Immunogen against bovine parainfluenza virus type 3 and uses thereof

Abstract

The present invention relates to new polypeptides derived from Bovine Parainfluenza Type 3 virus (BPI3V) showing immunogenic and / or antigenic properties and their uses thereof for prevention or treatment of mammals against BPI3V infection and / or BPI3V associated diseases.

Description

[0001] IMMUNOGEN AGAINST BOVINE PARAINFLUENZA VIRUS TYPE 3 AND USES

[0002] THEREOF

[0003] Field of the invention

[0004] The present invention relates to new polypeptides derived from Bovine Parainfluenza Type 3 virus (BPI3V) showing immunogenic and / or antigenic properties, and uses thereof. In particular, the present invention relates to compositions such as immunogenic compositions and vaccine compositions comprising one of said polypeptides and to the use of said polypeptide(s) and / or composition(s) for prevention or treatment of mammals against BPI3 V infection and / or BPI3V associated disease.

[0005] Description of the prior art

[0006] Bovine Respiratory Disease (BRD) affects calves and adult cattle in all parts of the world and accounts for considerable economic losses. Outbreaks are typically related to environmental stress factors (transport, crowding, unfavorable climate conditions) as the disease results from the interactions between microorganisms in the respiratory tract and the animal’s resistance, which is affected by such non-biological factors. Several viruses have established roles in BRD pathogenesis, including Bovine Parainfluenza Type 3 virus (BPI3 V).

[0007] BPI3 V belongs to the family of Paramyxoviridae genus paramyxovirus. Major transmembrane proteins are Haemmagglutinin-Neuraminidase (HN) and Fusion protein (F). Due to its role in the entry of the virons into the host cells, the BPI3V F protein is a key target for vaccine development. Class I Fusion proteins, such as BPI3 V F protein, are known to be metastable and likely require a prefusion (preF) stabilization for vaccine effectiveness. The pre-mature form F0 of the Fusion protein comprises 540 amino acids. Said pre-mature form has a peptide signal of 18 amino acid residues which is cleaved during maturation. The amino acid residues 19 to 495 of said F protein form the ectodomain. The amino acid residues 496 to 516 form the transmembrane domain and the amino acid residues from 517 to 540 form the cytoplasmic tail. During maturation, the inactive precursor F0 is proteolytically cleaved by host cell protease in two parts at amino acid residue 109, resulting in functionally active F2 glycoprotein (amino acid residue 19 to 109) and Fl glycoprotein (amino acid residue 110 to 540). Three monomers of disulfide-linked F1-F2 form a homotrimer. Worldwide, there are three types of genotypes, named type A, B and C. Whereas genotype B is predominantly occurring in the Americas and Asia, Type A is distributed in Americas and Europe. In recent years, type C is of increasing relevance in Europe, so that there is a co-circulation of genotype A and C strains. BPI3 V currently circulates worldwide in cattle herds. As this virus is exclusively a respiratory tract pathogen that rarely becomes systemic, the most important routes of transmission in susceptible animals are by aerosol and fomites resulting from nasal discharges. Clinical disease is usually mild to moderate, consisting of fever, lacrimation, serous nasal discharge, depression, dyspnea, and coughing with some animals developing bronchointerstitial pneumonia.

[0008] An uncomplicated bovine parainfluenza virus 3 infection runs a brief clinical course of 3-4 days that is usually followed by complete and uneventful recovery. In stressful circumstances (i.e. poor hygiene, crowding, transport, harsh climatic conditions), however, the infection, alone or in combination with other viral infections (e.g., bovine adenovirus, bovine coronavirus, bovine viral diarrhea virus, infectious bovine rhinotracheids virus, bovine respiratory syncytial virus), predisposes the animal to secondary bacterial infection due to Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and / or Mycoplasmopsis bovis. This syndrome is characterized by purulent nasal discharge, cough, polypnea / dyspnea, anorexia, fever, depression, and substantial mortality from bronchopneumonia.

[0009] Vaccine development is essential to prevent infection and / or reduce clinical signs associated with BPI3 V infection, and thus prevent secondary pulmonary bacterial infections. So far, there is no commercial vaccine against BPI3V based on a subunit approach. Current commercial vaccines are most often live attenuated or inactivated vaccine. These vaccines can lack high efficacy due to (i) the single BPI3V genotype used in these vaccines which could not provide cross protection against other circulating BPI3V genotypes and (ii) low immunogenicity induced by attenuation or inactivation processes. In addition, modified live BPI3 V vaccines are often administered intranasally to overcome maternally derived antibody (MDA) interference. Such administration route is not convenient for the producers and veterinarians and leads to a short duration of immunity. Therefore, there is a need for an alternative vaccine that will give a high protection against the main current circulating BPI3V genotypes even in presence of MDA.

[0010] Summary of the invention

[0011] The present invention overcomes at least partially one or more of the problems inherent in the prior art by providing new immunogens able to trigger an immune response against BPI3 V.

[0012] It is therefore an object of the present invention to provide an immunogen comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence corresponding to amino acid residues 19 to 481 of SEQ ID NO: 1.

[0013] It is a further object of the present invention to provide immunogens derived from the polypeptide having the amino acid sequence corresponding to amino acid residues 19 to 481 of SEQ ID NO: 1 and comprising a combination of substitutions selected from K108E + I172C + N238C + A463V, K108E + Q162C + L168C + I213C + G230C + A463V + I474Y, K108E + R48C + S246C + Q162C + L168C + I213C + G230C + A463V + I474Y, K108E + R48C + S246C + Q162C + L168C + A463V + I474Y, K108E + A140C-I270C + Q162C-L168C + I213C-G230C + A463V + I474Y, K108E + R48C + S246C + A140C + I270C + Q162C + L168C + I213C + G230C + A463V + I474Y, and K108E + R48C + S246C + A140C + I270C + A463 V + I474Y, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0014] It is further object of the present invention to provide an immunogen derived from a polypeptide having an amino acid sequence comprising, consisting of, or consisting essentially to the sequence corresponding to amino acid residues 19 to 481 of SEQ ID NO: 1 and comprising a trimerization domain directly or indirectly linked to its C-terminal end. Preferably, said trimerization domain is the bacteriophage T4 fibritin foldon trimerization domain or a GCN4 based isoleucine zipper trimerization domain and is added with a linker sequence to the C- terminal end of said immunogen. Preferably, the added sequence with linker sequence and T4- fibritin Foldon sequence is of SEQ ID NO: 17 and the added sequence with a linker sequence and a GCN4 based isoleucine zipper domain is of SEQ ID NO: 18.

[0015] In particular, the immunogen may comprise, consist of, or consist essentially of an amino acid sequence selected from any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:22.

[0016] It is a further object of the present invention to provide a nucleic acid molecule encoding an immunogen of the invention. An example of such a nucleic acid is provided in SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23 which includes a nucleic acids sequence encoding for a signal peptide that will be cleaved before use of the corresponding polypeptide as the immunogen.

[0017] The invention also provides a vector comprising a nucleic acid molecule of the invention, preferably under the control of a promoter, a recombinant cell comprising a nucleic acid or vector of the invention, and an immunogenic composition comprising at least one immunogen of the invention, and optionally, an excipient and / or an adjuvant.

[0018] It is a further object to provide a vaccine composition comprising an immunologically effective amount of:

[0019] (i) at least one immunogen of the invention, or (ii) a nucleic acid molecule encoding an immunogen of the invention, or

[0020] (iii) a vector of the invention, or

[0021] (v) a recombinant cell of the invention and an excipient and / or an adjuvant.

[0022] Preferably, the vaccine composition is a subunit vaccine, comprising one or more immunogens of the invention.

[0023] In a particular embodiment, the immunogenic composition or the vaccine composition further comprises at least one additional antigen directed against another pathogen associated with Bovine Respiratory Disease (BRD) and / or calf diarrhea, such as Bovine Respiratory Syncytial virus (BRSV), Bovine Viral Diarrhea virus (BVDV), Bovine Herpesvirus type 1 (BHV1), Bovine Coronavirus (BCV), Bovine rotavirus, Mycoplasmopsis Bovis, Pasteurella mullocidci, Mannheimia haemolytica, Histophilus somni and Escherichia coli. In an embodiment, the additional antigen is an antigenic peptide or immunogen.

[0024] The present invention further provides an immunogen or composition as defined above, for use in a method to prevent or treat a Bovine Parainfluenza virus Type 3 (BPI3 V) associated disease in a non-human mammal.

[0025] The present invention also provides an immunogen or composition as defined above for use in a method to reduce or prevent one or more clinical symptoms caused by or associated to a Bovine Parainfluenza virus Type 3 (BPI3 V) associated infection in a non-human mammal.

[0026] For instance, the clinical symptoms are selected from the group consisting of increased core body temperature, cough, lung lesions, tachypnea, polypnea, dyspnea, orthopnea, nasal discharge, ocular discharge, dysorexia / anorexia, depression and reduced milk production.

[0027] The present invention also provides an immunogen or composition as defined above, for use in a method to reduce BPI3V viral shedding in a non-human mammal.

[0028] Advantageously, the prevention or treatment of an infection by BPI3 V is based on, comprises or consists of the induction of an immune response against said BPI3 V.

[0029] In an embodiment, the immunogen or composition is able to prevent or treat a BPI3 V type A infection, a BPI3V type C infection, or both a BPI3V type A and BPI3V type C infection. Alternatively or in addition, said immunogen or composition may allow to reduce or prevent one or more clinical symptoms caused by or associated to a BPI3 V type A infection, a BPI3 V type C infection, or both a BPI3 V type A and BPI3 V type C infection.

[0030] In a particular embodiment, the method comprises a single administration of said immunogen or composition to said non-human mammal, preferably by parenteral administration, more preferably by injection.

[0031] In a particular embodiment, the immunogen or composition is administered to non-human mammals having anti-BPI3 V antibodies, in particular maternally derived antibodies (MDA).

[0032] Preferably, the non-human mammal is livestock selected from bovine, sheep and goats, preferably bovine, including calf.

[0033] More preferably, the non-human mammal is bovine, and particularly a bull, a cow or a calf.

[0034] In a particular embodiment, said immunogen or composition is to be administered to cattle between 1 and 28 days of age, in particular between 7 and 21 days of age, + / - 3 days.

[0035] Advantageously, the immunogen or composition is to be administered to calves between 3 and 28 day of age, + / - 3 days, in particular between 7 and 21 day of age, + / - 3 days.

[0036] In a particular embodiment, the method comprises a single administration of the composition or immunogen to calves between 7 and 21 days of age, by intramuscular injection.

[0037] Legend to the figures

[0038] Figure l is a boxplot of the Area Under the Curve (AUC) values for the Rectal Temperatures of calves vaccinated with either the immunogenic composition (BPI3 V vaccine “candidate 9” described in Example 1) or the PBS solution (control), and subsequently challenged with BPI3 V. The rectal temperature was collected daily from the day prior to Challenge (D27) to 12 Days Post Challenge (D40).

[0039] Figure l is a boxplot of the Area Under the Curve (AUC) values for Consolidated Lung Lesion Scores of calves vaccinated with either the immunogenic composition (BPI3V vaccine “candidate 9” described in Example 1) or the PBS solution (control), and subsequently challenged with BPI3V 12 Days Post Challenge (D40). Lungs were removed from each animal at necropsy and percentage gross involvement of lesions for each lobe was weighted. The weighted lung lobe values were summed to yield the percentage of total lung with lesions for each animal. Scoring was determined based on visual observation of lesions and palpation of the lungs. Figure 3 is a boxplot of the Area Under the Curve (AUC) values for the nasopharyngeal swab Log Virus Isolation of calves vaccinated with either the immunogenic composition (BPI3V vaccine “candidate 9”) or the PBS solution (control), and subsequently challenged with BPI3 V. Nasopharyngeal swab samples were collected daily from the day prior to Challenge (D27) to 12 Days Post Challenge (D40) and were tested for the presence of BPI3 by use of an immunoassay for the detection of BRSV and PI3 virus antigen. The tissue culture infective dose 50 (TCID50) was calculated for each sample following the methods detailed in SOP / PRO / 033: Calculation of Virus Titre by the Karber Method (Karber G. Beitrag zur kollektiven behandlung pharmakologiseher reihenversuche. Arch Exp Path Pharmacol. 1931;162:480-484. doi: 10.1007 / BF01863914).

[0040] Figure 4 is a Size Exclusion Chromatography (SEC) profile showing the absorbance of the BPI3V F ectodomain trimer for each of the four BPI3V F vaccine candidates described in Examplel. Curve 1 corresponds to the vaccine “candidate 10” of example 1, curve 2 corresponds to the vaccine “candidate 15” of example 1, curve 3 corresponds to the vaccinate “candidate 9” of example 1, and curve 4 corresponds to the vaccine “candidate 13” of example 1.

[0041] Figure 5 A and figure 5B are Western blot profiles showing the expression of BPI3V F ectodomain in CHO cells in non-reducing gel conditions and reducing gel conditions respectively. The trimeric form appears as band at approximately 170 kDa under non-reducing conditions (figure 5A) and the monomeric form appears as band at approximately 57 kD under reducing conditions (figure 5B).

[0042] Figure 6 illustrates the Phylogenetic tree of BPIV3 serotypes A and C. The isolates used for the serum neutalization assay (SNT) shown in bold.

[0043] Figure 7 presents the SNT results which illustrate the ability of BPI3 V vaccine candidate to induce a neutralizing antibody response against both BPI3 V type A and C strains. The sera of three cattle vaccinated with the BPI3 V vaccine “candidate 9” of example 1 (vaccinated group) and sera of one non vaccinated cattle (negative control) were analysed in SNT at DO (before vaccination) and D161. Sera were analyzed in SNT assay against four different BPIV3 strains, two belonging to Serotype A (strain Jena 135 and Dargen) and two belonging to serotype C (strain Parral and Frankreich) respectively.

[0044] Figure 8 is a boxplot of ELISA results showing mean titer of antibodies against BPI3V measured in non-heparinized blood samples collected from calves within 24 hours of arrival on days 0 (prior to administration) and on days 14, 21, 28 and 40. Figure 9 is a barplot of the degree of positivity ELISA data showing the post challenge percentage of BPI3V antibody in the range of negative, low, medium or high titres.

[0045] Detailed description of the invention

[0046] The present invention is based on the development of a consensus sequence of a BPI3 V protein, namely a fusion (F) protein, and variants thereof. The antigenic polypeptides of the invention have been designed by the inventors to provide improved immunogenic and / or expression properties. They are distinct from naturally-occurring F protein of BPI3V. These polypeptides exhibit a specific and potent immunogenic capacity, allowing the generation of a protective immunity in animals. These polypeptides can be efficiently expressed in recombinant host cells. Therefore, these polypeptides, or antigenic fragments thereof, represent novel active agents which can be used to produce efficient vaccine compositions.

[0047] The consensus sequences and variants thereof may be used to vaccinate mammals, in particular bovine, against BPI3V infection. Indeed, the corresponding proteins are able to stimulate the formation of neutralizing and / or protective antibodies against BPI3V in vaccinated animals without risking viral infection. Interestingly, said proteins may be effective against several genotypes of BPI3V.

[0048] Definitions

[0049] As used herein, the terms “ treatment" or "therapy" of a subject refer to any type of intervention or process performed on, or the administration of an active agent to, a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of at least one symptom, complication, condition, or biochemical indicia associated with a disease.

[0050] As used herein, the terms "prevent", "preventing" , "prevention" , "prophylactic treatment" and the like, refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition. In particular, these terms refer to reducing the probability of developing at least one symptom, complication, condition, or biochemical indicia associated with said disease or condition in said subject.

[0051] As used herein, the term “therapeutic effect' refers to an effect induced by a vaccine composition according to the invention, capable to prevent or to delay the appearance of a disease, or to prevent, to reduce or to delay the appearance of at least one or more symptoms associated to said disease, or to cure or to attenuate the effects of said disease or of at least one or more symptoms associated to said disease. As used herein, an “immunogenic composition" is a composition that elicits an immune response when administered to an animal. An immunogenic composition comprises at least one antigen or immunogen and at least one pharmaceutically-acceptable excipient, stabilizer, solubilizer or diluent.

[0052] The terms "vaccine" or “vaccine composition" as used herein is an immunogenic composition for the prophylaxis and / or treatment of diseases. In particular, a vaccine designates a composition which may be used to cause, stimulate or amplify an immune response in an organism in order to develop protection from, resistance to, prevention of, or treatment for a disease symptom, wherein said symptom is caused by a pathogenic organism, for example a virus or a bacterium. A vaccine may include, without limitation, antigen(s) or immunogen(s) in combination with pharmaceutically-acceptable adjuvant, excipient, stabilizer, solubilizer and / or diluent. A vaccine may include one or several antigens from one or multiple pathogens.

[0053] The term “subunit vaccine" as used herein refers to a composition containing an antigenic polypeptide or immunogen derived from or homologous to an antigen from BPI3V. Such a composition is prepared from recombinant analogs of immunogenic polypeptides of BPI3V. A subunit vaccine can comprise the subunit antigen(s) of interest substantially free of other antigens or polypeptides from BPI3 V.

[0054] An "immune response" designates the development in a host of a cellular and / or antibody- mediated immune response. Usually, an "immune response" includes the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens of interest. Preferably, the immune response is protective such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. For the vaccine according to the invention, the immune response induced in the vaccinated target animal has for instance the effect of reducing infection by Bovine Parainfluenza Type 3 virus. This refers to a reduction of the level or the extent of the infection, for example by reducing the viral load or shortening the duration of viral replication in the host animal. This effect is obtained e.g., by preventing or reducing the establishment or the proliferation of a productive infection by Bovine Parainfluenza Type 3 virus in its target organs. In turn, this leads to a reduction in the target animal of the number, the intensity, or the severity of lesions and clinical signs that could be caused by the viral infection. The person skilled in the art is able to determine the effectiveness of a vaccine according to the invention for reducing infection by Bovine Parainfluenza Type 3 virus. For instance, this determination may be done by monitoring the immunological response following vaccination or after a challenge infection, e.g. by monitoring the targets' signs of disease, clinical scores, serological parameters, or by re-isolation of the pathogen, and comparing these results to a vaccination-challenge response seen in mock vaccinated animals. As used herein, an "effective amount" is an amount that prevents an infection, treats a disease or medical condition in an individual, or, more generally, reduces symptoms, manages progression of the disease, or attenuates the viral infection for a period of time.

[0055] The terms “quantity,” “amount” and “dose” are used interchangeably herein and may refer to an absolute quantification of a molecule.

[0056] In the present description, the terms “peptide”, “polypeptide” , “protein”, refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. The amino acids are herein represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (He); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp ) and Y: tyrosine (Tyr).

[0057] The terms "antigen" or “immunogen” as used herein refer to an amino acid sequence which elicits an immune response when administered to an animal.

[0058] An “immunogenic fragment” or “antigenic fragment” of an antigen, immunogen, polypeptide or protein designates any fragment which can elicit an immune response, preferably any fragment which contains an epitope, preferably an antigen-specific epitope. Immunogenic fragments generally contain from 5 to 50 consecutive amino acid residues of an antigen, such as from 5 to 40, or from 10 to 40, or 10-30, 10-25, or 10-20.

[0059] The term “recombinant” in relation to a sequence, designates a sequence, nucleic acid or unit which does not exist naturally and / or which has been engineered using recombinant DNA technology (also called gene cloning or molecular cloning).

[0060] As used herein, the term “sequence identity” or “identity” refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical 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 and Wunsch algorithm; Needleman and Wunsch, 1970) 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 and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pairwise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 11, Gap extend = 1.

[0061] In the context of the invention, the expression “between x and ” means that the values x and y are included.

[0062] In the context of the invention, the term “about” means the given value plus or minus 10%.

[0063] A "subject" is a mammal, preferably a non-human mammal, more preferably livestock, in particular bovine, including calf.

[0064] By working on optimization of antigens able to trigger an immune response against BPI3 V for inducing strong and improved immunogenic properties, the inventors have developed an immunogen derived from a consensus amino acid sequence of the BPI3 V fusion (F) protein of two different BPI3 V genotypes, namely of genotypes A and C, said consensus sequence being set forth in SEQ ID NO: 1.

[0065] It is therefore an object of the present invention to provide an immunogen comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence corresponding to amino acid residues 19 to 481 of the SEQ ID NO: 1.

[0066] The inventors have developed variants of SEQ ID NO: 1, which may advantageously present increased stability and / or increased immunogenicity compared to the protein of SEQ ID NO: 1 and which elicit an immune response when administered to an animal.

[0067] The term “variant” refers to a polypeptide derived from SEQ ID NO: 1, and more particularly derived from amino acid sequence corresponding to residues 19 to 481 of SEQ ID NO: 1, and comprising at least one modification or alteration, i.e., a substitution, insertion, and / or deletion, at one or more (e.g., several) positions as compared to SEQ ID NO: 1, and having immunogenic properties. A variant may be obtained by various techniques well known in the art. In particular, examples of techniques for altering the DNA sequence encoding the protein of SEQ ID NO: 1, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction. Thus, the terms “ modification" and “alteration" as used herein in relation to a particular position means that the amino acid in this particular position has been modified compared to the amino acid in this particular position in the parent protein.

[0068] A “substitution” means that an amino acid residue is replaced by another amino acid residue. Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine, N-ethyl asparagine, allo-isoleucine, N-methylisoleucine, N- methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non- naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine). Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign “+” indicates a combination of substitutions. In the present document, the following terminology is used to designate a substitution: M35 A denotes that amino acid residue (Methionine, M) at position 35 of the parent sequence is substituted by an Alanine (A). A168V / I / M denotes that amino acid residue (Alanine, A) at position 168 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M). The substitution can be a conservative or nonconservative substitution.

[0069] Conservative amino acid mutations may be made to generate polypeptide variants of the invention. Conservative amino acid mutations may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity). Such a conservative amino acid substitution may be a substitution of a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term "basic amino acid," it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (H), arginine (R), and lysine (K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (S), threonine (T), cysteine (C), tyrosine (Y), asparagine (N), and glutamine (Q). The term "hydrophobic amino acid" (also "non-polar amino acid"), is meant to include amino acids exhibiting a hydrophobicity index of greater than zero. Hydrophobic amino acids include proline (P), isoleucine (I), phenylalanine (F). valine (V), leucine (L), tryptophan (W), methionine (M), alanine (A), and glycine (G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7 which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (E), and aspartate (D),

[0070] A semi -conserved amino acid substitution replaces one residue with another one that has similar steric conformation, but does not share chemical properties. Examples of semi-conservative substitutions would include substituting cysteine for alanine or leucine; substituting serine for asparagine; substituting valine for threonine; or substituting proline for alanine.

[0071] The inventors have identified particular amino acid substitutions suitable to stabilize the BPI3 F protein according to the invention in the pre Fusion conformation.

[0072] In a particular embodiment, the immunogen comprises at least one substitution or combination of substitutions selected from I172C+N238C, Q162C+L168C, I213C+G230C, R48C+S246C, A140C+I270C, K108C, A463V and I474Y, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0073] In a particular embodiment, the immunogen comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% with the amino acid sequence corresponding to amino acid residues 19 to 481 of SEQ ID NO: 1 and a combination of substitutions selected from:

[0074] K108E + I172C + N238C + A463V,

[0075] K108E + Q162C + L168C + I213C + G230C + A463V + I474Y,

[0076] K108E + R48C + S246C + Q162C + L168C + I213C + G230C + A463V + I474Y,

[0077] K108E + R48C + S246C + Q162C + L168C + A463V + I474Y,

[0078] K108E + A140C-I270C + Q162C-L168C + I213C-G230C + A463V + I474Y,

[0079] K108E + R48C + S246C + A140C + I270C + Q162C + L168C + I213C + G230C + A463V + I474Y, and

[0080] K108E + R48C + S246C + A140C + I270C + A463 V + I474Y, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1. According to the present invention, the immunogen may further comprise a trimerization domain directly or indirectly linked at its C-terminus in order to promote the trimeric form of the immunogen.

[0081] In a particular embodiment, the immunogen comprises at its C-terminal end a GCN4-based isoleucine zipper (IZ) trimerization domain (Harbury P. B. et al. Science 262, 1401-1407 1993) or the T4 bacteriophage fibritin foldon (Fd) trimerization domain (Harbury P. B. et al., Nature 371, 80-83 1994; Guthe S. et al., J. Mol. Biol. 337, 905-915, 2004).

[0082] In a preferred embodiment, the immunogen comprises at the C-terminal end the amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0083] In a particular embodiment, the immunogen comprises, consists of, or consists essentially of an amino acid sequence selected from any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8

[0084] In a particular embodiment, said immunogen further comprises a trimerization domain at the C-term end, preferably the trimerization domain comprises the sequence of SEQ ID NO: 17 or SEQ ID NO: 18. For instance, the immunogen comprises, consists of, or consists essentially of an amino acid sequence selected from any one of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 22.

[0085] In a preferred embodiment, the immunogen comprises, consists of, or consists essentially of an amino acid sequence selected from any one of SEQ ID NO:3 and SEQ ID NO: 10.

[0086] In a particular embodiment, the polypeptide comprises, at the N-terminal end of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:22, an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the full length amino acid sequence as set forth in SEQ ID NO: 16 (MEFWLSWVFLVAILKGVQC), corresponding to a signal peptide of particular interest for intracellular trafficking and posttranslational modification. This signal peptide is particularly adapted for expression in CHO cells. The skilled person is able to adapt said amino acid sequence to the cell line in which the polypeptide will be expressed.

[0087] Production o f the antigenic polypeptides

[0088] The polypeptides of the present invention may be produced by recombinant techniques, or they may be artificially produced. An example of such techniques includes the case when DNA encoding the expressed polypeptide is inserted into a suitable expression vector, which is in turn used to transform a host cell to produce the protein or polypeptide encoded by the DNA.

[0089] The term "recombinant polypeptide" , as used herein, thus in particular refers to a protein molecule which is expressed from a recombinant DNA molecule.

[0090] They may be in soluble form, or on solid phase. In particular, they may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filter, membranes, e.g., in the form of beads, columns, plates and the like.

[0091] The polypeptides of the invention are typically provided in an isolated form. The term "isolated" is meant to refer to a polypeptide which is in other than a native environment. For example, the polypeptide may be a component of a cell culture or other artificial medium; a component of a pharmaceutical composition; or partially or completely purified from its native environment.

[0092] In a particular embodiment, a composition is used which comprises the polypeptide as a partially purified cell supernatant. The cell supernatant may contain cell proteins, DNA fragments, and the like. The supernatant is preferably enriched for the polypeptide of the invention and, even more preferably, treated to inactivate or destroy any nucleic acid or virus present in the composition.

[0093] The polypeptides of the invention may be purified by techniques known per se in the art and stored under conventional techniques. They may be used as such, in purified form, either alone or in combinations.

[0094] The invention also relates to a nucleic acid molecule encoding a polypeptide as defined above.

[0095] It is therefore an object of the present invention to provide nucleic acid sequences encoding for a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:22 or amino acids residues 19 to 481 of SEQ ID NO: 1.

[0096] It is another object of the present invention to provide a nucleic acid sequence encoding for a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:22 or amino acids residues 19 to 481 of SEQ ID NO: 1 and, at the Nter of said SEQ IDs, the amino acid sequence as set forth in SEQ ID NO: 16, which encodes for a signal peptide. For instance, the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 10 and at the Nter, the amino acid sequence as set forth in SEQ ID NO: 16, may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 19.

[0097] It is another object of the present invention to provide a recombinant vector comprising a nucleic acid as defined above. In some embodiments, a "recombinant vector" as used herein refers to a genetic material, for example a virus or a plasmid, used as a vehicle to artificially carry foreign genetic material into a host cell where it can be replicated and / or expressed.

[0098] The nucleic acid molecules according to the invention may be provided in the form of a nucleic acid molecule per se such as naked nucleic acid molecules; a vector; virus or host cell etc., either from prokaryotic or eukaryotic origin. Vectors include expression vectors that contain a nucleic acid molecule of the invention. The vectors of the present invention may, for example, comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.

[0099] In a further embodiment of the invention, there is provided a host cell transformed with a nucleic acid molecule or vector according to the invention. Suitable examples of host cells will be known to those skilled in the art or can be readily selected by those skilled in the art. Host cells may, for example, include eukaryotic and prokaryotic cells. Examples of eukaryotic cells include mammalian (e.g., hamster), fungal, insect and plant cells. In a particular embodiment, the host cells are CHO-cells.

[0100] The invention also relates to a method for preparing a polypeptide of the invention, the method comprising culturing a host cell containing a nucleic acid or vector as defined above under conditions suitable for expression of the nucleic acid and recovering the polypeptide. As indicated above, the polypeptide may be purified according to techniques known per se in the art. The invention also provides expression kits comprising (a) a host cell (preferably CHO- cells), (b) means of expressing a polypeptide of the invention, e.g. comprising a vector system capable of being replicated in said cell, and (c) means of recovering the polypeptide of the invention.

[0101] Immunogenic compositions

[0102] It is a further object of the present invention to provide immunogenic composition, in particular vaccine compositions comprising one or more of the polypeptides as defined above. Advantageously, the composition is a vaccine composition comprising one or more of the polypeptides as defined above in an immunological effective amount, i.e., an amount capable of stimulating the immune system of the target animal sufficiently to at least reduce one or more negative effects of a post-vaccination challenge with a BPI3 V.

[0103] According to the invention, the composition may further comprise suitable ingredients, such as excipient(s) and / or adjuvant(s). In particular, vaccines may comprise other ingredients, known per se by one of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration.

[0104] Examples of pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as light liquid paraffin oil, or heavy liquid paraffin oil; squalene; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or iso- propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1 ,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the vaccine composition and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.

[0105] Examples of stabilizer may be for example carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.

[0106] The compositions of the invention may be liquid formulations such as an aqueous solution, water-in-oil or oil-in-water emulsion, syrup, an elixir, a tincture.

[0107] In a particular embodiment, the composition is a vaccine composition, in particular a subunit vaccine, comprising at least one of the polypeptides of the invention. The vaccine composition may comprise an adjuvant. An “adjuvant” is a compound or combination of compounds that enhance, activate, potentiate, or modulate the quality and / or the quantity of the immune response to an antigen. The choice and amount of adjuvant(s) may be easily determined by the man skilled in the art. Examples of adjuvants include, but are not limited to, oil in water emulsions, such as MontanidelSA®, aluminum hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL- 1 , IL-2, IL-7, IFN-[alpha], IFN-[beta], IFN-y, etc.), saponins, monophosphoryl lipid A (MLA) optionally combined with aluminum salts or other adjuvants, muramyl dipeptides (MDP) and the like, saponin-based adjuvants, such as Quil-A. Other suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat-stable enterotoxin(s) isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanus toxin and pertussis toxin may be inactivated prior to use, for example, by treatment with formaldehyde.

[0108] Advantageously, the vaccine composition is in a form suitable for systemic administration, in particular in a form suitable for parenteral administration, such as intramuscular or subcutaneous injection. Such formulations are known in the art and are typically prepared by dissolution of the immunogen and other typical additives in the appropriate carrier or solvent systems. Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents.

[0109] Alternatively, the vaccine composition is in a form suitable for intranasal instillation or inhalation, for instance by nebulization of the composition. An aerosol delivery system may be used to generate the vaccine particles in a size range suitable for inhalation by the subject. For instance, a specific canula or a nebulizer may be used.

[0110] According to the present invention, the vaccine composition comprises an effective amount of at least one polypeptide of the invention.

[0111] In practice, the exact amount required for an immunologically effective dose may vary from subject to subject depending on factors such as the age and general condition of the subject, the nature of the formulation and the mode of administration. Appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. For instance, methods are known in the art for determining or titrating suitable dosages of a vaccine to find minimal effective dosages based on the weight of the non-human animal subject, concentration of the vaccine and other typical factors.

[0112] In a particular embodiment, the vaccine composition comprises between 10 and 500 pg / dose of a polypeptide of the invention, preferably between 20 and 200pg / dose, between 25 and 150 pg / dose, between 50 and 100 pg / dose. In particular, the vaccine composition may comprise between 10 and 100 pg / dose of polypeptide of the invention, between 20 and 200 pg / dose, between 50 and 250pg / dose, between 100 and 350 pg / dose, between 150 and 400 pg / dose, between 200 and 450 pg / dose, between 250 and 500 pg / dose, such as 20 pg / dose, 25pg / dose, 50 pg / dose, 60 pg / dose, 80 pg / dose, 100 pg / dose, 150 pg / dose, 200 pg / dose, 250 pg / dose, 300 pg / dose, 350 pg / dose, 400 pg / dose, 450 pg / dose, 500 pg / dose. In a particular embodiment, the vaccine composition comprises 25 pg / dose, + / - 10%, or 50 pg / dose, + / - 10% or 100 pg / dose, + / - 10%.

[0113] In a particular embodiment, the vaccine composition comprises betweenlO and 500 pg / dose of the polypeptide of SEQ ID NO:3, such as 20 pg / dose, 25pg / dose, 50 pg / dose, 60 pg / dose, 80 pg / dose, 100 pg / dose, 150 pg / dose, 200 pg / dose, 250 pg / dose, 300 pg / dose, 350 pg / dose, 400 pg / dose, 450 pg / dose, 500 pg / dose.

[0114] In an embodiment, the vaccine composition comprises two or more homologous or heterologous polypeptides of the invention. In a particular embodiment, the vaccine composition comprises a BPI3 V F ectodomain dimer comprising two polypeptides of SEQ ID NO: 10. In a preferred embodiment, said vaccine composition comprises a BPI3VF ectodomain trimer comprising three polypeptides of SEQ ID NO: 10. Advantageously, the total amount of polypeptides, e.g., polypeptides of SEQ ID NO: 10, is between 10 and 500 pg / dose.

[0115] In another particular embodiment, the vaccine composition comprises a BPI3 V F ectodomain dimer comprising two polypeptides of SEQ ID NO: 11. In a preferred embodiment, said vaccine composition comprises a BPI3V F ectodomain trimer comprising three polypeptides of SEQ ID NO: 11. Advantageously, the total amount of polypeptides, e.g., polypeptides of SEQ ID NO: 11, is between 10 and 500 pg / dose.

[0116] In another particular embodiment, the vaccine composition comprises a BPI3 V F ectodomain dimer comprising two polypeptides of SEQ ID NO: 14. In a preferred embodiment, said vaccine composition comprises a BPI3V F ectodomain trimer comprising three polypeptides of SEQ ID NO: 14. Advantageously, the total amount of polypeptides, e.g., polypeptides of SEQ ID NO: 14, is between 10 and 500 pg / dose.

[0117] In another particular embodiment, the vaccine composition comprises a BPI3 V F ectodomain dimer comprising two polypeptides of SEQ ID NO:22. In a preferred embodiment, said vaccine composition comprises a BPI3V F ectodomain trimer comprising three polypeptides of SEQ ID NO: 22. Advantageously, the total amount of polypeptides, e.g., polypeptides of SEQ ID NO: 22, is between 10 and 500 pg / dose.

[0118] It is another object of the present invention to provide an immunogenic composition, in particular a vaccine composition, comprising an immunologically effective amount of any one of the nucleic acids encoding the polypeptides of the invention, and / or a vector comprising one of said nucleic acids, and / or a recombinant cell comprising said nucleic acid or said vector. The vaccine compositions of the invention may additionally comprise at least one immunogen from at least one additional pathogen. It is therefore an object of the present invention to provide a multivalent vaccine comprising at least one immunogen as described above, directed against BPI3 V, and at least one additional antigen or immunogen directed against another pathogen.

[0119] In a particular embodiment, the at least one additional antigen or immunogen is directed against a pathogen associated to Bovine Respiratory Disease (BRD) and / or calf diarrhea, such as Bovine Respiratory Syncytial virus (BRSV), Bovine Viral Diarrhea virus (BVDV), Bovine Herpesvirus type 1 (BHV1), Bovine Coronavirus (BCV), Bovine rotavirus, Mycoplasmopsis Bovis, Pasteurella mullocidci, Mannheimia haemolytica, Histophilus somni and Escherichia coli. In an embodiment, the additional antigen is an antigenic peptide or immunogen.

[0120] In a particular embodiment, the vaccine composition is a subunit vaccine, comprising at least one polypeptide of the invention and at least one additional polypeptide able to elicit or enhance an immune response against at least one pathogen associated to BRD, and optionally, an excipient and / or an adjuvant.

[0121] Administration o f the vaccine composition

[0122] According to the present invention, the vaccine composition may be administered to any nonhuman mammal. The vaccine composition of the present invention is particularly suited for farm animals, in particular livestock such as bovine, sheep, goats, etc., preferably bovine.

[0123] In a preferred embodiment, the vaccine composition is administered to bovine such as a bull, a cow or a calf. In a particular embodiment, the vaccine composition is administered to a pregnant cow.

[0124] In a preferred embodiment, the vaccine composition is administered to a calf.

[0125] In an embodiment, the vaccine is administered to a subject who has not yet been exposed to a BPI3 virus. Preferably, the subject is a bovine which is in need of vaccination against BPI3V infection and / or BRD.

[0126] In an embodiment, the vaccine composition of the invention is administered to an animal susceptible to or otherwise at risk for BPI3V infection to enhance the animal own immune response capabilities. The animal to which the vaccine is administered is in one embodiment a bovine, in particular a calf. The animal may be susceptible to infection by BPI3V or a closely related virus. The vaccine composition may be administered to cattle from herd free of BPI3V. The bovines may be either BPI3V seronegative or BPI3V seropositive. Alternatively, the vaccine composition may be administered to cattle from herd with an outbreak of BPI3 V. By definition, such herd comprises infected animals, e.g., clinically or subclinically infected, or noninfected animals. That is to say that the animals may be infected at the time of the vaccination. The vaccine composition may be administered indistinctly to infected and noninfected animals. Said animals, in particular said bovines, may be either BPI3 V seronegative or BPI3 V seropositive.

[0127] According to the present invention, the vaccination allows to protect vaccinated animals against BPI3 V, i.e., to prevent infection and / or to reduce the severity of at least one symptom associated with BPI3V infection. The vaccination may further treat BPI3V infection in already infected animals, at least by reducing the severity of one or more symptoms associated with BPI3V infection.

[0128] In an embodiment, the vaccine composition is administered to calves having maternally derived anti-BPI3V antibodies. For instance, the vaccine composition can be administered to calves from a herd having experienced or experiencing an outbreak of BPI3V.

[0129] In particular, the vaccine composition may be administered to bovine, preferably calves not older than 5 weeks. In the context of the present invention, a week refers to about 7 consecutive days.

[0130] The calve may be less than 5, 4, 3, 2 or 1 week old; 1 to 4 weeks old; 3 to 28 days old; or 7 to 21 days old. For instance, the vaccine composition is administered to calves between 1 and 5 weeks of age, between 1 and 2 weeks of age, between 2 and 3 weeks of age, etc.

[0131] In a particular embodiment, the vaccine composition is administered to calves between 3 and 28 days of age or older, + / - 3 days, such as between 7 and 21 days of age, + / - 7 days.

[0132] In a particular embodiment, the vaccine composition is administered to calves of 7 day of age, 10 day of age, 15 day of age, 21 day of age, or 28 day of age, + / - 1 day. When several calves from a herd are vaccinated, the age of vaccination may correspond to the mean age of the population of calves.

[0133] The dosage of the vaccines of the present invention will depend on the species, breed, age, size, vaccination history, health status of the animal to be vaccinated, as well as of the route of administration, e.g., subcutaneous, intradermal, oral, intramuscular or intravenous administration. In a particular embodiment, the vaccine composition is administered by intramuscular route. Depending on the desired duration and effectiveness of the treatment, the vaccine composition may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

[0134] Preferably, the vaccine composition is a single shot vaccine. In a particular embodiment, the vaccine composition is administered once (i.e., single shot) to the target animal.

[0135] In a preferred embodiment, the vaccine composition is administered once to calves, preferably by intramuscular injection. For instance, the vaccine composition is administered once to calves between 1 and 5 weeks of age, between 1 and 2 weeks of age, between 2 and 5 weeks of age, between 3 and 4 weeks of age, between 2 and 3 weeks, etc.

[0136] In a particular embodiment, the vaccine composition is administered once to calves between 1 and 28 days of age or older, such as between 7 and 21 days of age, + / - 3 days.

[0137] In a particular embodiment, the vaccine composition is administered once to calves of 1 day of age, of 7 days of age, 10 days of age, 15 days of age, 21 days of age, or 28 days of age, + / - 1 day.

[0138] In another particular embodiment, the vaccine composition is administered once to calves between 21 and 35 days of age or older, + / - 3 days, such as between 22 and 31 days of age, + / - 3 days.

[0139] In a particular embodiment, the vaccine composition is administered once to calves of 21 days of age, 25 days of age, 28 days of age, 30 days of age, 35 days of age, + / - 3 days.

[0140] It is therefore an object of the present invention to provide an immunogen or composition as described above, for use in a method to reduce or prevent one or more clinical symptoms caused by or associated to a BPI3V infection, said method comprising a single administration of the composition or immunogen to calves between 1 and 5 weeks of age, by intramuscular injection, in particular between 1 and 3 weeks of age, or between 3 and 5 weeks of age. In an embodiment, the calves are at least 1 weeks old. In another embodiment, the calves are at least 3 weeks old. In an embodiment, the calves are at most 5 weeks old. In another embodiment, the calves are at most 3 weeks old. The calves may have anti-BPI3V antibodies, in particular maternally derived anti-BPI3V antibodies.

[0141] It is therefore another object of the present invention to provide an immunogen or composition as described above, for use in a method to treat or prevent a BPI3V infection, said method comprising a single administration of the composition or immunogen to calves between 1 and 5 weeks of age, in particular between 1 and 3 weeks of age, or between 3 and 5 weeks of age, by intramuscular injection. In an embodiment, the calves are at least 1 weeks old. In another embodiment, the calves are at least 3 weeks old. In an embodiment, the calves are at most 5 weeks old. In another embodiment, the calves are at most 3 weeks old.

[0142] In another embodiment, the vaccine composition is administered twice to the target animal, e.g., between 1 and 7 weeks of age, + / - 7 days. For instance, the first administration is performed at 1 weeks of age. And the second administration is performed at about 3 weeks of age, 4 weeks of age or 5 weeks of age.

[0143] Vaccination of pregnant females may also be envisioned as it may confer passive immunity to the newborns via the transmission of maternal antibodies.

[0144] The dosage of the vaccine, concentration of components therein and timing of administering the vaccine, which elicit a suitable immune response, can be determined by methods such as by antibody titrations of sera, e.g., by ELISA and / or seroneutralization assay analysis and / or by vaccination challenge evaluation.

[0145] The vaccines of the invention can be administered alone or can be administered simultaneously or sequentially administered with one or more further compositions, such as for example other bovine immunogenic or vaccine compositions. Where the compositions are administered at different times the administrations may be separate from one another or overlapping in time.

[0146] According to the present invention, the vaccine composition comprising a polypeptide as described above is administered to livestock, in particular cattle, such as to calves, in order to treat BPI3V infection, to prevent BPI3 V infection, or at least to reduce the severity of at least one symptom associated to BPI3 V infection.

[0147] An embodiment of the invention relates to the method above, where the vaccination results in prevention or reduction of one or more symptoms associated with BPI3 V infection.

[0148] As used herein, “prevention / reduction of the incidence and / or severity of clinical signs" or "reduction of clinical symptoms" means, but is not limited to, reducing the number of infected animals in a group, reducing or eliminating the number of animals exhibiting clinical signs of infection, or reducing the severity of one or more clinical signs that are present in one or more animals, in comparison to wild-type infection. For example, it should refer to any reduction of pathogen load, pathogen shedding, reduction in pathogen transmission, or reduction of any clinical sign symptomatic of BPI3 V infection. Preferably these clinical signs are reduced in one or more animals receiving the vaccine composition of the present invention by at least 10% in comparison to animals not receiving the vaccine composition and that become infected. More preferably one or more clinical signs are reduced in animals receiving a composition of the present invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.

[0149] The term "reduction of viremia" or “reduction of pathogen load' means, but is not limited to, the reduction of BPI3 virus entering the bloodstream of an animal, wherein the viremia level, i.e. the number of BPI3 V RNA copies per mL of blood serum or the number of plaque forming colonies per deciliter of blood serum, is reduced in the blood serum of subjects receiving the composition of the present invention by at least 50% in comparison to subjects not receiving the composition and may become infected. More preferably, the viremia level is reduced in subjects receiving the composition of the present invention by at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999%.

[0150] The term "increased protection" herein means, but is not limited to, a statistically significant reduction of one or more symptoms which are associated with infection by BPI3V in a vaccinated group of subjects vs. a non-vaccinated control group of subjects. The term "statistically significant reduction of clinical symptoms" means, but is not limited to, the frequency in the incidence of at least one clinical symptom in the vaccinated group of subjects is at least 10%, preferably 20%, more preferably 30%, even more preferably 50%, and even more preferably 70% lower than in the non-vaccinated control group after infection by BPI3 V.

[0151] In a particular embodiment, the method of the invention is to reduce or prevent BPI3V viral shedding in a non-human mammal, in particular in bovine. For instance, the vaccine composition of the invention allows to reduce nasal shedding, or nasal discharge, in bovine.

[0152] In a particular embodiment, the method of the invention is to reduce or prevent at least one symptom associated to BPI3 V infection. Preferably, the composition of the invention allows to reduce or prevent one or more of the symptoms selected from the group consisting of increased core body temperature, depression, dysorexia / anorexia, reduced milk production, tachypnea, polypnea, dyspnea, orthopnea, nasal discharge, ocular discharge, cough, lung lesions.

[0153] Preferably, in the context of the present disclosure, the treatment or prevention of an infection with BPI3 V is based on or comprises or consists of the induction of an immune response against BPI3V.

[0154] In a particular embodiment, the composition allows to protect a subject in need thereof against an infection with BPI3 V type A. In another embodiment, the composition allows to protect a subject in need thereof against an infection with BPI3V type C. In another embodiment, the composition allows to protect a subject against both an infection with BPI3V type A and an infection with BPI3 V type C. The infection with BPI3 V type A and the infection with BPI3 V type C may be concurrent or separated in time.

[0155] According to the invention, the subject may be at risk to be infected by BPI3 V type A, by BPI3 V type C, with BPI3V type A and BPI3V type C. When infections by both BPI3V type A and BPI3 V type C are expected, said risks of infections or said infections may be concomitant or at different time, from days to months or years.

[0156] Further aspects and advantages of the invention are provided in the following section, which should be considered as illustrative only.

[0157] EXAMPLES

[0158] Example 1: Preparation of the BPI3V vaccine candidates

[0159] The BPI3V vaccine candidates tested in the examples hereafter comprised the polypeptide of SEQ ID NO: 10 (candidate 9), SEQ ID NO: 11 (candidate 10), , SEQ ID NO: 14 (candidate 13) or SEQ ID NO: 22 (candidate 15) as active ingredient. The corresponding polypeptides were produced in recombinant CHO cells by use of the nucleic acid sequence as set forth in SEQ ID NO: 19 (candidate 9), SEQ ID NO: 20 (candidate 10), SEQ ID NO:23 (candidate 15) , SEQ ID NO: 21 (candidate 13) or SEQ ID NO:23 (candidate 15). The recombinant CHO cells stably expressing the BPI3 V antigen were cultured at 500 mL scale in fed-batch process over 14 days. Supernatant containing the secreted antigen was collected, clarified by centrifugation and diafiltrated against PBS in tangential flow filtration resulting in the final antigen. The obtained polypeptide was quantified in an ELISA using polypeptide specific antibody and formulated. Resulting vaccine contained 1000 EU, i.e. ELISA units, of protein in phosphate buffered saline (PBS) mixed with the adjuvant Montanide ISA 61 VG (manufacturer: SEPPIC, France) at 60% (w / w) to a final volume of 2ml.

[0160] As Control material, a PBS solution was used.

[0161] Example 2: Efficacy study of a BPI3V vaccine in calves

[0162] An experiment was conducted to ascertain the safety of administering a BPI3 V vaccine of the invention to young calves, and to evaluate its efficacy in providing protection against an experimental BPI3V challenge.

[0163] Experimental design On arrival, all animals were colostrum-fed and held in quarantine. Each calf was housed in individual pens with no contact between animals.

[0164] In order to prevent occurrence of bacteria causing respiratory disease or diarrhea before the experimental BPI3V challenge, animals were treated with the following antibiotics:

[0165] Synulox Amoxicillin trihydrate, clavulanic acid; Zoetis; Lot No. D2006600; Expiry: 08 / 2024

[0166] Baytril Max Enrofloxacin; Bayer; Lot: KV03X5B; Expiry: 06 / 2024

[0167] Gabbrovet Paromomycin sulphate; Ceva; Batch No. 53 A2; Expiry: 03 / 2024

[0168] All animals were administered 3 intramuscular injections of 2.5 mL of Synulox RTU Injectable: on arrival, 24 hours, and 48 hours after initial administration. Five days after arrival and daily for five days, the animals were administered 7.5 mL of Gabbrovet via their milk per day. Seven days after arrival, the animals were administered 4 mL of a subcutaneous injection of Baytril Max. No impact on the BPI3V vaccine was expected as these antibiotics have a short-term action.

[0169] All enrolled calves have been tested negative for Bovine Viral Diarrhea Virus (BVDV) antigen in blood by RT-PCR analysis. Additionally, nasopharyngeal swabs were collected upon arrival and one week later to confirm the absence of BPI3V using PCR analyses. BVDV and BPI3V seropositive animals were excluded from the study. Furthermore, all animals must be in good general health prior to vaccination and fall within the specified age range of 22 to 31 days on Day 0.

[0170] Based on the analysis outcomes, all animals were deemed suitable for inclusion in the study. The calves were allocated to two groups of 11 and 12 animals, that were respectively administered with the BPI3V vaccine candidate 9 described in example 1 (BPI3V vaccine Group) and PBS (Control Group) on Day 0. Both administrations were injected intramuscularly in a 2ml volume.

[0171] On Day 28, all calves were challenged with approximately 8mL of challenge material homogenized with sterile PBS solution (1.56xl07TCID50 total of BPI3V), for a minimum of 8 minutes, via a nebulizer (Flexineb®).

[0172] Challenge material was a type C BPI3V strain (GenBankNo. OQ302284.1 - Respirovirus bovis isolate ICSA-ll / France / 2014), identified by Pr. Gilles MEYER on 19 / 03 / 2014 (Gaudino et al., Infect Genet Evol. 2023 "Molecular and genetic characterization of bovine parainfluenza type 3 European field and vaccine strains" . Blood samples were collected upon arrival to test the presence of BVDV and BPI3 V antibodies, and on Days 0, 14, 21, 28 and 40 to test the presence of BPI3V antibodies, using commercially available ELISA (e.g., BIOX BIO K 061 - Monoscreen AbELISA BPI3V / indirect). As the animals underwent quarantine upon arrival, were colostrum-fed, and tested negative for BPI3 V virus through PCR analysis, the presence of BPI3V antibodies prior to the injection of either BPI3 V vaccine or Control Material is inferred to be Maternally Derived Antibodies (MDA).

[0173] From Day 27 to Day 40, daily nasopharyngeal swab samples were systematically collected from all animals to test the presence of BPI3 V virus by an immunoassay according to the method well-known in the art. In brief, at Day 0, the nasal swabs are split into several aliquots that are further sonicated, diluted if necessary, and put into a 96 well microtiter plate. Then, host cells are added to the microtiter plates and incubated according to standard procedures. At day 4 / 7, the plates are read, and the results recorded. The TCID50 was calculated for each sample (calculation of Virus Titre by the Karber Method).

[0174] In order to assess BPI3V vaccine administration effects and injection site reactions, clinical observations and injection site assessments were conducted on all calves prior to administration on Day 0, 4hrs post administration and on Day 1.

[0175] To assess BPI3V vaccine efficacy to protect calves against BPI3V infection, post-challenge clinical observations were conducted on Day 27, prior to challenge on Day 28 and 4 hours (± 1 hour) post challenge on Day 28. Clinical observations were then conducted once daily on all animals from Day 29 to 34 and then twice daily from Day 35 to 39. Clinical observations consisted in assessing several clinical parameters including rectal temperature.

[0176] Rectal temperatures were assessed as outlined in Table 1.

[0177] Table 1 : Rectal Temperatures scoring

[0178] On Day 40, all calves were euthanized and necropsied. Lungs were removed from all animals and scored for the presence of lesions. More particularly, the percentage of lung damage, as evidenced by the presence of lesions, for each lung lobe, was recorded. More particularly, percentage gross involvement of lesions for each lobe was weighted using the following percentages (based on ratios of individual lung lobes to total lung mass): left apical 5%, left T1 cardiac 6%, left diaphragmatic 32%, right apical 6%, right accessory 5%, right cardiac 7%, right diaphragmatic 35% and intermediate 4%. The weighted lung lobe values were summed to yield the percentage of total lung with lesions for each animal. Scoring was determined based on visual observation of lesions but also following palpation of the lungs. The scoring system is based on Jericho, KWF and Langford, EV; 1982; Aerosol Vaccination of Calves with Pasteurella haemolytica Against Experimental Respiratory Disease; Can. J. comp. Med. 46; pp 287- 292. Weighting of lung scores was carried out using Excel.

[0179] Throughout this study, all standard procedures, including weighing, blood sampling, nasopharyngeal sampling, and challenge administration, adhered strictly to established clinical protocols and Moredun Scientific standard operating procedures.

[0180] Results

[0181] The results confirmed that BPI3V vaccine was safe when administered to calves, as no abnormal clinical signs were observed post administration. Similarly, no injection site reactions were observed.

[0182] Animals vaccinated with the BPI3V vaccine demonstrated reductions in all tested parameters compared to their corresponding challenge control group.

[0183] Regarding the Rectal Temperatures, statistical analysis indicated a significant difference between BPI3V vaccine Group and Control Group. Rectal temperatures were significantly reduced in favor of BPI3V vaccine Group (p=0.06) (Figure 1).

[0184] When comparing the Consolidated Lung Lesion Score on Day 40, statistical analysis revealed a significant reduction of lung lesions in favor of BPI3 V vaccine Group (p=0.016), as illustrated in Figure 2. Table 2 below further summarized the results for lung lesions.

[0185] Table 2: Lung Lesion Summary

[0186] Finaly, regarding the Virus Isolation Data collected, statistical analysis revealed a significant reduction of the viral load in favor of BPI3V vaccine Group (p=0.016) (Figure 3). Virus isolation was highest in Control Group with a peak of 2.32xl06TCIDso / ml on Day 33 (i.e., 5 days post-challenge) and a duration of 7 days (first to last positive sample). As compared, the BPI3V vaccine Group had a peak on Day 33 of 1.18xl05TCIDso / ml and a duration of 6 days. From the results depicted in Figures 1-3, a clear clinical advantage is evident for the calves that received vaccination with the immunogenic composition of the invention. Consequently, the data derived from this study suggest that the tested vaccine composition was efficacious, exerting a substantial protective effect on key parameters assessed, including rectal temperatures, lung lesions, and virus isolation.

[0187] Example 3: Expression of BPI3V F ectodomain in CHO cells

[0188] Detection of the BPI3V F ectodomain trimeric conformation in four vaccines candidates

[0189] The presence of the BPI3 V F ectodomain trimeric conformation in the four vaccines candidates described before (example 1) was identified by size exclusion chromatography (SEC). The SEC was performed using a S200 increase 10 / 300 GL column (manufacturer: Cytivia; product reference #28990944) equilibrated in 1.5 Column Volumes (CV) of dPBS (Dulbecco’s phosphate-buffered saline). A volume of 400 pl of respective samples at a concentration of 300 Units of BPIV3 were loaded into a 500 pl capillary loop. The samples were applied to the column by emptying the loop with 1 ml of DPBS. Fractions of 500 pl were collected after 0.2 CV.

[0190] Results

[0191] As shown in figure 4, a BPI3 V trimer peak fraction was observed, proving the detection of the BPI3 V F ectodomain trimer in the vaccine candidates.

[0192] Evaluation of the BPI3VF expression

[0193] The expression of the BPI3 V F trimeric / monomer forms in CHO cells by Western Blot analysis using both non-reducing and reducing conditions, has been performed.

[0194] Western Blots in non-reducing gel conditions was conducted using samples prepared in Non- Reducing Lane Marker Sample buffer (#39001; Thermo Fisher), and gels were run in IX Tris- Glycine buffer (#1610734; Bio-Rad) at 125V for 90 min. PAGERulerPlus prestained (#26619; Thermo Fisher) was used as marker. Nitrocellulose membranes (#88025; Thermo Fisher) were blotted in Native transfer buffer (25 mM Tris; 192 mM Glycine; 10% Ethanol) at 100V for 40 min at 4°C. Next, membranes were blocked in IX ROTIBlock (#A151.4; Roth), for Ih at RT. Membranes were then incubated overnight at 4°C with 1 : 10,000 dilution of pooled immunized rabbit sera (BioMedVet) in IX ROTIBlock. Membranes were washed with PBS 0.05% Tween- 20 (#28352; Thermo Fisher), and incubated for Ih at RT with 1 :5,000 dilution of goat antirabbit HRP secondary antibodies (#GTX213110-01; GeneTex). Membranes were then washed with PBS 0.05% Tween-20, and developped using the Clarity ECL substrate (#170-5060; BioRad).

[0195] Western Blots analysis in reducing conditions was conducted with samples prepared in Laemmli buffer (2% SDS; 375 mM Tris; 10% glycerol; 0.005% Bromophenol blue) with 20 mM DTT, and boiled for 5 min at 97°C. SDS PAGE gels were run with SDS-PAGE buffer (#3060.1; Roth) at 200V for 40 min. PAGERulerPlus prestained (#26619; Thermo Fisher) was used as marker. Nitrocellulose membranes (#88025; Thermo Fisher) were blotted in Towbin buffer (25 mM Tris; 192 mM Glycine; 20% Methanol), at 100V for Ih at 4°C. Next, membranes were blocked in IX ROTIBlock (#A151.4; Roth), for 1 hour at RT. Membranes were then incubated overnight at 4°C with 1 : 10,000 dilution of pooled immunized rabbit sera (BioMedVet) in IX ROTIBlock. Membranes were washed with PBS 0.05% Tween-20 (#28352; Thermo Fisher), and incubated for 1 hour at RT with 1 :5,000 dilution of goat anti-rabbit HRP secondary antibodies (#GTX213110-01; GeneTex). Membranes were then washed with PBS 0.05% Tween-20 and developped using the Clarity ECL substrate (#170-5060; Bio-Rad).

[0196] Results

[0197] A shown in figure 5 BPI3V F ectodomain expressed in CHO cells appears as band at approximately 170 kDa representing trimeric form in SDS-PAGE under non-reducing conditions (figure 5A) and approximately 57 kD under reducing conditions (figure 5B) representing monomeric form.

[0198] Example 4: Seroneutralization assays with cattle sera

[0199] The objective of this example is to demonstrate the ability of a BPI3V vaccine candidate to induce a neutralizing antibody response against both BPI3 V type A and C strains detectable in seroneutralization assays (SNT).

[0200] The sera of three cattle vaccinated with the BPI3V vaccine candidate 9 described before in example 1 (vaccinated group) and sera of one non vaccinated cattle (negative control) were analysed in SNT at DO (before vaccination) and D161 (after vaccination). Sera were analyzed in serum neutralization assay against four different BPIV3 strains, two belonging to Serotype A (strain Jena 135 and Dargen) and two belonging to serotype C (strain Parral and Frankreich) respectively.

[0201] BPI3V vaccine candidate

[0202] A BPI3V vaccine comprising the polypeptide of SEQ ID NO: 10 was prepared according to example 1.

[0203] Preparation of BPIV3 isolates for SNT assays Figure 6 illustrates the Phylogenetic tree of BPIV3 serotypes A and C and isolates used for SNT assays are shown in bold.

[0204] BPI3V Isolate sequencing: Viral genomic RNA was extracted using the QIAamp viral RNA mini kit (#52904; Qiagen), according to the manufacturer’s instructions. cDNA were synthetised using SuperScript™ III (#18080051 ; Invitrogen) and specific primers according the manufacturer’s instructions. After digestion with RNAse H (#M0297S; NEB), cDNA were used as templates for PCR using the Hot start High-Fidelity Q5 polymerase (#M0494S; NEB) and specific primers. PCR products were run on a 0.8% agarose gel and extracted using the GeneJET Gel Extraction Kit (#K0692; Thermo Fisher) according to the manufacturer’s instructions and analyzed by Eurofins with Sanger sequencing. A phylogenetic tree for BPIV3 serotypes A and C was built in Geneious using the Juke-Cantor model and the Neighbor-joining method.

[0205] Table 3 below summarizes the list of primers used for BPIV3 isolate cDNA synthesis, PCR, and sequencing Table 4 below shows the list of BPIV3 Fusion protein sequences from serotype A and C isolates or strains used to establish a phylogenetic tree. Results

[0206] As shown in figure 7, the results clearly demonstrate that all vaccinated animals have higher neutralizing antibody titers after vaccination against all tested BPIV3 strains (2 serotype A and 2 serotype C). For the vaccinated animals, the antibodies at DO are likely maternally derived. Although the animals were not free of MDA, SNT titres increased after vaccination. The negative control stays negative, proving that the increase in SNT is specific due to vaccination and not natural infection. Example 5: Specific antibody response induction in MDA positive calves vaccinated with BPI3 vaccine

[0207] Non-heparinised blood samples collected from all animals within 24 h of arrival and on Days 0, 14, 21, 28 and 40 were tested for the presence of BPi3 V antibodies, by ELISA for screening purposes. This was performed using a commercially available ELISA (BIOX BIO K 061 - Monoscreen AbELISA BPI3V / indirect) following the manufacturers recommandations. Boxplot of the ELISA results (Day 0 to 40) is shown in figure 8.

[0208] The mean titer of antibodies against BPI3V of each group decreased between Day 0 and Day 28, but increased to Day 40. A barplot of the degree of positivity ELISA data is shown in figure 9.

[0209] Post challenge on Day 28 the percentage of BPI3V antibody for each group was variable with a range of negative (“0”), low (“+”), medium (“++” ; “+++”) or high (“++++” ; “++++++”) titres. On Day 40 (12 days post challenge), the Control Group titers remained variable with a mix of low, medium and high titres, while in the BPI3V vaccinated Group there were no low titers, only medium or high titers.

[0210] Sequences listing of the invention

[0211] SEP ID NO:1

[0212] MITIVATTVILLLPLSLCQIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQS CGDQQINQYKKLLDRLIIPLYDGLKLQRDVIVVSHESHNSTNSRTKRFFGEIIGTIAIGIA TSAQITAAVALVEAKQARSDIDKLKEAIRDTNKAVQSIQSSVGNLIVAVKSVQDYVNNE IVPSITRLGCEAAGLQLGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTNITEIFT TSTVDQYDIYDLLFTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQ GKEWYIPLPNHIMTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCP KTVVTSDIVPRYAFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGING

[0213] MLFRTNKEGTLATYTFDDIVLNNSVALNPIDISMELNKAKLELEESKEWIKKSNQKLD SVGNWYQSSATIIIIIVMIVFLFIINITIIVVIIKYHRVQEKNPNYKNNEPYVLTNRT

[0214] SEO ID NO:2 / Candidate 1

[0215] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0216] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAK

[0217] QARSDIDKLKEAIRDTNKAVQSIQSSVGNLIVACKSVQDYVNNEIVPSITRLGCEAAG

[0218] LQLGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTCITEIFTTSTVDQYDIYDLL

[0219] FTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHIM

[0220] TKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRYAF

[0221] VNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLAT

[0222] YTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWIKKSNQKL

[0223] SEO ID NO:3 / Candidate 2

[0224] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0225] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAKQ

[0226] ARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAGLQ

[0227] LGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTSTVDQYDIYDLLFT

[0228] ESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHIMTK

[0229] GAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRYAFVN GGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLATYTF

[0230] DDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKL

[0231] SEO ID NO:4 / Candidate 3

[0232] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0233] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAK

[0234] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0235] LQLGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTCTVDQYDIYDL

[0236] LFTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0237] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0238] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0239] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKL

[0240] SEO ID NO:5 / Candidate 4

[0241] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0242] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAK

[0243] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0244] LQLGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTNITEIFTTCTVDQYDIYDL

[0245] LFTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0246] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0247] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0248] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKL

[0249] SEO ID NO:6 / Candidate 5

[0250] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0251] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVECK

[0252] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0253] LQLGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTSTVDQYDIYDL

[0254] LFTESIKMRVCDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0255] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0256] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKL

[0257] SEO ID NO:7 / Candidate 6

[0258] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0259] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVECK

[0260] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0261] LQLGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTCTVDQYDIYDL

[0262] LFTESIKMRVCDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0263] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0264] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0265] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKL

[0266] SEO ID NO:8 / Candidate 7

[0267] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0268] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVECK

[0269] QARSDIDKLKEAIRDTNKAVQSIQSSVGNLIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0270] LQLGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTNITEIFTTCTVDQYDIYDL

[0271] LFTESIKMRVCDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0272] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0273] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0274] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKL

[0275] SEO ID NO:9 / Candidate 8

[0276] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0277] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAK

[0278] QARSDIDKLKEAIRDTNKAVQSIQSSVGNLIVACKSVQDYVNNEIVPSITRLGCEAAG

[0279] LQLGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTCITEIFTTSTVDQYDIYDLL

[0280] FTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHIM

[0281] TKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRYAF

[0282] VNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLAT YTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWIKKSNQKLSAIGGYIPEAPRDGQ

[0283] AYVRKDGEWVLLSTFLGGLVPRGSH

[0284] SEO ID NO: 10 / Candidate 9

[0285] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQSCGDQQINQYKKLLDR LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAKQ ARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAGLQ LGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTSTVDQYDIYDLLFT ESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHIMTK

[0286] GAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRYAFVN GGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLATYTF DDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRDGQAY VRKDGEWVLLSTFLGGLVPRGSH

[0287] SEO ID NO: 11 / Candidate 10

[0288] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAK QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG LQLGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTCTVDQYDIYDL LFTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRD GQAYVRKDGEWVLLSTFLGGLVPRGSH

[0289] SEO ID NO: 12 / Candidate 11

[0290] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0291] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVEAK

[0292] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0293] LQLGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTNITEIFTTCTVDQYDIYDL

[0294] LFTESIKMRVIDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0295] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0296] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRD

[0297] GQAYVRKDGEWVLLSTFLGGLVPRGSH

[0298] SEO ID NO: 13 / Candidate 12

[0299] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0300] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVECK

[0301] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0302] LQLGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTSTVDQYDIYDL

[0303] LFTESIKMRVCDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0304] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0305] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0306] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRD

[0307] GQAYVRKDGEWVLLSTFLGGLVPRGSH

[0308] SEO ID NO: 14 / Candidate 13

[0309] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0310] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVECK

[0311] QARSDIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSITRLGCEAAG

[0312] LQLGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTCTVDQYDIYDL

[0313] LFTESIKMRVCDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHI

[0314] MTKGAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRY

[0315] AFVNGGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLA

[0316] TYTFDDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRD

[0317] GQAYVRKDGEWVLLSTFLGGLVPRGSH

[0318] SEO ID NO: 15 / Candidate 14

[0319] QIDITKLQRVGVLVNNPKGMKISQNFETCYLILSLIPKIENSQSCGDQQINQYKKLLDR

[0320] LIIPLYDGLKLQRDVIVVSHESHNSTNSRTERFFGEIIGTIAIGIATSAQITAAVALVECKQ

[0321] ARSDIDKLKEAIRDTNKAVQSIQSSVGNLIVAVKSVQDYVNNEIVPSITRLGCEAAGLQ LGIALTQHYSELTNIFGDNIGTLKEKGIKLQGIASLYRTNITEIFTTCTVDQYDIYDLLFT

[0322] ESIKMRVCDVDLSDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHIMTK

[0323] GAFLGGADIKECIEAFSSYICPSDPGFILNHEIENCLSGNITQCPKTVVTSDIVPRYAFVN

[0324] GGLIANCIPTTCTCNGIDNRINQAPDQGIKIITHKECQVIGINGMLFRTNKEGTLATYTF

[0325] DDIVLNNSVALNPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRDGQAY

[0326] VRKDGEWVLLSTFLGGLVPRGSH

[0327] SEQ ID NO: 16 / Signal peptide

[0328] MEFWLSWVFLVAILKGVQC

[0329] SEQ ID NO: 17 / SA-Linker and T4-Foldon

[0330] SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSH

[0331] SEQ ID NO: 18 / SA-Linker and GCN4-Zipper

[0332] SAIEDKIEEILSKIYHIENEIARIKKLIGEAP

[0333] SEQ ID NO: 19 / nucleic acid sequence

[0334] ATGGAATTTTGGCTCAGTTGGGTGTTCTTGGTGGCCATCCTGAAGGGGGTGCAGTG

[0335] TCAGATAGACATAACCAAGCTGCAGAGAGTAGGTGTCCTTGTCAATAACCCAAAA

[0336] GGCATGAAGATCTCTCAGAATTTCGAAACGCGGTACCTCATACTGAGCCTGATTCC

[0337] CAAGATCGAAAACAGTCAGTCTTGTGGAGACCAGCAGATAAATCAGTATAAGAAG

[0338] CTCCTCGATCGACTGATTATCCCACTGTACGACGGTCTGAAACTGCAGAGGGATGT

[0339] CATAGTGGTGTCCCATGAGTCCCACAACAGTACTAATAGCCGGACCGAACGGTTCT

[0340] TTGGCGAGATCATAGGGACCATAGCCATCGGGATAGCCACATCAGCCCAAATAACC

[0341] GCCGCCGTGGCTCTGGTCGAAGCTAAGCAGGCCAGATCAGATATTGATAAATTGAA

[0342] GGAAGCCATTAGGGACACTAACAAGGCCGTGCAGTCTATTTGTTCTTCAGTTGGCA

[0343] ACTGTATCGTTGCTGTGAAGTCTGTCCAGGATTATGTGAATAACGAAATTGTCCCTA

[0344] GTATTACACGGCTTGGGTGCGAGGCAGCTGGACTCCAGTTGGGAATTGCTCTGAC

[0345] ACAGCACTACAGTGAACTCACGAATTGTTTTGGCGATAACATCGGTACTTTGAAGG

[0346] AAAAGGGGATTAAACTTCAATGTATTGCTTCACTGTACCGTACAAACATCACTGAG

[0347] ATCTTTACTACAAGTACCGTAGACCAGTACGACATCTACGATCTGTTGTTCACAGA AAGCATAAAGATGAGAGTCATCGATGTGGATCTCAGTGACTACAGCATCACCCTAC

[0348] AAGTGCGCCTGCCCCTGCTGACAAAGCTGTCTAACACACAAATCTATAGAGTTGAC

[0349] TCTATCTCCTACAACATTCAGGGCAAAGAATGGTACATTCCACTGCCTAATCACATC

[0350] ATGACCAAAGGTGCATTTCTTGGTGGAGCAGATATTAAAGAGTGCATCGAGGCCTT

[0351] TAGCAGTTATATCTGTCCCTCTGATCCCGGCTTCATTCTTAATCACGAAATTGAGAA

[0352] CTGTCTCTCCGGCAACATCACTCAGTGTCCCAAGACTGTCGTGACCAGCGACATCG

[0353] TCCCGCGCTACGCATTTGTGAATGGGGGTTTGATCGCCAATTGTATTCCAACAACAT

[0354] GCACGTGCAACGGGATTGACAACCGGATCAACCAAGCTCCCGATCAGGGCATCAA

[0355] GATCATCACCCACAAAGAATGCCAAGTCATCGGGATTAACGGGATGCTTTTCAGGA

[0356] CTAATAAGGAGGGGACCCTGGCTACATACACCTTTGACGACATAGTACTCAACAAC

[0357] TCCGTTGCTTTGAACCCTATCGACATTAGCATGGAACTGAACAAGGTGAAGTTGGA

[0358] GTTGGAGGAAAGTAAAGAATGGTATAAGAAATCTAACCAGAAACTGAGTGCTATC

[0359] GGCGGTTACATTCCAGAGGCTCCCCGAGATGGCCAGGCATACGTCCGAAAAGACG

[0360] GAGAGTGGGTATTGTTGTCTACCTTTCTGGGTGGACTGGTGCCACGGGGGTCCCAT TAA

[0361] SEQ ID NQ:20 / nucleic acid / candidate 10

[0362] CAAATTGATATTACAAAGTTGCAGCGGGTCGGCGTACTTGTTAACAATCCCAAAGG

[0363] AATGAAGATCTCCCAAAACTTTGAGACCTGTTATCTGATCCTGTCCCTGATCCCAA

[0364] AGATCGAGAATTCCCAGTCCTGTGGCGACCAGCAGATTAATCAGTACAAAAAACTT

[0365] CTAGACCGGCTGATAATCCCTCTGTACGACGGTCTGAAGCTGCAGAGAGATGTCAT

[0366] AGTCGTGTCACATGAGAGTCATAACTCCACCAACTCACGTACCGAACGTTTTTTCG

[0367] GCGAGATCATTGGGACCATAGCCATCGGAATCGCTACATCTGCACAAATCACCGCA

[0368] GCCGTCGCTCTTGTGGAGGCTAAACAGGCTCGATCAGACATTGATAAGCTTAAGGA

[0369] GGCAATTCGGGACACCAATAAGGCTGTCCAGAGCATTTGTTCTTCAGTTGGGAATT

[0370] GTATTGTCGCAGTGAAGTCCGTGCAGGATTATGTAAATAATGAGATCGTGCCTAGC

[0371] ATCACCCGCCTTGGCTGTGAAGCCGCAGGCCTCCAACTGGGCATCGCCTTGACTC

[0372] AGCACTATTCTGAGCTAACTAACTGTTTCGGAGACAACATCGGTACACTGAAGGAA

[0373] AAGGGGATTAAACTTCAGTGCATCGCCTCACTGTACCGGACCAACATCACCGAGAT

[0374] CTTTACAACCTGTACCGTCGACCAGTATGATATATATGATCTGCTATTTACAGAGTCC

[0375] ATCAAGATGCGCGTAATTGACGTCGACTTGAGCGACTACAGTATTACATTGCAGGT

[0376] CAGACTACCCCTACTGACAAAACTCTCTAATACCCAGATCTACCGCGTGGATTCAA

[0377] TCAGTTACAACATCCAAGGTAAAGAGTGGTATATACCTCTGCCCAACCACATCATG

[0378] ACAAAGGGCGCATTTCTGGGCGGTGCTGATATCAAGGAGTGTATTGAGGCCTTTTC

[0379] AAGCTACATTTGTCCATCCGATCCCGGGTTCATCCTCAATCACGAGATTGAGAACT

[0380] GCCTGAGCGGTAAT SEQ ID NO:21 / nucleic acid sequence candidate 13

[0381] CAGATAGATATCACTAAATTGCAGCGCGTTGGTGTGCTGGTTAACAACCCCAAGGG

[0382] TATGAAGATTTCTCAGAATTTCGAAACTAGATACCTGATACTGTCTCTGATTCCCAA

[0383] GATTGAGAACTCCCAGCCATGCGGTGACCAACAGATAAATCAATACAAGAAGCTG

[0384] CTTGACCGTCTGATCATCCCACTCTACGATGGCCTTAAACTGCAGAGGGATGTCAT

[0385] CGTGGTCCGGCACGAGTCCAATAATTCCACATCATCCAGGACTGAAAGATTTTTTG

[0386] GCGAGATAATAGGCACTATTGCAATTGGGATAGCTACCTCAGCCCAAATCACCGCC

[0387] GCCGTGGCCCTCGTCGAGGCTAAACAGGCAAGAGCCGATATCGATAAACTGAAGG

[0388] AAGCCATCCGAGACACTAACAAAGCCGTTCAGTCCATCTGTAGCTCTGTAGGTAAT

[0389] TGCATCGTCGCCGTTAAGAGCGTGCAGGATTATGTGAACAACGAGATTGTACCATC

[0390] TATCGCCCGTTTGGGATGCGAAGCCGCCGGACTGCAGCTTGGCATTGCACTCACAC

[0391] AGCACTATTCTGAATTGACGAATTGCTTTGGAGATAATATTGGGACTCTTAAGGAGA

[0392] AGGGCATTAAACTTCAGTGTATCGCTTCTCTGTACAGGACAAATATTACTGAAATAT

[0393] TTACAACGAGCACTGTTGATCAATATGATATCTACGACCTGCTTTTCACTGAATCTAT

[0394] CAAAATGCGGGTCATTGATGTCGATCTAAATGACTACTCCATAACACTCCAGGTTC

[0395] GGCTTCCCCTTTTGACCAAACTTTCCAATACCCAGATTTACCGGGTGGACAGTATTT

[0396] CTTACAACATCCAGGGCAAGGAGTGGTATATCCCCCTGCCAAACCACATAATGACT

[0397] AAAGGAGCCTTCCTGGGAGGTGCCGATATTAAAGAATGTATCGAGGCCTTCTCATC

[0398] CTATATATGCCCATCCGATCCCGGCTTTATCCTGAATCGGGAAATAGAAAATTGTCTT

[0399] AGCGGAAATATTACTCAGTGCCCTAAGACCGTAGTCACCTCCGACATTGTGCCCAG

[0400] ATATGCCTTTGTGAACGGCGGCGTGATCGCCAATTGCATCCCGACTACCTGTACCTG

[0401] TGATGGCATCGACAACCGCATTAACCAGGCACCTGACCAAGGAATACGTATCATCA

[0402] CCCATAAAGAGTGCCAGGTAATCGGGATAAACGGCATGCTGTTCCGCCCCAATAAA

[0403] GAGGGTACACTCGCTACATACACATACGACGATATTGTGCTGAATAACTCCGTTGCT

[0404] TTGGACCCTATCGATATCTCAATGGAGTTGAATAAAGTGAAGCTGGAACTGGAGGA

[0405] GTCCAAAGAATGGTATAAAAAGTCAAATCAGAAGCTATCTGCTATCGAGGACAAA

[0406] ATCGAGGAGATTCTTTCCAAGATCTACCACATTGAAAATGAGATCGCTCGAATTAA

[0407] GAAGCTGATCGGCGAGGCCCCCTGA

[0408] SEQ ID NO:22 / amino acid sequence / Candidate 15

[0409] QIDITKLQRVGVLVNNPKGMKISQNFETRYLILSLIPKIENSQPCGDQQINQYKKLLDR

[0410] LIIPLYDGLKLQRDVIVVRHESNNSTSSRTERFFGEIIGTIAIGIATSAQITAAVALVEAKQ

[0411] ARADIDKLKEAIRDTNKAVQSICSSVGNCIVAVKSVQDYVNNEIVPSIARLGCEAAGLQ

[0412] LGIALTQHYSELTNCFGDNIGTLKEKGIKLQCIASLYRTNITEIFTTSTVDQYDIYDLLFT

[0413] ESIKMRVIDVDLNDYSITLQVRLPLLTKLSNTQIYRVDSISYNIQGKEWYIPLPNHIMTK GAFLGGADIKECIEAFSSYICPSDPGFILNREIENCLSGNITQCPKTVVTSDIVPRYAFVN

[0414] GGVIANCIPTTCTCDGIDNRINQAPDQGIRIITHKECQVIGINGMLFRPNKEGTLATYTY

[0415] DDIVLNNSVALDPIDISMELNKVKLELEESKEWYKKSNQKLSAIGGYIPEAPRDGQAY

[0416] VRKDGEWVLLSTFLGGLVPRGSH

[0417] SEQ ID NO:23 / nucleic acid sequence / Candidate 15

[0418] CAGATTGACATTACTAAGCTGCAGCGTGTGGGAGTGCTGGTGAACAACCCTAAAG

[0419] GCATGAAGATTAGTCAGAACTTTGAAACCTGTTACCTGATTTTGTCCCTCATTCCTA

[0420] AAATCGAAAACTCCCAATCATGTGGCGATCAGCAGATCAACCAGTACAAGAAGCT

[0421] GCTAGATAGGCTGATTATCCCGCTCTATGATGGACTCAAGCTCCAGCGAGACGTGA

[0422] TAGTCGTCTCTCACGAAAGTCACAACTCCACGAATTCTCGGACAGAACGGTTTTTC

[0423] GGCGAGATAATCGGTACCATTGCCATTGGAATCGCAACCAGCGCTCAGATCACTGC

[0424] CGCTGTGGCACTTGTTGAATGCAAGCAAGCTAGGAGCGACATCGACAAACTAAAA

[0425] GAGGCTATCCGTGACACTAACAAGGCTGTACAATCCATATGCTCTAGCGTGGGTAA

[0426] CTGTATAGTGGCCGTGAAGAGCGTCCAGGATTACGTTAACAACGAGATCGTACCAT

[0427] CAATTACGCGTCTTGGCTGCGAGGCCGCAGGCCTGCAGCTGGGAATCGCTCTCAC

[0428] ACAACACTACTCAGAGCTGACTAATTGTTTTGGGGACAACATAGGGACACTTAAA

[0429] GAGAAGGGAATCAAACTCCAGTGTATAGCATCCCTCTACCGGACTAATATTACCGA

[0430] GATTTTCACTACCTGCACTGTGGACCAGTACGACATTTATGACCTGCTGTTTACCGA

[0431] GTCCATTAAGATGCGGGTGTGTGACGTGGATCTTAGTGATTATAGCATCACTCTGCA

[0432] GGTGAGGTTGCCATTGCTCACAAAACTGAGCAATACACAGATCTACAGGGTTGATA

[0433] GCATCTCTTATAACATCCAGGGTAAGGAATGGTACATCCCTCTCCCAAACCACATTA

[0434] TGACCAAGGGAGCATTCCTGGGCGGCGCAGATATCAAGGAATGCATCGAGGCCTT

[0435] TTCTTCTTATATTTGCCCATCTGACCCCGGTTTTATCCTGAACCACGAGATCGAAAA

[0436] CTGTCTGTCCGGAAACATTACACAGTGTCCCAAGACTGTGGTGACCAGCGATATTG

[0437] TTCCCAGATATGCATTTGTTAACGGAGGACTGATAGCTAACTGCATCCCCACTACGT

[0438] GTACCTGCAACGGCATTGATAATCGGATTAACCAGGCTCCAGATCAGGGAATCAAG

[0439] ATCATTACTCATAAAGAGTGTCAGGTCATCGGTATTAACGGAATGCTGTTCCGCACT

[0440] AATAAGGAAGGAACCCTAGCCACATATACTTTTGACGACATCGTGTTGAACAATTC

[0441] CGTGGCCCTGAACCCAATCGACATTTCCATGGAACTCAACAAAGTGAAACTAGAA

[0442] CTAGAGGAAAGCAAGGAGTGGTACAAGAAGTCCAATCAAAAACTG

Claims

CLAIMS1. An immunogen comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence corresponding to the amino acid residues 19 to 481 of SEQ ID NO: 1.

2. The immunogen of claim 1, comprising a combination of substitutions, as compared to SEQ ID NO: 1, selected fromK108E + I172C + N238C + A463V,K108E + Q162C + L168C + I213C + G230C + A463V + I474Y,K108E + R48C + S246C + Q162C + L168C + I213C + G230C + A463V + I474Y,K108E + R48C + S246C + Q162C + L168C + A463V + I474Y,K108E + A140C-I270C + Q162C-L168C + I213C-G230C + A463V + I474Y,K108E + R48C + S246C + A140C + I270C + Q162C + L168C + I213C + G230C + A463V + I474Y, andK108E + R48C + S246C + A140C + I270C + A463V + I474Y, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

3. The immunogen of claim 1 or 2, further comprising at the C-terminal end a trimerization domain, preferably a GCN4-based isoleucine zipper trimerization domain or a T4 bacteriophage fibritin foldon (Fd) trimerization domain, more preferably a trimerization domain as set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

4. The immunogen of any one of claims 1 to 3, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO:15, more preferably consisting of SEQ ID NO: 10.

5. An immunogenic composition comprising the immunogen of any one of claims 1 to 4 and, optionally, an excipient and / or an adjuvant.

6. A vaccine composition comprising an immunologically effective amount of:(i) an immunogen of any one of claims 1 to 4, and / or(ii) a nucleic acid molecule encoding an immunogen of any one of claims 1 to 4 , and / or(iii) a vector comprising a nucleic acid molecule encoding an immunogen of any one of claims 1 to 4, preferably under the control of a promoter, and / or(v) a recombinant cell comprising a nucleic acid encoding an immunogen of any one of claims 1 to 4, or vector comprising a nucleic acid molecule encoding an immunogen of any one of claims 1 to 4, preferably under the control of a promoter and an excipient and / or an adjuvant.

7. The immunogenic composition of claim 5, or the vaccine composition of claim 6, further comprising at least one additional antigen directed against another pathogen, preferably at least one additional antigen directed against a pathogen associated to Bovine Respiratory Disease (BRD) and / or caff diarrhea, such as Bovine Respiratory Syncytial virus (BRSV), Bovine Viral Diarrhea virus (BVDV), Bovine Herpesvirus type 1 (BHV1), Bovine Coronavirus (BCV), Bovine rotavirus, Mycoplasmopsis Bovis, Pasteurella mullocidci, Mannheimia haemolytica, Histophilus somni and Escherichia coli.

8. An immunogen of any one of claims 1 to 4, or composition according to claim 6 or 7, for use in a method to prevent and / or treat a Bovine Parainfluenza virus Type 3 (BPI3V) associated disease in a non-human mammal.

9. An immunogen of any one of claims 1 to 4, or composition according to claim 6 or 7, for use in a method to reduce or prevent one or more clinical symptoms caused by or associated to a Bovine Parainfluenza virus Type 3 (BPI3V) associated infection in a non-human mammal, wherein the clinical signs are preferably selected from the group consisting of increased core body temperature, depression, dysorexia / anorexia, reduced milk production, tachypnea, polypnea, dyspnea, orthopnea, nasal discharge, ocular discharge, cough, lung lesions.

10. An immunogen of any one of claims 1 to 4, or composition according to claim 6 or 7, for use in a method to reduce BPI3V viral shedding in a non-human mammal.

11. The immunogen or composition, for use according to any one of claims 8 to 10, wherein said immunogen or composition is able to prevent and / or treat a BPI3V type A infection, a BPI3 V type C infection, or both a BPI3 V type A and BPI3 V type C infection, or wherein said immunogen or composition is able to reduce or prevent one or more clinical symptoms causedby or associated to a BPI3 V type A infection, a BPI3 V type C infection, or both a BPI3 V type A and BPI3 V type C infection.

12. The immunogen or composition, for use according to any one of claims 8 to 11, wherein said method comprises a single administration of said immunogen or composition to said non- human mammal.

13. The immunogen or composition, for use according to any one of claims 8 to 12, wherein said immunogen or composition is to be administered parenterally, preferably by injection, and / or wherein said immunogen or composition is to be administered to non-human mammal having anti-BPI3 V antibodies, in particular maternally derived antibodies (MDA).

14. The immunogen or composition, for use according to any one of claims 8 to 13, wherein said non-human animal is livestock selected from bovine, sheep and goats, preferably a bovine.

15. The immunogen or composition for use according to any one of claims 8 to 14, wherein said immunogen or composition is to be administered to cattle between 1 and 28 days of age, in particular between 7 and 21 days of age + / - 3 days.

16. The immunogen or composition, for use according to any one of claims 8 to 15, wherein said method comprises a single administration of the composition or immunogen to calves between 7 and 21 days of age, by intramuscular injection.

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