TW201533055A - Peptides derived from lawsonia intracellularis and their use in vaccination - Google Patents

Abstract

Novel proteins from Lawsonia intracellularis are disclosed herein, as well as immunogenic fragments of the proteins. The invention further provides methods for immunization and vaccination, as well as immunogenic compositions that utilize the novel proteins or nucleic acids encoding the proteins.

Description

a peptide derived from L. intracellularis and its use for vaccination

The present invention relates to the field of microbial peptides and vaccines. In detail, the present invention is based on a novel protein derived from L. intracellularis Listeria (Lawsonia intracellularis) and the polynucleotide. The invention further relates to vectors comprising the polynucleotides, transformed host organisms expressing the polynucleotides, antibodies (single or multiple plants) recognizing the multi-peptides, and diagnosis, prevention, and Therapeutic uses and methods. Finally, the preparation method is also part of the invention.

Proliferative enteropathy (also known as enteritis or ileitis) presents clinical signs and pathological syndromes involving mucosal hyperplasia of immature crypt epithelial cells in many animals (specifically, pigs), primarily at the end of the ileum. Other intestinal parts that can be affected include the jejunum, cecum, and colon. The main diseases are weaned pigs and early mature pigs. The typical clinical manifestations are rapid weight loss and dehydration. The natural clinical disease of pigs occurs worldwide. The disease has always been associated with the presence of intracellular curved bacteria, currently known as L. intracellularis.

The first complete genome sequence of L. intracellularis was published in 2010 (strain PHE/MN1-00 ); the bacterial chromosome consists of 1457619 base pairs.

Vaccination is considered an extremely effective way to prevent infectious diseases in human and veterinary health care. Vaccination is the administration of an immunogenic effective amount of antigenic material (vaccine) to produce immunity to disease/pathogenic pathogens. The vaccine has led to the eradication of smallpox, the near eradication of polio and the control of various diseases, including rubella, measles, mumps, chickenpox and typhoid fever.

Prior to the "genome era", vaccines were based on killed or attenuated live microorganisms or parts purified from them. Subunit vaccines (a newer type of vaccine) contain one or more protective antigens. In order to develop subunit vaccines, it is critical to identify proteins that are important for inducing protection.

Antigens are considered protective if they are protected from subsequent pathogenic infections in an appropriate animal model following immunization.

The empirical approach to subunit vaccine development involves several steps, starting with pathogen incubation, followed by identification and purification of key components, and subsequent testing of the antigen for protection. In addition to time and effort, this method has several limitations that can lead to failure. For microorganisms that may not be easily cultured, it is difficult to develop a vaccine using this method, and it is generally only suitable for obtaining a sufficient amount of antigen. The empirical method has a tendency to focus on the most abundant proteins, which in some cases are not immunoprotective. In other cases, antigens that are expressed during infection in vivo are not expressed during in vitro culture. In addition, antigen discovery involving large amounts of protein and empirical methods to detect protective antigens can be extremely time consuming. This makes it an extremely expensive method and may limit the development of vaccines for certain diseases caused by pathogens with large genomes.

The object of the invention

The object of particular embodiments of the present invention is to provide an intracellular L. sonnei-derived antigenic multi-peptide which acts as a vaccine against intracellular L. sonnei infection and intracellular L. solana infection The ingredients in the diagnosis. The goal is also to provide nucleic acids, vectors, transformed cells, vaccine compositions, and other useful means for molecular selection and for the treatment and diagnosis associated with L. intracellularis.

The inventors of the present invention have found that L. intracellularis exhibits a number of hitherto unknown surface-exposed proteins which are candidates for vaccine targets and are used as immunizing agents for the preparation of intracellular Laussonia. A candidate for antibodies to bacteria.

In a first aspect, the invention relates to a multi-peptide comprising: a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOS: 1-17 and 54, or b) at least 5 consecutive sequences from any one of SEQ ID NOS: 1-17 and 54 An amino acid sequence consisting of an amino acid residue, or c) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of a), and at least 60 of the amino acid sequences of d) and b) a sequence-equilibrium amino acid sequence of %, or e) an amino acid assembly derived from any one of SEQ ID NOS: 1-17 and 54, which has the effect of deriving the assembly to form a B cell antigen The base protein is substantially identical in the 3D configuration and the multi-peptide is antigenic in mammals.

In a second aspect, the invention relates to an isolated nucleic acid fragment comprising: i) a nucleotide sequence encoding a multi-peptide of the invention, or ii) from SEQ ID NOs: 18-51 and 55 a nucleotide sequence consisting of any one, iii) a nucleotide sequence consisting of at least 10 contiguous nucleotides of any one of SEQ ID NOS: 18-51 and 55, iv) and i) or ii) a nucleotide sequence having a sequence identity of at least 60%, v) a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence of iii), vi) and i) a nucleotide sequence complementary to the nucleotide sequence in -v), or a nucleotide sequence which vii hybridizes under stringent conditions to the nucleotide sequence in i)-vi).

In a third aspect, the invention relates to a vector comprising a nucleic acid of the invention, such as a selection vector or expression vector.

In a fourth aspect, the invention relates to cells that have been transformed to carry the vector of the invention.

In a fifth aspect, the invention relates to a pharmaceutical composition comprising a multi-peptide of the invention, a nucleic acid fragment of the invention, a vector of the invention, or a transformed cell of the invention, and a pharmaceutically An acceptable carrier, vehicle or diluent.

In a sixth aspect, the present invention relates to a method for inducing immunity of an animal by administering at least one immunogenic effective amount of the multi-peptide of the present invention, the nucleic acid fragment of the present invention, and the present invention The vector of the invention, the transformed cell of the invention or the pharmaceutical composition of the invention is used to induce adaptive immunity of the animal against L. intracellularis.

In the seventh and eighth aspects, the present invention relates to 1) a plurality of antibodies which specifically bind to at least one multipeptide of the present invention and 2) an isolated single species which specifically binds to the multi-peptide of the present invention Strain antibody or antibody analog. In a related ninth aspect, the invention relates to a pharmaceutical composition comprising the multi-strain or monoclonal antibody and a pharmaceutically acceptable carrier, vehicle or diluent.

In a tenth aspect, the present invention relates to a method for preventing, treating or ameliorating an intracellular L. solani infection comprising administering a therapeutically effective amount of the seventh or third aspect of the present invention to an animal in need thereof The antibody of the 8th aspect or the pharmaceutical composition of the ninth aspect.

In an eleventh aspect, the invention relates to a method for quantitatively or qualitatively determining the presence of L. intracellularis in a sample, the method comprising subjecting the sample to the eighth or ninth aspect of the invention The antibody is contacted and the presence of an antibody that detects the substance bound to the sample.

In a twelfth aspect, the present invention provides a method for quantitatively or qualitatively determining the presence of an antibody specific for a strain of L. intracellularis in a sample, the method comprising: ligating the sample with the peptide of the present invention Contacting and detecting the presence of antibodies that specifically bind to the multi-peptide.

In a thirteenth aspect, the invention relates to a method for quantitatively or qualitatively determining the presence of a nucleic acid specific to L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment of the invention, and The presence of a nucleic acid that hybridizes to the nucleic acid fragment in the sample is detected.

In a fourteenth aspect, the invention relates to a method for the preparation of a multi-peptide of the invention comprising: - under conditions conducive to the performance of a nucleic acid fragment of the invention encoding a multi-peptide of the invention, Incubating the transformed cells of the present invention capable of expressing the nucleic acid of the present invention, and subsequently recovering the multi-peptide; or - Preparation of the multi-peptide by means of solid phase or liquid phase peptide synthesis.

In a fifteenth aspect, the invention relates to a method for determining whether a substance, such as an antibody, is potentially suitable for treating an intracellular L. solani infection, the method comprising contacting a multi-peptide of the invention with the substance And subsequently determining whether the substance has at least one of the following characteristics: 1) the ability to specifically bind to the multi-peptide, and 2) the ability to compete with the multi-peptide for specific binding to the ligand/receptor , or 3) the ability to make the multi-peptide specific for inactivation.

Finally, in a sixteenth aspect, the invention relates to a method for determining whether a substance, such as a nucleic acid, is likely to be suitable for treating an intracellular L. sonnei infection, the method comprising administering the substance to the patent application of the invention A range of nucleic acid fragments is contacted and subsequently determined whether the substance has any of the following capabilities: 1) specifically binding to the nucleic acid fragment, or 2) specifically binding to a nucleic acid that hybridizes to the nucleic acid fragment.

Figure 1 shows a schematic model of the N-terminal passenger domain, the alpha-helical linker (the helix of the central marker N), and the C-terminal beta barrel domain anchored to the outer membrane.

Figure 2 shows a schematic representation showing the predicted characteristics of the Lausonia bacterium autotransporter protein antigen (LatA).

Figure 3 illustrates six selected autotransporter proteins of the invention that are predicted to be embedded in the outer membrane in different ways.

Figure 4 shows a density map of hot spot B cell epitopes in LIC037.

Figure 5 shows a density map of hot spot B cell epitopes in LI0890.

Figure 6 shows a density map of hot spot B cell epitopes in LIC058.

Figure 7 shows a density map of the general B cell epitopes in LIC037.

Figure 8 shows a density map of the general B cell epitopes in LI0890.

Figure 9 shows a density map of the general B cell epitope in LIC058.

Figure 10 shows a CD4+ epitope density map of LIC037.

Figure 11 shows a CD8+ epitope density map for LIC037.

Figure 12 shows a CD4+ epitope density map of LI0890.

Figure 13 shows a CD8+ epitope density map of LI0890.

Figure 14 shows a CD4+ epitope density map for LIC058.

Figure 15 shows a CD8+ epitope density map for LIC058.

Figure 16 shows an SDS-PAGE gel image of purified TRX-HIS-LIC058-Trunc after purification on a Superdex 200 loaded column.

Lane M contains a molecular weight marker. Lane R: reduction conditions. Lane N: non-reducing conditions.

Figure 17 shows an SDS-PAGE gel image of purified LIC091-A after purification on a Superdex 75 loaded column.

Lane M contains a molecular weight marker. Lane R: reduction conditions. Lane N: non-reducing conditions.

Figure 18 shows a density map of hot spot B cell epitopes in amino acid 1-4000 of LIC091.

Figure 19 shows a density map of a general B cell epitope in amino acid 1-4000 of LIC091.

Figure 20 shows a CD4+ epitope density map of amino acids 1-2550 in LIC091.

Figure 21 shows a CD8+ epitope density map of amino acids 1-2550 in LIC091.

definition

The term "polypeptide" means a short peptide of 2 to 10 amino acid residues, an oligopeptide of 11 to 100 amino acid residues, and a polypyrene of more than 100 amino acid residues. Peptide. Furthermore, the term is also intended to include proteins, ie biomolecules comprising at least one multi-peptide. When the protein comprises at least two multi-peptides, the multi-peptides can form a complex and are covalently or non-covalently linked. The multi-peptide in the protein may be glycosylated and/or lipidated, and/or comprise a prosthetic group.

The term "subsequence" means directly derived from a naturally occurring amino acid At least 3 amino acids of the sequence or nucleic acid sequence or, if relevant, any continuous extension of at least 3 nucleotides.

The term "amino acid sequence" means the sequence of amino acid residues linked by a peptide bond in the peptide and protein chains.

The term "adjuvant" has its usual meaning in vaccine technology, that is, 1) itself cannot initiate a specific immune response against a vaccine immunogen, but nevertheless 2) enhances the immune response against the immunogen. Substance or substance composition. In other words, only vaccination adjuvants do not provide an immune response against the immunogen. The vaccination immunogen may or may not produce an immune response against it, but the combination vaccination is an immune response against the immunogen that is stronger than the immunogen-induced immune response compared to the adjuvant induction.

"Sequence identity" is determined in the context of the present invention by comparing two optimal alignment sequences (eg, DNA, RNA or amino acid) having comparable lengths according to the following formula: (N _ref -N _Dif ). 100/N _ref , where N _ref is the number of residues in one of two sequences, and N _dif is the number of residues in which the two sequences are inconsistent over their entire length and in the same direction. For example, the two sequences 5'-ATTCGGAACC-3' and 5'-ATACGGGACC-3' will yield 80% sequence identity (N _ref = 10 and N _dif = 2).

"Assembly of amino acids" means two or more amino acids bonded together by physical or chemical means.

"3D conformation" is a three-dimensional structure of a biomolecule such as a protein. In the monomeric peptide/protein, the 3D configuration is also referred to as a "tertiary structure" and represents the spatial relative position of the amino acid residue to form a multi-peptide.

An "immunogenic carrier" is a molecule or moiety that can couple an immunogen or hapten to enhance or enable the elicitation of an immune response against an immunogen/hapten. Immunogenic vectors are, in the classical case, relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid, etc.) that can be fused or bound to an immunogen or hapten (which itself is not sufficiently immunogenic). Typically, The immunogenic carrier is capable of eliciting a strong T helper lymphocyte response against the combined immunogen and immunogenic carrier, followed by a modified response by B lymphocytes and cytotoxic T-lymphocytes. Recently, these large vector molecules have been replaced to some extent by so-called promiscuous T helper cell epitopes (ie, shorter peptides recognized by most HLA haplotypes in the population), triggering T helper lymphocytes reaction.

A "T-helper lymphocyte response" is an immune response elicited by an antigen-presenting cell based on a peptide capable of binding to an MHC class II molecule (eg, an HLA class II molecule). T helper lymphocytes are produced in animal species due to the complex between the T cell receptor recognition peptide and the presented MHC class II molecule.

An "immunogen" is a substance capable of inducing an adaptive immune response in a host in which the immune system encounters the immunogen. Thus, an immunogen is a subset of a larger class of "antigens" that are specifically recognized by the immune system (eg, when bound by an antibody, or when an antigenic fragment that binds to an MHC molecule is recognized by a T cell receptor) , but not necessarily capable of inducing immunity. However, an antigen is always capable of eliciting immunity, meaning that a host with established memory immunity against the antigen will initiate a specific immune response against the antigen.

"Hapten" is a small molecule that may not induce or elicit an immune response. However, when bound to an immunogenic carrier, antibodies or TCRs that recognize the hapten can be induced when the immune system is exposed to the hapten-carrier conjugate.

An "adaptive immune response" is an immune response against an antigen or immunogen that is specific for the epitope of the antigen/immunogen. An example of an adaptive immune response is the induction of antigen-specific antibody production or antigen-specific induction/activation of T helper lymphocytes or cytotoxic lymphocytes.

"Protective, adaptive immune response" is an antigen-specific immune response elicited in an individual as an exposure (artificial or natural) to an antigen, wherein the immune response protects the individual against subsequent Antigen or pathological changes including the antigen Attack of the pharmacy. Typically, prophylactic vaccination is directed to establishing a protective adaptive immune response against one or more pathogens.

"Stimulation of the immune system" means a substance or substance composition that exhibits an immunostimulatory effect. Many adjuvants and specific cytokines share the ability to stimulate the immune system. The result of using an immunostimulant is to increase the "alertness" of the immune system, meaning that subsequent vaccination with the immunogen induces a more pronounced immune response.

Hybridization under "stringent conditions" is defined herein as hybridization where the probe hybridizes to its target sequence to a greater detectable extent than the other sequences. Stringent conditions are target sequence dependent and will vary depending on the structure of the polynucleotide. By controlling the stringency and/or washing conditions of the hybridization, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, the stringency condition can be adjusted to allow for some mismatches in the sequence to detect lower similarities (hetero-source detection). Specificity typically varies with post-hybridization washing, with the key factors being the ionic strength and temperature of the wash liquor. Generally, highly stringent wash temperature conditions are selected to be 5 ° C to about 2 ° C below the melting point (Tm) of the specific sequence at the defined ionic strength and pH. The melting point or denaturation of DNA occurs in a narrower temperature range and represents a double helix break into its complementary single strand. This process is described by the temperature Tm (also referred to as the melting temperature) at the midpoint of the transition. The formula can be used in this technique to determine the melting temperature.

The term "animal" is intended to mean an animal species (preferably a mammal) in the context of the present invention, rather than a single animal. The term also refers to a population of such animal species.

As used herein, the term "antibody" refers to a multi-peptide or a group of multi-peptides composed of at least one antibody binding site. An "antibody combining site" is a three-dimensional binding space having an inner surface shape and a charge distribution complementary to the characteristics of an antigenic epitope of an antigen, so that the antibody can bind to the antigen. Antibodies include, for example, porcine antibodies, vertebrate antibodies, human antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, monovalent antibodies, single domain antibodies, and Fab proteins.

"Specific binding" means that the binding between two substances exceeds the random binding of the two. It also exceeds the simple association between substances that tend to aggregate (because they share the same total hydrophobicity or hydrophilicity). Thus, specific binding typically involves a combination of electrostatic interactions and other interactions between two conformational complementary regions on two species, meaning that such materials can "recognize" each other in a complex mixture.

The term "vector" is used to mean a vector nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted and which is used to introduce the heterologous nucleic acid sequence into a cell which can be replicated and expressed. The term additionally refers to certain biological vehicles, such as viral vectors and bacteriophages, that are suitable for the same purpose. Each of these infectious agents is capable of incorporating a heterologous nucleic acid sequence.

The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product and capable of being transcribed. In some cases, when the transcript is an mRNA molecule, this is then translated into a protein, a multi-peptide or a peptide.

Specific specific examples of the invention

Multi-peptide of the present invention

In some embodiments, in the definition of the first aspect of the invention, at least 5 consecutive amino acids mentioned in option b) constitute at least 6, such as at least 7, at least 8, at least 9, At least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 , at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, At least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48 At least 49, at least 50 and at least 51 contiguous amino acid residues.

When applicable, the number can be higher, such as at least 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 At least 124 and at least 125 contiguous amino acid residues. Another way of expressing this in the phrase is that for each of SEQ ID NOS: 1-17 and 54, the number of contiguous amino acid residues is at least Nn, where N is the length of the sequence ID in question and n is Any integer between 6 and N-1; that is, at least 5 consecutive amino acids can be at least any number between 5 and the length of the reference sequence minus one in increments of one.

In some embodiments, the multipeptide of the invention also has at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the amino acid sequence of a) as defined above. , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. Similarly, in some embodiments, the multipeptide of the present invention also has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, of the amino acid sequence of b) as defined above. Sequence identity of at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined above for option b) and if at least 5 amines The length of the base acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 1, 2, 3, 4, 5 of any of SEQ ID NOS: 1-17 and 54. , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, Any of 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to any of the amino acid residues 122, 123 and 124 of any of SEQ ID NOS: 2-17 and 54. One. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 125, 126, 127, 128 of any of SEQ ID NOS: 3-17 and 54, Any of 129, 130, 131, 132, 133, 134, 135, 136, and 137. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 138, 139, 140, 141 of any of SEQ ID NOS: 4-17 and 54, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, Any of 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to amino acid residues 256, 257, 258, 259 in any of SEQ ID NOS: 5-17 and 54, 260,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, Any of 381, 382, 383, 384, 385, and 386. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In the specific example defined by option b) above, the multi-peptide of the present invention is also a multi-peptide of at least 5 contiguous amino acid residues as defined in option b), and if the length of at least 5 amino acid residues is such, the N-terminal amino acid residue is corresponding to SEQ ID NO: amino acid residues 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, in any of 6-17 and 54 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473 One. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to amino acid residues 477, 478, 479, 480 in any of SEQ ID NOS: 7-17 and 54, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, Any of 621, 622, 623, 624, and 625. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 626, 627, 628, 629 of any of SEQ ID NOS: 8-17 and 54, Any of 630, 631, 632, 633, 634, 635, 636, 637, and 638. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 639, 640, 641, 642 of any of SEQ ID NOS: 9-17 and 54, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 671, 672, 673, 674, 675, 676, 677, 678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702, Any of 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, and 721. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 722, 723, 724, 725 of any of SEQ ID NOS: 10-17 and 54, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755,756,757,758,759,760,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785 790, 791, 792, 793, 794, 795, 796, 798, 799, 800, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 810, 811, 812, 813, Any of 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, and 828. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to amino acid residues 829, 830, 831, 832 in any of SEQ ID NOS: 11-17 and 54, Any of 833, 834, 835 or 836. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to amino acid residues 837, 838, 839, 840 of any of SEQ ID NOS: 12-17 and 54, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, Any of 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, and 930. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 931, 932, 933, 934 of any of SEQ ID NOS: 13-17 and 54, Any of 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, and 956. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 957, 958, 959, 960, 971, 972, 973 of SEQ ID NOS: 14-17 and 54. 974,975,976,977,978,979,980,981,982,983,984,985,986,987,988,989 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024 Any of 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In the specific example defined by option b) above, the multi-peptide of the present invention is also a multi-peptide of at least 5 contiguous amino acid residues as defined in option b), and if the length of at least 5 amino acid residues is such, the N-terminal amino acid residue is corresponding to SEQ ID NO: 15-17 and 54 in the amino acid residues 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062 Any of 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, and 1072. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 1073, 1074, 1075, 1076, 1077, 1078, 1079 of SEQ ID NOS: 16, 17 and 54. , 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104 1105, 1106, 1107, 1108, 1109, 1110, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128 , 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153 , 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1 183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, Any of 1334, 1335, and 1336. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In a specific example defined by option b) above, the multi-peptide of the invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above, and if at least 5 The length of the amino acid residue is such that its N-terminal amino acid residue corresponds to the amino acid residues 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344 of SEQ ID NOS: 17 and 54. , 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369 , 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394 , 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419 , 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1481, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, Any of 1600, 1601, 1602, and 1603. If the length of at least 5 amino acids is greater than 5, the first residue at the N-terminus will not be numbered greater than N-L+1, where N is the number of amino acid residues of the reference sequence and L is the option b The number of amino acids defined.

In the specific example defined by option b) above, The multi-peptide of the present invention is also a multi-peptide having at least 5 contiguous amino acid residues as defined in option b) above. And if the length of at least 5 amino acid residues allows this, Also having its N-terminal amino acid residue corresponding to SEQ ID NO: 54 amino acid residue 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701 1702 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801 1802 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2031 2022 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041 2042 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061 2062 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201 2202 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301 2302 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401 2402 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501 2502 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778, 2779, 2780, 2781 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896, 2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911, 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7253, 7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271 7272, 7273, 7274, 7275, 7276, 7277, 7278, 7279, 7280, 7281, 7282, 7283, 7284, 7285, 7286, 7287, 7288, 7289, 7290, 7291 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7299, 7300, 7301, 7302, 7303, 7304, 7305, 7306, 7307, 7308, 7309, 7310, 7311, 7312, 7313, 7314, 7315, 7316, 7317, 7318, 7319, 7320, 7321, 7322, 7323, 7324, 7325, 7326, 7327, 7328, 7329, 7330, 7331, 7332, 7333, 7334, 7335, 7336, 7337, 7338, 7339, 7340, 7341 7342, 7343, 7344, 7345, 7346, 7347, 7348, 7349, 7350, 7351, 7352, 7353, 7354, 7355, 7356, 7357, 7358, 7359, 7360, 7361 7362, 7363, 7364, 7365, 7366, 7367, 7368, 7369, 7370, 7371 7372, 7373, 7374, 7375, 7376, 7377, 7378, 7379, 7380, 7381 7382, 7383, 7384, 7385, 7386, 7387, 7388, 7389, 7390, 7391 7392, 7393, 7394, 7395, 7396, 7397, 7398, 7399, 7400, 7401, 7402, 7403, 7404, 7405, 7406, 7407, 7408, 7409, 7410, 7411, 7412, 7413, 7414, 7415, 7416, 7417, 7418, 7419, 7420, 7421, 7422, 7423, 7424, 7425, 7426, 7427, 7428, 7429, 7430, 7431 7432, 7433, 7434, 7435, 7436, 7437, 7438, 7439, 7440, 7441 7442, 7443, 7444, 7445, 7446, 7447, 7448, 7449, 7450, 7451 7452, 7453, 7454, 7455, 7456, 7457, 7458, 7459, 7460, 7461 7462, 7463, 7464, 7465, 7466, 7467, 7468, 7469, 7470, 7471 7472, 7473, 7474, 7475, 7476, 7477, 7478, 7479, 7480, 7481 7482, 7483, 7484, 7485, 7486, 7487, 7488, 7489, 7490, 7491 7492, 7493, 7494, 7495, 7496, 7497, 7498, 7499, 7500, 7501 7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511, 7512, 7513, 7514, 7515, 7516, 7517, 7518, 7519, 7520, 7521, 7522, 7523, 7524, 7525, 7526, 7527, 7528, 7529, 7530, 7531, 7532, 7533, 7534, 7535, 7536, 7537, 7538, 7539, 7540, 7541 7542, 7543, 7544, 7545, 7546, 7547, 7548, 7549, 7550, 7551 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560, 7561 7562, 7563, 7564, 7565, 7566, 7567, 7568, 7569, 7570, 7571 7572, 7573, 7574, 7575, 7576, 7577, 7578, 7579, 7580, 7581 7582, 7583, 7584, 7585, 7586, 7587, 7588, 7589, 7590, 7591 7592, 7593, 7594, 7595, 7596, 7597, 7598, 7599, 7600, 7601 7602, 7603, 7604, 7605, 7606, 7607, 7608, 7609, 7610, 7611, 7612, 7613, 7614, 7615, 7616, 7617, 7618, 7619, 7620, 7621, 7622, 7623, 7624, 7625, 7626, 7627, 7628, 7629, 7630, 7631 7632, 7633, 7634, 7635, 7636, 7637, 7638, 7639, 7640, 7641 7642, 7643, 7644, 7645, 7646, 7647, 7648, 7649, 7650, 7651 7652, 7653, 7654, 7655, 7656, 7657, 7658, 7659, 7660, 7661 7662, 7663, 7664, 7665, 7666, 7667, 7668, 7669, 7670, 7671 7672, 7673, 7674, 7675, 7676, 7677, 7678, 7679, 7680, 7681 7682, 7683, 7684, 7685, 7686, 7687, 7688, 7689, 7690, 7691 7692, 7693, 7694, 7695, 7696, 7697, 7698, 7699, 7700, 7701 7702, 7703, 7704, 7705, 7706, 7707, 7708, 7709, 7710, 7711, 7712, 7713, 7714, 7715, 7716, 7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731 7732, 7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740, 7741 7742, 7743, 7744, 7745, 7746, 7747, 7748, 7749, 7750, 7751 7752, 7753, 7754, 7755, 7756, 7757, 7758, 7759, 7760, 7761 7762, 7763, 7764, 7765, 7766, 7767, 7768, 7769, 7770, 7771 7772, 7773, 7774, 7775, 7776, 7777, 7778, 7779, 7780, 7781 7782, 7783, 7784, 7785, 7786, 7787, 7788, 7789, 7790, 7791 7792, 7793, 7794, 7795, 7796, 7797, 7798, 7799, 7800, 7801 7802, 7803, 7804, 7805, 7806, 7807, 7808, 7809, 7810, 7811, 7812, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820, 7821 7822, 7823, 7824, 7825, 7826, 7827, 7828, 7829, 7830, 7831 7832, 7833, 7834, 7835, 7836, 7837, 7838, 7839, 7840, 7841 7842, 7843, 7844, 7845, 7846, 7847, 7848, 7849, 7850, 7851 7852, 7853, 7854, 7855, 7856, 7857, 7858, 7859, 7860, 7681 7862, 7863, 7864, 7865, 7866, 7867, 7868, 7869, 7870, 7871 7872, 7873, 7874, 7875, 7876, 7877, 7878, 7879, 7880, 7881 7882, 7883, 7884, 7885, 7886, 7887, 7888, 7889, 7890, 7891 7892, 7893, 7894, 7895, 7896, 7897, 7898, 7899, 7900, 7901 7902, 7903, 7904, 7905, 7906, 7907, 7908, 7909, 7910, 7911, 7912, 7913, 7914, 7915, 7916, 7917, 7918, 7919, 7920, 7921 7922, 7923, 7924, 7925, 7926, 7927, 7928, 7929, 7930, 7931 7932, 7933, 7934, 7935, 7936, 7937, 7938, 7939, 7940, 7941 7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951 7952, 7953, 7954, 7955, 7956, 7957, 7958, 7959, 7960, 7961 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971 7972, 7973, 7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981 7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991 7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999, 8000, 8001 8002 8003, 8004, 8005, 8006, 8007, 8008, 8009, 8010, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030, 8031, 8032, 8033, 8034, 8035, 8036, 8037, 8038, 8039, 8040, 8041, 8042, 8043, 8044, 8045, 8046, 8047, 8048, 8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8058, 8059, 8060, 8061, 8062, 8063, 8064, 8065, 8066, 8067, 8068, 8069, 8070, 8071 8072, 8073, 8074, 8075, 8076, 8077, 8078, 8079, 8080, 8081 8082, 8083, 8084, 8085, 8086, 8087, 8088, 8089, 8090, 8091 8092, 8093, 8094, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8108, 8109, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8128, 8129, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8137, 8138, 8139, 8140, 8141, 8142, 8143, 8144, 8145, 8146, 8147, 8148, 8149, 8150, 8151, 8152, 8153, 8154, 8155, 8156, 8157, 8158, 8159, 8160, 8161, 8162, 8163, 8164, 8165, 8166, 8167, 8168, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181 8182, 8183, 8184, 8185, 8186, 8187, 8188, 8189, 8190, 8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201, 8202, 8203, 8204, 8205, 8206, 8207, 8208, 8209, 8210, 8211, 8212, 8213, 8214, 8215, 8216, 8217, 8218, 8219, 8220, 8221, 8222, 8223, 8224, 8225, 8226, 8227, 8228, 8229, 8230, 8231 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241 8242, 8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251 8252, 8253, 8254, 8255, 8256, 8257, 8258, 8259, 8260, 8261 8262, 8263, 8264, 8265, 8266, 8267, 8268, 8269, 8270, 8271 8272, 8273, 8274, 8275, 8276, 8277, 8278, 8279, 8280, 8281 8282, 8283, 8284, 8285, 8286, 8287, 8288, 8289, 8290, 8291 8292, 8293, 8294, 8295, 8296, 8297, 8298, 8299, 8300, 8301, 8302, 8303, 8304, 8305, 8306, 8307, 8308, 8309, 8310, 8311, 8312, 8313, 8314, 8315, 8316, 8317, 8318, 8319, 8320, 8321 8322, 8323, 8324, 8325, 8326, 8327, 8328, 8329, 8330, 8331 8332, 8333, 8334, 8335, 8336, 8337, 8338, 8339, 8340, 8341 8342, 8343, 8344, 8345, 8346, 8347, 8348, 8349, 8350, 8351, 8352, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361 8362, 8363, 8364, 8365, 8366, 8367, 8368, 8369, 8370, 8371 8372, 8373, 8374, 8375, 8376, 8377, 8378, 8379, 8380, 8381 8382, 8383, 8384, 8385, 8386, 8387, 8388, 8389, 8390, 8391 8392, 8393, 8394, 8395, 8396, 8397, 8398, 8399, 8400, 8401 8402, 8403, 8404, 8405, 8406, 8407, 8408, 8409, 8410, 8411, 8412, 8413, 8414, 8415, 8416, 8417, 8418, 8419, 8420, 8411 8422, 8423, 8424, 8425, 8426, 8427, 8428, 8429, 8430, 8431 8432, 8433, 8434, 8435, 8436, 8437, 8438, 8439, 8440, 8441 8442, 8443, 8444, 8445, 8446, 8447, 8448, 8449, 8450, 8451 8452, 8453, 8454, 8455, 8456, 8457, 8458, 8459, 8460, 8461 8462, 8463, 8464, 8465, 8466, 8467, 8468, 8469, 8470, 8471 8472, 8473, 8474, 8475, 8476, 8477, 8478, 8479, 8480, 8481 8482, 8483, 8484, 8485, 8486, 8487, 8488, 8489, 8490, 8491 8492, 8493, 8494, 8495, 8496, 8497, 8498, 8499, 8500, 8501 8502, 8503, 8504, 8505, 8506, 8507, 8508, 8509, 8510, 8511, 8512, 8513, 8514, 8515, 8516, 8517, 8518, 8519, 8520, 8511 8522, 8523, 8524, 8525, 8526, 8527, 8528, 8529, 8530, 8531 8532, 8533, 8534, 8535, 8536, 8537, 8538, 8539, 8540, 8541 8542, 8543, 8544, 8545, 8546, 8547, 8548, 8549, 8550, 8551 8552, 8553, 8554, 8555, 8556, 8557, 8558, 8559, 8560, 8561 8562, 8563, 8564, 8565, 8566, 8567, 8568, 8569, 8570, 8571 8572, 8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580, 8581 8582, 8583, 8584, 8585, 8586, 8587, 8588, 8589, 8590, 8591 8592, 8593, 8594, 8595, 8596, 8597, 8598, 8599, 8600, 8601, 8602, 8603, 8604, 8605, 8606, 8607, 8608, 8609, 8610, 8611, 8612, 8613, 8614, 8615, 8616, 8617, 8618, 8619, 8620, 8621 8622, 8623, 8624, 8625, 8626, 8627, 8628, 8629, 8630, 8631 8632, 8633, 8634, 8635, 8636, 8637, 8638, 8639, 8640, 8641 8642, 8643, 8644, 8645, 8646, 8647, 8648, 8649, 8650, 8651 8652, 8653, 8654, 8655, 8656, 8657, 8658, 8659, 8660, 8661 8662, 8663, 8664, 8665, 8666, 8667, 8668, 8669, 8670, 8671 8672, 8673, 8674, 8675, 8676, 8677, 8678, 8679, 8680, 8681 8682, 8683, 8684, 8685, 8686, 8687, 8688, 8689, 8690, 8691 8692, 8693, 8694, 8695, 8696, 8697, 8698, 8699, 8700, 8701 8702, 8703, 8704, 8705, 8706, 8707, 8708, 8709, 8710, 8711, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731 8732, 8733, 8734, 8735, 8736, 8737, 8738, 8739, Any of 8740 and 8741. If the length of at least 5 amino acids is greater than 5, The first residue at the N-terminus will not be a number greater than N-L+1. Wherein N is the number of amino acid residues of the reference sequence, And L is the number of amino acids defined by option b).

The multi-peptides of the invention (i.e., any of SEQ ID NOS 1-17 and 54) are also fused or conjugated to an immunogenic carrier molecule in certain embodiments. Immunogenic carrier molecules are typically multi-peptides that induce T-helper lymphocyte responses in mammals. The immunogenic carrier molecule may be selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, a tetanus toxoid or a fragment thereof, or a diphtheria toxoid or a fragment thereof. Other suitable carrier molecules are discussed below.

The multi-peptides of the invention may also be fused to other amino acid sequences that confer the desired function. For example, a multi-peptide can be fused to an amino acid sequence that facilitates purification (eg, a "His tag" polyhistidine group), or a fusion partner can be useful for facilitating export or other cellular transport mechanisms and fusion. The protein localizes to a (heterologous) signal peptide at a given cell location.

In a preferred embodiment, the multi-peptide of the invention detailed above is capable of eliciting an adaptive immune response against the multi-peptide in a mammal, in particular a pig. Preferably, the adaptive immune response is a protective adaptive immune response against an intracellular L. sonnei infection. The multi-peptide can be here In other cases, induce humoral and/or cellular immune responses.

Antigenic determinant

SEQ ID NOS: 1-17 and 54 include an epitope (antigenic determinant) which is itself recognized by the antibody or recognized by the T cell receptor when bound to the MHC molecule. For the purposes of the present invention, a B cell epitope (i.e., an antibody binding epitope) has a particular relevance.

Identification of linear B cell epitopes is relatively easy. One method entails testing for antibodies raised against the Lactobacillus intracellularis or Lactobacillus intracellularis-derived proteins disclosed herein, which are superimposed and derived from any of SEQ ID NOS: 1-17 and 54 The combination of polypeptides. Thus, the intracellular L. sonnei peptide can be identified as responsible for or contributing to the region of binding to the antibody.

Alternatively or additionally, a human can produce a mutant version of a multi-peptide of the invention, such as a version of each of the individual non-alanine residues in SEQ ID NOS: 1-17 and 54 mutated to alanine. This method is suitable for the identification of complex, non-constructed B cell epitopes. This is the case when the binding of the same antibody is changed by mutating the amino acid in a different region of the full-length polypeptide.

Computer-based methods can also be used to predict B cell epitopes. Modern systems for predicting beta-turn are provided in Petersen et al. (November 2010), Plos One 5 (11): e15079; prediction of linear B cell epitopes, see: Larsen et al. (April 2006), Immunome Research, 2: 2; prediction of solvent exposed amino acid of: Petersen B et al. (July 2009), BMC Structural Biology, 9: 51.

Certain truncated versions of the multi-peptides of the invention are of particular interest as they appear to include the desired amount of B cell and T cell epitopes. Therefore, a multi-peptide that is of particular interest is a multi-peptide consisting of the following fragments, consisting of or including the epitopes of the following fragments: LIC058-trunc, LIC037-A, LIC037-B, LIC037-C, LI0890-A , LI0890-B, LIC091-A, LIC091-B, LIC091-C, LIC091-D or LIC091-E. (See the example for details.)

Nucleic acid fragment of the invention

The nucleic acid fragment of the present invention is preferably a DNA fragment (such as SEQ ID NO: 18-34) or RNA. Fragments (such as SEQ ID NOs 35-51).

The nucleic acid fragment of the invention typically consists of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 of any one of SEQ ID NOs: 18-51 and 55. , at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, At least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42 , at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, At least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67 , at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76 At least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 101 At least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118, at least 119, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126 At least 127, at least 128, at least 129, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141, at least 142, at least 143, at least 144, to 145 fewer, at least 146, at least 147, at least 148, at least 149, at least 150, at least 151, at least 152, at least 153, at least 154, at least 155, at least 156, at least 157 At least 158, at least 159, at least 160, at least 161, at least 162, at least 163, at least 164, at least 165, at least 166, at least 167, at least 168, at least 169, at least 170, at least 171, at least 172, at least 173, at least 174, at least 175, at least 176, at least 177, at least 178, at least 179, at least 180, at least 181, at least 182 At least 183, at least 184, at least 185, at least 186, at least 187, at least 188, at least 189, at least 190, at least 191, at least 192, at least 193, at least 194, at least 195, at least 196, at least 197, at least 198, at least 199, at least 200, at least 201, at least 202, at least 203, at least 204, at least 205, at least 206, at least 207 At least 208, at least 209, at least 210, at least 211, 212 fewer, at least 213, at least 214, at least 215, at least 216, at least 217, at least 218, at least 219, at least 220, at least 221, at least 222, at least 223, at least 224 , at least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236, At least 237, at least 238, at least 239, at least 240, at least 241, at least 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least 249 , at least 250, at least 251, at least 252, at least 253, at least 254, at least 255, at least 256, at least 257, at least 258, at least 259, at least 260, at least 261, At least 262, at least 263, at least 264, at least 265, at least 266, at least 267, at least 268, at least 269, at least 270, at least 271, at least 272, at least 273, at least 274 , at least 275, at least 276, at least 277, at least 278 , At least 279, at least 280, at least 281, at least 282, to 283 fewer, at least 284, at least 285, at least 286, at least 287, at least 288, at least 289, at least 290, at least 291, at least 292, at least 293, at least 294, at least 295 , at least 296, at least 297, at least 298, at least 299, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, At least 308, at least 309, at least 310, at least 311, at least 312, at least 313, at least 314, at least 315, at least 316, at least 317, at least 318, at least 319, at least 320 , at least 321 , at least 322 , at least 323 , at least 324 , at least 325 , at least 326 , at least 327 , at least 328 , at least 329 , at least 330 , at least 331 , at least 332 , At least 333, at least 334, at least 335, at least 336, at least 337, at least 338, at least 339, at least 340, at least 341, at least 342, at least 343, at least 344, at least 345 At least 346, at least 347, at least 348, at least 349 At least 350, at least 351, at least 352, at least 353, at least 354, at least 355, at least 356, at least 357, at least 358, at least 359, at least 360, at least 361, at least 362 , at least 363, at least 364, at least 365, at least 366, at least 367, at least 368, at least 369, at least 370, at least 371, at least 372, at least 373, at least 374 and Composition of at least 375 contiguous nucleotides. Covering longer segments, ie having at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, of SEQ ID NOS: 18-51 and 55, Fragments of at least 2000, at least 2500, at least 3000, at least 3500, and at least 4000 nucleotides.

The nucleic acid fragment of the invention typically has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91% of the nucleotide sequence as defined in i) or ii) above. At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity.

The nucleic acid fragment of the invention may also have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 of the nucleotide sequence as defined in iii) above. Sequence identity of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

Carrier of the present invention

The vectors of the present invention are divided into several categories as discussed below. A preferred vector of the present invention comprises an activatable linkage and an expression control region comprising a enhancer/promoter suitable for driving the expression of the nucleic acid fragment defined in the above option i) in the 5'-3' direction. The signal peptide sequence used in the case, the nucleotide sequence defined in option i) and the terminator selected as appropriate. Thus, the vector constitutes a performance vector suitable for effecting the production of the multi-peptide of the invention in a cell. Since the multi-peptide of the present invention is derived from bacteria, recombinant production is suitably carried out in a bacterial host cell. Therefore, it is preferred that the expression control region promotes expression in prokaryotic cells (such as bacteria), such as in Escherichia coli . However, if the vector is to be used to promote expression in mammalian cells (as would be the case for DNA vaccine vectors), the expression control region should be suitable for this particular use.

In any event, certain vectors of the invention are capable of autonomous replication.

The vector of the present invention may also be a vector capable of integration into the host cell genome. This applies in particular if the vector is used to produce a stably transformed cell, wherein the progeny will also include genetic information introduced via the vector. Alternatively, vectors that are not capable of integration into the genome of a mammalian host cell are suitable, for example, for DNA vaccination.

Typically, the vectors of the invention are selected from the group consisting of, but not limited to, viruses, such as attenuated viruses (which may themselves be suitable as vaccines); phage; plastids; pocket chromosomes and viscous bodies.

A more detailed discussion of the vectors of the present invention is provided below: The multi-peptide of the present invention can be encoded by a nucleic acid molecule contained in a vector. A nucleic acid sequence can be "heterologous", meaning that it is foreign to the cell into which the vector is introduced. It may also include sequences that are homologous to sequences in the cell but are located in locations that are not normally found in the host cell. Carrier package These include DNA, RNA, plastids, vesicles, viruses (phage, animal viruses, and plant viruses) and artificial chromosomes (such as YAC). Those skilled in the art should be well equipped to construct vectors via standard recombinant techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). In addition to encoding a multi-peptide of the invention, the vectors of the invention may encode a multi-peptide sequence, such as a tag or an immunogenic enhancing peptide (eg, an immunogenic carrier or fusion partner that stimulates the immune system, such as a cytokine or Its active fragment). Suitable vectors encoding such fusion proteins include the pIN vector (Inouye et al., 1985), a vector encoding an extended histidine, and a pGEX vector for the production of a glutathione S transferase (GST) soluble fusion protein for use in the latter. Purification and separation or lysis.

The vector of the present invention can be used in a host cell to produce a multi-peptide of the present invention, which can be subsequently purified for administration to an individual, or the vector can be purified for direct administration to an individual for expression of the protein in the individual (when This is the case when a nucleic acid vaccine is administered.

A expression vector can contain a variety of "control sequences" which refer to the transcription of a coding sequence operably linked in a particular host organism and the nucleic acid sequences required for translation. In addition to the control sequences that govern transcription and translation, the vector and expression vector can contain nucleic acid sequences that also provide additional functions and are described below.

Promoter and enhancer

"Promoter" is a control sequence. A promoter is typically a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain genetic elements that regulate proteins and molecules, such as RNA polymerase and other transcription factors. The phrases "operatively positioned", "operatively linked", "under control" and "under transcriptional control" mean a promoter. The nucleic acid sequence is in an appropriate functional position and/or orientation to control the transcriptional initiation and expression of the sequence. The promoter may or may not be used in conjunction with an "enhancer", which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be a promoter that is naturally associated with a gene or sequence and can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment or exon. This promoter can be referred to as "endogenous". Similarly, a enhancer can be a enhancer that is naturally associated with a nucleic acid sequence, either downstream or upstream of the sequence. Alternatively, certain advantages will be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which means that it is not normally associated with the nucleic acid sequence in its natural environment. The promoter. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a nucleic acid sequence in their native state. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and not "naturally occurring" promoters. Or a enhancer, that is, a different element containing a different transcriptional regulatory region and/or a mutation that alters performance. In addition to a promoter and synthetically produced nucleic acid sequences of enhancers, may be on the compositions disclosed herein, using recombinant cloning and / or nucleic acid amplification techniques (including PCR ^TM) produced sequences (see U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906 , each of which is incorporated herein by reference.

Naturally, it can be important to use a promoter and/or enhancer to effectively direct the DNA segment to behave in a cell type or organism selected for expression. The skilled artisan is generally known to use promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al., 2001, incorporated herein by reference). The promoter used can be constitutive, tissue specific or inducible, and in certain embodiments, can direct high level performance of the introduced DNA segment under specific conditions, such as large scale production of recombinant proteins or peptides.

Examples of inducible elements that are regions of a nucleic acid sequence that are responsive to specific stimulation include, but are not limited to, immunoglobulin heavy chains (Banerji et al, 1983; Gilles et al, 1983; Grosschedl et al, 1985; Atchinson Et al, 1986, 1987; toilet et al, 1987; Weinberger et al, 1984; Kiledjian et al, 1988; Porton et al; 1990), immunoglobulin light chain (Queen et al, 1983; Picard et al, 1984) , T cell receptors (Luria et al, 1987; Winoto et al, 1989; Redondo et al; 1990), HLA DQ alpha and / or DQ beta (Sullivan et al, 1987), --interferon (Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988), interleukin-2 (Greene et al., 1989), interleukin-2 receptor (Greene et al., 1989). ; Lin et al., 1990), MHC class II 5 (Koch et al., 1989), MHC class II HLA-DR alpha (Sherman et al., 1989), β-actin (Kawamoto et al., 1988; Ng et al. 1989), muscle creatine kinase (MCK) (Jaynes et al, 1988; Horlick et al, 1989; Johnson et al, 1989), prealbumin (transthyretin) (Costa et al, 1988), elastase I (Omitz et al., 1987), metallothionein (MTII) (Karin et al., 1987; Culotta et al., 1989), collagenase (Pinkert et al., 1987; Angel et al., 1987), albumin (Pinkert et al.). Human, 1987; Tranche et al., 1989, 1990), alpha-fetoprotein (Godbout et al., 1988; Campere et al., 1989), gamma-globulin (Bodine et al., 1987; Perez-Stable et al., 1990). , β-globulin (Trudel et al., 1987), c-fos (Cohen et al., 1987), c-HA-ras (Triesman, 1986; Deshamps et al., 1985), insulin (Edlund et al., 1985), Nerve cell adhesion molecule (NCAM) (Hirsh et al Human, 1990), α 1-antitrypsin (Larimer et al., 1990), H2B (TH2B) histone (Hwang et al., 1990), mouse and/or type I collagen (Ripe et al., 1989), Glucose regulatory proteins (GRP94 and GRP78) (Chang et al., 1989), rat growth hormone (Larsen et al., 1986), human serum amyloid A (SAA) (Edbrooke et al., 1989), troponin I ( TN I) (Yutzey et al., 1989), platelet-derived growth factor (PDGF) (Pech et al., 1989), Duchenne muscle dystrophy (Klamut et al., 1990), SV40 (Banerji et al., 1981; Moreau) Et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al. , 1987; Schaffner et al., 1988), polyoma (Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al. 1984; Hen et al., 1986; Satake et al., 1988; Campbell et al., 1988), retrovirus (Kriegler et al., 1982, 1983; Levinson et al., 1982; Kri Egler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al, 1987; Thiesen et al, 1988; Celander et al, 1988; Choi et al, 1988; Reisman et al, 1989), papillomavirus (Campo et al, 1983; Lusky et al, 1983; Spandidos and Wilkie, 1983; Spalholz et al, 1985; Lusky et al, 1986; Cripe et al, 1987; Gloss et al, 1987; Hirochika et al, 1987; Stephens et al, 1987), hepatitis B virus (Bulla et al, 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988), Human immunodeficiency virus (Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989, Cellular Humor (CMV) IE. (Weber et al, 1984; Boshart et al, 1985; Foecking et al, 1986), gibbon leukemia virus (Holbrook et al, 1987; Quinn et al, 1989).

Inducible elements include, but are not limited to, MT II-phorbol ester (TFA)/heavy metals (Palmiter et al, 1982; Haslinger et al, 1985; Searle et al, 1985; Stuart et al, 1985; Imagawa et al, 1987) Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989); MMTV (mouse breast cancer virus)-glucocorticoid (Huang et al., 1981; Lee et al., 1981; Majors et al., 1983; Chandler et al, 1983; Lee et al, 1984; Ponta et al, 1985; Sakai et al, 1988); beta-interferon-poly(rl)x/poly(rc) (Tavernier et al, 1983); adenovirus 5 E2-ElA (Imperiale et al., 1984); collagenase-phorbol ester (TPA) (Angel et al., 1987a); matrix lysin-phorbol ester (TPA) (Angel et al., 1987b); SV40-Buddha Peryl ester (TPA) (Angel et al., 1987b); murine MX gene-interferon, Newcastle chicken prion (Hug et al., 1988); GRP78 gene-A23187 (Resendez et al., 1988); Protein-IL-6 (Kunz et al., 1989); vimentin-serum (Rittling et al., 1989); MHC class I gene H-2 κ b-interferon (Blanar et al., 1989); HSP70-E1A/SV40 Large T antigen (Taylor et al., 1989, 1990a, 1990b); Protein - phorbol ester / TPA (Mordacq et al., 1989); tumor necrosis factor -PMA (Hensel et al., 1989); and thyrotropin α gene - thyroid hormone (Chatterjee et al., 1989).

Also suitable for use in the present invention are the dectin-1 and dectin-2 promoters. In addition, any promoter/enhancer combination (according to the eukaryotic promoter database, EPDB) can also be used to drive structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or heterologous proteins of interest. Performance.

The specific promoter of the salty credit to control the expression of the polynucleotide encoding the peptide or protein of the present invention is not critical as long as it is capable of directing the expression of the polynucleotide in the target cell, preferably a bacterial cell. When targeting a porcine cell, the polynucleotide coding region is preferably placed adjacent to a promoter capable of behaving in a porcine cell and under the control of the promoter. In general, the promoter may comprise a bacterial promoter, a porcine promoter or a viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain a high level of the polynucleotide of the present invention. which performed. Other viral promoter or mammalian cell promoters or bacteriophage promoters well known in the art are also contemplated to achieve the performance of the polynucleotide.

In a specific example of administering a vector to an individual for expression of a protein, it is contemplated that the desired promoter for use with the vector is a promoter that is not downregulated by the cytokine, or that is sufficiently powerful to produce an effective amount in the individual even if the assay is reduced. The protein/polypeptide of the invention is a promoter that elicits an immune response. Non-limiting examples of such promoters are CMVIE and RSV LTR. In other embodiments, a promoter that is elevated in the presence of a cytokine is employed. The MHC I promoter increased performance in the presence of IFN-γ.

Tissue-specific promoters can be used, particularly if expressed in cells that require antigen expression, such as dendritic cells or macrophages. Mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters.

2. Initiation signal and internal ribosome binding site (IRES)

The specific initiation signal can also be required for efficient translation of the coding sequence. These signals include ATG Start codon or similar sequence. It may be necessary to provide an exogenous translation control signal, including the ATG start codon. Those skilled in the art should be readily able to determine this and provide the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signal and initiation codon can be natural or synthetic and can be operable in bacterial or mammalian cells. The efficacy of performance can be enhanced by the inclusion of appropriate transcriptional enhancer elements.

In certain embodiments of the invention, an internal ribosome entry site (IRES) element is used to generate a polygenic or polycistronic message. The IRES element is able to bypass the 5' methylated cap-dependent translation of the ribosome scanning model and begin translation at the internal site (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988) and IRES from mammalian sources (Macejak and Sarnow, 1991). The IRES element can be connected to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, producing a polycistronic message. With the aid of the IRES element, each open reading frame can be entered for efficient translation. A plurality of genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, the disclosures of each of which are incorporated herein by reference).

3. Multiple selection sites

The vector may include a multiple selection site (MCS), which is a nucleic acid region containing a plurality of restriction enzyme sites, any of which may be used in conjunction with standard recombinant techniques to digest the vector. (See Carbonelli et al, 1999, Levenson et al, 1998, and Cocea, 1997, which is incorporated herein by reference.) Often, the use of restriction enzymes to cleave within the MCS linearizes or fragments the vector such that The source sequence can be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombination.

4. Splice sites

Most transcriptional eukaryotic RNA molecules will undergo RNA splicing to remove inclusions from the primary transcript child. If relevant in the context of the vectors of the present invention, vectors containing the somatic eukaryotic sequences may require donor and/or acceptor splice sites to ensure proper processing of the transcript for protein expression. (See Chandler et al., 1997, incorporated herein by reference.)

5. Termination signal

The vector or construct of the invention typically comprises at least one termination signal. A "termination signal" or "terminator" includes a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, termination signals that end RNA transcript production are contemplated. Terminators are necessary to achieve the desired level of information in the body.

In eukaryotic systems, the terminator region can also contain specific DNA sequences that allow site-specific cleavage of new transcripts to expose polyadenylation sites. This signaling specialized endogenous polymerase adds approximately 200 A residue (poly A) extensions to the 3' end of the transcript. The RNA molecules modified by this multiple A tail appear to be more stable and can be translated more efficiently. Thus, in other specific embodiments involving eukaryotes, the terminator preferably comprises a signal to cleave the RNA, and the terminator signal is better to facilitate polyadenylation of the message.

Terminators contemplated for use in the present invention include any of the known transcription terminators described herein or generally known to those skilled in the art, including but not limited to, for example, bovine growth hormone terminator or viral termination sequences, such as the SV40 terminator. . In some embodiments, the termination signal may lack a sequence that is transcribed or translatable, such as due to sequence truncation.

6. Polyadenylation signal

In protein expression, particularly eukaryotic protein expression (associated with nucleic acid vaccination), we will typically include a polyadenylation signal to achieve correct polyadenylation of the transcript. Polyadenylation can also increase the stability of the transcript and/or can help cytoplasmic transport. The nature of the salty polyadenylation signal is not critical to the successful practice of the invention and any such sequence can be employed. Preferred specific examples include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal.

7. Copying starting point

To allow the vector to proliferate in a host cell, the vector may contain the origin of one or more replication sites (often referred to as "ori"), which is the specific nucleic acid sequence at which replication begins. Alternatively, if the host cell is yeast, an autonomously replicating sequence (ARS) can be employed.

8. Selectable and filterable markers

In certain embodiments of the invention, cells comprising a nucleic acid construct of the invention can be identified in vitro or in vivo by encoding a selectable or selectable marker in the expression vector. When transcribed and translated, the marker confers identifiable changes to the cell, allowing easy identification of cells containing the expression vector. In general, a mark can be selected that is marked to impart characteristics that allow selection. A positive selectable marker is the marker whose presence is allowed to be selected, while a negative selectable marker is a marker for which there is a choice to prevent it from being selected. An example of a positive selectable marker is a drug resistance marker.

The inclusion of a drug selection marker usually facilitates the selection and identification of the transformant. For example, markers that confer neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histamine resistance are optional. mark. In addition to the labeling that allows for the phenotype that allows the identification of the transformant based on conditions, other types of markers include a selectable marker such as GFP for colorimetric analysis. Alternatively, a screenable enzyme such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art are also aware of how to use immunological markers that can be used in conjunction with FACS analysis. The label used in the letter is not important as long as it can be expressed simultaneously with the nucleic acid encoding the protein of the present invention. Other examples of selectable and screenable markers are well known to those skilled in the art.

Transduced cells of the invention

The transformed cells of the present invention are useful in the production of the multi-peptide of the present invention and as a simple "container" for the nucleic acids and vectors of the present invention.

Certain transformed cells of the invention are capable of replicating a nucleic acid fragment as defined in option i) of the second aspect of the invention. Preferred transmorphic cells of the invention are capable of expressing a nucleic acid fragment as defined in option i) segment.

For recombinant production, transformed cells are suitable (but not necessarily) prokaryotic, such as bacteria. However, both prokaryotic and eukaryotic cells can be used.

Suitable prokaryotic cells are selected from the group consisting of Escherichia (Escherichia) (such as Escherichia coli (E.coli)), Bacillus (Bacillus) (e.g. Bacillus subtilis (Bacillus subtilis)), the genus Salmonella (Salmonella) and points Zhigan species (Mycobacterium) (preferably non-pathogenic, e.g. M. bovis (M.bovis) BCG) the group consisting of.

Eukaryotic cells can be in the form of yeast (such as Saccharomyces cerevisiae) and protozoa. Alternatively, the transduced eukaryotic cells are derived from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells.

For production purposes, it is advantageous for the transformed cells of the present invention to be stably transformed by the stable integration of the nucleic acid defined by the above option i) into its genome. In certain embodiments, it is also preferred that the transfected cells secrete or carry the multi-peptide of the invention on their surface as this facilitates recovery of the multi-peptide produced. A specific version of this specific example is a version in which the transformed cells are bacteria and the multi-peptide of the present invention is secreted into the periplasmic space.

Further details regarding cells and cell lines are presented below: Cells expressing recombinant nucleic acids suitable for use in the nucleic acid fragments of the invention are prokaryotic and eukaryotic. Examples of prokaryotic cells include E. coli; Staphylococcus (Staphylococcus) of the members, such as grape skin fungus ball (S.epidermidis); member of the genus Lactobacillus (Lactobacillus) of, such as Lactobacillus plantarum (L.plantarum); Lactococcus (Lactococcus) of a member, such as Lactococcus lactis (the L. lactis); members of the genus Bacillus, such as Bacillus subtilis; member of the genus Corynebacterium (Corynebacterium), the glutamic acid as Corynebacterium (C. glutamicum); and Pseudomonas (Pseudomonas) of the members, such as P. fluorescens (P.fluorescens). Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells, such as members of the genus Saccharomyces (eg, Saccharomyces cerevisiae), members of the genus Pichia (eg Pichia pastoris ( P .pastoris )), a member of the Hansenula (eg H. polymorpha ), a member of the genus Kluyveromyces (eg K. lactis ( K.) lactis) or Kluyveromyces fragilis (K.fragilis)), and members of the genus fission yeast (Schizosaccharomyces) of (such as S. pombe (S.pombe)).

Techniques for recombinant gene production, introduction into cells, and expression of recombinant genes are well known in the art. Examples of such techniques are provided in the following references, such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002; and Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989.

As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably. All such terms also include their descendants, and the progeny are any and all descendants. It should be understood that all progeny may be different due to intentional or accidental mutations. In the context of the expression of a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. The host cell can be used as a recipient of a vector or virus. A host cell can be "transfected" or "transformed", which refers to a method of transferring or introducing an exogenous nucleic acid, such as a recombinant protein coding sequence, into a host cell. Transduced cells include nascent individual cells and their progeny.

Host cells can be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells, for vector replication or for the performance of some or all of the nucleic acid sequences. Many cell lines and cultures can be used as host cells; they are available through the American Type Culture Collection (ATCC), an organization that serves as an archive of live cultures and genetic material (www.atcc.org) Or from other hosting agencies such as Deutsche Sammlung vor Micrroorganismen und Zellkulturen (DSM). Suitable hosts can be determined by those skilled in the art based on the vector backbone and the desired result. A plastid or a vesicle can, for example, be introduced into a prokaryotic host cell for the production of multiple copies or for expression of the encoded protein. For vector replication and / or expression of the bacterial cells include strains of Staphylococcus and E. coli host many commercially available, such as SURE ^® Competent Cells and ^{SoloPack TM Gold Cells (Stratagene ®;} La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 can be used as host cells for phage. Suitable yeast cells may include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.

Examples of eukaryotic host cells for vector replication and/or expression include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many cell types and many host cells of the organism are available and should be known to those skilled in the art. Similarly, viral vectors can be used in conjunction with eukaryotic or prokaryotic host cells, particularly host cells that permit replication or expression of the vector.

Some vectors may employ a control sequence such that they can be replicated and/or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will be further aware of the conditions under which all of the above host cells are grown to maintain their growth and permit replication of the vector. Techniques and conditions that permit large-scale production of vectors and production of nucleic acids encoded by vectors and their homologous peptides, proteins or peptides are also known and known.

Performance system

There are many performance systems that include at least some or all of the compositions discussed above. Systems based on prokaryotes and/or eukaryotes can be used with the present invention to produce nucleic acid sequences or homologous peptides, proteins and peptides thereof. Many of these systems are commercially available and generally available.

The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patent Nos. 5,871,986, 4,879, 236, the entireties of each of Such systems are commercially available, for example under the trademarks MaxBac ^® 2.0 (Invitrogen ^® ) and BacPAK ^TM (Clontech ^® ).

In addition to the disclosed expression systems of the present invention, other examples of expression systems include COMPLETE CONTROL ^TM-inducible mammalian expression system (Stratagene ^®), which relates to a synthetic ecdysone-inducible receptor; or pET expression system (Stratagene ^®), That is, the E. coli expression system. Another example of inducible expression systems for the T-REx ^TM system (Invitrogen ^®), which involves the tetracycline-regulated expression. The Pichia methanolica expression system (Invitrogen ^® ) is a yeast expression system designed to produce recombinant proteins at a high level in methylotrophic yeast P. methanolica. Those skilled in the art will know how to present a vector (such as a representation construct) to produce a nucleic acid sequence or a homologous multipeptide, protein or peptide thereof.

Nucleic acid amplification

Nucleic acids used as amplification templates can be isolated from cells, tissues or other samples according to standard methodology (Sambrook et al., 2001). In some embodiments, whole cell, tissue homogenate, or biological fluid samples are analyzed without substantial purification of the template nucleic acid. The nucleic acid can be genomic DNA or fractionated or whole cell RNA. When using RNA, it may be necessary to first convert the RNA to complementary DNA (cDNA).

As used herein, the term "primer" is intended to encompass any nucleic acid capable of eliciting nascent nucleic acid synthesis in a template dependent method. Typically, the primers are ten to twenty and/or thirty base long oligonucleotides, but longer sequences can be employed. The primers may be provided in double-stranded and/or single-stranded form, but the single-stranded form is preferred.

A primer pair designed to selectively hybridize to a nucleic acid corresponding to the gene sequence identified herein is contacted with a template nucleic acid under conditions that permit selective hybridization. Depending on the desired application, high stringency hybridization conditions can be selected, allowing hybridization only to sequences that are fully complementary to the primer. In other embodiments, hybridization can occur under reduced stringency conditions to allow amplification of a nucleic acid containing one or more mismatches to the primer sequence. After hybridization, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification (also known as "cycles") are performed until a sufficient amount of amplification product is produced.

The amplified product can be detected or quantified. In some applications, detection can be performed by visual means. Alternatively, detection may involve radioactive scintigraphy via chemiluminescence, incorporated radioactive or fluorescent labeling, or even indirect identification of the product via a system using electrical pulse signals and/or thermal pulse signals (Bellus, 1994).

Many template-dependent methods can be used to amplify the presence of oligonucleotides in a given template sample Column. One well-known method of amplification is the polymerase chain reaction (PCR), which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800, 159, each to each of each of

An alternative method for amplifying a target nucleic acid sequence useful in the practice of the present invention is disclosed in U.S. Patent Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 And 5,942,391, GB Application No. 2,202, 328, and PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety.

Gene transfer method

Methods suitable for nucleic acid delivery to achieve the performance of the compositions of the present invention include introduction of nucleic acids (eg, DNA, including viral and non-viral vectors) into cells, tissues, as described herein or as would be known to those of ordinary skill in the art. Or the method in the organism. Such methods include, but are not limited to, direct delivery of DNA, such as by injection (U.S. Patent Nos. 5,994,624, 5,981, 274, 5, 945, 100, 5, 780, 448, 5, 736, 524, 5, 702, 932, 5, 656, 610, 5, 589, 466, and 5, 580, 859, each incorporated herein by reference) Microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5, 789, 215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); Precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al. Human, 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneta et al, 1989; Kato et al, 1991 Blasting by microprojectiles (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patent Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, each incorporated herein by reference); By Of agitation with silicon fibers (Kaeppler et al., 1990; U.S. Patent No. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each Incorporate herein by reference; or by dry/inhibition-mediated DNA uptake (Potrykus et al., 1985). By applying techniques such as these, organelles, cells, tissues or organisms can be stably or transiently transformed.

Antibody of the invention

Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and passive immunoassays and therapies.

Multiple strains and monoclonal antibodies against the proteins of the present invention can be prepared by conventional methods. Typically, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of multiple strains of serum due to available serum volume and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally carried out by mixing or emulsifying the protein in physiological saline, preferably in an adjuvant such as Freund's complete adjuvant, and parenterally (generally subcutaneously or intramuscularly) by injecting the mixture or emulsion. A dose of 50-200 μg per injection is typically sufficient. Immunization is generally boosted by one or more injections of the protein in physiological saline (preferably Freund's incomplete adjuvant) after 2-6 weeks. Alternatively, antibodies can be produced by in vitro immunization using methods known in the art, which are considered to be immunologically equivalent in vivo for the purposes of the present invention. A plurality of antiserums are obtained by culturing the immunized animals into a glass or plastic container, culturing the blood at 25 ° C for one hour, followed by incubating at 4 ° C for 2-18 hours. Serum is recovered by centrifugation (eg, 1,000 g for 10 minutes). Approximately 20-50 ml of each exsanguination can be obtained from the rabbit.

Monoclonal antibodies (MAb) were prepared using the standard method of Köhler & Milstein [Nature (1975) 256:495-96] or a modification thereof. Typically, the mouse or rat is immunized as described above. However, instead of letting the animals bleed to extract serum, it is better to remove the spleen (and several large lymph nodes, as appropriate) and discretize them into single cells. If desired, spleen cells (after removal of non-specific adherent cells) can be screened by applying the cell suspension to a plate or well coated with protein antigen. Have an antigen Specific B cells express membrane-bound immunoglobulin bound to the plate and are not washed away with the rest of the suspension. The resulting B cells or all dissociated spleen cells are then induced to fuse with myeloma cells to form a fusion tumor and cultured in a selective medium (eg, hypoxanthine, aminopterin, thymidine medium, or "HAT"). The resulting fusion tumors were coated by limiting dilution and analyzed for the production of antibodies that specifically bind to the immune antigen (and which does not bind to an unrelated antigen). The selected MAb secreting fusion tumor is then cultured in a test tube (e.g., in a tissue culture flask or a hollow fiber reactor) or in vivo (e.g., ascites of a mouse).

If desired, antibodies can be labeled using conventional techniques (whether multi-plant or individual). Suitable labels include fluorophores, chromophores, radioactive atoms (especially ³² P and ¹²⁵ I), electron-dense reagents, enzymes, and ligands with specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is typically quantitatively detected by a spectrophotometer by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment. "Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, such as in the case of an antigen and a monoclonal antibody specific thereto. Other specific binding partners include biotin, avidin, streptavidin, IgG, and protein A, as well as many receptor-ligand conjugates known in the art. It should be understood that the above description is not intended to classify multiple tags into different categories, as the same tag may function in several different modes. For example, ¹²⁵ I can act as a radioactive label or as an electron-dense reagent. HRP can act as an enzyme or as an antigen of MAb. In addition, we can combine multiple markers for the desired effect. For example, MAb and avidin also require labeling in the practice of the present invention: therefore, we can label MAb with biotin and detect its resistance to ¹²⁵ I-labeled avidin or to HRP-labeled Biotin MAbs are present together. Other variations and possibilities should be readily apparent to those of ordinary skill in the art and are considered equivalents within the scope of the invention.

According to the present invention, the isolated monoclonal antibody or antibody analog is preferably selected from the group consisting of a multidomain antibody (such as a murine antibody), a chimeric antibody (such as a humanized antibody), a fully human antibody, and a llama or camel. A monoclonal antibody to a domain antibody. The antibody analog can be selected from antibody fragments, such as Fab, F(ab') ₂ or scFV; also reference to the definition of the term "antibody" provided above.

Composition of the invention; vaccine

The pharmaceutical composition of the present invention (in detail, the vaccine) may be prophylactic (i.e., prevent infection) or therapeutic (i.e., treat the disease after infection).

Such vaccines comprise an immunizing antigen, an immunogen, a multi-peptide, a protein or a nucleic acid, usually in combination with a "pharmaceutically acceptable carrier", which includes antibodies which do not themselves induce harmful to the individual receiving the composition. Any carrier produced. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) And inactive virions.

Such carriers are well known to those of ordinary skill in the art. In addition, such carriers can act as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be coupled to the bacterial toxoid, such as diphtheria, tetanus, cholera, or H. pylori (of H.pylori.) The toxoid.

The pharmaceutical compositions of the present invention thus typically comprise an immunological adjuvant which may be one of an aluminum based adjuvant or any number of other adjuvants, as described below: Preferred adjuvants for enhancing the effectiveness of the composition include (but not limited to) (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents, such as cell walls) Indole peptide (see below) or bacterial cell wall component), such as (a) MF59 (WO 90/14837; Vaccine design: the subunit and adjuvant approach, Chapter 10, edited Powell and Newman, Plenum Press 1995), which contains 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (depending on the amount of MTP-PE (see below), although not required), using a microfluidizer unit such as the Model 110Y Microfluidizer (Microfluidics) , Newton, MA) formulated into submicron particles, (b) SAF containing 10% squalane, 0.4% Tween 80, 5% Plonik block copolymer L121 and thr-MDP (see below), microfluidics Formation into submicron emulsions or vortexing to produce larger particle size emulsions, and (c) Ribi adjuvant system (RAS) (Ribi Immunochem, Hamilt On, MT), which contains 2% squalene, 0.2% Tween 80, and one or more of a group consisting of monophosphorus lipid A (MPL), trehalose dimylate (TDM), and cell wall skeleton (CWS) bacterial cell wall component, preferably ^{MPL + CWS (Detox TM);} (3) saponin adjuvants may be used, such as ^{Stimulon TM (Cambridge Bioscience; Worcester,} MA) or particles generated therefrom of, such as the ISCOM (immunostimulating complexes) (4) Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) Cytokines such as interleukin (eg IL-1, IL-2, IL-4, IL-5) , IL-6, IL-7, IL-12, etc.), interferons (such as gamma interferon), macrophage community stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) act as an immune A stimulant to enhance the effectiveness of the composition.

As described above, cell wall purine peptides include, but are not limited to, N-acetyl group-cell wall-L-threonyl-D-iso-bromide (thr-MDP), N-acetyl group -Demethylcellulose-L-alaninyl-D-isoglutamic acid (nor-MDP), N-acetylthiol-mercapto-L-alaninyl-D-isosorbide Base-L-alanine-2"-2'-dipalmitoyl-sn-glyceryl-3-hydroxyphosphonium-ethylamine (MTP-PE) or the like.

An immunogenic composition (eg, an immunogenic or immunogen or multi-peptide or protein or nucleic acid, a pharmaceutically acceptable carrier and adjuvant) will typically contain a diluent such as water, physiological saline, glycerol, ethanol Wait. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in the vehicles.

Typically, the immunogenic composition is prepared as an injectable preparation (in the form of a liquid solution or suspension); solid forms suitable for dissolving or suspending in a liquid vehicle before injection may also be prepared. The formulation may also be emulsified or encapsulated in liposomes for enhancing the adjuvant effect, as discussed above.

An immunogenic composition for use as a vaccine comprises an immunologically effective amount of an antigenic or immunogenic polypeptide and any other of the above components (if desired). By "immunologically-effective amount" is meant that the amount is administered to an individual in a single dose or as part of a series of doses effective for treatment or prevention. This amount depends on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (eg, non-human primates, primates, etc.), the individual's immune system, synthetic antibodies, or universally initiated immunity. The ability of the epidemic response, the degree of protection required, the formulation of the vaccine, the assessment of the medical condition and other relevant factors. This amount is expected to be a relatively broad range that can be determined by routine experimentation. However, for the purpose of protein vaccination, the amount administered per immunization is typically in the range between 0.5 μg and 500 mg (often not higher than 5,000 μg), and often between 10 μg and 200 μg. Within the scope.

Immunogenic compositions are conventionally administered parenterally, for example by subcutaneous, intramuscular or transdermal injection (see, for example, W098/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, intravenous or intra-arterial routes may also be available.

Dosage therapy can be a single dose regimen or a multiple dose regimen. Vaccines can also be administered in combination with other immunomodulators.

As an alternative to protein-based vaccines, DNA vaccination (also known as nucleic acid vaccination or genetic vaccination) can be used [eg Robinson and Torres (1997) Seminars in Immunology 9: 271-283; Donnelly et al. (1997) Ann Rev Immunol 15:617-648; later].

Another aspect of the invention provides for the recognition of a combination vaccine wherein two or more of the Laussum antigens disclosed herein are combined to enhance the immune response of the vaccinated animal, including the initial immune response and The duration of immunity is optimized. For the purposes of this aspect of the invention, multiple antigenic fragments derived from the same L. sonnei protein may also be used, such as a combination of polypeptide sequence fragments of different lengths using one protein.

Use of multiple types

Another aspect of the invention provides for the recognition of a combination vaccine wherein two or more of the Laussian antigens (or nucleic acids encoding the same) disclosed herein are combined to enhance the immune response of the vaccinated animal, This includes optimizing the initial immune response and duration of immunization. For the purposes of this aspect of the invention, it is also possible to use a protein derived from the same antigenic Lawsonia species. An antigen, such as a combination of multi-peptide sequence antigens of different lengths using one protein. Representative examples of combination antigen formulations are combined antigen formulations comprising any of LIC037 or a fragment thereof disclosed herein and a combination of a multi-peptide derived from at least two other multi-peptides of the invention and fragments thereof. .

The treatment method of the invention

The method of the sixth aspect of the present invention is generally related to the induction of immunity, and therefore also requires methods for the treatment, prevention and improvement of diseases.

When the immunization method requires administration of the multi-peptide of the present invention or a composition comprising the multi-peptide, the animal (e.g., pig) typically receives from 0.5 to 5,000 μg of the multi-peptide of the present invention per administration.

In a preferred embodiment of the sixth aspect, the immunization procedure includes the animal (e.g., pig) receiving a first-time administration and one or more additional administrations.

A preferred embodiment of the sixth aspect of the present invention comprises administration for the purpose of inducing protective immunity against L. intracellularis. In this particular embodiment, it is particularly preferred that the protective immunization is effective to reduce the risk of intracellular L. sonnei infection, or to effectively treat or ameliorate the intracellular L. sonnei infection.

As mentioned herein, the preferred vaccine of the invention induces humoral immunity. Therefore, it is preferred to administer an antibody for inducing specificity against L. intracellularis, and wherein the antibodies or B lymphocytes producing the antibodies are subsequently recovered automatically.

As also mentioned, the sixth aspect of the method can also be applied to antibody production. Therefore, in other specific examples, the purpose of inducing antibodies specific for Lawsonia intracellularis is administered, and the B lymphocytes in which the antibodies are produced are subsequently automatically recovered and used for monoclonal antibodies. preparation.

The pharmaceutical composition as described above may also comprise the multi-peptide, antibody or nucleic acid of the invention. The pharmaceutical composition will contain a therapeutically effective amount thereof.

As used herein, the term "therapeutically-effective amount" or "pre- "Prophylactically-effective amount" means an amount of a therapeutic agent used to treat, ameliorate or prevent a desired disease or condition or to exhibit a detectable therapeutic or prophylactic effect. The effect can be detected by, for example, chemical labeling or antigen content. Therapeutic effects also include a reduction in physical symptoms, such as a decrease in body temperature. The precise and effective amount for an individual will depend on the individual's size and condition, the nature and extent of the condition, and the combination of therapeutic or therapeutic agents selected for administration. Therefore, it does not apply to specifying an accurate and effective amount in advance. However, reference is made to the above range of doses for immunologically effective amounts of the multi-peptide.

However, an effective amount for a given situation can be determined by routine experimentation and within the judgment of the clinician.

For the purposes of the present invention, an effective dosage will be from about 0.01 mg/kg to 50 mg/kg, or from 0.05 mg/kg to about 10 mg/kg of DNA construct in the individual to be administered.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically-acceptable carrier" refers to a carrier used to administer a therapeutic agent, such as an antibody or multi-peptide, a gene, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies that are detrimental to the individual receiving the composition and that can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virions. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, including, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts such as acetates, propionates , malonate, benzoate and the like. A thorough discussion of pharmaceutically acceptable salts is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

The pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, physiological saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in the vehicles. Typically, the therapeutic composition is prepared as an injectable, liquid In the form of a body solution or suspension. Solid forms suitable for dissolution or suspension in the vehicle prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

The invention also includes specific examples of treatments and preventions disclosed herein, as will be apparent from the scope of the invention. The present invention also includes the following specific examples, wherein: - the multi-peptide of the present invention is used as a medicament, in particular, as a medicament for treating, preventing or ameliorating the infection of L. intracellularis; - a nucleic acid fragment or the present invention The carrier of the invention is used as a medicament, in particular as a medicament for treating, preventing or ameliorating the infection of L. intracellularis; - the transformed cell of the invention is used as a medicament, in particular for treatment, prevention or A drug for improving intracellular Lactobacillus infection; - an antibody, antibody fragment or antibody analog of the present invention is used as a medicament, and is used in detail as a medicament for treating, preventing or ameliorating an intracellular L. sonnei infection.

An additional numbered statement of the invention

Representative aspects of the invention include the following specific examples of the invention and the following examples.

Specific Example 1. A multi-peptide comprising a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOS: 1-17 and 54, or b) SEQ ID NOS: 1-17 and An amino acid sequence consisting of at least 5 contiguous amino acid residues of any of 54 or c) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of a), d) An amino acid sequence having at least 60% sequence identity to the amino acid sequence of b), or e) an amino acid assembly derived from any one of SEQ ID NOS: 1-17 and 54 having The multi-peptide is antigenic in mammals, in contrast to the 3D conformation that derivatizes the assembly to form a B cell epitope.

Specific Example 2. The multi-peptide of the specific example 1, wherein the at least 5 consecutive amino acids are at least 6, such as at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, At least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34 and at least 35 contiguous amino acid residues.

Specific Example 3. The peptide of Specific Example 1 or 2, wherein the sequence identity to the amino acid sequence of a) is at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85% At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

Specific Example 4. The peptide of Specific Example 1 or 2, wherein the sequence identity to the amino acid sequence of b) is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80% At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

The multi-peptide of any one of the preceding embodiments, wherein the at least 5 contiguous amino acid residues have an amino acid residue corresponding to any one of SEQ ID NOS: 1-17 and 54 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, N-terminal amine of any of 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121 Base acid residue.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 2-17 and 54 An N-terminal amino acid residue of any of residues 122, 123, and 124.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 3-17 and 54 An N-terminal amino acid residue of any of residues 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, and 137.

The multi-peptide of any one of the embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 4-17 and 54 Residues 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, N-terminal amino acid residues of any of 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255 .

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 5-17 and 54 Residues 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, An N-terminal amino acid residue of any of 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, and 386.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 6-17 and 54 Residues 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457, An N-terminal amino acid residue of any of 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, and 476.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 7-17 and 54 Residues 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, Any of 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, and 625 N-terminal amino acid residue.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 8-17 and 54 An N-terminal amino acid residue of any of residues 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, and 638.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 9-17 and 54 Residues 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, An N-terminal amino acid residue of any of 713, 714, 715, 716, 717, 718, 719, 720, and 721.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 10-17 and 54 Residues 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, An N-terminal amino acid residue of any of 820, 821, 822, 823, 824, 825, 826, 827, and 828.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 11-17 and 54 An N-terminal amino acid residue of any of residues 829, 830, 831, 832, 833, 834, 835, and 836.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 12-17 and 54 Residues 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,880,881 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, N-terminal amino acid of any of 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, and 930 Residues.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 13-17 and 54 Residues 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, N-terminal amino acid residue of any of 955 and 956.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 14-17 and 54 Residues 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, Any of 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, and 1046 N-terminal amino acid residue.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 15-17 and 54 Residues 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, An N-terminal amino acid residue of any of 1071 and 1072.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have an amino acid corresponding to any one of SEQ ID NOS: 16, 17, and 54 Residues 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 11 91, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, An N-terminal amino acid residue of any of 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, and 1336.

The multi-peptide of any one of embodiments 1 to 4, wherein the at least 5 contiguous amino acid residues have amino acid residues 1337, 1338 corresponding to SEQ ID NO: 17 or 54, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, An N-terminal amino acid residue of any of 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, and 1603.

Specific example 22.  As the polypeptide of any one of the specific examples 1 to 4, Wherein the at least 5 contiguous amino acid residues have a corresponding SEQ ID NO: 54 amino acid residue 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701 1702 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801 1802 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2031 2022 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041 2042 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061 2062 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201 2202 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301 2302 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401 2402 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501 2502 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778, 2779, 2780, 2781 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896, 2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920, 2921, 2922, 2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944, 2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956, 2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965, 2966, 2967, 2968, 2969, 2970, 2971, 2972, 2973, 2974, 2975, 2976, 2977, 2978, 2979, 2980, 2981, 2982, 2983, 2984, 2985, 2986, 2987, 2988, 2989, 2990, 2991, 2992, 2993, 2994, 2995, 2996, 2997, 2998, 2999, 3000, 3001 3002 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3011 3022 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041 3042 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3052, 3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3061, 3062, 3063, 3064, 3065, 3066, 3067, 3068, 3069, 3070, 3071, 3072, 3073, 3074, 3075, 3076, 3077, 3078, 3079, 3080, 3081, 3082, 3083, 3084, 3085, 3086, 3087, 3088, 3089, 3090, 3091, 3092, 3093, 3094, 3095, 3096, 3097, 3098, 3099, 3100, 3101 3102, 3103, 3104, 3105, 3106, 3107, 3108, 3109, 3110, 3111, 3112, 3113, 3114, 3115, 3116, 3117, 3118, 3119, 3120, 3121, 3122, 3123, 3124, 3125, 3126, 3127, 3128, 3129, 3130, 3131, 3132, 3133, 3134, 3135, 3136, 3137, 3138, 3139, 3140, 3141, 3142, 3143, 3144, 3145, 3146, 3147, 3148, 3149, 3150, 3151, 3152, 3153, 3154, 3155, 3156, 3157, 3158, 3159, 3160, 3161, 3162, 3163, 3164, 3165, 3166, 3167, 3168, 3169, 3170, 3171, 3172, 3173, 3174, 3175, 3176, 3177, 3178, 3179, 3180, 3181, 3182, 3183, 3184, 3185, 3186, 3187, 3188, 3189, 3190, 3191, 3192, 3193, 3194, 3195, 3196, 3197, 3198, 3199, 3200, 3201 3202 3203, 3204, 3205, 3206, 3207, 3208, 3209, 3210, 3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220, 3221, 3222, 3223, 3224, 3225, 3226, 3227, 3228, 3229, 3230, 3231, 3232, 3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244, 3245, 3246, 3247, 3248, 3249, 3250, 3251, 3252, 3253, 3254, 3255, 3256, 3257, 3258, 3259, 3260, 3261, 3262, 3263, 3264, 3265, 3266, 3267, 3268, 3269, 3270, 3271, 3272, 3273, 3274, 3275, 3276, 3277, 3278, 3279, 3280, 3281, 3282, 3283, 3284, 3285, 3286, 3287, 3288, 3289, 3290, 3291, 3292, 3293, 3294, 3295, 3296, 3297, 3298, 3299, 3300, 3301 3302 3303, 3304, 3305, 3306, 3307, 3308, 3309, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3319, 3320, 3321, 3322, 3323, 3324, 3325, 3326, 3327, 3328, 3329, 3330, 3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342, 3343, 3344, 3345, 3346, 3347, 3348, 3349, 3350, 3351, 3352, 3353, 3354, 3355, 3356, 3357, 3358, 3359, 3360, 3361, 3362, 3363, 3364, 3365, 3366, 3367, 3368, 3369, 3370, 3371, 3372, 3373, 3374, 3375, 3376, 3377, 3378, 3379, 3380, 3381, 3382, 3383, 3384, 3385, 3386, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3394, 3395, 3396, 3397, 3398, 3399, 3400, 3401, 3402, 3403, 3404, 3405, 3406, 3407, 3408, 3409, 3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3456, 3457, 3458, 3459, 3460, 3461, 3462, 3463, 3464, 3465, 3466, 3467, 3468, 3469, 3470, 3471, 3472, 3473, 3474, 3475, 3476, 3477, 3478, 3479, 3480, 3481, 3482, 3483, 3484, 3485, 3486, 3487, 3488, 3489, 3490, 3491, 3492, 3493, 3494, 3495, 3496, 3497, 3498, 3499, 3500, 3501 3502 3503, 3504, 3505, 3506, 3507, 3508, 3509, 3510, 3511, 3512, 3513, 3514, 3515, 3516, 3517, 3518, 3519, 3520, 3521, 3522, 3523, 3524, 3525, 3526, 3527, 3528, 3529, 3530, 3531, 3532, 3533, 3534, 3535, 3536, 3537, 3538, 3539, 3540, 3541, 3542, 3543, 3544, 3545, 3546, 3547, 3548, 3549, 3550, 3551, 3552, 3553, 3554, 3555, 3556, 3557, 3558, 3559, 3560, 3561, 3562, 3563, 3564, 3565, 3566, 3567, 3568, 3569, 3570, 3571, 3572, 3573, 3574, 3575, 3576, 3577, 3578, 3579, 3580, 3581, 3582, 3583, 3584, 3585, 3586, 3587, 3588, 3589, 3590, 3591, 3592, 3593, 3594, 3595, 3596, 3597, 3598, 3599, 3600, 3601 3602 3603, 3604, 3605, 3606, 3607, 3608, 3609, 3610, 3611, 3612, 3613, 3614, 3615, 3616, 3617, 3618, 3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3640, 3641, 3642, 3643, 3644, 3645, 3646, 3647, 3648, 3649, 3650, 3651, 3652, 3653, 3654, 3655, 3656, 3657, 3658, 3659, 3660, 3661, 3662, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700, 3701, 3702, 3703, 3704, 3705, 3706, 3707, 3708, 3709, 3710, 3711, 3712, 3713, 3714, 3715, 3716, 3717, 3718, 3719, 3720, 3721, 3722, 3723, 3724, 3725, 3726, 3727, 3728, 3729, 3730, 3731, 3732, 3733, 3734, 3735, 3736, 3737, 3738, 3739, 3740, 3741, 3742, 3743, 3744, 3745, 3746, 3747, 3748, 3749, 3750, 3751, 3752, 3753, 3754, 3755, 3756, 3757, 3758, 3759, 3760, 3761, 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3776, 3777, 3778, 3779, 3780, 3871 3782, 3783, 3784, 3785, 3786, 3787, 3788, 3789, 3790, 3791, 3792, 3793, 3794, 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, 3810, 3811, 3812, 3813, 3814, 3815, 3816, 3817, 3818, 3819, 3820, 3821, 3822, 3823, 3824, 3825, 3826, 3827, 3828, 3829, 3830, 3831, 3832, 3833, 3834, 3835, 3836, 3837, 3838, 3839, 3840, 3841, 3842, 3843, 3844, 3845, 3846, 3847, 3848, 3849, 3850, 3851, 3852, 3853, 3854, 3855, 3856, 3857, 3858, 3859, 3860, 3861, 3862, 3863, 3864, 3865, 3866, 3867, 3868, 3869, 3870, 3871, 3872, 3873, 3874, 3875, 3876, 3877, 3878, 3879, 3880, 3881, 3882, 3883, 3884, 3885, 3886, 3887, 3888, 3889, 3890, 3891, 3892, 3893, 3894, 3895, 3896, 3897, 3898, 3899, 3900, 3901, 3902, 3903, 3904, 3905, 3906, 3907, 3908, 3909, 3910, 3911, 3912, 3913, 3914, 3915, 3916, 3917, 3918, 3919, 3920, 3921, 3922, 3923, 3924, 3925, 3926, 3927, 3928, 3929, 3930, 3931, 3932, 3933, 3934, 3935, 3936, 3937, 3938, 3939, 3940, 3941, 3942, 3943, 3944, 3945, 3946, 3947, 3948, 3949, 3950, 3951, 3952, 3953, 3954, 3955, 3956, 3957, 3958, 3959, 3960, 3961, 3962, 3963, 3964, 3965, 3966, 3967, 3968, 3969, 3970, 3971, 3972, 3973, 3974, 3975, 3976, 3977, 3978, 3979, 3980, 3981, 3982, 3983, 3984, 3985, 3986, 3987, 3988, 3989, 3990, 3991, 3992, 3993, 3994, 3995, 3996, 3997, 3998, 3999, 4000, 4001 4002 4003, 4004, 4005, 4006, 4007, 4008, 4009, 4010, 4011, 4012, 4013, 4014, 4015, 4016, 4017, 4018, 4019, 4020, 4011 4022 4023, 4024, 4025, 4026, 4027, 4028, 4029, 4030, 4031, 4032, 4033, 4034, 4035, 4036, 4037, 4038, 4039, 4040, 4041, 4042, 4043, 4044, 4045, 4046, 4047, 4048, 4049, 4050, 4051, 4052, 4053, 4054, 4055, 4056, 4057, 4058, 4059, 4060, 4061, 4062, 4063, 4064, 4065, 4066, 4067, 4068, 4069, 4070, 4071, 4072, 4073, 4074, 4075, 4076, 4077, 4078, 4079, 4080, 4081, 4082, 4083, 4084, 4085, 4086, 4087, 4088, 4089, 4090, 4091, 4092, 4093, 4094, 4095, 4096, 4097, 4098, 4099, 4100, 4101 4102, 4103, 4104, 4105, 4106, 4107, 4108, 4109, 4110, 4111, 4112, 4113, 4114, 4115, 4116, 4117, 4118, 4119, 4120, 4121, 4122, 4123, 4124, 4125, 4126, 4127, 4128, 4129, 4130, 4131, 4132, 4133, 4134, 4135, 4136, 4137, 4138, 4139, 4140, 4141, 4142, 4143, 4144, 4145, 4146, 4147, 4148, 4149, 4150, 4151, 4152, 4153, 4154, 4155, 4156, 4157, 4158, 4159, 4160, 4161, 4162, 4163, 4164, 4165, 4166, 4167, 4168, 4169, 4170, 4171, 4172, 4173, 4174, 4175, 4176, 4177, 4178, 4179, 4180, 4181, 4182, 4183, 4184, 4185, 4186, 4187, 4188, 4189, 4190, 4191, 4192, 4193, 4194, 4195, 4196, 4197, 4198, 4199, 4200, 4201, 4202, 4203, 4204, 4205, 4206, 4207, 4208, 4209, 4210, 4211, 4212, 4213, 4214, 4215, 4216, 4217, 4218, 4219, 4220, 4221, 4222, 4223, 4224, 4225, 4226, 4227, 4228, 4229, 4230, 4231, 4232, 4233, 4234, 4235, 4236, 4237, 4238, 4239, 4240, 4241, 4242, 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8091 8092, 8093, 8094, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8108, 8109, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8128, 8129, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8137, 8138, 8139, 8140, 8141, 8142, 8143, 8144, 8145, 8146, 8147, 8148, 8149, 8150, 8151, 8152, 8153, 8154, 8155, 8156, 8157, 8158, 8159, 8160, 8161, 8162, 8163, 8164, 8165, 8166, 8167, 8168, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181 8182, 8183, 8184, 8185, 8186, 8187, 8188, 8189, 8190, 8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201, 8202, 8203, 8204, 8205, 8206, 8207, 8208, 8209, 8210, 8211, 8212, 8213, 8214, 8215, 8216, 8217, 8218, 8219, 8220, 8221, 8222, 8223, 8224, 8225, 8226, 8227, 8228, 8229, 8230, 8231 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241 8242, 8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251 8252, 8253, 8254, 8255, 8256, 8257, 8258, 8259, 8260, 8261 8262, 8263, 8264, 8265, 8266, 8267, 8268, 8269, 8270, 8271 8272, 8273, 8274, 8275, 8276, 8277, 8278, 8279, 8280, 8281 8282, 8283, 8284, 8285, 8286, 8287, 8288, 8289, 8290, 8291 8292, 8293, 8294, 8295, 8296, 8297, 8298, 8299, 8300, 8301, 8302, 8303, 8304, 8305, 8306, 8307, 8308, 8309, 8310, 8311, 8312, 8313, 8314, 8315, 8316, 8317, 8318, 8319, 8320, 8321 8322, 8323, 8324, 8325, 8326, 8327, 8328, 8329, 8330, 8331 8332, 8333, 8334, 8335, 8336, 8337, 8338, 8339, 8340, 8341 8342, 8343, 8344, 8345, 8346, 8347, 8348, 8349, 8350, 8351, 8352, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361 8362, 8363, 8364, 8365, 8366, 8367, 8368, 8369, 8370, 8371 8372, 8373, 8374, 8375, 8376, 8377, 8378, 8379, 8380, 8381 8382, 8383, 8384, 8385, 8386, 8387, 8388, 8389, 8390, 8391 8392, 8393, 8394, 8395, 8396, 8397, 8398, 8399, 8400, 8401 8402, 8403, 8404, 8405, 8406, 8407, 8408, 8409, 8410, 8411, 8412, 8413, 8414, 8415, 8416, 8417, 8418, 8419, 8420, 8411 8422, 8423, 8424, 8425, 8426, 8427, 8428, 8429, 8430, 8431 8432, 8433, 8434, 8435, 8436, 8437, 8438, 8439, 8440, 8441 8442, 8443, 8444, 8445, 8446, 8447, 8448, 8449, 8450, 8451 8452, 8453, 8454, 8455, 8456, 8457, 8458, 8459, 8460, 8461 8462, 8463, 8464, 8465, 8466, 8467, 8468, 8469, 8470, 8471 8472, 8473, 8474, 8475, 8476, 8477, 8478, 8479, 8480, 8481 8482, 8483, 8484, 8485, 8486, 8487, 8488, 8489, 8490, 8491 8492, 8493, 8494, 8495, 8496, 8497, 8498, 8499, 8500, 8501 8502, 8503, 8504, 8505, 8506, 8507, 8508, 8509, 8510, 8511, 8512, 8513, 8514, 8515, 8516, 8517, 8518, 8519, 8520, 8511 8522, 8523, 8524, 8525, 8526, 8527, 8528, 8529, 8530, 8531 8532, 8533, 8534, 8535, 8536, 8537, 8538, 8539, 8540, 8541 8542, 8543, 8544, 8545, 8546, 8547, 8548, 8549, 8550, 8551 8552, 8553, 8554, 8555, 8556, 8557, 8558, 8559, 8560, 8561 8562, 8563, 8564, 8565, 8566, 8567, 8568, 8569, 8570, 8571 8572, 8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580, 8581 8582, 8583, 8584, 8585, 8586, 8587, 8588, 8589, 8590, 8591 8592, 8593, 8594, 8595, 8596, 8597, 8598, 8599, 8600, 8601, 8602, 8603, 8604, 8605, 8606, 8607, 8608, 8609, 8610, 8611, 8612, 8613, 8614, 8615, 8616, 8617, 8618, 8619, 8620, 8621 8622, 8623, 8624, 8625, 8626, 8627, 8628, 8629, 8630, 8631 8632, 8633, 8634, 8635, 8636, 8637, 8638, 8639, 8640, 8641 8642, 8643, 8644, 8645, 8646, 8647, 8648, 8649, 8650, 8651 8652, 8653, 8654, 8655, 8656, 8657, 8658, 8659, 8660, 8661 8662, 8663, 8664, 8665, 8666, 8667, 8668, 8669, 8670, 8671 8672, 8673, 8674, 8675, 8676, 8677, 8678, 8679, 8680, 8681 8682, 8683, 8684, 8685, 8686, 8687, 8688, 8689, 8690, 8691 8692, 8693, 8694, 8695, 8696, 8697, 8698, 8699, 8700, 8701 8702, 8703, 8704, 8705, 8706, 8707, 8708, 8709, 8710, 8711, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731 8732, 8733, 8734, 8735, 8736, 8737, 8738, 8739, N-terminal amino acid residues of any of 8740 and 8741.

A polypeptide according to any one of the preceding embodiments, which is fused or conjugated to an immunogenic carrier molecule.

Specific Example 24. The multi-peptide of the specific example 23, wherein the immunogenic carrier molecule is a multi-peptide that induces a T-helper lymphocyte reaction in a majority of mammals, such as selected from the group consisting of keyhole spirulina or a fragment thereof, An immunogenic carrier protein of a group consisting of tetanus toxoid or a fragment thereof, or a diphtheria toxoid or a fragment thereof.

A polypeptide according to any one of the preceding embodiments, which is capable of inducing an adaptive immune response against a multi-peptide of a mammal, in particular a pig.

Specific Example 26. A multi-peptide of the specific example 25 which is capable of eliciting a protective adaptive immune response against a strain of L. intracellularis in a mammal.

Specific Example 27. A multi-peptide of the specific example 25 or 26 which induces a humoral and/or cellular immune response.

Specific Example 28. An isolated nucleic acid fragment comprising i) a nucleotide sequence encoding a multi-peptide of any of the preceding specific examples, or ii) any one of SEQ ID NOs: 18-51 and 55 a nucleotide sequence consisting of a partial amino acid coding portion, iii) at least 10 of the amino acid coding portion of any one of SEQ ID NOs: 18-51 and 55 a nucleotide sequence consisting of contiguous nucleotides, iv) a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence in i) or ii), v) and nucleotides in iii) a nucleotide sequence having a sequence identity of at least 60%, vi) a nucleotide sequence complementary to the nucleotide sequence in i)-v), or vii) under stringent conditions and i)-vi) A nucleotide sequence in which the nucleotide sequence hybridizes.

Specific Example 29. A nucleic acid fragment according to the specific example 28 which is a DNA or RNA fragment.

The nucleic acid fragment of the specific example 28 or 29, wherein the nucleotide sequence is at least 11 and at least 12 of the amino acid-encoding portion of any one of SEQ ID NOS: 18-51 and 55, At least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 , at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, At least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 , at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, At least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71 At least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84 , at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, At least 97, at least 98, at least 99, at least 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, At least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118, at least 119, at least 120 , at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 131, at least 132, At least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141, at least 142, at least 143, at least 144, at least 145 , at least 146, at least 147, at least 148, at least 149, at least 150, at least 151, at least 152, at least 153, at least 154, at least 155, at least 156, at least 157, At least 158, at least 159, at least 160, at least 161, at least 162, at least 163, at least 164, at least 165, at least 166, at least 167, at least 168, at least 169, at least 170 , at least 171, at least 172, at least 173, at least 174 At least 175, at least 176, at least 177, at least 178, at least 179, at least 180, at least 181, at least 182, at least 183, at least 184, at least 185, at least 186, at least 187, at least 188, at least 189, at least 190, at least 191, at least 192, at least 193, at least 194, at least 195, at least 196, at least 197, at least 198, at least 199 At least 200, at least 201, at least 202, at least 203, at least 204, at least 205, at least 206, at least 207, at least 208, at least 209, at least 210, at least 211, at least 212, at least 213, at least 214, at least 215, at least 216, at least 217, at least 218, at least 219, at least 220, at least 221, at least 222, at least 223, at least 224 At least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236, at least 237, at least 238, at least 239, at least 240, to 241, at least 242, at least 243, at least 244, at least 245 , at least 246, at least 247, at least 248, at least 249, at least 250, at least 251, at least 252, at least 253, at least 254, at least 255, at least 256, at least 257, At least 258, at least 259, at least 260, at least 261, at least 262, at least 263, at least 264, at least 265, at least 266, at least 267, at least 268, at least 269, at least 270 At least 271, at least 272, at least 273, at least 274, at least 275, at least 276, at least 277, at least 278, at least 279, at least 280, at least 281, at least 282, At least 283, at least 284, at least 285, at least 286, at least 287, at least 288, at least 289, at least 290, at least 291, at least 292, at least 293, at least 294, at least 295 , at least 296, at least 297, at least 298, at least 299, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, At least 308, at least 309, at least 310, at least 311, to 312, at least 313, at least 314, at least 315, at least 316, at least 317, at least 318, at least 319, at least 320, at least 321 , at least 322, at least 323, at least 324 At least 325, at least 326, at least 327, at least 328, at least 329, at least 330, at least 331, at least 332, at least 333, at least 334, at least 335, at least 336, at least 337, at least 338, at least 339, at least 340, at least 341, at least 342, at least 343, at least 344, at least 345, at least 346, at least 347, at least 348, at least 349 At least 350, at least 351, at least 352, at least 353, at least 354, at least 355, at least 356, at least 357, at least 358, at least 359, at least 360, at least 361, at least 362, at least 363, at least 364, at least 365, at least 366, at least 367, at least 368, at least 369, at least 370, at least 371, at least 372, at least 373, at least 374 and Composition of at least 375 contiguous nucleotides.

The nucleic acid fragment of any one of the examples 28 to 30, wherein the Or the nucleotide sequence of ii) has a sequence identity of at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

The nucleic acid fragment of any one of embodiments 28 to 30, wherein the nucleotide sequence of the nucleotide sequence of iii) is at least 65%, such as at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

Specific Example 33. A vector comprising a nucleic acid according to any one of embodiments 28 to 32, such as a selection vector or expression vector.

Specific Example 34. The vector of Specific Example 33, comprising an operably linked and comprising a enhancer/promoter in the 5'-3' direction for promoting expression of the nucleic acid fragment as defined in the specific example 27-i) The expression control region, the signal peptide coding sequence selected as appropriate, the nucleotide sequence defined in the specific example 27-i), and the terminator selected as appropriate.

Embodiment 35. The vector of embodiment 33 or 34, wherein the expression control region promotes expression in a prokaryotic cell such as a bacterium, such as in E. coli.

The carrier of any one of embodiments 33 to 35, which is capable of autonomous replication.

The vector of any of embodiments 33 to 36, which is capable of integration into the genome of a host cell.

The vector of any of embodiments 33 to 37, which is incapable of integrating into the genome of a mammalian host cell.

The vector of any one of embodiments 33 to 38, which is selected from the group consisting of a virus (such as an attenuated virus), a phage, a plastid, a pocket chromosome, and a viscous body.

Specific Example 40. A cell transformed to carry the vector of any of Examples 33 to 39.

Specific Example 41. The transformed cell of Example 40, which is capable of replicating a specific example A nucleic acid fragment as defined in 28-i).

A specific example 42. The transformed cell of embodiment 40, which is capable of exhibiting the nucleic acid fragment as defined in specific example 28-i).

The transformed cell of any one of the examples 40 to 42 which is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

The transduced cell of any one of embodiments 40 to 42 which is selected from the group consisting of Escherichia (such as Escherichia coli), Bacillus (such as Bacillus subtilis), Salmonella and A bacterial cell of the group consisting of Mycobacterium (preferably not pathogenic, such as M. bovis BCG).

The transduced cell of any one of embodiments 40 to 44, which is stably transformed by stably integrating the nucleic acid defined in the specific example 42-i) into its genome.

The transfected cells of any one of the specific examples 40 to 45 which secrete or carry on their surface the multi-peptide of any one of the specific examples 1 to 27.

Specific example 47. The transformed cell of embodiment 46, wherein the cell is a bacterium and the secretion enters the periplasmic space.

A 48. A cell line derived from a transduced cell of any of embodiments 40-47.

A pharmaceutical composition comprising the multi-peptide of any one of the specific examples 1 to 27, the nucleic acid fragment according to any one of the specific examples 28 to 32, as in any one of the specific examples 33 to 39. A carrier, or a cell of any one of embodiments 40 to 48, and a pharmaceutically acceptable carrier, vehicle or diluent.

Specific Example 50. The pharmaceutical composition of Embodiment 49, which additionally comprises an immunological adjuvant.

A pharmaceutical composition according to the specific example 50, wherein the adjuvant is an aluminum-based adjuvant.

Specific Example 52. A method for inducing immunity of an animal by administering at least one A multi-peptidic peptide according to any one of the embodiments 1 to 27, a nucleic acid fragment according to any one of the specific examples 28 to 32, a carrier according to any one of the specific examples 33 to 39, as specified The cells of any one of Examples 40 to 48, or the pharmaceutical composition according to any one of Examples 49 to 51 to induce adaptive immunity of the animal against L. intracellularis.

The method of embodiment 52, wherein when the multi-peptide of any one of the specific examples 1 to 27 or the composition comprising the multi-peptide is administered, the animal receives 0.5 to each administration. 5,000 μg of the multi-peptide of any of Examples 1 to 27.

The method of embodiment 52 or 53, wherein the animal is subjected to a first administration and one or more additional administrations.

The method of any one of embodiments 52 to 54, wherein the animal is a pig.

The method of any one of embodiments 52 to 55, wherein the administration is for the purpose of inducing protective immunity against L. intracellularis.

The method of embodiment 56, wherein the protective immunization is effective to reduce the risk of causing an intracellular L. sonnei infection or to effectively treat or ameliorate the intracellular L. sonnei infection.

The method of embodiment 52, wherein the administration is for the purpose of eliciting an antibody specific for L. intracellularis and wherein the antibodies or B lymphocytes producing the antibodies are subsequently The animal is recycled.

The method of embodiment 52, wherein the administration is for the purpose of inducing an antibody specific for L. intracellularis and wherein the B lymphocytes producing the antibody are subsequently recovered from the animal and For the preparation of monoclonal antibodies.

Specific Example 60. A multi-strain antibody, wherein the antibodies specifically bind to at least one multi-peptide as in any one of embodiments 1 to 27, and which substantially binds non-specifically to other intracellular Lawsonia An antibody to a multi-peptide.

Specific Example 61. An isolated monoclonal antibody or antibody analog that specifically binds to A multi-peptide of any one of embodiments 1 to 27.

Specific Example 62. The isolated monoclonal antibody or antibody analog of Example 61, which is selected from the group consisting of a multi-domain antibody (such as a murine antibody), a chimeric antibody (such as a humanized antibody), a fully human antibody, and a A monoclonal antibody to a single domain antibody of a horse or camel, or an antibody analog thereof selected from antibody fragments such as Fab, F(ab') ₂ or scFV.

A pharmaceutical composition comprising an antibody according to any one of embodiments 60 to 62 and a pharmaceutically acceptable carrier, vehicle or diluent.

Specific Example 64. A method for preventing, treating or ameliorating an infection of L. intracellularis comprising administering to a mammal in need thereof a therapeutically effective amount of an antibody according to any one of embodiments 60 to 62 or as The pharmaceutical composition of the specific example 63.

Specific Example 65. A method for quantitatively or qualitatively determining the presence of L. intracellularis in a sample, the method comprising contacting and detecting the sample with an antibody of any one of embodiments 60 to 62 The presence of antibodies to the substance in the sample.

Specific Example 66. A method for quantitatively or qualitatively determining the presence of an antibody specific for a strain of L. intracellularis in a sample, the method comprising subjecting the sample to any of the specific examples 1 to 27 The peptide contacts and detects the presence of the antibody to the multi-peptide.

Specific Example 67. A method for quantitatively or qualitatively determining the presence of a nucleic acid specific to L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment of any of Examples 28 to 32 and The presence of a nucleic acid that hybridizes to the nucleic acid fragment in the sample is detected.

The method of embodiment 67, which comprises molecular amplification of at least one step of the nucleic acid to be detected in the sample, such as a PCR amplification step.

Specific Example 68. A method for the preparation of the multi-peptide of any of Examples 1 to 27, which comprises - as in the specific example 41 and the specific examples 43 to 48 (as long as the specific examples are specific The transduced cells of any of Example 42 behaved as in Example 28-i) and specific The transformed cells are cultured under conditions of the nucleic acid fragments of any of Examples 29 to 32 (as long as such specific examples are based on the specific example 28-i), and then the multi-peptide is recovered, or - The multi-peptide is prepared by solid phase or liquid phase peptide synthesis.

Specific Example 69. A method for determining whether a substance such as an antibody is suitable for treating an intracellular L. sonnei infection, the method comprising contacting a multi-peptide of any one of the specific examples 1 to 27 with the substance And subsequently determining whether the substance has at least one of the following characteristics: 1) the ability to specifically bind to the multi-peptide, and 2) the ability to compete with the multi-peptide for specific binding to the ligand/receptor, 3) The ability to specifically deactivate the polypeptide.

Specific Example 70. A method for determining whether a substance, such as a nucleic acid, is suitable for use in treating an intracellular L. sinensis infection, the method comprising contacting the substance with a nucleic acid fragment of any one of embodiments 28 to 32 and It is then determined whether the substance has any of the following capabilities: 1) specifically binding to the nucleic acid fragment, or 2) specifically binding to a nucleic acid that specifically hybridizes to the nucleic acid fragment.

The polypeptide of any one of the specific examples 1 to 27, which is used as a medicament.

The multi-peptide of any one of the specific examples 1 to 27, which is useful as a medicament for treating, preventing or ameliorating an intracellular L. sonicia infection.

The nucleic acid fragment according to any one of the specific examples 28 to 32, or the carrier according to any one of the specific examples 32 to 38, which is used as a medicament.

The nucleic acid fragment according to any one of the examples 28 to 32, or the carrier according to any one of the specific examples 32 to 38, which is used as a medicament for treating, preventing or ameliorating an intracellular L. sonnei infection.

The cell of any one of the specific examples 39 to 48, which is for use as a medicament.

Specific example 76. The cell of any one of embodiments 39 to 48 for use as a treatment, pre- A drug that prevents or improves the infection of L. intracellularis.

The antibody, antibody fragment or antibody analog of any of embodiments 60 to 62, for use as a medicament.

The antibody, antibody fragment or antibody analog of any one of the specific examples 60 to 62 for use as a medicament for the treatment, prevention or amelioration of an intracellular L. sonnei infection.

Before the example

In the following examples and figures, the L. sonnei protein is identified by its locus identifier (LocusID) ia. The following table lists the LocusIDs used in this application along with the corresponding SEQ ID NO:

The amino acid sequence of the protein discussed herein is provided in the following list: LIC037-SEQ ID NO: 17

Double underline: LIC037-A

Lowercase: LIC037-B

Underline: LIC037-C

LI0890-SEQ ID NO: 16

Underline: LI0890-A

Lowercase: LI0890-B

LIC058-SEQ ID NO: 14

Underline: LIC058-trunc

LIB022-SEQ ID NO: 12

LIC041-SEQ ID NO:10

LI0996-SEQ ID NO: 3

LI0668-SEQ ID NO: 5

LI1077-SEQ ID NO: 4

LIC047-SEQ ID NO: 11

LI1093-SEQ ID NO: 15

LI0494-SEQ ID NO:1 MKKWMVICFLVWFVSPVMAGSRFDDIDLNKDGYISWEEFQTVFPNLKKVAFDAIDTNKDGKISREEWERFQKSHEQNVQSEEGKKEMDNVKMHQRSPEELTDSEQPKTTSTKSHMKPLVQPPVTK

LI0041-SEQ ID NO:8

LI1114-SEQ ID NO:7

LI0535-SEQ ID NO: 13

LI0855-SEQ ID NO:6

LIC048-SEQ ID NO: 9

LI0138-SEQ ID NO: 2 MKVHAVIIRSGILSINLFTIPLAVAHNFSDQFVVETSITQSIKDGYTNLKSNVEETMSSLTDEKNTDAEKYLEQHDKDLKAYQNEIKKADQNYMKARKEAQRNYMKNHSQLPMEEDIEKDMEHIKSGL

Trx-His-Lic058-Trunc-SEQ ID NO: 52

His-tag LIC091-A-SEQ ID NO: 53

LIC091-SEQ ID NO: 54

Underline: LIC091-A

Lowercase: LIC091-B

Double underline: LIC091-C

Gray background: LIC091-D

Underlined: LIC091-E

Example 1

Host cell interaction, pathogenicity and immune response

The following proteins for host cell interaction, pathogenicity, and immune response have been searched for literature, domain analysis, protein interaction analysis, and additional bioinformatics analysis.

Literature search in PubMed. By using Pfam (Punta M et al. (2012); The Pfam protein families database; Nucleic Acids Res. Jan; 40 (Database issue), D290-301; PMID: 22127870) and InterProScan (Mulder N and Apweiler R. (2007) ); InterPro and InterProScan: tools for protein sequence classification and comparison. Methods Mol Biol. 396: 59-70; PMID: 18025686) Domain analysis. A STRING database of known and predicted protein interactions (Szklarczyk D et al. (2011); The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.Nucleic Acids Res.2011 Jan; 39 (Database issue): D561-8. Epub 2010 Nov 2; PMID: 21045058) in order to predict the function and function of the protein. Predictive signal peptides were used (SignalP-4.1 - see Petersen TN et al. (2011); SignalP 4.0: discriminating signal peptides from transmembrane regions; Nat Methods. Sep 29; 8(10): 785-6; PMID: 21959131), cross Membrane region (TmHmm-2.0 - see Käll L et al. (2004); A combined transmembrane topology and signal peptide prediction method; J Mol Biol. May 14; 338 (5): 1027-36; PMID: 15110065), lipoprotein ( LipoP-1.0 - see Juncker AS et al. (2003); Prediction of lipoprotein signal peptides in Gram-negative bacteria; Protein Sci. Aug; 12(8): 1652-62; PMID: 12876315), 2D structure (NetSurfP - see Petersen BI et al; A generic method for assignment of reliability scores applied to solvent accessibility predictions.; BMC Struct Biol.2009 Jul 31;9:51.doi:10.1186/1472-6807-9-51;PMID:19646261), non-classic Secreted proteins (Secretome P-2.0 - see Bendtsen JD et al. (2005); Non-classical protein secretion in bacteria; BMC Microbiol. Oct 7; 5:58; PMID: 16212653) to expand knowledge of proteins.

The results of domain analysis showed that 6 were autotransporter proteins, one was a metal binding domain, and one serine-rich protein had a functionally ambiguous domain (common bacterial SprA-associated domain) and one unidentified domain. . Additional analysis results for these proteins are provided below.

Autotransporter (LIC037, LI0890, LIC058, LIB022, LIC041, LIC047, LIC091): The autotransporter is a C-terminal beta-tubular domain containing an N-terminal extracellular ("passenger") domain and anchoring the protein to the outer membrane. Bacterial virulence factors (see Figures 1 and 2) (Nishimura K et al. (2010); Autotransporter passenger proteins: virulence factors with comrnon structural themes; J Mol Med (Berl). May; 88(5): 451-8 ;PMID: 20217035). The passenger domain of the autotransporter protein is exported to the bacterial surface where it is cleavable and appears in the supernatant, as in the case of EatA (Patel SK et al. (2004); Identification and molecular characterization of EatA, an autotransporter Protein of enterotoxigenic Escherichia coli; Infect Immun 72: 1786-1794; PMID: 14977988), or to maintain most of the association with bacterial cell surfaces, as described for antigen 43 (van der Woude MW and Henderson IR (2008); Regulation and function of Ag43(flu); Annu Rev Microbiol 62: 153-169; PMID: 18785838). Since the passenger domain of the autotransporter protein is exported to the bacterial surface, it often triggers the host's immune response during infection (see Al-Hasani K et al. (2009); The immunogenic SigA enterotoxin of Shigella flexneri 2a binds to HEp- 2 cells and induces fodrin redistribution in intoxicated epithelial cells. PLoS One 4:e8223;PMID:20011051,Turner DP et al. (2006);Characterization of MspA,an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis;Infect Immun 74: 2957-2964; PMID: 16622234, and Litwin CM et al. (2007); Characterization of an immunogenic outer membrane autotransporter protein, Arp, of Bartonella henselae; Infect Immun 75: 5255-5263; PMID: 17785470). In addition, the latest immunoproteomic studies have indicated the identification of autotransporters during experimental infection in mice (Roy K et al. (2010); Enterotoxigenic Escherichia coli elicits immune responses to multiple surface proteins; Infect Immun 78: 3027-3035; PMID :20457787).

Autotransporter proteins are widely distributed in Gram-negative bacteria, several of which have been associated with virulence-related functions, including adhesins such as AIDA-I (E. coli) and whooping cough Bacillus adhesin (Bordetella pertussis) (see Benz, I. and MASchmidt. (1992); Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia Coli strain 2787 (O126: H27); Infect. Immun. 60: 13-18; PMID: 1729177 and Leininger, E. et al., (1991). Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes Adherence of mammalian cells; Proc. Natl. Acad. Sci.; 88: 345-349; PMID: 1988935) and proteolytic enzymes (see Dutta, PR et al. (2002); Functional comparison of serine protease Autotransporters of Enterobacteriaceae; Infect. Immun. 70: 7105-7113; PMID: 12438392). Several autotransporters have been identified as immunodominant antigens, perhaps most notably B. pertussis adhesin, which forms a component of the pertussis vaccine (EP0527753A).

Additional bioinformatics analysis demonstrated that the six protein-based domain autotransporters were annotated by the identified beta cartridge, alpha helix linker, and passenger domain. Figure 3A illustrates the manner in which the autotransporters LIC037, LI0890, LIC058, LIB022, LIC041, LIC047 are embedded in the outer membrane, while Figure 3B illustrates the same for LIC091. Note that it is considered that LIC091 makes the hairpin-like structure extended beyond the β-tube into the extracellular space (Nishimura K et al; Autotransporter passenger proteins:virulence factors with common structural themes.; J Mol Med(Berl).2010 May;88( 5): 451-8.doi: 10.1007/s00109-010-0600-y.Epub 2010 Mar 9; PMID: 20217035). This uncertainty is due to the identification of an additional putative alpha helix linker in the N-terminus.

The STRING database does not contain any association proteins with high confidence in the seven autotransporter proteins.

Unknown protein (LI0668):

In the STRING database, the unidentified domain protein (LI0668) has a high confidence interaction with the autotransporter protein (LIC060). This indicates that the protein can be associated to the outer membrane. This protein is also predicted to have a signal peptide and is a non-classical secreted protein.

Unknown protein rich in serine (LI1077):

The serine-rich protein (LI1077) does not have an associated protein in the STRING database and is predicted to be a non-classical secreted protein.

Metal binding protein (LI0996):

Metal-binding proteins with zink strip domains are predicted to be non-classical secreted proteins.

Additional bioinformatics analysis to ensure successful performance of vaccine candidates

Additional bioinformatics analysis is performed, including truncation of the protein sequence to ensure folding and performance of the native recombinant protein in the protective/toxic region. Signal peptide, transmembrane region and beta tube are not included in the protein In the truncated part. The alpha helix linker region serves as a natural truncation site.

LIC037, LI0890, LIC058, LIB022, LIC041, LIC047 (self-transporter)

Only the passenger domain should be represented. The N-terminal transmembrane region, the C-terminal beta tube, and the C-terminal alpha linker region are not included.

LIC091 (self-transporter)

This large protein consists of 8746 amino acids and has a C-terminal alpha helix linker sequence of about 6000 amino acids. Likewise, the protein has an additional putative alpha helix linker of about 600 amino acids at the N-terminus that allows for the formation of hairpin-like structures. Considering the two structural possibilities of this autotransporter, two truncated parts have been produced.

Hairpin passenger structural structure (1500-2500 amino acids):

It is assumed that the hairpin passenger domain is represented together with a small portion of the two alpha-helical connectors for the purpose of stabilizing the hairpin passenger domain structure. Does not include the signal peptide, beta tube and extremely long C-terminal alpha-helical linker.

Non-hairpin passenger structure (40-2068 amino acids):

The observed N-terminal alpha helix linker is now considered to be part of the passenger domain. Only the passenger domain should be represented, and thus the transmembrane region, the beta cartridge, and the very long C-terminal alpha linker region have been removed.

LI0668 (associated to outer membrane protein)

Does not include the signal peptide.

LI1077 (protein rich in serine)

Full-length performance is recommended because the protein lacks the area to be excluded.

LI0996 (metal binding protein)

Full-length performance is recommended because the protein lacks the area to be excluded.

Example 2

Identification of immunogenic truncated parts

Based on six antigenic fragments truncated from three intracellular L. serovar vaccine candidate proteins It is predicted to trigger a multiplicity of protective and immunogenic antibody responses to be ranked. Specific feature predictions such as epitope density and structural/functional prediction are used herein to rank these fragments. The six fragments were additionally identified in Table 2A below. Five fragments of LIC091 were identified in a similar manner for the following predictions:

1) Prediction of B cell epitope density (hot spot epitopes and general epitopes).

2) Prediction of T cell CD4+ epitope density for 47 available SLA dual genes.

3) Prediction of T cell CD8+ epitope density for 40 available SLA dual genes.

B cell epitope density

Prediction of B cell epitopes for four vaccine candidate proteins was performed using Evaxion's BepiFinder computer predictor. General B cell epitopes and hot B cell epitopes are predicted. The hot-spot epitope region is distinguished from the general epitope by its high-density β-turn, so the probability of antibody binding is extremely high. BepiFinder uses the algorithm to process the research subroutine BepiPred (see: Larsen JE, Lund O, Nielsen M.; Improved method for predicting linear B-cell epitopes; Immunome Res. 2006 Apr 24; 2:2, PMID: 16635264), NetSurfP (See: Petersen B1, Petersen TN, Andersen P, Nielsen M, Lundegaard C.; A generic method for assignment of reliability scores applied to solvent accessibility predictions.; BMC Struct Biol. 2009 Jul 31; 9:51.doi:10.1186/ 1472-6807-9-51; PMID: 19646261) and NetTurnP (see: Petersen B, Lundegaard C, Petersen TN1 (2010); NetTurnp-Neural Network Prediction of Beta-turns by Use of Evolutionary Information and Predicted Protein Sequence Features; PLoS ONE 5 (11): e15079 doi: 10.1371/journal.pone.0015079; PMID: 21152409) output. The B cell epitope determinants of the four proteins are shown in Figures 4-9; Figures 4-6 show the hot cell B cell epitope regions of the first three proteins, while Figures 7-9 The B cell epitopes of the same three proteins are shown. Figures 18 and 19 show the hot B cell epitopes and the general B cell epitopes of the large truncated portion of LIC091 (amino acids 1-4000).

T cell CD4+ and CD8+ epitope density

T cell epitope predictions for four vaccine candidate proteins were performed using Evaxion's TepiFinder computer predictor. TepiFinder processes the research subroutine netMHCIIpan by using an algorithm (see: Karosiene E, Rasmussen M, Blicher T, Lund O, Buus S, and Nielsen M (2013); NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including All three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ; Immunogenetics. 2013 Oct; 65(10): 711-24.doi:10.1007/s00251-013-0720-y.Epub 2013 Jul 31 ;PMID: 23900783) and netMHCcons (see Karosiene E, Lundegaard C, Lund O, Nielsen M.; NetMHCcons: a consensus method for the major histocompatibility complex class I predictions.; Immunogenetics. 2012 Mar; 64(3): 177-86 .doi: 10.1007/s00251-011-0579-8.Epub 2011 Oct 20; PMID: 22009319) Output. TepiFinder has predicted the epitopes of each of the SLA dual genes listed in Table 1 below (including 47 porcine MHC class II miRNAs and 40 porcine MHC class I miRNAs):

The T cell CD4+ and CD8+ epitope density maps for the first three proteins are shown in Figures 10-15, and the truncated portion of LIC091 (amino acid 1-2550, which corresponds to the discovery of hot spot B cell epitopes) The same information for one of the 4000 amino acid truncated portions is shown in Figures 20 and 21.

in conclusion

Antigen fragments are ranked in Table 2 below based on their likelihood of eliciting a protective and immunogenic antibody response. The ranking is based on the highest calculated score, which includes a primary focus on B cell epitope density (protective antibody binding) and a secondary focus on T cell CD4+ epitope density (T helper cell immunogenicity). All fragments are truncated by the autotransporter passenger acceptor region; thus the B cell epitope region is predicted to be a possible acceptor region. The T cell CD8+ epitope density listed in the table below is not included in the ranking due to concentration in the antibody reaction.

Table 2A - Sorting of fragments: "Priority" is directly related to "score". The score was calculated by adding the three normalized values of "number of B cell epitope hotspots", "number of B cell epitopes", and "number of T cell CD4 + epitopes". [ Normalization = (amino acid count - average of amino acid count) / standard deviation of amino acid count].

Table 2 - Sorting of fragments: "Priority" is directly related to "score". The score was calculated by adding the three normalized values of "number of B cell epitope hotspots", "number of B cell epitopes", and "number of T cell CD4 + epitopes". [ Normalization = (amino acid count - average of amino acid count) / standard deviation of amino acid count].

Example 3

Recombination produces the multi-peptide of the present invention

A. Protein purification protocol for TRX-HIS-LIC058-trunc protein:

Performance of TRX-HIS-LIC058-Trunc

The gene encoding the synthetic polypeptide TRX-HIS-LIC058 (SEQ ID NO: 52) (SEQ ID NO: 56) was synthesized and cloned into pET32a using KpnI and XhoI restriction sites (available from Novagen, catalog number 69015- 3) Medium. The recombinant plasmid was transformed into the host strain BL21 (DE3). The transformants were grown in TB broth medium with appropriate antibiotics to an OD600 value of 0.6. IPTG was added to the culture to a final concentration of 0.1 mM to induce performance. The induction was carried out for 3 hours at 30 ° C and 230 rpm.

Purification of LIC058-Trunc

15 g of bacteria (wet weight) were resuspended in 200 mL of binding buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) on ice and subjected to sonication. The resulting lysate was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was immediately placed on ice. The sample was applied to a Ni ²⁺ chelated agarose flow (GE Healthcare) column (25/100) with at least 2 column volumes of wash buffer (20 mM Tris-HCl, 300 mM NaCl, 60 mM imidazole, pH 8.0). washing. The column was subsequently eluted with 30 mL of elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0) while monitoring the OD at 280 nm.

The resulting eluted fraction was then applied to a HiLoadTM 26/600 SuperdexTM 200 pg column (GE Healthcare) equilibrated in equilibration buffer (1 x PBS, pH 7.4) at a flow rate of 3.5 mL/min. The resulting fraction is then concentrated and lyophilized. Figure 16 shows purified proteins in an SDS-PAGE gel.

B. Protein Purification Protocol for LIC091-A Protein:

Performance of LIC091-A

The gene encoding the His-tagged LIC091-A protein (SEQ ID NO: 53) (SEQ ID NO: 57) was synthesized and cloned into pET-32a using NdeI and XhoI restriction sites. The recombinant plasmid was transformed into the host strain BL21 (DE3). The transformants were grown in TB broth medium with appropriate antibiotics to an OD600 value of 0.6. IPTG was added to the culture to a final concentration of 0.1 mM to induce performance. The inducing conditions were maintained at 30 ° C and 230 rpm for 3 hours.

Purification of LIC091-A

10 g of bacteria (wet weight) were resuspended in 200 mL of extraction buffer (20 mM Tris-HCl, 300 mM NaCl, 1% Triton X-100, pH 8.0) on ice and subjected to sonication, and the mixture was centrifuged at 10,000 rpm. minute. The insoluble centrifugation block was dissolved in 200 mL of denaturing buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 8 M urea, 20 mM b-mercaptoethanol, pH 8.0) and stirred overnight at 4 ° C, and then centrifuged at 10,000 rpm. 30 minutes. The supernatant was applied to a Ni ²⁺ chelated agarose flow (GE Healthcare) column (35/80) and relative to binding buffer (20 mM Tris-HCl, 7 M guanidine hydrochloride, 20 mM imidazole, 20 mM b-mercaptoethanol) , pH 8.0) Dialysis was performed in the downstream, and the dialysis buffer was replaced twice. The dialysis is applied to a Ni ²⁺ chelated agarose flow (GE Healthcare) column (35/80) with at least 2 column volumes of wash buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, Wash with 8 M urea, pH 8.0). The column was subsequently eluted with 50 mL of elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, 8 M urea, pH 8.0) while monitoring the OD at 280 nm. Additional b-mercaptoethanol was then added to the elution fraction to bring the reducing agent concentration to 20 mM.

LIC091-A was then applied to a HiLoadTM 26/600 SuperdexTM equilibrated in equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 8 M urea, 20 mM b-mercaptoethanol, pH 8.0) at a flow rate of 3.0 mL/min. 75 pg column (GE Healthcare). The LIC091-A was then dialyzed against 30% acetonitrile containing 0.1% TFA, with the dialysis buffer replaced twice. The dialyzed LIC091-A was then lyophilized. An image of an SDS-PAGE gel with purified protein is shown in Figure 17.

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Claims (16)

A multi-peptide comprising: a) an amino acid sequence selected from the group consisting of any of SEQ ID NOS: 1-17 and 54, or b) SEQ ID NOS: 1-17 and 54 An amino acid sequence consisting of at least 5 contiguous amino acid residues of either, or c) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of a), d) and b) An amino acid sequence having at least 60% sequence identity, or e) an amino acid assembly derived from any one of SEQ ID NOS: 1-17 and 54 having The multi-peptide is antigenic in mammals, in contrast to the 3D conformation that derivatizes the assembly to form a B cell epitope. An isolated nucleic acid fragment comprising: i) a nucleotide sequence encoding a multi-peptide of any of claims 1 or ii) by any one of SEQ ID NOs: 18-51 and 55 a nucleotide sequence comprising a partial encoding of the amino acid, iii) a nucleotide sequence consisting of at least 10 contiguous nucleotides of the amino acid encoding portion of any one of SEQ ID NOs: 18-51 and 55, Iv) a nucleotide sequence having a sequence identity of at least 60% with the nucleotide sequence of i) or ii), v) a nucleoside having at least 60% sequence identity with the nucleotide sequence of iii) The acid sequence, vi) a nucleotide sequence complementary to the nucleotide sequence in i)-v), or vii) a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence in i)-vi). A vector comprising a nucleic acid as in claim 2, such as a selection vector or expression vector. A cell transformed to carry a vector as in claim 3 of the patent application. A pharmaceutical composition comprising a multi-peptide of claim 1 of the patent application, a nucleic acid fragment of claim 2, a carrier of claim 3, or a cell of claim 4 And a pharmaceutically acceptable carrier, vehicle or diluent. A method for inducing immunity of an animal by administering at least one immunogenic effective amount of a multi-peptide as in claim 1 of the patent application, such as a nucleic acid fragment of claim 2, as claimed The carrier of item 3, such as the cell of claim 4, or the pharmaceutical composition of claim 5, is for inducing adaptive immunity of the animal against L. intracellularis. A multi-strain antibody, wherein the antibody specifically binds to a multi-peptide of the first aspect of the patent application, and which is substantially non-specifically bound to other antibodies of the intracellular L. sonnei peptide. An isolated monoclonal antibody or antibody analog that specifically binds to a multi-peptide as in claim 1 of the scope of the patent application. An isolated monoclonal antibody or antibody analog according to claim 8 which is a multi-domain antibody selected from, for example, a murine antibody, a chimeric antibody such as a humanized antibody, a fully human antibody, and a llama or camel A monoclonal antibody of a single domain antibody, or an antibody analog thereof selected from antibody fragments such as Fab, F(ab') ₂ and scFV. A method for preventing, treating or ameliorating an infection of L. intracellularis comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition as claimed in claim 5 or as claimed in claim 7 The antibody of any one of item 9 to claim 9. A method for quantitatively or qualitatively determining the presence of L. intracellularis in a sample, the method comprising contacting and detecting the sample with an antibody according to any one of claims 7 to 9 of the patent application The presence of antibodies to the substance in the sample. A method for quantitative or qualitative determination of specificity of L. intracellularis in a sample A method of the presence of an antibody, the method comprising contacting the sample with a multi-peptide of claim 1 and detecting the presence of the antibody. A method for quantitatively or qualitatively determining the presence of a nucleic acid specific to L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment of claim 2 and detecting the sample with the The presence of a nucleic acid that hybridizes to the nucleic acid fragment. A method for the preparation of a multi-peptide of the first aspect of the patent application, which comprises the conditions of a nucleic acid fragment which contributes to the transformation of a cell as described in claim 4 of the patent application, such as the nucleic acid fragment of claim 2 The transduced cells are cultured, and then the multi-peptide is recovered, or the multi-peptide is prepared by solid phase or liquid phase peptide synthesis. A method for determining whether a substance such as an antibody is suitable for treating an intracellular L. solani infection, the method comprising contacting a multi-peptide of the first aspect of the patent application with the substance and subsequently determining whether the substance has At least one of the following features: 1) the ability to specifically bind to the multi-peptide, 2) the ability to compete with the multi-peptide to specifically bind to the ligand/receptor, and 3) specificity The ability to deactivate the multi-peptide. A method for determining whether a substance such as a nucleic acid is suitable for treating an intracellular L. sonnei infection, the method comprising contacting the substance with a nucleic acid fragment as in claim 2 and subsequently determining whether the substance has the following Either ability: 1) specifically binds to the nucleic acid fragment, or 2) specifically binds to a nucleic acid that specifically hybridizes to the nucleic acid fragment.