New vaccine

Abstract

Several novel engineered variants of Protein Q, or fragments thereof, are provided, mediating protective immune responses in the treatment and prevention of leishmaniasis in animal and in human subjects.

Description

[0001] New vaccine

[0002] Field

[0003] The present invention relates to novel engineered polypeptide variants of Protein Q, peptide modules thereof, nucleic acid molecules and nucleic acid constructs encoding said polypeptide variants and said peptide modules, cells expressing them and compositions comprising them. The invention is useful for treatment and prevention of parasitic diseases, in particular leishmanises, in animal and human subjects.

[0004] Background of the invention

[0005] The parasitic protozoan of the Leishmania genus causes a range of diseases collectively known as leishmaniasis. There are 3 main forms of leishmaniasis: visceral, cutaneous, and mucocutaneous. The most severe form, Visceral leishamaniasis (VL), is a zoonotic disease caused by the parasite L. infantum. Furthermore, this form of leishmaniasis is associated with a high rate of mortality and morbidity in symptomatic patients. By exhibiting severe cutaneous parasitism, dogs play a key role in the transmission and maintenance of VL.

[0006] Therefore, the development of prophylactic vaccines against canine visceral leishmaniasis (CVL) has been proposed as a key strategy to prevent the transmission of the disease to humans. The development of recombinant protein-based vaccines, which elicit a potent and sustained immune response due to their diverse antigens, is considered advantageous because of their cost-effective production, ease of standardization, and improved efficacy.

[0007] LetiFend® (Laboratorios LETI, Spain, which became LETI Pharma as of December 2020), which was granted marketing authorization as a veterinary product for medicinal use in Europe in 2016, is currently the only commercial vaccine for CVL based on a recombinant protein termed Protein Q. Protein Q is formed by five antigenic fragments isolated from four different L. infantum proteins (ribosomal proteins LiP2a, LiP2b and LiPO and the N- and C-terminal portions of histone H2A). The sequence and functional characterization of Protein Q are disclosed in patent application WO 00 / 039298, which is incorporated herein by reference in its entirety. Protein Q is represented by SEQ ID NO:1 in the current application. According to the results of a pre-licensing phase III trial involving 549 dogs exposed to natural infection in two CVL-endemic areas in France and Spain over a two-year period, LetiFend showed a 72% vaccine efficacy in preventing clinical signs of CVL. The vaccine also reduced the likelihood of confirmed CVL cases and the development of clinical signs in vaccinated dogs by 5 and 9.8 times, respectively, compared to dogs treated with a placebo (Fernandez Cotrina J, Iniesta V, Monroy I et al. A large-scale field randomized trial demonstrates safety and efficacy of the vaccine LetiFend against canine leishmaniosis. Vaccine 2018: 36: 1972-1982). Furthermore, no general or local adverse effects were observed after administration during laboratory or field studies and no false-positive results were detected in L. infantum serological diagnostic tests (Iniesta V, Fernandez-Cotrina J, Solano-Gallego L, Monroy I, Gomez-Luque A, Munoz-Madrid R. Vaccination with LetiFend, a novel canine leishmaniosis vaccine, does not interfere with serological diagnostic tests. Proceedings of the X Southern European Veterinary Conference / 51 Congreso Nacional Avepa, 20-22 October 2016, Granada (Spain). Poster SEVC00678, 2016).

[0008] Despite its proven efficacy in preventing CVL, the activity, stability, yield and purity of Protein Q may be further improved. The present invention focuses on developing strategies for rational design of Protein Q derivatives with improved yield, purity, stability, and / or antigenicity and immunogenicity compared to Protein Q.

[0009] Brief description of the figures

[0010] Figure 1A: Cysteine (C) pattern of Protein Q (SEQ ID NO: 1), Protein Q-TEV (SEQ ID NO: 44) and the novel polypeptide variants derived from Protein Q. Variant 2 corresponds to SEQ ID NO: 5 with Cysteine replaced by Alanine (A) at position 22, Variant 3 corresponds to SEQ ID NO: 6 with Cysteine replaced by Alanine (A) at position 68, Variant 4 corresponds to SEQ ID NO: 7 with Cysteine replaced by Alanine (A) at position 158, and Variant 5 corresponds to SEQ ID NO: 8 with rcysteines at positions 22, 68 and 158.

[0011] Figure 1 B: Protein Q-TEV (SEQ ID NO: 44) and the novel polypeptide variants derived from Protein Q have a C-terminal cleavage site for Tobaco Etch Protease (TEV). The amino acid sequence of TEV recognition and cleavage site is illustrated here and is represented by SEQ ID NO:39. Proteolytic processing by TEV allows for removal of the Histidine tag, which is used during protein purification.

[0012] Figure 2A: The protein stability of the novel polypeptide variants derived from Protein Q is improved after one week storage at room temperature (RT) relative to the original Protein Q. t=0 denotes an ultrafrozen aliquot kept at -80°C, which acted as an internal baseline for protein stability.

[0013] Figure 2B: The protein stability of novel polypeptide variants 3, 4 and 5 derived from Protein Q is improved after one month storage at room temperature (RT) relative to the original Protein Q. t=0 denotes an ultrafrozen aliquot kept at -80°C, which acted as an internal baseline for protein stability. Black arrows indicate smearing indicative of protein degradation, which is observed for Protein Q and for Variant 2 stored at RT.

[0014] Figure 2C: The intensity of the protein bands shown on Figure 2A and Figure 2B indicates that novel polypeptide variant 5 (SEQ ID NO: 8) with replaced cysteines at positions 22, 68 and 158 remains stable for up to one month when stored at RT. Band intensity is relative to Protein Q (PQ). Figure 2D: Novel Protein Q Variants 4 (SEQ ID NO: 7) and 5 (SEQ ID NO: 8) remain stable after 2 months’ storage (60 days) at RT. In the graph, a 50% limit is marked for clarity (dashed horizontal line). Variants 4 (SEQ ID NO: 7) and 5 (SEQ ID NO: 8) are above such limit after 2 months at RT (60 days), whereas the other variants and Protein Q (SEQ ID NO: 1) are below the limit.

[0015] Figure 3A: Oriole staining of Protein Q and novel polypeptide variants 2-5 separated under reducing conditions. Lanes 1 and 5 contain molecular weight markers, Lane 2 contains Protein Q, and Lanes 3, 4, 6, and 7 contain the novel polypeptide variants (Variant 2, 3, 4, and 5, respectively).

[0016] Figure 3B: Oriole staining of Protein Q and novel polypeptide variants 2-5 separated under non-reducing conditions, s. Lanes 1 and 5 contain molecularweight markers, Lane 2 contains Protein Q, and Lanes 3, 4, 6, and 7 contain the novel polypeptide variants (Variant 2, 3, 4, and 5, respectively).

[0017] Figure 3C: Western blot with an antibody against Protein Q showing Protein Q and novel polypeptide variants 2-5 separated under reducing conditions. Lane 1 contains a molecular weight marker, Lane 2 contains Protein Q, and Lanes 3, 4, 5, and 6 contain the novel polypeptide variants (Variant 2, 3, 4, and 5, respectively).

[0018] Figure 3D: Western blot with an antibody against Protein Q showing Protein Q and novel polypeptide variants 2-5 separated under non-reducing conditions. Lane 1 contains a molecularweight marker, Lane 2 contains Protein Q, and Lanes 3, 4, 5, and 6 contain the novel polypeptide variants (Variant 2, 3, 4, and 5, respectively)

[0019] Figure 4A: ELISA assay using sera of dog sera collected from dogs at various clinical stages of Canine Leishmaniosis (CL) shows that the novel polypeptide variants retain their antigenicity. Based on clinical signs, clinical-pathologic abnormalities and serologic status CL is classified in the following stages shown on the figure legend: healthy non-infected (H, n=5); healthy infected (HI, n=5); sick stage I (SS1 , n=5); sick stage II (SS2, n=5); sick stage lll / IV (SS3 / SS4, n=5).

[0020] Figure 4B: The median values of Figure 4A measured for antibody binding to all of the novel polypeptide variants were higher at the late stages of infection (sick stage II (SS2); sick stage lll / IV) relative to the same values measured for Protein Q. These promising averages, though not statistically significant due to large variability, mark a positive trend and point to the possibility of further improving the antigenicity of all novel polypeptide variants. Based on clinical signs, clinical-pathologic abnormalities and serologic status CL is classified in the following stages shown on the figure legend: healthy non-infected (H, n=5); healthy infected (HI, n=5); sick stage I (SS1 , n=5); sick stage II (SS2, n=5); sick stage lll / IV (SS3 / SS4, n=5).

[0021] Figure 5: A schematic representation of the mice immunization assay used to assess the immunogenicity of the novel polypeptide variants derived from Protein Q. Mice are immunized with Protein Q, with control buffer and with the novel variants 3-5. Humoral response is evaluated at three time points post-vaccination: Day 0, Day 14 and Day 28. After 28 days, some mice are euthanized for additional tests. AB denotes antibody

[0022] Figure 6A: Antigen-specific lgG1 response following immunization of mice with Protein Q (SEQ ID NO: 1), Protein Q-TEV (SEQ ID NO: 44) and with novel Protein Q variants 3-5 (SEQ ID NOs: 6-8). All immunized groups showed increased lgG1 counts on Day 14 that were maintained through Day 28, while the negative control group treated with vehicle buffer showed no increase in lgG1 count. No statistical differences were observed between treatment groups.

[0023] Figure 6B: Antigen-specific lgG2a response following immunization of mice with Protein Q (SEQ ID NO: 1), Protein Q-TEV (SEQ ID NO: 44) and with novel Protein Q variants 3-5 (SEQ ID NOs: 6-8). All immunized groups showed increased lgG2a counts on Day 14 that were maintained through Day 28, while the negative control group treated with vehicle buffer showed no increase in lgG2a count. No statistical differences were observed between treatment groups.

[0024] Figure 7A. A schematic representation of the mice immunization assay used to assess the cellular responses in mice immunized with with Protein Q (SEQ ID NO: 1), Protein Q-TEV (SEQ ID NO: 44) and with novel Protein Q variants 3-5 (SEQ ID NOs: 6-8).

[0025] Figure 7B. Elevated counts of CD4+ T cells following splenocyte stimulation with novel Protein Q variants 4 and 5 (SEQ ID NO: 7 and 8) at 16 hours post-stimulation. Variants 4 and 5 (SEQ ID NO: 7 and 8) show markedly higher activation of CD4+ T cells compared with Protein Q (SEQ ID NO: 1).

[0026] Figure 7C. Elevated counts of CD8+ T cells following splenocyte stimulation with novel Protein Q variant 5 (SEQ ID NO: 8) at 16 hours post-stimulation. Variant 5 (SEQ ID NO: 8) shows markedly higher activation of CD8+ T cells compared with Protein Q (SEQ ID NO: 1).

[0027] Summary of the invention

[0028] In a first aspect, the invention provides a peptide or a polypeptide derived from SEQ ID NO:1 , said peptide or polypeptide being represented by an amino acid sequence having at least 80% identity or similarity with any one of SEQ ID NO:4, wherein: • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof,

[0029] In a preferred embodiment, a polypeptide disclosed in a first aspect of the invention is provided, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with any one of SEQ ID NO:7 or SEQ ID NO:8, wherein position 158 of SEQ ID NO:7 or of SEQ ID NO:8 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof.

[0030] In some embodiments, the cysteine of a peptide or polypeptide provided in a first aspect herein, is replaced or substituted by proline, glycine, serine, valine and threonine, and most preferably is alanine.

[0031] In some embodiments, the identity or the similarity of a peptide or polypeptide disclosed in a first aspect of the invention is assessed over the full length of said peptide’s or polypeptide’s aminoacid sequence.

[0032] In some embodiments, the peptide or polypeptide disclosed in a first aspect of the invention comprises a C-terminal recognition site for proteolytic cleavage mediated by a protease, wherein said protease is preferably a cysteine protease.

[0033] In some embodiments, the peptide or polypeptide disclosed in a first aspect of the invention does not comprise a protein purification tag, wherein said protein purification tag is preferably a Polyhistidine (His) tag.

[0034] In a preferred embodiment, a polypeptide disclosed in a first aspect of the invention is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 7, 42 or 48 wherein the aminoacid at position 158 of SEQ ID NO:7, 42 or 48 is not cysteine and may be glycine, serine, valine and threonine, and most preferably by alanine.

[0035] In a preferred embodiment, a polypeptide disclosed in a first aspect of the invention is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 8, 43 or 49 wherein the aminoacids at positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 are not cysteine and may be glycine, serine, valine and threonine, and most preferably is Alanine.

[0036] In a preferred embodiment, the invention provides a polypeptide according to the first aspect of the invention, said polyppetide comprising a Leishmania antigenic determinant and preferably inducing a protective immune response against leishmaniasis.

[0037] In a second aspect, the invention provides a nucleic acid molecule encoding any of the peptides or polypeptides disclosed in a first aspect of the invention.

[0038] In a preferred embodiment, the nucleic acid molecule disclosed in a second aspect of the invention is an RNA molecule, and preferably a messenger RNA (mRNA) molecule. In a third aspect, the invention provides a nucleic acid construct comprising the nucleic acid molecules disclosed in a second aspect of the invention.

[0039] In a fourth aspect, the invention provides a cell comprising the nucleic acid molecule and / or, the nucleic acid construct disclosed, respectively, in a second and third aspect of the invention, and / or expressing any of the peptides or polypeptides disclosed in a first aspect of the invention. In a particular embodiment, a cell disclosed in the fourth aspect of the invention is provided, wherein said cell is an eukaryotic cell, preferably an insect, yeast, fungal, mammalian or a plant cell.

[0040] In a fifth aspect, the invention provides a composition comprising any of the peptides or polypeptides of the first aspect, the nucleic acid of the second aspect, the nucleic acid construct of the third aspect and the cell of the fourth aspect of the invention, respectively.

[0041] In a preferred embodiment, the composition provided in a fifth aspect of the invention is a pharmaceutical composition.

[0042] In a specific embodiment, the composition disclosed in a fifth aspect of the invention comprises a physiological adjuvant appropriate for intraperitoneal, subcutaneous, intradermal, epidermal or intramuscular administration to a human or an animal subject.

[0043] In a sixth aspect, the invention provides any of the peptides or polypeptides disclosed in its first aspect, the nucleic acid molecule disclosed in its second aspect, the nucleic acid construct disclosed in its third aspect, the cell disclosed in its fourth aspect or the pharmaceutical composition disclosed in its fifth aspect, for use as a medicament.

[0044] In a particular embodiment, the medicament for use disclosed in a sixth aspect of the invention is for generating an immune response.

[0045] In a particular embodiment, the medicament for use disclosed in a sixth aspect of the invention is a vaccine.

[0046] In a particular embodiment, the medicament for use disclosed in a sixth aspect of the invention is a therapeutic and / or prophylactic vaccine.

[0047] In a particular embodiment, the medicament disclosed in a sixth aspect of the invention is a medicament for use in the treatment and / or prevention of leishmaniasis in a human or an animal subject.

[0048] Detailed Description of The Invention

[0049] Various features of the aspects and embodiments disclosed herein are further described below. It is noted that headings used throughout this specification are to assist navigation only and should not be interpreted as definitive, and that features described in different sections may be relevant for all aspects and embodiments described herein and may thus be combined as appropriate.

[0050] Peptide In a first aspect, the invention provides a peptide or a polypeptide derived from SEQ ID NO:1 , said peptide or polypeptide being represented by an amino acid sequence having at least 80% identity or similarity with with any one of SEQ ID NO:2-8, 40-43, 46-49, wherein:

[0051] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is alanine,

[0052] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is an alanine,

[0053] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is alanine,

[0054] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is alanine,

[0055] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is alanine,

[0056] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is alanine,

[0057] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, and most preferably is alanine

[0058] Herein, the term “peptide” is defined as a short chain consisting of or essentially consisting of 2-50 amino acids connected by peptide bonds.

[0059] A peptide may serve as a modular building block of a “polypeptide”, wherein the term “polypeptide” signifies an aminoacid chain of at least 51 aminoacids in length. In an embodiment, a polypeptide consists or essentially consists of 51 to 450 or 51 to 400 or 51 to 350 or 51 to 300 or 51 to 250 or 51 to 200 or 51 to 150 or 51 to 100 amino acids. Therefore, in the context of the present invention, the terms “peptides”, “peptide building blocks” or “modules” are used interchangeably.

[0060] In the context of the present invention, a peptide may induce an immune response in a subject, preferably an animal subject, and more preferably a human subject by virtue of said peptide’s antigenic determinant. Herein, the terms “antigenic determinant”, “antigen”and “epitope” are used interchangeably and refer to a sequence comprised in a peptide or a polypeptide disclosed herein, wherein said sequence is recognized and bound by an antibody or immune cell receptor. A detailed definition of “antigen” is provided later herein. The term “linear antigen” is used herein to refer to sequential aminoacid residues comprised in peptides disclosed in the invention. The term “conformational antigen” as used herein, refers to patches of solvent-exposed atoms from aminoacid residues that are not necessarily sequential. Polypeptides of the invention may comprise both linear and conformational antigens. Said conformational antigens may arise as a result of steric interactions of aminoacid residues from different linear antigens, wherein said linear antigens may be juxtaposed or distant from one another. Alternatively, said steric interactions may occur when the sequence context surrounding a linear antigen changes or when the sequenctial order of said linear antigen’s own aminoacids is altered.

[0061] It is therefore understood that each of the peptide sequences disclosed in the present invention may be used alone or when present together in a polypeptide (or combined together to form a polypeptide) to induce an immune response in a subject, preferably an animal, more preferably a human subject. When combined, for example by recombinant gene technology or chemical synthesis, the peptides of the invention may form a polypeptide, wherein said polypeptide harbours the linear antigens of their respective peptide modules. When combined in a polypeptide, said linear antigens may contribute to the formation of conformational antigens.

[0062] As described herein the term “peptide” also encompasses peptidomimetics, polypeptides, as well as variants (mutants) of peptides and polypeptides. A detailed definition of the term “peptidomimetic” is provided in the part of the description entitled “General definitions”.

[0063] As used herein, the term "derived" is to be understood as meaning that the peptides or polypeptides of the invention are obtained from, originate from, or are based upon the amino acid sequence, structural characteristics, and / or functional properties of a precursor molecule. However, the peptide or polypeptide disclosed herein, which is derived from the polypeptide represented by SEQ ID NO:1 and / or by SEQ ID NO: 44, but it is not identical to SEQ ID NO: 1 and / or SEQ ID NO: 44. In other words, the peptide or polypeptide disclosed herein is represented by an amino acid sequence which does not comprise SEQ ID NO:1 and / or of SEQ ID NO: 44 and which does not consist of SEQ ID NO:1 and / or of SEQ ID NO: 44.

[0064] Derivation may occur through any process known to the skilled person as long as the amino acid sequence of the peptide or polypeptide disclosed herein is not identical with SEQ ID NO:1 and / or to SEQ ID NO: 44 (and / or is not identical with a sequence comprising SEQ ID NO:1 and / or SEQ ID NO:44).

[0065] The peptide or polypeptide disclosed herein may be obtained via proteolytic cleavage, where enzymatic or chemical processes cleave a larger precursor protein to produce shorter peptides or fragments that retain structural or functional characteristics of the precursor sequence. The peptide or polypeptide derived from SEQ ID NO:1 and / or from SEQ ID NO: 44 may also be chemically synthesized, for example, via solid-phase peptide synthesis, or produced recombinantly in host cells to replicate the sequence, or a portion of the sequence, of a precursor molecule. Post-translational modifications, such as phosphorylation, glycosylation, or acetylation, are also included within the scope of peptides or polypeptides of the invention. In a preferred embodiment, the term "derived from" encompasses modified or variant forms of the original precursor polypeptide, including amino acid substitutions, deletions, or insertions, provided that such variants preserve essential structural or functional characteristics of the precursor. In a preferred embodiment, the peptide or polypeptide disclosed herein is produced recombinantly in a host cell as defined in the general part of the description entitled “general definitions”.

[0066] In an embodiment, a precursor polypeptide is protein Q represented by SEQ ID NO:1 and / or by SEQ ID NO: 44.

[0067] An activity of the peptide modules of the invention and of the polypeptides that comprise them (hereinafter, sometimes referred to as (poly)peptides) is their ability to induce an immune response (preferably a protective immune response) against a Leishmania species in an immunized subject, preferably an animal or, most preferably, a human subject. The protective immune response induced by the peptide or polypeptide disclosed herein may be assessed by measuring a set of biomarkers. The term “biomarker” is described in the section of the description entitled “General definitions”. Such biomarkers may comprise pro- and antiinflammatory cytokines, levels and populations of circulating T- and B-cells, levels and subclasses of circulating IgG antibodies, levels of Nitric Oxide and levels of Circulating Immune Complexes (CICs) measured at selected time points after administration of the peptide or polypeptide disclosed herein to an animal or human subject.

[0068] Said immune response is elicited by an antigen or by several antigens comprised in the peptide or polypeptide disclosed herein. Herein, an “antigen” is a molecule or molecular structure that is recognized (for example, bound to) by an antigen receptor or an antigen-binding protein. An antigen may be or may comprise, for example, a peptide, a polypeptide, a carbohydrate, a chemical, a moiety, a non-peptide antigen, a peptide-based antigen, a lipid-based antigen, or any combination thereof. In some embodiments, an antigen is capable of inducing an immune response. In some examples, an antigen binds to an antigen receptor or antigen-binding protein, or induces an immune response, when present in a complex e.g., presented by major histocompatibility complex (MHC). In some cases, an antigen adopts a certain conformation in order to bind to an antigen receptor or antigen-binding protein, and / or to induce an immune response, e.g., adopts a conformation in response to the presence or absence of one or more metabolites. Antigen can refer to a whole target molecule, a whole complex, or a fragment of a target molecule or complex that binds to an antigen receptor or an antigen-binding protein.

[0069] An antigen capable of eliciting an immune response may trigger a specific humoral and cellular response of the adaptive and / or innate immune system. Herein, the adaptive humoral response is represented by antibodies produced by antibody-secreting B-cells.

[0070] B cells are activated by antigens and their differentiation into antibody-producing cells is assisted by helper T cells. While a "helper T cell" typically refers to Th2 cells, some Th1 cells can also assist in B-cell activation. The balance between the Th1 and Th2 responses, along with regulatory mechanisms, is a determinant of the outcome of leishmaniasis (Costa-da-Silva AC, Nascimento DO, Ferreira J RM, Guimaraes-Pinto K, Freire-de-Lima L, Morrot A, Decote- Ricardo D, FilardyAA, Freire-de-Lima CG. Immune Responses in Leishmaniasis: An Overview. Trop Med Infect Dis. 2022 Mar 31;7(4):54. doi: 10.3390 / tropicalmed7040054. PMID: 35448829; PMCID: PMC9029249). Specifically, the host's immune response to the Leishmania parasite can manifest as either a resistant or susceptible immune response. In the resistant immune response, a Th1-type immune response dominates, leading to the production of cytokines such as interferon-gamma (IFN-y), interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-a), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Nitric oxide (NO), produced by macrophages activated by IFN-y or TNF-a, acts as a toxic agent against Leishmania parasites. Conversely, in a susceptible response, a Th2-type immune response prevails, and is characterized by the production of cytokines like IL-4, IL-10, and transforming growth factor-beta (TGF-p), which inhibit phagocytic cell activation and promote parasite proliferation and disease progression.

[0071] In an embodiment, a (poly)peptide of the invention is or comprises a Leishmania antigen or is or comprises a Leishmania antigenic determinant. It means that it is specifically recognized (or specifically bound to) by serum from dogs infected with leishmania. In an embodiment, a (poly)peptide presented herein is not specifically recognized by serum from healthy dogs. In an embodiment, the serum of infected dogs is collected from dogs classified into the following leishmaniosis stages according to the LeishVet classification, which is based on clinical signs, clinical-pathologic abnormalities and serologic status: healthy non-infected (H); healthy infected (HI); sick stage I (SS1); sick stage II (SS2); sick stage lll / IV (SS3 / SS4) (Solano-Gallego et al., 2009, 2011).

[0072] The binding affinity of the (poly)peptide presented herein may be assessed using ELISA by measuring the OD450 as described in the experimental part (Figure 4 in Example 4). The value of the binding affinity may correlate with the stage of the disease. In an embodiment, the binding affinity is higher for serum of dogs in sick stage lll / IV than for the serum of dogs in sick stage II, I or the serum of healthy infected dogs (Figure 4 in Example 4). A detectable OD450 may be seen as an evidence of the antigenicity of the (poly)peptide presented herein.

[0073] In an embodiment, the OD450 value obtained using the (poly)peptide presented herein may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the OD450 value induced by protein Q (SEQ ID NO:1), both OD450 being assessed under the same conditions. In an embodiment, the OD450 value obtained using the (poly)peptide presented herein may be similar with the OD450 value induced by protein Q (SEQ ID NO:1), both OD450 being assessed under the same conditions.

[0074] In another embodiment, the OD450 value obtained using the (poly)peptide may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% higher than the OD450 value induced by protein Q (SEQ ID NO:1), both QD450 being assessed under the same conditions.

[0075] In an embodiment, a (poly)peptide presented herein induces an immune response against leishmania, preferably a B cell response (or humoral response) characterized by the production of an antibody (or IgG). In a preferred embodiment, such B cell response is a protective immune response against leishmaniasis. A protective immune response may be characterized by an increased lgG2a / lgG1 ratio (meaning an increased Th1 cellular immune response). Additionally, the Th1 or Th1-type protective immune response can be correlated with increased production of IFNy and IL-12 by splenocytes stimulated with any of the poly (peptides) of the invention.

[0076] The promotion of a Th1 cellular immune response may be further characterized in the absence (or the absence of an induction) of a Th2 immune response.

[0077] A Th2 immune response is characterised by a detectable increase in IL-4, IL-10 induction and / or the production of detectable lgG1 immunoglobulines when compared with non-treated splenocytes. The assessment of the induction of IL-4 and / or IL-10 is preferably carried out by ELISA on splenocytes. The elicitation or promotion of a Th1 immune response may further be defined by the generation of an increase in IFNy / IL-10 ratio and / or IFNy I IL-4 ratio and / or a decrease in lgG1 / lgG2a ratio against a defined antigen.

[0078] Therefore in an embodiment, the (poly)peptide presented herein induces a protective immune response against Leishamaniasis by inducing an increased lgG2a / lgG1 ratio, an increase of IFNy and / or an increase of IL-12 all assessed by ELISA in the supernatant of splenocytes stimulated with the (poly)peptides provided herein. Methods for measuring lg2a / lgG1 ratios in cultured splenocytes are known in the art and have been described previously (Parody, N., et al. "Adjuvant guided polarization of the immune humoral response against a protective multicomponent antigenic protein (Q) from Leishmania infantum. A CpG+ Q mix protects Balb / c mice from infection."; Parasite immunology 26.6-7 (2004): 283-293; Iborra, Salvador, et al. "Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice." Microbes and infection 10.10-11 (2008): 1133-1141).

[0079] In an embodiment, the increase of the lgG2a / lgG1 ratio obtained using the (poly)peptide presented herein may be an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the value of said ratio with no (poly)peptide presented herein (control solution).

[0080] In an embodiment, the increase of the lgG2a / lgG1 ratio obtained using the (poly)peptide presented herein may be similar with the increase of the lgG2a / lgG1 ratio induced by protein Q (SEQ ID NO:1), both ratio being assessed under the same conditions.

[0081] In another embodiment, the increase of the lgG2a / lgG1 ratio obtained using the (poly)peptide presented herein may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% higher than the increase of the lgG2a / lgG1 ratio induced by protein Q (SEQ ID NO:1), both ratio being assessed underthe same conditions

[0082] In an embodiment, the increase of IFNy and / or IL-12 obtained using the (poly)peptide presented herein may be an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the amount of IFNg and / or IL-12 induced with no (poly)peptide presented herein (control solution). In an embodiment, the increase of IFNy and / or IL-12 obtained using the (poly)peptide presented herein may be similar with the increase induced by protein Q (SEQ ID NO:1), both IFNy and / or IL-12 being assessed under the same conditions.

[0083] In another embodiment, the increase of IFNy and / or IL-12 obtained using the (poly)peptide presented herein may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% higherthan the increase induced by protein Q (SEQ ID NO:1), both IFNy and / or IL-12 being assessed under the same conditions.

[0084] In an embodiment, a peptide or polypeptide disclosed herein comprises a Leishmania antigenic determinant and preferably induces a protective immune response against leishmaniasis.

[0085] In an embodiment, an activity of protein Q is preserved at least to some extent in a peptide or polypeptide disclosed herein. In an embodiment, such activity of protein Q is to induce an immune response (preferably a protective immune response) against a Leishmania species in an animal subject, most preferably a human subject. In this context, “to some extent” may mean at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 150%, of the immune response induced by SEQ ID NO:1 .

[0086] In one embodiment of the present invention, altering at least one, at least two and preferably three cysteine residues of the precursor molecule represented by SEQ ID NO:1 (or SEQ ID NO:44), has resulted in surprising improvements of the derived peptides and polypeptides that comprise them. Such improvements pertain to improved polypeptide or peptide yield and / or polypeptide or peptide stability when the polypeptide or peptide is recombinantly produced by a host cell, without compromising the immunogencity of said molecules.

[0087] A precursor molecule used to derive a peptide or polypeptide disclosed herein described herein may be represented by the aminoacid sequence of SEQ ID NO:1 or 44.

[0088] In one embodiment, at least one of the cysteine residues present in SEQ ID NO:1 (or in 44) is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine, and most preferably by alanine.

[0089] In one embodiment, at least two of the cysteine residues that are present in SEQ ID NO:1 (or 44) is replaced or substituted by an amino acid comprising a side chain lacking disulfide bondforming capability or a derivative thereof, preferably proline, glycine, serine, valine and threonine, and most preferably by alanine.

[0090] In one embodiment, the three cysteine residues present in SEQ ID NO:1 (or 44) is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine and most preferably by alanine. A peptide or polypeptide disclosed herein is distinct from SEQ ID NO:1 (and from SEQ ID NO:44) and has at least 80% identity or similarity with any one of SEQ ID NO: 2-8, 40-43, 46- 49, wherein:

[0091] Position 20 of SEQ ID NO:2 is not a cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine, and most preferably by alanine. In one embodiment, the peptide of the invention consists of SEQ ID NO: 9 (ATPRSAKKAVRKSGSKSA) or may comprise an amino acid sequence having at least 80% identity or similarity with SEQ ID NO: 9 (ATPRSAKKAVRKSGSKSA), with the proviso that this sequence is not SEQ ID NO:1 or 44. In one embodiment, the peptide of the invention comprises an aminoacid sequence having at least 80% identity or similarity with SEQ ID NO: 10 (ASKSGSKRVAKKASRPTA).

[0092] Position 13 of SEQ ID NO:3 is not a cysteine, is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine, and most preferably by alanine. In one embodiment, the peptide of the invention consists of SEQ ID NO: 11 (HSGVVPNISKAMAKKKGGKKGKATPSA) or may comprise an amino acid sequence having at least 80% identity or similarity with SEQ ID NO: 11 (HSGVVPNISKAMAKKKGGKKGKATPSA), with the proviso that this sequence is not SEQ ID NO:1 or 44. In one embodiment, the peptide of the invention comprises an aminoacid sequence having at least 80% identity or similarity with SEQ ID NO 12 (ASPTAKGKKGGKKKAMAKSINPVVGSH).

[0093] Position 28 of SEQ ID NO:4 is not a cysteine, is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine, and most preferably by alanine. In one embodiment, the peptide of the invention consists of SEQ ID NO: 13 (AAKKDEPEEEA) or may comprise an amino acid sequence having at least 80% identity or similarity with SEQ ID NO: 13 (AAKKDEPEEEA), with the proviso that this sequence is not SEQ ID NO:1 or 44. In one embodiment, the peptide of the invention comprises an aminoacid sequence having at least 80% identity or similarity with SEQ ID NO 14:. (AEEEPEDKKAA).

[0094] Position 22 of SEQ ID NO:5, 40 and 46 is not a cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine, and most preferably by alanine. Position 68 of SEQ ID NO:6, 41 and 47 is not a cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine, and most preferably by alanine.

[0095] Position 158 of SEQ ID NO: 7, 42 and 48 is not a cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine, and most preferably by alanine.

[0096] Positions 22, 68 and 158 of SEQ ID NO: 8, 43 and 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably by proline, glycine, serine, valine and threonine, and most preferably by alanine.

[0097] The terms "sequence identity," and “sequence similarity” have been defined in details in the part of the description entitled “General definitions”. The identity or similarity defined above may be at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0098] SEQ ID NO:1 is protein Q and may be considered as a precursor polypeptide, the polypeptide (or peptide) of the invention derives from.

[0099] SEQ ID NO:1 :

[0100] MRGSHHHHHHTDPHASSNNNNNNNNNNLGIEGRPLATPRSAKKAVRKSGSKSAKCGLIFPV GRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLL KNVTLSHSGVVPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYALASLSKASPSQ ADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGVTASAAG DAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSKADVQAVLKAAGVA VDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGAVSTAGAGAGAVAEAKKEEPEE EEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDDFGMGGLF

[0101] In some embodiments, Protein Q may be represented by a variant having a SEQ ID NO:44, wherein a C-terminal recognition site for clevage mediated by Tobaco Etch Virus protease (TEV) has been added (ENLYFQJ.G, SEQ ID NO: 39). Hereinafter, this variant is referred to as Protein Q-TEV and it serves as a control to standardize the activity of novel variants derived from Protein Q. This standardization is necessary for evaluating the effect of TEV protease processing following protein purification, and for compensating for variations in the cloning and expression systems used to produce the novel variants, which are described later herein. SEQ ID NO 44:

[0102] MRGSHHHHHHTDPHASSNNNNNNNNNNLGIEGRPLATPRSAKKAVRKSGSKSAKCGLIFPV GRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLL KNVTLSHSGVVPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYALASLSKASPSQ ADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGVTASAAG DAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSKADVQAVLKAAGVA VDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGAVSTAGAGAGAVAEAKKEEPEE EEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDDFGMGGLFSSGENLYFQG

[0103] The present invention has identified three distinct peptides or peptide building blocks derived from SEQ ID NO: 2, 3 or 4 respectively and that can be used as a standalone peptide or as a building block to form a polypeptide derived from SEQ ID NO:1 or 44.

[0104] In one embodiment, a peptide is derived from SEQ ID NO:2:

[0105] ATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGA (so called peptide module 1 or peptide 1), wherein the aminoacid at position 20 of SEQ ID NO:2 is not cysteine. In an embodiment, the amino acid at position 20 of SEQ ID NO:2 has been replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine and threonine, and most preferably alanine.

[0106] In one embodiment, a peptide of the invention (so called peptide module 1 or peptide 1) has at least 80% identity with SEQ ID NO:2 and has an aminoacid at position 20 of SEQ ID NO:2, which is not cysteine. In an embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine and threonine, and most preferably is by alanine.

[0107] The identity or similarity may be of at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 2.

[0108] In an embodiment, a peptide of the invention comprises a linear antigen 1 , wherein said linear antigen 1 is represented by an aminoacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO 9: ATPRSAKKAVRKSGSKSA and / or to SEQ ID NO 10: ASKSGSKRVAKKASRSPTA, with the proviso that a polypeptide comprising said peptide, (i.e. said peptide comprising said linear antigen 1) is not represented by or is not identical with SEQ ID NO:1 or 44. In embodiments, the aminoacid sequences representing linear antigen 1 may be used as a standalone sequence or combined in (tandem) repeats of 1 , 2, x, and preferably x copies comprised in a peptide or in a polypeptide. In embodiments, any of amino acid sequences representing linear antigen 1 may be positioned upstream or downstream of the substituted cysteine at position 20 of a peptide represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 2. In one embodiment, a peptide is derived from SEQ ID NO: 3 SVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKG KATPSA (so called building block 2 or peptide 2) wherein the aminoacid at position 13 is not cysteine. In an embodiment, the amino acid at position 13 may be represented by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 13 of SEQ ID NO:3 is proline, glycine, serine, valine and threonine, and most preferably by alanine. In one embodiment, a peptide of the invention (so called peptide module 2 or peptide 2) has at least 80% identity with SEQ ID NO:3 and has aminoacid at position 13 of SEQ ID NO:3 which is not cysteine. In an embodiment, the amino acid at position 13 of SEQ ID NO:3 is an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine and threonine, and most preferably is alanine.

[0109] The identity or similarity of such a peptide may be of at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 3.

[0110] In an embodiment, a peptide of the invention comprises a linear antigen 2, wherein said linear antigen 2 is represented by an aminoacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO 11 : HSGVVPNISKAMAKKKGGKKGKATPSA and / or to SEQ ID NO 12:. ASPTAKGKKGGKKKAMAKSINPVVGSH, with the proviso that a polypeptide comprising said peptide, (i.e. said peptide comprising said linear antigen 2) is not represented by or is not identical with SEQ ID NO:1 or 44. In embodiments, the aminoacid sequences representing linear antigen 2 may be used as a standalone sequence or combined in (tandem) repeats of 1 , 2, x, and preferably x copies comprised in a peptide or in a polypeptide. In embodiments, any of the aminoacid sequences representing linear antigen 2 may be positioned upstream or downstream of the substituted cysteine at position 13 in a peptide represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 3.

[0111] In one embodiment, a peptide is derived from SEQ ID NO:4

[0112] MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKM SAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMG (so called building block 3 or peptide 3), wherein the aminoacid at position 28 is not cysteine. In an embodiment, the amino acid at position 28 may be represented by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine and threonine, and most preferably by alanine. In one embodiment, a peptide of the invention (so called peptide module 3 or peptie 3) has at least 80% identity with SEQ ID NO:4 and has aminoacid at position 28 of SEQ ID NO:4 which is not cysteine. In an embodiment, the amino acid at position 28 of SEQ ID NO:4 is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine and threonine, and most preferably alanine. The identity or similarity may be of at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 4.

[0113] In an embodiment, a peptide of the invention comprises a linear antigen 3, wherein said linear antigen 3 is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO 13: AAKKDEPEEEA and / or to SEQ ID NO 14: AEEEPEDKKAA , with the proviso that a polypeptide comprising said peptide, (i.e. said peptide comprising said linear antigen 3) is not represented by or is not identical with SEQ ID NO:1 or 44. In embodiments, the aminoacid sequences representing linear antigen 3 may be used as a standalone sequence or combined in (tandem) repeats of 1 , 2, x, and preferably x copies comprised in a peptide or in a polypeptide. In embodiments, any of the aminoacid sequences representing linear antigen 3 may be positioned upstream or downstream of the substituted cysteine at position 28 in a peptide represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 4.

[0114] In a preferred embodiment, a peptide is represented by an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with any one of SEQ ID NO:4, wherein position 28 of SEQ ID NO: 4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the cysteine at position 28 of SEQ ID NO: 28 is substituted with alanine. Surprisingly, by virtue of the replaced cysteine at position 28 of SEQ ID NO: 4, the respective peptide or a polypeptide comprising it is capable of more potent immune cell activation relative to Protein Q or to variants comprising an alternative cysteine replacement (e.g. Variant 3 / SEQ ID NO: 6 with replaced cysteine at position 68). As understood herein, immune cell activation is represented as the percentual difference between the subset of activated CD4+ and / or CD8+ T-cells measured by flow cytometry in splenocytes isolated from immunized mice and stimulated with Protein Q (SEQ ID NO:1), Protein Q-TEV (SEQ ID NO: 44) and with novel Protein Q variants 3-5 (SEQ ID NOs: 6-8 and / or SEQ ID NOs: 47-49 corresponding to variants lacking HIS-Tag, respectively). Specifically, Example 7 and Figure 7 show that splenocytes obtained from mice immunized with Variant 4 (SEQ ID NO: 7 and / or SEQ ID NO. 48 lacking a HIS-Tag) and / or 5 (SEQ ID NO: 8 and / or SEQ ID NO. 49 lacking a HIS-Tag) showed higher activation of CD4+ and / or CD8+ T cells compared to other Protein Q variants as early as 16 hours post-induction (see Figures 7B-C). Thus, in one embodiment a peptide or polypeptide comprising or consisting of an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with SEQ ID NO:4 with replaced cysteine at position 28 is capable of inducing a 10%, 20%, 30%, 40%, and preferably 50% more potent immune cell activation relative to Protein Q or to other Protein Q variants that lack the same cysteine replacement. In a particular embodiment, the immune cell is a CD4+ and / or a CD8+ T-cell.

[0115] In yet another embodiment, a peptide or polypeptide comprising or consisting of an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with SEQ ID NO:4 with replaced cysteine at position 28 exhibits an improved protein stability during prolonged storage at room temperature relative to Protein Q (SEQ ID NO: 1) or to a variant with an alternative or no cysteine substitution. As shown in Example 1 , Figures 2A-D, such peptide or polypeptide is at least 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and preferably, 100% more stable than Protein Q (SEQ ID NO: 1) when stored at RT for up to 2 months.

[0116] In yet another embodiment, a peptide or polypeptide comprising or consisting of an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with SEQ ID NO:4 with replaced cysteine at position 28 exhibits an improved protein stability during prolonged cold storage relative to Protein Q (SEQ ID NO: 1) or to a variant with an alternative or no cysteine substitution. Herein, prolonged cold storage is understood to encompass at least 1 week, at least 1 month, at least 2 months, 3 months, 4 months, 5 months, and up to 6 months, wherein the polypeptide is stored at 6°C, 5°C, 4 °C, 3 °C, 2 °C, 1 °C, 0 °C, -1 °C, -5 °C, -10 °C, -15 °C, or down to -20 °C.

[0117] Each of the peptide building blocks derived from SEQ ID NO: 2, 3 or 4 may be used as a stand alone peptides. Alternatively, each of these peptide building blocks may be combined with each other to form a polypeptide as described below, with the proviso that a polypeptide comprising each of said peptide is not represented by or is not identical with SEQ ID NO:1 or 44. Furthermore, each of these peptide building blocks may be used in combination with other peptides to form a polypeptide.

[0118] In a preferred embodiment, each of these peptides building blocks may be comprised in a polypeptide and may be combined together, with the proviso that a polypeptide comprising said peptide is not represented by or is not identical with SEQ ID NO:1 or 44. Such a polypeptide may comprise, consist or essentially consist of:

[0119] The peptide module 1 and the peptide module 2,

[0120] The peptide module 1 and the peptide module 3, The peptide module 3 and the peptide module 2, The peptide module 1 , the peptide module 2 and the peptide modules. Therefore, in one embodiment the invention relates to a polypeptide comprising peptide module 1 and peptide module 2, said polypeptide having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 15-18, wherein:

[0121] • Said polypeptide comprises SEQ ID NO: 2 (peptide module 1) and wherein the aminoacid residue at position 20 of SEQ ID NO:2 (module 1) is not a cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bondforming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0122] • Said polypeptide comprises SEQ ID NO:3 (peptide module 2) and wherein the aminoacid residue at position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 13 of SEQ ID NO:3 is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0123] • And optionally at least one of the following is met:

[0124] • Said polypeptide comprises or essentially consists of or consists of 51 to 300 or 51 to 250 or 51 to 200 or 51 to 150 amino acids

[0125] • Said Peptide Module 1 may comprise SEQ ID NO 9:

[0126] ATPRSAKKAVRKSGSKSA and / or SEQ ID NO 10 ASKSGSKRVAKKASRPTA

[0127] • Said Peptide Module 2 may comprise SEQ ID NO 11 :

[0128] HSGVVPNISKAMAKKKGGKKGKATPSA and / or to SEQ ID NO 12:. ASPTAKGKKGGKKKAMAKSINPVVGSH

[0129] • Both peptide building blocks can be positioned in two different orientations relative to each other. By way of example and not limitation, peptide module 1 may be positioned either N- or C-terminally with respect to peptide module 2. Alternatively, peptide module 2 may be positioned either N- or C-terminally with respect to peptide modulel . Both polypeptide building blocks can be directly fused to each other or separated by a linker. Non-limiting examples of such linkers may be represented by the following sequences: SEQ ID NO: 52 (PRISEF), SEQ ID NO: 53 (PSRVDP), SEQ ID NO: 54 (PVDLQ). In some instances, a linker may be represented by a triplet of the flexible Glycine-Serine linker (SEQ ID NO: 55: (GGGGS)s, wherein said linker may provide flexibility and prevent steric hindrance of the resulting fusion polypeptide. The term linker is described in detail in the part of the descriptions entitled “General Definitions”.

[0130] SEQ ID NO: 15 is represented below: HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGASVKAAAQSGKK

[0131] RARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA

[0132] SEQ ID NO: 16 is represented below:

[0133] HMSVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGK

[0134] KGKATPSA ATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGA

[0135] SEQ ID NO: 17 is represented below:

[0136] HMASKSGSKRVKAKKASRPTAKAGLIFPVGRVGGMMRRGQYARRIGASGASVKAAAQSGK

[0137] KRARLNPRTVMLAARHDDDIGTLLKNVTLSASPTAKGKKGGKKKAMAKSINPVVGSH

[0138] SEQ ID NO: 18 is represented below:

[0139] HMSVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSASPTAKGKKGGKKKAMAKS

[0140] INPVVGSH ASKSGSKRVKAKKASRPTAKAGLIFPVGRVGGMMRRGQYARRIGASGA

[0141] In another embodiment, the invention relates to a polypeptide comprising peptide module 1 and peptide module 3, said polypeptide having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 19-22 wherein:

[0142] • Said polypeptide comprises SEQ ID NO: 2 (peptide module 1) and wherein the aminoacid residue at position 20 of SEQ ID NO:2 (module 1) is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine, threonine, and most preferably is alanine .

[0143] • Said polypeptide comprises SEQ ID NO:4 (peptide module 3) and wherein the aminoacid residue at position 28 of SEQ ID NO:4 is not a is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0144] • And optionally at least one of the following is met:

[0145] • Said polypeptide comprises or essentially consists of or consists of 51 to 300 or 51 to 250 or 51 to 200 or 51 to 150 amino acids

[0146] • Said Peptide Module 1 may comprise SEQ ID NO 9: ATPRSAKKAVRKSGSKSA and / or SEQ ID NO 10 ASKSGSKRVAKKASRPTAT

[0147] • Said Peptide Module 3 may comprise SEQ ID NO 13: AAKKDEPEEEA and / or to SEQ ID NO 14:AEEEPEDKKAA • Both peptide building blocks can be positioned in two different orientations relative to each other. By way of example and not limitation, peptide module 1 may be positioned either N- or C- terminally with respect to peptide module 3. Alternatively, peptide module 3 may be positioned either N- or C-terminally with respect to peptide modulel . Both polypeptide building blocks can be directly fused to each other or separated by a linker. The term linker is described in detail in the part of the descriptions entitled “General Definitions”. Non-limiting examples of such linkers may be represented by the following sequences: SEQ ID NO: 52 (PRISEF), SEQ ID NO: 53 (PSRVDP), SEQ ID NO: 54 (PVDLQ). In some instances, a linker may be represented by a triplet of the flexible Glycine-Serine linker (SEQ ID NO: 55: (GGGGS)3, wherein said linker may provide flexibility and prevent steric hindrance of the resulting fusion polypeptide.

[0148] SEQ ID NO: 19 is represented below: HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAMSTKYLAAYALA SLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAA AGVTASAAGDAAPAAAAAKKDEPEEEADDDMG

[0149] SEQ ID NO: 20 is represented below:

[0150] MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKM SAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGATPRSAKKAVRKSGSKS AKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEF

[0151] SEQ ID NO: 21 is represented below:

[0152] HMASKSGSKRVAKKASRSPTAKAGLIFPVGRVGGMMRRGQYARRIGASGAMSTKYLAAYAL ASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGA AAGVTASAAGDAAPAAA AEEEPEDKKAA DDDMG

[0153] SEQ ID NO: 22 is represented below:

[0154] MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKM SAMPAASSGAAAGVTASAAGDAAPAAAAEEEPEDKKAADDDMGASKSGSKRVAKKASRSP TAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEF

[0155] In another embodiment, the invention relates to a polypeptide comprising peptide module 2 and peptide module 3, said polyppetide having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 23- 26, wherein: • Said polypeptide comprises SEQ ID NO: 3 (peptide module 2) and wherein the aminoacid residue at position 13 of SEQ ID NO:3 (module 2) is not cysteine and is replaced or substituted by is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 13 of SEQ ID NO:3 is proline, glycine, serine, valine, threonine, and most preferably is alanine .

[0156] • Said polypeptide comprises SEQ ID NO:4 (peptide module 3) and wherein the aminoacid residue at position 28 of SEQ ID NO:4 is not is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0157] • And optionally at least one of the following is met:

[0158] • Said polypeptide comprises or essentially consists of or consists of 51 to 300 or 51 to 250 or 51 to 200 or 51 to 150 amino acids

[0159] • Said Peptide Module 2 may comprise SEQ ID NO 11 : HSGVVPNISKAMAKKKGGKKGKATPSA and / or SEQ ID NO 12: ASPTAKGKKGGKKKAMAKSINPVVGSH

[0160] • Said Peptide Module 3 may comprise SEQ ID NO 13: AAKKDEPEEEA and / or to SEQ ID NO 14: AEEEPEDKKAA

[0161] • Both peptide building blocks can be positioned in two different orientations relative to each other. By way of example and not limitation, peptide module 2 may be positioned either N- or C-terminally with respect to peptide module 3. Alternatively, peptide module 3 may be positioned either N- or C-terminally with respect to peptide module 2. Both polypeptide building blocks can be directly fused to each other or separated by a linker. The term linker is described in detail in the part of the descriptions entitled “General Definitions”. Non-limiting examples of such linkers may be represented by the following sequences: SEQ ID NO: 52 (PRISEF), SEQ ID NO: 53 (PSRVDP), SEQ ID NO: 54 (PVDLQ). In some instances, a linker may be represented by a triplet of the flexible Glycine-Serine linker (SEQ ID NO: 55: (GGGGS)3, wherein said linker may provide flexibility and prevent steric hindrance of the resulting fusion polypeptide.

[0162] SEQ ID NO: 23 is represented below:

[0163] SVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGWPNISKAMAKKKGGKKGKATP SAMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAK MS AMPAAS S GAAAGVTAS AAGDAAPAAAAAKKDE PE EE ADDDMG SEQ ID NO: 24 is represented below:

[0164] MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMS

[0165] AMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGSVKAAAQSGKKRARLNPRTVM

[0166] LAARHDDD I GTLLKNVTLSHS GWPNI SKAMAKKKGGKKGKATPSA

[0167] SEQ ID NO: 25 is represented below:

[0168] SVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSASPTAKGKKGGKKKAMAKSINPWG

[0169] SHMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAK

[0170] MSAMPAAS S GAAAGVTASAAGDAAPAAAAEEE PEDKKAADDDMG

[0171] SEQ ID NO: 26 is represented below: MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMS AMPAASSGAAAGVTASAAGDAAPAAAAEEEPEDKKAADDDMGSVKAAAQSGKKRARLNPRTVM LAARHDDD I GTLLKNVTLSAS PTAKGKKGGKKKAMAKS INPWGSH

[0172] In another embodiment, the invention relates to a polypeptide comprising peptide module 1 , peptide module 2 and peptide module 3, said polyppetide having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 27-38 wherein:

[0173] • Said polypeptide comprises SEQ ID NO: 2 (peptide module 1) wherein the aminoacid residue at position 20 of SEQ ID NO:2 (module 1) is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0174] • Said polypeptide comprises SEQ ID NO: 3 (peptide module 2) and wherein the aminoacid residue at position 13 of SEQ ID NO: 3 (module 2) is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 13 of SEQ ID NO:2 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0175] • Said polypeptide comprises SEQ ID NO:4 (peptide module 3) and wherein the aminoacid residue at position 28 of SEQ ID NO:4 is not a is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine, threonine, and most preferably is alanine. • And optionally at least one of the following is met:

[0176] • Said polypeptide comprises or essentially consists of or consists of 51 to 300 or 51 to 250 or 51 to 200 or 51 to 150 amino acids

[0177] • Said Peptide Module 1 may comprise SEQ ID NO 9:

[0178] ATPRSAKKAVRKSGSKSA and / or SEQ ID NO 10 ASKSGSKRVAKKASRPTA

[0179] • Said Peptide Module 2 may comprise SEQ ID NO 11 :

[0180] HSGVVPNISKAMAKKKGGKKGKATPSA and / or SEQ ID NO 12: ASPTAKGKKGGKKKAMAKSINPVVGSH

[0181] • Said Peptide Module 3 may comprise SEQ ID NO 13: AAKKDEPEEEA and / or to SEQ ID NO 14:AEEEPEDKKAA

[0182] • All the three peptide building blocks can be represented in any of the following orientations relative to each other: peptide module 1 , peptide module 2, and peptide module 3 (1-2-3); peptide module 1 , peptide module 3, and peptide module 2 (1-3-2); peptide module2, peptide modulel , and peptide module 3 (2-1-3); peptide module2, peptide modules, and peptide module 1 (2-3-1); peptide modules, peptide module 1 , and peptide module 2 (3-1-2); and peptide module 3, peptide module 2, and peptide module 1 (3-2-1). The three peptide modules can be directly fused to each other or separated by a linker. The term linker is described in detail in the section of the description entitled “General definitions”. Non-limiting examples of such linkers may be represented by the following sequences: SEQ ID NO: 52 (PRISEF), SEQ ID NO: 53 (PSRVDP), SEQ ID NO: 54 (PVDLQ). In some instances, a linker may be represented by a triplet of the flexible Glycine-Serine linker (SEQ ID NO: 55: (GGGGS)3, wherein said linker may provide flexibility and prevent steric hindrance of the resulting fusion polypeptide.

[0183] SEQ ID NO: 27 is represented below:

[0184] HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGASVKAAAQSGKKRARLN PRTVMLAARHDDDIGTLLKNVTLSHSGWPNISKAMAKKKGGKKGKATPSAMSTKYLAAYALASL SKASPSQADVEAlAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGV TASAAGDAAPAAAAAKKDEPEEEADDDMG

[0185] SEQ ID NO: 28 is represented below:

[0186] HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAMSTKYLAAYALASLSK AS P SQADVEAI AKAVH I DVDQATLAFVME S VTGRDVATL I AEGAAKMS AMPAAS SGAAAGVT A SAAGDAAPAAAAAKKDEPEEEADDDMGSVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNV TLSHSGVVPNI SKAMAKKKGGKKGKATPSA

[0187] SEQ ID NO: 29 is represented below: SVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGWPNISKAMAKKKGGKKGKATP SAATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAMSTKYLAAYALASLSK

[0188] AS P SQADVEAI AKAVH I DVDQATLAFVME S VTGRDVATL I AEGAAKMS AMPAAS SGAAAGVT A SAAGDAAPAAAAAKKDEPEEEADDDMG

[0189] SEQ ID NO: 30 is represented below:

[0190] SVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGWPNISKAMAKKKGGKKGKATP

[0191] SAMST KYL AAYAL ASL S KAS P S QADVE Al AKAVH I DVDQATLAFVME S VTGRDVATL I AEGAAK MSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGATPRSAKKAVRKSGSKSAK AGL I FPVGRVGGMMRRGQ YARRI GAS GA

[0192] SEQ ID NO: 31 is represented below:

[0193] MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMS

[0194] AMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGATPRSAKKAVRKSGSKSAKAG

[0195] LIFPVGRVGGMMRRGQYARRIGASGASVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTL SHSGWPNISKAMAKKKGGKKGKATPSA

[0196] SEQ ID NO: 32 is represented below:

[0197] MSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMS

[0198] AMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGSVKAAAQSGKKRARLNPRTVM LAARHDDDIGTLLKNVTLSHSGVVPNI SKAMAKKKGGKKGKATPSAATPRSAKKAVRKSGSKSAK

[0199] AGL I FPVGRVGGMMRRGQ YARRI GAS GA

[0200] SEQ ID NO: 33 is represented below:

[0201] HMASKSGSRKVAKKASRPTAKAGLIFPVGRVGGMMRRGQYARRIGASGASVKAAAQSGKKRARLN PRTVMLAARHDDDIGTLLKNVTLSASPATKGKKGGKKKAMAKSINPWGSMSTKYLAAYALASLS

[0202] KASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGVT ASAAGDAAPAAAAE EE PE DKKAADDDMG

[0203] SEQ ID NO: 34 is represented below:

[0204] HMASKSGSRKVAKKASRPTAKAGLIFPVGRVGGMMRRGQYARRIGASGAMSTKYLAAYALASLSK

[0205] AS P SQADVEAI AKAVH I DVDQATLAFVME S VTGRDVATL I AEGAAKMS AMPAAS SGAAAGVT A

[0206] SAAGDAAPAAAAEEEPEDKKAADDDMGSVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNV TLSASPATKGKKGGKKKAMAKS INPWGS

[0207] SEQ ID NO: 35 is represented below:

[0208] HMSVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSASPATKGKKGGKKKAMAKSINPV

[0209] VGSASKSGSRKVAKKASRPTAKAGLIFPVGRVGGMMRRGQYARRIGASGAMSTKYLAAYALASLS KAS PSQADVEAlAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGVT

[0210] AS AAG DAAP AAAAE E E P E D KKAADD DMG

[0211] SEQ ID NO: 36 is represented below:

[0212] HMSVKAAAQSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSASPATKGKKGGKKKAMAKS INPV VGSMSTKYLAAYALASLSKASPSQADVEAlAKAVHIDVDQATLAFVMESVTGRDVATLIAEGAA KMSAMPAASSGAAAGVTASAAGDAAPAAAAEEEPEDKKAADDDMGASKSGSRKVAKKASRPTA

[0213] KAGL I FPVGRVGGMMRRGQ YARRI GAS GA

[0214] SEQ ID NO: 37 is represented below:

[0215] HMMST KYL AAYAL ASL S KAS P S QADVE Al AKAVH I DVDQATLAFVME S VTGRDVATL I AEGAAK MSAMPAASSGAAAGVTASAAGDAAPAAAAEEEPEDKKAADDDMGASKSGSRKVAKKASRPTAK AGLI FPVGRVGGMMRRGQYARRIGASGASVKAAAQSGKKRARLNPRTVMLAARHDDDI GTLLKNV TLSASPATKGKKGGKKKAMAKS INPWGS

[0216] SEQ ID NO: 38 is represented below:

[0217] HMMST KYL AAYAL ASL S KAS P S QADVE Al AKAVH I DVDQATLAFVME S VTGRDVATL I AEGAAK MSAMPAASSGAAAGVTASAAGDAAPAAAAEEEPEDKKAADDDMGSVKAAAQSGKKRARLNPRT VMLAARHDDDI GTLLKNVTLSAS PATKGKKGGKKKAMAKS INPWGSASKSGSRKVAKKASRPTA KAGL I FPVGRVGGMMRRGQ YARRI GAS GA

[0218] In a preferred embodiment, a polypeptide disclosed herein is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 5, 40 or 46 wherein the aminoacid at position 22 of SEQ ID NO:5, 40 or 46 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 22 of SEQ ID NO:5, 40 or 46 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0219] Such a polypeptide may comprise the amino acid sequence of SEQ ID NO:2 (peptide modulel), wherein the aminoacid at position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0220] Such a polypeptide may comprise an amino acid sequence having at least 80% (or at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity or similarity with SEQ ID NO:2 (peptide module 1), wherein the aminoacid at position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0221] Surprisingly, the substitution of a single cysteine residue at position 22 of SEQ ID NO: 5, 40 or 46 resulted in a significant improvement of the stability of the corresponding polypeptide during prolonged storage for one week at room temperature (RT) over Protein Q (i.e. SEQ ID NO:1) As used herein, RT is defined as a range from 20°C to 25°C. In this instance, protein stability is evaluated by measuring the band intensity of an ultrafrozen aliquot (- 80C, referred as control t=0), which is compared to the band intensities of aliquots stored at RT, 4°C, and -20°C over a 7-day period. To assess their protein stability over prolonged storage at different temperatures, liquid compositions comprising the control Protein Q (i.e. SEQ ID NO:1), or the novel polypeptide variants (i.e. SEQ ID NOs: 5-8) were dissolved in buffered saline contaning arginine and boric acid as described previously (Cacheiro-Llaguno, Cristina, et al. "Vaccination with LetiFend® reduces circulating immune complexes in dogs experimentally infected with L. infantum." Vaccine 38.4 (2020): 890-896). The samples were first subjected to electrophoretic separation under reducing conditions using SDS-PAGE, followed by visualization via Oriole staining or by Western blot with an antibody against Protein Q, as described in the experimental part (Example 1). The band intensities of all tested samples were quantified as ratios relative to the intensity of the t=0 control for each respective group, wherein the band intensity of each t=0 control was represented as 100%. The sample groups represented either Protein Q or any of the novel polypeptide Variants 2-5. Additionally, protein degradation across various storage times and temperatures was measured by subtracting the percentage intensity of each sample from the initial control (t=0) for each sample group. Thus, protein stability was expressed as the percentual difference between the levels of degradation measured for a control protein (i.e. Protein Q) and for a polypeptide disclosed herein.

[0222] After one week of storage at RT, Variant 2 represented by SEQ ID NO: 5, (or, equivalently by SEQ ID NOs: 40 or 46) with substituted cysteine residue at position 22 displayed 101.6 % intensity relative to its t=0 control. In comparison, the band intensity of Protein Q stored at RT in the course of one week was only 96% relative to its control (t=0). (Example 1 , table 9). In contrast, the level of degradation of Protein Q with respect to its t=0 control amounted to 3.8% (Example 1 , table 10).

[0223] This means that by virtue of its substituted cysteine residue at position 22, the polypeptide disclosed herein, which is represented by SEQ ID NO: 5 (or, equivalently by SEQ ID NOs: 40 or 46) and referred to as Variant 2, manifested with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and preferably 200% improvement in stability over Proten Q during one-week storage at RT. Interestingly, the immunogenic properties of said polypeptide were not compromised by said cysteine substitution as shown in the ELISA assay in Example 4. Specifically, Variant 2 was successfully bound by antibodies present in sera of dogs infected with Canine Leishmaniasis. This effect is unexpected as the substituted cysteine residue at position 22 is located in immediate proximity to one of the linear antigens comprised in the peptide represented by SEQ ID NO: 2, wherein said linear antigen is represented by SEQ ID NO 9: ATPRSAKKAVRKSGSKSA. Thus, contrary to the expectation that any alteration in the amino acid sequence context surrounding the antigenic region would negatively impact the polypeptide’s antigenicity, this property of said polypeptide was successfully preserved.

[0224] SEQ ID NO 5 is given below:

[0225] HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVAT LIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQ YLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVG SGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEP EESDEDDFGMGGLFSSGENLYFQGLEHHHHHH

[0226] In a preferred embodiment, a polypeptide disclosed herein is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 6, 41 or 47, wherein the aminoacid at position 68 of SEQ ID NO:6, 41 or 47 is not is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bondforming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 68 of SEQ ID NO:6, 41 or 47 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0227] Such a polypeptide may comprise the amino acid sequence of SEQ ID NO:3 (peptide module2), wherein the aminoacid at position 13 of SEQ ID NO:3 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 13 of SEQ ID NO:3 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0228] Such a polypeptide may comprise an amino acid sequence having at least 80% (or at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity or similarity with SEQ ID NO:3 (peptide module 2), wherein the aminoacid at position 13 of SEQ ID NO:3 is not is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 13 of SEQ ID NO:3 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0229] The substitution of a single cysteine residue at position 68 of SEQ ID NO:6 (or, equivalently by SEQ ID NOs: 41 or 47) resulted in two unexpected improvements over Protein Q (i.e. SEQ ID NO:1) and / or Protein Q-TEV (i.e. SEQ ID NO:44), namely improved stability during prolonged storage at RT (Example 1), and improved yield following recombinant protein production (Example 2). As described earlier herein, protein stability was expressed as the percentual difference between the levels of degradation measured for a control protein (i.e. Protein Q) and for a novel polypeptide variant.

[0230] After one month of storage at RT, Variant 3 represented by SEQ ID NO: 6 (or, equivalently by SEQ ID NOs: 41 or 47) and with substituted cysteine residue at position 68 exhibited 79.2% band intensity when normalized to the t=0 control. After one month of storage at RT, the equivalent value measured for Protein Q was 75%, (Example 1 , Table 9). These band intensities corresponded to 20.7% protein degradation calculated for Variant 3 with respect to its t=0 control) . In comparison, Protein Q exhibited 24.4% degradation (Example 1 , Table 10). Therefore, Variant 3 with substituted cysteine at Position 68 of SEQ ID NO: 6 (or, equivalently by SEQ ID NOs: 41 or 47) was at least 2.5%, 5.0%, 7.5%, 10.0%, 12.5%, 15.0%, and preferably 16.5% more stable than Protein Q.

[0231] Furthermore, said cysteine substitution at position 68 of SEQ ID NO:6 (or, equivalently by SEQ ID NOs: 41 or 47) led to increased yields of the recombinant polypeptide obtained after host cell lysis and a first round of affinity purification on a Nickel column as described in Example 2. In this case, recombinant polypeptide yields were calculated as a percentage, representing the ratio of the total polypeptide recovered in the eluate after the first round of affinity purification (mg) to the total biomass (g) used for host cell lysis. When measured, the total yield of the polypeptide represented by SEQ ID NO: 6, 41 or 47 with a cysteine substitution at position 68, averaged 0.34%, while the yield obtained from Protein Q-TEV was only 0.09% (n=1).

[0232] For all experiments related to protein yield, Protein Q-TEV (i. e. SEQ ID NO: 44) was used as a control representing the same cysteine pattern of Protein Q (i. e. SEQ ID NO: 1). This is due to the fact that the labscale production process used to obtain the novel polypeptide variants of the invention differs from the commercial scale process employed for Protein Q. However, both Protein Q and Protei Q-TEV share the same cysteine pattern that differentiates them from the novel polypeptide variants. Therefore, Variant 3 represented by SEQ ID NO: 6 (or, equivalently by SEQ ID NOs: 41 or 47) with substituted cysteine at position 68 resulted in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and preferably 90%improved yield over Protein Q-TEV, which is a C-terminally tagged variant of the original Protein Q.

[0233] In addition, as shown in the ELISA test in Example 4, the antigenicity of said polypeptide was not negatively affected by the substitution of the cysteine residue at position 68. Specifically, Variant 3 represented by SEQ ID NO: 6 (or, equivalently by SEQ ID NOs: 41 or 47) with substituted cysteine at position 68, was successfully bound by antibodies present in sera of dogs infected with Canine Leishmaniasis (Example 4).

[0234] Furthermore, the median values measured for antibody binding to all of the polypeptides of the invention were higher at the late stages of infection (sick stage II (SS2); sick stage lll / IV) relative to the same values measured for Protein Q. These promising averages, though not statistically significant due to large variability, mark a positive trend and point to the possibility of further improving the antigenicity of all novel polypeptide variants.

[0235] SEQ ID NO 6 is given below: HMATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVAT LIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQ YLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVG SGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEP EESDEDDFGMGGLFSSGENLYFQGLEHHHHHH

[0236] In a preferred embodiment, a polypeptide disclosed herein is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 7, 42 or 48 wherein the aminoacid at position 158 of SEQ ID NO:7, 42 or 48 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 158 of SEQ ID NO:7, 42 or 48 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0237] Such a polypeptide may comprise the amino acid sequence of SEQ ID NO:4 (peptide module 3), wherein the aminoacid at position 28 of SEQ ID NO:4 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0238] Such a polypeptide may comprise an amino acid sequence having at least 80% (or at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity or similarity with SEQ ID NO:4 (peptide module 3), wherein the aminoacid at position 28 of SEQ ID NO:4 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 28 of SEQ ID NO:4 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0239] The substitution of a single cysteine residue at position 158 of SEQ ID NO: 7 (or equivalently, SEQ ID NOs: 42 or 48) resulted in three unexpected improvements over Protein Q (i.e. SEQ ID NO:1) or Protein Q-TEV (i.e. SEQ ID NO:44). First, the stability of the encoded polypeptide was increased during prolonged storage at RT (Example 1). When assessed as described earlier herein, the normalized band intensity of Variant 4 with a substituted cysteine residue at position 158 equaled 80% after one month storage at RT. The respective values for Protein Q were 75.5% respectively (Example 1 , Table 9). These band intensities corresponded to 20% protein degradation calculated for Variant 4 with respect to its t=0 control. In comparison, Protein Q exhibited 24.5% degradation (Example 1 , Table 10). Thus, based on the percentual difference between the measured protein degradation values, Variant 4 represented by SEQ ID NO: 7 (or equivalently by SEQ ID NOs: 42 or 48) with a substituted cysteine at position 158 manifested with at least 5%, 10%, 15%, and preferably 20% improvement in stability over Proten Q during one month storage at RT. This trend continued up to two months’ storage of Variant 4 at RT and up to 6 months’ storage at 4° and at -20°C. In an embodiment, a polypeptide comprising or consisting of a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity or similarity with SEQ ID NO: 7 (SEQ ID NO: 48 lacking a HIS-Tag), wheren the cysteine at position 158 is replaced, preferably by alanine, exhibits an improved protein stability during prolonged storage at RT relative to Protein Q (SEQ ID NO: 1) or to a Protein Q variant with an alternative or no cysteine substitution. Herein, prolonged storage at RT is understood to encompasses at least 1 week, at least 1 month, and preferably up to 2 months. As shown in Example 1 , Figures 2A-D, such polypeptide is at least 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and preferably, 100% more stable than Protein Q (SEQ ID NO: 1) when stored at RT for up to 2 months.

[0240] In another embodiment, a polypeptide comprising or consisting of a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity or similarity with SEQ ID NO: 7 (SEQ ID NO: 48 lacking a HIS-Tag), wherein the cysteine at position 158 is replaced, preferably by alanine, exhibits an improved protein stability during prolonged cold storage relative to Protein Q (SEQ ID NO: 1) or to a Protein Q variant with an alternitivce or no cysteine substitution. Herein, prolonged cold storage is understood to encompass at least 1 week, at least 1 month, at least 2 months, 3 months, 4 months, 5 months, and up to 6 months, wherein the polypeptide is stored at 6°C, 5°C, 4 °C, 3 °C, 2 °C, 1 °C, 0 °C, -1 °C, -5 °C, -10 °C, -15 °C, or down to -20 °C.

[0241] Next, the yield of the recombinant polypeptide after host cell lysis and first round of affinity purification was also improved compared to the counterpart control, Protein Q-TEV, which retains the original cysteine pattern of Protein Q. (Example 2). Recombinant polypeptide yield was calculated as described earlier herein and amounted to an average of 0.26% for Variant 4 (SEQ ID NO: 7, 42 or 48 Cys158Ala), whereas the total yield obtained from Protein Q-TEV amounted to only 0.09%. Therefore, the polypeptide disclosed herein represented by SEQ ID NO: 7, 42 or 48 with substituted cysteine at position 158 resulted in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and preferably 110% improved yield over Protein Q- TEV, which is a C-terminally tagged variant of the original Protein Q and it retains the cysteine pattern of Protein Q.

[0242] Additionally, Variant 4 exhibited reduced oligomerization, as indicated by the protein band profile observed during electrophoresis under non-reducing conditions (Example 3). Oligomerization is often undesirable in recombinant protein production because it can reduce solubility, complicate purification, and introduce heterogeneity. As described in detail in Example 3, to assess protein oligomerization, samples are typically separated electrophoretically under both non-reducing and reducing conditions, to allow for comparison of protein structures in their native and denatured forms. Visualization can be achieved through staining methods, such as whole-protein Oriole staining, or Western blotting with antibodies specific for Protein Q. Under non-reducing conditions, disulfide bonds and other non-covalent interactions are preserved, maintaining the native state of the protein. However, under reducing conditions, these bonds are disrupted, causing supramolecular assemblies to dissociate. By comparing the band intensities of the different monomer fractions obtained under non-reducing conditions, the degree of protein oligomerization can be quantitatively assessed. Specifcally, the fraction of monomeric protein corresponding to Protein Q, or the novel variants 2-5 with substituted cysteines has a molecular weight of 37-50kDA, which distinguishes is from supramolecular assemblies exceeding 100kDA.

[0243] When assessed in such way, the band intensity of the monomer fraction corresponding to Variant 4 with a substituted cysteine residue at position 158 equaled 94%, whereas the band intensity of the monomer fraction of Protein Q and equaled 17%. Based on the percentual difference in intensity bands of monomer fractions, Variant 4 with substituted cysteine at position 158 represented by SEQ ID NO:7 (or equivalently SEQ ID NOs; 42 or 48) was at least 10%, 20%, 30%, 40%, 50%, 60%, 70% and preferably 80% times purer than Protein Q.

[0244] Despite reducing the purity of recombinant proteins, oligomerization may positively impact the antigenicity and immunogenicity of certain (poly)peptide antigens, mainly by increasing their affinity to T / B cell receptors and the potency of antibody responses as described previously (Metz, S. I / IZ, Thomas, A., Brackbill, A. et al. Oligomeric state of the ZIKV E protein defines protective immune responses. Nat Commun 10, 4606 (2019). Unexpectedly, the reduced oligomerization of Variant 4 with substituted cysteine at position 158 and represented by SEQ ID NO: 7 (or equivalently SEQ ID NOs: 42 or 48) did not negatively impact the polypetide’s antigenicity as demonstrated by the ELISA assay described in Example 4.

[0245] SEQ ID NO:7 is given below:

[0246] HMATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATL IAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQY LAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGS GSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPE ESDEDDFGMGGLFSSGENLYFQGLEHHHHHH

[0247] In a preferred embodiment, a polypeptide disclosed herein is represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NO: 8, wherein the aminoacids at positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 are not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bondforming capability or a derivative thereof. In a preferred embodiment, the amino acid at positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 is proline, glycine, serine, valine, threonine, and most preferably is alanine. Such a polypeptide may comprise the amino acid sequences represented by SEQ ID NO:2, 3 and 4 (peptide building blocks 1 , 2 and 3), wherein the aminoacids at position 20 of SEQ ID NO:2, position 13 of SEQ ID NO:3 and position 28 of SEQ ID NO:4 are not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2, position 13 of SEQ ID NO:3 and position 28 of SEQ ID NO:4 are proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0248] Such a polypeptide may comprise an amino acid sequence having at least 80% (or at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity or similarity with the aminoacid sequences represented by SEQ ID NO:2, 3 and 4 (peptide building blocks 1 , 2 and 3), wherein the aminoacids at position 20 of SEQ ID NO:2, position 13 of SEQ ID NO:3 and position 28 of SEQ ID NO:4 are not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bondforming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 20 of SEQ ID NO:2, position 13 of SEQ ID NO:3 and position 28 of SEQ ID NO:4 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0249] The substitution of at least three cysteine residues at positions 22, 68 and 158 of SEQ ID NO: 8 (or equivalently SEQ ID NOs: 43 or 49) resulted in three unexpected improvements over Protein Q and / or Protein Q-TEV. First, the stability of the corresponding Variant 5 was dramatically increased during prolonged storage at RT. When assessed as described earlier herein, the normalized band intensity of Variant 5 with substituted cysteine residues at positions 22, 68 and 158 equaled 110% after one month storage at RT. The value for Protein Q was 75.5%. These band intensities corresponded to 0% protein degradation calculated for Variant 5 with respect to its t=0 control (Example 1 , Table 9). In comparison, Protein Q exhibited 24.5% degradation (Example 1 , Table 10). Thus, based on the percentual difference between the measured protein degradation values, Variant 5 represented by SEQ ID NO: 8 (or equivalently by SEQ ID NOs: 43 or 49) with substituted cysteine residues at positions 22, 68 and 158 manifested with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and preferably 100% improvement in stability over Proten Q during one month storage at RT. In addition, Variant 5 manifested with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and preferably 100% more stable than Protein Q-TEV after one month of storage at RT. This trend continued up to two months’ storage of Variant 5 at RT and up to six-month storage at 4° and at -20°C. In an embodiment, a polypeptide comprising or consisting of a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity or similarity with SEQ ID NO: 8 (SEQ ID NO: 49 lacking a HIS-Tag), wheren the cysteine at positions 22, 68 and 158 is replaced, preferably by alanine, exhibits an improved protein stability during prolonged storage at RT relative to Protein Q (SEQ ID NO: 1) or to a Protein Q variant with an alternative or no cysteine substitution. Herein, prolonged storage at RT is understood to encompass at least 1 week, at least 1 month, and preferably up to 2 months. As shown in Example 1 , Figures 2A-D, such polypeptide is at least 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and preferably, 100% more stable than Protein Q (SEQ ID NO: 1) when stored at RT for up to 2 months.

[0250] In another embodiment, a polypeptide comprising or consisting of a sequence having at least at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity or similarity with SEQ ID NO: 8, wheren the cysteine at positions 22, 68 and 158 is replaced, preferably by alanine, exhibits an improved protein stability during prolonged cold storage relative to Protein Q (SEQ ID NO: 1) or to a variant with an alternative or no cysteine substitution. Herein, prolonged cold storage is understood to encompass at least 1 week, at least 1 month, at least 2 months, 3 months, 4 months, 5 months, and up to 6 months, wherein the polypeptide is stored at 6 °C, 5 °C, 4 °C, 3 °C, 2 °C, 1 °C, 0 °C, -1 °C, -5 °C, -10 °C, -15 °C, or down to -20 °C. Next, the yield of the recombinant polypeptide after host cell lysis and first round of affinity purification was also improved over the control Protein Q-TEV (Example 2). Recombinant polypeptide yield was calculated as described earlier herein and amounted to an average of 0.18% for the polypeptide disclosed herein (SEQ ID NO: 8, 43 or 49 Cys22Ala, Cys22Ala, Cys158Ala), whereas the total yield obtained from Protein Q-TEV amounted to only an average of 0.09%. Therefore, the polypeptide disclosed herein represented by SEQ ID NO: 8 with at least three substituted cysteine residues resulted in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and preferably 67% improved recombinant protein yield over Protein Q-TEV. Additionally, Variant 5 exhibited no detectable oligomerization, as indicated by the protein band profile observed during electrophoresis under non-reducing conditions. (Example 3).

[0251] When monomer band intensity was measured on gel under non-reducing conditions, the monomer fraction of Variant 5 equaled 100%, whereas the monomer fractions of Protein Q 17% and. In addition, Variant 5 also outperformed Variants 2-4, which forwhich the monomer fraction equaled 73%, 36%, 94%, respectively. Therefore, Variant 5 represented by SEQ ID NO:8 (or equivalently by SEQ ID NOs: 43 or 49) with substituted cysteines at positions 22, 68 and 158 was at least 10%, 20%, 30%, 40%, 50%, 60%, 70% and preferably 83% purer than Protein Q. Unexpectedly, the reduced oligomerization of Variant 5 did not negatively impact antigenicity as demonstrated by the ELISA assay described in Example 4.

[0252] SEQ ID NO:8 is given below: HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATL IAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQY LAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGS GSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPE ESDEDDFGMGGLFSSGENLYFQGLEHHHHHH In a preferred embodiment, the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with any one of SEQ ID NO:7 orSEQ ID NO:8, wherein position 158 of SEQ ID NO: 7 or of SEQ ID NO: 8 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the cysteine at position 158 of of SEQ ID NO: 7 or of SEQ ID NO: 8 is substituted with alanine.

[0253] As described earlier herein, cysteine replacement at position 158 of SEQ ID NO: 7 and / or of SEQ ID NO: 8 does not result in a decreased immunogenicicty of the corresponding polypeptides. On the contrary, Example 6 and Figures 6A and B show that in vivo immunisation of test mice with Variant 4 (SEQ ID NO: 7and / or SEQ ID NO: 48 lacking a HIS-Tag) and Variant 5 (SEQ ID NO:8 and / or SEQ ID NO: 49 lacking a HIS-Tag), wherein the cysteine at position 158 in both polypeptides is replaced, preferably with alanine, elicits a humoral response comparable to the one induced by Protein Q (SEQ ID NO: 1). Surprisingly, by virtue of the replaced cysteine at position 158, both polypeptides (e. g. represented by SEQ ID NO: 7 (SEQ ID NO: 48 lacking a HIS-Tag) and SEQ ID NO: 8 (SEQ ID NO: 49 lacking a HIS-Tag) were capable of more potent immune cell activation relative to Protein Q or to variants comprising an alternative cysteine replacement (e.g. Variant 3 / SEQ ID NOs: 6 and / or 47 with replaced cysteine at position 68). As understood herein, immune cell activation is represented as the percentual difference between the subset of activated CD4+ and / or CD8+ T-cells measured by flow cytometry in splenocytes isolated from immunized mice and stimulated with Protein Q (SEQ ID NO:1), Protein Q-TEV (SEQ ID NO: 44) and with novel Protein Q variants 3-5 (SEQ ID NOs: 6-8 and / or versions lacking the HIS-Tag represented by SEQ ID NOs 47-48, respectively). Specifically, Example 7 and Figure 7 show that splenocytes obtained from mice immunized with Variant 4 (SEQ ID NO: 7 and / or SEQ ID NO: 48 lacking a HIS-Tag) and 5 (SEQ ID NO: 8 and / or SEQ ID NO: 49 lacking a HIS-Tag) showed higher activation of CD4+ and / or CD8+ T cells compared to other Protein Q variants as early as 16 hours post-induction (see Figures 7B-C).

[0254] Thus, in one embodiment a polypeptide comprising or consisting of an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with any one of SEQ ID NOT (and / or SEQ ID NO: 48 lacking a HIS-Tag) or SEQ ID NO:8 (and / or SEQ ID NO: 49 lacking a HIS-Tag) with replaced cysteine at position 158 is capable of inducing a 10%, 20%, 30%, 40%, and preferably 50% more potent immune cell activation relative to Protein Q or to other Protein Q variants that lack the same cysteine replacement. In a particular embodiment, the immune cell is a CD4+ and / or a CD8+ T-cell.

[0255] In yet another embodiment, a peptide or polypeptide comprising or consisting of an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with SEQ ID NOT or 8 with replaced cysteine at position 158 exhibits an improved protein stability during prolonged storage at room temperature relative to Protein Q (SEQ ID NO: 1) or to a variant with an alternative or no cysteine substitution. As shown in Example 1 , Figures 2A-D, such polypeptide is at least 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and preferably, 100% more stable than Protein Q (SEQ ID NO: 1) when stored at RT for up to 2 months.

[0256] In yet another embodiment, a polypeptide comprising or consisting of an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with any one of SEQ ID NO:7 (SEQ ID NO: 48 lacking a HIS-Tag) or SEQ ID NO:8 (SEQ ID NO: 49 lacking a HIS-Tag) with replaced cysteine at position 158 is exhibits an improved protein stability during prolonged cold storage relative to Protein Q (SEQ ID NO: 1) or to a variant with an alternative or no cysteine substitution. Herein, prolonged cold storage is understood to encompass at least 1 week, at least 1 month, at least 2 months, 3 months, 4 months, 5 months, and up to 6 months, wherein the polypeptide is stored at 6°C, 5°C, 4 °C, 3 °C, 2 °C, 1 °C, 0 °C, -1 °C, -5 °C, -10 °C, -15 °C, or down to -20 °C.

[0257] In some embodiments, the identity or the similarity of the peptide or polypeptide disclosed herein is assessed over the full length of their sequence. The terms “sequence identity”, “sequence similarity” and the methods for assessment thereof are explained in the section of the description entitled “General definitions”.

[0258] In some embodiments, the peptide or polypeptide described in a first aspect of the invention may comprise a recognition site for proteolytic cleavage mediated by a protease, preferably a cysteine protease, and more preferably a protease derived from Tobacco Etch Virus (TEV). A protease recognition site is a sequence within a (poly)peptide that directs the cleavage activity of a specific protease, enabling controlled processing or removal of protein segments, preferably controlled removal of purification tags. A consensus recognition site for such protase may be represented by the aminoacid sequence ENLYFQJ.G (SEQ ID NO: 39), wherein the cleavage occurs between the glutamine (Q) and glycine (G) residues. Such recognition site may be positioned at the N- or at the C-terminus of the peptide or polypeptide, most preferably at the C-terminus. A person skilled in the art will appreciate that variations derived from said recognition site might be used interchangeably. In a preferred embodiment, the polypeptides of the invention comprise a C-terminal recognition site for TEV protease and are represented by an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NOs 40, 41 , 42 and 43, wherein:

[0259] • Position 22 of SEQ ID NQ:40 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 22 of SEQ ID NQ:40 is proline, glycine, serine, valine, threonine, and most preferably is alanine , • Position 68 of SEQ ID NO:41 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 68 of SEQ ID NO:41 is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0260] • Position 158 of SEQ ID NO: 42 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 1588 of SEQ ID NO:42 is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0261] • Positions 22, 68 and 158 of SEQ ID NO: 43 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at positions 22, 68 and 158 of SEQ ID NO:43 is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0262] SEQ ID NO 40 is given below:

[0263] HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVAT LIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQ YLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVG SGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEP EESDEDDFGMGGLFSSGENLYFQG

[0264] SEQ ID NO 41 is given below:

[0265] HMATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVAT LIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQ YLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVG SGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEP EESDEDDFGMGGLFSSGENLYFQG

[0266] SEQ ID NO 42 is given below:

[0267] HMATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATL IAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQY LAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGS GSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPE ESDEDDFGMGGLFSSGENLYFQG SEQ ID NO 43 is given below:

[0268] HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATL IAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQY LAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGS GSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPE ESDEDDFGMGGLFSSGENLYFQG

[0269] In some embodiments, the peptide or polypeptide described in a first aspect of the invention does not comprise a protein purification tag, most preferably a Polyhistidine (His) tag. As used herein, a “protein purification tag” or “affinity tag” refers to a short aminoacid sequence that is fused to a target peptide or polypeptide to assist in its isolation and purification from a complex mixture. The tag enables the target protein to be selectively bound and separated using affinity chromatography or other specific purification methods. Protein purification tags are typically fused to either the N- or C-terminus of the target protein and can greatly enhance the efficiency of purification, yielding a more concentrated and pure form of the protein of interest. A His-tag (Polyhistidine tag) comprises a sequence of several histidine residues. Histidine residues may bind to metal ions such as nickel or cobalt, allowing purification through immobilized metal affinity chromatography (IMAC). In one embodiment, a His-tag may be represented by the aminoacid sequence HHHHHH (SEQ ID NO: 45). Other, non-limiting examples of protein purification tags include a GST-tag (Glutathione S-transferase) binding to glutathione, for purification using glutathione affinity chromatography, a FLAG-tag that binds to specific anti- FLAG antibodies for antibody-based purification, and a Strep-tag or Strep-Tactin for purification using streptavidin-based affinity systems. In a preferred embodiment, the polypeptides of the invention lack a C-terminal HIS-tag and are represented by an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to SEQ ID NOs 46, 47, 48 and 49, wherein:

[0270] • Position 22 of SEQ ID NO:46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 22 of SEQ ID NO:46 is is replacedproline, glycine, serine, valine, threonine, and most preferably is alanine,

[0271] • Position 68 of SEQ ID NO: 47 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 68 of SEQ ID NO:47 is proline, glycine, serine, valine, threonine, and most preferably is alanine , • Position 158 of SEQ ID NO: 48 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at position 158 of SEQ ID NO:48 is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0272] • Positions 22, 68 and 158 of SEQ ID NO: 49 are not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof. In a preferred embodiment, the amino acid at positions 22, 68 and 158 of SEQ ID NO:49 are proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0273] SEQ ID NO 46 is given below:

[0274] HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVAT LIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQ YLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVG SGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEP EESDEDDFGMGGLFSSGENLYFQ

[0275] SEQ ID NO 47 is given below

[0276] HMATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVAT LIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQ YLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVG SGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEP EESDEDDFGMGGLFSSGENLYFQ

[0277] SEQ ID NO 48 is given below

[0278] HMATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATL IAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQY LAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGS GSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPE ESDEDDFGMGGLFSSGENLYFQ

[0279] SEQ ID NO 49 is given below HMATPRSAKKAVRKSGSKSAKAGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAA QSGKKRARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSA PEFGSSRPMSTKYLAAYALASLSKASPSQADVEAIAKAVHIDVDQATLAFVMESVTGRDVATL IAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQY LAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGS GSAAPAGAVSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPE ESDEDDFGMGGLFSSGENLYFQ

[0280] A nucleic acid molecule

[0281] In a second aspect, the invention provides a nucleic acid encoding the peptide or polypeptide described in the first aspect. In the context of the present invention, the terms Gene or coding sequence or “nucleic acid molecule”, “gene” and “coding sequence” are used interchangeably and are defined in the part of the description entitled “General definitions”.

[0282] In a particular embodiment, the nucleic acid molecule described in the second aspect of the invention is an RNA molecule, preferably messenger RNA (mRNA).

[0283] Such mRNA molecule may comprise ribonucleic acid sequence variants encoding the (poly)peptide(s) represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, or any combination thereof, wherein:

[0284] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0285] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0286] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0287] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0288] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0289] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0290] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine.

[0291] Such mRNA molecule may be comprised of functional regions, including the 5' cap, the 3' poly(A) tail, the open reading frame (ORF) of a coding sequence, and 3' untranslated regions (UTRs). The mRNA molecule may be produced in a cell-free system, preferably by in vitro transcription.

[0292] The mRNA molecule may be modified or unmodified. A modified (m)RNA may also comprise modified sugar, base and / or or linkage that (i) are synthetic, naturally occurring, and non- naturally occurring, and (ii) have similar binding properties as a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and morpholino structures.

[0293] A modified (m)RNA may be self-amplifying and may comprise nonstructural polyproteins isolated from alphavirus. Said non-structural polyproteins function as replicases allowing the saRNA to replicate to a high titer. A modified mRNA may have a circular conformation, wherein the circular mRNA is generated by back-splicing, in which the 3'-end of an exon ligates to the 5'-end of its own or an upstream exon through a 3', 5' phosphodiester bond, thus forming a closed structure.

[0294] Nucleic acid construct

[0295] In a third aspect, the invention provides a nucleic acid construct comprising the nucleic acid molecules described in the second aspect of the invention.

[0296] In the context of the invention, the terms “construct,” “vector,” and “plasmid” are used interchangeably to refer to a polynucleotide vehicle introducing genetic material into a cell. Vectors may be linear or circular and may integrate into a target genome of a host cell or replicate episomally. A vector may comprise a recombinant or synthetic "expression cassette," which refers to a polynucleotide containing regulatory sequences operably linked to a selected polynucleotide to facilitate its expression in a host cell.

[0297] A “targeting vector” refers to a recombinant DNA construct that generally includes specific DNA regions homologous to the genomic DNA surrounding critical parts of a target gene or sequence. Once introduced into a host cell, the targeting vector is integrated into the cell's genome by means of homologous recombination. Optionally, a targeting vector may include a selection cassette with a selectable marker inserted into the target gene. Targeting regions near or within the target gene may be used to regulate gene expression.

[0298] An “episomal plasmid” is a type of plasmid that can replicate independently of the host cell's chromosomal DNA and exists as an extrachromosomal circular DNA molecule within the cell. Unlike “targeting vectors”, which integrate into the host genome, episomal plasmids remain separate but can still replicate autonomously, relying on the host’s cellular machinery.

[0299] Episomal plasmids may carry genes of interest, such as antibiotic resistance markers or recombinant (poly)peptides, and maintain high copy numbers in the host cell. Antibiotic resistance markers may include genes encoding reistsance against antibiotics used as selection markers such as ampicillin, kanamycin, chloramphenicol, tetracycline, hygromycin B, zeocin, streptomycin, blasticidin, puromycin, and carbenicillin. Some episomal plasmids can be maintained stably through multiple cell divisions without integrating into the host genome, making them useful for protein expression.A cell-free in vitro transcription construct may be used for the production of mRNA. Such a construct typically comprises a promoter suitable for in vitro transcription (e.g., the T7, T3, or SP6 promoter), operably linked to a nucleotide sequence encoding an mRNA of interest. This construct may be used in a cell-free system where RNA polymerase is employed to transcribe the mRNA in vitro. The in vitro transcribed mRNA may then be used for further applications such as translation or direct administration into cells for gene expression.

[0300] A cell

[0301] In a fourth aspect, the invention provides a cell comprising the nucleic acid molecule and / or, the nucleic acid construct described in previous aspects of the invention, and / or expressing the (poly)peptide(s) disclosed in a first aspect of the invention.

[0302] In the context of the present invention, a cell generally refers to a biological cell as the basic structural and functional unit of a living organism. As described herein, a “cell” might have the meaning of a “host cell” , “cell factory” or “expression chassis”, wherein said cell is used to produce the (poly)peptides represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, wherein:

[0303] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine, • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0304] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0305] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0306] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0307] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0308] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof , preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0309] In a preferred embodiment, a prokaryotic cell carries an episomal plasmid with an expression cassette comprising a selection marker and a nucleotide sequence encoding any one of SEQ ID NOs: 2-8, 40-43, 46-49, operably linked to a regulatory element facilitating (poly)peptide expression, wherein:

[0310] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0311] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0312] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0313] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0314] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0315] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0316] Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine. Said prokaryotic cell may be a bacterial cell, preferably an engineered bacterial strain including but not limited to engineered strains of the Escherichia coli species. The term “engineered” encompasses all means of bacterial strain engineering including classical mutagenesis, genomic engineering and / or strain screening and selection that are used to optimize the synthesis and purification of recombinant (poly)peptides. Non-limiting examples of such bacterial strains may include E. coli XL1 Blue and E.coli BL21 DE3. A selection marker can be represented by an antibiotic resistance gene conferring resistance of the host cell to a range of antibiotics such as ampicillin, kanamycin, chloramphenicol, tetracycline, hygromycin B, zeocin, streptomycin, blasticidin, puromycin, and carbenicillin.

[0317] In a particular embpodiment, the invention provides a cell, wherein said cell is an eukaryotic cell, preferably an insect, yeast, fungal, mammalian or a plant cell.

[0318] An eukaryotic cell may be an yeast cell including but not limited to Saccharomyces cerevisiae, Pichia pastoris (also Komagataella phaffii), Yarrowia lipolytica, Kluyveromyces lactis, Schizosaccharomyces pombe, Candida utilis, Hansenula polymorpha, Zygosaccharomyces bailii, Ogataea polymorpha. An eukaryotic cell may be a fungal cell including but not limited to Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, Rhizopus oryzae, Fusarium venenatum, Mucor circinelloides, Neurospora crassa. An eukaryotic cell may be an insect cell including but not limited to Spodoptera frugiperda (Sf9, Sf21), Trichoplusia ni (Tni), Drosophila melanogaster (S2), Bombyx mori (Bm5). An eukaryotic cell may be a mammalian cell culture cell including but not limited to Chinese Hamster Ovary (CHO), Human Embryonic Kidney (HEK293), Baby Hamster Kidney (BHK), Vero (African green monkey kidney), Madin-Darby Canine Kidney (MDCK), NSO (mouse myeloma), Jurkat (human T lymphocyte).

[0319] A plant cell may be selected from, but is not limited to, a leaf cell, a callus cell, a suspension cell, or a hairy root cell, derived from various plant species. Non-limiting examples of such plant species include soybean (Glycine max), maize (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), tobacco (Nicotiana tabacum), and wheat (Triticum aestivum). For instance, leaf cells, such as those from Nicotiana benthamiana, are frequently used for transient expression systems, particularly in agroinfiltration, allowing rapid and high-yield recombinant protein production. In another non-limiting example, suspension-cultured cells, such as those derived from Nicotiana tabacum, may be grown in bioreactors for scalable and continuous recombinant protein production. Callus cells, which are dedifferentiated plant cells, offer a versatile system for genetic manipulation and sustained production, while hairy root cells, induced by Agrobacterium rhizogenes, provide high metabolic activity and stable expression. Additionally, in some instances, chloroplasts extracted from these plant cells may also be employed, offering high recombinant protein yield.

[0320] An eukaryotic cell of the invention may carry an episomal plasmid with an expression cassette comprising a selection marker and a sequence encoding or having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with any one of SEQ ID NOs: 2-8, 40-43, 46-49, which are operably linked to a regulatory element facilitating (poly)peptide expression, wherein:

[0321] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0322] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0323] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0324] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0325] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0326] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0327] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0328] An eukaryotic cell of the invention may be modified with a targeting vector carrying an expression cassette that is integrated into the cell's genome by means of homologous recombination. Said expression cassette may comprise a sequence encoding or having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity with any one of SEQ ID NOs: 2-8, 40-43, 46- 49, operably linked to a regulatory element or located in the proximity of an endogenous regulatory element, and / or a selection marker, wherein:

[0329] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0330] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0331] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0332] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0333] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine, • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0334] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereo, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0335] Said selection marker may be an antibiotic resistance gene or a visual repoter. An eukaryotic cell of the invention might be transfected with a mRNA or with a DNA molecule encoding a (poly)peptide represented by anaminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, wherein:

[0336] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0337] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0338] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0339] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0340] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0341] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine, • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0342] An eukaryotic cell of the invention may be used for molecular farming purposes, wherein molecular farming is defined the use of plants or plant cells as bioreactors for the large-scale production of recombinant proteins, including but not limited to, therapeutic proteins, vaccines, antibodies, and industrial enzymes. Plant species commonly utilized in molecular farming include, but are not limited to, soybean (Glycine max), maize (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), tobacco (Nicotiana tabacum), and wheat (Triticum aestivum). In a non-limiting example, transient expression systems utilizing Nicotiana benthamiana leaf cells enable the rapid production of target proteins, particularly in agroinfiltration-based methods. In another embodiment, stable transformation of plant species such as rice or maize allows for the accumulation of recombinant proteins in seeds, providing the advantage of long-term storage and stability. Additionally, suspension-cultured plant cells, such as those from Nicotiana tabacum, are employed in bioreactors for scalable and controlled production of recombinant proteins. Chloroplast transformation offers yet another method, where chloroplasts from plants like tobacco are engineered to produce high levels of protein with the added benefit of genetic containment.

[0343] A composition

[0344] In a fifth aspect, the invention provides a composition comprising the (poly)peptide, the nucleic acid, the nucleic acid construct and / or the cell of the second, third and fourth aspect of the invention, respectively.

[0345] In a preferred embodiment, the composition is a pharmaceutical composition.

[0346] A composition may comprise the (poly)peptide represented by an aminoacid sequence having having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, or a combination thereof, wherein:

[0347] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0348] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine, • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0349] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0350] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0351] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0352] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0353] Such composition may comprise an adjuvant. Within the context of the invention, an "adjuvant" refers to a substance or combination of substances that is co-delivered with another molecule to an animal or human subject, where it functions to enhance or modulate the activity of said molecule.

[0354] A composition may comprise an mRNA molecule encoding the (poly)peptide represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, or a combination thereof, wherein:

[0355] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0356] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine, • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0357] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0358] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0359] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0360] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0361] Said mRNA may be comprised in a lipid nanoparticle including but not limited to cationic lipid nanoparticles, ionizable lipid nanoparticles or lipid nanoparticles with immunostimulatory potency. Said composition may comprise an mRNA molecule and a (lipid)carrier such as a nucleoside lipid or a protamine.

[0362] In an embodiment, the composition comprises one or more adjuvants to enhance the immune response to the peptide or polypeptide disclosed herein

[0363] In a specific embodiment, an adjuvant may stimulate and enhance the immune response triggered by the (poly)peptide represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, or a combination thereof, wherein:

[0364] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0365] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0366] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0367] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0368] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0369] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0370] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0371] Preferably, such an adjuvant is a Th1 -promoting adjuvant, which is able to elicit a T-helper 1 type of cellular immune response as described later herein. A Th1 -promoting adjuvant may be a self-adjuvating peptide., a Toll-like receptor ligand (TLR), it may be of the type of immunostimulating complexes (ISCOMs), oil emulsion, an extract of a bacterial protein, and / or a particulate. Preferably, a Th-1 promoting adjuvant may comprise an oligodeoxynucleotide (ODN), wherein said ODN comprises a non-methylated CpG, or a 3’purine-CpG-5’pyrimidine. A preferred oligodeoxynucleotide may comprise a phosphorothioate modified ODN sequence. The use of oligodeoxynucleotides with this modification is advantageous because they are more stable than unmodified oligonucleotides and are less likely to degrade once introduced into the bloodstream. An adjuvant may comprise at least one CpG motif, at least two, or at least three. Preferred sequences of the immunostimulatory ODN (5’ to 3’) may be represented by TCAACGTTGA (SEQ ID NO: 50), by GCTAGCGTTAGCGT (SEQ ID NO: 51) or by any variations thereof.

[0372] When any of the (poly)peptides of the invention are encoded by an mRNA molecule, various commonly used immunostimulatory adjuvants can be employed, including: (1) the self- adjuvanting effect of in vitro transcribed (IVT) mRNA; (2) immune-activating proteins encoded by the mRNA, such as CD70, CD40L, and TriMix-DC; (3) direct-acting adjuvants like pathogen- and danger-associated molecular patterns (e.g., TLRs, helicases, NODs, and inflammasome agonists); (4) mRNAs complexed with delivery reagents, such as protamine or lipid reagents; and (5) adjuvants that enhance dendritic cell (DC) recruitment, proliferation, and crosspresentation, such as GM-CSF and FLT3L.

[0373] In a specific embodiment, the invention provides a composition comprising a physiological adjuvant appropriate for intraperitoneal, subcutaneous or intramuscular administration to a human or animal.

[0374] A physiological adjuvant may be used in a composition to administer the mRNA or the (poly)peptide represented by an aminoacid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, or a combination thereof, wherein:

[0375] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0376] • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0377] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0378] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0379] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0380] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0381] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0382] As used herein, the term “physiological adjuvant” may include pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients.

[0383] Accordingly, the one or more pharmaceutically acceptable ingredients may be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients may be found in standard handbooks such as in Remington: The Science and Practice of Pharmacy (supra).

[0384] A medical use

[0385] In a sixth aspect, the invention provides a (poly)peptide, a nucleic acid, or a nucleic acid construct encoding said (poly)peptide, and a cell or a pharmaceutical composition comprising said (poly)peptide, for use as a medicament.

[0386] A medicament as defined herein is preferably administered parenterally, e.g. by injection or infusion by an intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial or intralesional route. A preferred administration mode of the (poly)peptide presented herein, or of a nucleic acid or a nucleic acid construct encoding said polypeptide, or of a composition comprising it is subcutaneous. The (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it is not limited to a specific mode of administration and may be administered orally, for example using a capsule or a tablet. Alternatively, the the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it might be locally administered via a catheter or a pump, or a suppository. Alternatively, the the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it may be topically administered. The formulation of the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it depends on the intended mode of administration and (therapeutic) application. A pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliverthe (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it to a subject. Such carriers may be represented by sterile water, or inert solids or excipients may be used as the carrier, usually complemented with pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like. Compositions will either be in liquid, e.g. a stabilized suspension of the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it, or in solid and / or dry forms: e.g. powder.

[0387] For oral and rectal administration, the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it can be administered in solid dosage forms, such as capsules, tablets, suppositories, and powders, or in liquid dosage forms, such as elixirs, syrups, cream, ointment and suspensions.

[0388] Another form may be a semi-solid or semi-liquid form wherein the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it is present in a liquid form in or on a solid support such as a patch.

[0389] A medicament may be combined with a pharmaceutically acceptable medium or delivery vehicle by conventional techniques known in the art. For example, the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it and optionally an adjuvant may be dissolved in Phosphate buffer saline (PBS). Methods for preparing parenterally administrable compositions are well known in the art and described in more details in various sources, including, for example, Remington’s Pharmaceutical Sciences, Ed. AR Gennaro, 20thedition, 2000, Williams & Wilkins, PA, USA.

[0390] A medicament comprising the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it can be administered intra- or epidermally. The intradermal administration route involves delivery into the dermis, whereas the epidermal administration route relies on delivery to the epidermis using microneedle arrays, typically in the form of a patch.

[0391] In a particular embodiment, the invention provides a medicament for use, wherein the medicament is for generating an immune response.

[0392] The immune response induced by the medicament of the invention is represented by the protective type of immune response as described earlier herein, and it is assessed by a set of biomolecular markers measured in immunized subjects. Said biomarkers and methods for assessment thereof are also described earlier herein.

[0393] In a particular embodiment, the invention provides a medicament for use, wherein the medicament is a vaccine.

[0394] This embodiment relates to a (poly)peptide- or an mRNA-based vaccine, wherein said vaccine is used to induce a protective immune response when administered to a human or animal subject. An antigen or antigens triggering a protective immune response may be comprised in the (poly)peptide represented by an aminoacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or similarity to any one of SEQ ID NOs: 2-8, 40-43, 46-49, or a combination thereof, wherein

[0395] • Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine, • Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is an alanine,

[0396] • Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0397] • Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0398] • Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0399] • Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine,

[0400] • Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by anamino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, preferably is proline, glycine, serine, valine, threonine, and most preferably is alanine

[0401] In another instance, said antigen or antigens may be comprised in a pharmaceutical composition. Alternatively, an immune response may be triggered by a mRNA molecule encoding the antigenic determinats of said (poly)peptide(s), and / or by a pharmaceutical composition comprising said mRNA molecule.

[0402] As defined earlier herein, a protective immune response may be represented by the presence of circulating antibody (humoral immunity), the actions of activated T-and B cells(cell-mediated immunity) or a combination of these factors as defined earlier herein.

[0403] In a particular embodiment, the invention provides a medicament for use, wherein the medicament is a therapeutic and / or prophylactic vaccine.

[0404] A therapeutic vaccine is preferably administered in a therapeutically effective dose, i.e. one that will increase the ability of the human or animal immune system to fight an infection and / or a disease. Preferably, a therapeutically effective dose of a medical preparation of the (poly)peptide presented herein, or of a nucleic acid or a nucleic acid construct encoding said polypeptide, or of a composition comprising it is capable of eliciting a protective immune response as defined earlier herein. A dose comprising the (poly)peptide presented herein, or a nucleic acid or a nucleic acid construct encoding said polypeptide, or a composition comprising it is therapeutically effective when it is able to elicit an immune response in a treated subject. Preferably, this means that it is able to:

[0405] • promote or trigger a immune response against a given antigen comprised in the invention and / or that

[0406] • it is able to prevent and / or delay the development of associated symptomatology and / or

[0407] • it induces a significant reduction of the parasite load in a dermal and / or mucosal lesion and / or

[0408] • it induces a significant reduction of the parasite load in an ear and / or in a draining lymph node (DLN), which preferably draines any of these infected regions (dermal, mucosal region, ear), as well as in an internal organ such as liver, spleen, bone marrow, kidney, brain, etc

[0409] In a particular embodiment, the invention provides a medicament for use in the treatment and / or prevention of leishmaniasis in a human or animal. An animal which is encompassed within the scope of the invention includes a mammal, preferably a dog. The invention provides a use of the mRNA or (poly)peptide represented presented herein as a 1) preventive (prophylactic), 2) post exposure / infection or 3) therapeutic / curative vaccine.

[0410] A leishmaniasis may encompass cutaneous, mucocutaneous and, most preferably, visceral leishmaniasis. Furthermore, a leishmaniasis infection may be caused by several Leishmania species, which are highly conserved. Such species may be represented by L. major, L. infantum, L. chagasi, L amazonensis, L. braziliensis or L. Mexicana.

[0411] General definitions:

[0412] Proteins and amino acids

[0413] The terms "protein" or "peptide" or "polypeptide" or “peptde” or “amino acid sequence” refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. In amino acid sequences as described herein, amino acids or “residues” are denoted by three-letter symbols. These three-letter symbols as well as the corresponding one-letter symbols are well known to a person of skill in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (He) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine. A residue may be any proteinogenic amino acid, but also any non-proteinogenic amino acid such as D-amino acids and modified amino acids formed by post-translational modifications, and also any non-natural amino acid.

[0414] Peptidomimetic

[0415] As used herein, a “peptidomimetic” (alternatively referred to as a ‘’mimetic”) is understood to encompass all compounds whose essential elements mimic a natural peptide and which retain the ability to interact with the biological target and exert the natural peptide’s biological activity. Peptidomimetics typically arise either from modification of an existing peptide, or by designing similar systems that mimic peptides, such as peptoids and p-peptides. Structures and synthesis of peptidomimetics are for instance described in William D. Lubell (ed.), Peptidomimetics I and II, Topics in Heterocyclic Chemistry (Book 48), Springer 1 st ed., XVI, 310 p (2017); Trabocchi A. Chapter 6 Principles and applications of small molecule peptidomimetics, Small Molecule Drug Discovery Methods, Molecules and Applications, pp 163-195, Elsevier (2020); Vagner et al., Curr OpinChem Biol. 12(3): 292-296 (2008), all of which incorporated herein by reference in their entireties.

[0416] Biomarker

[0417] In the context of the present invention, a "biomarker" refers to a measurable biological molecule, substance, or characteristic that is indicative of a normal or pathological biological process, condition, or disease, or the response of an organism to a therapeutic intervention. Biomarkers can be proteins, peptides, nucleic acids, metabolites, or cells that are detected and quantified in biological samples such as blood, tissue, or other bodily fluids. For example, a biomarker may be used to assess disease progression, diagnose a condition, predict therapeutic response, or monitor the effectiveness of a treatment. Biomarkers may also serve as indicators of immune responses, cellular processes, or molecular signatures specific to a condition or treatment.

[0418] Linker

[0419] In the context of the invention, the term “linker" refers to a short sequence of amino acids or nucleotides used to connect two or more protein domains, peptides, or polyppetides in a recombinant construct. A linker may provide flexibility and spatial separation between functional elements to ensure proper folding and activity. For example, a linker is used in fusion proteins, such as in green fluorescent protein (GFP)-tagged proteins, where a flexible Glycine-Serine (Gly-Ser) linker connects GFP to a target protein, permitting fluorescence without disrupting the target protein’s function. Aletrnatively, a linker may tether an effector molecule to an antibody, as in antibody-drug conjugates (ADCs). Additionally, a linker may provide flexibility and prevent steric hindrance, as seen in multidomain proteins such as Modular Polyketide Synthases (PKSs), where a Gly-Ser linker allows independent folding and function of the catalytic domains. Furthermore, a linker may incorporate a proteolytic cleavage site, facilitating precise separation of components, as demonstrated in recombinant insulin production, where a thrombin- cleavable linker connects the A and B chains of insulin, allowing post-synthetic cleavage to produce functional insulin. Linkers are thus used in recombinant biotechnology to optimize the spatial orientation, stability, and functionality of connected biomolecules.

[0420] Gene or coding sequence or nucleic acid molecule

[0421] “Gene” or “coding sequence” or “nucleic acid” or “nucleic acid molecule” refers to a DNA or RNA region (the transcribed region) which “encodes” a particular polypeptide such as protein Q or a polypeptide derived therefrom or a peptide that can be used in a variant of protein Q. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide when placed under the control of an appropriate regulatory region, such as a promoter. A gene may optionally comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, an intron, a coding sequence and a 3’ nontranslated sequence, comprising a polyadenylation site or a signal sequence. A chimeric or recombinant gene (such as the ones described herein) is a gene not normally found in nature, such as a gene in which for example the promoter is not associated in nature with part or all of the transcribed DNA region, or genes comprising nucleotide sequences encoding domains from multiple polypeptides. “Expression of a gene” refers to the process wherein a gene is transcribed into an RNA and / or translated into an active protein, polypeptide or peptide.

[0422] Such nucleotides forming the nucleic acid may comprise “codons”, wherein the term “codon” refers to a triplet of nucleotides that corresponds to a specific amino acid or a stop signal during (poly)peptide synthesis.

[0423] Codon optimization

[0424] “Codon optimization”, as used herein, refers to the processes employed to modify an existing coding sequence, or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host cell. For example, to suit the codon preference of mammalian, insect, plant, or microbial cells. Codon optimization also eliminates elements that potentially impact negatively RNA stability and / or translation (e. g. termination sequences, TATA boxes, splice sites, ribosomal entry sites, repetitive and / or GC rich sequences and RNA secondary structures or instability motifs). Codon optimization may be done according to standard methods available to skilled person.

[0425] In the context of the disclosure, a polypeptide comprises an amino acid sequence. In the context of the disclosure, a nucleic acid such as a nucleic acid molecule encoding a polypeptide or fragment thereof or a peptide. A nucleic acid molecule may comprise a regulatory region.

[0426] It is to be understood that each nucleic acid molecule or polypeptide or construct as identified herein by a given Sequence Identity Number (SEQ ID NO) is not limited to this specific sequence as disclosed. Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given polypeptide or peptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% or at least 80% sequence identity with SEQ ID NO: X; ii. a nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i);

[0427] Hi. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) or (ii) due to the degeneracy of the genetic code; or, iv. a nucleotide sequence that encodes an amino acid sequence that has at least 60% or at least 80% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.

[0428] Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide or peptide comprising an amino acid sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. In the context of the application, the minimum identity or similarity in relation to a polypeptide or peptide may mean an identity or a similarity of at least 60%. In the context of the application, the minimum identity or similarity in relation to a polypeptide or peptide may mean an identity or a similarity of at least 60%.

[0429] Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage (e.g. at least 60%) with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity (or a similarity where applicable) of at least 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the given nucleotide or amino acid sequence respectively. In a preferred embodiment, sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. In other words, sequence identity is preferably calculated based on the full length of two given sequences being compared (for example of a sequence represented by a SEQ ID NO herein and of another sequence it is being compared to). Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole).

[0430] Sequence identity

[0431] "Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid or two nucleic acid sequences is typically defined by assessing their identity within a whole length SEQ ID NO as identified herein or part thereof. Part thereof in terms of comparing the identity or similarity of two or more sequences may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0432] In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference in its entirety.

[0433] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide (or polypeptide) or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water (EMBLI-EBI) using default parameters share at least a certain minimal percentage of sequence identity (as described herein).

[0434] A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) I 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) I 0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for amino acid sequences the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

[0435] Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleotide and amino acid sequences of some embodiments of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences having a certain identity with nucleic acid molecules of the disclosure. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences having a certain identity or similarity with polypeptides of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov / .

[0436] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

[0437] Table 1 : amino acid residues for conservative substitutions:

[0438] Table 2 : alternative amino acid residues for conservative substitutions

[0439] Table 3: physical and functional classifications of amino acid residues

[0440] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cysteine to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

[0441] Promoter

[0442] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes (or coding sequence), located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated depending on physiological or developmental conditions.

[0443] Operably linked “Operably linked” is defined herein as a configuration in which a control sequence such as a promoter sequence or regulating sequence is appropriately placed at a position relative to the nucleotide sequence of interest. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being “underthe control of’ the promoter.

[0444] Nucleic acid construct

[0445] An "expression construct” or "nucleic acid construct” comprises a nucleic acid molecule, such as the ones described herein, which may be expressed in a host cell. In some cases, such a construct is a viral expression construct. A viral expression construct comprises parts of a virus’ genome, as further described later herein.

[0446] Within the context of the disclosure, a host cell encompasses a cell used to make the construct or a cell to which the construct will be administered. Alternatively, a nucleic acid construct is capable of integrating into a cell's genome, e.g., through homologous recombination or otherwise. An expression construct may comprise a nucleotide sequence encoding the polypeptide or peptide described herein.

[0447] A preferred expression construct is one wherein a nucleotide sequence encoding polypeptide or a pepetide as described herein, is operably linked to a promoter as defined herein wherein said promoter is capable of directing expression of said nucleotide sequence (i.e., coding sequence) in a cell. Preferred cells are described herein.

[0448] Such a preferred expression construct is said to comprise an expression cassette.

[0449] Expression constructs disclosed herein could be prepared using recombinant techniques which result in nucleotide sequences being expressed in a suitable cell, e.g., cultured cells or cells of a multicellular organism, such as described in Ausubel et al., and in Sambrook and Green (supra).

[0450] Typically, a nucleic acid molecule or construct is used in a vector. A vector may alternatively be called an expression vector. The phrase "expression vector" generally refers to a nucleotide sequence that is capable of effecting expression of a gene in a host compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as described herein. An expression vector may optionally be suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or may be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines (such as HEK293, HEK293T (e.g., HEK293T / 17 (ATCC CRL-11268)), and HEK293F cells, and the like).

[0451] A nucleic acid molecule, construct, or vector, prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term “operably linked” has already been defined herein. DNA signal sequences may be included. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide. Generally, DNA sequences that are operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.

[0452] The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g., Sambrook and Green, supra). Additional examples have been provided earlier herein.

[0453] A transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognised by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Green, supra). For example, an expression vector which includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. In most cases, the replication system is only functional in the cell that is used to make the vector (e.g., bacterial cell as E. coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are for example described in Sambrook and Green (supra). For example, suitable expression vectors can be expressed in, yeast, e.g. S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. A cell may thus be a prokaryotic or eukaryotic host cell. A cell may be a cell that is suitable for culture in liquid or on solid media. A cell may be a cell line, e.g. a HEK293, HEK293T (e.g., HEK293T / 17 (ATCC CRL-11268)), or HEK293F cell line or a derivative thereof. In some cases, a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal. Commercial kits comprising cells for vector expression are available, for example the LV-Max system from Thermo Fisher Scientific (Waltham, MA, USA).

[0454] A vector as described herein may be selected from any genetic element known in the art which can facilitate transfer of nucleic acids between cells, such as, but not limited to, plasmids, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, and the like. A vector may also be a chemical vector, such as a lipid complex or naked DNA. "Naked DNA” or "naked nucleic acid” refers to a nucleic acid molecule that is not contained in encapsulating means that facilitates delivery of a nucleic acid into the cytoplasm of a target host cell. Naked DNA may be circular or linear (linearized DNA sequence). Optionally, a naked nucleic acid can be associated with standard means used in the art for facilitating its delivery of the nucleic acid to the target host cell, for example to facilitate the transport of the nucleic acid through the cell membrane. Transformed or engineered cells

[0455] The term "engineered cells" refers herein to cells having been engineered, e.g., by the introduction of an exogenous nucleic acid sequence as defined herein. Such a cell has been genetically modified for example by the introduction of for example one or more mutations, insertions and / or deletions in an endogenous gene and / or insertion of a nucleic acid construct in the genome. The modification may have been introduced using recombinant DNA technology. An engineered cell may refer to a cell in isolation or in culture. Engineered cells may be "transduced cells" wherein the cells have been infected with e.g. a modified virus, for example, a retrovirus may be used but other suitable viruses may also be contemplated such as lentiviruses. Non-viral methods may also be used, such as transfections. Engineered cells may thus also be "stably transfected cells" or "transiently transfected cells". Transfection refers to non-viral methods to transfer DNA (or RNA) to cells such that a gene is expressed. Transfection methods are widely known in the art, such as calcium phosphate transfection, PEG transfection, and liposomal or lipoplex transfection of nucleic acids. Such a transfection may be transient, but may also be a stable transfection wherein cells can be selected that have the gene construct integrated in their genome.

[0456] In some embodiments, an “engineered cell” has been transformed, modified or transduced to comprise a heterologous or exogenous nucleic acid molecule. In the application, the wording “engineered cell” is replaced by “modified cell” or “transformed cell” or “transduced cell”.

[0457] General

[0458] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” is replaced by “to consist essentially of’ meaning that a method, respectively component as defined herein may comprise additional step(s), respectively component(s) than the ones specifically identified, said additional step(s), respectively component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0459] The word “about” when used in association with an integer (about 10) preferably means that the value may be the given value of 10 more or less 1 of the value: about 10 preferably means from 9 to 11. The word “about” when used in association with a numerical value (about 10.6) preferably means that the value may be the given value of 10.6 more or less 0.1 of the value 10.6: about 10.6 preferably means from 10.5 to 10.7.

[0460] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included or contained, but items not specifically mentioned are not excluded. Thus, the terms 'comprising', 'comprises', 'comprised of and the like as used herein are synonymous with 'including', 'includes' or 'containing', 'contains', and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[0461] In addition, the verb “to consist” is replaced by “to consist essentially of meaning that subject matter as described herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of this disclosure. In addition, the verb “to consist” is replaced by “to consist essentially of meaning that a method as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of this disclosure.

[0462] Throughout this disclosure, the term “comprising” is replaced with the term “consisting essentially of or “consisting of.

[0463] As used herein, the singular forms 'a', 'an', and 'the' include both singular and plural referents unless the context clearly dictates otherwise. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0464] As used herein, with "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.

[0465] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

[0466] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 1 % of the value.

[0467] As used herein, the term "and / or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0468] Various embodiments are described herein. Each embodiment as identified herein may be combined together unless otherwise indicated. Titles, subtitles, headings and the likes are used herein solely for ease of reading and are not intended to limit or restrict the disclosure in any way.

[0469] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the specific methods and materials described.

[0470] Table 4: listing of the sequences present in the sequence listing:

[0471] No Name Type

[0472] Examples

[0473] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. The generalization of certain aspects and features disclosed in the below examples to the foregoing description is part of this disclosure.

[0474] General procedures to the Examples

[0475] Gene synthesis

[0476] Synthetic genes for individual proteins, each with a hexahistidine purification tag and TEV protease cleavage site, were optimized for E. coli expression and purchased from Genscript ligated into the pET-29b (+) vector at the Ndel and Xhol restriction sites. For clone verification, a small-scale DNA purification (miniprep) from 5 ml of bacterial culture from isolated colonies of each clone was performed followed by a PCR for Sanger sequencing and fragment analysis. The sequencing reaction was performed with a purified amplification product and primer at a concentration of 5uM. The enzymatic reaction was carried out with the BigDye terminator V.3.1 kit (Life Technologies), purified to remove unincorporated ddNTPs by magnetic beads, and the fluorescent products were analyzed by capillary electrophoresis with the ABI PRISM 3730 DNA analyzer (Applied Biosystems).

[0477] Protein Purification

[0478] Bacterial lysates were clarified by centrifugation at 10,000 g for 20 min and applied to a 5 mL column bed of Ni-NTA resin (Qiagen) for purification by Immobilized Metal Affinity Chromatography (IMAC). Resin was prewashed with the buffer containing 10 mM imidazole. The protein of interest was eluted using a phosphate buffer supplemented with 250 mM imidazole (pH 8) The eluates were combined, subjected to buffer exchange with PBS, and concentrated using 3-1 OK molecular weight cut-off (MWCO) centrifugal filters. The eluates were incubated overnight with TEV protease (GenScript) at 4C using a ratio of 18 pg protein to 12 III of protease. A second chromatography was performed using the same protocol described previously. The protein of interest in the flow through fraction, the non-digested leftovers in the eluted fractions and the in-process samples were collected and analyzed by SDS-PAGE 4-12% Bis-Tris under reducing conditions and visualized using Oriole fluorescent gel staining according to the manufacturer’s instructions (Biorad 161-0496)or by Western Blot with two sets of specific antibodies: 1) against PQ (i.e. SEQ ID NO: 1) and 2) against the histidine tail to confirm the purity of the proteins and the removal of the histidine tail, respectively. For the Western Blot, the primary antibodies mouse anti-PQ (house made) and anti-His (PentaHis mAb mouse IgG, Qiagen) at 1 / 5,000 and 1 / 600 dilution respectively were used and the secondary antibody antimouse IgG PA y chain specific (Sigma) at 1 / 20,000 dilution was used. The protein concentrations were determined by MicroBCA assay employing BSA as the standard.

[0479] Assessment of endotoxin content

[0480] Endotoxins content (EU / ml) was determined by a colorimetric technique based on the Limulus 25 Amebocyte Lysate assay and using an Endoscan V system (Charles River Laboratories).

[0481] Table 5: Determination of endotoxin levels in purified protein samples shows that all samples comply with established threshold values for endotoxin contamination (pass limit < 10 EU / mL.

[0482] Host Cell protein content

[0483] Host Cell protein content (ng / mL) was determined by ELISA (Cynus Tecnologies E. coli HCP #F1020) following manufacturer’s instructions.

[0484] Table 6: Determination of host cell protein content (HCP) shows that samples have overall low contamination with E. coli (Coefficient of Variation: CV).

[0485] Protein digestion

[0486] In-Gel Digestion (Stacking gel):

[0487] The protein extracts, were suspended in a volume up to 50 pl of sample buffer, and then applied onto 1 ,2-cm wide wells of a conventional SDS-PAGE gel (0.75 mm-thick, 4% stacking, and 10% resolving). Then run was stopped as soon as the front entered 3 mm into the resolving gel, so that the whole proteome became concentrated in the stacking / resolving gel interface. The unseparated protein bands were visualized by Coomassie staining, excised, cut into cubes (2 x 2 mm), and placed in 0.5 ml microcentrifuge tubes (Sanchiz A, Morato E, Rastrojo A, Camacho E, Gonzalez-de la Fuente SG, Marina A, Aguado B, Requena JM. The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations. Genes (Basel). 2020 Sep 2;11(9):1036. doi: 10.3390 / genes11091036. PMID: 32887454; PMCID: PMC7563732). The gel pieces were destained in acetonitrile:water (ACN:H2O, 1 :1), were reduced and alkylated (disulfide bonds from cysteinyl residues were reduced with 10 mM DTT for 1 h at 56 °C, and then thiol groups were alkylated with 10 mM iodoacetamide for 30 min at room temperature in darkness) and digested in situ with sequencing grade trypsin (Promega, Madison, Wl) as described by Shevchenko et al. with minor modifications (Shevchenko A, Wilm M, Vorm O, Mann M: Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996, 68:850-858). The gel pieces were shrunk by removing all liquid using sufficient ACN. Acetonitrile was pipetted out and the gel pieces were dried in a speedvac. The dried gel pieces were re-swollen in 100 mM Tris-HCI pH 8, 10mM CaCh with 60 ng / pl trypsin at 5:1 protein:enzyme (w / w) ratio. The tubes were kept in ice for 2 h and incubated at 37°C for 12 h. Digestion was stopped by the addition of 1 % TFA. Whole supernatants were dried down and then desalted onto ZipTip C18 Pipette tips (Millipore) until the mass spectrometric analysis.

[0488] In-Gel Digestion (Bands or Spots):

[0489] After drying, gel bands or spots were destained in acetonitrile:water (ACN:H2O, 1 :1), were reduced and alkylated (disulfide bonds from cysteineinyl residues were reduced with 10 mM DTT for 1 h at 56 °C, and then thiol groups were alkylated with 10 mM iodoacetamide for 30 min at room temperature in darkness) and digested in situ with sequencing grade trypsin (Promega, Madison, Wl) as described by Shevchenko et al. (Shevchenko A, Wilm M, Vorm O, Mann M: Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996, 68:850-858) with minor modifications (Santos Al, Louren o AS, Simao S, Marques da Silva D, Santos DF, Onofre de Carvalho AP, Pereira AC, Izquierdo-Alvarez A, Ramos E, Morato E, Marina A, Martinez-Ruiz A, Araujo IM. “Identification of new targets of S-nitrosylation in neural stem cells by thiol redox proteomics”. Redox Biology. 2020 May; 32:101457. DOI: 10. 1016 / j.redox.2020. 101457. PMID: 32088623; PMCID: PMC7038503). The gel pieces were shrunk by removing all liquid using sufficient ACN. Acetonitrile was pipetted out and the gel pieces were dried in a speedvac. The dried gel pieces were re-swollen in 100 mM Tris-HCI pH 8, 10mM CaChwith 12.5 ng / pl trypsin for 1 hr in an ice-bath. The digestion buffer was removed and gels were covered again with 100 mM Tris-HCI pH 8, 10mM CaCh and incubated at 37°C for 12 hr. Digestion was stopped by the addition of 1 % TFA. Whole supernatants were dried down and then desalted onto ZipTip C18 Pipette tips (Millipore) until the mass spectrometric analysis.

[0490] Reverse phase-liquid chromatography RP-LC-MS / MS analysis (Dynamic Exclusion Mode):

[0491] The desalted protein digest was dried, resuspended in 10 pl of 0.1 % formic acid and analyzed by RP-LC-MS / MS in an Easy nLC 1200 system coupled to an ion trap LTQ-Orbitrap-Velos-Pro hybrid mass spectrometer (Thermo Scientific). The peptides were concentrated (on-line) by reverse phase chromatography using a 0.1 mm x 20 mm C18 RP precolumn (Thermo Scientific), and then separated using a 0.075mm x 250 mm bioZen 2.6 pm Peptide XB-C18 RP column (Phenomenex) operating at 0.25 pl / min. Peptides were eluted using a 60-min dual gradient. The gradient profile was set as follows: 5-25% solvent B for 45 min, 25-40% solvent B for 15min, 40-100% solvent B for 2min and 100% solvent B for 18 min (Solvent A: 0,1 % formic acid in water, solvent B: 0,1 % formic acid, 80% acetonitrile in water). ESI ionization was done using a Nano-bore emitters Stainless Steel ID 30 pm (Proxeon) interface at 2.1 kV spray voltage with S-Lens of 60%. The Orbitrap resolution was set at 30.000 (Ruth Alonso, Diana Pisa, Ana Isabel Marina, Esperanza Morato, Alberto Rabano, Izaskun Rodal and Luis Carrasco: Evidence for Fungal Infection in Cerebrospinal Fluid and Brain Tissue from Patients with Amyotrophic Lateral Sclerosis. International Journal of Biological Sciences 2015; 11(5): 546- 558).

[0492] Peptides were detected in survey scans from 400 to 1600 amu (1 pscan), followed by twenty data dependent MS / MS scans (Top 20), using an isolation width of 2 u (in mass-to-charge ratio units), normalized collision energy of 35%, and dynamic exclusion applied during 60 seconds periods. Charge-state screening was enabled to reject unassigned and singly charged protonated ions.

[0493] Data processing:

[0494] PEAKS Studio 11.5: Peptide identification from raw data was carried out using PEAKS Studio v11 .5 search engine (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada). Database search was performed against Uniprot-Escherichia coli + Protein Q (SEQ ID NO: 1), PQ-TEV (SEQ ID NO: 44) and the novel polypeptide variants (v2-v5, corresponsing to SEQ ID NO: 5, Cys22Ala, SEQ ID NO: 6, Cys68Ala, SEQ ID NO: 7, Cys158Ala, and SEQ ID NO: 8, Cys22Ala, Cys68Ala, Cys158Ala, respectively) sequences fasta (4405 entries; UniProt releaseOl / 2023); decoy-fusion database). The proteins identified by LC-MS / MS after first on-column purification and after TEV treatment and second on-colum purification are shown on Table 7 and 8, respectively. The following constraints were used for the searches: tryptic cleavage after Arg and Lys (semispecific), up to two missed cleavage sites, and tolerances of 20 ppm for precursor ions and 0.6 Da for MS / MS fragment ions and the searches were performed allowing optional Met oxidation and Cys carbamidomethylation. False discovery rates (FDR) for peptide spectrum matches (PSM) and for protein was limited to 0.01 . Only those proteins with at least two unique peptides being discovered from LC / MS / MS analyses were considered reliably identified.

[0495] Table 7: Protein sequence coverage of control Protein Q-TEV and novel variants 2-5 (SEQ ID NOs: 5-8) after first on-column purification (His-tag protein fraction) identified by LC-MS / MS.

[0496] Table 8: Protein sequence coverage and protein purity of novel variants 3-5 (SEQ ID NOs: 6-

[0497] 8) identified by LC-MS / MS:

[0498] * The number of amino acids in a specific protein sequence that were found in the peptides sequenced in the LC-MS / MS study

[0499] ** All samples present E.coli protein contamination, although in different proportions. Quantification based on the sum of the areas of the peptides of a protein is an approximation ,but it can be used to give an idea of the relationship between the recombinant protein and the contaminating proteins of each preparation. The areas of the contaminating proteins have been added together to compare them with that of the protein variants and fractions.

[0500] ***V5 68% : The V5 digestion with TEV protease gave a much higher yield, hence what is retained from V5 in the leftover fraction is negligible compared to theE.coli proteins, that is why the V5 / protein ratio of E.coli isso unfavorable for Variant 5.

[0501] ELISA assay with sera of infected dogs

[0502] High-binding ELISA plates (96 well, Costar®) were coated for 30 min at 37 °C with 10OpI of sterile PBS containing 2pg / ml of Protein Q (control reference) or Protein Q-TEV and novel variants 2-5 (corresponsing to SEQ ID NO: 5, Cys22Ala, SEQ ID NO: 6, Cys68Ala, SEQ ID NO: 7, Cys158Ala, and SEQ ID NO: 8, Cys22Ala, Cys68Ala, Cys158Ala, respectively). Sera from dogs that were naturally infected with the Leshamania parasite and from healthy control dogs were diluted 1 / 200 and tested in duplicate in each plate. A 1 / 100,000 dilution goat antidog IgG H+L horseradish peroxidase conjugated from Bethyl Laboratories was used as a secondary antibody. Plates were developed with OPD substrate (with H2O2 in acetic buffer) and the optical density (OD) was read at 450 nm.

[0503] Results

[0504] Example 1 : Novel Protein Q variants exhibit better stability during prolonged storage at room temperature

[0505] All proteins, including the controls Protein Q (SEQ ID NO: 1), as well as the novel variants 2-5 (corresponsing to SEQ ID NO: 5, Cys22Ala, SEQ ID NO: 6, Cys68Ala, 4 (SEQ ID NO: 7, Cys158Ala, and 5 SEQ ID NO: 8, Cys22Ala, Cys68Ala, Cys158Ala, respectively) are shown on Figure 1A. Protein Q-TEV (SEQ ID NO: 44) and the novel polypeptide variants derived from Protein Q have a C-terminal cleavage site for Tobaco Etch Protease (TEV). The amino acid sequence of TEV is illustrated here. Proteolytic processing by TEV allows for removal of the Histidine tag, which is used during protein purification (Figure 1 B).

[0506] To evaluate the impact of substituting cysteine on protein stability during extended storage at different temperatures, a series of experiments were conducted with Protein Q, and with novel variants 2 (SEQ ID NO: 5, Cys22Ala), 3 (SEQ ID NO: 6, Cys68Ala), 4 (SEQ ID NO: 7, Cys158Ala), and 5 (SEQ ID NO: 8, Cys22Ala, Cys68Ala, Cys158Ala).

[0507] All tested proteins were incubated at room temperature (RT), 4°C, and 20°C at the following time points: one day (data not shown), one week and one month,. At each time point, aliquots stored at these temperatures were assessed against an ultrafrozen aliquot kept at -80°C, which acted as an internal baseline for protein stability (referred to as t=0).

[0508] All protein samples underwent electrophoretic separation under reducing conditions and were subsequently analyzed via Western Blot using an antibody against Protein Q, as detailed in the "General procedures to the Examples" section.

[0509] Surprisingly, the novel variants 2-5 demonstrated improved stability when stored at room temperature for extended periods of time. As early as one week, aliquots of Protein Q stored at RT began showing signs of degradation, evident as smearing when compared to their respective t=0 controls (Figure 2A). In contrast, such smearing was absent or decreased in aliquots of variants 2, 4, and 5 stored under at RT for one week (Figure 2B).

[0510] The stability of the novel variants was even more pronounced after one month storage at RT, with the exception of variant 2, whose stability began to decline under these conditions, as evidenced by smearing (Figure 2B). Meanwhile, variants 3, 4, and 5 showed no significant changes in stability up to the one-month mark at RT (Figure 2B and 2C). This suggests that most of the novel variants are more resistant to degradation during prolonged storage at room temperature compared to Protein Q.

[0511] In the case of Variant 4 (SEQ ID NO: 7) and Variant 5 (SEQ ID NO: 8), the protein is highly stable with 100% -80% intensities measured after 1 month at RT. In the case of Variant 4 (SEQ ID NO: 7), the % intensity remains at 80% even after two months’ storage at RT (see Figure 2D) . This is in sharp contrast to the intensity measured in the Protein Q reference (SEQ ID NO: 1), with values of 76% and 30% after 1 and 2 months, respectively. After longer incubation times (e.g. 3 months and 6 months) both original and novel variants stored at RT showed a marked decrease in their stability with band intensity ranges between 4 % and 40%.

[0512] At the 6-month mark, all Variants 2-5 (corresponding to SEQ ID NOs 5-8) were still stable when stored 4°C or at -20° C, albeit degraded at RT.

[0513] Table 9: Novel Variants 2-5 exhibit higher band intensity during prolonged storage at RT.

[0514] Table 10: Novel Variants 2-5 exhibit less protein degradation during prolonged storage at RT.

[0515] Example 2: Novel Protein Q variants exhibit improved yield of intermediate (His-tagged) protein:

[0516] Protein Q-TEV (i.e. SEQ ID NO: 44) as well as the novel polypeptide variants (i.e. SEQ ID NOs:5-8) were expressed in bacterial strain BL21 (DE3) grown in 1 L LB medium in baffled shake flasks. Cells were grown at 37 °C to an QD600 ~ 0.6 and then induced with 1 mM IPTG. Bacteria were harvested and subsequently lysed by sonication for 10 min with 30s pulses at 60% amplitude in a phosphate buffer supplemented with 10 mM imidazole (pH 8) .

[0517] Next, all protein variants were purified from the bacterial lysate using a 2-step Ni-NTA chromatography, combined with TEV protease treatment as detailed in the "General procedures to the Examples" section.

[0518] During the initial purification phase, the bacterial lysate was passed through a Ni-NTA column to separate the His-tagged protein fraction (+His; intermediate protein) from E. coli impurities. The purified fraction was subsequently digested with a protease to remove the histidine tail and further purified on-column by a second round of Ni-NTA

[0519] Of note, after TEV protease treatment, the cleaved final protein that lacks a His tail is collected in the flow-through whereas the uncut protein and other impurities are retained in the column due to the histidine tail. The uncut protein that carries a His tail is collected in the elution fraction for analysis. Overall, the process gave clean fractions (see supporting data: HOP and endotoxin levels on Tables 5 and 6 for protein purity) that showed protein sequence coverages >80% (Tables 7 and 8). A side-to-side analysis of the flow through and of the eluate fractions showed that novel Variant 5 (SEQ ID NO: 8) had the highest TEV cleavage efficiency. For this reason, no Protein Q signal was observed in the eluted fraction. This also correlates with the unexpected high percentage of contaminant protein calculated after protein analysis due to the low amount of V5 present in the eluate fraction, Table 8).

[0520] When assessed by microBCA after lysis and first IMAC purification, the total intermediate protein (His+) measured for Variant 3 was highest (2 mg protein / 1 L), followed by Variants 4 and 5. When expressed as a percentage of total protein obtained after first IMAC purification to host cell biomass used for lysis, the yield of Variants 3, 4 and 5 was better than values obtained for Variant 2 and for the control Protein Q-TEV (Table 11). Therefore, novel variants 3-5 resulted in improved yield over the control Protein Q-TEV.

[0521] Table 11 : Increased yield of recombinant protein for novel Protein Q variants.

[0522] Example 3: Novel Protein Q variants are less prone to oligomerization (Originally submitted Figure 3A, B and Figure 4, currently proposed figures 5A, B, C and D)

[0523] To investigate the effect of cysteine replacement on protein oligomerization, the samples were electrophoretically separated under non-reducing and reducing conditions and visualized by Oriole staining (Figure 3A, B) or by Western blot using an antibody against Protein Q (Figure 3C,D). Of note, protein oligomerization is only observable under non-reductive conditions (Figure 3B,D) and not under reductive conditions (Figure 3A,C). This is likely due to the preservation of disulfide bonds and possibly other non-covalent interactions that are essential for oligomer stability. Reductive conditions disrupt these bonds, often leading to the disassembly of oligomeric protein structures into monomers or smaller subunits, which can be visualised. Novel polypeptide variants 2-5 with cysteines partially or completely substituted with alanines showed an enrichment of monomeric forms and reduction of oligomeric forms compared to Protein Q.

[0524] Variant 4 had the highest monomer-to-oligomer ratio (Figure 3B, lane 8; Figure 3D, lane 6). In addition, similar to variant 3 the molecular weight (MW) of the oligomers of variant 4 is 80kDa- 75 kDa, whereas the oligomers formed by Protein Q have bigger sizes starting at 100kDa up to 250 kDa (Figure 3B, lane 2, 3 and 5; Figure 3D, lane 2,3 and 5).

[0525] Variant 5 does not form oligomers or any kind of high MW assemblies as evidenced by the absence of bands with MW > 50 kDa under all analysed conditions (both non-reductive and reductive conditions alike; Figure 3A,B, lane 9; Figure 3C,D, lane 7).

[0526] By comparing the band intensities of the monomer protein fractions under non-reducing conditions, the degree of protein oligomerization can be quantitatively assessed. Specifically, the fraction of monomeric protein corresponding to Protein Q or the novel variants 2-5 with substituted cysteines has a molecular weight of 37-50kDA, which distinguishes is from oligomeric assemblies exceeding 100kDA. Values representing total band intensity and percentage of the monomer and oligomer fraction for Protein Q are shown on Table 12 below. Taken together, these results seem to suggest that the novel variants are less prone to oligomerization compared to Protein Q.

[0527] Table 12: Novel variants 4 and 5 show significantly decreased oligomerization. Band intensities representing monomer fraction of PQ or the novel variants with substituted cysteines exhibit a molecular weight between 37-50kDA.

[0528] Example 4: Novel Protein Q variants retain their antigenicity

[0529] To assess their antigenicity — or their capacity to bind antibodies generated in response to Leishmania antigens — both Protein Q, and novel variants 2-5 were subjected to Enzyme- Linked Immunosorbent Assay (ELISA) using sera collected from dogs at various clinical stages of Canine Leishmaniosis (Figure 4A, B). Serum samples were collected from animals classified into the following leishmaniosis stages according to the LeishVet classification (Solano-Gallego et al., 2009, 2011) based on clinical signs, clinical-pathologic abnormalities and serologic status: healthy non-infected (H, n=5); healthy infected (HI, n=5); sick stage I (SS1 , n=5); sick stage II (SS2, n=5); sick stage lll / IV (SS3 / SS4, n=5). The ELISA assay was performed as described in the "General procedures to the Examples" section.

[0530] The data show the O.D corresponding to the specific binding of each protein variant to the IgG antibodies in the sera of infected dogs that have been in contact with Leishmania parasites (Figure 4A) .

[0531] Neither the changes in cysteine pattern, nor the modifications associated with protein expression and purification (expression system, TEV treatment) affect the ability of the novel variants to be recognized by IgG in the sera of infected dogs. Therefore, the novel variants retain the capacity to recognize sera from infected dogs in the same way as the original Protein Q.

[0532] Furthermore, the median values measured for antibody binding to all of the polypeptides of the invention were higher at the late stages of infection (sick stage II (SS2); sick stage lll / IV) relative to the same values measured for Protein Q. These promising averages, though not statistically significant due to large variability, mark a positive trend and point to the possibility of further improving the antigenicity of all novel polypeptide variants (Figure 4B).

[0533] Example 5: In-vivo immunization assays to assess the immune response elicited by the novel Protein Q variants

[0534] A protocol for in-vivo immunization assays to assess the immune response elicited by the novel Protein Q variants is presented on Figure 5.

[0535] To evaluate the humoral and cellular immune response elicited by the novel Protein Q variants, mice are divided in treatment groups and immunized as follows: Vehicle buffer (negative control; Group 1), Protein Q (positive control; Group 2), Protein Q-TEV (second positive control; Group 3) and with variant 3-5 (treatment; Group 4-7, respectively). Each protein is administered via a subcutaneous injection, with 8pg protein resuspended in a total volume of 180pl vehicle buffer, without an adjuvant. Each treatment group consists of 10 animals that are followed at several time points during 4 weeks post-immunization: at Day 0, Day 14 and Day 28. At each of these time points, blood is drawn to assess the antibody response, which is represented by lgG1 and lgG2a immunoglobulin count. In addition, at Day 28 of each treatment group are euthanized to obtain samples from the spleen. This serves to assess the elicitation of cellular protective immune responses resulting from immunization. To this end, splenocytes are cultured and stimulated with a soluble leishmanial antigen (SLA) and the protein used to immunize each group. Following this, the supernatant is collected and the amount of secreted interferon-gamma and interleukin 12 (IL-12) and others are assessed by ELISA as described in the “General procedures to the Examples" section.

[0536] References:

[0537] Genes (Basel). 2020 Sep 2;11 (9):1036. doi: 10.3390 / genesl 1091036. PMID: 32887454; PMCID: PMC7563732.

[0538] Nat Commun 13, 3108 (2022). https: / / doi.org / 10.1038 / s41467-022-30867-7

[0539] Example 6: Cysteine replacements in novel Protein Q variants do not negatively impact humoral immune response in immunized mice

[0540] Mice immunization protocol

[0541] To evaluate the humoral response elicited by the novel Protein Q variants, mice were divided into treatment groups and immunized as follows: with Vehicle buffer (negative control; Group 1), with Protein Q / SEQ ID NO: 1 (positive control; Group 2), Protein Q-TEV / SEQ ID NO: 44 (second positive control; Group 3) and with novel Protein Q Variants 3-5 (SEQ ID NOs: 6-8 and / or SEQ ID NOs: 47-49 corresponding to versions lacking a HIS-Tag; treatment; Groups 4- 6, respectively). Each protein was administered via a subcutaneous injection, with 8pg protein resuspended in a total volume of 180pl vehicle buffer, without an adjuvant. Each treatment group consisted of 10 animals that were followed at several time points during 4 weeks postimmunization: at Day 0, Day 14 and Day 28. At each of these points, blood was drawn to assess the antibody response, which is represented by lgG1 and lgG2a count. An updated schematic drawing of the immunization protocol is shown on Figure 7A.

[0542] Detection of lgG1 and lgG2a in mice serum by ELISA

[0543] Serum samples were prepared from blood collected from at Day 0, Day 14 and Day 28. The samples were incubated at room temperature (RT) for at least 30 min and afterwards centrifuged for 5 min, 10000 ref, RT. The serum was pipetted into 0,5 mL-microtubes and stored at <-70°C until measurement. High-binding ELISA plates (96 well, Costar®) were coated with 2pg / ml of Protein Q (control reference) and the variants overnight at 4C°. Sera from the mice from each treatment group were diluted 1 / 100 and tested in duplicate. 1 / 100,000 dilution Goat anti-Mouse lgG2a-HRP conjugated (Southern Biotech: E0524-SB24) and anti-Mouse lgG1- HRP (Southern Biotech: A0022-ZG33D) used as secondary antibodies. Sera of mice were analyzed in duplicates, in each plate. Plates were developed with OPD substrate (with H2O2 in acetic buffer) and the optical density (OD) was read at 450 nm.

[0544] Results

[0545] The production level of lgG1 and lgG2a isotypes was analyzed in the sera of all treated mice groups. No significant differences were found in the levels of IgG 1 and lgG2a generated by the novel Protein Q variants in comparison with Protein Q. Protein specific IgG antibodies were detected 14 days and remained stable in the blood after immunization until day 28. No statistical differences were observed between the treatment groups and time points tested (Figure 6A and B). Our findings confirm that none of the modifications introduced in the novel Protein Q variants (cysteine pattern, the removal of the His-Tag nor the elimination of the poly-Ans fragment in the N-terminal) had detrimental effects in the humoral response of the different vaccine treatments.

[0546] Example 7: In-vivo immunization with novel Protein Q variants 4 and 5 leads to more potent CD4+ and CD8+ T-cell activation in immunized mice

[0547] Mice immunization protocol

[0548] To assess the cellular immune response elicited by the novel Protein Q variants, mice were immunized as follows: with Vehicle buffer (negative control; Group 1), with Protein Q / SEQ ID NO: 1 (positive control; Group 2), Protein Q-TEV / SEQ ID NO: 44 (second positive control; Group 3) and with novel Protein Q Variants 3-5 (SEQ ID NOs: 6-8 and / or SEQ ID NOs: 47-49 corresponding to versions lacking a HIS-Tag) (treatment; Groups 4-6, respectively). At Day 28, each treatment group was euthanized to obtain samples from the spleen. This serves to assess the elicitation of cellular protective immune responses resulting from immunization. To this end, splenocytes are cultured and stimulated with a soluble leishmanial antigen (SLA) and the protein used to immunize each group (Figure 7A).

[0549] Cell stimulation and immunophenotyping

[0550] Splenocytes were isolated from each animal 28 days after dose administration and were frozen until use. In day 0 cells were seeded at 5x106total cells / ml in supplemented RPMI with 10% FBS in a 96-well culture plate and stimulated with protein Q (SEQ ID NO: 1), Variant 1 / Protein Q TEV (SEQ ID NO: 44), Variant 3 (SEQ ID NO: 6 and / or SEQ ID NO: 47 lacking a HIS-Tag), Variant 4 (SEQ ID NO: 7 and / or SEQ ID NO: 48 lacking a HIS-Tag) or Variant 5 (SEQ ID NO: 8 and / or SEQ ID NO: 49 lacking a HIS-Tag) at 12 pg / ml during 12 or 36h at 37 °C in a humidified chamber with 5% CO2. As a positive control, cells were stimulated with Concanavalin A (ConA) at 3 pg / ml and as a negative control unstimulated cells (NE) were incubated with culture media. For immunophenotyping, cells were incubated with fluorochrome-conjugated monoclonal antibodies specific to CD4, CD8 and CD69 (CD4-APC; CD8-FITC; CD69-PE; BD ref 553051 , 553031 , 561932 respectively). Antibodies were added at the supplier’s recommended concentrations and incubated for 30 minutes at 4 °C in the dark. Following incubation, cells were washed twice with staining buffer to remove unbound antibodies.

[0551] To exclude dead cells, a viability dye (Ghost Dye 540, ref 13-0879 CYTEK) was added according to the manufacturer’s instructions prior to acquisition.

[0552] Samples were acquired on a flow cytometer (BD FACSCanto II) calibrated daily using standard beads. Compensation was performed using single-stained controls. Data was analyzed using FlowJo software (version V10.10), applying gating strategies to identify CD4+CD69+and CD8+CD69+populations.

[0553] Results

[0554] The novel Protein Q variants exhibited differential cellular responses. Using CD69 as a marker of activation, we investigated the percentage of activated CD4+ and CD8+ T cell populations upon ex vivo stimulation of splenocytes in the treatment groups at 16 hours post stimulation. CD4+ T cells showed a pronounced activation in Variant 4 (SEQ ID NO: 7 and / or SEQ ID NO: 48 lacking a HIS-Tag) and Variant 5 (SEQ ID NO: 8 and / or SEQ ID NO: 49 lacking a HIS-Tag), with significantly higher percentage of CD4+CD69+ T cell population in Variant 4 (SEQ ID NO: 7 and / or SEQ ID NO: 48 lacking a HIS-Tag) group (mean ± SE, 7.96 ± 1.41 p = 0.025; see Figure 7B) and in Variant 5 group (SEQ ID NO: 8 and / or SEQ ID NO: 49 lacking a HIS-Tag) (mean ± SE, 8, 72 ± 1.9, p = 0.003; see Figure 7B) compared to Protein Q (mean ± SE, 0.67 ± 0.19). There was also a significantly higher percentage of CD8+CD69+ T cell (mean ± SE, 6.27 ± 0.9, p = 0.0047) in V5 compared to to Protein Q (mean ± SE 0.46 ± 0.07, see Figure 7C).

[0555] Statistical analysis

[0556] Statistical analysis of the obtained results from each group of mice was performed by one-way ANOVA using GraphPad Prism 5.04 for Windows (GraphPad Software, San Diego, CA, USA). For all the data, differences were considered statistically significant when P < 0.05. Additional aspects and embodiments

[0557] In a first aspect, presented herein is a peptide or polypeptide derived from SEQ ID NO:1 , which is represented by an amino acid sequence having at least 80% identity or similarity with any one of SEQ ID NO: 8, 2, 3, 4, 5, 6, and 7, and SEQ ID NOs: 40-43, 46-49, wherein:

[0558] Positions 22, 68 and 158 of SEQ ID NO: 8, 43 or 49 are not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof

[0559] Position 20 of SEQ ID NO:2 is not cysteine and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, Position 13 of SEQ ID NO:3 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, Position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof, Position 22 of SEQ ID NO:5, 40 or 46 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof,

[0560] Position 68 of SEQ ID NO:6, 41 or 47 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof

[0561] Position 158 of SEQ ID NO:7, 42 or 48 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof.

[0562] In one embodiment, presented herein is a peptide or polypeptide according to a first aspect, wherein the cysteine is replaced or substituted by proline, glycine, serine, valine and threonine, and most preferably is alanine.

[0563] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect, wherein the identity or similarity is assessed over the full length of said sequence.

[0564] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect, , wherein the peptide or polypeptide comprises a C-terminal recognition site for proteolytic cleavage mediated by TEV protease.

[0565] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect,, wherein the peptide or polypeptide does not comprise a His tag. In an embodiment, presented herein is a peptide or polypeptide according to a first aspect,, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:5, 40 or 46 said identity or similarity being assessed over the whole length of SEQ ID NO:5, 40 or 46 and wherein position 22 of SEQ ID NO:5, 40 or 46 is not a cysteine, is preferably replaced or substituted by proline, glycine, serine, valine and threonine, and more preferably alanine is present at said position.

[0566] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect,, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:6, 41 or 47 said identity or similarity being assessed over the whole length of SEQ ID NO:6, 41 or 47 and wherein position 68 of SEQ ID NO:6, 41 or 47 is not a cysteine, is preferably replaced or substituted by proline, glycine, serine, valine and threonine, and more preferably an alanine is present at said position.

[0567] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect,, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:7, 42 or 48 said identity or similarity being assessed over the whole length of SEQ ID NO:7, 42 or 48 and wherein position 158 of SEQ ID NO:7, 42 or 48 is not a cysteine, wherein preferably position 48 of SEQ ID NO:7, 42 or 48 is replaced or substituted by proline, glycine, serine, valine and threonine, and more preferably an alanine is present at said positions.

[0568] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect,, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:8, 43 or 49 said identity or similarity being assessed over the whole length of SEQ ID NO:8, 43 or 49 and wherein positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 are not cysteine, wherein preferably positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 is replaced or substituted by proline, glycine, serine, valine and threonine, and more preferably alanine residues are present at these positions.

[0569] In an embodiment, presented herein is a peptide or polypeptide according to a first aspect,, said peptide or polypeptide comprising a Leishmania antigenic determinant and preferably inducing a protective immune response against leishmaniasis.

[0570] In a second aspect, presented herein is a nucleic acid molecule encoding the peptide or polypeptidedisclosed in a first aspect herein. . In an embodiment, presented herein is a nucleic acid molecule according toa second aspect , wherein the nucleic acid molecule is RNA, preferably messenger RNA (mRNA).

[0571] In a third aspect, presented herein is a nucleic acid construct comprising the nucleic acid molecule of a second aspect presented herein .

[0572] In a fourth aspect, presented herein is a cell comprising the nucleic acid molecule disclosed in a second aspect, the nucleic acid construct disclosed in a third aspect and / or expressing the peptide or polypeptide of any one of claims 1-10, preferably wherein said cell is a bacterial cell or an eukaryotic cell, more preferably, wherein said eukaryotic cell is an insect, yeast, fungal or mammalian cell.

[0573] In a fifth aspect presented herein is a pharmaceutical composition comprising the peptide or polypeptideaccording to a first aspect , the nucleic acidaccording to a second aspect , the nucleic acid construct according to a third aspect or the cell aaccording to a fourth aspect , preferably wherein said composition comprises one or more adjuvants to enhance the immune response to the peptide or polypeptidedisclosed in a first aspect .

[0574] In an embodiment, presented herein is a composition according toa fifth aspect disclosed herein , said composition comprising a physiological adjuvant appropriate for intraperitoneal, subcutaneous, intradermal, epidermal or intramuscular administration to a human or animal.

[0575] In a sixth aspect, presented herein is a peptide or polypeptide according to a fist aspect , a nucleic acid molecule according toa second aspect , a nucleic acid construct according to a third aspect , a cell according to a fourth aspect or a pharmaceutical composition according to a fifth aspect disclosed herein for use as a medicament.

[0576] In an embodiment, presented herein are a peptide or polypeptide, a nucleic acid molecule, a nucleic acid construct, a cell or a pharmaceutical composition for use as a medicament according to a sixth aspect disclosed herein, wherein the medicament is for generating an immune response, wherein the medicament is a vaccine, preferably wherein the vaccine is therapeutic and / or prophylactic and / or wherein the medicament is for the treatment and / or prevention of leishmaniasis in a human or animal.

Claims

87Claims1 . A peptide or polypeptide derived from SEQ ID NO:1 , which is represented by an amino acid sequence having at least 80% identity or similarity with any one of SEQ ID NO: 4 wherein position 28 of SEQ ID NO:4 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof.

2. A polypeptide according to claim 1 , wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with any one of SEQ ID NO:7 or SEQ ID NO:8, wherein position 158 of SEQ ID NO:7 or of SEQ ID NO:8 is not cysteine, and is replaced or substituted by an amino acid comprising a side chain lacking disulfide bond-forming capability or a derivative thereof.

3. A peptide or polypeptide according to claim 1 or 2, wherein the cysteine is replaced or substituted by proline, glycine, serine, valine and threonine, and most preferably is alanine.

4. A peptide or polypeptide according to any one of claims 1 to 3, wherein the identity or similarity is assessed over the full length of said sequence.

5. A peptide or polypeptide according to any one of claims 1 to 4, wherein the peptide or polypeptide comprises a C-terminal recognition site for proteolytic cleavage mediated by TEV protease.

6. A peptide or polypeptide according to any one of claims 1 to 5, wherein the peptide or polypeptide does not comprise a His tag.

7. A polypeptide according to any one of claims 1 to 6, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:7, 42 or 48 said identity or similarity being assessed over the whole length of SEQ ID NO:7, 42 or 48 and wherein position 158 of SEQ ID NO:7, 42 or 48 is not a cysteine, wherein preferably position 48 of SEQ ID NO:7, 42 or 48 is replaced or substituted by proline, glycine, serine, valine and threonine, and more preferably an alanine is present at said positions.

8. A polypeptide according to any one of claims 1 to 7, wherein the polypeptide is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:8, 43 or 49 said identity or similarity being assessed over the whole length of SEQ ID NO:8, 4388 or 49 and wherein positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 are not cysteine, wherein preferably positions 22, 68 and 158 of SEQ ID NO:8, 43 or 49 is replaced or substituted by proline, glycine, serine, valine and threonine, and more preferably alanine residues are present at these positions.

9. A polypeptide according to any one of claims 1 to 8, comprising a Leishmania antigenic determinant and preferably inducing a protective immune response against leishmaniasis.

10. A nucleic acid molecule encoding the peptide or polypeptide of any one of claims 1 to 9.11 . A nucleic acid molecule according to claim 10, wherein the nucleic acid molecule is RNA, preferably messenger RNA (mRNA).

12. A nucleic acid construct comprising the nucleic acid molecule of any one of claims 10 or 11.

13. A cell comprising the nucleic acid molecule of any one of claims 10 or 11 , the nucleic acid construct of claim 11 and / or expressing the peptide or polypeptide of any one of claims 1 - 9, preferably wherein said cell is a bacterial cell or an eukaryotic cell, more preferably, wherein said eukaryotic cell is an insect, yeast, fungal or mammalian cell.

14. A pharmaceutical composition comprising the peptide or polypeptide of any one of claims 1 to 9, the nucleic acid molecule of claim 10 or 11 , the nucleic acid construct of claim 12 or the cell of claim 13, preferably wherein said composition comprises one or more adjuvants to enhance the immune response to the peptide or polypeptide of any one of claims 1 -to 9.

15. A composition according to claim 14, said composition comprising a physiological adjuvant appropriate for intraperitoneal, subcutaneous, intradermal, epidermal or intramuscular administration to a human or animal.

16. A peptide or polypeptide according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10 or 11 , a nucleic acid construct according to claim 12, a cell according to claim 13 or a pharmaceutical composition according to claim 14 or 15 for use as a medicament.8917. A peptide or polypeptide, a nucleic acid molecule, a nucleic acid construct, a cell or a pharmaceutical composition according to claim for use as a medicament according to claim 16, wherein the medicament is for generating an immune response, wherein the medicament is a vaccine, preferably wherein the vaccine is therapeutic and / or prophylactic and / or wherein the medicament is for the treatment and / or prevention of leishmaniasis in a human or animal.

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