokine is linked to at least one T cell epitope by one or more spacers (e.g., 1, 2, 3, 4 or 5). A spacer may also be present to link T cell epitopes or cytokine fragments.

[0242] In one embodiment, the spacer is cleavable. In one embodiment, the spacer is cleavable, for example, by a protease, such as a cathepsin.

[0243] In one embodiment, the spacer is not cleavable.

[0244] Examples of spacers include, but are not limited to, PMGLP (SEQ ID NO: 86), a cathepsin cleavage site, an amino acid doublet, GP, LG, GS, GPGPG (SEQ ID NO: 45), GGSGGGGSGG (SEQ ID NO: 98), (GGGGS) n (wherein n ranges from 1 to 4 (SEQ ID NO: 139) n )), ASG, KG, and RR.

[0245] In one embodiment, the spacer comprises or consists of the amino acid sequence of PMGLP (SEQ ID NO:86), which may be encoded by the nucleic acid sequence of SEQ ID NO:87 (CCCAUGGGCCUGCCC), or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90% or 99% identity to SEQ ID NO:87.

[0246] In one embodiment, a short oligo- or polypeptide spacer, for example having a length in the range of 2 to 10 amino acids, may form the spacer. In one embodiment, the linker consists of 2, 3, 4, or 5 amino acids.

[0247] Examples of short spacers include, but are not limited to, GP, LG, ASG, KG, and RR.

[0248] In one embodiment, each of the short spacers GP, LG, ASG, RR and KG can be encoded by an RNA sequence listed in Table 3.

[0249] Table 3: Spacer amino acid sequences [Table 3]

[0250] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-4 (preferably having the sequence of SEQ ID NO: 1) and CRM197 (SEQ ID NO: 48), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 103). In one embodiment, at least one RNA molecule of the invention encodes, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 103, human IL-4, preferably having the sequence of SEQ ID NO: 1, a GP spacer, and a CRM197, preferably having the sequence of SEQ ID NO: 48. 197 The nucleic acid sequence encoding the nucleic acid sequence of the present invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of:

[0251] In one embodiment, the at least one RNA molecule comprises human IL-4 (preferably the RNA sequence of SEQ ID NO: 2), and CRM 197(preferably the RNA sequence of SEQ ID NO: 49), and optionally an RNA sequence encoding a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 104). In one embodiment, an RNA molecule of the invention comprises from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 104, an RNA sequence encoding human IL-4, preferably having the sequence of SEQ ID NO: 2, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and a CRMP, preferably having the sequence of SEQ ID NO: 49. 197 , comprising or consisting of an RNA sequence encoding the

[0252] In one embodiment, at least one RNA molecule of the invention comprises human IL-4 (preferably having the sequence of SEQ ID NO: 1) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0253] In one embodiment, at least one RNA molecule of the invention comprises human IL-4 (preferably having the sequence of SEQ ID NO: 1) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0254] In one embodiment, at least one RNA molecule of the invention comprises human IL-4 (preferably having the sequence of SEQ ID NO: 1) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0255] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-4 (preferably having the sequence of SEQ ID NO: 1) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 103). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 103, human IL-4, preferably having the sequence of SEQ ID NO: 1, a GP spacer, and TpD, preferably having the sequence of SEQ ID NO: 54.

[0256] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding human IL-4 (preferably the RNA sequence of SEQ ID NO: 2), and TpD (preferably the RNA sequence of SEQ ID NO: 55), and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 104). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 104, an RNA sequence encoding human IL-4, preferably having the sequence of SEQ ID NO: 2, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding TpD, preferably having the sequence of SEQ ID NO: 55.

[0257] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-4 (preferably having the sequence of SEQ ID NO:1) and p2-RR-p30(TT) (SEQ ID NO:56), and optionally a spacer.

[0258] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of human IL-4 (preferably having the sequence of SEQ ID NO:1) and an N10 construct (preferably having the sequence of SEQ ID NO:80), and optionally a spacer.

[0259] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-4 (preferably having the sequence of SEQ ID NO: 1) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 103). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 103, human IL-4, preferably having the sequence of SEQ ID NO: 1, a GP spacer, and an N19 construct, preferably having the sequence of SEQ ID NO: 82.

[0260] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding human IL-4 (preferably the RNA sequence of SEQ ID NO: 2), and an N19 construct (preferably having the sequence of SEQ ID NO: 83) and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 104). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 104, an RNA sequence encoding human IL-4, preferably having the sequence of SEQ ID NO: 2, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding an N19 construct, preferably having the sequence of SEQ ID NO: 83.

[0261] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-4 (preferably having the sequence of SEQ ID NO: 1) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), optionally with a spacer.

[0262] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and a CRMP. 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0263] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and a CRMP. 197(299-312)(SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0264] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and a CRMP. 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0265] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and a CRMP. 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0266] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0267] In one embodiment, at least one RNA molecule of the invention encodes a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0268] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0269] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 3, 4, 5, or 6) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0270] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 3, 4, 5, or 6) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), and optionally a spacer.

[0271] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and CRM 197 (SEQ ID NO: 48), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide, preferably having the sequence of SEQ ID NO: 140. In one embodiment, at least one RNA molecule of the invention encodes, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 140, murine IL-4, preferably having the sequence of SEQ ID NO: 11, a GP spacer, and a CRMP, preferably having the sequence of SEQ ID NO: 48.197 The nucleic acid sequence encoding the nucleic acid sequence of the present invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of:

[0272] In one embodiment, the at least one RNA molecule comprises mouse IL-4 (preferably the RNA sequence of SEQ ID NO: 12), and CRM 197 (preferably the RNA sequence of SEQ ID NO: 49), and optionally an RNA sequence encoding a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 141). In one embodiment, an RNA molecule of the invention comprises from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 141, an RNA sequence encoding murine IL-4, preferably having the sequence of SEQ ID NO: 12, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and a CRMP, preferably having the sequence of SEQ ID NO: 49. 197 The nucleic acid sequence comprises or consists of an RNA sequence encoding

[0273] In one embodiment, at least one RNA molecule of the invention comprises or consists of the sequence of SEQ ID NO:146.

[0274] In one embodiment, at least one RNA molecule is a variant of SEQ ID NO: 146, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to SEQ ID NO: 146 from which it is derived.

[0275] SEQ ID NO:146

[0276] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0277] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0278] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0279] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 140). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 140, mouse IL-4, preferably having the sequence of SEQ ID NO: 11, a GP spacer, and TpD, preferably having the sequence of SEQ ID NO: 54.

[0280] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding mouse IL-4 (preferably the RNA sequence of SEQ ID NO: 12), and TpD (preferably the RNA sequence of SEQ ID NO: 55), and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 141). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 141, an RNA sequence encoding mouse IL-4, preferably having the sequence of SEQ ID NO: 12, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding TpD, preferably having the sequence of SEQ ID NO: 55.

[0281] In one embodiment, at least one RNA molecule of the invention comprises or consists of the sequence of SEQ ID NO:144.

[0282] In one embodiment, at least one RNA molecule is a variant of SEQ ID NO: 144, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO: 144 from which it is derived.

[0283] SEQ ID NO:144 AUGGGCCUGAACCCCCAGCUGGUGGUGAUCCUGCUGUUCUUCCUGGAGUGCACCAGGAGCCACAUCCACGGCUGCGACAAGAACCACCUGAGGGAGAUCAUCGGCAUCCUGAACGAGGUGACCGGCGAGGG CACCCCCUGCACCGAGAUGGACGUGCCCAACGUGCUGACCGCCACCAAGAACACCACCGAGAGCGAGCUGGUGUGCAGGGCCAGCAAGGUGCUGAGGAUCUUCUACCUGAAGCACGGCAAGACCCCCUGCC UGAAGAAGAACAGCAGCGUGCUGAUGGAGCUGCAGAGGCUGUUCAGGGCCUUCAGGUGCCUGGACAGCAGCAUCAGCUGCACCAUGAACGAGAGCAAGAGCACCAGCCUGAAGGACUUCCUGGAGAGCCUG AAGAGCAUCAUGCAGAUGGACUACAGCGGCCCCAUCCUGAUGCAGUACAUCAAGGCCAACAGCAAGUUCAUCGGCAUCCCCAUGGGCCUGCCCCAGAGCAUCGCCCUGAGCAGCCUGAUGGUGGCCCAGUAG

[0284] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of mouse IL-4 (preferably having the sequence of SEQ ID NO:11) and p2-RR-p30(TT) (SEQ ID NO:56), and optionally a spacer.

[0285] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0286] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of mouse IL-4 (preferably having the sequence of SEQ ID NO: 11) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 140). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 140, mouse IL-4, preferably having the sequence of SEQ ID NO: 11, a GP spacer, and an N19 construct, preferably having the sequence of SEQ ID NO: 82.

[0287] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding mouse IL-4 (preferably the RNA sequence of SEQ ID NO: 12), and an N19 construct (preferably having the sequence of SEQ ID NO: 83) and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 141). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 141, an RNA sequence encoding mouse IL-4, preferably having the sequence of SEQ ID NO: 12, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding an N19 construct, preferably having the sequence of SEQ ID NO: 83.

[0288] In one embodiment, at least one RNA molecule of the invention comprises or consists of the sequence of SEQ ID NO:145.

[0289] In one embodiment, at least one RNA molecule is a variant of SEQ ID NO: 145, where the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO: 145 from which it is derived.

[0290] SEQ ID NO:145

[0291] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of murine IL-4 (preferably having the sequence of SEQ ID NO: 11) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), optionally with a spacer.

[0292] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 13, 14, 15, or 16) and a CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0293] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 13, 14, 15, or 16) and a CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0294] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 13, 14, 15, or 16) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0295] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 13, 14, 15, or 16) and a CRM 197(300-450)(SEQ ID NO: 46), and optionally a spacer.

[0296] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 13, 14, 15, or 16) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0297] In one embodiment, at least one RNA molecule of the invention encodes a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 13, 14, 15, or 16) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0298] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 13, 14, 15, or 16) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0299] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 13, 14, 15, or 16) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0300] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 13, 14, 15, or 16) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), and optionally a spacer.

[0301] In one embodiment, at least one RNA molecule of the invention comprises canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0302] In one embodiment, at least one RNA molecule of the invention comprises canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0303] In one embodiment, at least one RNA molecule of the invention comprises canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0304] In one embodiment, at least one RNA molecule of the invention comprises canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0305] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0306] In one embodiment, at least one RNA molecule of the present invention encodes canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0307] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0308] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0309] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of canine IL-4 (preferably having the sequence of SEQ ID NO: 21) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), optionally with a spacer.

[0310] In one embodiment, at least one RNA molecule of the present invention comprises a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 94, 95, 96, or 97) and a CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0311] In one embodiment, at least one RNA molecule of the present invention comprises a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 94, 95, 96, or 97) and a CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0312] In one embodiment, at least one RNA molecule of the present invention comprises a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 94, 95, 96, or 97) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0313] In one embodiment, at least one RNA molecule of the present invention comprises a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 94, 95, 96, or 97) and a CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0314] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 94, 95, 96, or 97) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0315] In one embodiment, at least one RNA molecule of the present invention encodes a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 94, 95, 96, or 97) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0316] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 94, 95, 96, or 97) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0317] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 94, 95, 96, or 97) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0318] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 94, 95, 96, or 97) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), and optionally a spacer.

[0319] In one embodiment, at least one RNA molecule of the invention comprises equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0320] In one embodiment, at least one RNA molecule of the invention comprises equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0321] In one embodiment, at least one RNA molecule of the invention comprises equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0322] In one embodiment, at least one RNA molecule of the invention comprises equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0323] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0324] In one embodiment, at least one RNA molecule of the invention encodes equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0325] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0326] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0327] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-4 (preferably having the sequence of SEQ ID NO: 99) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), optionally with a spacer.

[0328] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 109, 110, 111, 112) and a CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0329] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 109, 110, 111, 112) and a CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0330] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 109, 110, 111, 112) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0331] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 109, 110, 111, 112) and a CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0332] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 109, 110, 111, 112) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0333] In one embodiment, at least one RNA molecule of the invention encodes a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 109, 110, 111, 112) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0334] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 109, 110, 111, 112) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0335] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-4 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 109, 110, 111, 112) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0336] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-4 (preferably having or comprising a sequence selected from SEQ ID NOs: 109, 110, 111, 112) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), and optionally a spacer.

[0337] In one embodiment, at least one RNA molecule of the invention comprises human IL-13 (preferably having the sequence of SEQ ID NO: 23) and CRM 197 (SEQ ID NO: 48), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide, preferably having the sequence of SEQ ID NO: 117. In one embodiment, at least one RNA molecule of the invention encodes, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 117, human IL-13, preferably having the sequence of SEQ ID NO: 23, a GP spacer, and a CRM, preferably having the sequence of SEQ ID NO: 48. 197 The nucleic acid sequence encoding the nucleic acid sequence of the present invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of:

[0338] In one embodiment, the at least one RNA molecule comprises human IL-13 (preferably the RNA sequence of SEQ ID NO: 24), and CRM 197(preferably the RNA sequence of SEQ ID NO: 49), and optionally an RNA sequence encoding a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 118). In one embodiment, an RNA molecule of the invention comprises from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 118, an RNA sequence encoding human IL-13, preferably having the sequence of SEQ ID NO: 24, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding a CRMP, preferably having the sequence of SEQ ID NO: 49. 197 The nucleic acid sequence comprises or consists of an RNA sequence encoding

[0339] In one embodiment, at least one RNA molecule of the invention comprises human IL-13 (preferably having the sequence of SEQ ID NO: 23) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0340] In one embodiment, at least one RNA molecule of the invention comprises human IL-13 (preferably having the sequence of SEQ ID NO: 23) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0341] In one embodiment, at least one RNA molecule of the invention comprises human IL-13 (preferably having the sequence of SEQ ID NO: 23) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0342] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-13 (preferably having the sequence of SEQ ID NO: 23) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 117). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 117, human IL-13, preferably having the sequence of SEQ ID NO: 23, a GP spacer, and TpD, preferably having the sequence of SEQ ID NO: 54.

[0343] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding human IL-13 (preferably the RNA sequence of SEQ ID NO: 24), and TpD (preferably the RNA sequence of SEQ ID NO: 55), and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 118). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 118, an RNA sequence encoding human IL-13, preferably having the sequence of SEQ ID NO: 24, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding TpD, preferably having the sequence of SEQ ID NO: 55.

[0344] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of human IL-13 (preferably having the sequence of SEQ ID NO: 23) and p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0345] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of human IL-13 (preferably having the sequence of SEQ ID NO: 23) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0346] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-13 (preferably having the sequence of SEQ ID NO: 23) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 117). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide having the sequence of SEQ ID NO: 117, preferably human IL-13 having the sequence of SEQ ID NO: 23, a GP spacer, and an N19 construct, preferably having the sequence of SEQ ID NO: 82.

[0347] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding human IL-13 (preferably the RNA sequence of SEQ ID NO: 24), and an N19 construct (preferably having the sequence of SEQ ID NO: 83) and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 118). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 118, an RNA sequence encoding human IL-13, preferably having the sequence of SEQ ID NO: 24, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding an N19 construct, preferably having the sequence of SEQ ID NO: 83.

[0348] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of human IL-13 (preferably having the sequence of SEQ ID NO: 23) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), optionally with a spacer.

[0349] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 25, 26, 27 or 28) and a CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0350] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 25, 26, 27 or 28) and a CRM 197(299-312)(SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0351] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 25, 26, 27 or 28) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0352] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 25, 26, 27 or 28) and a CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0353] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 25, 26, 27 or 28) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0354] In one embodiment, at least one RNA molecule of the invention encodes a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 25, 26, 27 or 28) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0355] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 25, 26, 27 or 28) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0356] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 25, 26, 27 or 28) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0357] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of human IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 25, 26, 27 or 28) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), and optionally a spacer.

[0358] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and CRM 197(SEQ ID NO: 48), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide, preferably having the sequence of SEQ ID NO: 142. In one embodiment, at least one RNA molecule of the invention encodes, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 142, murine IL-13, preferably having the sequence of SEQ ID NO: 33, a GP spacer, and a CRM, preferably having the sequence of SEQ ID NO: 48. 197 The nucleic acid sequence encoding the nucleic acid sequence of the present invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of:

[0359] In one embodiment, the at least one RNA molecule comprises mouse IL-13 (preferably the RNA sequence of SEQ ID NO: 34), and CRM 197 (preferably the RNA sequence of SEQ ID NO: 49), and optionally an RNA sequence encoding a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 143). In one embodiment, an RNA molecule of the invention comprises from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 143, an RNA sequence encoding murine IL-13, preferably having the sequence of SEQ ID NO: 34, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and a CRMP, preferably having the sequence of SEQ ID NO: 49. 197 The nucleic acid sequence comprises or consists of an RNA sequence encoding

[0360] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and CRM 197(299-312)(SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0361] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0362] In one embodiment, at least one RNA molecule of the invention comprises mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0363] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 142). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 142, mouse IL-13, preferably having the sequence of SEQ ID NO: 33, a GP spacer, and TpD, preferably having the sequence of SEQ ID NO: 54.

[0364] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding mouse IL-13 (preferably the RNA sequence of SEQ ID NO: 34), and TpD (preferably the RNA sequence of SEQ ID NO: 55), and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 143). In one embodiment, the RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 143, an RNA sequence encoding mouse IL-13, preferably having the sequence of SEQ ID NO: 34, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding TpD, preferably having the sequence of SEQ ID NO: 55.

[0365] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0366] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of murine IL-13 (preferably having the sequence of SEQ ID NO: 33) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0367] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of mouse IL-13 (preferably having the sequence of SEQ ID NO: 33) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer. In one embodiment, at least one RNA molecule encodes a GP spacer. In one embodiment, at least one RNA molecule further encodes a signal peptide (preferably having the sequence of SEQ ID NO: 142). In one embodiment, at least one RNA molecule of the invention encodes a protein construct (preferably a single chain protein construct) comprising or consisting of, from N-terminus to C-terminus, a signal peptide, preferably having the sequence of SEQ ID NO: 142, mouse IL-13, preferably having the sequence of SEQ ID NO: 33, a GP spacer, and an N19 construct, preferably having the sequence of SEQ ID NO: 82.

[0368] In one embodiment, at least one RNA molecule comprises or consists of an RNA sequence encoding mouse IL-13 (preferably the RNA sequence of SEQ ID NO: 34), and an N19 construct (preferably having the sequence of SEQ ID NO: 83) and optionally a spacer. In one embodiment, at least one RNA molecule further comprises a sequence encoding a GP spacer (preferably the RNA sequence GGCCCC). In one embodiment, at least one RNA molecule further comprises a sequence encoding a signal peptide (preferably the RNA sequence of SEQ ID NO: 143). In one embodiment, an RNA molecule of the invention comprises or consists of, from 5' to 3': an RNA sequence encoding a signal peptide, preferably having the sequence of SEQ ID NO: 143, an RNA sequence encoding mouse IL-13, preferably having the sequence of SEQ ID NO: 34, an RNA sequence encoding a GP spacer, preferably having the sequence GGCCCC, and an RNA sequence encoding an N19 construct, preferably having the sequence of SEQ ID NO: 83.

[0369] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of murine IL-13 (preferably having the sequence of SEQ ID NO: 33) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), optionally with a spacer.

[0370] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 35, 36, 37 or 38) and a CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0371] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 35, 36, 37 or 38) and a CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0372] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 35, 36, 37 or 38) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0373] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 35, 36, 37 or 38) and a CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0374] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 35, 36, 37 or 38) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0375] In one embodiment, at least one RNA molecule of the invention encodes a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 35, 36, 37 or 38) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0376] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 35, 36, 37 or 38) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0377] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 35, 36, 37 or 38) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0378] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of mouse IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 35, 36, 37 or 38) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), and optionally a spacer.

[0379] In one embodiment, at least one RNA molecule of the invention comprises canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0380] In one embodiment, at least one RNA molecule of the invention comprises canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0381] In one embodiment, at least one RNA molecule of the invention comprises canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0382] In one embodiment, at least one RNA molecule of the invention comprises canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0383] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0384] In one embodiment, at least one RNA molecule of the present invention encodes canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0385] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0386] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0387] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of canine IL-13 (preferably having the sequence of SEQ ID NO: 43) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58 to 67), optionally with a spacer.

[0388] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 119, 120, 121 or 122) and a CRM 197(SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0389] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 119, 120, 121 or 122) and a CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0390] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 119, 120, 121 or 122) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0391] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 119, 120, 121 or 122) and a CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0392] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 119, 120, 121 or 122) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0393] In one embodiment, at least one RNA molecule of the present invention encodes a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 119, 120, 121 or 122) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0394] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 119, 120, 121 or 122) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0395] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 119, 120, 121 or 122) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0396] In one embodiment, at least one RNA molecule of the present invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of canine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 119, 120, 121 or 122) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), and optionally a spacer.

[0397] In one embodiment, at least one RNA molecule of the invention comprises equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and CRM 197(SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0398] In one embodiment, at least one RNA molecule of the invention comprises equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0399] In one embodiment, at least one RNA molecule of the invention comprises equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0400] In one embodiment, at least one RNA molecule of the invention comprises equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0401] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0402] In one embodiment, at least one RNA molecule of the invention encodes equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0403] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0404] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0405] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of equine IL-13 (preferably having the sequence of SEQ ID NO: 101) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), optionally with a spacer.

[0406] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 129, 130, 131 or 132) and a CRM 197 (SEQ ID NO: 48), and optionally comprising or consisting of a spacer.

[0407] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 129, 130, 131 or 132) and a CRM 197(299-312) (SEQ ID NO:50), and optionally comprising or consisting of a spacer.

[0408] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 129, 130, 131 or 132) and a CRM 197(425-440) (SEQ ID NO:52), and optionally comprising or consisting of a spacer.

[0409] In one embodiment, at least one RNA molecule of the invention comprises a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 129, 130, 131 or 132) and a CRM 197(300-450) (SEQ ID NO: 46), and optionally a spacer.

[0410] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 129, 130, 131 or 132) and TpD (preferably having the sequence of SEQ ID NO: 54), and optionally a spacer.

[0411] In one embodiment, at least one RNA molecule of the invention encodes a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 129, 130, 131 or 132) and an amino acid sequence comprising or consisting of p2-RR-p30(TT) (SEQ ID NO: 56), and optionally a spacer.

[0412] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 129, 130, 131 or 132) and an N10 construct (preferably having the sequence of SEQ ID NO: 80), and optionally a spacer.

[0413] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NO: 129, 130, 131 or 132) and an N19 construct (preferably having the sequence of SEQ ID NO: 82), and optionally a spacer.

[0414] In one embodiment, at least one RNA molecule of the invention encodes an amino acid sequence comprising or consisting of a peptide comprising at least one epitope of equine IL-13 (preferably a peptide having or comprising a sequence selected from SEQ ID NOs: 129, 130, 131 or 132) and at least one CD4 polyepitope (preferably having a sequence selected from the group consisting of SEQ ID NOs: 58-67), and optionally a spacer.

[0415] In one embodiment, at least one RNA molecule of the present invention comprising a chemical structure capable of promoting stability and / or translation efficiency may also be used. In one embodiment, at least one RNA molecule preferably comprises a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR is 0-3000 nucleotides in length. The 5'UTR and 3'UTR may be the naturally occurring endogenous 5' and 3'UTR of the gene to be expressed (e.g., IL-4 or IL-13). Alternatively, UTR sequences that are not endogenous to the gene of interest may be added by incorporating UTR sequences in the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene to be expressed may be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3'UTR sequence may reduce mRNA stability.

[0416] In one embodiment, the 5'UTR may comprise a Kozak sequence of an endogenous gene. In one embodiment, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in the cell.

[0417] In one embodiment, at least one RNA molecule of the present invention has both a 5' cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in the cell.

[0418] In one embodiment, at least one RNA molecule of the present invention comprises a poly(A) tail. The poly(A) tail provides stability to RNAs and reduces their degradation. In general, the length of the poly(A) tail positively correlates with the stability of the RNA. In one embodiment, the poly(A) tail is 100-5000 adenosines.

[0419] Furthermore, attachment of different chemical groups to the 3' end can increase the stability of mRNA. Such attachments can include modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of RNA.

[0420] In one embodiment, at least one RNA molecule of the present invention comprises a 5' cap. A 5' cap on an RNA can provide stability to the RNA molecule.

[0421] In one embodiment, at least one RNA molecule of the present invention comprises an internal ribosome entry site (IRES) sequence, in particular a viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation.

[0422] GC content also participates in regulating the stability of mRNA or reducing secondary structure.Enriching GC content can optimize and increase mRNA stability and in vivo protein translation.In one embodiment, at least one RNA molecule of the present invention comprises optimized GC content.In one embodiment, at least one RNA molecule comprises high GC content.

[0423] In one embodiment, the RNA sequence is modified to replace at least one, and preferably all, uridine residues with a 1-methylpseudouridine residue.

[0424] In one embodiment, the RNA is formulated for delivery into a target cell, rather than being naked RNA.

[0425] The compositions may be delivered into target cells by well-known methods such as lipofection, sonoporation, microinjection, biolistics, virosomes, liposomes, lipid nanoparticles, immunoliposomes, polycation or lipid:nucleic acid conjugates, virus-like particles and artificial virions.

[0426] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acid into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, enclosed in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in a suspension in lipid, contained in or complexed with micelles, or otherwise associated with lipid. The lipid / RNA association composition is not limited to any particular structure in solution. For example, they can exist as micelles or in a bilayer structure with a "collapsed" structure. They can also simply be interspersed in the solution and possibly form aggregates that are not uniform in size or shape. Lipid is a fatty substance that can be naturally occurring lipid or synthetic lipid.For example, lipid includes the lipid droplets that naturally occur in cytoplasm, and the class of compounds that include long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.In one embodiment, delivery vehicle is lipid nanoparticles, which can be called LNP.

[0427] Lipids suitable for use are available from commercial sources, for example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO, dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY), cholesterol ("Choi") is available from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL).

[0428] "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components rearrange themselves before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers. However, compositions that have structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes and lipidots are also contemplated.

[0429] In one embodiment, the composition is a pharmaceutical composition and further comprises at least one pharma- ceutically acceptable excipient.Thus, the present invention further relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of at least one RNA molecule and at least one pharma- ceutically acceptable excipient, wherein the at least one RNA molecule encodes the amino acid sequence of at least one cytokine or cytokine fragment, at least one T-cell epitope, and optionally at least one spacer.

[0430] Pharmaceutically acceptable excipients that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g. sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0431] In one embodiment, the composition of the invention is a pharmaceutical product or for use as a pharmaceutical product.Thus, the present invention further relates to a pharmaceutical product comprising, consisting essentially of, or consisting of at least one RNA molecule, wherein at least one RNA molecule codes for at least one cytokine or cytokine fragment as described above, at least one T cell epitope, and optionally at least one spacer.

[0432] The present invention further relates to at least one RNA molecule for use as a medicament, wherein the at least one RNA molecule encodes at least one cytokine or cytokine fragment, at least one T cell epitope, and optionally at least one spacer.

[0433] As used herein, with respect to a pharmaceutical composition or medicament, the term "consisting essentially of" means that the composition or at least one RNA molecule of the invention is the only therapeutic agent or agent having biological activity within the pharmaceutical composition or medicament.

[0434] In one embodiment, the pharmaceutical composition or medicament of the invention comprises or consists essentially of at least one RNA molecule encoding IL-4 or a fragment thereof and at least one T cell epitope, optionally linked by at least one spacer.

[0435] In one embodiment, the pharmaceutical composition or medicament of the invention comprises or consists essentially of at least one RNA molecule encoding IL-13 or a fragment thereof and at least one T cell epitope, optionally linked by at least one spacer.

[0436] In one embodiment, the pharmaceutical composition or medicament of the invention comprises or consists essentially of at least one RNA molecule encoding both IL-4 and IL-13 (or fragments thereof) and at least one T cell epitope, optionally linked by a spacer. In one embodiment, the pharmaceutical composition or medicament of the invention comprises or consists essentially of (i) at least one RNA molecule encoding IL-4 (or a fragment thereof) and at least one T cell epitope, optionally linked by a spacer, and (ii) at least one RNA molecule encoding IL-13 (or a fragment thereof) and at least one T cell epitope, optionally linked by a spacer.

[0437] In one embodiment, the composition comprising at least one RNA molecule of the present invention is a vaccine composition.In one embodiment of the present invention, the vaccine composition of the present invention comprises at least one adjuvant.In one embodiment of the present invention, the vaccine composition of the present invention does not comprise any adjuvant.

[0438] The present invention further relates to formulations of the compositions, pharmaceutical compositions, medicaments or vaccines of the invention, which compositions, pharmaceutical compositions, medicaments or vaccines are adjuvanted. In one embodiment, the compositions, pharmaceutical compositions, medicaments or vaccines of the invention thus comprise one or more adjuvants.

[0439] In one embodiment, at least one RNA molecule, composition, pharmaceutical composition, or vaccine composition of the present invention may be administered (or be for administration) by injection, topically (e.g., by transdermal delivery), rectally, nasally, or vaginally.

[0440] In one embodiment, at least one RNA molecule, composition, pharmaceutical composition, medicament or vaccine composition of the present invention is in a form suitable for injection.Thus, in one embodiment, the composition, pharmaceutical composition, medicament or vaccine composition of the present invention is injected (or is injectable) into a subject by intramuscular, intraperitoneal or subcutaneous injection.

[0441] Examples of suitable forms for injection include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of injectable sterile solutions or dispersions.Prevention of microbial contamination can be achieved by adding preservatives, such as various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.), to the composition.In one embodiment, it may be preferable to include an isotonic agent, such as sugar or sodium chloride, to reduce pain during injection.In one embodiment, prolonged absorption of the injectable composition can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0442] In one embodiment, the freeze-dried vaccine composition of the present invention is dissolved in water for injection and mixed gently.The above-mentioned immune adjuvant is then added.The mixture is mixed gently and filled into a suitable syringe.The present invention therefore also relates to a medical device that includes a syringe filled or prefilled with the vaccine composition of the present invention.

[0443] In one embodiment, at least one RNA molecule, composition, pharmaceutical composition, medicament or vaccine composition of the present invention is in a form adapted for local administration. Examples of forms adapted for local administration include, but are not limited to, polymeric patches, or controlled release patches.

[0444] In another embodiment, at least one RNA molecule, composition, pharmaceutical composition, medicament or vaccine composition of the invention is in a form adapted for rectal administration. Examples of forms adapted for rectal administration include, but are not limited to, suppositories, microenemas, enemas, gels, bowel foams, creams, ointments, etc.

[0445] The present invention further relates to at least one RNA molecule of the invention, or a composition, pharmaceutical composition, medicament, or vaccine composition of the invention, for treating an inflammatory disorder in a subject.

[0446] Thus, the present invention further relates to a method for treating an inflammatory disorder in a subject, the method comprising administering to the subject at least one RNA molecule, composition, pharmaceutical composition, medicament or vaccine composition of the present invention.

[0447] The present invention further relates to a method for inducing an immune response to IL-4, IL-13, or both in a subject, the method comprising administering to the subject at least one RNA molecule, composition, pharmaceutical composition, medicament, or vaccine composition of the present invention.

[0448] The present invention further relates to a method for inducing in a subject the production of an antibody that inhibits or neutralizes the biological activity of IL-4, IL-13, or both, comprising administering to the subject at least one RNA molecule, composition, pharmaceutical composition, medicament, or vaccine composition of the invention. In one embodiment, the antibody is a polyclonal antibody.

[0449] In one embodiment, the subject is suffering from, preferably diagnosed with, an inflammatory disorder, particularly a disorder associated with aberrant IL-4 and / or IL-13 expression or activity.

[0450] In one embodiment, the subject is a human. Preferably, according to this embodiment, at least one cytokine included in the composition of the invention is human.

[0451] In one embodiment, the subject in need thereof is a livestock or animal of agricultural value, including, but not limited to, cattle, cows, bison, pigs, swine, sheep, goats, horses, donkeys, alpacas, llamas, deer, elk, moose, ostriches, emus, ducks, geese, chickens, partridges, quail, pheasants, minks, salmon, cod, catfish, herring, trout, bass, perch, flounder, etc. non-human animals, including fish (e.g., whales, sharks, tuna, crabs, lobsters, crayfish, snails, shellfish, oysters, etc.), companion animals (e.g., dogs, cats, rabbits, rodents, fish, snakes, etc.), and non-human primates (e.g., great apes, including chimpanzees, gorillas, and orangutans; lesser apes, including gibbons; Old World monkeys; New World monkeys; and prosimians, including tarsiers, lemurs, and lorises, etc.).

[0452] In one embodiment, the inflammatory disorder is a disorder associated with aberrant IL-4 and / or IL-13 expression or activity.

[0453] Examples of inflammatory disorders include, but are not limited to, asthma (either allergic or non-allergic), allergic conditions (e.g., food allergies, venom allergies, cat allergies, drug allergies, hyper-IgE syndrome, allergic rhinitis, allergic conjunctivitis, and allergic enterogastritis), atopic disorders (e.g., atopic dermatitis, urticaria (including chronic idiopathic urticaria and chronic spontaneous urticaria), eczema, and the like), bullous pemphigoid, respiratory disorders (allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), and the like), nasal polyposis and other conditions involving airway inflammation (e.g., eosinophilia, fibrosis, and excess mucus production, e.g., cystic fibrosis and pulmonary fibrosis, systemic sclerosis (SSc); inflammatory and / or autoimmune disorders or conditions, gastrointestinal disorders or conditions (e.g., inflammatory bowel disease (IBD) and eosinophilic esophagitis (EE), as well as eosinophil-mediated gastrointestinal diseases, ulcerative colitis, Crohn's disease, and systemic lupus erythematosus); systemic lupus erythematosus, liver disorders or conditions (e.g., cirrhosis, and hepatocellular carcinoma), scleroderma; fibrotic diseases or disorders (e.g., fibrosis of the liver (e.g., fibrosis caused by hepatitis B and / or C viruses), scleroderma; solid tumors or cancers such as leukemia (e.g., B-cell chronic lymphocytic leukemia), glioblastoma, lymphoma (e.g., Hodgkin's lymphoma), and mastocytosis.

[0454] In one embodiment, the inflammatory disorder is selected from the group including asthma (e.g., allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergies, nasal polyposis, and eosinophilic esophagitis.

[0455] In one embodiment, the inflammatory disorder is selected from the group including asthma (eg, allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and food allergies.

[0456] In one embodiment, the inflammatory disorder is allergy, asthma, or atopic dermatitis.

[0457] In one embodiment, the inflammatory disorder is allergic asthma.

[0458] In one embodiment, the inflammatory disorder is a solid tumor. In one embodiment, the method of the invention is for preventing metastasis from a solid tumor.

[0459] In one embodiment, the treatment consists of a single dose or multiple doses over a period of time.

[0460] In one embodiment of the invention, the subject to be treated is administered at least one therapeutically effective amount of the above composition.

[0461] In another embodiment of the present invention, the treated subject may be further administered a therapeutically effective amount of the composition described herein above when the amount of antibodies against IL-4 is undetectable in a serum sample obtained from the subject.

[0462] In another embodiment of the present invention, the treated subject may be further administered a therapeutically effective amount of the composition described herein above when the amount of antibodies against IL-13 is undetectable in a serum sample obtained from the subject.

[0463] In another embodiment of the present invention, the treated subject may be further administered a therapeutically effective amount of the composition described herein above when the amount of antibodies against IL-4 and IL-13 is undetectable in a serum sample obtained from the subject. [Brief description of the drawings]

[0464] [Figure 1] 1 is a map of an IL-4 RNA construct according to the invention.EP: epitope;EPX: multiple epitopes present (X epitopes present);FL: full length. [Diagram 2] 1 is a map of an IL-4 RNA construct according to the invention.EP: epitope;EPX: multiple epitopes present (X epitopes present);FL: full length. [Diagram 3]1 is a map of the IL-13 RNA construct of the invention. EP: epitope; EPX: multiple epitopes present (X epitopes present); FL: full length. [Figure 4] 1 is a map of the IL-13 RNA construct of the invention. EP: epitope; EPX: multiple epitopes present (X epitopes present); FL: full length. [Diagram 5] 1 is a composite of graphs showing in vitro protein expression of muIL4 and CRM in transfected HEK293 cells. Protein expression was measured by flow cytometry 24 hours after LNP / mRNA transfection. LNP / mRNA-muIL4 (white boxes), LNP / mRNA-CRM (hatched boxes), LNP / mRNA-muIL4-TpD (light grey boxes), LNP / mRNA-muIL4-N19 (dark grey) and LNP / mRNA-muIL4-CRM (black boxes) were tested. [Figure 5A] Graph showing percentage of cells expressing muIL4. [Figure 5B] Graph showing the percentage of cells expressing CRM. [Figure 5C] Graph showing median fluorescence intensity (MFI) of muIL4 in expressing cells (n=2) Mean range. [Figure 6] Figure 1 shows the in vitro activity of muIL4 in the supernatant of HEK293 cells transfected with at least one RNA construct of the invention. Protein activity was measured in a proliferation bioassay in HEK293 cell supernatant 24 hours after LNP / mRNA transfection. Supernatants of mRNA-muIL4 (white), mRNA-muIL4-TpD (light grey), mRNA-muIL4-CRM (black), mRNA-muIL4-N19 (dark grey) and mRNA-CRM (hatched circle) transfections were tested. Optical density (OD) at 490 nm is shown corresponding to sample dilution. (n=2) Mean range. [Figure 7]A scheme of the in vivo study is shown. BALB / c mice received three intramuscular (IM) immunizations with 10 μg LNP / mRNA (arrows) on D0, D7, and D28. Blood samples were taken at the induced time points. [Figure 8] Induction of neutralizing antibodies against muIL4 in serum samples after intramuscular injection of LNP / mRNA in BALB / c mice. BALB / c mice received three IM immunizations with 10 μg of mRNA-muIL4 (open circles), mRNA-CRM (hatched circles), mRNA-muIL4-TpD (light grey circles), mRNA-muIL4-N19 (dark grey circles), or mRNA-muIL4-CRM (filled circles) in LNP on D0, D7, and D28. Bars indicate median values. Neutralizing capacity as indicated by half-maximal neutralizing titers against muIL4 was measured on D21, D41, D62, and D80 after the first dose. For CRM, muIL4-N19, and muIL4-CRM, n=10 / group. For muIL4-TpD, n=9, for muIL4, n=2. [Figure 9] Animal survival curves after intramuscular injection of LNP / mRNA in BALB / c mice. BALB / c mice received three IM immunizations at D0, D7, and D28 with 10 μg of mRNA-muIL4 (stop line), mRNA-muIL4-TpD (gray line), mRNA-CRM, mRNA-muIL4-N19, and mRNA-muIL4-CRM (black line) in LNP. Kaplan-Meier survival curves are shown (n=10 / group).

[0465] Working Example RNA construction

[0466] Constructs according to the invention corresponding to an RNA molecule encoding IL-4 (full length or an epitope thereof), a spacer and at least one T cell epitope are shown in FIGS.

[0467] Table 4: RNA constructs encoding IL-4 full length cytokine [Table 4]

[0468] Table 5: RNA constructs encoding IL-4 epitopes [Table 5]

[0469] Constructs according to the invention corresponding to an RNA molecule encoding IL-13 (full length or an epitope thereof), a spacer and at least one T cell epitope are shown in FIGS.

[0470] Table 6: RNA constructs encoding IL-13 full length cytokine [Table 6]

[0471] Table 7: RNA constructs encoding IL-13 epitopes [Table 7]

[0472] Example 1 material and method mouse IL4RA / IL13 / IL4 humanized mouse strain (hIL-4 / hIL-13KI; hIL-4RαKI) and female BALB / cJRj mice were used for the following experiments (Conde et al., 2021).

[0473] Intranasal challenge with IL-4 and IL-13. hIL-4 / hIL-13KI;hIL-4RαKI mice were exposed to mRNA constructs of the present invention containing sequences encoding IL-4 or IL-13, or to PBS as a control. After sacrifice, analysis of BAL eosinophil counts was performed.

[0474] Quantification of mouse and human IL-4 and IL-13, and antibodies against T cell epitopes. The immunogenicity of the mRNA vaccine of the present invention was determined by assessing antibodies against mouse IL-4, human IL-4, mouse IL-13, human IL-13, and T cell epitope(s) in sera collected at different time points after vaccination using an ELISA system.

[0475] Assessment of neutralizing capacity against IL-4 and IL-13 in sera from vaccinated mice. The neutralizing capacity of anti-mouse IL-4, anti-mouse IL-13, anti-human IL-4 and anti-human IL-13 antibodies was assessed using, for example, a CTLL-2 cell proliferation assay (ECACC, ref. 93042610, batch number: 12K006) or a HEK-Blue IL-4 / IL-13 reporter gene cell line bioassay (In vivoGen, hkb-il413, batch number: X14-37-01), adapted from the manufacturer's instructions.

[0476] Example 2 material and method In vitro functionality of mRNA Three micrograms of mRNA encoding murine IL4 (mu-IL4), CRM, or the fusion proteins muIL4-GP-CRM, muIL4-GP-TpD, or muIL4-GP-N19 encapsulated in LNPs were diluted in OptiMEM serum-free medium to 6x10 5 After overnight incubation, cells were harvested, fixed, and permeabilized with an internal staining kit (Miltenyi). muIL4 and CRM 197 Intracellular expression of was assessed by staining cells with monoclonal anti-mouse IL-4PE conjugate (clone BVD4-1D11) and anti-diphtheria toxin IgG1 (clone 8G1) detected by anti-mouse IgG1-APC conjugate. Expression was assessed by flow cytometry.

[0477] In vitro activity of muIL4 in the supernatants of transfected HEK293 cells. The supernatant of HEK293 cells transfected with the mRNA of the present invention was collected 24 hours after transfection, and muIL4 proliferation activity was evaluated using the CTLL-2 cell assay. Briefly, CTLL-2 cells were grown in complete RPMI-1640 medium in the presence of 10 ng / mL human IL-2. For activity bioassay, human IL-2 was replaced with latent mu-IL4 present in the supernatant. Thus, latent mu-IL4 induces the growth of CTLL-2.

[0478] Supernatant samples were added into culture plates at selected dilutions and 2-fold serial dilutions in RPMI-1640 medium + FBS [10% (v / v)]. Mu-IL4 protein was used as a positive control with a standard curve starting at 10 ng / ml. 20,000 CTLL-2 cells were then added to all wells. After 48 hours of incubation, cell viability was quantified by MTS / PMS assay by adding 40 μl of substrate. Optical density (OD) was read at 490 nm after 4 hours of incubation.

[0479] In vivo generation of neutralizing antibodies against muIL4 after intramuscular injection of LNP / mRNA. Five-week-old female BALB / cByJ mice were obtained from Janviers labs (Saint Berthevin, France). Pre-challenge bleeds were performed 7 days before the first immunization. Animals were injected by intramuscular (IM) route with 50 μl of LNPs containing 10 μg of mRNA encoding muIL4, CRM, or the fusion proteins muIL4-GP-TpD, muIL4-GP-N19, and muIL4-GP-CRM on days 0, 7, and 28. Blood was collected on days 21, 41, 62, and 80. Neutralizing capacity against muIL-4 was evaluated using a bioassay test performed on CTLL-2 cells.

[0480] Briefly, cells were grown in the presence of human IL2, which was replaced with mu-IL4 for the neutralization bioassay. Thus, potential anti-muIL-4 neutralizing antibodies induced after immunization would prevent the growth of CTLL-2.

[0481] Serum samples were added at a final dilution of 1 / 200, and a positive control polyclonal anti-mu-IL4 antibody (AF-404-NA, Biotechne) was added at a final concentration of 1 μg / mL. All samples were serially diluted 2-fold in RPMI-1640 medium + FBS [10% (v / v)]. Mu-IL4 was added to serum samples and controls at 2 ng / mL and then incubated for 1 h at room temperature. 20,000 CTLL-2 cells were then added to the pre-incubated samples (positive control with serum or muIL-4). After 48 h of incubation, cell viability was quantified by MTS / PMS assay.

[0482] NC50 results were expressed as the serum dilution factor (dil-1) that neutralized 50% of mu-IL4 activity, whose maximum value was defined by incubation of CTLL-2 cells with 2 ng / mL mu-IL4. NC50 was determined by interpolating the serum dilution that resulted in 50% mu-IL4 activity.

[0483] Constructs Five different mRNA constructs were used. Murine IL-4 was expressed with three different T cell epitopes: TpD (SEQ ID NO: 144), N19 (SEQ ID NO: 145) and CRM 197 (SEQ ID NO: 146). RNA-IL4 (SEQ ID NO: 147) and RNA-CRM (SEQ ID NO: 148) were both used as controls.

[0484] SEQ ID NO:147 AUGGGCCUGAACCCCCAGCUGGUGUGAUCCUGCUGUUCUUCCUGGAGUGCACCAGGAGCCACAUCCACGGCUGCGACAAGAACCACCUGAGGGAGAUCAUCGGCAUCCUGAACGAGGUGACCGGCGAGGGCACCCCCUGCACCGAGAUGGACGUGCCCAACGUGCUGACCGCCCCAAGAACACCACCGAGAGCGAGCUGGUGCAGGG CCAGCAAGGUGCUGAGGAUCUUCUACCUGAAGCACGGCAAGACCCCGCCUGAAGAAGAACAGCAGCGUGCUGAUGGAGCUGCAGAGGCUGUUCAGGGCCUUCAGGUGCCUGGACAGCAGCAUCAGCUGCACCAUGAACGAGAGCAAGAGCACCAGCCUGAAGGACUUCCUGGAGGCCUGAAGACAUCAUGCAGAUGGACUACAGCUAG

[0485] அக்க்கு நுர்க்கு:148

[0486] result In vitro protein expression of muIL4 and CRM in transfected HEK293 cells

[0487] MuIL4 expression was observed in all tested conditions except for the condition transfected with mRNA-CRM alone (hatched boxes), where the percentage of cells expressing IL4 was greater than 80% (Figure 5A). CRM expression was observed in CRM and muIL4-CRM conditions (Figure 5B). The MFI of muIL4 in HEK293 cells was 2-4 times higher in muIL4-CRM (black boxes) compared to muIL4, muIL4-TpD, and muIL4-N19 (Figure 5C).

[0488] In vitro activity of muIL4 in the supernatant of transfected HEK293 cells As shown in Figure 6, the highest mu-IL-4 activity was observed in cells transfected with mRNA-IL4. The muIL4 activity measured in the supernatant of HEK293 cells transfected with mRNA-muIL4-N19, mRNA-muIL4-CRM, and mRNA-muIL4-TpD was lower. Finally, mu-IL4 activity was not detected in the supernatant of HEK293 cells transfected with mRNA-CRM. As observed by Western blot (data not shown), substantially equal amounts of mIL4 protein were quantified in the supernatant of transfected HEK293 cells in the different conditions (except for the mRNA-CRM condition). Thus, the in vitro differences in muIL4 activity observed in Figure 6 are not due to differences in the amount of muIL4 protein, but instead due to the lower activity of the protein obtained with the RNA construct of the present invention compared to native mouse IL4.

[0489] Analysis of neutralizing antibodies against muIL4 in serum samples after intramuscular injection of LNP / mRNA in BALB / c mice Five-week-old female BALB / c ByJ mice were injected by the intramuscular (IM) route with 50 μl of LNPs containing 10 μg of mRNA encoding muIL4, CRM, or the fusion proteins muIL4-GP-TpD, muIL4-GP-N19, and muIL4-GP-CRM on days 0, 7, and 28. Blood was collected on days 21, 41, 62, 80, and at the end of the study (day 104) (FIG. 7), and the presence of neutralizing antibodies was assessed in the serum of these mice.

[0490] Neutralizing antibodies against muIL4 were detected in mice immunized three times with 10 μg of mRNA encoding muIL4-TpD, muIL4-N19, and muIL4-CRM in LNP from day 41 onwards. On day 41, 7 / 9, 6 / 10, and 8 / 10 responses were detected in groups administered mRNA encoding muIL4-TpD, muIL4-N19, and muIL4-CRM in LNP, respectively (NC50>200 dil-1). On day 62, one additional mouse was found to be positive in the muIL4-CRM group, but the median values ​​were starting to decline in all groups, possibly indicating that the peak of the response was reached around day 41. Except for mice with high neutralizing capacity, NC50 did not decrease between days 62 and 80, and the medians were similar from day 62 to day 80, ranging from 726 to 775 in the muIL4-CRM group, 302 to 266 in the muIL4-N19 group, and 481 to 605 in the muIL4-TpD group. MuIL4 neutralizing antibodies were not detected in the muIL4 and CRM groups at any time point (Figure 8 and Table 8).

[0491] Table 8: Median NC50(Dil-1) of ability to neutralize muIL4 for CRM, muIL4, muIL4-CRM, muIL4-N19 and muIL4-CRM groups at days 21, 41, 62 and 80. [Table 8]

[0492] All animals injected with LNPs encapsulating mRNA-CRM, mRNA-muIL4-CRM, and mRNA-muIL4-N19 were alive at day 80 (100%). The survival rate of mice administered mRNA-muIL4-TpD in LNPs drops to 90% with the death of one isolated mouse. Finally, only 20% survival rate was observed in the group receiving mRNA-muIL4 in LNPs at day 80, with 60% of the mice dying after two rounds of immunization (D0 and D7) (Figure 9). This result indicates the toxicity of mRNA-muIL4, which was not observed with the mRNA constructs described in the present invention.

Claims

1. 1. A composition comprising at least one RNA molecule, said at least one RNA molecule comprising: at least one cytokine, or at least one fragment or epitope thereof, at least one T cell epitope, A composition encoding at least one amino acid sequence comprising:

2. The composition described in claim 1, wherein the at least one cytokine is interleukin-4 (IL-4) and / or interleukin-13 (IL-13).

3. The composition described in claim 1, wherein the at least one RNA molecule encodes at least one amino acid sequence further comprising at least one spacer.

4. The composition of claim 1, wherein the at least one cytokine is IL-4.

5. 3. The composition of claim 2, wherein the IL-4 fragment is selected from the group consisting of SEQ ID NOs: 7-10, 13-16, 94-97 and 109-112.

6. The composition of claim 1, wherein the at least one cytokine is IL-13.

7. 3. The composition of claim 2, wherein the IL-13 fragment is selected from the group consisting of SEQ ID NOs: 25-28, 35-38, 119-122 and 129-132.

8. 2. The composition of claim 1, wherein the at least one RNA molecule encodes IL-4 or at least one fragment or epitope thereof and IL-13 or at least one fragment or epitope thereof.

9. The at least one T cell epitope is a CRM １９７ , a combination of diphtheria and tetanus epitopes (TpD), an epitope of tetanus toxin (TT), a universal CD4 polyepitope, variants and fragments thereof.

10. The at least one spacer is selected from the group consisting of PMGLP (SEQ ID NO: 86), a cathepsin cleavage site, an amino acid doublet, GP, GPGPG (SEQ ID NO: 45), GGSGGGGGSGG (SEQ ID NO: 98), (GGGGGS) ｎ 4. The composition of claim 3, wherein the amino acid sequence is selected from the group consisting of ((SEQ ID NO: 139) n ), where n ranges from 1 to 4, LG, ASG, KG, and RR.

11. The composition of claim 1 , wherein the at least one RNA molecule is encapsulated.

12. The composition described in claim 1, wherein the at least one RNA molecule is encapsulated in a nanoparticle, a liposome, or a virus-like particle.

13. A pharmaceutical composition comprising the composition of claim 1 and at least one pharmaceutically acceptable excipient.

14. A vaccine composition comprising the composition of claim 1.

15. The vaccine composition of claim 14, further comprising at least one adjuvant.

16. A pharmaceutical comprising at least one RNA molecule, wherein the at least one RNA molecule is: at least one cytokine, or at least one fragment or epitope thereof, at least one T cell epitope, A pharmaceutical product encoding at least one amino acid sequence comprising:

17. 14. The pharmaceutical composition of claim 13 for use in treating an inflammatory disorder.

18. The pharmaceutical composition for use according to claim 17, wherein the inflammatory disorder is associated with abnormal IL-4 and / or IL-13 expression or activity.

19. 18. The pharmaceutical composition for use according to claim 17, wherein the inflammatory disorder is selected from the group consisting of asthma, allergic conditions, atopic disorders, bullous pemphigoid, respiratory disorders, nasal polyposis and other conditions involving airway inflammation; inflammatory and / or autoimmune disorders or conditions, gastrointestinal disorders or conditions; systemic lupus erythematosus, liver disorders or conditions, scleroderma; fibrotic diseases or disorders; solid tumors or cancers, and mastocytosis.

20. The pharmaceutical composition for use according to claim 17, wherein the inflammatory disorder is selected from the group consisting of allergic asthma, non-allergic asthma, food allergy, venom allergy, animal allergy, drug allergy, anaphylaxis, hyper-IgE syndrome, allergic rhinitis, allergic conjunctivitis, allergic enterogastritis, atopic dermatitis, urticaria, eczema, chronic obstructive pulmonary disease (COPD), eosinophilia, fibrosis, excessive mucus production, systemic sclerosis (SSc), inflammatory bowel disease (IBD), eosinophilic esophagitis (EE), eosinophil-mediated gastrointestinal disease, ulcerative colitis, Crohn's disease, cirrhosis, hepatocellular carcinoma, fibrosis of the liver, leukemia, glioblastoma, and lymphoma.

21. The pharmaceutical composition for use according to claim 17, wherein the inflammatory disorder is selected from the group consisting of chronic idiopathic urticaria, chronic spontaneous urticaria, cystic fibrosis, pulmonary fibrosis, fibrosis caused by hepatitis B virus, fibrosis caused by hepatitis C virus, B-cell chronic lymphocytic leukemia, and Hodgkin's lymphoma.

22. 18. The pharmaceutical composition for use according to claim 17, wherein the inflammatory disorder is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergy, nasal polyposis and eosinophilic esophagitis.

23. The pharmaceutical composition for use according to claim 17, wherein the inflammatory disorder is allergy, asthma, or atopic dermatitis.

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