WO2014191465A1 - Peptides derived from lawsonia intracellularis and their use in vaccination - Google Patents

Peptides derived from lawsonia intracellularis and their use in vaccination Download PDF

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Publication number
WO2014191465A1
WO2014191465A1 PCT/EP2014/061078 EP2014061078W WO2014191465A1 WO 2014191465 A1 WO2014191465 A1 WO 2014191465A1 EP 2014061078 W EP2014061078 W EP 2014061078W WO 2014191465 A1 WO2014191465 A1 WO 2014191465A1
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Prior art keywords
antibody
polypeptide
amino acid
nucleic acid
intracellularis
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PCT/EP2014/061078
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French (fr)
Inventor
Niels Iversen MØLLER
Andreas Mattsson
Flemming Velin
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Møller Niels Iversen
Andreas Mattsson
Flemming Velin
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Application filed by Møller Niels Iversen, Andreas Mattsson, Flemming Velin filed Critical Møller Niels Iversen
Publication of WO2014191465A1 publication Critical patent/WO2014191465A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to the field of microbial peptides and vaccines.
  • the present invention relates to novel proteins and polynucleotides derived from Lawsonia intracellularis.
  • the invention further relates to vectors comprising the polynucleotides, transformed host organisms expressing the polynucleotides, antibodies (monoclonal or polyclonal) recognizing the polypeptides, as well as diagnostic, prophylactic and therapeutic uses and methods. Finally, also methods of preparation are part of the invention.
  • Proliferative enteropathy also called enteritis or ileitis
  • enteritis in many animals, in particular pigs, presents clinical signs and a pathological syndrome involving mucosal hyperplasia of immature crypt epithelial cells, primarily in the terminal ileum.
  • Other sites of the intestines that can be affected include the jejunum, caecum and colon.
  • Weanling and young adult pigs are principally affected, with typical clinical manifestations of rapid weight loss and dehydration.
  • Natural clinical disease in pigs occurs worldwide. The disease is consistently associated with the presence of an intracellular curved bacterium, presently known as Lawsonia intracellularis.
  • the first full-genome sequence of L. intracellularis was published in 2010 (strain PHE/MN1- 00); the bacterium's chromosome consists of 1457619 base pairs.
  • Vaccination is considered to be a very effective method of preventing infectious diseases in human and veterinary health care.
  • Vaccination is the administration of immunogenically effective amounts of antigenic material (the vaccine) to produce immunity to a
  • Vaccines have contributed to the eradication of smallpox, the near eradication of polio, and the control of a variety of diseases, including rubella, measles, mumps, chickenpox, typhoid fever.
  • vaccines were based on killed or live-attenuated microorganisms, or parts purified from them.
  • Subunit vaccines a more recent type of vaccine, contains one or more protective antigens.
  • An antigen is said to be protective if it is able to induce protection from subsequent challenge by a disease-causing infectious agent in an appropriate animal model following immunization.
  • the empirical approach to subunit vaccine development begins with pathogen cultivation, followed by identification and purification of critical components, and then testing of the antigens for protection. Apart from being time and labour consuming, this approach has several limitations that can lead to failure. It is difficult to develop vaccines using this approach for microorganisms which cannot easily be cultured, and it is generally only useful for antigens which can be obtained in sufficient quantities.
  • the empirical approach has a tendency to focus on the most abundant proteins, which in some cases are not immuno-protective. In other cases, the antigen expressed during in vivo infection is not expressed during in vitro cultivation.
  • antigen discovery involving a large number of proteins and an empirical approach in order to discover the protective antigens may be very time consuming. This renders it a very expensive approach, and can limit vaccine development for certain diseases caused by pathogens with large genomes.
  • L. intracellularis-der ' wed antigenic polypeptides that may serve as constituents in vaccines against L. intracellularis infections, and in diagnosis of L. intracellularis infections. It is also an object to provide nucleic acids, vectors, transformed cells, vaccine compositions, and other useful means for molecular cloning, as well as for therapy and diagnosis with relevance for L. intracellularis.
  • L. intracellularis expresses a number of hitherto unknown putatively surface-exposed proteins which are candidates as vaccine targets, and candidates as immunizing agents for the preparation of antibodies that target L. intracellularis.
  • the present invention relates to a polypeptide comprising :
  • the invention relates to an isolated nucleic acid fragment, which comprises:
  • nucleotide sequence encoding a polypeptide of the invention
  • nucleotide sequence consisting of any one of SEQ ID NOs: 18-51 and 55.
  • nucleotide sequence consisting of at least 10 consecutive nucleotides in any one of SEQ ID NOs: 18-51 and 55,
  • nucleotide sequence having a sequence identity of at least 60% with the nucleotide sequence in i) or ii),
  • the invention relates to a vector comprising the nucleic acid of the invention, such as a cloning vector or an expression vector.
  • the invention relates to a cell which is transformed so as to carry the vector of the invention.
  • the invention in a fifth aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide 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 acceptable carrier, vehicle or diluent.
  • the invention in a sixth aspect, relates to a method for inducing immunity in an animal by administering at least once an immunogenically-effective amount of a polypeptide of the invention, a nucleic acid fragment of the invention, a vector of the invention, a transformed cell of the invention, or a pharmaceutical composition of the invention, so as to induce adaptive immunity against L. intracellularis in the animal.
  • the invention relates to 1) a polyclonal antibody which specifically binds to at least one polypeptide of the invention, and to 2) an isolated monoclonal antibody or antibody analogue which binds specifically to a polypeptide of the invention.
  • the invention relates to a pharmaceutical composition comprising such a polyclonal or monoclonal antibody, and a pharmaceutically-acceptable carrier, vehicle or diluent.
  • the invention relates to a method for prophylaxis, treatment or amelioration of infection with L. intracellularis, comprising administering a therapeutically effective amount of an antibody of the 7 th or 8 th aspect of the invention or a pharmaceutical composition of the 9 th aspect to an animal in need thereof.
  • the invention relates to a method for determining, quantitatively or qualitatively, the presence of L. intracellularis in a sample, the method comprising contacting the sample with an antibody of the 8 th or 9 th aspects of the invention, and detecting the presence of antibody bound to material in the sample.
  • the invention provides a method for determining, quantitatively or qualitatively, the presence of antibodies specific for L. intracellularis in a sample, the method comprising contacting the sample with a polypeptide of the invention, and detecting the presence of antibody that specifically binds said polypeptide.
  • the invention in a 13 th aspect, relates to a method for determining, quantitatively or qualitatively, the presence of a nucleic acid characteristic of L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment of the invention, and detecting the presence of nucleic acid in the sample that hybridizes to said nucleic acid fragment.
  • the invention relates to a method for the preparation of the polypeptide of the invention, comprising:
  • the invention relates to a method for determining whether a substance, such as an antibody, is potentially useful for treating infection with L. intracellularis, the method comprising contacting the polypeptide of the invention with the substance, and subsequently establishing whether the substance has at least one of the following characteristics:
  • the invention relates to a method for determining whether a substance, such as a nucleic acid, is potentially useful for treating infection with L.
  • the method comprising contacting the substance with the nucleic acid fragment of claim of the invention and subsequently establishing whether the substance has either the ability to:
  • Fig.l shows a schematic model of an N-terminal passenger domain, alpha helical linker (central helix markd N) and C-terminal beta barrel domain anchored to the outer membrane.
  • Fig. 2 shows aschematic diagram showing the predicted features of Lawsonia Autotransporter Protein Antigen (LatA).
  • Fig. 3 illustrates the 6 selected autotransporter proteins of the present invention predicted to be differently embedded in the outer membrane.
  • Fig. 4 shows a density plot of hotspot B-cell epitopes in LIC037.
  • Fig. 5 shows a density plot of hotspot B-cell epitopes in LI0890.
  • Fig. 6 shows a density plot of hotspot B-cell epitopes in LIC058.
  • Fig. 7 shows a density plot of general B-cell epitopes in LIC037.
  • Fig. 8 shows a density plot of general B-cell epitopes in LI0890.
  • Fig. 9 shows a density plot of general B-cell epitopes in LIC058.
  • Fig. 10 shows a CD4+ epitope density plot for LIC037.
  • Fig. 11 shows a CD8+ epitope density plot for LIC037.
  • Fig. 12 shows a CD4+ epitope density plot for LI0890.
  • Fig. 13 shows a CD8+ epitope density plot for LI0890.
  • Fig. 14 shows a CD4+ epitope density plot for LIC058.
  • Fig. 15 shows a CD8+ epitope density plot for LIC058.
  • Fig. 16 shows a picture of a SDS-PAGE gel of purified TRX-HIS-LIC058-Trunc after purification on a Superdex 200 loaded column.
  • the M lane contains molecular weight markers. Lane R: reducing conditions. Lane N : non- reducing conditions.
  • Fig. 17 shows a picture of a SDS-PAGE gel of purified LIC091-A after purification on Superdex 75 loaded column.
  • the M lane contains molecular weight markers. Lane R: reducing conditions. Lane N : non- reducing conditions.
  • Fig. 18 shows a density plot of hotspot B-cell epitopes in amino acids 1-4000 of LIC091.
  • Fig. 19 shows a density plot of general B-cell epitopes in amino acids 1-4000 of LIC091.
  • Fig. 20 shows a CD4+ epitope density plot for amino acids 1-2550 in LIC091.
  • Fig. 21 shows a CD8+ epitope density plot for amino acids 1-2550 in LIC091.
  • polypeptide means both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e.
  • polypeptide in a protein can be glycosylated, and/or lipidated, and/or comprise prosthetic groups.
  • sequence means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally-occurring amino acid sequence or nucleic acid sequence, respectively.
  • amino acid sequence means the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins.
  • adjuvant has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is nevertheless 2) capable of enhancing the immune response against the immunogen. In other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen.
  • Vaccination with the immunogen may or may not give rise to an immune response against it, but the combined vaccination with it and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • An “assembly of amino acids” means two or more amino acids bound together by physical or chemical means.
  • the "3D conformation” is the three dimensional structure of a biomolecule, such as a protein.
  • the 3D conformation is also termed the "tertiary structure", and denotes the relative locations in space of the amino acid residues forming the polypeptide.
  • An immunogenic carrier is a molecule or moiety to which an immunogen or a hapten can be coupled in order to enhance or enable the elicitation of an immune response against the immunogen/hapten.
  • Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen or hapten (which is not sufficiently immunogenic in its own right).
  • the immunogenic carrier is capable of eliciting a strong T-helper lymphocyte response against the combined immunogen and immunogenic carrier, which in turn provides for improved responses by B-lymphocytes and cytotoxic T-lymphocytes.
  • T-helper lymphocyte response is an immune response elicited on the basis of a peptide which is able to bind to an MHC class II molecule (e.g. an HLA class II molecule) in an antigen-presenting cell.
  • MHC class II molecule e.g. an HLA class II molecule
  • immunogen is a substance of matter which is capable of inducing an adaptive immune response in a host whose immune system is confronted with the immunogen.
  • immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system (e.g. when bound by antibodies, or
  • an antigen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against it.
  • a "hapten” is a small molecule which can neither induce or elicit an immune response.
  • 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 to an antigen or immunogen which is specific for antigenic determinants of the antigen/immunogen.
  • adaptive immune responses are induction of antigen-specific antibody production, or antigen-specific induction/activation of T helper lymphocytes or cytotoxic lymphocytes.
  • a "protective, adaptive immune response” is an antigen-specific immune response induced in a subject as a reaction to exposure (artificial or natural) to an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen, or a pathology-related agent that includes the antigen.
  • prophylactic vaccination aims at establishing a protective adaptive immune response against one or more pathogens.
  • Stimulation of the immune system means that a substance or composition of matter exhibits an immunostimuiatory effect. A number of adjuvants and certain cytokines share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased “alertness" of the immune system, meaning that subsequent vaccination with the immunogen induces a more significant immune response.
  • Hybridization under "stringent conditions” is herein defined as hybridization performed under conditions by which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences.
  • Stringent conditions are target sequence-dependent, and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to a probe (homologous probing) . Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences, so that lower degrees of similarity are detected (heterologous probing). Specificity is typically a function of post-hybridization washes, with critical factors being the ionic strength and temperature of the wash solutions. Generally, highly stringent wash temperature conditions are selected to be about 5°C to about 2°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow
  • Tm temperature of the midpoint of transition
  • animal is in the present context intended to denote an animal species (preferably mammalian), and not just a single animal. The term also denotes a population of such an animal species.
  • antibody refers to a polypeptide or group of polypeptides composed of at least one antibody combining site.
  • An “antibody combining site” is the three- dimensional binding space having an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows binding of the antibody to the antigen.
  • Antibody includes, for example, porcine antibodies, vertebrate antibodies, human antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, univalent antibodies, , single domain antibodies, and Fab proteins.
  • Specific binding denotes binding between two substances which goes beyond random binding of the two. It also goes beyond simple association between substances that tend to aggregate because they share the same overall hydrophobicity or hydrophilicity. As such, specific binding usually involves a combination of electrostatic and other interactions between two conformationally complementary areas on the two substances, meaning that the substances can "recognize” each other in a complex mixture.
  • the term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted, and is used to introduce it into a cell, where it can be replicated and expressed.
  • the term further denotes certain biological vehicles useful for the same purpose, e.g. viral vectors and phage. Both of these infectious agents are capable of incorporating a heterelogous nucleic acid sequence.
  • expression vector refers to a vector containing a nucleic acid sequence(s) coding for at least part of a gene product(s), and is capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide. Specific embodiments of the invention
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention 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.
  • the number may, where applicable, be higher, such as at least 52, 53, 54, 55, 56, 57, 58,
  • the number of the contiguous amino acid residues is at least N-n, where N is the length of the sequence ID in question and n is any integer between 6 and N-l; that is, the at least 5 contiguous amino acids can be at least any number between 5 and the length of the reference sequence minus one, in increments of one.
  • the polypeptide of the invention also has a sequence identity with the amino acid sequence of a) defined above 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%.
  • polypeptide of the invention in some embodiments also has a sequence identity with the amino acid sequence of b) defined above of 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 polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 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, 40,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 26 ,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,
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 122, 123 and 124 in any on of SEQ ID NOs: 2-17 and 54, if the length of the at least 5 amino acid residues so permits If the length of the at least 5 amino acids are higher than 5, the N- terminal first residue will not be higher numbered than N-L+ l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 and 137 in any one of SEQ ID NOs: 3-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b).
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 138, 139, 140, 141, 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
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 256, 257, 258, 259, 260, 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, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 45
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 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
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637 and 638 in any one of SEQ ID NOs: 8-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 639, 640, 641, 642, 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, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 771, 772, 773, 774, 775, 776, 777
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 829, 830, 831, 832, 833, 834, 835 og 836 in any one of SEQ ID NOs: 11-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 955, and 956 ,in any one of SEQ ID NOs: 13-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 957, 958, 959, 960, 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, 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, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035,
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 1071 and 1072 in any one of SEQ ID NOs: 15-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will be higher numbered than N-L+1, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 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,
  • N- terminal first residue will not be higher numbered than N-L+ l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b) .
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1337,
  • the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1604,
  • polypeptide of the invention that is, any one of SEQ ID NOs. 1-17 and 54- is in certain embodiments, also fused or conjugated to an immunogenic carrier molecule.
  • immunogenic carrier molecule is typically a polypeptide that induces a T-helper lymphocyte response in a mammal.
  • immunogenic carrier molecules can be selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, or dipththeria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra.
  • the polypeptides of the invention may also be fused to other amino acid sequences that confer desired a functionality. For instance, the polypeptides may be fused to amino acid sequences that facilitate purification (e.g.
  • His tags a polyhistidinyl group
  • the fusion partner may be a (heterologous) signal peptide that facilitates export or other cellular transport mechanisms, and localization of the fusion protein in the intended cellular location.
  • the polypeptide of the invention detailed above is capable of inducing an adaptive immune response against the polypeptide in a mammal, in particular in a pig.
  • the adaptive immune response is a protective adaptive immune response against infection with L. intracellularis.
  • the polypeptide may, in these cases, induce a humeral and/or a cellular immune response.
  • SEQ ID NOs: 1-17 and 54 include antigenic determinants (epitopes) that are as such recognized by antibodies, or by T-cell receptors when bound to MHC molecules.
  • B-cell epitopes i .e. antibody-binding epitopes
  • L. intracellularis or L. intracellularis-der ' wed proteins disclosed herein are tested for binding to overlapping oligomeric peptides derived from any one of SEQ ID NO: 1-17 and 54. Thereby, the regions of the L. intracellularis polypeptide which are responsible for, or contribute to, binding to the antibodies can be identified .
  • mutated versions of the polypeptides of the invention e.g. versions where each single non-alanine residue in SEQ ID NOs. : 1-17 and 54 are point mutated to alanine. This method is useful in identifying complex non- conformational B-cell epitopes. This is the case when binding of the same antibody is altered by mutating amino acids in different areas of the full-length polypeptide.
  • polypeptides of particular interest are those that include, consist of, or include epitopes from, the 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 Examples for details.)
  • the nucleic acid fragment of the invention is preferably is a DNA fragment (such as SEQ ID NOs: 18-34), or an RNA fragment (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 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
  • fragments 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, at least 2000, at least 2500, at least 3000, at least 3500, and at least 4000 nucleotides from those of SEQ ID NOs: 18-51 and 55.
  • the nucleic acid fragment of the invention typically has a sequence identity with the nucleotide sequence defined for i) or ii) above, which is 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 nucleic acid fragment of the invention may also have a sequence identity with the nucleotide sequence defined for iii) above, which is 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%.
  • Vectors of the invention fall into several categories discussed infra.
  • One preferred vector of the invention comprises in operable linkage, and in the 5'-3' direction, an expression control region comprising an enhancer/promoter, useful for driving expression of the nucleic acid fragment defined for option i) above, optionally a signal peptide coding sequence, a nucleotide sequence defined for option i), and optionally a terminator.
  • an expression control region comprising an enhancer/promoter, useful for driving expression of the nucleic acid fragment defined for option i) above, optionally a signal peptide coding sequence, a nucleotide sequence defined for option i), and optionally a terminator.
  • an expression vector useful for effecting production in cells of the polypeptide of the invention. Since the polypeptides of the invention are bacterial in origin, recombinant production is conveniently effected in bacterial host cells.
  • the expression control region drives expression in prokaryotic cells, such as a bacterium, e.g. in Escherichia coli.
  • prokaryotic cells such as a bacterium, e.g. in Escherichia coli.
  • the expression control region should be adapted to this particular use. At any rate, certain vectors of the invention are capable of autonomous replication.
  • the vector of the invention may be one that is capable of being integrated into the genome of a host cell. This is particularly useful if the vector is used in the production of stably-transformed cells, where the progeny will also include the genetic information introduced via the vector.
  • vectors incapable of being integrated into the genome of a mammalian host cell are useful in e.g. DNA vaccination.
  • the vector of the invention is selected from the group consisting of, but not limited to, a virus, such as an attenuated virus (which may in itself be useful as a vaccine agent), a bacteriophage, a plasmid, a minichromosome, and a cosmid.
  • a virus such as an attenuated virus (which may in itself be useful as a vaccine agent)
  • a bacteriophage such as a bacteriophage, a plasmid, a minichromosome, and a cosmid.
  • Polypeptides of the invention may be encoded by a nucleic acid molecule comprised in a vector.
  • a nucleic acid sequence can be "heterologous,” which means that it is foreign to the cell into which the vector is being introduced. It can also include a sequence homologous to a sequence in the cell, but be located in a position within the host cell where it is ordinarily not found.
  • Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • a vector of the present invention may encode polypeptide sequences such as a tag or immunogenicity-enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof).
  • polypeptide sequences such as a tag or immunogenicity-enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof).
  • Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al, 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST)-soluble fusion proteins, for later purification and separation or cleavage.
  • GST glutathione S-transferase
  • Vectors of the invention may be used in a host cell to produce a polypeptide of the invention, that may subsequently be purified for administration to a subject, or the vector may be purified for direct administration to a subject for expression of the protein in the subject (as is the case when administering a nucleic acid vaccine).
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably-linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well, and are described infra.
  • a “promoter” is a control sequence.
  • the promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence.
  • a 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 one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural state.
  • 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 promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference).
  • promoter and/or enhancer that effectively direct(s) the expression of the DNA segment in the cell type or organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al, 2001, incorporated herein by reference).
  • the promoters employed may be constitutive, tissue-specific, or inducible, and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.
  • inducible elements which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus
  • examples of inducible elements include but are not limited to Immunoglobulin Heavy Chain (Banerji et al, 1983; Gilles et al, 1983; Grosschedl et al, 1985; Atchinson et al, 1986, 1987; toiler 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 Receptor (Luria et al, 1987; Winoto et al, 1989; Redondo et al ; 1990), HLA DQa and/or 0( ⁇ (Sullivan et al, 1987), ⁇ -Interferon (Goodbourn et al, 1986; Fujita et al, 1987; Goodbourn et al,
  • MHC Class II 5 Koch et al, 1989
  • MHC Class II HLA-DRa Sherman et al, 1989
  • ⁇ - Actin Kawamoto et al, 1988; Ng et al ; 1989
  • Muscle Creatine Kinase MK
  • Prealbumin Transthyretin
  • Troponin I Troponin I
  • PDGF Platelet-Derived Growth Factor
  • Duchenne Muscular Dystrophy Klamut et al, 1990
  • SV40 Bonerji et al, 1981 ; Moreau et al, 1981 ; Sleigh et al, 1985; Firak et al, 1986; Herr et al, 1986; Imbra et al,
  • 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 mammary tumor virus) - Glucocorticoids (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); ⁇ - Interferon - poly(rl)x/poly(rc) (Tavernier et al, 1983); Adenovirus 5 E2 - EIA (Imperiale et al, 1984); Collagenase -
  • dectin-1 and dectin-2 promoters are also contemplated as useful in the present invention. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base, EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins, or a heterologous protein of interest.
  • EPDB Eukaryotic Promoter Data Base
  • the particular promoter that is employed to control the expression of a peptide- or protein- encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of directing expression of the polynucleotide in a targeted cell, preferably a bacterial cell.
  • a porcine cell it is preferable to position the polynucleotide coding region adjacent to, and under the control of, a promoter that is capable of being expressed in a porcine cell.
  • a promoter might include either a bacterial, porcine or viral promoter.
  • 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 high level expression of a related polynucleotide to this invention.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well.
  • a desirable promoter for use with the vector is one that is not down- regulated by cytokines, or one that is strong enough that even if down-regulated, it produces an effective amount of the protein/polypeptide of the current invention in a subject to elicit an immune response.
  • cytokines Non-limiting examples of these are CMV IE and RSV LTR.
  • a promoter that is up-regulated in the presence of cytokines is employed.
  • the MHC I promoter increases expression in the presence of IFN- ⁇ .
  • Tissue-specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages.
  • mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters.
  • a specific initiation signal may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon, or analogous sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this, and providing 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 signals and initiation codons can be either natural or synthetic, and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated cap-dependent translation, and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian source (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • each open reading frame is accessible to ribosomes for efficient translation.
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). 3. Multiple Cloning Sites
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS, enabling exogenous sequences to be ligated to the vector.
  • Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. If relevant in the context of vectors of the present invention, vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al, 1997, incorporated herein by reference.) 5. Termination Signals
  • the vectors or constructs of the present invention will generally comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
  • the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable, and are translated more efficiently.
  • terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
  • Terminators contemplated for use in the invention include any known transcription terminator described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator, or viral termination sequences, such as the SV40 terminator.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • protein expression particularly eukaryotic protein expression (as is relevant in nucleic acid vaccination)
  • polyadenylation of the transcript may also increase the stability of the transcript, and/or may facilitate cytoplasmic transport.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed.
  • Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously-replicating sequence
  • cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector.
  • a marker When transcribed and translated, a marker confers an identifiable change to the cell, permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker. Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants.
  • markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions include screenable markers, such as GFP, for colorimetric analysis.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk), or chloramphenicol acetyltransferase (CAT), may be utilized.
  • tk herpes simplex virus thymidine kinase
  • CAT chloramphenicol acetyltransferase
  • One of skill in the art would also know how to employ immunologic markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a protein of the invention. Further examples of selectable and screenable markers are
  • Transformed cells of the invention are useful for producing the polypeptide of the invention, but also as simple "containers" of nucleic acids and vectors of the invention.
  • Certain transformed cells of the invention are capable of replicating the nucleic acid fragment defined for option i) of the second aspect of the invention.
  • Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment defined for option i).
  • the transformed cell is prokaryotic, such as a bacterium.
  • prokaryotic cells such as a bacterium
  • eukaryotic cells may be used.
  • Suitable prokaryotic cells are bacterial cells selected from the group consisting of Escherichia (such as E. coli), Bacillus (e.g. Bacillus subtilis) , Salmonella, and Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG).
  • Escherichia such as E. coli
  • Bacillus e.g. Bacillus subtilis
  • Salmonella e.g. M. bovis BCG
  • Mycobacterium preferably non-pathogenic, e.g. M. bovis BCG.
  • Eukaryotic cells can be in the form of yeasts (such as Saccharomyces cerevisiae) and protozoans.
  • the transformed eukaryotic cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell.
  • the transformed cell of the invention is stably transformed by having the nucleic acid defined above for option i) stably integrated into its genome.
  • the transformed cell secretes or carries on its surface the polypeptide of the invention, since this facilitates recovery of the polypeptides produced.
  • a particular version of this embodiment is one where the transformed cell is a bacterium, and secretion of the polypeptide of the invention is into the periplasmic space.
  • Suitable cells for recombinant nucleic acid expression of the nucleic acid fragments of the present invention are prokaryotic and eukaryotic.
  • prokaryotic cells include E. coli; members of the Staphylococcus genus, such as S. epidermidis; members of the
  • Lactobacillus genus such as L. plantarum
  • members of the Lactococcus genus such as L. lactis
  • members of the Bacillus genus such as B. subtilis
  • members of the Corynebacterium genus such as C. glutamicum
  • members of the Pseudomonas genus such as P.
  • eukaryotic cells include mammalian cells; insect cells; yeast cells, such as members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastoris), members of the Hansenula genus (e.g. H. polymorpha), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis), and members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastoris), members of the Hansenula genus (e.g. H. polymorpha), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis), and members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastor
  • Schizosaccharomyces genus e.g. S. pombe.
  • Techniques for recombinant gene production, introduction into a cell, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002; and
  • host cell refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector.
  • a host cell can be used as a recipient for vectors or viruses.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell; they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials
  • DSM Micrroorganismen und Zellkulturen
  • An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for the generation of multiple copies, or expression of encoded proteins.
  • Bacterial cells used for vector replication and/or expression include Staphylococcus strains, as well as a number of commercially available E. coli hosts, such as SURE ® Competent Cells and SoloPackTM Gold Cells (Stratagene ® ; La Jolla, CA).
  • E. coli LE392 could be used as host cells for bacteriophage.
  • Appropriate yeast cells can include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.
  • eukaryotic host cells for replication and/or expression of a vector examples include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells of various cell types and organisms are available, and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above-described host cells to maintain them, and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors, and their cognate polypeptides, proteins, or peptides.
  • Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely 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. Patents 5,871,986, 4,879,236, both herein incorporated by reference. These are commercially available, for example, under the names MaxBac ® 2.0 (Invitrogen ® ), and BacPAKTM (Clontech ® ).
  • expression systems include COMPLETE CONTROLTM Inducible Mammalian Expression System (Stratagene ® ), which involves a synthetic ecdysone-inducible receptor, or the pET Expression System (Stratagene ® ), an E. coli expression system.
  • An inducible expression system is the T-RExTM System ( Invitrogen ® ), which involves tetracycline- regulated expression.
  • Pichia methanolica Expression System is a yeast expression system designed for high-level production of recombinant proteins in the methylotrophic yeast, Pichia methanolica.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 2001). In certain aspects,
  • analysis is performed on whole cells, tissue homogenates, or biological fluid samples, without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA, or fractionated or whole-cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA (cDNA).
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty bases in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single- stranded form, although the single-stranded form is preferred.
  • Pairs of primers designed to selectively hybridize to nucleic acids corresponding to sequences of genes identified herein, are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency conditions to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template- dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label, or even via a system using electrical and/or thermal impulse signals (Bellus, 1994).
  • PCR polymerase chain reaction
  • Suitable methods for nucleic acid delivery to effect expression of compositions of the present invention include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue, or an organism, as described herein, or as would be known to one of ordinary skill in the art.
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 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), including microinjection (Harland and Weintraub, 1985; U.S.
  • Patent 5,789,215 incorporated herein by reference
  • electroporation U.S. Patent No. 5,384,253, incorporated herein by reference
  • calcium phosphate precipitation Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990
  • DEAE dextran followed by polyethylene glycol (Gopal, 1985)
  • direct sonic loading Fechheimer et al, 1987
  • liposome-mediated transfection Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et al, 1989; Kato et al,
  • organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.
  • Antibodies directed against the proteins of the invention are useful for affinity
  • Antibodies to the proteins of the invention may be prepared by conventional methods.
  • 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 polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti- rabbit and anti-goat antibodies.
  • Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly).
  • Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant.
  • Polyclonal antiserum is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25 C for one hour, followed by incubating at 4 C for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.
  • Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256 : 495-96], or a modification thereof.
  • a mouse or rat is immunized as described above.
  • the spleen (and optionally several large lymph nodes) is removed, and dissociated into single cells.
  • the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen.
  • B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension.
  • Resulting B-cells, or all dissociated spleen cells are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g. hypoxanthine, aminopterin, thymidine medium, or "HAT").
  • a selective medium e.g. hypoxanthine, aminopterin, thymidine medium, or "HAT”
  • the resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens).
  • the selected MAb-secreting hybridomas are then cultured either in vitro (e.g. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
  • the antibodies may be labelled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32 P and 125 I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3', 5,5'- tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB 3,3', 5,5'- tetramethylbenzidine
  • Specific binding partner refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor.
  • Other specific binding partners include biotin, avidin, streptavidin, IgG and protein A, and numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 125 I may serve as a radioactive label, or as an electron-dense reagent. HRP may serve as enzyme, or as antigen for a MAb. Further, one may combine various labels for desired effect.
  • MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with, 125 I, or with an anti-biotin MAb labeled with HRP.
  • the isolated monoclonal antibody or antibody analogue is preferably a monoclonal antibody selected from a multi-domain antibody such as a murine antibody, a chimeric antibody such as a humanized antibody, a fully human antibody, and single-domain antibody of a llama or a camel.
  • the antibody analogue may be selected from a fragment of an antibody such as a Fab, a F(ab') 2 , or a scFV; cf. also the definition of the term "antibody” presented above.
  • compositions of the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e, to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from Diphtheria, tetanus, cholera, or H. pylori..
  • the pharmaceutical compositions of the invention thus typically contain an immunological adjuvant, which can be an aluminium-based adjuvant, or one of any number of other adjuvants, as described in the following :
  • Preferred adjuvants to enhance effectiveness of the composition include, but are 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 muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
  • CFA Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.
  • M-CSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • muramyl peptides include, but are not limited to, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2"-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy-ethylamine (MTP-PE), etc.
  • the immunogenic compositions e.g.
  • the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier, and adjuvant typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above.
  • Immunogenic compositions used as vaccines comprise an immunologically-effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed.
  • immunologically-effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors.
  • the amount administered per immunization is typically in the range between 0.5 ⁇ g and 500 mg (often not higher than 5,000 ⁇ g), and very often in the range between 10 and 200 ⁇ g.
  • the immunogenic compositions are conventionally administered parenterally, e.g, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (e.g. see W0 98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intra-arterial routes may be applicable.
  • Dosage treatment may be a single dose schedule, or a multiple dose schedule.
  • the vaccine may also be administered in conjunction with other immunoregulatory agents.
  • DNA vaccination also termed nucleic acid vaccination, or gene vaccination
  • nucleic acid vaccination also termed nucleic acid vaccination, or gene vaccination
  • a further aspect of the invention is the recognition that combination vaccines can be provided, wherein 2 or more Lawsonia antigens disclosed herein are combined to enhance the immune response by the vaccinated animal, including to optimize initial immune response and duration of immunity.
  • multiple antigenic fragments derived from the same Lawsonia protein can also be used, such as the use a combination of different lengths of polypeptide sequence fragments from one protein.
  • a further aspect of the invention is the recognition that combination vaccines can be provided wherein 2 or more Lawsonia antigens disclosed herein (or nucleic acids encoding the same) are combined to enhance immune response by the vaccinated animal, including to optimize initial immune response and duration of immunity.
  • multiple antigens derived from the same antigenic Lawsonia protein can also be used, such as to use a combination of different lengths of polypeptide sequence fragments from one protein.
  • Representative examples of combination antigen formulations are those that include LIC037 or any of its fragments disclosed herein in combination with polypeptides and fragments thereof derived from at least 2 of the other polypeptides of the present invention..
  • Treatment methods of the invention generally relates to induction of immunity, and as such, also entails methods that relate to treatment, prophylaxis and amelioration of disease.
  • immunization methods entail that a polypeptide of the invention, or a composition comprising such a polypeptide, is administered, the animal (e.g. the pig) typically receives between 0.5 and 5,000 ⁇ g of the polypeptide of the invention per administration.
  • the immunization scheme includes that the animal (e.g. the pig) receives a priming administration, and one or more booster
  • Preferred embodiments of the 6 th aspect of the invention comprise that the administration is for the purpose of inducing protective immunity against L. intracellularis.
  • the protective immunity is effective in reducing the risk of infection with L. intracellularis, or is effective in treating or ameliorating infection with L. intracellularis.
  • the preferred vaccines of the invention induce humoral immunity.
  • the administration is for the purpose of inducing antibodies specific for L. intracellularis, and wherein said antibodies or B-lymphocytes producing said antibodies are subsequently recovered from the animal.
  • the method of the 6 th aspect may also be useful in antibody production.
  • the administration is for the purpose of inducing antibodies specific for L. intracellularis, and wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal, and used for preparation of monoclonal antibodies.
  • compositions as mentioned above can also comprise polypeptides, antibodies, or nucleic acids of the invention.
  • the pharmaceutical compositions will comprise a
  • therapeutically effective amount refers to an amount of a therapeutic agent used to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the effect can be detected by, for example, chemical markers or antigen levels.
  • Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.
  • the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. Reference is however made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above. However, the effective amount for a given situation can be determined by routine
  • an effective dose will be from about 0.01 mg/kg to 50 mg/kg, or 0.05 mg/kg to about 10 mg/kg of the DNA constructs, in the individual to which it is administered.
  • a pharmaceutical composition can also contain a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. 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 the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically-acceptable salts is available in Remington's
  • compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH-buffering substances, and the like, may be present in such vehicles.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection, may also be prepared. Liposomes are included within the definition of a
  • the invention also includes related embodiments to the treatment and prophylaxis disclosed herein.
  • the invention also includes embodiments wherein:
  • polypeptide of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L.
  • nucleic acid fragment of the invention or the vector of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L. intracellularis;
  • the transformed cell of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L.
  • the antibody, antibody fragment or antibody analogue of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L. intracellularis.
  • Embodiment 1 A polypeptide comprising
  • polypeptide being antigenic in a mammal.
  • Embodiment 2 The polypeptide according to embodiment 1, wherein the at least 5 contiguous 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.
  • Embodiment 3 The polypeptide according to embodiment 1, wherein the at least 5 contiguous 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 contig
  • polypeptide according to embodiment 1 or 2 wherein the sequence identity with 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%.
  • sequence identity with 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%.
  • polypeptide according to embodiment 1 or 2 wherein the sequence identity with 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%.
  • Embodiment 5 The polypeptide according to any one of the preceding
  • the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 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, 101, 102, 103, 104, 105, 106, 107
  • Embodiment 6 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 122, 123, and 124 in any on of SEQ ID NOs: 2-17 and 54.
  • Embodiment 7 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, and 137 in any one of SEQ ID NOs: 3-17 and 54.
  • Embodiment 8 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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,
  • Embodiment 9 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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
  • Embodiment 10 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 458, 459, 460, 461, 462,
  • Embodiment 11 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 477, 478, 479, 480, 481, 482, 483, 484,
  • Embodiment 12 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, and 638 in any one of SEQ ID NOs: 8-17 and 54.
  • Embodiment 13 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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
  • Embodiment 14 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 790, 791, 7
  • Embodiment 15 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 829, 830, 831, 832, 833, 834, 835, and 836 in any one of SEQ ID NOs: 11-17 and 54.
  • polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 9
  • Embodiment 17 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 955, and 956 in any one of SEQ ID NOs: 13-17 and 54.
  • Embodiment 18 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 1024, 1025, 1026, 1027, 1028
  • Embodiment 19 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 1071, and 1072 in any one of SEQ ID NOs: 15-17 and 54.
  • Embodiment 20 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 1073, 1074, 1075, 1076, 1077, 1078, 1079,
  • Embodiment 21 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 1337, 1338, 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, 140
  • Embodiment 22 The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 1604, 1605, 1606, 1607, 1608, 1609, 1610,

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

PEPTIDES DERIVED FROM LAWSONIA INTRACELLULARS AND THEIR USE IN VACCINATION FIELD OF THE INVENTION
The present invention relates to the field of microbial peptides and vaccines. In particular the present invention relates to novel proteins and polynucleotides derived from Lawsonia intracellularis. The invention further relates to vectors comprising the polynucleotides, transformed host organisms expressing the polynucleotides, antibodies (monoclonal or polyclonal) recognizing the polypeptides, as well as diagnostic, prophylactic and therapeutic uses and methods. Finally, also methods of preparation are part of the invention.
BACKGROUND OF THE INVENTION Proliferative enteropathy (also called enteritis or ileitis) in many animals, in particular pigs, presents clinical signs and a pathological syndrome involving mucosal hyperplasia of immature crypt epithelial cells, primarily in the terminal ileum. Other sites of the intestines that can be affected include the jejunum, caecum and colon. Weanling and young adult pigs are principally affected, with typical clinical manifestations of rapid weight loss and dehydration. Natural clinical disease in pigs occurs worldwide. The disease is consistently associated with the presence of an intracellular curved bacterium, presently known as Lawsonia intracellularis.
The first full-genome sequence of L. intracellularis was published in 2010 (strain PHE/MN1- 00); the bacterium's chromosome consists of 1457619 base pairs. Vaccination is considered to be a very effective method of preventing infectious diseases in human and veterinary health care. Vaccination is the administration of immunogenically effective amounts of antigenic material (the vaccine) to produce immunity to a
disease/disease-causing pathogenic agent. Vaccines have contributed to the eradication of smallpox, the near eradication of polio, and the control of a variety of diseases, including rubella, measles, mumps, chickenpox, typhoid fever.
Before the "genomic era", vaccines were based on killed or live-attenuated microorganisms, or parts purified from them. Subunit vaccines, a more recent type of vaccine, contains one or more protective antigens. In order to develop subunit vaccines, it is critical to identify the proteins which are important for inducing protection. An antigen is said to be protective if it is able to induce protection from subsequent challenge by a disease-causing infectious agent in an appropriate animal model following immunization.
The empirical approach to subunit vaccine development, which includes several steps, begins with pathogen cultivation, followed by identification and purification of critical components, and then testing of the antigens for protection. Apart from being time and labour consuming, this approach has several limitations that can lead to failure. It is difficult to develop vaccines using this approach for microorganisms which cannot easily be cultured, and it is generally only useful for antigens which can be obtained in sufficient quantities. The empirical approach has a tendency to focus on the most abundant proteins, which in some cases are not immuno-protective. In other cases, the antigen expressed during in vivo infection is not expressed during in vitro cultivation. Furthermore, antigen discovery involving a large number of proteins and an empirical approach in order to discover the protective antigens may be very time consuming. This renders it a very expensive approach, and can limit vaccine development for certain diseases caused by pathogens with large genomes. OBJECT OF THE INVENTION
It is an object of embodiments of the invention to provide L. intracellularis-der'wed antigenic polypeptides that may serve as constituents in vaccines against L. intracellularis infections, and in diagnosis of L. intracellularis infections. It is also an object to provide nucleic acids, vectors, transformed cells, vaccine compositions, and other useful means for molecular cloning, as well as for therapy and diagnosis with relevance for L. intracellularis.
SUMMARY OF THE INVENTION
It has been found by the present inventor(s) that L. intracellularis expresses a number of hitherto unknown putatively surface-exposed proteins which are candidates as vaccine targets, and candidates as immunizing agents for the preparation of antibodies that target L. intracellularis.
In a first aspect, the present invention relates to a polypeptide comprising :
a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1- 17 and 54, or
b) an amino acid sequence consisting of at least 5 contiguous amino acid residues from any one of SEQ ID NOs: 1-17 and 54, or
c) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of a), d) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of b), or
e) an assembly of amino acids derived from any one of SEQ ID NOs: 1-17 and 54 which has essentially the same 3D conformation as in the protein from whicht said assembly is derived so as to constitute a B-cell epitope, said polypeptide being antigenic in a mammal.
In a second aspect, the invention relates to an isolated nucleic acid fragment, which comprises:
i) a nucleotide sequence encoding a polypeptide of the invention, or
ii) a nucleotide sequence consisting of any one of SEQ ID NOs: 18-51 and 55.
iii) a nucleotide sequence consisting of at least 10 consecutive nucleotides in 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 in i) or ii),
v) a nucleotide sequence having a sequence identity of at least 60% with the nucleotide sequence in iii),
vi) a nucleotide sequence complementary to the nucleotide sequence in i)-v), or
vii) a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence in i)-vi).
In a third aspect, the invention relates to a vector comprising the nucleic acid of the invention, such as a cloning vector or an expression vector.
In fourth aspect, the invention relates to a cell which is transformed so as to carry the vector of the invention.
In a fifth aspect, the invention relates to a pharmaceutical composition comprising a polypeptide 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 acceptable carrier, vehicle or diluent.
In a sixth aspect, the invention relates to a method for inducing immunity in an animal by administering at least once an immunogenically-effective amount of a polypeptide of the invention, a nucleic acid fragment of the invention, a vector of the invention, a transformed cell of the invention, or a pharmaceutical composition of the invention, so as to induce adaptive immunity against L. intracellularis in the animal.
In a 7th and 8th aspect, the invention relates to 1) a polyclonal antibody which specifically binds to at least one polypeptide of the invention, and to 2) an isolated monoclonal antibody or antibody analogue which binds specifically to a polypeptide of the invention. In a related 9th aspect, the invention relates to a pharmaceutical composition comprising such a polyclonal or monoclonal antibody, and a pharmaceutically-acceptable carrier, vehicle or diluent. In a 10th aspect, the invention relates to a method for prophylaxis, treatment or amelioration of infection with L. intracellularis, comprising administering a therapeutically effective amount of an antibody of the 7th or 8th aspect of the invention or a pharmaceutical composition of the 9th aspect to an animal in need thereof.
In an 11th aspect, the invention relates to a method for determining, quantitatively or qualitatively, the presence of L. intracellularis in a sample, the method comprising contacting the sample with an antibody of the 8th or 9th aspects of the invention, and detecting the presence of antibody bound to material in the sample.
In a 12th aspect, the invention provides a method for determining, quantitatively or qualitatively, the presence of antibodies specific for L. intracellularis in a sample, the method comprising contacting the sample with a polypeptide of the invention, and detecting the presence of antibody that specifically binds said polypeptide.
In a 13th aspect, the invention relates to a method for determining, quantitatively or qualitatively, the presence of a nucleic acid characteristic of L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment of the invention, and detecting the presence of nucleic acid in the sample that hybridizes to said nucleic acid fragment.
In a 14th aspect, the invention relates to a method for the preparation of the polypeptide of the invention, comprising:
- culturing a transformed cell of the present invention which is capable of expressing the nucleic acid of the invention, under conditions that facilitate expression of the nucleic acid fragment of the invention which encodes a polypeptide of the invention, and subsequently recovering said polypeptide; or
- preparing said polypeptide by means of solid or liquid phase peptide synthesis.
In a 15th aspect, the invention relates to a method for determining whether a substance, such as an antibody, is potentially useful for treating infection with L. intracellularis, the method comprising contacting the polypeptide of the invention with the substance, and subsequently establishing whether the substance has at least one of the following characteristics:
1) the ability to bind specifically to said polypeptide, 2) the ability to compete with said polypeptide for specific binding to a ligand/receptor, or
3) the ability to specifically inactivate said polypeptide.
Finally, in a 16th aspect, the invention relates to a method for determining whether a substance, such as a nucleic acid, is potentially useful for treating infection with L.
intracellularis, the method comprising contacting the substance with the nucleic acid fragment of claim of the invention and subsequently establishing whether the substance has either the ability to:
1) bind specifically to the nucleic acid fragment, or
2) bind specifically to a nucleic acid that hybridizes with the nucleic acid fragment. LEGENDS TO THE FIGURES
Fig.l shows a schematic model of an N-terminal passenger domain, alpha helical linker (central helix markd N) and C-terminal beta barrel domain anchored to the outer membrane.
Fig. 2 shows aschematic diagram showing the predicted features of Lawsonia Autotransporter Protein Antigen (LatA). Fig. 3 illustrates the 6 selected autotransporter proteins of the present invention predicted to be differently embedded in the outer membrane.
Fig. 4 shows a density plot of hotspot B-cell epitopes in LIC037.
Fig. 5 shows a density plot of hotspot B-cell epitopes in LI0890.
Fig. 6 shows a density plot of hotspot B-cell epitopes in LIC058. Fig. 7 shows a density plot of general B-cell epitopes in LIC037.
Fig. 8 shows a density plot of general B-cell epitopes in LI0890.
Fig. 9 shows a density plot of general B-cell epitopes in LIC058.
Fig. 10 shows a CD4+ epitope density plot for LIC037.
Fig. 11 shows a CD8+ epitope density plot for LIC037. Fig. 12 shows a CD4+ epitope density plot for LI0890.
Fig. 13 shows a CD8+ epitope density plot for LI0890.
Fig. 14 shows a CD4+ epitope density plot for LIC058.
Fig. 15 shows a CD8+ epitope density plot for LIC058. Fig. 16 shows a picture of a SDS-PAGE gel of purified TRX-HIS-LIC058-Trunc after purification on a Superdex 200 loaded column.
The M lane contains molecular weight markers. Lane R: reducing conditions. Lane N : non- reducing conditions.
Fig. 17 shows a picture of a SDS-PAGE gel of purified LIC091-A after purification on Superdex 75 loaded column.
The M lane contains molecular weight markers. Lane R: reducing conditions. Lane N : non- reducing conditions.
Fig. 18 shows a density plot of hotspot B-cell epitopes in amino acids 1-4000 of LIC091. Fig. 19 shows a density plot of general B-cell epitopes in amino acids 1-4000 of LIC091. Fig. 20 shows a CD4+ epitope density plot for amino acids 1-2550 in LIC091. Fig. 21 shows a CD8+ epitope density plot for amino acids 1-2550 in LIC091.
DETAILED DISCLOSURE OF THE INVENTION
Definitions
The term "polypeptide" means both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e.
functional biomolecules, comprising at least one polypeptide. When proteins comprise at least two polypeptides, these may form complexes, and be covalently or non-covalently linked. The polypeptide (s) in a protein can be glycosylated, and/or lipidated, and/or comprise prosthetic groups. The term "subsequence" means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally-occurring amino acid sequence or nucleic acid sequence, respectively.
The term "amino acid sequence" means the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins.
The term "adjuvant" has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is nevertheless 2) capable of enhancing the immune response against the immunogen. In other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen.
Vaccination with the immunogen may or may not give rise to an immune response against it, but the combined vaccination with it and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
"Sequence identity" is in the context of the present invention determined by comparing 2 optimally aligned sequences of comparable length (e.g. DNA, RNA or amino acid) according to the following formula : (Nref - Ndif)- 100/Nref , wherein Nref is the number of residues in one of the 2 sequences, and Ndif is the number of residues which are non-identical in the two sequences when they are aligned over their entire lengths and in the same direction. For example, two sequences, 5'-ATTCGGAACC-3' and 5'- ATACGGGACC-3', will yield a sequence identity of 80% (Nref=10 and Ndif=2).
An "assembly of amino acids" means two or more amino acids bound together by physical or chemical means.
The "3D conformation" is the three dimensional structure of a biomolecule, such as a protein. In monomeric polypeptides/proteins, the 3D conformation is also termed the "tertiary structure", and denotes the relative locations in space of the amino acid residues forming the polypeptide.
"An immunogenic carrier" is a molecule or moiety to which an immunogen or a hapten can be coupled in order to enhance or enable the elicitation of an immune response against the immunogen/hapten. Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen or hapten (which is not sufficiently immunogenic in its own right). Typically, the immunogenic carrier is capable of eliciting a strong T-helper lymphocyte response against the combined immunogen and immunogenic carrier, which in turn provides for improved responses by B-lymphocytes and cytotoxic T-lymphocytes. More recently, these large carrier molecules have, to a certain extent, been replaced by so-called promiscuous T-helper epitopes- i.e. shorter peptides that are recognized by a large fraction of HLA haplotypes in a population- which elicit T-helper lymphocyte responses. A "T-helper lymphocyte response" is an immune response elicited on the basis of a peptide which is able to bind to an MHC class II molecule (e.g. an HLA class II molecule) in an antigen-presenting cell. T-helper lymphocytes are produced in an animal species as a consequence of T-cell receptor recognition of the complex between the peptide and the MHC Class II molecule present. An "immunogen" is a substance of matter which is capable of inducing an adaptive immune response in a host whose immune system is confronted with the immunogen. As such, immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system (e.g. when bound by antibodies, or
alternatively, when fragments of the antigens bound to MHC molecules are recognized by T- cell receptors), but which are not necessarily capable of inducing immunity. An antigen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against it.
A "hapten" is a small molecule which can neither induce or elicit an immune response.
However, when conjugated 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 to an antigen or immunogen which is specific for antigenic determinants of the antigen/immunogen. Examples of adaptive immune responses are induction of antigen-specific antibody production, or antigen-specific induction/activation of T helper lymphocytes or cytotoxic lymphocytes. A "protective, adaptive immune response" is an antigen-specific immune response induced in a subject as a reaction to exposure (artificial or natural) to an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen, or a pathology-related agent that includes the antigen. Typically, prophylactic vaccination aims at establishing a protective adaptive immune response against one or more pathogens. "Stimulation of the immune system" means that a substance or composition of matter exhibits an immunostimuiatory effect. A number of adjuvants and certain cytokines share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system, meaning that subsequent vaccination with the immunogen induces a more significant immune response.
Hybridization under "stringent conditions" is herein defined as hybridization performed under conditions by which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences. Stringent conditions are target sequence-dependent, and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to a probe (homologous probing) . Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences, so that lower degrees of similarity are detected (heterologous probing). Specificity is typically a function of post-hybridization washes, with critical factors being the ionic strength and temperature of the wash solutions. Generally, highly stringent wash temperature conditions are selected to be about 5°C to about 2°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow
temperature range, and represents disruption of the double helix into its complementary single strands. The process is described by the temperature of the midpoint of transition, Tm, which is also called the melting temperature. Formulas are available in the art for the determination of melting temperatures.
The term "animal" is in the present context intended to denote an animal species (preferably mammalian), and not just a single animal. The term also denotes a population of such an animal species.
As used herein, the term "antibody" refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An "antibody combining site" is the three- dimensional binding space having an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows binding of the antibody to the antigen. Antibody includes, for example, porcine antibodies, vertebrate antibodies, human antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, univalent antibodies, , single domain antibodies, and Fab proteins.
"Specific binding" denotes binding between two substances which goes beyond random binding of the two. It also goes beyond simple association between substances that tend to aggregate because they share the same overall hydrophobicity or hydrophilicity. As such, specific binding usually involves a combination of electrostatic and other interactions between two conformationally complementary areas on the two substances, meaning that the substances can "recognize" each other in a complex mixture. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted, and is used to introduce it into a cell, where it can be replicated and expressed. The term further denotes certain biological vehicles useful for the same purpose, e.g. viral vectors and phage. Both of these infectious agents are capable of incorporating a heterelogous nucleic acid sequence.
The term "expression vector" refers to a vector containing a nucleic acid sequence(s) coding for at least part of a gene product(s), and is capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide. Specific embodiments of the invention
The polypeptides of the invention
In some embodiments the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention 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. The number may, where applicable, 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 to phrase this is that for each of SEQ ID NOs: 1-17 and 54, the number of the contiguous amino acid residues is at least N-n, where N is the length of the sequence ID in question and n is any integer between 6 and N-l; that is, the at least 5 contiguous 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 polypeptide of the invention also has a sequence identity with the amino acid sequence of a) defined above 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%. Similarly, the polypeptide of the invention in some embodiments also has a sequence identity with the amino acid sequence of b) defined above of 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%.
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 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, 40,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 26 ,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, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 and 121 in any one of SEQ ID NOs: 1-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 122, 123 and 124 in any on of SEQ ID NOs: 2-17 and 54, if the length of the at least 5 amino acid residues so permits If the length of the at least 5 amino acids are higher than 5, the N- terminal first residue will not be higher numbered than N-L+ l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 and 137 in any one of SEQ ID NOs: 3-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence and L is the number of amino acids defined for option b). In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 138, 139, 140, 141, 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, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 and 255 in any one of SEQ ID NOs: 4-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 256, 257, 258, 259, 260, 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, 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, 381, 382, 383, 384, 385 and 386 in any one of SEQ ID NOs: 5-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, and 476 in any one of SEQ ID NOs: 6-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 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, 621, 622, 623, 624, and 625 in any one of SEQ ID NOs: 7-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637 and 638 in any one of SEQ ID NOs: 8-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 639, 640, 641, 642, 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, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720 and 721 in any one of SEQ ID NOs: 9-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b). In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 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, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827 and 828 in any one of SEQ ID NOs: 10-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 829, 830, 831, 832, 833, 834, 835 og 836 in any one of SEQ ID NOs: 11-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 881, 882, 883, 884, 885, 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, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, and 930 in any one of SEQ ID NOs: 12-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N- terminal first residue will not be higher numbered than N-L+ l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b). In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 955, and 956 ,in any one of SEQ ID NOs: 13-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+1, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 957, 958, 959, 960, 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, 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, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046 in SEQ ID NOs: 14-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+1, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b). In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 1071 and 1072 in any one of SEQ ID NOs: 15-17 and 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+1, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b).
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid 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, 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, 1183, 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, 1334
1335, and 1336 in SEQ ID NO: 16, 17, , or 54 , if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N- terminal first residue will not be higher numbered than N-L+ l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b) .
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1337,
1338, 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, 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, 1600, 1601, 1602 and 1603 in SEQ ID NO: 17 or 54, if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N- terminal first residue will not be higher numbered than N-L+ l, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b) .
In the embodiments defined by option b) above, the polypeptide of the invention is also one that has at least 5 contiguous amino acid residues defined for option b) above, and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 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, 2021, 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, 3021, 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, 3781, 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, 4021, 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, 4243, 4244, 4245, 4246, 4247, 4248, 4249, 4250
4251, 4252, 4253, 4254, 4255, 4256, 4257, 4258, 4259, 4260, 4261, 4262, 4263, 4264
4265, 4266, 4267, 4268, 4269, 4270, 4271, 4272, 4273, 4274, 4275, 4276, 4277, 4278
4279, 4280, 4281, 4282, 4283, 4284, 4285, 4286, 4287, 4288, 4289, 4290, 4291, 4292
4293, 4294, 4295, 4296, 4297, 4298, 4299, 4300, 4301, 4302, 4303, 4304, 4305, 4306
4307, 4308, 4309, 4310, 4311, 4312, 4313, 4314, 4315, 4316, 4317, 4318, 4319, 4320
4321, 4322, 4323, 4324, 4325, 4326, 4327, 4328, 4329, 4330, 4331, 4332, 4333, 4334
4335, 4336, 4337, 4338, 4339, 4340, 4341, 4342, 4343, 4344, 4345, 4346, 4347, 4348
4349, 4350, 4351, 4352, 4353, 4354, 4355, 4356, 4357, 4358, 4359, 4360, 4361, 4362
4363, 4364, 4365, 4366, 4367, 4368, 4369, 4370, 4371, 4372, 4373, 4374, 4375, 4376
4377, 4378, 4379, 4380, 4381, 4382, 4383, 4384, 4385, 4386, 4387, 4388, 4389, 4390
4391, 4392, 4393, 4394, 4395, 4396, 4397, 4398, 4399, 4400, 4401, 4402, 4403, 4404
4405, 4406, 4407, 4408, 4409, 4410, 4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418
4419, 4420, 4421, 4422, 4423, 4424, 4425, 4426, 4427, 4428, 4429, 4430, 4431, 4432
4433, 4434, 4435, 4436, 4437, 4438, 4439, 4440, 4441, 4442, 4443, 4444, 4445, 4446
4447, 4448, 4449, 4450, 4451, 4452, 4453, 4454, 4455, 4456, 4457, 4458, 4459, 4460
4461, 4462, 4463, 4464, 4465, 4466, 4467, 4468, 4469, 4470, 4471, 4472, 4473, 4474
4475, 4476, 4477, 4478, 4479, 4480, 4481, 4482, 4483, 4484, 4485, 4486, 4487, 4488
4489, 4490, 4491, 4492, 4493, 4494, 4495, 4496, 4497, 4498, 4499, 4500, 4501, 4502
4503, 4504, 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4516
4517, 4518, 4519, 4520, 4521, 4522, 4523, 4524, 4525, 4526, 4527, 4528, 4529, 4530
4531, 4532, 4533, 4534, 4535, 4536, 4537, 4538, 4539, 4540, 4541, 4542, 4543, 4544
4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558
4559, 4560, 4561, 4562, 4563, 4564, 4565, 4566, 4567, 4568, 4569, 4570, 4571, 4572
4573, 4574, 4575, 4576, 4577, 4578, 4579, 4580, 4581, 4582, 4583, 4584, 4585, 4586
4587, 4588, 4589, 4590, 4591, 4592, 4593, 4594, 4595, 4596, 4597, 4598, 4599, 4600
4601, 4602, 4603, 4604, 4605, 4606, 4607, 4608, 4609, 4610, 4611, 4612, 4613, 4614
4615, 4616, 4617, 4618, 4619, 4620, 4621, 4622, 4623, 4624, 4625, 4626, 4627, 4628
4629, 4630, 4631, 4632, 4633, 4634, 4635, 4636, 4637, 4638, 4639, 4640, 4641, 4642
4643, 4644, 4645, 4646, 4647, 4648, 4649, 4650, 4651, 4652, 4653, 4654, 4655, 4656
4657, 4658, 4659, 4660, 4661, 4662, 4663, 4664, 4665, 4666, 4667, 4668, 4669, 4670
4671, 4672, 4673, 4674, 4675, 4676, 4677, 4678, 4679, 4680, 4681, 4682, 4683, 4684
4685, 4686, 4687, 4688, 4689, 4690, 4691, 4692, 4693, 4694, 4695, 4696, 4697, 4698
4699, 4700, 4701, 4702, 4703, 4704, 4705, 4706, 4707, 4708, 4709, 4710, 4711, 4712
4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725, 4726
4727, 4728, 4729, 4730, 4731, 4732, 4733, 4734, 4735, 4736, 4737, 4738, 4739, 4740 4741, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750, 4751, 4752, 4753, 4754
4755, 4756, 4757, 4758, 4759, 4760, 4761, 4762, 4763, 4764, 4765, 4766, 4767, 4768
4769, 4770, 4771, 4772, 4773, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782
4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796
4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4808, 4809, 4810
4811, 4812, 4813, 4814, 4815, 4816, 4817, 4818, 4819, 4820, 4821, 4822, 4823, 4824
4825, 4826, 4827, 4828, 4829, 4830, 4831, 4832, 4833, 4834, 4835, 4836, 4837, 4838
4839, 4840, 4841, 4842, 4843, 4844, 4845, 4846, 4847, 4848, 4849, 4850, 4851, 4852
4853, 4854, 4855, 4856, 4857, 4858, 4859, 4860, 4861, 4862, 4863, 4864, 4865, 4866
4867, 4868, 4869, 4870, 4871, 4872, 4873, 4874, 4875, 4876, 4877, 4878, 4879, 4880
4881, 4882, 4883, 4884, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894
4895, 4896, 4897, 4898, 4899, 4900, 4901, 4902, 4903, 4904, 4905, 4906, 4907, 4908
4909, 4910, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918, 4919, 4920, 4921, 4922
4923, 4924, 4925, 4926, 4927, 4928, 4929, 4930, 4931, 4932, 4933, 4934, 4935, 4936
4937, 4938, 4939, 4940, 4941, 4942, 4943, 4944, 4945, 4946, 4947, 4948, 4949, 4950
4951, 4952, 4953, 4954, 4955, 4956, 4957, 4958, 4959, 4960, 4961, 4962, 4963, 4964
4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978
4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992
4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002, 5003, 5004, 5005, 5006
5007, 5008, 5009, 5010, 5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020
5021, 5022, 5023, 5024, 5025, 5026, 5027, 5028, 5029, 5030, 5031, 5032, 5033, 5034
5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048
5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062
5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073, 5074, 5075, 5076
5077, 5078, 5079, 5080, 5081, 5082, 5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090
5091, 5092, 5093, 5094, 5095, 5096, 5097, 5098, 5099, 5100, 5101, 5102, 5103, 5104
5105, 5106, 5107, 5108, 5109, 5110, 5111, 5112, 5113, 5114, 5115, 5116, 5117, 5118
5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132
5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141, 5142, 5143, 5144, 5145, 5146
5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160
5161, 5162, 5163, 5164, 5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174
5175, 5176, 5177, 5178, 5179, 5180, 5181, 5182, 5183, 5184, 5185, 5186, 5187, 5188
5189, 5190, 5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202
5203, 5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215, 5216
5217, 5218, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, 5227, 5228, 5229, 5230
5231, 5232, 5233, 5234, 5235, 5236, 5237, 5238, 5239, 5240, 5241, 5242, 5243, 5244
5245, 5246, 5247, 5248, 5249, 5250, 5251, 5252, 5253, 5254, 5255, 5256, 5257, 5258
5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266, 5267, 5268, 5269, 5270, 5271, 5272
5273, 5274, 5275, 5276, 5277, 5278, 5279, 5280, 5281, 5282, 5283, 5284, 5285, 5286 5287, 5288, 5289, 5290, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300
5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, 5312, 5313, 5314
5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328
5329, 5330, 5331, 5332, 5333, 5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342
5343, 5344, 5345, 5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356
5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370
5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384
5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5394, 5395, 5396, 5397, 5398
5399, 5400, 5401, 5402, 5403, 5404, 5405, 5406, 5407, 5408, 5409, 5410, 5411, 5412
5413, 5414, 5415, 5416, 5417, 5418, 5419, 5420, 5421, 5422, 5423, 5424, 5425, 5426
5427, 5428, 5429, 5430, 5431, 5432, 5433, 5434, 5435, 5436, 5437, 5438, 5439, 5440
5441, 5442, 5443, 5444, 5445, 5446, 5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454
5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464, 5465, 5466, 5467, 5468
5469, 5470, 5471, 5472, 5473, 5474, 5475, 5476, 5477, 5478, 5479, 5480, 5481, 5482
5483, 5484, 5485, 5486, 5487, 5488, 5489, 5490, 5491, 5492, 5493, 5494, 5495, 5496
5497, 5498, 5499, 5500, 5501, 5502, 5503, 5504, 5505, 5506, 5507, 5508, 5509, 5510
5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519, 5520, 5521, 5522, 5523, 5524
5525, 5526, 5527, 5528, 5529, 5530, 5531, 5532, 5533, 5534, 5535, 5536, 5537, 5538
5539, 5540, 5541, 5542, 5543, 5544, 5545, 5546, 5547, 5548, 5549, 5550, 5551, 5552
5553, 5554, 5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566
5567, 5568, 5569, 5570, 5571, 5572, 5573, 5574, 5575, 5576, 5577, 5578, 5579, 5580
5581, 5582, 5583, 5584, 5585, 5586, 5587, 5588, 5589, 5590, 5591, 5592, 5593, 5594
5595, 5596, 5597, 5598, 5599, 5600, 5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608
5609, 5610, 5611, 5612, 5613, 5614, 5615, 5616, 5617, 5618, 5619, 5620, 5621, 5622
5623, 5624, 5625, 5626, 5627, 5628, 5629, 5630, 5631, 5632, 5633, 5634, 5635, 5636
5637, 5638, 5639, 5640, 5641, 5642, 5643, 5644, 5645, 5646, 5647, 5648, 5649, 5650
5651, 5652, 5653, 5654, 5655, 5656, 5657, 5658, 5659, 5660, 5661, 5662, 5663, 5664
5665, 5666, 5667, 5668, 5669, 5670, 5671, 5672, 5673, 5674, 5675, 5676, 5677, 5678
5679, 5680, 5681, 5682, 5683, 5684, 5685, 5686, 5687, 5688, 5689, 5690, 5691, 5692
5693, 5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5704, 5705, 5706
5707, 5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5718, 5719, 5720
5721, 5722, 5723, 5724, 5725, 5726, 5727, 5728, 5729, 5730, 5731, 5732, 5733, 5734
5735, 5736, 5737, 5738, 5739, 5740, 5741, 5742, 5743, 5744, 5745, 5746, 5747, 5748
5749, 5750, 5751, 5752, 5753, 5754, 5755, 5756, 5757, 5758, 5759, 5760, 5761, 5762
5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770, 5771, 5772, 5773, 5774, 5775, 5776
5777, 5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790
5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5804
5805, 5806, 5807, 5808, 5809, 5810, 5811, 5812, 5813, 5814, 5815, 5816, 5817, 5818
5819, 5820, 5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5832 5833, 5834, 5835, 5836, 5837, 5838, 5839, 5840, 5841, 5842, 5843, 5844, 5845, 5846
5847, 5848, 5849, 5850, 5851, 5852, 5853, 5854, 5855, 5856, 5857, 5858, 5859, 5860
5861, 5862, 5863, 5864, 5865, 5866, 5867, 5868, 5869, 5870, 5871, 5872, 5873, 5874
5875, 5876, 5877, 5878, 5879, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888
5889, 5890, 5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5900, 5901, 5902
5903, 5904, 5905, 5906, 5907, 5908, 5909, 5910, 5911, 5912, 5913, 5914, 5915, 5916
5917, 5918, 5919, 5920, 5921, 5922, 5923, 5924, 5925, 5926, 5927, 5928, 5929, 5930
5931, 5932, 5933, 5934, 5935, 5936, 5937, 5938, 5939, 5940, 5941, 5942, 5943, 5944
5945, 5946, 5947, 5948, 5949, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5957, 5958
5959, 5960, 5961, 5962, 5963, 5964, 5965, 5966, 5967, 5968, 5969, 5970, 5971, 5972
5973, 5974, 5975, 5976, 5977, 5978, 5979, 5980, 5981, 5982, 5983, 5984, 5985, 5986
5987, 5988, 5989, 5990, 5991, 5992, 5993, 5994, 5995, 5996, 5997, 5998, 5999, 6000
6001, 6002, 6003, 6004, 6005, 6006, 6007, 6008, 6009, 6010, 6011, 6012, 6013, 6014
6015, 6016, 6017, 6018, 6019, 6020, 6021, 6022, 6023, 6024, 6025, 6026, 6027, 6028
6029, 6030, 6031, 6032, 6033, 6034, 6035, 6036, 6037, 6038, 6039, 6040, 6041, 6042
6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6051, 6052, 6053, 6054, 6055, 6056
6057, 6058, 6059, 6060, 6061, 6062, 6063, 6064, 6065, 6066, 6067, 6068, 6069, 6070
6071, 6072, 6073, 6074, 6075, 6076, 6077, 6078, 6079, 6080, 6081, 6082, 6083, 6084
6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095, 6096, 6097, 6098
6099, 6100, 6101, 6102, 6103, 6104, 6105, 6106, 6107, 6108, 6109, 6110, 6111, 6112
6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121, 6122, 6123, 6124, 6125, 6126
6127, 6128, 6129, 6130, 6131, 6132, 6133, 6134, 6135, 6136, 6137, 6138, 6139, 6140
6141, 6142, 6143, 6144, 6145, 6146, 6147, 6148, 6149, 6150, 6151, 6152, 6153, 6154
6155, 6156, 6157, 6158, 6159, 6160, 6161, 6162, 6163, 6164, 6165, 6166, 6167, 6168
6169, 6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182
6183, 6184, 6185, 6186, 6187, 6188, 6189, 6190, 6191, 6192, 6193, 6194, 6195, 6196
6197, 6198, 6199, 6200, 6201, 6202, 6203, 6204, 6205, 6206, 6207, 6208, 6209, 6210
6211, 6212, 6213, 6214, 6215, 6216, 6217, 6218, 6219, 6220, 6221, 6222, 6223, 6224
6225, 6226, 6227, 6228, 6229, 6230, 6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238
6239, 6240, 6241, 6242, 6243, 6244, 6245, 6246, 6247, 6248, 6249, 6250, 6251, 6252
6253, 6254, 6255, 6256, 6257, 6258, 6259, 6260, 6261, 6262, 6263, 6264, 6265, 6266
6267, 6268, 6269, 6270, 6271, 6272, 6273, 6274, 6275, 6276, 6277, 6278, 6279, 6280
6281, 6282, 6283, 6284, 6285, 6286, 6287, 6288, 6289, 6290, 6291, 6292, 6293, 6294
6295, 6296, 6297, 6298, 6299, 6300, 6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308
6309, 6310, 6311, 6312, 6313, 6314, 6315, 6316, 6317, 6318, 6319, 6320, 6321, 6322
6323, 6324, 6325, 6326, 6327, 6328, 6329, 6330, 6331, 6332, 6333, 6334, 6335, 6336
6337, 6338, 6339, 6340, 6341, 6342, 6343, 6344, 6345, 6346, 6347, 6348, 6349, 6350
6351, 6352, 6353, 6354, 6355, 6356, 6357, 6358, 6359, 6360, 6361, 6362, 6363, 6364
6365, 6366, 6367, 6368, 6369, 6370, 6371, 6372, 6373, 6374, 6375, 6376, 6377, 6378 6379, 6380, 6381, 6382, 6383, 6384, 6385, 6386, 6387, 6388, 6389, 6390, 6391, 6392
6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6404, 6405, 6406
6407, 6408, 6409, 6410, 6411, 6412, 6413, 6414, 6415, 6416, 6417, 6418, 6419, 6420
6421, 6422, 6423, 6424, 6425, 6426, 6427, 6428, 6429, 6430, 6431, 6432, 6433, 6434
6435, 6436, 6437, 6438, 6439, 6440, 6441, 6442, 6443, 6444, 6445, 6446, 6447, 6448
6449, 6450, 6451, 6452, 6453, 6454, 6455, 6456, 6457, 6458, 6459, 6460, 6461, 6462
6463, 6464, 6465, 6466, 6467, 6468, 6469, 6470, 6471, 6472, 6473, 6474, 6475, 6476
6477, 6478, 6479, 6480, 6481, 6482, 6483, 6484, 6485, 6486, 6487, 6488, 6489, 6490
6491, 6492, 6493, 6494, 6495, 6496, 6497, 6498, 6499, 6500, 6501, 6502, 6503, 6504
6505, 6506, 6507, 6508, 6509, 6510, 6511, 6512, 6513, 6514, 6515, 6516, 6517, 6518
6519, 6520, 6521, 6522, 6523, 6524, 6525, 6526, 6527, 6528, 6529, 6530, 6531, 6532
6533, 6534, 6535, 6536, 6537, 6538, 6539, 6540, 6541, 6542, 6543, 6544, 6545, 6546
6547, 6548, 6549, 6550, 6551, 6552, 6553, 6554, 6555, 6556, 6557, 6558, 6559, 6560
6561, 6562, 6563, 6564, 6565, 6566, 6567, 6568, 6569, 6570, 6571, 6572, 6573, 6574
6575, 6576, 6577, 6578, 6579, 6580, 6581, 6582, 6583, 6584, 6585, 6586, 6587, 6588
6589, 6590, 6591, 6592, 6593, 6594, 6595, 6596, 6597, 6598, 6599, 6600, 6601, 6602
6603, 6604, 6605, 6606, 6607, 6608, 6609, 6610, 6611, 6612, 6613, 6614, 6615, 6616
6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630
6631, 6632, 6633, 6634, 6635, 6636, 6637, 6638, 6639, 6640, 6641, 6642, 6643, 6644
6645, 6646, 6647, 6648, 6649, 6650, 6651, 6652, 6653, 6654, 6655, 6656, 6657, 6658
6659, 6660, 6661, 6662, 6663, 6664, 6665, 6666, 6667, 6668, 6669, 6670, 6671, 6672
6673, 6674, 6675, 6676, 6677, 6678, 6679, 6680, 6681, 6682, 6683, 6684, 6685, 6686
6687, 6688, 6689, 6690, 6691, 6692, 6693, 6694, 6695, 6696, 6697, 6698, 6699, 6700
6701, 6702, 6703, 6704, 6705, 6706, 6707, 6708, 6709, 6710, 6711, 6712, 6713, 6714
6715, 6716, 6717, 6718, 6719, 6720, 6721, 6722, 6723, 6724, 6725, 6726, 6727, 6728
6729, 6730, 6731, 6732, 6733, 6734, 6735, 6736, 6737, 6738, 6739, 6740, 6741, 6742
6743, 6744, 6745, 6746, 6747, 6748, 6749, 6750, 6751, 6752, 6753, 6754, 6755, 6756
6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764, 6765, 6766, 6767, 6768, 6769, 6770
6771, 6772, 6773, 6774, 6775, 6776, 6777, 6778, 6779, 6780, 6781, 6782, 6783, 6784
6785, 6786, 6787, 6788, 6789, 6790, 6791, 6792, 6793, 6794, 6795, 6796, 6797, 6798
6799, 6800, 6801, 6802, 6803, 6804, 6805, 6806, 6807, 6808, 6809, 6810, 6811, 6812
6813, 6814, 6815, 6816, 6817, 6818, 6819, 6820, 6821, 6822, 6823, 6824, 6825, 6826
6827, 6828, 6829, 6830, 6831, 6832, 6833, 6834, 6835, 6836, 6837, 6838, 6839, 6840
6841, 6842, 6843, 6844, 6845, 6846, 6847, 6848, 6849, 6850, 6851, 6852, 6853, 6854
6855, 6856, 6857, 6858, 6859, 6860, 6861, 6862, 6863, 6864, 6865, 6866, 6867, 6868
6869, 6870, 6871, 6872, 6873, 6874, 6875, 6876, 6877, 6878, 6879, 6880, 6881, 6882
6883, 6884, 6885, 6886, 6887, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896
6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910
6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923, 6924 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935, 6936, 6937, 6938
6939, 6940, 6941, 6942, 6943, 6944, 6945, 6946, 6947, 6948, 6949, 6950, 6951, 6952
6953, 6954, 6955, 6956, 6957, 6958, 6959, 6960, 6961, 6962, 6963, 6964, 6965, 6966
6967, 6968, 6969, 6970, 6971, 6972, 6973, 6974, 6975, 6976, 6977, 6978, 6979, 6980
6981, 6982, 6983, 6984, 6985, 6986, 6987, 6988, 6989, 6990, 6991, 6992, 6993, 6994
6995, 6996, 6997, 6998, 6999, 7000, 7001, 7002, 7003, 7004, 7005, 7006, 7007, 7008
7009, 7010, 7011, 7012, 7013, 7014, 7015, 7016, 7017, 7018, 7019, 7020, 7021, 7022
7023, 7024, 7025, 7026, 7027, 7028, 7029, 7030, 7031, 7032, 7033, 7034, 7035, 7036
7037, 7038, 7039, 7040, 7041, 7042, 7043, 7044, 7045, 7046, 7047, 7048, 7049, 7050
7051, 7052, 7053, 7054, 7055, 7056, 7057, 7058, 7059, 7060, 7061, 7062, 7063, 7064
7065, 7066, 7067, 7068, 7069, 7070, 7071, 7072, 7073, 7074, 7075, 7076, 7077, 7078
7079, 7080, 7081, 7082, 7083, 7084, 7085, 7086, 7087, 7088, 7089, 7090, 7091, 7092
7093, 7094, 7095, 7096, 7097, 7098, 7099, 7100, 7101, 7102, 7103, 7104, 7105, 7106
7107, 7108, 7109, 7110, 7111, 7112, 7113, 7114, 7115, 7116, 7117, 7118, 7119, 7120
7121, 7122, 7123, 7124, 7125, 7126, 7127, 7128, 7129, 7130, 7131, 7132, 7133, 7134
7135, 7136, 7137, 7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148
7149, 7150, 7151, 7152, 7153, 7154, 7155, 7156, 7157, 7158, 7159, 7160, 7161, 7162
7163, 7164, 7165, 7166, 7167, 7168, 7169, 7170, 7171, 7172, 7173, 7174, 7175, 7176
7177, 7178, 7179, 7180, 7181, 7182, 7183, 7184, 7185, 7186, 7187, 7188, 7189, 7190
7191, 7192, 7193, 7194, 7195, 7196, 7197, 7198, 7199, 7200, 7201, 7202, 7203, 7204
7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213, 7214, 7215, 7216, 7217, 7218
7219, 7220, 7221, 7222, 7223, 7224, 7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232
7233, 7234, 7235, 7236, 7237, 7238, 7239, 7240, 7241, 7242, 7243, 7244, 7245, 7246
7247, 7248, 7249, 7250, 7251, 7252, 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, 7861, 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, 8421, 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
8521, 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, 8740, and 8741 in SEQ ID NO if the length of the at least 5 amino acid residues so permits. If the length of the at least 5 amino acids are higher than 5, the N-terminal first residue will not be higher numbered than N-L+ 1, where N is the number of amino acid residues of the reference sequence, and L is the number of amino acids defined for option b) .
The polypeptide of the invention- that is, any one of SEQ ID NOs. 1-17 and 54- is in certain embodiments, also fused or conjugated to an immunogenic carrier molecule. The
immunogenic carrier molecule is typically a polypeptide that induces a T-helper lymphocyte response in a mammal. Such immunogenic carrier molecules can be selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, or dipththeria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra. The polypeptides of the invention may also be fused to other amino acid sequences that confer desired a functionality. For instance, the polypeptides may be fused to amino acid sequences that facilitate purification (e.g. "His tags", a polyhistidinyl group), or the fusion partner may be a (heterologous) signal peptide that facilitates export or other cellular transport mechanisms, and localization of the fusion protein in the intended cellular location. In preferred embodiments, the polypeptide of the invention detailed above is capable of inducing an adaptive immune response against the polypeptide in a mammal, in particular in a pig. Preferably, the adaptive immune response is a protective adaptive immune response against infection with L. intracellularis. The polypeptide may, in these cases, induce a humeral and/or a cellular immune response. Epitopes
SEQ ID NOs: 1-17 and 54 include antigenic determinants (epitopes) that are as such recognized by antibodies, or by T-cell receptors when bound to MHC molecules. For the purposes of the present invention, B-cell epitopes (i .e. antibody-binding epitopes) are of particular relevance.
It is relatively uncomplicated to identify linear B-cell epitopes. One approach entails that antibodies raised against L. intracellularis or L. intracellularis-der'wed proteins disclosed herein are tested for binding to overlapping oligomeric peptides derived from any one of SEQ ID NO: 1-17 and 54. Thereby, the regions of the L. intracellularis polypeptide which are responsible for, or contribute to, binding to the antibodies can be identified .
Alternatively or additionally, one can produce mutated versions of the polypeptides of the invention, e.g. versions where each single non-alanine residue in SEQ ID NOs. : 1-17 and 54 are point mutated to alanine. This method is useful in identifying complex non- conformational B-cell epitopes. This is the case when binding of the same antibody is altered by mutating amino acids in different areas of the full-length polypeptide.
Also, in silico methods for B-cell epitope prediction can be employed . Useful state-of-the-art systems for β-turn prediction are provided in Petersen et al. (November 2010), Plos One 5(11) : el5079; prediction of linear B-cell epitopes, cf: Larsen et al. (April 2006), Immunome Research, 2: 2; prediction of solvent exposed amino acids: Petersen B et al. (July 2009), BMC Structural Biology, 9 : 51.
Certain truncated versions of the polypeptides of the invention are or particular interest, since they appear to include a desirable number of B-cell and T-cell epitopes. Hence, polypeptides of particular interest are those that include, consist of, or include epitopes from, the 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 Examples for details.)
The nucleic acid fragments of the invention
The nucleic acid fragment of the invention is preferably is a DNA fragment (such as SEQ ID NOs: 18-34), or an RNA fragment (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 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, 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, 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, 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 at least 375 consecutive nucleotides in any one of SEQ ID NOs: 18-51 and 55. Longer fragments are contemplated, i.e. fragments 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, at least 2000, at least 2500, at least 3000, at least 3500, and at least 4000 nucleotides from those of SEQ ID NOs: 18-51 and 55. The nucleic acid fragment of the invention typically has a sequence identity with the nucleotide sequence defined for i) or ii) above, which is 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 nucleic acid fragment of the invention may also have a sequence identity with the nucleotide sequence defined for iii) above, which is 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 vectors of the invention
Vectors of the invention fall into several categories discussed infra. One preferred vector of the invention comprises in operable linkage, and in the 5'-3' direction, an expression control region comprising an enhancer/promoter, useful for driving expression of the nucleic acid fragment defined for option i) above, optionally a signal peptide coding sequence, a nucleotide sequence defined for option i), and optionally a terminator. Hence, such a vector constitutes an expression vector useful for effecting production in cells of the polypeptide of the invention. Since the polypeptides of the invention are bacterial in origin, recombinant production is conveniently effected in bacterial host cells. Therefore, it is preferred that the expression control region drives expression in prokaryotic cells, such as a bacterium, e.g. in Escherichia coli. However, if the vector is to drive expression in mammalian cells (as would be the case for a DNA vaccine vector), the expression control region should be adapted to this particular use. At any rate, certain vectors of the invention are capable of autonomous replication.
Also, the vector of the invention may be one that is capable of being integrated into the genome of a host cell. This is particularly useful if the vector is used in the production of stably-transformed cells, where the progeny will also include the genetic information introduced via the vector. Alternatively, vectors incapable of being integrated into the genome of a mammalian host cell are useful in e.g. DNA vaccination.
Typically, the vector of the invention is selected from the group consisting of, but not limited to, a virus, such as an attenuated virus (which may in itself be useful as a vaccine agent), a bacteriophage, a plasmid, a minichromosome, and a cosmid. A more detailed discussion of vectors of the invention is provided in the following:
Polypeptides of the invention may be encoded by a nucleic acid molecule comprised in a vector. A nucleic acid sequence can be "heterologous," which means that it is foreign to the cell into which the vector is being introduced. It can also include a sequence homologous to a sequence in the cell, but be located in a position within the host cell where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al, 2001; Ausubel et al, 1996, both incorporated herein by reference). In addition to encoding the polypeptides of this invention, a vector of the present invention may encode polypeptide sequences such as a tag or immunogenicity-enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof). Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al, 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST)-soluble fusion proteins, for later purification and separation or cleavage.
Vectors of the invention may be used in a host cell to produce a polypeptide of the invention, that may subsequently be purified for administration to a subject, or the vector may be purified for direct administration to a subject for expression of the protein in the subject (as is the case when administering a nucleic acid vaccine). Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably-linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well, and are described infra.
1. Promoters and Enhancers
A "promoter" is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A 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 one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural 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 promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference). Naturally, it may be important to employ a promoter and/or enhancer that effectively direct(s) the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al, 2001, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, or inducible, and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.
Examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain (Banerji et al, 1983; Gilles et al, 1983; Grosschedl et al, 1985; Atchinson et al, 1986, 1987; toiler 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 Receptor (Luria et al, 1987; Winoto et al, 1989; Redondo et al ; 1990), HLA DQa and/or 0(} (Sullivan et al, 1987), β-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-DRa (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, 1987;
Tranche et al, 1989, 1990),a-Fetoprotein (Godbout et al, 1988; Campere et al, 1989), γ- Globin (Bodine et al, 1987; Perez-Stable et al, 1990), β- Globin (Trudel et al, 1987), c-fos (Cohen et al, 1987), c-HA-ras (Triesman, 1986; Deschamps et al, 1985), Insulin (Edlund et al, 1985), Neural Cell Adhesion Molecule (NCAM) (Hirsh et al, 1990), al-Antitrypain (Larimer et al, 1990), H2B (TH2B) Histone (Hwang et al, 1990), Mouse and/or Type I Collagen (Ripe et al, 1989), Glucose-Regulated 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 Muscular 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), Retroviruses (Kriegler et al, 1982, 1983; Levinson et al, 1982; Kriegler 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), Papilloma Virus (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), Cytomegalovirus (CMV) IE (Weber et al, 1984; Boshart et al, 1985; Foecking et al, 1986), Gibbon Ape 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 mammary tumor virus) - Glucocorticoids (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);β- Interferon - poly(rl)x/poly(rc) (Tavernier et al, 1983); Adenovirus 5 E2 - EIA (Imperiale et al, 1984); Collagenase - Phorbol Ester (TPA) (Angel et al, 1987a); Stromelysin - Phorbol Ester (TPA) (Angel et al, 1987b); SV40 - Phorbol Ester (TPA) (Angel et al, 1987b); Murine MX Gene - Interferon, Newcastle Disease Virus (Hug et al, 1988); GRP78 Gene - A23187 (Resendez et al, 1988);a-2-Macroglobulin - IL-6 (Kunz et al, 1989); Vimentin - Serum (Rittling et al, 1989); MHC Class I Gene H-2Kb - Interferon (Blanar et al, 1989); HSP70 - E1A/SV40 Large T Antigen (Taylor et al, 1989, 1990a, 1990b); Proliferin - Phorbol Ester/TPA (Mordacq et al, 1989); Tumor Necrosis Factor - PMA (Hensel et al, 1989); and Thyroid Stimulating Hormonea Gene - Thyroid Hormone (Chatterjee et al, 1989).
Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base, EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins, or a heterologous protein of interest.
The particular promoter that is employed to control the expression of a peptide- or protein- encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of directing expression of the polynucleotide in a targeted cell, preferably a bacterial cell. Where a porcine cell is targeted, it is preferable to position the polynucleotide coding region adjacent to, and under the control of, a promoter that is capable of being expressed in a porcine cell. Generally speaking, such a promoter might include either a bacterial, porcine or 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 high level expression of a related polynucleotide to this invention. The use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well. In embodiments in which a vector is administered to a subject for expression of the protein, it is contemplated that a desirable promoter for use with the vector is one that is not down- regulated by cytokines, or one that is strong enough that even if down-regulated, it produces an effective amount of the protein/polypeptide of the current invention in a subject to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, a promoter that is up-regulated in the presence of cytokines is employed. The MHC I promoter increases expression in the presence of IFN-γ.
Tissue-specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages. The
mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters.
2. Initiation Signals and Internal Ribosome Binding Sites (IRES)
A specific initiation signal may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon, or analogous sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this, and providing 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 signals and initiation codons can be either natural or synthetic, and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated cap-dependent translation, and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian source (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). 3. Multiple Cloning Sites
Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. (See Carbonelli et al, 1999, Levenson et al, 1998, and Cocea, 1997, incorporated herein by reference.) Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS, enabling exogenous sequences to be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
4. Splicing Sites Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. If relevant in the context of vectors of the present invention, vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al, 1997, incorporated herein by reference.) 5. Termination Signals
The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable, and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
Terminators contemplated for use in the invention include any known transcription terminator described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator, or viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
6. Polyadenylation Signals
In protein expression, particularly eukaryotic protein expression (as is relevant in nucleic acid vaccination), one will typically include a polyadenylation signal to effect proper
polyadenylation of the transcript. Polyadenylation may also increase the stability of the transcript, and/or may facilitate cytoplasmic transport. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal.
7. Origins of Replication
In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively, an autonomously-replicating sequence (ARS) can be employed if the host cell is yeast.
8. Selectable and Screenable Markers
In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell, permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers include screenable markers, such as GFP, for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk), or chloramphenicol acetyltransferase (CAT), may be utilized. One of skill in the art would also know how to employ immunologic markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a protein of the invention. Further examples of selectable and screenable markers are well known to one of skill in the art. The transformed cells of the invention
Transformed cells of the invention are useful for producing the polypeptide of the invention, but also as simple "containers" of nucleic acids and vectors of the invention.
Certain transformed cells of the invention are capable of replicating the nucleic acid fragment defined for option i) of the second aspect of the invention. Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment defined for option i).
For recombinant production it is convenient, but not a prerequisite, that the transformed cell is prokaryotic, such as a bacterium. However, both prokaryotic cells and eukaryotic cells may be used.
Suitable prokaryotic cells are bacterial cells selected from the group consisting of Escherichia (such as E. coli), Bacillus (e.g. Bacillus subtilis) , Salmonella, and Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG).
Eukaryotic cells can be in the form of yeasts (such as Saccharomyces cerevisiae) and protozoans. Alternatively, the transformed eukaryotic cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. For production purposes, it is advantageous that the transformed cell of the invention is stably transformed by having the nucleic acid defined above for option i) stably integrated into its genome. In certain embodiments, it is also preferred that the transformed cell secretes or carries on its surface the polypeptide of the invention, since this facilitates recovery of the polypeptides produced. A particular version of this embodiment is one where the transformed cell is a bacterium, and secretion of the polypeptide of the invention is into the periplasmic space.
Further details on cells and cell lines are presented in the following:
Suitable cells for recombinant nucleic acid expression of the nucleic acid fragments of the present invention are prokaryotic and eukaryotic. Examples of prokaryotic cells include E. coli; members of the Staphylococcus genus, such as S. epidermidis; members of the
Lactobacillus genus, such as L. plantarum; members of the Lactococcus genus, such as L. lactis; members of the Bacillus genus, such as B. subtilis; members of the Corynebacterium genus, such as C. glutamicum; and members of the Pseudomonas genus, such as P.
fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells, such as members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastoris), members of the Hansenula genus (e.g. H. polymorpha), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis), and members of the
Schizosaccharomyces genus (e.g. S. pombe). Techniques for recombinant gene production, introduction into a cell, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002; and
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can be used as a recipient for vectors or viruses. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell; they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials
( www.atcc.org') , or from other depository institutions, such as Deutsche Sammlung vor
Micrroorganismen und Zellkulturen (DSM). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for the generation of multiple copies, or expression of encoded proteins. Bacterial cells used for vector replication and/or expression include Staphylococcus strains, as well as a number of commercially available E. coli hosts, such as SURE® Competent Cells and SoloPack™ Gold Cells (Stratagene®; La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for bacteriophage. Appropriate yeast cells can include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.
Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells of various cell types and organisms are available, and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above-described host cells to maintain them, and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors, and their cognate polypeptides, proteins, or peptides. Expression Systems
Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely 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. Patents 5,871,986, 4,879,236, both herein incorporated by reference. These are commercially available, for example, under the names MaxBac® 2.0 (Invitrogen®), and BacPAK™ (Clontech®).
In addition to the disclosed expression systems of the invention, other examples of expression systems include COMPLETE CONTROL™ Inducible Mammalian Expression System (Stratagene®), which involves a synthetic ecdysone-inducible receptor, or the pET Expression System (Stratagene®), an E. coli expression system. Another example of an inducible expression system is the T-REx™ System ( Invitrogen®), which involves tetracycline- regulated expression. Pichia methanolica Expression System (Invitrogen®), is a yeast expression system designed for high-level production of recombinant proteins in the methylotrophic yeast, Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide. Amplification of Nucleic Acids
Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 2001). In certain
embodiments, analysis is performed on whole cells, tissue homogenates, or biological fluid samples, without substantial purification of the template nucleic acid. The nucleic acid may be genomic DNA, or fractionated or whole-cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA (cDNA).
The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty bases in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single- stranded form, although the single-stranded form is preferred.
Pairs of primers, designed to selectively hybridize to nucleic acids corresponding to sequences of genes identified herein, are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency conditions to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template- dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label, or even via a system using electrical and/or thermal impulse signals (Bellus, 1994).
A number of template-dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in U.S. Patents 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each of which is incorporated herein by reference in their entirety. Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Patents 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 in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety.
Methods of Gene Transfer
Suitable methods for nucleic acid delivery to effect expression of compositions of the present invention include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue, or an organism, as described herein, or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 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), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate 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, 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et al, 1989; Kato et al,
1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-med ated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); by PEG-mediated transformation of protoplasts (Omirulleh et al, 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); or by desiccation/inhibition-mediated DNA uptake (Potrykus et al, 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.
The antibodies of the invention
Antibodies directed against the proteins of the invention are useful for affinity
chromatography, immunoassays, as well as for passive immunisation and therapy. Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, 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 polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti- rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antiserum is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25 C for one hour, followed by incubating at 4 C for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.
Monoclonal antibodies (MAbs) are prepared using the standard method of Kohler & Milstein [Nature (1975) 256 : 495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed, and dissociated into single cells. If desired, the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g. hypoxanthine, aminopterin, thymidine medium, or "HAT"). The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (e.g. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
If desired, the antibodies (whether polyclonal or monoclonal) may be labelled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3', 5,5'- tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
"Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin, avidin, streptavidin, IgG and protein A, and numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 125I may serve as a radioactive label, or as an electron-dense reagent. HRP may serve as enzyme, or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with, 125I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
According to the invention, the isolated monoclonal antibody or antibody analogue is preferably a monoclonal antibody selected from a multi-domain antibody such as a murine antibody, a chimeric antibody such as a humanized antibody, a fully human antibody, and single-domain antibody of a llama or a camel. The antibody analogue may be selected from a fragment of an antibody such as a Fab, a F(ab')2, or a scFV; cf. also the definition of the term "antibody" presented above.
Compositions of the invention; vaccines Pharmaceutical compositions, in particular vaccines, according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e, to treat disease after infection).
Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from Diphtheria, tetanus, cholera, or H. pylori.. The pharmaceutical compositions of the invention thus typically contain an immunological adjuvant, which can be an aluminium-based adjuvant, or one of any number of other adjuvants, as described in the following :
Preferred adjuvants to enhance effectiveness of the composition include, but are 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 muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below), either microfluidized into a submicron emulsion, or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience; Worcester, MA) may be used, or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete
Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2"-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy-ethylamine (MTP-PE), etc. The immunogenic compositions (e.g. the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above. Immunogenic compositions used as vaccines comprise an immunologically-effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically-effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. However, for the purposes 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 very often in the range between 10 and 200 μg.
The immunogenic compositions are conventionally administered parenterally, e.g, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (e.g. see W0 98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intra-arterial routes may be applicable.
Dosage treatment may be a single dose schedule, or a multiple dose schedule. The vaccine may also be administered in conjunction with other immunoregulatory agents.
As an alternative to protein-based vaccines, DNA vaccination (also termed nucleic acid vaccination, or gene vaccination) may be used [e.g. Robinson & Torres (1997) Seminars in Immunology 9: 271-283; Donnelly et al. (1997) Ann Rev Immunol 15 : 617-648; later herein] . A further aspect of the invention is the recognition that combination vaccines can be provided, wherein 2 or more Lawsonia antigens disclosed herein are combined to enhance the immune response by the vaccinated animal, including to optimize initial immune response and duration of immunity. For the purposes of this aspect of the invention, multiple antigenic fragments derived from the same Lawsonia protein can also be used, such as the use a combination of different lengths of polypeptide sequence fragments from one protein.
Use of multiple species
A further aspect of the invention is the recognition that combination vaccines can be provided wherein 2 or more Lawsonia antigens disclosed herein (or nucleic acids encoding the same) are combined to enhance immune response by the vaccinated animal, including to optimize initial immune response and duration of immunity. For the purposes of this aspect of the invention, multiple antigens derived from the same antigenic Lawsonia protein can also be used, such as to use a combination of different lengths of polypeptide sequence fragments from one protein. Representative examples of combination antigen formulations are those that include LIC037 or any of its fragments disclosed herein in combination with polypeptides and fragments thereof derived from at least 2 of the other polypeptides of the present invention..
Treatment methods of the invention The method of the sixth aspect of the invention generally relates to induction of immunity, and as such, also entails methods that relate to treatment, prophylaxis and amelioration of disease.
When immunization methods entail that a polypeptide of the invention, or a composition comprising such a polypeptide, is administered, the animal (e.g. the pig) typically receives between 0.5 and 5,000 μg of the polypeptide of the invention per administration.
In preferred embodiments of the sixth aspect, the immunization scheme includes that the animal (e.g. the pig) receives a priming administration, and one or more booster
administrations.
Preferred embodiments of the 6th aspect of the invention comprise that the administration is for the purpose of inducing protective immunity against L. intracellularis. In this embodiment, it is particularly preferred that the protective immunity is effective in reducing the risk of infection with L. intracellularis, or is effective in treating or ameliorating infection with L. intracellularis.
As mentioned herein, the preferred vaccines of the invention induce humoral immunity.
Thus, it is preferred that the administration is for the purpose of inducing antibodies specific for L. intracellularis, and wherein said antibodies or B-lymphocytes producing said antibodies are subsequently recovered from the animal.
As also mentioned, the method of the 6th aspect may also be useful in antibody production. Thus, in other embodiments, the administration is for the purpose of inducing antibodies specific for L. intracellularis, and wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal, and used for preparation of monoclonal antibodies.
Pharmaceutical compositions as mentioned above can also comprise polypeptides, antibodies, or nucleic acids of the invention. The pharmaceutical compositions will comprise a
therapeutically effective amount thereof. The terms "therapeutically-effective amount", or "prophylactically-effective amount", as used herein, refer to an amount of a therapeutic agent used to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. Reference is however made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above. However, the effective amount for a given situation can be determined by routine
experimentation, and is within the judgement of the clinician.
For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg, or 0.05 mg/kg to about 10 mg/kg of the DNA constructs, in the individual to which it is administered. A pharmaceutical composition can also contain a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be
administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. 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 the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically-acceptable salts is available in Remington's
Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).
Pharmaceutically-acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH-buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection, may also be prepared. Liposomes are included within the definition of a
pharmaceutically acceptable carrier.
As is apparent from the claims, the invention also includes related embodiments to the treatment and prophylaxis disclosed herein. The invention also includes embodiments wherein:
- the polypeptide of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L.
intracellularis;
- the nucleic acid fragment of the invention or the vector of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L. intracellularis;
- the transformed cell of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L.
intracellularis. - the antibody, antibody fragment or antibody analogue of the invention is for use as a pharmaceutical, in particular for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L. intracellularis.
ADDITIONAL NUMBERED STATEMENTS OF THE INVENTION
Representative aspects of the invention include the following numbered embodiments of the invention, and the Examples section which follows. Embodiment 1. A polypeptide comprising
a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1- 17 and 54, or
b) an amino acid sequence consisting of at least 5 contiguous amino acid residues from any one of SEQ ID NOs: 1-17 and 54, or
c) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of a),
d) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of b), or
e) an assembly of amino acids derived from any one of SEQ ID NOs: 1-17 and 54, which has essentially the same 3D conformation as in the protein from which said assembly is derived so as to constitute a B-cell epitope,
said polypeptide being antigenic in a mammal.
Embodiment 2. The polypeptide according to embodiment 1, wherein the at least 5 contiguous 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. Embodiment 3. The polypeptide according to embodiment 1 or 2, wherein the sequence identity with 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%. Embodiment 4. The polypeptide according to embodiment 1 or 2, wherein the sequence identity with 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%. Embodiment 5. The polypeptide according to any one of the preceding
embodiments, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121 in any one of SEQ ID NOs: 1-17 and 54.
Embodiment 6. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 122, 123, and 124 in any on of SEQ ID NOs: 2-17 and 54.
Embodiment 7. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, and 137 in any one of SEQ ID NOs: 3-17 and 54.
Embodiment 8. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255 in any one of SEQ ID NOs: 4-17 and 54.
Embodiment 9. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, and 386 in any one of SEQ ID NOs: 5-17 and 54.
Embodiment 10. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, and 476 in any one of SEQ ID NOs: 6-17 and 54.
Embodiment 11. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 568, 569, 570, 571, 572, 573, 574, 575, 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, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, and 625 in any one of SEQ ID NOs: 7-17 and 54. Embodiment 12. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, and 638 in any one of SEQ ID NOs: 8-17 and 54.
Embodiment 13. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 713, 714, 715, 716, 717, 718, 719, 720, and 721 in any one of SEQ ID NOs: 9-17 and 54.
Embodiment 14. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, and 828 in any one of SEQ ID NOs: 10-17 and 54.
Embodiment 15. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 829, 830, 831, 832, 833, 834, 835, and 836 in any one of SEQ ID NOs: 11-17 and 54. Embodiment 16. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 881, 882, 883, 884, 885, 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, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, and 930 in any one of SEQ ID NOs: 12-17 and 54.
Embodiment 17. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 955, and 956 in any one of SEQ ID NOs: 13-17 and 54.
Embodiment 18. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, and 1046 in any one of SEQ ID NOs: 14-17 and 54.
Embodiment 19. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 1071, and 1072 in any one of SEQ ID NOs: 15-17 and 54.
Embodiment 20. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid 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, 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, 1334, 1335, and 1336 in any one of SEQ ID NOs: 16, 17 and 54.
Embodiment 21. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 1337, 1338, 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, 1595,
1596, 1597, 1598, 1599, 1600, 1601, 1602 and 1603 in SEQ ID NO: 17 or 54.
Embodiment 22. The polypeptide according to any one of embodiments 1-4, wherein the at least 5 contiguous amino acid residues have an N-terminal amino acid residue corresponding to any one of amino acid residues 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, 2021, 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, 3021, 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
3781, 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, 4021, 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
4243, 4244, 4245, 4246, 4247, 4248, 4249, 4250, 4251, 4252, 4253, 4254, 4255, 4256
4257, 4258, 4259, 4260, 4261, 4262, 4263, 4264, 4265, 4266, 4267, 4268, 4269, 4270
4271, 4272, 4273, 4274, 4275, 4276, 4277, 4278, 4279, 4280, 4281, 4282, 4283, 4284
4285, 4286, 4287, 4288, 4289, 4290, 4291, 4292, 4293, 4294, 4295, 4296, 4297, 4298
4299, 4300, 4301, 4302, 4303, 4304, 4305, 4306, 4307, 4308, 4309, 4310, 4311, 4312
4313, 4314, 4315, 4316, 4317, 4318, 4319, 4320, 4321, 4322, 4323, 4324, 4325, 4326
4327, 4328, 4329, 4330, 4331, 4332, 4333, 4334, 4335, 4336, 4337, 4338, 4339, 4340
4341, 4342, 4343, 4344, 4345, 4346, 4347, 4348, 4349, 4350, 4351, 4352, 4353, 4354
4355, 4356, 4357, 4358, 4359, 4360, 4361, 4362, 4363, 4364, 4365, 4366, 4367, 4368
4369, 4370, 4371, 4372, 4373, 4374, 4375, 4376, 4377, 4378, 4379, 4380, 4381, 4382
4383, 4384, 4385, 4386, 4387, 4388, 4389, 4390, 4391, 4392, 4393, 4394, 4395, 4396
4397, 4398, 4399, 4400, 4401, 4402, 4403, 4404, 4405, 4406, 4407, 4408, 4409, 4410
4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418, 4419, 4420, 4421, 4422, 4423, 4424
4425, 4426, 4427, 4428, 4429, 4430, 4431, 4432, 4433, 4434, 4435, 4436, 4437, 4438
4439, 4440, 4441, 4442, 4443, 4444, 4445, 4446, 4447, 4448, 4449, 4450, 4451, 4452
4453, 4454, 4455, 4456, 4457, 4458, 4459, 4460, 4461, 4462, 4463, 4464, 4465, 4466
4467, 4468, 4469, 4470, 4471, 4472, 4473, 4474, 4475, 4476, 4477, 4478, 4479, 4480
4481, 4482, 4483, 4484, 4485, 4486, 4487, 4488, 4489, 4490, 4491, 4492, 4493, 4494
4495, 4496, 4497, 4498, 4499, 4500, 4501, 4502, 4503, 4504, 4505, 4506, 4507, 4508
4509, 4510, 4511, 4512, 4513, 4514, 4515, 4516, 4517, 4518, 4519, 4520, 4521, 4522
4523, 4524, 4525, 4526, 4527, 4528, 4529, 4530, 4531, 4532, 4533, 4534, 4535, 4536
4537, 4538, 4539, 4540, 4541, 4542, 4543, 4544, 4545, 4546, 4547, 4548, 4549, 4550
4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, 4562, 4563, 4564
4565, 4566, 4567, 4568, 4569, 4570, 4571, 4572, 4573, 4574, 4575, 4576, 4577, 4578
4579, 4580, 4581, 4582, 4583, 4584, 4585, 4586, 4587, 4588, 4589, 4590, 4591, 4592
4593, 4594, 4595, 4596, 4597, 4598, 4599, 4600, 4601, 4602, 4603, 4604, 4605, 4606
4607, 4608, 4609, 4610, 4611, 4612, 4613, 4614, 4615, 4616, 4617, 4618, 4619, 4620
4621, 4622, 4623, 4624, 4625, 4626, 4627, 4628, 4629, 4630, 4631, 4632, 4633, 4634
4635, 4636, 4637, 4638, 4639, 4640, 4641, 4642, 4643, 4644, 4645, 4646, 4647, 4648
4649, 4650, 4651, 4652, 4653, 4654, 4655, 4656, 4657, 4658, 4659, 4660, 4661, 4662
4663, 4664, 4665, 4666, 4667, 4668, 4669, 4670, 4671, 4672, 4673, 4674, 4675, 4676
4677, 4678, 4679, 4680, 4681, 4682, 4683, 4684, 4685, 4686, 4687, 4688, 4689, 4690 4691, 4692, 4693, 4694, 4695, 4696, 4697, 4698, 4699, 4700, 4701, 4702, 4703, 4704
4705, 4706, 4707, 4708, 4709, 4710, 4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718
4719, 4720, 4721, 4722, 4723, 4724, 4725, 4726, 4727, 4728, 4729, 4730, 4731, 4732
4733, 4734, 4735, 4736, 4737, 4738, 4739, 4740, 4741, 4742, 4743, 4744, 4745, 4746
4747, 4748, 4749, 4750, 4751, 4752, 4753, 4754, 4755, 4756, 4757, 4758, 4759, 4760
4761, 4762, 4763, 4764, 4765, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4773, 4774
4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788
4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802
4803, 4804, 4805, 4806, 4807, 4808, 4809, 4810, 4811, 4812, 4813, 4814, 4815, 4816
4817, 4818, 4819, 4820, 4821, 4822, 4823, 4824, 4825, 4826, 4827, 4828, 4829, 4830
4831, 4832, 4833, 4834, 4835, 4836, 4837, 4838, 4839, 4840, 4841, 4842, 4843, 4844
4845, 4846, 4847, 4848, 4849, 4850, 4851, 4852, 4853, 4854, 4855, 4856, 4857, 4858
4859, 4860, 4861, 4862, 4863, 4864, 4865, 4866, 4867, 4868, 4869, 4870, 4871, 4872
4873, 4874, 4875, 4876, 4877, 4878, 4879, 4880, 4881, 4882, 4883, 4884, 4885, 4886
4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894, 4895, 4896, 4897, 4898, 4899, 4900
4901, 4902, 4903, 4904, 4905, 4906, 4907, 4908, 4909, 4910, 4911, 4912, 4913, 4914
4915, 4916, 4917, 4918, 4919, 4920, 4921, 4922, 4923, 4924, 4925, 4926, 4927, 4928
4929, 4930, 4931, 4932, 4933, 4934, 4935, 4936, 4937, 4938, 4939, 4940, 4941, 4942
4943, 4944, 4945, 4946, 4947, 4948, 4949, 4950, 4951, 4952, 4953, 4954, 4955, 4956
4957, 4958, 4959, 4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970
4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983, 4984
4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998
4999, 5000, 5001, 5002, 5003, 5004, 5005, 5006, 5007, 5008, 5009, 5010, 5011, 5012
5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024, 5025, 5026
5027, 5028, 5029, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037, 5038, 5039, 5040
5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054
5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068
5069, 5070, 5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081, 5082
5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090, 5091, 5092, 5093, 5094, 5095, 5096
5097, 5098, 5099, 5100, 5101, 5102, 5103, 5104, 5105, 5106, 5107, 5108, 5109, 5110
5111, 5112, 5113, 5114, 5115, 5116, 5117, 5118, 5119, 5120, 5121, 5122, 5123, 5124
5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138
5139, 5140, 5141, 5142, 5143, 5144, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152
5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166
5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178, 5179, 5180
5181, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193, 5194
5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205, 5206, 5207, 5208
5209, 5210, 5211, 5212, 5213, 5214, 5215, 5216, 5217, 5218, 5219, 5220, 5221, 5222
5223, 5224, 5225, 5226, 5227, 5228, 5229, 5230, 5231, 5232, 5233, 5234, 5235, 5236 5237, 5238, 5239, 5240, 5241, 5242, 5243, 5244, 5245, 5246, 5247, 5248, 5249, 5250
5251, 5252, 5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264
5265, 5266, 5267, 5268, 5269, 5270, 5271, 5272, 5273, 5274, 5275, 5276, 5277, 5278
5279, 5280, 5281, 5282, 5283, 5284, 5285, 5286, 5287, 5288, 5289, 5290, 5291, 5292
5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306
5307, 5308, 5309, 5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320
5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334
5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347, 5348
5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361, 5362
5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376
5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390
5391, 5392, 5393, 5394, 5395, 5396, 5397, 5398, 5399, 5400, 5401, 5402, 5403, 5404
5405, 5406, 5407, 5408, 5409, 5410, 5411, 5412, 5413, 5414, 5415, 5416, 5417, 5418
5419, 5420, 5421, 5422, 5423, 5424, 5425, 5426, 5427, 5428, 5429, 5430, 5431, 5432
5433, 5434, 5435, 5436, 5437, 5438, 5439, 5440, 5441, 5442, 5443, 5444, 5445, 5446
5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460
5461, 5462, 5463, 5464, 5465, 5466, 5467, 5468, 5469, 5470, 5471, 5472, 5473, 5474
5475, 5476, 5477, 5478, 5479, 5480, 5481, 5482, 5483, 5484, 5485, 5486, 5487, 5488
5489, 5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5500, 5501, 5502
5503, 5504, 5505, 5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516
5517, 5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530
5531, 5532, 5533, 5534, 5535, 5536, 5537, 5538, 5539, 5540, 5541, 5542, 5543, 5544
5545, 5546, 5547, 5548, 5549, 5550, 5551, 5552, 5553, 5554, 5555, 5556, 5557, 5558
5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568, 5569, 5570, 5571, 5572
5573, 5574, 5575, 5576, 5577, 5578, 5579, 5580, 5581, 5582, 5583, 5584, 5585, 5586
5587, 5588, 5589, 5590, 5591, 5592, 5593, 5594, 5595, 5596, 5597, 5598, 5599, 5600
5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5612, 5613, 5614
5615, 5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5624, 5625, 5626, 5627, 5628
5629, 5630, 5631, 5632, 5633, 5634, 5635, 5636, 5637, 5638, 5639, 5640, 5641, 5642
5643, 5644, 5645, 5646, 5647, 5648, 5649, 5650, 5651, 5652, 5653, 5654, 5655, 5656
5657, 5658, 5659, 5660, 5661, 5662, 5663, 5664, 5665, 5666, 5667, 5668, 5669, 5670
5671, 5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681, 5682, 5683, 5684
5685, 5686, 5687, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698
5699, 5700, 5701, 5702, 5703, 5704, 5705, 5706, 5707, 5708, 5709, 5710, 5711, 5712
5713, 5714, 5715, 5716, 5717, 5718, 5719, 5720, 5721, 5722, 5723, 5724, 5725, 5726
5727, 5728, 5729, 5730, 5731, 5732, 5733, 5734, 5735, 5736, 5737, 5738, 5739, 5740
5741, 5742, 5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751, 5752, 5753, 5754
5755, 5756, 5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768
5769, 5770, 5771, 5772, 5773, 5774, 5775, 5776, 5777, 5778, 5779, 5780, 5781, 5782 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796
5797, 5798, 5799, 5800, 5801, 5802, 5803, 5804, 5805, 5806, 5807, 5808, 5809, 5810
5811, 5812, 5813, 5814, 5815, 5816, 5817, 5818, 5819, 5820, 5821, 5822, 5823, 5824
5825, 5826, 5827, 5828, 5829, 5830, 5831, 5832, 5833, 5834, 5835, 5836, 5837, 5838
5839, 5840, 5841, 5842, 5843, 5844, 5845, 5846, 5847, 5848, 5849, 5850, 5851, 5852
5853, 5854, 5855, 5856, 5857, 5858, 5859, 5860, 5861, 5862, 5863, 5864, 5865, 5866
5867, 5868, 5869, 5870, 5871, 5872, 5873, 5874, 5875, 5876, 5877, 5878, 5879, 5880
5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892, 5893, 5894
5895, 5896, 5897, 5898, 5899, 5900, 5901, 5902, 5903, 5904, 5905, 5906, 5907, 5908
5909, 5910, 5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922
5923, 5924, 5925, 5926, 5927, 5928, 5929, 5930, 5931, 5932, 5933, 5934, 5935, 5936
5937, 5938, 5939, 5940, 5941, 5942, 5943, 5944, 5945, 5946, 5947, 5948, 5949, 5950
5951, 5952, 5953, 5954, 5955, 5956, 5957, 5958, 5959, 5960, 5961, 5962, 5963, 5964
5965, 5966, 5967, 5968, 5969, 5970, 5971, 5972, 5973, 5974, 5975, 5976, 5977, 5978
5979, 5980, 5981, 5982, 5983, 5984, 5985, 5986, 5987, 5988, 5989, 5990, 5991, 5992
5993, 5994, 5995, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005, 6006
6007, 6008, 6009, 6010, 6011, 6012, 6013, 6014, 6015, 6016, 6017, 6018, 6019, 6020
6021, 6022, 6023, 6024, 6025, 6026, 6027, 6028, 6029, 6030, 6031, 6032, 6033, 6034
6035, 6036, 6037, 6038, 6039, 6040, 6041, 6042, 6043, 6044, 6045, 6046, 6047, 6048
6049, 6050, 6051, 6052, 6053, 6054, 6055, 6056, 6057, 6058, 6059, 6060, 6061, 6062
6063, 6064, 6065, 6066, 6067, 6068, 6069, 6070, 6071, 6072, 6073, 6074, 6075, 6076
6077, 6078, 6079, 6080, 6081, 6082, 6083, 6084, 6085, 6086, 6087, 6088, 6089, 6090
6091, 6092, 6093, 6094, 6095, 6096, 6097, 6098, 6099, 6100, 6101, 6102, 6103, 6104
6105, 6106, 6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118
6119, 6120, 6121, 6122, 6123, 6124, 6125, 6126, 6127, 6128, 6129, 6130, 6131, 6132
6133, 6134, 6135, 6136, 6137, 6138, 6139, 6140, 6141, 6142, 6143, 6144, 6145, 6146
6147, 6148, 6149, 6150, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6158, 6159, 6160
6161, 6162, 6163, 6164, 6165, 6166, 6167, 6168, 6169, 6170, 6171, 6172, 6173, 6174
6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182, 6183, 6184, 6185, 6186, 6187, 6188
6189, 6190, 6191, 6192, 6193, 6194, 6195, 6196, 6197, 6198, 6199, 6200, 6201, 6202
6203, 6204, 6205, 6206, 6207, 6208, 6209, 6210, 6211, 6212, 6213, 6214, 6215, 6216
6217, 6218, 6219, 6220, 6221, 6222, 6223, 6224, 6225, 6226, 6227, 6228, 6229, 6230
6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238, 6239, 6240, 6241, 6242, 6243, 6244
6245, 6246, 6247, 6248, 6249, 6250, 6251, 6252, 6253, 6254, 6255, 6256, 6257, 6258
6259, 6260, 6261, 6262, 6263, 6264, 6265, 6266, 6267, 6268, 6269, 6270, 6271, 6272
6273, 6274, 6275, 6276, 6277, 6278, 6279, 6280, 6281, 6282, 6283, 6284, 6285, 6286
6287, 6288, 6289, 6290, 6291, 6292, 6293, 6294, 6295, 6296, 6297, 6298, 6299, 6300
6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313, 6314
6315, 6316, 6317, 6318, 6319, 6320, 6321, 6322, 6323, 6324, 6325, 6326, 6327, 6328 6329, 6330, 6331, 6332, 6333, 6334, 6335, 6336, 6337, 6338, 6339, 6340, 6341, 6342
6343, 6344, 6345, 6346, 6347, 6348, 6349, 6350, 6351, 6352, 6353, 6354, 6355, 6356
6357, 6358, 6359, 6360, 6361, 6362, 6363, 6364, 6365, 6366, 6367, 6368, 6369, 6370
6371, 6372, 6373, 6374, 6375, 6376, 6377, 6378, 6379, 6380, 6381, 6382, 6383, 6384
6385, 6386, 6387, 6388, 6389, 6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398
6399, 6400, 6401, 6402, 6403, 6404, 6405, 6406, 6407, 6408, 6409, 6410, 6411, 6412
6413, 6414, 6415, 6416, 6417, 6418, 6419, 6420, 6421, 6422, 6423, 6424, 6425, 6426
6427, 6428, 6429, 6430, 6431, 6432, 6433, 6434, 6435, 6436, 6437, 6438, 6439, 6440
6441, 6442, 6443, 6444, 6445, 6446, 6447, 6448, 6449, 6450, 6451, 6452, 6453, 6454
6455, 6456, 6457, 6458, 6459, 6460, 6461, 6462, 6463, 6464, 6465, 6466, 6467, 6468
6469, 6470, 6471, 6472, 6473, 6474, 6475, 6476, 6477, 6478, 6479, 6480, 6481, 6482
6483, 6484, 6485, 6486, 6487, 6488, 6489, 6490, 6491, 6492, 6493, 6494, 6495, 6496
6497, 6498, 6499, 6500, 6501, 6502, 6503, 6504, 6505, 6506, 6507, 6508, 6509, 6510
6511, 6512, 6513, 6514, 6515, 6516, 6517, 6518, 6519, 6520, 6521, 6522, 6523, 6524
6525, 6526, 6527, 6528, 6529, 6530, 6531, 6532, 6533, 6534, 6535, 6536, 6537, 6538
6539, 6540, 6541, 6542, 6543, 6544, 6545, 6546, 6547, 6548, 6549, 6550, 6551, 6552
6553, 6554, 6555, 6556, 6557, 6558, 6559, 6560, 6561, 6562, 6563, 6564, 6565, 6566
6567, 6568, 6569, 6570, 6571, 6572, 6573, 6574, 6575, 6576, 6577, 6578, 6579, 6580
6581, 6582, 6583, 6584, 6585, 6586, 6587, 6588, 6589, 6590, 6591, 6592, 6593, 6594
6595, 6596, 6597, 6598, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606, 6607, 6608
6609, 6610, 6611, 6612, 6613, 6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622
6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634, 6635, 6636
6637, 6638, 6639, 6640, 6641, 6642, 6643, 6644, 6645, 6646, 6647, 6648, 6649, 6650
6651, 6652, 6653, 6654, 6655, 6656, 6657, 6658, 6659, 6660, 6661, 6662, 6663, 6664
6665, 6666, 6667, 6668, 6669, 6670, 6671, 6672, 6673, 6674, 6675, 6676, 6677, 6678
6679, 6680, 6681, 6682, 6683, 6684, 6685, 6686, 6687, 6688, 6689, 6690, 6691, 6692
6693, 6694, 6695, 6696, 6697, 6698, 6699, 6700, 6701, 6702, 6703, 6704, 6705, 6706
6707, 6708, 6709, 6710, 6711, 6712, 6713, 6714, 6715, 6716, 6717, 6718, 6719, 6720
6721, 6722, 6723, 6724, 6725, 6726, 6727, 6728, 6729, 6730, 6731, 6732, 6733, 6734
6735, 6736, 6737, 6738, 6739, 6740, 6741, 6742, 6743, 6744, 6745, 6746, 6747, 6748
6749, 6750, 6751, 6752, 6753, 6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762
6763, 6764, 6765, 6766, 6767, 6768, 6769, 6770, 6771, 6772, 6773, 6774, 6775, 6776
6777, 6778, 6779, 6780, 6781, 6782, 6783, 6784, 6785, 6786, 6787, 6788, 6789, 6790
6791, 6792, 6793, 6794, 6795, 6796, 6797, 6798, 6799, 6800, 6801, 6802, 6803, 6804
6805, 6806, 6807, 6808, 6809, 6810, 6811, 6812, 6813, 6814, 6815, 6816, 6817, 6818
6819, 6820, 6821, 6822, 6823, 6824, 6825, 6826, 6827, 6828, 6829, 6830, 6831, 6832
6833, 6834, 6835, 6836, 6837, 6838, 6839, 6840, 6841, 6842, 6843, 6844, 6845, 6846
6847, 6848, 6849, 6850, 6851, 6852, 6853, 6854, 6855, 6856, 6857, 6858, 6859, 6860
6861, 6862, 6863, 6864, 6865, 6866, 6867, 6868, 6869, 6870, 6871, 6872, 6873, 6874 6875, 6876, 6877, 6878, 6879, 6880, 6881, 6882, 6883, 6884, 6885, 6886, 6887, 6888
6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899, 6900, 6901, 6902
6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916
6917, 6918, 6919, 6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929, 6930
6931, 6932, 6933, 6934, 6935, 6936, 6937, 6938, 6939, 6940, 6941, 6942, 6943, 6944
6945, 6946, 6947, 6948, 6949, 6950, 6951, 6952, 6953, 6954, 6955, 6956, 6957, 6958
6959, 6960, 6961, 6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972
6973, 6974, 6975, 6976, 6977, 6978, 6979, 6980, 6981, 6982, 6983, 6984, 6985, 6986
6987, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 6996, 6997, 6998, 6999, 7000
7001, 7002, 7003, 7004, 7005, 7006, 7007, 7008, 7009, 7010, 7011, 7012, 7013, 7014
7015, 7016, 7017, 7018, 7019, 7020, 7021, 7022, 7023, 7024, 7025, 7026, 7027, 7028
7029, 7030, 7031, 7032, 7033, 7034, 7035, 7036, 7037, 7038, 7039, 7040, 7041, 7042
7043, 7044, 7045, 7046, 7047, 7048, 7049, 7050, 7051, 7052, 7053, 7054, 7055, 7056
7057, 7058, 7059, 7060, 7061, 7062, 7063, 7064, 7065, 7066, 7067, 7068, 7069, 7070
7071, 7072, 7073, 7074, 7075, 7076, 7077, 7078, 7079, 7080, 7081, 7082, 7083, 7084
7085, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7093, 7094, 7095, 7096, 7097, 7098
7099, 7100, 7101, 7102, 7103, 7104, 7105, 7106, 7107, 7108, 7109, 7110, 7111, 7112
7113, 7114, 7115, 7116, 7117, 7118, 7119, 7120, 7121, 7122, 7123, 7124, 7125, 7126
7127, 7128, 7129, 7130, 7131, 7132, 7133, 7134, 7135, 7136, 7137, 7138, 7139, 7140
7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152, 7153, 7154
7155, 7156, 7157, 7158, 7159, 7160, 7161, 7162, 7163, 7164, 7165, 7166, 7167, 7168
7169, 7170, 7171, 7172, 7173, 7174, 7175, 7176, 7177, 7178, 7179, 7180, 7181, 7182
7183, 7184, 7185, 7186, 7187, 7188, 7189, 7190, 7191, 7192, 7193, 7194, 7195, 7196
7197, 7198, 7199, 7200, 7201, 7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210
7211, 7212, 7213, 7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224
7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237, 7238
7239, 7240, 7241, 7242, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250, 7251, 7252
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, 7861, 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, 8421, 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, 8521, 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, 8740, and 8741 in SEQ ID NO: 54.
Embodiment 23. The polypeptide according to any one of the preceding embodiments, which is fused or conjugated to an immunogenic carrier molecule. Embodiment 24. The polypeptide according to embodiment 23, wherein the immunogenic carrier molecule is a polypeptide that induces T-helper lymphocyte responses in a majority of mammals, such as immunogenic carrier proteins selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, dipththeria toxoid or a fragment thereof. Embodiment 25. The polypeptide according to any one of the preceding
embodiments, which is capable of inducing an adaptive immune response against the polypeptide in a mammal, in particular in a pig.
Embodiment 26. The polypeptide according to embodiment 25, which is capable of inducing, in the mammal, a protective adaptive immune response against infection with L. intracellularis.
Embodiment 27. The polypeptide according to embodiment 25 or 26, which induces a humoral and/or a cellular immune response. Embodiment 28. An isolated nucleic acid fragment, which comprises i) a nucleotide sequence encoding a polypeptide according to any one of the preceding embodiments, or
ii) a nucleotide sequence consisting of the amino acid encoding part of any one of SEQ ID NOs: 18-51 and 55.
iii) a nucleotide sequence consisting of at least 10 consecutive nucleotides in the amino acid encoding part 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 in i) or ii),
v) a nucleotide sequence having a sequence identity of at least 60% with the nucleotide sequence in iii),
vi) a nucleotide sequence complementary to the nucleotide sequence in i)-v), or
vii) a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence in i)-vi) . Embodiment 29. The nucleic acid fragment according to embodiment 28, which is a
DNA or an RNA fragment.
Embodiment 30. The nucleic acid fragment according to embodiment 28 or 29, wherein the nucleotide sequence consists of 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 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 east 156 a least 157 , at least 158, at least 159, at least 160, at least 161, at least 162 east 163 a least 164 , at least 165, at least 166, at least 167, at least 168, at least 169 east 170 a least 171 , at least 172, at least 173, at least 174, at least 175, at least 176 east 177 a least 178 , at least 179, at least 180, at least 181, at least 182, at least 183 east 184 a least 185 , at least 186, at least 187, at least 188, at least 189, at least 190 east 191 a least 192 , at least 193, at least 194, at least 195, at least 196, at least 197 east 198 a least 199 , at least 200, at least 201, at least 202, at least 203, at least 204 east 205 a least 206 , at least 207, at least 208, at least 209, at least 210, at least 211 east 212 a least 213 , at least 214, at least 215, at least 216, at least 217, at least 218 east 219 a least 220 , at least 221, at least 222, at least 223, at least 224, at least 225 east 226 a least 227 , at least 228, at least 229, at least 230, at least 231, at least 232 east 233 a least 234 , at least 235, at least 236, at least 237, at least 238, at least 239 east 240 a least 241 , at least 242, at least 243, at least 244, at least 245, at least 246 east 247 a least 248 , at least 249, at least 250, at least 251, at least 252, at least 253 east 254 a least 255 , at least 256, at least 257, at least 258, at least 259, at least 260 east 261 a least 262 , at least 263, at least 264, at least 265, at least 266, at least 267 east 268 a least 269 , at least 270, at least 271, at least 272, at least 273, at least 274 east 275 a least 276 , at least 277, at least 278, at least 279, at least 280, at least 281 east 282 a least 283 , at least 284, at least 285, at least 286, at least 287, at least 288 east 289 a least 290 , at least 291, at least 292, at least 293, at least 294, at least 295 east 296 a least 297 , at least 298, at least 299, at least 300, at least 301, at least 302 east 303 a least 304 , at least 305, at least 306, at least 307, at least 308, at least 309 east 310 a least 311 , at least 312, at least 313, at least 314, at least 315, at least 316 east 317 a least 318 , at least 319, at least 320, at least 321, at least 322, at least 323 east 324 a least 325 , at least 326, at least 327, at least 328, at least 329, at least 330 east 331 a least 332 , at least 333, at least 334, at least 335, at least 336, at least 337 east 338 a least 339 , at least 340, at least 341, at least 342, at least 343, at least 344 east 345 a least 346 , at least 347, at least 348, at least 349, at least 350, at least 351 east 352 a least 353 , at least 354, at least 355, at least 356, at least 357, at least 358 east 359 a least 360 , at least 361, at least 362, at least 363, at least 364, at least 365 east 366 a least 367 , at least 368, at least 369, at least 370, at least 371, at least 372 east 373 a least 374 and at least 375 consecutive nucleotides in the amino acid encoding part of a ny one of SEQ ID NOs: 18-51 and 55.
Embodiment 31. The nucleic acid fragment according to any one of embodiments
28-30, wherein the sequence identity with the nucleotide sequence in i) or ii) 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%. Embodiment 32. The nucleic acid fragment according to any one of embodiments
28-30, wherein the sequence identity with the nucleotide sequence in 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%.
Embodiment 33. A vector comprising the nucleic acid according to any one of embodiments 28-32, such as a cloning vector or an expression vector.
Embodiment 34. The vector according to embodiment 33, which comprises in operable linkage and in the 5'-3' direction, an expression control region comprising an enhancer/promoter for driving expression of the nucleic acid fragment defined in embodiment 27-i), optionally a signal peptide coding sequence, a nucleotide sequence defined in embodiment 27-i), and optionally a terminator.
Embodiment 35. The vector according to embodiment 33 or 34, wherein the expression control region drives expression in prokaryotic cell such as a bacterium, e.g. in E coli.
Embodiment 36. The vector according to embodiment any one of embodiments 33-
35, which is capable of autonomous replication.
Embodiment 37. The vector according to any one of embodiments 33-36, which is capable of being integrated into the genome of a host cell. Embodiment 38. The vector according to any one of embodiments 33-37, which is incapable of being integrated into the genome of a mammalian host cell.
Embodiment 39. The vector according to any one of embodiments 33-38, which is selected from the group consisting of a virus, such as a attenuated virus, a bacteriophage, a plasmid, a minichromosome, and a cosmid. Embodiment 40. A cell which is transformed so as to carry the vector according to any one of embodiments 33-39.
Embodiment 41. The transformed cell according to embodiment 40, which is capable of replicating the nucleic acid fragment defined in embodiment 28-i). Embodiment 42. The transformed cell according to embodiment 40, which is capable of expressing the nucleic acid fragment defined in embodiment 28-i).
Embodiment 43. The transformed cell according to any one of embodiments 40-42, which is selected from a prokaryotic cell and a eukaryotic cell. Embodiment 44. The transformed cell according to any one of embodiments 40-42, which is a bacterial cell selected from the group consisting of Escherichia (such as E. coli.), Bacillus (e.g. Bacillus subtilis), Salmonella, and Mycobacterium, preferably non-pathogenic, e.g. M. bovis BCG.
Embodiment 45. The transformed cell according to any one of embodiments 40-44, which is stably transformed by having the nucleic acid defined in embodiment 42-i) stably integrated into its genome.
Embodiment 46. The transformed cell according to any one of embodiments 40-45, which secretes or carries on its surface the polypeptide according to any one of embodiments 1-27. Embodiment 47. The transformed cell according to embodiment 46, wherein the cell is a bacterium and secretion is into the periplasmic space.
Embodiment 48. A cell line derived from a transformed cell according to any one of embodiments 40-47.
Embodiment 49. A pharmaceutical composition comprising a polypeptide according to any one of embodiments 1-27, a nucleic acid fragment according to any one of embodiments 28-32, a vector according to any one of embodiments 33-39, or a cell according to any one of embodiments 40-48, and a pharmaceutically acceptable carrier, vehicle or diluent.
Embodiment 50. The pharmaceutical composition according to embodiment 49, which further comprises an immunological adjuvant.
Embodiment 51. The pharmaceutical composition according to embodiment 50, wherein the adjuvant is an aluminium based adjuvant. Embodiment 52. A method for inducing immunity in an animal by administering at least once an immunogenically effective amount of a polypeptide according to any one of embodiments 1-27, a nucleic acid fragment according to any one of embodiments 28-32, a vector according to any one of embodiments 33-39, a cell according to any one of embodiments 40-48, or a pharmaceutical composition according to any one of embodiments 49-51 so as to induce adaptive immunity against L. intracellularis in the animal.
Embodiment 53. The method according to embodiment 52, wherein, when the polypeptide according to any one of embodiment 1-27 or a composition comprising said polypeptide is administered, the animal receives between 0.5 and 5,000 μg of the
polypeptide according to any one of embodiments 1-27 per administration.
Embodiment 54. The method according to embodiment 52 or 53, wherein the animal receives a priming administration and one or more booster administrations.
Embodiment 55. The method according to any one of embodiments 52-54, wherein the animal is a pig. Embodiment 56. The method according to any one of embodiments 52-55, wherein the administration is for the purpose of inducing protective immunity against L.
intracellularis.
Embodiment 57. The method according to embodiment 56, wherein the protective immunity is effective in reducing the risk of attracting infection with L. intracellularis or is effective in treating or ameliorating infection with L. intracellularis.
Embodiment 58. The method according to embodiment 52, wherein the
administration is for the purpose of inducing antibodies specific for L. intracellularis and wherein said antibodies or B-lymphocytes producing said antibodies are subsequently recovered from the animal. Embodiment 59. The method according to embodiment 52, wherein the
administration is for the purpose of inducing antibodies specific for L. intracellularis and wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies.
Embodiment 60. A polyclonal antibody in which the antibodies specifically bind to at least one polypeptide according to any one of embodiments 1-27, and which is essentially free from antibodies binding specifically to other L. intracellularis polypeptides. Embodiment 61. An isolated monoclonal antibody or antibody analogue which binds specifically to a polypeptide according to any one of embodiments 1-27.
Embodiment 62. The isolated monoclonal antibody or antibody analogue according to embodiment 61, which is a monoclonal antibody selected from a multi-domain antibody such as a murine antibody, a chimeric antibody such as a humanized antibody, a fully human antibody, and single-domain antibody of a llama or a camel, or which is an antibody analogue selected from a fragment of an antibody such as an Fab or an F(ab')2, and an scFV.
Embodiment 63. A pharmaceutical composition comprising an antibody according to any one of embodiments 60-62 and a pharmaceutically acceptable carrier, vehicle or diluent.
Embodiment 64. A method for prophylaxis, treatment or amelioration of infection with L. intracellularis comprising administering a therapeutically effective amount of an antibody according to any one of embodiments 60-62 or a pharmaceutical composition according to embodiment 63 to an animal in need thereof. Embodiment 65. A method for determining, quantitatively or qualitatively, the presence of L. intracellularis in a sample, the method comprising contacting the sample with an antibody according to any one of embodiments 60-62 and detecting the presence of antibody bound to material in the sample.
Embodiment 66. A method for determining, quantitatively or qualitatively, the presence of antibodies specific for L. intracellularis, , in a sample, the method comprising contacting the sample with a polypeptide according to any one of embodiments 1-27 and detecting the presence of antibody said polypeptide.
Embodiment 67. A method for determining, quantitatively or qualitatively, the presence of a nucleic acid characteristic of L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment according to any one of embodiments 28-32 and detecting the presence of nucleic acid in the sample that hybridized to said nucleic acid fragment.
The method according to embodiment 67, which includes at least one step of molecular amplification of the nucleic acid which is to be detected in the sample, for instance a step of PCR amplification. Embodiment 68. A method for the preparation of the polypeptide according to any one of embodiments 1-27, comprising
- culturing a transformed cell according to embodiment 41 and any one of embodiments 43- 48, insofar as these depend on embodiment 42 under conditions that facilitate that the transformed cell expresses the nucleic acid fragment according to embodiment 28-i) and any one of embodiments 29-32 insofar as these depend on embodiment 28-i) and subsequently recovering said polypeptide, or
- preparing said polypeptide by means of solid or liquid phase peptide synthesis.
Embodiment 69. A method for determining whether a substance, such as an antibody, is potentially useful for treating infection with L. intracellularis, the method comprising contacting the polypeptide according to any one of embodiments 1-27 with the substance and subsequently establishing whether the substance has at least one of the following characteristics:
1) the ability to bind specifically to said polypeptide,
2) the ability to compeed with said polypeptide for specific binding to a ligand/receptor, 3) the ability to specifically inactivate said polypeptide.
Embodiment 70. A method for determining whether a substance, such as a nucleic acid, is potentially useful for treating infection with L. intracellularis, the method comprising contacting the substance with the nucleic acid fragment of any one of embodiments 28-32 and subsequently establishing whether the substance has either the ability to
1) bind specifically to the nucleic acid fragment, or
2) bind specifically to a nucleic acid that hybridizes specifically with the nucleic acid fragment.
Embodiment 71. The polypeptide according to any one of embodiments 1-27 for use as a pharmaceutical. Embodiment 72. The polypeptide according to any one of embodiments 1-27 for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L. intracellularis.
Embodiment 73. The nucleic acid fragment according to any one of embodiments
28-32 or the vector according to any one of embodiments 32-38 for use as a pharmaceutical. Embodiment 74. The nucleic acid fragment according to any one of embodiments
28-32 or the vector according to any one of embodiments 32-38 for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L. intracellularis. Embodiment 75. The cell according to any one of embodiments 39-48 for use as a pharmaceutical.
Embodiment 76. The cell according to any one of embodiments 39-48 for use as a pharmaceutical in the treatment, prophylaxis or amelioration of infection with L.
intracellularis.
Embodiment 77. The antibody, antibody fragment or antibody analogue according to any one of embodiments 60-62 for use as a pharmaceutical.
Embodiment 78. The antibody, antibody fragment or antibody analogue according to any one of embodiments 60-62 for use as a pharmaceutical in the treatment, prophylaxi or amelioration of infection with L. intracellularis.
PREAMBLE TO EXAMPLES
In the following examples and in the figures, Lawsonia proteins are i.a. identified by their locus identifier (LocusID). The following tables list these LocusIDs together with the corresponding SEQ ID NOs used in the present application:
LocusID SEQ ID NO: LocusID SEQ ID NO:
LIC037 17 L11093 15
LI0890 16 LI0494 1
LIC058 14 LI0041 8
LIB022 12 LI1114 7
LIC041 10 LI0535 13
LI0996 3 LI0855 6
LI0668 5 LIC048 9
L11077 4 LI0138 2
LIC047 1 1 LIC091 54
The amino acid sequences of the proteins discussed herein are provided in the following list LIC037 - SEQ ID NO: 17
Double underline: LIC037-A
Lowercase: LIC037-B
Underline: LIC037-C MKIKNKIIFISIVIISMSIMLLILSKYSITKLYSIDSYITKNEYTKLYYFLDKKFFYDAEYVKKOANDNKLLKKM YKGGDDRSILSKKYIALLETHESTFEODINSOVNSVYMCPLKKNLNETPCSLPTAEIGNKKNDSEENHPTV EOIDDILOETEIEOTNDTLSKASPATNPKFATOKOOVVSILPSKHISLLKKIIKPSOHSSKNRPSSVAKAIIG IPGLPLYAVNLYGORGDAPSGGDKKPSGTEGNNSSSDDEEKEEKNHSHYDSEINEODKHTDKGEKSDLV GAVGGTSDNFTNLDGNKSDGSNGFEVLDLPSHNHSEPWQKLIVVDDESSHESHTENSTQKNESDNNAE PKPLKDLYELGEHYPPMFNQPKQDMSMGIIGPGDDLIRPLINFCEPQFQHYKYIHHHHHshhqhryhhqqqq hhhhplhhhykiqipllspnknkpqpljTnifilcqrhlqtknpsidnqqn
glswekdfgqssnrgknkhleeqndgehsnssnvsiaplmshssstgqqntsvennqhsngeqnppsvgcehrgpmstsnhs sscfvqlnksgsdfnnesnqpsgpkkeivqsinsspesehtpsnnssnssfekylhlcyssvgneashssgessknngntqeehtl mphnnsppvsqsfypkndneseneasseysssefsfekernfdhfssengnsldgfevvehgpgnsaevspfkptvqtgfkgtp sptrt lllppsssddsksleqslksqqdsnndessdwsssnsfdltsenkkiflvqtqqhphrppkkndtkqphsepsenpnqedq iqqnepsvqenylCFTKSSGSNHSHPSHRDHYNEHEONPPDETEDHSAHHOSOSSLNDKSKSSSKSRSS SIGSNSGSSIILLNGSSSGDSLLEYPGPEDDEGDCLGRECKRKRNSDSNSESDYSCSFERLGNSSASWIL RNHGGSHSSTKSNSSSSSSIIILEESSSEHYNNNRDSSYTDSNOGNNFNSYEEDTGHOSEDGGNKSSS SSSIOILSTSNDSIDWPNSRNNSESEYDGVNDGIAWPGSANNSGDEHNSDDGIDWPNPRNNSEPEHD DSDDGIAWPGSANNSEDEHNSDDEIDWPNPRNNSESEHDGSDDGIAWPGSANNSENGDDGSKSDSD SHPEGESNNNDTDGGNRSGNDELSDGYSNSSGSNGENSSLOYRRPVMLGAMPPARATPRRGNPSGAN VRQVLSKTPKNLQATQAKRPPISSLDSYRNACKALTQAIHAIAQSSQQAMRSLALATTTGYPDTTKRFRIF GSASTKKPYPKKHQPYAVTVGVIANPFSAINLGVAYTYTQHTTKDFHISNTDALFEGSAAGKLQTNHISLIT AWNPEGYGLTGFLEYAYGTGDATLIRKFAYDNLAFTAKGKTRTHTDGGTARIGYRFKVTETIAVTPYGELA AVRARWNNYSENKGKFPANISSTKELVREERLGIETRWQIVDNCQIYAWGAYISSKKKNGVGIEALLTID DHTENMTVSKYSRKQSMGEIGAAIAIKPTEHFGISFNTSIRTKSNSKVGLDSQYIGTSIWYAF
LI0890 - SEQ ID NO: 16
Underline: LI0890-A
Lowercase: LI0890-B
MNNTCVLSLINYIKLLYIIKFLNRYRLVKYINNINNNSKYLTKINIKSIPCILILLFSIVCIKVTITFAMDKNSD MNDKEASTPNISNFLKGLVPKKLHCYLDDEOTELDINVTAIKESOKTKNPSOKSIIINCTIIHPOLREPLIFG SYTOVNOSELELLKTLVOOPITIROLAOLGLSGAESSNKLOTAAOLTSPLGSKRKYAPVSSTETLTTRTSSS IETSSIPOSTOSASSSIITTSNLEEPVVRPKIPSTKOGKNKAPLDKPOTLVTASKRSVFSLNLOLLENLGLOK IKAIYEEGOSTLHILLOTIKDLHREGOADKELTEKYRSMLEOOVELAKFIKEKEEEEEOOOIKOVMKRHEN VODLLEMLKKNISCSTSTPEOINFDLLSLPVDOLSOLDLTTLKNIFEPVESKRRTLEDOIROOIKEGKOVDP TLOOKYOYLVTLOTDVLMATKNFSEEKLSOLPPTSGLAYRLOEVKAITOLLKEKIPPSPKSEDGNNIOFIILE
kkddrrsetskttyrinqqqiqttntkektkkknliatldkssslletdiepphsseqqsssssnstkkkkkekktspvkthtfsanipse evitttsisseqttvqmpleaktlvatgdkqqpsssshhkqkkkqtqtstlssnqsllsihanspspsiestsqqsnamplktkeaien ectpdiprkkkekkkhlkhtpknpsssiptcceeektstsnedstipyptlnrpefeelkipdtpkkkpkkkkkpglgtlvgdsllsegr ttstsdttpiqppsgfinqdhkdkkealkncplsppdefsdddntkedsgpmyipgvfcgnnldlesprpkkphtylpniigdvtsitt etletldiynsplgegeednedpqknvgeneeiqvavreseqsqaqqqqqpqctnntgtsplgqntpqhpivaqtpaqptplpgn grrarrqqtrqtqlltkkhpstnsliaellpegqfsSLQSLQFALSDTSEFISETLKSLALKAHFSSKHLSTTTHKKLQLC LNNRKTSGSLNKSTIENTNIIEALQPTKSYTVFTSINKYTTNLENKLRSAQAGIMLNFSPTTNIGLAYNFNKK EYKEYSGTQLNSSNGSVKSKSTTDGLAAIFTLNPEKKGITGTMIGFYSWGKVTNSRRVTHGEKHIVTKGS PDITLTGGLIHLGYNIPAIKTLILTPYIGCLVSNVRWSPYTEEQSPVVCKISENRENLLEKSIGLKTKWELTEY SQLQTWIVGSSGLKQTNSLSSNSMITPLFKYTISAPSYKKKYTKAEGGLSYEVQLSPTLTLDIDGILSFEKR KKIEKKQHISFHFNYYY
LIC058 - SEQ ID NO: 14
Underline: LIC058-trunc
MLTIFIYFILIFLFFCRRYTHMYMYETYCIKSLTWDMYGFYSWLVQYSIKVFSRNKVFIIFSSFLCVYLTAGEA FSISSIKRGYNSPSPPPPSLVDLSLGDSODLGSDSSSSGSLLGSFODLSSESSFDDGHNPSPPPPSLVDLS LGDSODLGSDSSSSGSLLGSFODLSSESSFDDGHNPSPPPPSLVDLSLGDSODLGSDSSSSGSLLGSFO DLSSESSFDDGHNPSPPPPSLVDLSLGDSODLGSDSSSSGSLLGSFODLSSESSFDDGHNPSPPPPSLV DLSLGDSODLGSDSSSSGSLLGSFODLSSESSFDDGHNPSPPPPSLVDLSLGDSODLGSDSSSSGSLLG SFODLSSESSFDDGHNPSPPPPSLVDLSLGDSODLGSDSSSSGSLLGSFODLSSESSFDDGHNPSPPPP SLVDLSLGDSODLGSDSSSSGSLLGSFODLSSESSFDDGHNPSPPPPSLVDLSLGDSODLGSDLSLGDS OYLGSDSPSSDLSLGDSOYLGSDSPYSSDSGEEDNSSFSYTHDGISRRPCILSEFDGVNSSTSSIDTVVV TOPKGFSENHACLDTVKAVCVGATAGGAIGCGIDWICSCLGISSNVGLYSACGTCGCSVGAGVGCVICC VGHYWKELYKKDRLCLASPNSVLGKCDSHTDSRSSEGSSSEKNHSTITQTQQCSNTEKNKLLGVDKTTA FLLPGYNYKGQVCSLFGIQHMLLDTSKRISTTVKMLGMKACSSNITANEHTRFGSHFRRLASKKSKHIFSG ITNNIPYRITTSLDSYCSNMEYKAASGQMGVLFDLWPTLTAGFIYGRHNDRVQKTNGMQGDLKSSSVST RKKLDSLSAVVAWNTKKNGFIGHIVTYYGWGTVTNIRSVPHADCMVKTKGTSEIQSIAGLIQLGYNLQLS SEIVCIPYVEYMFSSIRCAGYQEYTGILPSKFTRNNEQTQEKSIGLRSQYKVSQNSQLQGWISYVSGQYHF AEFKSTLLGIPNIRYSVSIPSYKKEYSGTEIGMIHESKLTSRCTIGAHMIIHLYHNSVYNMYTGAQIRYHY
LIB022 - SEQ ID NO: 12
MIHLFKDINKHFFTISLIYTYIYISIIIHNIKSIVNIAILSMFSILICYTNAFCINNTKESGGLIKHNVRAQIAEE RKKAKAEGRLEEFEKNLKAKRAEEQKQRVAAWKAKKREEKKLSQTEEERKKSEAHRKGQLKRIERRQEV NKRIEIAIREGNEDEVRRTISQEEIQIKKERDLKYKEEKKAKILIKNPPLTEFEKLRRVAISETQTERHANDKI LAKKEEELIKIESADIVKEKIKREKLQRIKQNRARWSSKNRETKRAELSEEERKKSEARSQGQKERQQQH KEIKKKLQKALKEGKLEEVKKEIELSKLERKKFLKNRQLIRNATKHAEKQMAMWLSLPEDEKNRRMKISQR CSEYHSSMKKIRKKSKSAEESGCMEQFLEAFSEELQLKEETVKRKKKIQKEQKKKKKKEKQSKKQNIDTG QEPEPPLMEDEYCFLLGDLQPSFLPSEEILLEPDQTFLLDLETIDPSSLDLQIFQEESTSTYREIAFSNSNSEG DNSNNIPSLKDNALQQSEEIQCTETAISECNNTIKELDLIEGDSISSQQPKGKQSSLTASIVINQTHSNEL QILSYIDGYNKKSQLSSLKSLQNLFYSPKEQMLARLKTFVLQSSFMPSDVNYYEYDSEDSTNTMLQYPTML SNIVKNSCSNLKQYNFFGYSKNNSKNLELKPYSYQIGIITNIKSGLSLSLAYNRDKDKISSFSHMIMESFCS PAQSTISSDCLSSLFIWDPSYSGFKGYIVSAYGWGQMKNIRYAIHNKKEICTKGETTVTIYGSLYRLGYTFP LGKSIMFTPYIENSFVQIAWESYNEHFGVLPCKISSYKEKMYERNIGVHISWTDNDCSLLQAWTVINNKR HISSKIEAIPLSSPKSFFYLSQIPKNKKEYTQKEIGISYKRKIIEEMAITFDSKFFYAKHNNGKEVECSIHYIF LIC041 - SEQ ID NO: 10
MDLSHQYNIIH NIYEHTH NYLSLFPKKCKNYCIIILLCMCYILFNYEYSFSVKPTRQLSTNSDSSVEQQSEES SSSSEEEEGNPGSDSRNQFLLLWEQETSNVKEQVFPLQEESSSSKEFIDKAVQTGEDSSQSSSQSSESS TSSTSAALGTSEMFFLSESEEVQEVIITSSEEQMQQEVISNLKKKVKKLKEKLQQERKRHQQQMDKLKTC AVRQVQFLQSMVKMAQHQIQKVEIEKLEAQQVAALFQEALLGQEAQSQSSTSRSSVIISPPHGEEN KSPC SPLSREDEGSDDSMPPLEGEEGDIWRDEYVSQEQRSQFFLQQQQQQSSSLSEVSQTGVVGSSRGLPTI QEEDEDGSSCSSIEGYLSSPTPSVLIAEEGSGSDLEESVKSPSEH PSGVDSDTLEEGEVIGEEVQGPLTTK RPAPSSSSDSSDDESLSPVVKRLKQSSSSPPKSPGSPSSRGDMSSMLIPGYNH ESQYGSLKGLQCVLFG AVEQMSANLKTLVLKTSLYQAETKDKKTSSLIAKESKKACMSLCGKERSVKKVMSDSSNLLSFLNTCH FF GYTDSYLANQENKVSSGKVGLLMN PISNVSIGLTYN RH KN HGKEYHGAQLGTSFGSAKAN MELDGFSAV VAWNSEEKGLTGYLSGCHNWGQMKNTRQVMQAGKEMRTKGSPDVQLSGVLGQLGYTFPLSKKVSCVP YIEATHIVVKWNQYREDSGSLPCVISGYTEKFQEKTIGLRTSM KITDKAILQTWVAGGTVQHTTSNITSMP LASPLLLYKTSVPGNKKKSFYSECGTLYEMNVTDFCKVDLHGSLRFKKDQKLDAGQMRLHFQYLF
LI0996 - SEQ ID NO: 3
MPIYEYYCTQCDNTFEEWSKNASSH EH KKCPNCGGDAKQLISNTAFILKGSGWYVTEYGNKKSEEQNKK NTSDKTEVSSDSSSTTLESSGDGSSSTETH KTPSISEPQKTSKEKPKVQQNTKSAVTASSEVTSSN EATI SK
LI0668 - SEQ ID NO: 5
MRAPFASASPSSLVSSSVISTATLSSVVSSSSVVKVTQEVDKPLTKSSSSKMSVKIDSTSNISTTTTTTTTT TTTQDSSIKQAPLVPQKPQRLSIPLTTSVKLTTSNNSDNDEDWGDDGWDAPH PESSAVEHGM KQDFLE QGQSLFSSVLNGTYSRNTISERGERDQSSASPSASSYQNGRLVDSSSSCPLSLHQEGEPGDSFDTAKAK LEVFMQKRH EPPQQPTKKPIGPRDEPNNAFKEAQSKLGAFFFKEEGWNAQSLKEPLKPTGRQSEDYKKN R AAMELLFAGVIAQHQDIQDYQRDRLSSPSSLSSSCLVENYEQGRQKWLKLKEDLEKGERVYEGMYGFFQI SNQN EELRAKLFAIHKKN ESLKSELESLGQELEDMWH RGLV LI1077 - SEQ ID NO: 4
MIEGISSSSISFSGCSGCSVHSDSQKSKKENTNTPIQN NLSKEKDTDATGLRLTPEQKKEVEELKKRDQE VRTH EQAH MTAGGDLTSPASYTTTKGPDNKSYATGGEVQINVSPEEGN PEKTIEKARKIRSAALAPAN PS SQDMQVASQANQMEAQAKIEKNSIENAENGKNKLSEKNTITTKKQEKKSIN KIGN FSVN ESLIGFLASPF MFSSAKNTTIDGNMIGAH LPPSQKAVASLYSYN DKLAQETLPSSLSLMI LIC047 - SEQ ID NO: 11
MIYIYITICYSYTLTVLFLFQKKLLKTYYIFVAGVFIFLCTAKH DHAIAMDRLSGIPQRDQVETDSSSSEGEEE EQGATGIDMGFYNTLDSIEFAAEVTGRNHSEECNYRYENRRYRKKKEREEEIQEEKKRCEMIEKQCREVT HYQNVKQKVESFIAYPSLVAEQKCWMDTYKEEALDRLN NVSQERH EKERSIEEKQQQILRSQEINQRLIA EGQSESFFNIFEQSESRILRPVFRTRFQLRVRREELNAYFN RMMDFLYGSDAFLQEIIDEEEEAECLEREER VALDALEQLRVELEH N LVQQHQEMQRRIPDNILNH REELILQELTWSYTEVLSNAFIAYDTHCQTLSAQQS RPPFFPYLEEAMRQKRSKAEIVASVDMALLSLDDSREDSESSERGEIHDVSWFGGNALLDETLGEVEEIEH SESSKSDVSQKSTACTEGESEEASSTQQSEQSISLEQRYLLQGARPRQPSKGGASDSSLSFIPGFSFGGQ ISSLQSMQYILCDVSEKVSTELKSLVQHARQQQYDKDN ECCLTN LISRKRYSSVPQYDTVQHTYNISSPY RVFGGVDSYSKNLEH KIRSSQAGVMFTPH NGYTVKLLYDRH KKDVQEHSGLQLGTAIGSVKSYIDTDGL STIFSWYPCGSGFTGH LAGYYGWGQLKNTRFFTHADNQACSKGSTDIH LNGGMIQIGYIFLLSKKLAIIPY IESILSRVGWHSYKETIGVLPCTISSH KEVCLEKSIGLFCQWNISASTQLQCWGIRTSGTSNVDKVTSQPT RGPNTYTIVVPNYKKKYM RTELGLSYSSKLTDFFTIVLNSNAQFNSN KRHIQNATVH LRYT
LI 1093 - SEQ ID NO: 15
MKFFIYSWLFQFFLITGGMILFTVEYVEAVSWKWSMLPGRERIIISYDTPNQGKKASRISTTQLEIPLSTPA KDLSRIGASPSPESLVSDLSLDGSKLHINLRDAAFGYITTSTNPNQLIIDVFSDPLGNRWRNLGVPAPASA PSRASSALPEVPKNISKQIKVSNNGQRLNKDQEKTSLSQEQKQVAKAPAESITSEGKAEKKQSIKADTSK DNVKKSQDVKGTLPVDTKDKQQVSKKENQEAISKEEKIETVPTVPPPSVDELTM LTAKVEKSQLASDEYS WSKKAKSPAQEDNTEEKLEKSQN NN NQKVNILENSSSSTEGTVQSKLTETPIVEAPIEETVNSIHSKLNFK GPDAWPKEKALSTLDKLEEVLN KN KLMQQPETRDSLQPLSKTNEEKPVSEPVVVYVDEKGN PVGKPPDTT AIIEEAKKNM RAGQVQKAKDLLATLKGHALVQEQHEEVLYLFSELNEKIYKDRWIEGYEPIITSTN KAM NF NLRSPRVAEALMRLGMVNLRIGNQDEAAGYFGALRRKFPQSEFIPEAYLALGKDQFSKGEYADAVKTFQLI LDNYPESKAVQDASRFMAEALFKQGHYSRALILVDFVDRRWPRLYLEDPNYLKMVGDLYSRENRLDDALK AYWTYYN LVPEAKDSH DTLFKIGTSYFKKGLMQGGKDVFEELLKKFPKSDSAPKALLALGEEQVIKENPTI QELVTIFENPSSTIPEIYYKKILDEYPNSPEAQQAAIRLAAWKLWHRDIPTAMTMAQQFLDKYPESPYAPRA EEIIARGFDQSFALALQEENYERILSLWEKYPYLQIAYKDMTDELRVALARAYLN RGDEEKGMDLLNQFLE SPQDPNYGDYVYNLYLAHYLRKEDWTNILKLGEKVSSWKFPVSARAQLDYALAIASEN LGLGN KALPLWE RLYQREDIPLYQKAYANYFMARDAERRRDLNAAYKLN LNTLKLFVTLQEERSDKADPERVRESLAALM DVT EVANRFVESLEWADQYAAFVPETSPDYAGLLFRKARLYRKMGDLSKWKQLLDEIVRREPESVFGRMAASE LRTYEVSRDLSRFTQ LI0494 - SEQ ID NO: 1
MKKWMVICFLVWFVSPVMAGSRFDDIDLN KDGYISWEEFQTVFPNLKKVAFDAIDTN KDGKISREEWER FQKSH EQNVQSEEGKKEM DNVKM HQRSPEELTDSEQPKTTSTKSH MKPLVQPPVTK
LI0041 - SEQ ID NO: 8
MESN MKVGPSDPGINRVQIEEQPSTESISSSSSGSSSGIRTREQSSNLDQRRMTSSN LRMSLRLN PVVS DKVLKFFGLRSASGSYSPSTSSSSSSSSATRSSRYSSTDSLSSRSTIGSNITFSESDESYYSTIEEDSESIY ESVSSSSSSDIYTYDSNSNSSSVSNSTQSQAPELPPRPNVLQSEAPEPPLRPTSTSSQHSIQSQDSVGSD DVFWSESEPESLYADKEQVGEGAYGPIRSSLEEPIVLESNYISFGSDGVVYSDFAPMPQVTSKSSQSDSV STPPPVSPREQVAGSLGVGSRSKLSSTN NVASSLFDPN MQRIARQAIDKTIHGNFGSPNGPKVDFPYQVG GYSKTGYIAGTDLIIQQHVN PTEQEVYTFMSQVGEWLKDPQGGPENAPEVLSGLTYPQLSVIHSYRDRLL GSLPIPVAMGGTSDNSAVVLLNPN KQPDGSGGVMN LSGFESTDVKVGPILVSKSELSAHYSKQAGGLGW ARKVVTGRIGPGM RGATIRGYDMKLGKN ERGFSRLQTAVGNSDDAFPKRLGGLTSNQLKN LEKTMEGLQ QAAHFLPVTFVGSTLQFVLPNPGDTTTMPRVM LGDIGH PIFKDEATGDGPISPRIYDKYKSN FTSGMDSIK ETISNIVKSKIE
LI1114 - SEQ ID NO: 7
MLDFIRSNAQSWGVKIAFGLIIIVFIFWGIGTLSGGPEVEVVSVNGQSITIQELQKRCEEIEQNIRQNFPTI SAAEINTFRIKQLATQQLILETLVLQEAN RIGLIITPIELRKTIESFPAFHNTEGKFDPAIYLQFLKERNDTPGH FESQIRTN LLINKIQQEITAGAYISPTEAYDVYMYESATRTIQYLLFPTEDYLN KVNISADEVAKFYN ESSEQ FKTPTELNLEYLLITPRSLAN NQTIDDTTVSEYYQKHKEDFKSPEQIHAQHIVVFAPENSEPEVLKKAQEKIN QAANAIKKGEDFSSVAKKFSQDNVAQNGGDLGWFTYEQAVPAFADVAFSLTPGEISQPIQTPVGYHIIKLI DKKPEEIKSFDEVSKTIQQRLAEDKASIELQDVLENVQLALIEN KSLTEAGKPYNLSPISTGLFPIKDIASQL ELAKPDELSKLTHVSPGTILEN PLITKTGYVIIKVKDIKPESIKSIEVVENQISSLLKKQKAN ELAM KAAEEQL TILKNSSIPPAVAKN LKTSPAIIRSATIENLKNPQLVPDTFKATPGSWLTTPYTVEQGVLIAYVSKEVVYPSK EQWDNIKEAFMSAIVKSKREQMFKGFLTMLEKQAKITIKEEKIIN
LI0535 - SEQ ID NO: 13
MINDKIKVKDLAIELGVSTKDLLRVLRELEISAKSTISNISIEDLPKIRAQFNTPNTNKEEERRQIQPGVILRR KRQQPSTNQTDIKLNPVDTNVSESTIQTENLILEN KNTLSPHIEETTEKIPATTN EILYNSQIAKIVQYPTPS VPNKTLTTTPHQTKKN HSEKDVLESH DSSN KNIKQSSSQNTEKTN RKPAASAIPEGSSAPSLLPPVSEQIR RLH DEETENSSSEEKNVDIQQKEIPSTQVRVISKPNITQSHSWDANNTRSSSGQRTETEKQSNTAPHTD SREHTGH NKRPVSYQGQNRNN FIATPDTIPNVEHDGQN KKKRHTSRRSTEFNH KFQYNN EDDDISRQN RGRKRH KQKTTSQVTTQPIKLTKRKIRVEEAIRVADMAHQMGLKANEIIKVLFN LGVMATIN MSLDIDTAT LVAAEFGYEVEKIGFTEEDYLVATAPEQSESLKRRPPVVTIMGHVDHGKTSLLDAIRKTNVTGGEAGGITQ HIGAYHVTTKSGEIVFLDTPGHEAFTTMRARGAQVTDIVVLVVAADDGVMEQTREAVNHARAANVPIMVA VNKMDKPEAN PDRVLRELSDIGLVPEDWGGDTIVTKVSAKSLDGIDELLELLALQTDILELKAN PDKPARG HIVEAKLDKGRGPIATVLIQEGTLHQGDTFVCGVFSGRVRAMFNDQGKKVKDAGPSM PIEVQGFEGVPE AGEAFICLPDEKLARRIAESRAIKQREKELAKESRVTLETFLSKTSNEKEAQVLN LVVKSDVQGSLEAILEA LRKLSTAKVRINIIHGGSGAITESDILLASASDAIVIGFNVRPTAKVKEVAEQENVDIRFYDIIYKLVEEIKSA MAGLLAPISREVYLGQADVREIFNVPKIGTIAGSHVSDGKVLRNAGVRLLREGVVVYTGGIASLRRFKEDV REVQKGYECGISLEN FN DIKLGDVIESFETVEEAAS
LI0855 - SEQ ID NO: 6
MN NKKFSTDEKN H PKN KLNTGTVFMGPSPDKESSLKQVIGTH EQEVWN RRTVSEYMTRVRERATIHVQA MLDKARANAETIRKTAKQWAEKTKKESDKLH KQAQQALKEAERIREEADHIRELAHEEGYRLGIEQAQEE IKEQIKSIHMTAASILKTIERQYTVIFDNWRSDLVKLLHTATEVATDWILSKEHTAILDSVLNKAVQQLEER QRVTIRVNPN N KNTIIDLITNIKNQFPELKNWEIKIDVTMGENDVIVESRSGMVDSNSQLRKEEVRAILQQ LIIPESEIDIVAAQSIADIADITGITALSKEAAILQHTPVQN DSSEKTPSSKETLSPNDKKPISPAEIDLIN PPS EEVEFTFPELEGIELPPDIPQYEIN KELSSGLSSTDESTITN PSLSTSLTDSFTSDISTDNNSILTSH HTDIN D NIERDIFSELPKNLQMEEFSSAMVEQGSN HELN MDNTPSQVTLTNFTPS LIC048 - SEQ ID NO: 9
MYDRKFKNVKYVLQEPIYQHKDEYPKVSKQLQEKEEELKVYDAELEKMYGASVKVLQKKLSEIQNVGGEL DREMLCLLQKLGDRDVITQEKTELQSRLSIMKIDVSPNEFKIDLIDDASIRSPEIYRPFTLYTKKNAQDWVQ EKSRERNIKEAAEIERNCRLVLEKRGYVEEGVKKETVAKVSLEECEFSDVDIDDSEDESESEEQPIISSRVT QIDNRYLTRIHHHPTPLRDDSLRPWNDSTPSSTTGQKEEETTATTSEAGSHSLPDNVLDQSGEGSSQATA SDQEQEVTHQESPGEVGPQEDSQQQQQQQQVQQTPGNQAQTSQSGETQPQAEGGDSHDYSTEGARP KLRGKDTSRGGPGGKATFIPGYSLEGHISSLYGMQAILLDVSEQVAFTLKGLALKVQNNAMVKDDVLQLN DSSASIKKKKQIGMQALQQESAFQELMPYQVIGFTDTKDTNLECKLGSSQVGLLVTPKDGLSVGLAYNRY KDTGKEYQGISFSAGSGSAKSKAELDGLSMLVAWNPRGQGFTGHVVGYYGWGELKNERSFTHDGAQVT SKGTPGISLSGGLIQLGYTIPITKQVSFTPYVECMVSNVTWNGYKENSGPLPCEVSSNREKVFEKSIGLRS RWDVTERSCIQSWVAGVSSYRSTNKVHVKPLIAAYDSKYEASIPSHGRQSTKAELGMSYKSKVTRNVEI GLQGKAQLDKSNKRPQHQVNVHFQYMF
LI0138 - SEQ ID NO: 2
MKVHAVIIRSGILSINLFTIPLAVAHNFSDQFVVETSITQSIKDGYTNLKSNVEETMSSLTDEKNTDAEKYL EQHDKDLKAYQNEIKKADQNYMKARKEAQRNYMKNHSQLPMEEDIEKDMEHIKSGL
Trx-His-Lic058-Trunc - SEQ ID NO: 52
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPK YGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAA KFERQHMDSPDLGTDDDDKIKRGYNSPSPPPPSLVDLSLGDSQDLGSDSSSSGSLLGSFQDLSSESSFD DGHNPSPPPPSLVDLSLGDSQDLGSDSSSSGSLLGSFQDLSSESSFDDGHNPSPPPPSLVDLSLGDSQD LGSDSSSSGSLLGSFQDLSSESSFDDGHNPSPPPPSLVDLSLGDSQDLGSDSSSSGSLLGSFQDLSSES SFDDGHNPSPPPPSLVDLSLGDSQDLGSDSSSSGSLLGSFQDLSSESSFDDGHNPSPPPPSLVDLSLGD SQDLGSDSSSSGSLLGSFQDLSSESSFDDGHNPSPPPPSLVDLSLGDSQDLGSDSSSSGSLLGSFQDLS SESSFDDGHNPSPPPPSLVDLSLGDSQDLGSDSSSSGSLLGSFQDLSSESSFDDGHNPSPPPPSLVDLSL GDSQDLGSDLSLGDSQYLGSDSPSSDLSLGDSQYLGSDSPYSSDSGEEDNSSFSYTHDGISRRPCILSE FDGVNSSTSSIDTVVVTQPKGFSENHA
His tagged LIC091-A - SEQ ID NO: 53
MHHHHHHADKDSEKNINSSKENIAAPLGESGNTEDFQQAETSSNTQQPQVPTSPSGMPVTSGSQVVLD LFSPPPPPPPSSTIFSPSFLCAPLTLYGPPAMSYIPIPNMGVRRSYSSPDINCNFERKNEHEKSGSLSVPVLL EKCIYDKNSESKRCTNICLTIESIIRGHEETVKQKEKKKKSKKYDKRNIKEQQRRIEKRQQEEKVALLKQYE EKLQALQSQQASQRLEALNNGTDMYTLFSQQYNEEQDCHREKEKKLEELLMLHKQERDIFFIPVSDSTEK GTVEGRQEDYSDINKDRRHSTPSIPVTALNIFIEDSSKSTPGRLEHQDSYSNISNEPKSLSSDLDVYNNRD VVSSIPLSLINPDEEATVEVSASESLTETSIQVGPFPSKTSLLKYFNRVMNEEGFGSIQKYYRDDLYLPGCA VYQTAKKEHKRKHSGIRLPWLKRFNLSSKTKTDLQDESLQESSPPKSLREDGIYPPLLQHKRPSVYYTKEV GAGDDTPHSLKHVKYRKKRVGGILQSPEIPDFCKIEKIFRS LIC091 - SEQ ID NO: 54
Underline: LIC091-A
Lowercase: LIC091-B
Double underline: LIC091-C
Grey background: LIC091-D
Dotted underline: LIC091-E
MSIYITKYVINTFLSKWRAIVALFLCICIILNSVTVTISADKDSEKNINSSKENIAAPLGESGNTEDFOOAET SSNTOOPOVPTSPSGMPVTSGSOVVLDLFSPPPPPPPSSTIFSPSFLCAPLTLYGPPAMSYIPIPNMGVRRS YSSPDINCNFERKNEHEKSGSLSVPVLLEKCIYDKNSESKRCTNICLTIESIIRGHEETVKOKEKKKKSKKY DKRNIKEOORRIEKROOEEKVALLKOYEEKLOALOSOOASORLEALNNGTDMYTLFSOOYNEEODCHRE KEKKLEELLMLHKOERDIFFIPVSDSTEKGTVEGROEDYSDINKDRRHSTPSIPVTALNIFIEDSSKSTPGR LEHODSYSNISNEPKSLSSDLDVYNNRDVVSSIPLSLINPDEEATVEVSASESLTETSIOVGPFPSKTSLLK YFNRVMNEEGFGSIOKYYRDDLYLPGCAVYOTAKKEHKRKHSGIRLPWLKRFNLSSKTKTDLODESLOES SPPKSLREDGIYPPLLOHkrpsvvytkevaaaddtphslkhvkyrkkrvaailqspeipdfckiekifrslpsseeeqeselcps gpqgkiylftlgtqgsepgycimrdpfestscyypsgsdaksesseqgfyssnletenntflfnrvsppeelqygkkkstlplqkrtsc kknkkwkssyvvddkqraqskmelqkhqyyitdcllrleklhkhqyevakktgddlcslekkqakd kerlqatlneqyldlltkhqql iarnthpseeslseisegniedaessesdlerkhqakkkrlnkklkkllvenytigrnhrqsveetttkidkrhaqerchwttkkgpfsq clmlqteelwafynekekerekieaekkenlkkykqekaakqsylgwlfgwkkaasesekeksssedfktssdseleikevvslsd kehsefdhsdnyeydkskpkcyistkemkrhkkslqrdsrltaglslgaprqimyqdkkdeksdddkkvsqqnespqvtkest dakkevsafeenlksdealsvaditfeLSVLSGATGGDEDPLGKDKGSHEOEKEOKKDOKKWSDKKKKKWLTF KADOKKCPILTKEKYWSICCDNLLELHNRHEKEYKEAEKNASNIELLLEKOSOEOECLRSELEELWKOTRE AOASSSSESSGSIVSSDIRSRGGYDIESDVDOETSNKRARDKOISKLOKKOORERKTLLAMORTKOEOL DEIOEERRRKAKRLGMSLVELNLMOOEOKEMLHNOOOOOVNRLVEROROOMNELLASLHOPTMSESTM YTSCYSGIFGYLFGWGSQKVSSGKEENDVEQTPEKITEEKVKAVAGIGTLGSDAELEEGIFSDGADFSSTI LOVMSDSELSNDIEOVTEOVEYKGVVSRGSECDEIGEPMDESHVPEKIMRLFERATFVDKDSSSGKEGD FSSEERKSSSYRNRYYGVEKRRKLRKIHLKEKAOLEKOLKEPEEKIRLOLEOROOERRRKNLMFGYHISED FLDKLEEDEYKTSFEPFNKLRAELEO OAQERLSLOAAL KEFEESCQODSEDISDPEVKQKKKEERDLLV ALIEEAAGDIQDVEESEEEYIIEWK NERARRAALLASARESGKPLCFQFTAQSSSANAASSMHGSHPET QEGQSSD ASATTVEPSNITCPGDTTSTALT ETSSVLLQSSSSSIVTSQDPEEIQHTDENKQDTSLFDG ENR EQEKDSTPLGTGVSGGYHDASDHYGQGRAGEGGGGNNDPPVPSDSTQPSSSTSSTGVQTCQS SLIENAQDSSLQSSLLGYLLPSSITDKNIST STLKTEGVDQSVSSSRCPEALTASVTSLFSSSYLKVLFS G GVTNVEAAVTSDPNES N NS DIDILGENSGNTSIEGNQDSLSDSDGSGQSPSGATGGGSGESLG EPDDDKDKKESSGHKKKKKKKHKRKK EQGTSTET^
V G.eftDGKHDKDNNTVSPSE
DELECGFAIDAMKL^
QLEELRES^LQKKNKTLRKNIKDYKKKK
.QACSLYRKDGDQVEVLLLQQKEEYEK
.QPTFDS^SQGTTNS^WMF^RLDFS„D
LDS„NGNSSESGDLFPFSDGDY .GADVVSVIASSTASCGGGEEE
LKAiCKEEH NNATSLEEQQELAKQH ERKFGALSHMGMAQH LFLQEKLGKKKKRWLKGEDVSSEEECSLW CN FVFEPRGDDSDTISSEDETNVAVEVIVDAGTNTETDTKGQPKEVRTGGENDPLEGESSGTVSSKSKEL QEKH ECEIQCFWEEYKEQLVTLH DNQEQRLQKWIN DGKPEH DLKNTFGEEEAALHQTQGCFLKSLQEKH KREEEEERRQKGQEDSGTSLEQPTSKSKSKVLQEKHASEMQCFWVGYKEKLTILREKQKQH HQEWVES GKPEDELKDTFEKEEEALHQMQKCLLASIQEKH KREEEEERKQKEQKDKDSESSIIQGPLEEQN EETPAS RDSITSSQGDTPH RENTSLVTFKELGVESGEEEINKEDKKDSSEEQTSESKSSNGLLQKVISFFQSVGSSF NKEEGAVGGIVTESCYVKSKELQEKH ESEIECFWGEYGIQCRLLRDFQDAQRQSKGLRNTAEALENQLDK EREDFRKKKKKEWQN LQEKH KQEEEEERRRQEEGQPSKGGISGAKEKESDELGDETDGEKVFSLTFFGP PTQFDDN NAPPPFPYDPPQDSPQNGEDSSEINPN PEGSSQQESPSESSTEDTSSSSEDEDIVTLLSGNIK QARPIGQSTITTTTTLVTSVEGAEGLFPIEEETEQNLEDVQGSGATQGMVGLSTIEEEVEEAPGASEDLDS NQGATGEVSSPGSKEEVVYLSSSGERAIH KWLESSISM PVTMEEGSLSQGGGKTSTLYSLMVSSGILSFG GDGNGEKASSSN KQSKRSPEECKAFEEKQVQQRH NLLQSIEEKKKELLLTFEKQCPPGLPSYKKDKLVKE QKVTMEDFLCVSCYQYETLEQKQNEQRRRWEEGEDVAEEEDLILLWDTLDNEAEEGTKEEHAEVKVEGV EGEVFDGISEEDKPKKDDKEEQEQKATLGDSSGETIEESQQPQQEEEEKKENSPSGSNESPSPQQEEES VDETSSVVTSSPLLSIN EVKQTEDKSAKKKRSEKELDDFN RKQAEQKDCFLGQVQQAKNELEEKYGEAC CSASLDELLELEKKFIKQNQKLDSTSEAQFKKLERRQEDKRNRWEEGEDVSSDEEICFIWSSDESYEKSD RLH EKHTQELADFWRNYKKKLSTLKEIQQERRDQSSEDSKEKQAM EEMFLAEQEAMYKSQMNTFAELKQ QQKKEEELEELGWTLEN MQGAVGGVEPEDEDKIESPLDRKHQQERKSFWEKCRVDSKFIEQEQSERRD RWRKQGKPIASLEEIFIEEQQAVFETKM LFWESLQERQKKEREEKERREEQKRIAELEKQLSPFWKAFKD DQARMRENH DMRRKEWEESGKSLVVLEQLLAEEEEIFFQDKMQM LEELERKKYFEEKQSSRQKERQGRF STKDDSDEDEDDIRYHTVFVQGEGEPPGN EAPPPLPFPPPVLGDSGEMQSGDTGGAGPPSEELSLGNILS SSDEDDNIIVSSTESSSSSSSSSDESDVSSFETSSDEGPDCVDGVYGAVGGEEPEKKKGNGYH KKKFKK VKKKLH NVALNQKLKKLKDKLQQLEKEYH KSIADCQN RYQDVKCN LLMAQAEELIGKSREEQDTLCKKN SEKIKKIEDGYRRQYEQLTESYYTKKQVVLN KLSAVMEKKALLKQKIQEYDSVDGKHRVSKGYKKKASKL KKESKKLEKELEALKEQQTKELQDFDQKSDACRQQILFFQKEEVNSIKDAGGDLCALQN KHTEEDCKVEE LFTSQKELLIKEHESQRNEKEKRKKELEKQQQATREKDTPTCYEHGIDNIVKLLEEEIESLEKELEELEEKQ EEEWKAFELQTEACRKQVLFLQKAKPKDSAREGGEEDRRAFESMVASQQELLLKEQERQRTEKLIRLTEA KEKRKQH KKKQQQKQEEKEREKQKKKQEQVKREKERELERQKQQLKKEHVQDRKCFVTTFN FCKEQLQ RLHQENLKVVH EKGSDDTSLMQEYEKEKECFLTAQKSQYEKLLHQQRQEMKTLIQGSHGADVSSSQKEH EKSHAKTLEKLLQRQESCLKQLEDAQKQERQEAEKLGKDLSKLDEKH RKEKEDFFDH HKGILEQCLLQN L SETVCLLGRDESSSDEGSFDLEGFYSPDRCMGRVGEPIFTFVGSDFVRSQEASGIYPLEQQVEAFDEVDT GRKKEEDSYTDKEKKKKKKSKKERKLESELEKLKKEQAKAKSEFEVKVTSCREELFKSQRDEFVTAENKK QDLKALEKKH EDENVQFTSMCEEQFQILCKNQEEEKVRKEQELQKAKEDH KKKKEKKKEKKEKKKEKEIS KLQQQHIKAREQFLQSYKNCKEKMEM LDQEELLIVEASGGDVEAKVKELQAQAEIFFKIQKDQYKLLFHR QEQEMKALEASFYGDEVDAPTGARSIEEFLVDRMLKPQLSEDSEEELKPLEDPSTFYNPSKYGEGSFIFFT QLSSEQTRDQYGSDDENGFRYHTVIVQGEGEPKGDEAPPPFPFYSFVSGDSGEMQSGDTGGAGPPSEEL SLGNTSSSSDDDIITSSIESSSSSSSSGDEADVSSFETSLDEGSDCVDGVYGAVGGEEPEKKKGKAVDD VDGGRKKEEDSYTDKEKKKKKKSKKERKLESELEKLKKEQAKARSEFEVKVTFCKEELFKSQRDEFVTAE NKKQDLKALEKKH EDENAQFTSMCEEQFQILCKNQEEEKVRKEQELQKAKEDH KKKKEKKKEKKEKKKE KEISKLQQQHIKAREQFLQSYKNCKEKMEMLDQEELLIVEASGGDVEAKVKELQAQAEIFFKIQKDQYKLL FHRQEQEMKALEASFYGDEVDAPSGARSIDEVLSSEDSEEEPKPLEDPSTFYN PFKYGEGSFIFFTQSSSE QTRDQYGSDDENGFRYHTVIVQGEGEPKGDEAPPPPVKLRVPPSLTADLEDVGDGNTEVDSRCFNQGVT SSGISSSRSSFTTEEGSQGSGSMSSLVRLQVSSSLITDLEDTENGNTKTGTQQWLGNTGNDNSCN EST QYTDNSSHHSSDDDGTDGHSNDNINGAEGGSGAAGGSGGAEGGSGGSDDDN NPPDDNSSNEEEEEE SILLVSETDLTDTKAEDGDSEGEGGIKDQRKKRKH KKVSKRKQRQH ERQLLEKKSLAHSQWN EKKKQQ EKQRNRALLFSIQRKEDLSEQIRKEEREQQEFESNQH HERILLEAKH KRQNAEHSTDSDGEVH FPSKRPK KSH KKKSQQVGKQSSQVEQQVEKKDSKKQREEKDVPSCLLTDISYKRLLSSSQQEEKIHCVTSQQKEKE ELQKKQEKEKEEYKKEAAKVQKCLAKLSSQQQPGASRQQKQEEEEILQQQTKEGNQYILKSKVKLN NKQ LAEKDKLIRKHQEEN LKLSKKH RKEKTEKEKKRKKKEEIKKKKHTITPEEKELQEYCSGRRRTLYSMQIDE RDLLEEKH KRELGQILDM EEVEVLEEQH EEEVKELFEKQSKDRGLLEEQIKILLQQQSSHTTSGDEKKECV ESSDEFDSIPSSSTYYDSTSSSYSTGDFYSSMPSGEPEVEIGIEIDENFGVGTSENSNNTQDETH RPIGGA RKYQPKEYKDVSDTSKKQKKLTRSTPKLM LLSKKEKEALSH NVTQSYHNIYKKIRKKEIELEDVLRTEQEK DRRGEDADYQLILLLKDELQHLLSLFEQTLEEERQALAAVDAM DEQTELEQQSIEESEN KKKEEPRRKSSL LERIFKRKQEGDEPSKKGKEN HH ENQKRLEEEPLVGLKKRPKRTSTTSRLSLTLTSIGESKIPQCEKSDES NLVGPVVSFFKKELRTRKSSMIHGVCFPQTGGVLQSLDVNSQKN LVSRDDSKKLESSGAPGGGDGSGD DSH FKKDKGSKRDGSSDSKKKKKKHSH KRFCRKRLAH ERN KLRRKHQEEKELYKEQFTRGVQQVFALQ DKERAEVAKKGGDLEALAERHMQDEEEHYVRN RREVADLHDKQN KERCRLYLISKGGGESANTTVTSDE SSVSEILESGSEAAVYKEPVTQEVEELRRQQRKELAVLMFCCEDEKTALQTMH EKNKESLVGTILLETLEK VH KQEEDDLAAEHQKKKEELLKKH KQEQKDLENKHKRSSSKRGPKRKLH PKVKAVLLEH KQAKN RQKII HQN RKTH LEKKQEEEKRMISQGQVPSVLLKKVSEYHGGQEGTSLVELVALYH EGEMSALLDEQKRQREE REKRQRQDLIRAKVGAFFTQRTPSVRKKEGLSLSQSSLH DSSPQKYTH PSH KTCGRLAAEQAIEKGFFEN HKANCSFILGIEQKKELKQLEEECEKELKQLEEECDAAYAIFLQQLPFLCDEDRQTQEQH FVSQRQQIEKY REQQKEQLTCLQH KHQQEAKELEKLLEVQAKELEKRQKEERQKQRDVVSSYPERKAQQERLITQSKH LTA LKEALSEEDSIFQKDLTSSTIVKQQLSKVEQQKELEALQEDFVLDLCFTESDYTSVSEESSEESSVPEGISR KPHGPSGGGFRHPRREISSSDSDTEGKSDDQSVRRIEQEIQTEQTGPQGTEESQTYAVQQKTSQFVEVK EEEIQVLEEKVQIVEERGDSDKEKVLTREEKKRKKEEVQRKHEKELN LWMSQYETTVQKIEQKYKEKKAN RHILGCTLEELQEQEEKESKVAVGNFTVLLEKMREKQQKELQQFPSSDEDESDVDRRKKKKVIKTKAERN AQREQRKH H HTPHPYHPEESTSHLVLDTKQIVTLTPSSSDQETPVQSKETATN ETPIPSLPSTVKGLTLEEV TVTVLPQPTQLSSLLSYFSSEPLQEQPCQVEEQQVSFESSQVQASTDEATIDPEVQLVLDEYSSKVACLQQ EMDKKLQEIEEKGTDSSASSDTEWSWPKKDMPREIKTLKGSDSEGKDQQEVPKIPSAGAHSLSSMAESE DVGAVSHIEKKKRKRH KKHKRSQREKISRAKRALMAQYFAKVTLLGQECSEKVSEIQEQKKQKCEIEEKQ RKEREEFFDQHQAARNALLAKQQQELIGVAPEEVGPLQDKH RQEQQAQSRQWGLN LHSLIKQQRKERG LSSSISSSSSDEDIIDEGSQSDDQEDSKSLSSPISPPPSPPVSGADSQCIGGASSSDTDATM KSDGH KSP EVPVSSDKKEETGGNQSSKVTTYLLSVFTGKGGTAAGAQSSSSEHTGSKRQQPSGSDQTSKSSRQGPS TPFEGQGTSGVEGASGGAGDPGDGEDKKSKKLDDSHGKDQKSSLKKHKDKKKHTTEEEEAKKRLQEKV EQKRKN RELLKQKCKEEEEAFLRNREKCKKQFLQEQEEEYEAARQSGKDLELLLVEQRERQVEFYNH H ES QHAKM KKKHEALLKGKRKKKVKKYGTSLEEQQAKERAEARQKYQKGIEVMEKRQQEELDEASAQGKNL EDIKKKHRQEEEDFYREQMLGELGNLLQRQNQERQSVGLPPIEGSSSSGVTTYSDTEYSSLEMDLETKEE VYTSGNESTEQEVVTEQEVVVTEAEEVTEEKLLVKDESFSYQGSLTEYLSQAIPEEVNKPCIDDTSSSTSTT GRKSSFFEKFFSKVKGRKHSQEETESKSGTGSTASYGSKSSTTMQKDEVQQHSDRKLKHKQKKKRKKE KQLEEKHQQQQQDVYKDFKKRRLLMETRHQKELEEAISKGEDITKLQEKHHEEASVQHPLLFSQAMSVL REQQKVERENLIVSSGDSDTTSDTSDLEETGVSSKDHSPEKKGGKDGKPDPDGGSTSSSQYQGLVYYVT ADSAPVFPYGSEASLPFSSDKGKNEGKQQTLQCGDSGYESDNQESGDGSGGSSTENQVCVSQESNSSL GGGPHSDDGYESSDENKNGKGSSGPQSVCCVNGVGATGAANGGGSSVASGVGSGNTGSSGSSSSSS GVSGNVGTPGNGGSSAGSPGGSSGSPGGSGGNVGGSGNSAPGGNGLVSVTSSMGNGSNPPDPNDPL TMSLLADDEDTLSDEAKEELLVPLDYNENAIMGSLDGLQQVLHTPAKSVQSAMSSLALQSRTGHMSPTK RFQAFASIEGEVSSQSGNQNAYTSQFGIVIHPVPNIRVGLAYNYSRDHTGDLYGIRSKKDIGFAKSKVSTH ILSSMLVWNAEDSGFTAHIGISYGLGQVKNTRYISYLDDVIHTKGRSDTSILGGIAQLGYTLRLPKFLLTPYI ELSTVNAWWKAYGEEYGLLASDISRGEERIWEKSMGLRTRWSITDDIQWRVWSKVISGQRKSKELTATL KAPFVPWETSTPDKKKTYSQLELGTAFDIQLSKQFGIGVNGSIRFSKKKEMRQQNAGMRFWYNF
EXAMPLE 1 Host cell interaction, pathogenicity and immune response
Literature search, domain analysis, protein interaction analysis and additional bioinformatic analysis has been performed of selected proteins below with regards to host cell interaction, pathogenicity and immune response.
LIC037 lAutotransporter signal peptide
L10890 Autotransporter hypothetical protein
LIC058 Autotransporter hypothetical protein
LIB022 Autotransporter hypothetical protein
LIC041 Autotransporter hypothetical protein
110668 Unknown (associated to outer membrane proteins) hypothetical protein
LI1077 Unknown (serine rich protein) hypothetical protein
UC047 Autotransporter hypothetical protein
LI0996 i Metal-binding protein hypothetical protein Literature search was done in PubMed. The domain analysis was done by use of 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). The STRING database (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) of known and predicted protein interactions was used in order to predict the the function and role of the proteins. Additional bioinformatic analysis tools predicting signal peptides (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), transmembrane regions (TmHmm-2.0 - see Kail L et al. (2004); A combined transmembrane topology and signal peptide prediction method; J Mol Biol. May
14;338(5) : 1027-36; PMID: 15111065), lipoproteins (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 structures (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-classical secretion proteins (SecretomeP-2.0 - see Bendtsen JD et al.
(2005); Non-classical protein secretion in bacteria; BMC Microbiol. Oct 7; 5: 58; PMID:
16212653) were used to expand on the knowledge of the proteins. The result of the domain analysis shows that 6 are autotransporter proteins, 1 is a metal- binding domain, 1 serine rich protein has a functionally blurred domain (common bacteria SprA-related domain) and 1 with no identified domains. The results of the additional analysis on these proteins is found below.
The autotransporters fLIC037. LI0890. LIC058. LIB022. LIC041. LIC047. LIC0911 : Autotransporters are bacterial virulence factors that contain an N-terminal extracellular
("passenger") domain and a C-terminal beta barrel domain that anchors the protein to the outer membrane (see Figs. 1 and 2) (Nishimura K et al. (2010); Autotransporter passenger proteins: virulence factors with common structural themes; J Mol Med (Berl). May;88(5) :451- 8; PMID: 20217035). Passenger domains of autotransporter proteins are exported to the bacterial surface where they may be cleaved and appear 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 remain largely associated with bacterial cell surface as has been 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). Because passenger domains of autotransporter proteins are exported to the bacterial surface, they often elicit an immunologic response in the host during the course of 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). Furthermore, recent immuno-proteomic studies have indicated that autotransporters are recognized during the course of 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 widespread among Gram-negative bacteria, with several having been associated with functions related to virulence, including adhesins such as AIDA-I (E. coli) and pertactin (Bordetella pertussis) (see Benz, I., and M. A. Schmidt. (1992); Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (0126: 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, P. R. 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, pertactin, which forms a component of the pertussis vaccine (EP0527753A). Additional bioinformatic analysis supported the domain based autotransporter annotation of the 6 proteins by identified beta barrels, alpha helical linkers and passenger domains. Fig. 3A illustrates how the autotransporters LIC037, LI0890. LIC058. LIB022, LIC041, LIC047 are embedded in the outer membrane, whereas Fig. 3B illustrates the same for LIC091. Note that it is considered uncertain whether or not LIC091 is making a hairpin-like structure sticking out of the beta barrel 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 extra putative alpha helical linker in the N-terminal. The STRING database did not comprise any associated proteins with high confidence to the 7 autotransporter proteins.
The unknown protein (LI0668) :
In the STRING database the protein (LI0668) with no identified domain has a high confidence interaction to the autotransporter protein (LIC060). This indicates that the protein may be associated to the outer membrane. Also, this protein is predicted to have a signal peptide and being a non-classical secreted protein.
The unknown serine rich protein ('LI1077') :
The serine rich protein (LI1077) has no associated proteins in the STRING database and is predicted to be a non-classical secretion protein.
The metal-binding protein (^0996) :
The metal-binding protein, which has a zink ribbon domain is predicted to be a non-classical secretion protein.
Additional bioinformatic analysis to secure successful vaccine candidate expression Additional bioinformatic analysis was performed including truncation of the protein sequences in order to secure the native recombinant protein fold and expression of protective/virulent regions. Signal peptides, transmembrane regions and beta barrels were excluded in truncation of the proteins. The alpha helical linker regions were used as natural truncation sites. LIC037. LI0890. LIC058. LIB022. LIC041. LIC047 fautotransportersl
Only the passenger domains should be expressed. N-terminal transmembrane region/regions, C-terminal beta barrel and C-terminal alpha-linker regions are excluded.
LIC091 (autotransporter)
This large protein consists of 8746aa and has a C-terminal alpha helical linker sequence, which is ~6000aa . Also, the protein has an extra putative alpha helical linker of ~600aa in the N-terminal, which allows the formation of a hairpin-like structure. Given the two structural possibilities for this autotransporter two truncations have been made.
Hairpin passenger structure construct (1500-2500aa) :
The putative hairpin passenger domain is expressed together with minor parts of the two alpha-helical linkers aiming at stabilizing the hairpin passenger domain structure. The signal peptide, beta barrel and extremly long C-terminal alpha helical linker are excluded. Non-hairpin passenger struture (40-2068aa) :
The observed putative N-terminal alpha helical linker is now considered to be a part of the passenger domain. Only the passenger domain should be expressed, and so the
transmembran region, beta barrels and the extremly long C-terminal alpha-linker region have been removed.
LI0668 (association to outer membran proteins')
Signal peptide is excluded.
LI1077 (serine rich protein)
Full length expression is recommeded, as the protein lacks regions to exclude. LI0996 (metal-binding protein)
Full length expression is recommended, as the protein lacks regions to exclude
EXAMPLE 2
Identification of immunogenic truncates
6 antigen fragments truncated from 3 Lawsonia intracellulars vaccine candidate proteins were ranked based on their predicted potential to elicit a protective and immunogenic antibody response. Specific feature predictions like epitope density and structural/functional prediction where used to rank these fragments. The 6 fragments are identified in Table 2A further below. In a similar manner, 5 fragments of LIC091
The following predictions were performed : 1) B cell epitope density prediction (hotspot epitopes and general epitopes).
2) T cell CD4+ epitope density prediction on 47 available SLA alleles.
3) T cell CD8+ epitope density prediction on 40 available SLA alleles. B-cell epitope density
The B cell epitope prediction on the 4 vaccine candidate proteins was done by use of
Evaxion's BepiFinder /n silico predictor. General B cell epitopes and Hotspot B cell epitopes were predicted. The hotspot epitope areas differentiate from general epitopes by their high density of beta-turns, hence a very high probability of antibody binding. BepiFinder works by using algorithmic processing on the output of the sub programs 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 Bl, 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 TNI (2010); NetTurnp - Neural Network Prediction of Beta-turns by Use of Evolutionary
Information and Predicted Protein Sequence Features; PLoS ONE 5(ll) :el5079
doi : 10.1371/journal. pone.0015079; PMID: 21152409). B cell epitope density plots of the four proteins are listed in Figs. 4-9; Figs. 4-6 shows hot spot B-cell epitotope areas in the 3 first proteins, whereas Figs. 7-9 generally show B-cell epitopes from the same 3 proteins, Figs. 18 and 19 show the hot spot B-cell epitopes and general B-cell epitopes in a large truncate (amino acids 1-4000) of LIC091.
T-cell CD4+ and CD8+ epitope density The T cell epitope prediction on the 4 vaccine candidate proteins was done by use of
Evaxion's TepiFinder in silico predictor. TepiFinder works by using algorithmic processing on the output of the sub programs netMHCIIpan (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). TepiFinder has predicted epitopes of each SLA allele listed in Table 1 below, including 47 swine MHC class II alleles and 40 swine MHC class I alleles:
Figure imgf000096_0001
T cell CD4+ arj§ CD8+ epitope density plots of the 3 first proteins are shown in Figs. 10-15, whereas the same data are shown in Figs. 20 and 21 for a truncate of LIC091 (amino acids 1-2550, i.e. corresponding to the part of the 4000 amino acid truncate where hot spot B-cell epitopes are found). Conclusion
The antigen fragments are ranked in Table 2 below based on their predicted potential to elicit a protective and immunogenic antibody response. The ranking is based on the highest calculated score, which includes primary focus on B cell epitope densities (protective antibody binding) and secondary focus on T cell CD4+ epitope density (T helper cell immunogenicity) . All the fragments are truncated from autotransporter passenger receptor regions; hence the predicted B cell epitope areas are potential receptor areas. The T cell CD8+ epitope density listed in the table below was left out of the ranking due to the focus on antibody response.
Figure imgf000097_0001
Table 2A - Ranking of Fragments: The rder of Priority' correlated directly with the 'SCORE'. The score is calculated by adding the three normalized values of 'No of B cell epitope hotspots', 'No of B cell epitopes' and 'No of T-cell CD4+ epitopes'. [Normalization (AA_count - mean_of_AA_count) / standard_deviation_of_AA count].
Figure imgf000097_0002
Table 2 - Ranking of Fragments: The 'Order of Priority' correlated directly with the 'SCORE'. The score is calculated by adding the three normalized values of 'No of B cell epitope hotspots', 'No of B cell epitopes' and 'No of T-cell CD4+ epitopes'. [Normalization = (AA_count - mean_of_AA_count) / standard_deviation_of_AA count]. EXAMPLE 3
Recombinant production of polypeptides of the invention
A. Protein Purification Protocol for TRX-HIS-LIC058-trunc Protein:
Expression of TRX-HIS-LIC058-Trunc The gene (SEQ ID NO: 56) encoding the synthetic polypeptide TRX-HIS-LIC058 (SEQ ID NO: 52) was synthesized and cloned into pET32a (available from Novagen, catalogue number 69015-3) using Kpnl and Xhol restriction sites. Recombinant plasmid was transformed into host stain 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 expression. Induction was done at 30°C, 230 rpm for 3h.
Purification of LIC058-Trunc.
15 g bacteria (wet weight) were resuspended and sonicated in 200 mL of binding buffer (20 mM Tris-HCI, 300 mM NaCI, 20 mM imidazole, pH 8.0) on ice. The resulting lysate was centrifuged at 10,000 rpm for 30 min, and the supernatant was immediately put on ice. The sample was applied to a Ni2+ chelating sepharose fast flow (GE Healthcare) column (25/100) and washed with at least 2 column volumes of the wash buffer (20 mM Tris-HCI, 300 mM NaCI, 60 mM imidazole, pH 8.0). The column was subsequently eluted with 30 mL of elution buffer (20 mM Tris-HCI, 300 mM NaCI, 300 mM imidazole, pH 8.0) while monitoring OD at 280 nm. The resulting elution fraction was subsequently applied to a HiLoadTM 26/600 SuperdexTM 200 pg column (GE Healthcare) equilibrated in equilibration buffer (1 x PBS, pH 7.4) with a flow rate of 3.5 mL/min. The resulting fractions were then pooled and lyophilized. Fig. 16 shows the purified protein in an SDS-PAGE gel.
B. Protein Purification Protocol for LIC091-A Protein: Expression of LIC091-A.
The gene (SEQ ID NO: 57) encoding a His-tagged LIC091-A protein (SEQ ID NO: 53) was synthesized and cloned into pET-32a using Ndel and Xhol restriction sites. Recombinant plasmid was transformed into host stain 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 expression. The induction condition was 30 °C, 230 rpm for 3 h.
Purification of LIC091-A. 10 g bacteria (wet weight) were resuspended and sonicated in 200 mL of extraction buffer (20 mM Tris-HCI, 300 mM NaCI, 1% Triton X-100, pH 8.0) on ice and the mixture was centrifuged at 10,000 rpm for 30 min. Insoluble pellets were dissolved in 200 mL of denaturing buffer (20 mM Tris-HCI, 300 mM NaCI, 20 mM imidazole, 8 M urea, 20 mM bmercaptoethanol, pH 8.0) and stirred overnight at 4°C and then centrifuged at 10,000 rpm for 30 min. The supernatant was applied to a Ni2+ chelating sepharose fast flow (GE
Healthcare) column (35/80) and the flowthrough was dialyzed against binding buffer (20 mM Tris-HCI, 7 M guanidine hydrochloride, 20 mM imidazole, 20 mM b-mercaptoethanol, pH 8.0), with 2 changes of dialysis buffer. The dialyzed flowthrough was applied to a Ni2+- chelating sepharose fast flow (GE Healthcare) column (35/80) and washed with at least 2 column volumes of the wash buffer (20 mM Tris-HCI, 300 mM NaCI, 20 mM imidazole, 8 M urea, pH 8.0). The column was subsequently eluted with 50 mL of elution buffer (20 mM Tris-HCI, 300 mM NaCI, 300 mM imidazole, 8 M urea, pH 8.0) while monitoring OD at 280 nm. Additional b- mercaptoethanol was then added to the elution fraction to bring up the concentration of the reductant to 20 mM. LIC091-A was subsequently applied to a HiLoadTM 26/600 SuperdexTM 75 pg column (GE Healthcare) equilibrated in equilibration buffer (20 mM Tris-HCI, 300 mM NaCI, 20 mM imidazole, 8 M urea, 20 mM b-mercaptoethanol, pH 8.0) with a flow rate of 3.0 mL/min. Then LIC091-A was dialyzed against 30% acetonitrile containing 0.1% TFA, with 2 changes of dialysis buffer. The dialyzed LIC091-A was then lyophilized. A picture of an SDS-PAGE gel with the purified protein is shown in Fig. 17.
1. A polypeptide comprising:
a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1- 17 and 54, or
b) an amino acid sequence consisting of at least 5 contiguous amino acid residues from any one of SEQ ID NOs: 1-17 and 54, or
c) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of a),
d) an amino acid sequence having a sequence identity of at least 60% with the amino acid sequence of b), or
e) an assembly of amino acids derived from any one of SEQ ID NOs: 1-17 and 54, which has essentially the same 3D conformation as in the protein from which said assembly is derived so as to constitute a B-cell epitope,
said polypeptide being antigenic in a mammal. 2. An isolated nucleic acid fragment, which comprises:
i) a nucleotide sequence encoding a polypeptide according to any claim 1, or
ii) a nucleotide sequence consisting of the amino acid encoding part of any one of SEQ ID NOs: 18-51 and 55.
iii) a nucleotide sequence consisting of at least 10 consecutive nucleotides in the amino acid encoding part 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 in i) or ii),
v) a nucleotide sequence having a sequence identity of at least 60% with the nucleotide sequence in iii),
vi) a nucleotide sequence complementary to the nucleotide sequence in i)-v), or
vii) a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence in i)-vi).
3. A vector comprising the nucleic acid according to claim 2, such as a cloning vector or an expression vector. 4. A cell which is transformed so as to carry the vector according to claim 3.
5. A pharmaceutical composition comprising a polypeptide according to claim 1, a nucleic acid fragment according to claim 2, a vector according to claim 3, or a cell according to claim 4, and a pharmaceutically acceptable carrier, vehicle or diluent.

Claims

6. A method for inducing immunity in an animal by administering at least once an immunogenically-effective amount of a polypeptide according to claim 1, a nucleic acid fragment according to claim 2, a vector according to claim 3, a cell according to claim 4, or a pharmaceutical composition according to claim 5, so as to induce adaptive immunity against L. intracellularis in the animal.
7. A polyclonal antibody in which the antibody specifically binds to a polypeptide of claim 1, and which is essentially free from antibodies binding specifically to other L. intracellularis polypeptides.
8. An isolated monoclonal antibody or antibody analogue which specifically binds to a polypeptide of claim 1.
9. The isolated monoclonal antibody or antibody analogue according to claim 8, which is a monoclonal antibody selected from a multi-domain antibody such as a murine antibody, a chimeric antibody such as a humanized antibody, a fully human antibody, and single-domain antibody of a llama or a camel, or which is an antibody analogue selected from a fragment of an antibody, such as a Fab, a F(ab')2, and a scFV.
10. A method for prophylaxis, treatment or amelioration of infection with L. intracellularis comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 5, or an antibody according to any one of claims 7-9, to an animal in need thereof. 11. A method for determining, quantitatively or qualitatively, the presence of L. intracellularis in a sample, the method comprising contacting the sample with an antibody according to any one of claims 7-9 and detecting the presence of antibody bound to material in the sample.
12. A method for determining, quantitatively or qualitatively, the presence of antibodies specific for L. intracellularis in a sample, the method comprising contacting the sample with a polypeptide according to claim 1, and detecting the presence of antibody said polypeptide.
13. A method for determining, quantitatively or qualitatively, the presence of a nucleic acid characteristic of L. intracellularis in a sample, the method comprising contacting the sample with a nucleic acid fragment according to claim 2, and detecting the presence of nucleic acid in the sample that hybridized to said nucleic acid fragment. 14. A method for the preparation of the polypeptide according to claim 1, comprising culturing a transformed cell according to claim 4 under conditions that facilitate that the transformed cell expressing the nucleic acid fragment according to claim 2, and subsequently recovering said polypeptide, or preparing said polypeptide by means of solid or liquid phase peptide synthesis.
15. A method for determining whether a substance, such as an antibody, is potentially useful for treating infection with L. intracellularis, the method comprising contacting the polypeptide according to claim 1 with the substance and subsequently establishing whether the substance has at least one of the following characteristics:
1) the ability to bind specifically to said polypeptide,
2) the ability to compeed with said polypeptide for specific binding to a ligand/receptor,
3) the ability to specifically inactivate said polypeptide.
16. A method for determining whether a substance, such as a nucleic acid, is potentially useful for treating infection with L. intracellularis, the method comprising contacting the substance with the nucleic acid fragment of claim 2, and subsequently establishing whether the substance has either the ability to
1) bind specifically to the nucleic acid fragment, or
2) bind specifically to a nucleic acid that hybridizes specifically with the nucleic acid fragment.
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Publication number Priority date Publication date Assignee Title
US20190055298A1 (en) * 2013-11-22 2019-02-21 The Board Of Trustees Of The University Of Illinois Engineered high-affinity human t cell receptors

Citations (2)

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WO2004033631A2 (en) * 2002-10-04 2004-04-22 Regents Of The University Of Minnesota Nucleic acid and polypeptide sequences from lawsonia intracellularis and methods of using
WO2011125015A2 (en) * 2010-04-05 2011-10-13 Bar-Ilan University Protease-activatable pore-forming polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004033631A2 (en) * 2002-10-04 2004-04-22 Regents Of The University Of Minnesota Nucleic acid and polypeptide sequences from lawsonia intracellularis and methods of using
WO2011125015A2 (en) * 2010-04-05 2011-10-13 Bar-Ilan University Protease-activatable pore-forming polypeptides

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190055298A1 (en) * 2013-11-22 2019-02-21 The Board Of Trustees Of The University Of Illinois Engineered high-affinity human t cell receptors

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