WO2017220787A1 - Vaccines against aearomonas salmonicida infection - Google Patents

Vaccines against aearomonas salmonicida infection Download PDF

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Publication number
WO2017220787A1
WO2017220787A1 PCT/EP2017/065557 EP2017065557W WO2017220787A1 WO 2017220787 A1 WO2017220787 A1 WO 2017220787A1 EP 2017065557 W EP2017065557 W EP 2017065557W WO 2017220787 A1 WO2017220787 A1 WO 2017220787A1
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WIPO (PCT)
Prior art keywords
exactly
amino acid
residue
acid residues
seq
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PCT/EP2017/065557
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French (fr)
Inventor
Niels IVERSEN MØLLER
Andreas HOLM MATTSSON
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Evaxion Biotech Aps
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Publication of WO2017220787A1 publication Critical patent/WO2017220787A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • the present invention relates to the field of prophylactic vaccination of fish .
  • the present invention relates to prophylactic vaccines that induce immunity against A.
  • Aeromonas salmonicida subsp. salmonicida ⁇ A. Salmonicida or AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality.
  • Furunculosis induced by A. salmonicida subsp. salmonicida has been known as a disease for more than 100 years. Its virulence is contributed to the T3SS virulence system, which causes disease in salmonid fish by circumventing all barriers imposed by the host and the environment. (Bergh PV and Frey J, 2013, Microbial Biotechnol.7, 381-400.
  • the present invention relates to a method of inducing immunity in freshwater fish against A. salmonicida comprising administering an effective amount of a polypeptide comprising
  • the present invention relates to method of inducing immunity in freshwater fish against A. salmonicida comprising administering an effective amount of 1) a nucleic acid encoding a polypeptide used in the first aspect of the invention and the embodiments thereof disclosed herein or 2) a vector comprising and being capable of expressing the nucleic acid in freshwater fish.
  • a third aspect of the present invention relates to a composition
  • a composition comprising a polypeptide used in the first aspect of the invention and the embodiments thereof disclosed herein in admixture with a diluent, carrier, excipient or vehicle.
  • a fourth aspect of the present invention relates to a composition
  • a composition comprising a nucleic acid or vector of the third aspect of the invention and the embodiments thereof disclosed herein in admixture with a diluent, carrier, excipient or vehicle.
  • a fifth aspect of the invention relates to any polypeptide disclosed herein as a polypeptide derived from SEQ ID NOs: 1-20, i.e. any one of the polypeptides referred to in options a-e in the first aspect of the invention and the embodiments thereof.
  • the invention relates to the polypeptides defined in the context any of options b-e. Consequently, when referring to a "polypeptide of the invention” is meant that the polypeptide is a polypeptide derived from any one of SEQ ID NOs: 1-20, which is disclosed in the context of the method of the first aspect of the invention.
  • a sixth aspect of the invention relates to a polypeptide of the fifth aspect for use as a pharmaceutical .
  • a sixth aspect of the invention relates to a polypeptide of the fifth aspect for use in a method of the various embodiments of the first aspect of the invention.
  • Fig . 1 Graph showing cumulative mortality results from infection challenge of rainbow trout. Duplicate tanks were pooled after survival curve comparison with log-rank test showed no significant difference in mortalities.
  • Fig . 2 Levels of A. salmonicida-spec ⁇ f ⁇ c antibodies in serum were measured from 10 fish per group (5 fish per duplicate tank) by ELISA at 7 weeks post-vaccination (wpv) and at 3 weeks post- challenge (wpc) . Sera were diluted 1 : 500.
  • Fig . 3 Levels in fish serum of specific antibodies against individual proteins from the VacB vaccine.
  • A levels measured from 10 fish per group (5 fish per duplicate tank) by ELISA at 7 weeks post- vaccination (wpv) .
  • Sera were diluted 1 : 100.
  • Fig . 4 Levels of specific antibodies in serum from fish against individual proteins from the VacC vaccine.
  • Sera were diluted 1 : 100.
  • Fig . 5 Correlation (Pearson) between mean antibody levels (dilution 1 : 100) at 7 weeks post vaccination (wpv) and survival in different experimental groups at 24 days post challenge (dpc) .
  • Antibody levels were measured from 10 fish per group (5 fish per duplicate tank) by
  • A Protein #8; B: Protein #10; C: Protein # 11.
  • polypeptide is in the present context intended to mean 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 comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked.
  • the polypeptide (s) 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 reference amino acid sequence or nucleic acid sequence, respectively
  • amino acid sequence is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins.
  • adjuvant or "immunological 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 2) nevertheless capable of enhancing the immune response against the immunogen.
  • 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 the immunogen, but the combined vaccination with immunogen 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 3 dimensional structure of a biomolecule such as a protein.
  • the 3D conformation is also termed “the tertiary structure” and denotes the relative locations in 3 dimensional 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/hapten, which is not sufficiently immunogenic in its own right - typically, the immunogenic carrier is capable of eliciting a strong cellular immune response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen antibody producing cells and cytotoxic cells.
  • the large carrier molecules have to a certain extent been substituted by so-called promiscuous epitopes, i.e. shorter peptides that are recognized by a large fraction of MHC-haplotypes in a population, and which elicit antigen specific cellular immune responses.
  • immunogen is a substance of matter which is capable of inducing an adaptive immune response in a host, whose immune system is exposed to the immunogen.
  • immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system 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 the antigen.
  • a "hapten” is a (typically) small molecule, which can neither induce nor elicit an immune response, but if conjugated to an immunogenic carrier, a specific adaptive immune response can be induced against a hapten upon exposure of the immune system with the hapten carrier conjugate.
  • An “adaptive immune response” is an immune response in response to exposure to an antigen or immunogen, where the immune response 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 cellular immune responses.
  • a "protective, adaptive immune response” is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with 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 several pathogens.
  • Stimulation of the immune system means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect.
  • a number of adjuvants and putative adjuvants (such as 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 simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.
  • 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 the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. Generally, 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
  • an 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 with an internal surface shape and charge distribution
  • Antibody includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
  • Specific binding denotes binding between two substances which goes beyond binding of either substance to randomly chosen substances and also goes beyond simple association between substances that tend to aggregate because they share the same overall
  • vector is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction 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 and bacterial vectors - both these infectious agents are capable of introducing a heterologous nucleic acid sequence into a host and effect subsequence expression of a nucleic acid in the host.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product 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.
  • the at least or exactly 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention constitute at least or exactly 5, at least or exactly or at most 6, at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32
  • the number of contiguous amino acids can be higher, for all of SEQ ID NOs: 2-20. Another way to phrase this is that for each of SEQ ID NOs: 1-20, the number of the contiguous amino acid residues is at least or exactly or at most N-n, where N is the length of the particular sequence ID in question and n is 1, 2, 3, 4, or 5; 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 at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 94, at least or exactly or at most 95, at least or exactly or at most 96, at least or exactly or at most 97, at least or exactly or at most 98, at least or exactly or at most 99, at least or exactly or at most 100, at least or exactly or at most 101, at least or exactly or at most 102, at least or exactly or at most 103, at least or exactly or at most 104, at least or exactly or at most 105, at least or exactly or at most 106, at least or exactly or at most 107, at least or exactly or at most 108, at least or exactly or at most 109, at least or exactly or at most 110, at least or exactly or at most 111, at least or exactly or at most 112, at least or exactly or at most 113, at least or exactly or at most 110, at least or exactly or at most 111, at least or exactly or at most 112, at least
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 242, at least or exactly or at most 243, at least or exactly or at most 244, at least or exactly or at most 245, at least or exactly or at most 246, at least or exactly or at most 247, at least or exactly or at most 248, at least or exactly or at most 249, at least or exactly or at most 250, at least or exactly or at most 251, at least or exactly or at most 252, at least or exactly or at most 253, at least or exactly or at most 254, at least or exactly or at most 255, at least or exactly or at most 256, at least or exactly or at most 257, at least or exactly or at most 258, at least or exactly or at most 259, at least or exactly or at most 260, at least or exactly or at most 261, at least or exactly or at most 262, at least or exactly or at most
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 389, at least or exactly or at most 390, at least or exactly or at most 391, at least or exactly or at most 392, at least or exactly or at most 393, at least or exactly or at most 394, at least or exactly or at most 395, at least or exactly or at most 396, at least or exactly or at most 397, at least or exactly or at most 398, at least or exactly or at most 399, at least or exactly or at most 400, at least or exactly or at most 401, or at least or exactly or at most 402 contiguous amino acid residues.
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 403, at least or exactly or at most 404, at least or exactly or at most 405, at least or exactly or at most 406, at least or exactly or at most 407, at least or exactly or at most 408, at least or exactly or at most 409, at least or exactly or at most 410, at least or exactly or at most 411, at least or exactly or at most 412, at least or exactly or at most 413, at least or exactly or at most 414, at least or exactly or at most 415, at least or exactly or at most 416, at least or exactly or at most 417, at least or exactly or at most 418, at least or exactly or at most 419, at least or exactly or at most 420, at least or exactly or at most 421, at least or exactly or at most 422, at least or exactly or at most 423, at
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 469, at least or exactly or at most 470, at least or exactly or at most 471, at least or exactly or at most 472, at least or exactly or at most 473, at least or exactly or at most 474, at least or exactly or at most 475, at least or exactly or at most 476, at least or exactly or at most 477, at least or exactly or at most 478, at least or exactly or at most 479, or at least or exactly or at most 480 contiguous amino acid residues.
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 481, at least or exactly or at most 482, at least or exactly or at most 483, at least or exactly or at most 484, at least or exactly or at most 485, at least or exactly or at most 486, at least or exactly or at most 487, at least or exactly or at most 488, at least or exactly or at most 489, at least or exactly or at most 490, at least or exactly or at most 491, at least or exactly or at most 492, at least or exactly or at most 493, at least or exactly or at most 494, at least or exactly or at most 495, at least or exactly or at most 496, at least or exactly or at most 497, at least or exactly or at most 498, at least or exactly or at most 499, at least or exactly or at most 500, at least or exactly or at most 501, at least or
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 557, at least or exactly or at most 558, at least or exactly or at most 559, at least or exactly or at most 560, at least or exactly or at most 561, at least or exactly or at most 562, at least or exactly or at most 563, at least or exactly or at most 564, at least or exactly or at most 565, at least or exactly or at most 566, at least or exactly or at most 567, at least or exactly or at most 568, at least or exactly or at most 569, at least or exactly or at most 570, at least or exactly or at most 571, at least or exactly or at most 572, at least or exactly or at most 573, at least or exactly or at most 574, at least or exactly or at most 575, at least or exactly or at most 576, at least or exactly or at most 557, at least or exactly or at most 558, at least or exactly or
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 579, at least or exactly or at most 580, at least or exactly or at most 581, at least or exactly or at most 582, at least or exactly or at most 583, at least or exactly or at most 584, at least or exactly or at most 585, at least or exactly or at most 586, at least or exactly or at most 587, at least or exactly or at most 588, at least or exactly or at most 589, at least or exactly or at most 590, at least or exactly or at most 591, at least or exactly or at most 592, at least or exactly or at most 593, at least or exactly or at most 594, at least or exactly or at most 595, at least or exactly or at most 596, at least or exactly or at most 597, at least or exactly or at most 598, at least or exactly
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 640 contiguous amino acid residues
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 641, at least or exactly or at most 642, at least or exactly or at most 643, at least or exactly or at most 644, at least or exactly or at most 645, at least or exactly or at most 646, at least or exactly or at most 647, at least or exactly or at most 648, at least or exactly or at most 649, at least or exactly or at most 650, at least or exactly or at most 651, at least or exactly or at most 652, at least or exactly or at most 653, at least or exactly or at most 654, at least or exactly or at most 655, at least or exactly or at most 656, at least or exactly or at most 657, at least or exactly or at most 658, at least or exactly or at most 659, at least or exactly or at most 660, or at least or exactly or at most 661, at least or exactly or at most 641, at least or exactly or at most 642, at least or exactly or
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 681, at least or exactly or at most 682, at least or exactly or at most 683, at least or exactly or at most 684, at least or exactly or at most 685, at least or exactly or at most 686, at least or exactly or at most 687, at least or exactly or at most 688, at least or exactly or at most 689, at least or exactly or at most 690, at least or exactly or at most 691, at least or exactly or at most 692, at least or exactly or at most 693, at least or exactly or at most 694, at least or exactly or at most 695, at least or exactly or at most 696, or at least or exactly or at most 697 contiguous amino acid residues.
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 698, at least or exactly or at most 699, at least or exactly or at most 700, at least or exactly or at most 701, at least or exactly or at most 702, at least or exactly or at most 703, at least or exactly or at most 704, at least or exactly or at most 705, at least or exactly or at most 706, at least or exactly or at most 707, at least or exactly or at most 708, at least or exactly or at most 709, at least or exactly or at most 710, at least or exactly or at most 711, or at least or exactly or at most 712 contiguous amino acid residues.
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 713, at least or exactly or at most 714, at least or exactly or at most 715, at least or exactly or at most 716, at least or exactly or at most 717, at least or exactly or at most 718, at least or exactly or at most 719, at least or exactly or at most 720, at least or exactly or at most 721, at least or exactly or at most 722, at least or exactly or at most 723, at least or exactly or at most 724, at least or exactly or at most 725, at least or exactly or at most 726, at least or exactly or at most 727, at least or exactly or at most 728, at least or exactly or at most 729, at least or exactly or at most 730, at least or exactly or at most 731, at least or exactly or at most 732, at least or exactly or at most
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 755, at least or exactly or at most 756, at least or exactly or at most 757, at least or exactly or at most 758, at least or exactly or at most 759, at least or exactly or at most 760, at least or exactly or at most 761, at least or exactly or at most 762, at least or exactly or at most 763, at least or exactly or at most 764, at least or exactly or at most 765, at least or exactly or at most 766, at least or exactly or at most 767, at least or exactly or at most 768, at least or exactly or at most 769, at least or exactly or at most 770, at least or exactly or at most 771, at least or exactly or at most 772, at least or exactly or at most 773, at least or exactly or at most 774, at least or exactly or at most
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 878, at least or exactly or at most 879, at least or exactly or at most 880, at least or exactly or at most 881, at least or exactly or at most 882, at least or exactly or at most 883, at least or exactly or at most 884, at least or exactly or at most 885, at least or exactly or at most 886, at least or exactly or at most 887, at least or exactly or at most 888, at least or exactly or at most 889, at least or exactly or at most 890, at least or exactly or at most 891, at least or exactly or at most 892, at least or exactly or at most 893, at least or exactly or at most 894, at least or exactly or at most 895, at least or exactly or at most 896, at least or exactly or at most 897, at least or exactly
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 916, at least or exactly or at most 917, at least or exactly or at most 918, at least or exactly or at most 919, at least or exactly or at most 920, at least or exactly or at most 921, at least or exactly or at most 922, at least or exactly or at most 923, at least or exactly or at most 924, at least or exactly or at most 925, at least or exactly or at most 926, at least or exactly or at most 927, at least or exactly or at most 928, at least or exactly or at most 929, at least or exactly or at most 930, at least or exactly or at most 931, at least or exactly or at most 932, at least or exactly or at most 933, at least or exactly or at most 934, at least or exactly or at most 935, at least or
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 1021, at least or exactly or at most 1022, at least or exactly or at most 1023, at least or exactly or at most 1024, at least or exactly or at most 1025, at least or exactly or at most 1026, at least or exactly or at most 1027, at least or exactly or at most 1028, at least or exactly or at most 1029, at least or exactly or at most 1030, at least or exactly or at most 1031, at least or exactly or at most 1032, at least or exactly or at most 1033, at least or exactly or at most 1034, at least or exactly or at most 1035, at least or exactly or at most 1036, at least or exactly or at most 1037, at least or exactly or at most 1038, at least or exactly or at most 1039, at least or exactly or at most 1040, at least or exactly
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 1157, at least or exactly or at most 1158, at least or exactly or at most 1159, at least or exactly or at most 1160, at least or exactly or at most 1161, at least or exactly or at most 1162, at least or exactly or at most 1163, at least or exactly or at most 1164, at least or exactly or at most 1165, at least or exactly or at most 1166, at least or exactly or at most 1167, at least or exactly or at most 1168, at least or exactly or at most 1169, at least or exactly or at most 1170, at least or exactly or at most 1171, at least or exactly or at most 1172, at least or exactly or at most 1173, at least or exactly or at most 1174, at least or exactly or at most 1175, at least or exactly or at most 1176, at least or exactly or exactly or
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 1266, at least or exactly or at most 1267, at least or exactly or at most 1268, at least or exact y or at most 1269, at least or exactly or at most 1270, at least or exactly or at most 1271, at least or exactly or at most 1272, at least or exactly or at most 1273, at least or exact y or at most 1274, at least or exactly or at most 1275, at least or exactly or at most 1276, at least or exactly or at most 1277, at least or exactly or at most 1278, at least or exact y or at most 1279, at least or exactly or at most 1280, at least or exactly or at most 1281, at least or exactly or at most 1282, at least or exactly or at most 1283, at least or exact y or at most 1284, at least or exactly or at most 1285, at
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 1345, at least or exactly or at most 1346, at least or exactly or at most 1347, at least or exact y or at most 1348, at least or exactly or at most 1349, at least or exactly or at most 1350, at least or exactly or at most 1351, at least or exactly or at most 1352, at least or exact y or at most 1353, at least or exactly or at most 1354, at least or exactly or at most 1355, at least or exactly or at most 1356, at least or exactly or at most 1357, at least or exact y or at most 1358, at least or exactly or at most 1359, at least or exactly or at most 1360, at least or exactly or at most 1361, at least or exactly or at most 1362, at least or exact y or at most 1363, at least or exactly or at most 1364
  • 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 used in the invention is also one that has at least or exactly or at most 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, 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, or 89 in any one of SEQ ID NOs: 1-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 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
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, or 398 in any one of SEQ ID NOs: 4-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, or 464 in any one of SEQ ID NOs: 5
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, or 476 in any one of SEQ ID NOs: 6-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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, 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,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, or 574 in any one of SEQ ID NOs: 8-20, if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L” is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635 in any one of SEQ ID NOs: 9-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to amino acid residue 636 in any one of SEQ ID NOs: 10-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 637, 638, 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, or 676 in any one of SEQ ID NOs: 11-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, or 693 in any one of SEQ ID NOs: 12-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, or 708 in any one of SEQ ID NOs: 13-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 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, or 750 in any one of SEQ ID NOs: 14-20, if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, or 911 in any one of SEQ ID NOs: 16-20,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 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, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 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, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 10
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163,
  • the polypeptide used in the invention is also one that has at least or exactly or at most 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 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
  • Particularly preferred polypeptides of the invention are the following : Each and every of SEQ ID NO: 1-20.
  • ASA_0744-30-388 (SEQ ID NO: 3, residues 30-388) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-29 of SEQ ID NO: 3.
  • ASA_4042-19-402 (SEQ ID NO: 4, residues 19-402) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-18 of SEQ ID NO: 4.
  • ASA_4105-35-468 (SEQ ID NO: 5, residues 35-468) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-34 of SEQ ID NO: 5.
  • ASA_2321-22-480 SEQ ID NO: 6, residues 22-480
  • ASA_3943-18-556 SEQ ID NO: 7, residues 18-556
  • fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-17 of SEQ ID NO: 7.
  • ASA_3320-24-640 (SEQ ID NO: 10, residues 24-640) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-23 of SEQ ID NO: 10.
  • ASA_3883-25-680 (SEQ ID NO: 11, residues 25-680) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-24 of SEQ ID NO: 11.
  • ASA_3328-25-697 (SEQ ID NO: 12, residues 25-697) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-24 of SEQ ID NO: 12.
  • ASA_2532-23-334 (SEQ ID NO: 13, residues 23-334) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-22 and/or do not include the amino acid sequence constituted by residues 335-712 of SEQ ID NO: 13.
  • ASA_3455-25-754 (SEQ ID NO: 14, residues 25-754) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-24 of SEQ ID NO: 14.
  • ASA_P5G035-24-629 SEQ ID NO: 15, residues 24-629) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-23 and/or do not include the amino acid sequence constituted by residues 630-877 of SEQ ID NO: 15.
  • ASA_3723-210-915 (SEQ ID NO: 16, residues 210-915) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-209 of SEQ ID NO: 16.
  • ASA_1675-310-1156 (SEQ ID NO: 18, residues 310-1156) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-309 of SEQ ID NO: 18.
  • ASA_0826-2340-3195 (SEQ ID NO: 20, residues 2340-3195) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-2340 of SEQ ID NO: 20.
  • the polypeptide of the invention is in certain embodiments also fused or conjugated to an immunogenic carrier molecule; or, phrased otherwise, the polypeptide of the invention also comprises such an immunogenic carrier molecule in addition to the amino acid sequence derived from SEQ ID NOs: 1-20.
  • the immunogenic carrier molecule is typically a polypeptide that induces cellular immune responses in freshwater fish in a manner similar to induction of T helper lymphocyte responses in mammals; since it has been demonstrated (See Nakanishi T et al.
  • An immunogenic carrier protein can be selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, diphtheria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra.
  • One further fusion partner which is preferably incorporated is a "His tag", i.e. a stretch of amino acids, which is rich in or only consists of histidinyl residues so as to facilitate protein purification subsequent to recombinant production.
  • the polypeptide of the invention detailed above is capable of inducing an adaptive immune response against the polypeptide in fish, in particular in Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, and Halibut, and where the polypeptide in particular is capable of inducing a prophylactic immune response that reduces the risk of furunculosis in fish.
  • SEQ ID NOs: 1-20 include antigenic determinants (epitopes) that are as such recognized by antibodies and/or T cells in fish.
  • epitopes that are as such recognized by antibodies and/or T cells in fish.
  • B-cell epitopes I.e. antibody binding epitopes
  • mutated versions of the polypeptides of the invention e.g . version where each single non-alanine residue in any one of SEQ ID NOs: 1- 20 are point mutated to alanine - this method also assists in identifying complex assembled B-cell epitopes; this is the case when binding of the same antibody is modified by exchanging amino acids in different areas of the full-length polypeptide.
  • Methods of the invention modes, routes and timing of administration
  • the administration of a vaccine to induce an immune response in fish - whether this is done according to the first or second aspect of the invention - is typically via oral administration (providing the polypeptide or the composition as an ingredient in food), immersion (such as by bathing, dipping or spraying), or injection (such as intraperitoneal or intramuscular) .
  • oral administration providing the polypeptide or the composition as an ingredient in food
  • immersion such as by bathing, dipping or spraying
  • injection such as intraperitoneal or intramuscular
  • the nucleic acid fragment used in the second aspect of the invention referred to above is preferably a DNA fragment (of a sequence such as SEQ ID NOs: 21-40) or an RNA fragment (of a sequence such as SEQ ID NOs 1-60) .
  • the nucleic acid may vary compared to SEQ ID NOs: 21-60 (e.g . by being a fragment since it encodes a fragment of any one of SEQ ID NOs: 1-20 disclosed above) and may have nucleotide sequence with at least 60% sequence identity with any one of SEQ ID NOs: 21-60. Certain of these sequence variants are particularly useful due to choice of codons that optimize expression of the nucleic acid in a particular host cell.
  • These nucleic acids are considered patentable in their own right and are termed nucleic acids of the invention.
  • the nucleic acid fragment of the invention discussed above typically has a sequence identity with the nucleotide sequence defined for i) or ii) above, which 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%.
  • nucleic acid fragment of the invention discussed above may also have a sequence identity with the nucleotide sequence defined for iii) above, which 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%.
  • the nucleic acid fragment of the invention described above comprises in certain embodiments at least or exactly or at most X distinct nucleic acid sequences, which each encode a polypeptide of the invention, where each of said X distinct nucleic acid sequences encodes at least or exactly or at most one immunogenic amino acid sequence present in or derived from any one of SEQ ID NOs: 1-20 and wherein said X distinct nucleic acid sequences together encode immunogenic amino acid sequences present in or derived from at least or exactly or at most X of SEQ ID NOs: 1-20, wherein X is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • such a nucleic acid fragment encodes several polypeptides of the invention.
  • the X nucleic acid sequences are expressed as separate encoded proteins and in other embodiments as "pearls on a string", i.e. fused proteins.
  • immunogenic amino acid sequences from any one of SEQ ID NOs: 1-20 are only present in one of said X nucleic acid sequences.
  • nucleic acid fragments of the invention may be used for both production, carrier and vaccine purposes - the latter will require that the sequences are included in expression vectors that may lead to production of immunogenic proteins in the fish receiving the vector.
  • the nucleic acid is comprised in a vector capable of expressing the nucleic acid in the freshwater fish upon administration.
  • a vector of the invention often 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, an optional signal peptide coding sequence, a nucleotide sequence of the invention, and optionally a terminator.
  • 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 protozoan of origin, recombinant production has to be effected in host cells that can express the coding nucleic acid. Bacterial host cells may be used. However, if the vector is to drive expression in eurkaryotic cell (as would be the case for a nucleic acid vaccine vector), the expression control region should be adapted to this particular use.
  • the expression control region drives expression in a prokaryotic cell such as a bacterium, e.g. in E. coli, or in a eukaryotic cell such as a plant cell, an insect cell, or a mammalian cell.
  • a prokaryotic cell such as a bacterium, e.g. in E. coli
  • a eukaryotic cell such as a plant cell, an insect cell, or a mammalian cell.
  • the expression control region has to be able to drive expression in a fish cell.
  • the vector is capable of integrating the nucleic acid into the genome of a host cell - this is particularly useful if the vector is use 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 piscine host cell are useful in e.g . nucleic acid vaccination.
  • proteins can be produced at low cost in plants using an Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest.
  • Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest.
  • One commercially available platform are those provided by iBio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovatoin Way, Suite 100, Newark, DE 19711, USA) and disclosed in e.g. EP 2 853 599, EP 1 769 068, and EP 2 192 172.
  • the vector is an Agrobacterium vector or other vector suitable for transfection of plants.
  • the vector is typically selected from the group consisting of a virus, such as a virus which is non-pathogenic in fish, a bacterium such as a bacterium which is non-pathogenic in fish, a plasmid, a minichromosome, and a cosmid.
  • viral vectors are viral vectors (in particular those useful as vaccine agents in fish). These may be selected from the group consisting of a retrovirus vector, such as a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a pox virus vector. Certain pox virus vectors are preferred, in particular vaccinia virus vectors. A particularly preferred vaccinia virus vector is a modified vaccinia Ankara (MVA) vector.
  • VVA modified vaccinia Ankara
  • Polypeptides of the invention may as indicated be encoded by a nucleic acid molecule comprised in a vector.
  • a nucleic acid sequence can be "heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced, which includes a sequence homologous to a sequence in the cell but in a position within the host cell where it is ordinarily not found.
  • Vectors include naked 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).
  • Useful vectors encoding such fusion proteins include pIN vectors, 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 or the vector may be purified for direct administration for expression of the protein (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.
  • control sequences refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism.
  • 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
  • an "enhancer” 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.
  • 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.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction in connection with the compositions disclosed herein.
  • 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.
  • 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, include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light Chain, T Cell Receptor, HLA DQa and/or DQ , ⁇ - Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRa, ⁇ - Actin, Muscle Creatine Kinase (MCK), Prealbumin (Transthyretin), Elastase I, Metallothionein (MTII), Collagenase, Albumin, a-Fetoprotein, ⁇ -Globin, ⁇ -Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), al-Antitrypain, H2B (TH2B) Histone, Mouse and/or Type I Collagen, Glucose-Regulated Protein
  • Inducible Elements include MT II - Phorbol Ester (TFA)/Heavy metals; MMTV (mouse mammary tumor virus) - Glucocorticoids; ⁇ -Interferon - poly(rl)x/poly(rc); Adenovirus 5 E2 - EIA; Collagenase - Phorbol Ester (TPA); Stromelysin - Phorbol Ester (TPA); SV40 - Phorbol Ester (TPA); Murine MX Gene - Interferon, Newcastle Disease Virus; GRP78 Gene - A23187; ⁇ -2-Macroglobulin - IL-6; Vimentin - Serum; MHC Class I Gene H-2Kb - Interferon; HSP70 - E1A/SV40 Large T Antigen; Proliferin - Phorbol Ester/TPA; Tumor Necrosis Factor - PMA; and Thyroid Stimulating Hormonea Gene - Thyroid Hormon
  • 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.
  • the particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell.
  • a piscine cell is targeted (as is the case in nucleic acid vaccination)
  • a promoter might include either a bacterial, piscine or viral promoter as long as the promoter is effective in piscine cells.
  • 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 human cytomegalovirus
  • SV40 early promoter the SV40 early promoter
  • 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.
  • 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 fish 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 the fish counterparts of dendritic cells and macrophages.
  • the mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters in man and it is contemplated that corresponding piscine promoters will be effective.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent 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 internal ribosome entry sites
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites.
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating
  • 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).
  • 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. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable 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
  • vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression.
  • 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 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 terminator of transcription 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.
  • polyadenylation signal In expression, particularly eukaryotic expression (as is relevant in nucleic acid vaccination), one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed.
  • Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "on"), which is a specific nucleic acid sequence at which replication is initiated.
  • an autonomously replicating sequence can be employed if the host cell is yeast.
  • 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.
  • 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 other types of markers including 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.
  • Transformed cells are useful as organisms 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 1) of the second aspect of the invention.
  • Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment defined for option 1).
  • the transformed cell according is prokaryotic, such as a bacterium, but generally 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).
  • 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 stably integrated into its genome, and 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.
  • stably transformed cells are preferred - these i.a. allows that cell lines comprised of transformed cells as defined herein may be established - such cell lines are partilucarly preferred aspects of the invention.
  • Suitable cells for recombinant nucleic acid expression of the nucleic acid fragments of the present invention are prokaryotes and eukaryotes.
  • 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 Ps.
  • 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. pastoris
  • Schizosaccharomyces genus e.g. S. pombe.
  • 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, and has been, 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, and 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
  • a plasmid or cosmid can be introduced into a prokaryote host cell for replication of many vectors or expression of encoded proteins.
  • Bacterial cells used as host cells for vector replication and/or expression include Staphylococcus strains, DH5a, JMI 09, and KC8, as well as a number of commercially available bacterial hosts such as SURE(R) Competent Cells and SOLOP ACK(TM) Gold Cells (STRATAGENE®, La Jolla, CA).
  • bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Appropriate yeast cells 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 from 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, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTM Baculovirus expression system from CLONTECH®
  • expression systems include STRATAGENE®'s COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • INVITROGEN® which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is 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 acid delivery to effect expression of compositions of the present invention are believed to 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), including microinjection (U.S. Patent 5,789,215); by electroporation (U.S. Patent No.
  • Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055); or by PEG mediated transformation of protoplasts (U.S. Patents 4,684,611 and 4,952,500); by desiccation/inhibition mediated DNA uptake.
  • organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently
  • 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). A dose of 50-200 ⁇ g/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.
  • 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 x 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 nonspecifically 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 I aedium (elg. hypexanthine, aminopterin, thymidine medium, "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 32p and 1251), 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 and avidin or streptavidin, IgG and protein A, and the 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, 1151 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, 1251, or with an anti-biotin MAb labeled with HRP.
  • a MAb with biotin and detect its presence with avidin labeled with, 1251, 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, or which is an antibody analogue selected from a fragment of an antibody such as an Fab or an F(ab') 2 , an scFV; cf. also the definition of the term "antibody” presented above.
  • 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 , an scFV; cf. also the definition of the term "antibody” presented above.
  • Antibodies binding the polypeptides of the invention are in their own right useful as treatment agents, but will not be able to confer lasting immunity if administered to fish. Nevetheless, it may be of relevance to also - e.g. as a supplement - immunize fish passively by
  • compositions of the invention comprising
  • compositions in particular vaccines, according to the invention are prophylactic.
  • Generic disclosures relating to such vaccines are provided in "Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470- 67455-0, in particular chapters 3-7, 10 and 11.
  • 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.
  • the pharmaceutical compositions such as vaccines include merely one single antigen, immunogen, polypeptide, protein, nucleic acid or vector of the invention, but in other embodiments, the pharmaceutical compositions comprise
  • the pharmaceutical composition is a vector mentioned herein, which encodes and can effect expression of at least 2 nucleic acid fragments of the invention.
  • RNA as the active principle, i.e. at least one mRNA encoding a polypeptide of the invention.
  • An embodiment of a pharmaceutical composition of the invention comprises Y or at least Y or at most Y distinct polypeptides of the invention described above, where each of said Y or at least Y or at most Y distinct polypeptides comprises an immunogenic amino acid sequence present in or derived from any one of SEQ ID NOs: 1-20 and wherein said Y or at least Y or at most Y distinct polypeptides together comprise immunogenic amino acid sequences present in or derived from Y or at least Y or at most Y of SEQ ID NOs: 1-20, wherein Y is an integer selected from 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, and 30.
  • composition of the invention comprises Z or at least Z or at most Z distinct nucleic acid molecules (such as DNA and RNA) each encoding a polypeptide of the invention, where each of said Z or at least Z or at most Z distinct nucleic acid molecules encodes an immunogenic amino acid sequence present in or derived from any one of SEQ ID NOs: 1-20 and wherein said at Z or least Z distinct nucleic acid molecules together encode immunogenic amino acid sequences present in or derived from Z or at least Z or at most Z of SEQ ID NOs: 1-20, wherein Z is an integer selected from 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, and 30.
  • 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, H. pylori, etc. pathogen, cf. the description of immunogenic carriers supra.
  • compositions of the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants 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.
  • Span 85 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 (MPL), trehalose dimycolate (TDM), and cell wall skeleton (MPL), trehalose dimycol
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • cytokines such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg.
  • Alum and MF59TM adjuvants are preferred together with CFA and IFA.
  • 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.
  • thr-MDP N-acetyl-muramyl-L- threonyl-D-isoglutamine
  • nor- MDP N-acetyl-normuramyl-L-alanyl-D-isoglutamine
  • MTP-PE N-acetylmuramyl-L-alanyl-D-
  • polypeptide vaccine formulation Another possibility for a polypeptide vaccine formulation is to include the vaccine
  • polypeptide(s) of the present invention in a virus-like particle, i.e. a non-infectious self- assembling structure composed of envelope or capsid proteins, where the protein(s) of the invention are incorporated.
  • the effect is multiple presentations of the polypeptides of the invention on the surface of the VLP, which in turn provides for improved immune recognition of the polypeptides.
  • VLPs exert immunological adjuvant effects, too.
  • the immunogenic compositions 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 under pharmaceutically acceptable carriers.
  • 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 (eg. nonhuma 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 treating doctor's assessment of the medical situation, and other relevant factors.
  • the amount of immunogen will fall in a relatively broad range that can be determined through routine trials.
  • the amount administered per immunization is typically in the range between 0.5 ⁇ g and 500 mg (however, often not higher than 5,000 ⁇ g).
  • the amount of polypeptide of the invention can therefore be between 1 and 400 ⁇ g, between 2 and 350 ⁇ g, between 4 and 300 ⁇ g, between 5 and 250 ⁇ g, and between 10 and 200 ⁇ g.
  • the composition will typically contain between 0.1-500 ⁇ g of protein of the invention per g of vaccine composition.
  • the immunogenic compositions are conventionally administered parenterally, eg, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. W098/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intraarterial routes may be applicable.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the vaccine may be administered in conjunction with other immunoregulatory agents.
  • DNA vaccination also termed nucleic acid vaccination or gene vaccination
  • nucleic acid vaccination may be used [eg. Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al. (1997) Avnu Rev Innnunol 15 : 617-648; later herein] .
  • a further aspect of the invention is as mentioned above the recognition that combination vaccines can be provided, wherein 2 or more antigens disclosed herein are combined to enhance the immune response by the vaccinated fish, including to optimize initial immune response and duration of immunity.
  • combination vaccines can be provided, wherein 2 or more antigens disclosed herein are combined to enhance the immune response by the vaccinated fish, including to optimize initial immune response and duration of immunity.
  • multiple antigenic fragments derived from the same, longer protein can also be used, such as the use of a combination of different lengths of polypeptide sequence fragments from one protein.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 1 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 2 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 3 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 4 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 5 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 6 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 7 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 8 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 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, and 30 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 9 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 10 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 11 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16.
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 12 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 13 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 14 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 15 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 16 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 17 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 18 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 19 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 20 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
  • the method of the first and second aspects of the invention generally relate to induction of immunity and as such also entails methods that are prophylactic.
  • immunization methods entail that a polypeptide of the invention or a composition comprising such a polypeptide is administered the animal (e.g. the human) typically receives between 0.5 and 5,000 ⁇ g of the polypeptide of the invention per administration, cf. the above indications concerning dosages.
  • the immunization scheme includes that the fish receives one single administration of the polypeptide(s) or composition(s) of the invention, but it may be necessary to follow up with one or more booster administrations.
  • Preferred embodiments comprise that the administration is for the purpose of inducing protective immunity against A.salmonicida.
  • the protective immunity is effective in reducing the risk of attracting infection with A.salmonicida.
  • the preferred vaccines of the invention induce humoral immunity, so it is preferred that the administration is for the purpose of inducing antibodies specific for A.salmonicida.
  • the immunization method may also be useful in antibody production, so in other embodiments the administration is for the purpose of inducing antibodies specific for A.salmonicida wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies.
  • compositions for immunization can as mentioned above comprise polypeptides, antibodies, nucleic acids, or vectors of the invention.
  • the pharmaceutical compositions will comprise a therapeutically effective amount thereof.
  • terapéuticaally effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable preventative effect in a group of fish.
  • the effect can be detected by, for example, chemical markers or antigen levels.
  • 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 fish 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, 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.
  • 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
  • InterPro provides functional analysis of proteins by classifying them into families, predicting domains and important sites.
  • the subcellular localization of the individual proteins was predicted by the Wolf pSORT prediction server (available at http://www.genscript.com/wolf-psort.html). It is common that protective B-cell protein antigens are located in the outer membrane and extracellular environment; consequently, these predicted subcellular locations are a target of selection.
  • Fragments of the 14 proteins were constructed based on conventional in silico analysis such as prediction of signal peptides (SignalP-4.1, see: Petersen T. N., et al., SignalP 4.0:
  • the rationale of expressing the protein as fragments was due to: 1) enhanced probability of expressing the native protein structure, 2) expressing the protective part of the protein only, and 3) establishing a successful recombinant expression. Predicted signal peptide, transmembrane and intracellular regions were removed. Proteins larger than 1000 amino acid residues were split in N- and C-terminal fragments and only the fragment comprising the predicted active functional site domains were expressed. The splitting point of these fragments where decided based on structural domain predictor aiming at keeping the native structure of each structural domain. The rationale of splitting the > 1000 amino acid residue protein was to increase the probability of a successful recombinant expression in E. coli.
  • VacA and VacB comprised of 5 proteins
  • vacC comprised of 4 proteins. See Table B below.
  • each protein solution in vaccine formulations was calculated considering each fish to receive 25 ⁇ g of an individual protein in the mixture corresponding to a total of 200- 225 ⁇ g of mixed protein per fish.
  • Disinfected eyed rainbow trout eggs originating from Fousing Trout Farm, Jutland, Denmark were translocated and hatched in a pathogen-free rearing facility at Bornholm Salmon Hatchery (AquaBaltic, Nexo, Denmark). Fish were vaccinated and then reared for 6 weeks (568 degree-days) in the system containing recirculated municipal water at 14°C in 700 I (1 m 3 ) tanks and fed 1% biomass per day with dry pellet feed (BioMar A/S, Brande, Denmark). Fish were then transported to the fish keeping facility at the University of Copenhagen, Frederiksberg, Denmark and acclimatized by gradually raising the water temperature to 19°C for 1 week before challenge (exposure to AS) was performed. The studies were approved under the license no. 2015-15-0201-00655 issued by the Animal Experiments Inspectorate, Ministry of Environment and Food, Denmark.
  • a total of 360 rainbow trout (average 30 g) were randomly divided into 6 groups of 60 fish and each group was further subdivided into duplicate tanks each containing 30 fish.
  • the fish were anaesthetized (75 mg MS222/I) and i.p. injected with 0.1 ml of the different vaccine formulations (room temperature) (Table B).
  • 10 fish from each group (5 fish per duplicate tank) were sampled for ELISA.
  • the remaining 25 fish (now 40 g) in all the duplicate tanks were challenged with 5.4 x 10 8 CFU/ml AS strain 090710-1/23 by a challenge method using a multi-puncture device.
  • Blood samples for ELISA were collected by caudal vein puncture from euthanized (300 mg MS222/I) fish at 7 wpv and 3 wpc. Blood was allowed to clot at 4 °C overnight whereafter serum was separated by centrifugation at 3000G and 4°C for 10 min and stored at -80° C until further analysis.
  • Enzyme-linked immunosorbent assay was performed according to previously established protocol (Chettri JK et al.; Comparative evaluation of administration methods for a vaccine protecting rainbow trout against Yersinia ruckeri 01 biotype 2 infections; Vet Immunol Immunopathol. 2013 Jul 15; 154(l-2) :42-7. doi: 10.1016/j.vetimm.2013.04.001. Epub 2013 Apr 8).
  • the 96-well microtiter plates MaxiSorpTM, Nunc, Denmark
  • Working dilutions were chosen based on the calibration trials/preliminary results. Dilutions 1 : 5 X 10 2 and 1 : 5 X 10 3 were chosen for testing specific antibodies against sonicated bacteria and dilutions 1 : 10 2 - 1 : 10 4 were chosen for testing specific antibodies against individual proteins. All serum samples were diluted with assay diluent (wash buffer with 0.1% BSA) whereafter 100 ⁇ _ of sample was added to each duplicate ELISA plate well and incubated at 4 °C overnight. After a washing step the plates were incubated for 1 h with 100 ⁇ mouse anti- salmonid Ig (cat. no. MCA2182 AbD Serotec, Germany, diluted 1 : 500).
  • RPS relative percentage of survival
  • This positive control is an injectable formulation of inactivated AS in admixture with inactivated Listonella angullarum serotype 01 and Listonella anguillarum serotype 02a, see.
  • ASA_3328 haemoglobin/transferrin/lactoferrin receptor outer membrane outer membrane 100% [4/4] 98% 3%
  • Table X Recombinant construct design, protein type and vaccine formulation: The column “ACTION” lists which part of the sequence have been removed. The column “aa-region” illustrates which amino acid region of the protein has been expressed. E.g. the signal peptide, transmembrane
  • the full length polypeptides disclosed herein having SEQ ID NOs: 1-20 also have the following designations, which correspond to their accession numbers in the KEGG database (http://www.genome.jp/), where also the corresponding DNA sequences are available:
  • ASA_3883_25-680 this means that the sequence is constituted by amino acid residues 25-680 in ASA_3883 (amino acid residues in SEQ ID NO: 11).

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Abstract

Disclosed is a method of inducing immunity in fish against Aeromonas salmonicida comprising induction of immunity against peptides and polypeptides derived from any one of SEQ ID NOs 1-20. Also disclosed are compositions and agents useful in the method of the invention.

Description

VACCINES AGAINST AEAROMONAS SALMONICIDA INFECTION FIELD OF THE INVENTION
The present invention relates to the field of prophylactic vaccination of fish . In particular, the present invention relates to prophylactic vaccines that induce immunity against A.
salmonicida as well as to related methods and immunogenic agents.
BACKGROUND OF THE INVENTION
Aeromonas salmonicida subsp. salmonicida {A. Salmonicida or AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality.
Furunculosis induced by A. salmonicida subsp. salmonicida has been known as a disease for more than 100 years. Its virulence is contributed to the T3SS virulence system, which causes disease in salmonid fish by circumventing all barriers imposed by the host and the environment. (Bergh PV and Frey J, 2013, Microbial Biotechnol.7, 381-400.
Bergh PV et al., 2013, Proteome Science 11: 44, discloses the in vitro exoproteome of hypervirulent AS and points to potential vaccine candidate proteins.
Commercially available vaccines against AS infection exist. All commercial vaccines include as the immunogen killed (inactivated) AS. Commercially available vaccines include the
AlphaJect® vaccines from Pharmaq/Zoetis.
OBJECT OF THE INVENTION
It is an object of embodiments of the invention to provide a prophylactic subunit vaccine against Aeromonas salmonicida, which is based on recombinant technology. SUMMARY OF THE INVENTION
It has been found by the present inventor(s) that a number of proteins expressed by A. salmonicida are excellent vaccine agents that can induce protective immunity in freshwater fish.
So, in a first aspect the present invention relates to a method of inducing immunity in freshwater fish against A. salmonicida comprising administering an effective amount of a polypeptide comprising
a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1- 20, or
b) an amino acid sequence consisting of at least or exactly or at most 5 contiguous amino acid residues from any one of SEQ ID NOs: 1-20, 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: 1-20, 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.
In a second aspect, the present invention relates to method of inducing immunity in freshwater fish against A. salmonicida comprising administering an effective amount of 1) a nucleic acid encoding a polypeptide used in the first aspect of the invention and the embodiments thereof disclosed herein or 2) a vector comprising and being capable of expressing the nucleic acid in freshwater fish.
A third aspect of the present invention relates to a composition comprising a polypeptide used in the first aspect of the invention and the embodiments thereof disclosed herein in admixture with a diluent, carrier, excipient or vehicle.
A fourth aspect of the present invention relates to a composition comprising a nucleic acid or vector of the third aspect of the invention and the embodiments thereof disclosed herein in admixture with a diluent, carrier, excipient or vehicle.
A fifth aspect of the invention relates to any polypeptide disclosed herein as a polypeptide derived from SEQ ID NOs: 1-20, i.e. any one of the polypeptides referred to in options a-e in the first aspect of the invention and the embodiments thereof. In particular, the invention relates to the polypeptides defined in the context any of options b-e. Consequently, when referring to a "polypeptide of the invention" is meant that the polypeptide is a polypeptide derived from any one of SEQ ID NOs: 1-20, which is disclosed in the context of the method of the first aspect of the invention.
A sixth aspect of the invention relates to a polypeptide of the fifth aspect for use as a pharmaceutical .
A sixth aspect of the invention relates to a polypeptide of the fifth aspect for use in a method of the various embodiments of the first aspect of the invention.
LEGENDS TO THE FIGURE
Fig . 1 : Graph showing cumulative mortality results from infection challenge of rainbow trout. Duplicate tanks were pooled after survival curve comparison with log-rank test showed no significant difference in mortalities.
Fig . 2: Levels of A. salmonicida-spec\f\c antibodies in serum were measured from 10 fish per group (5 fish per duplicate tank) by ELISA at 7 weeks post-vaccination (wpv) and at 3 weeks post- challenge (wpc) . Sera were diluted 1 : 500.
Fig . 3 : Levels in fish serum of specific antibodies against individual proteins from the VacB vaccine.
A: levels measured from 10 fish per group (5 fish per duplicate tank) by ELISA at 7 weeks post- vaccination (wpv) .
B: levels measured from 10 fist per group (5 fish per duplicate tank) by ELISA at 3 weeks post-challenge (wpc) .
Sera were diluted 1 : 100.
Fig . 4: Levels of specific antibodies in serum from fish against individual proteins from the VacC vaccine.
A. Levels were measured from 10 fish per group (5 fish per duplicate tank) by ELISA at 7 weeks post vaccination (wpv)
B: Levels were measured from 10 fish per group (5 fish per duplicate tank) by ELISA at 3 weeks post challenge (wpc) .
Sera were diluted 1 : 100.
Fig . 5 : Correlation (Pearson) between mean antibody levels (dilution 1 : 100) at 7 weeks post vaccination (wpv) and survival in different experimental groups at 24 days post challenge (dpc) . Antibody levels were measured from 10 fish per group (5 fish per duplicate tank) by
ELISA one day before challenge.
A: Protein #8; B: Protein #10; C: Protein # 11.
DETAILED DISCLOSURE OF THE INVENTION
Definitions
The term "polypeptide" is in the present context intended to mean 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 comprising at least two polypeptides, these may form complexes, be covalently linked, or may be 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 reference amino acid sequence or nucleic acid sequence, respectively
The term "amino acid sequence" is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins.
The term "adjuvant" or "immunological 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 2) nevertheless capable of enhancing the immune response against the immunogen. Or, 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 the immunogen, but the combined vaccination with immunogen 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 aligned sequences of equal 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. So, two sequences 5'-ATTCGGAAC-3' and 5'- ATACGGGAC-3' will provide the sequence identity 77.78% (Nref=9 and Ndif=2). It will be understood that such a sequence identity determination requires that the two aligned sequences are aligned so that there are no overhangs between the two sequences: each amino acid in each sequence will have to be matched with a counterpart in the other sequence.
An "assembly of amino acids" means two or more amino acids bound together by physical or chemical means.
The "3D conformation" is the 3 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 3 dimensional 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/hapten, which is not sufficiently immunogenic in its own right - typically, the immunogenic carrier is capable of eliciting a strong cellular immune response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen antibody producing cells and cytotoxic cells. More recently, the large carrier molecules have to a certain extent been substituted by so-called promiscuous epitopes, i.e. shorter peptides that are recognized by a large fraction of MHC-haplotypes in a population, and which elicit antigen specific cellular immune responses.
An "immunogen" is a substance of matter which is capable of inducing an adaptive immune response in a host, whose immune system is exposed to 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 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 the antigen.
A "hapten" is a (typically) small molecule, which can neither induce nor elicit an immune response, but if conjugated to an immunogenic carrier, a specific adaptive immune response can be induced against a hapten upon exposure of the immune system with the hapten carrier conjugate.
An "adaptive immune response" is an immune response in response to exposure to an antigen or immunogen, where the immune response 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 cellular immune responses.
A "protective, adaptive immune response" is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with 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 several pathogens.
"Stimulation of the immune system" means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as 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 simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.
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 the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. Generally, 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 the 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.
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 with an internal surface shape and charge distribution
complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. "Antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
"Specific binding" denotes binding between two substances which goes beyond binding of either substance to randomly chosen substances and 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 for introduction 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 and bacterial vectors - both these infectious agents are capable of introducing a heterologous nucleic acid sequence into a host and effect subsequence expression of a nucleic acid in the host.
The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product 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 used in the first and third aspects of the invention
In some embodiments the at least or exactly 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention constitute at least or exactly 5, at least or exactly or at most 6, at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or at most 33, at least or exactly or at most 34, at least or exactly or at most 35, at least or exactly or at most 36, at least or exactly or at most 37, at least or exactly or at most 38, at least or exactly or at most 39, at least or exactly or at most 40, at least or exactly or at most 41, at least or exactly or at most 42, at least or exactly or at most 43, at least or exactly or at most 44, at least or exactly or at most 45, at least or exactly or at most 46, at least or exactly or at most 47, at least or exactly or at most 48, at least or exactly or at most 49, at least or exactly or at most 50, at least or exactly or at most 51, at least or exactly or at most 52, at least or exactly or at most 53, at least or exactly or at most 54, at least or exactly or at most 55, at least or exactly or at most 56, at least or exactly or at most 57, at least or exactly or at most 58, at least or exactly or at most 59, at least or exactly or at most 60, at least or exactly or at most 61, at least or exactly or at most 62, at least or exactly or at most 63, at least or exactly or at most 64, at least or exactly or at most 65, at least or exactly or at most 66, at least or exactly or at most 67, at least or exactly or at most 68, at least or exactly or at most 69, at least or exactly or at most 70, at least or exactly or at most 71, at least or exactly or at most 72, at least or exactly or at most 73, at least or exactly or at most 74, at least or exactly or at most 75, at least or exactly or at most 76, at least or exactly or at most 77, at least or exactly or at most 78, at least or exactly or at most 79, at least or exactly or at most 80, at least or exactly or at most 81, at least or exactly or at most 82, at least or exactly or at most 83, at least or exactly or at most 84, at least or exactly or at most 85, at least or exactly or at most 86, at least or exactly or at most 87, at least or exactly or at most 88, at least or exactly or at most 89, at least or exactly or at most 90, at least or exactly or at most 91, or at least or exactly or at most 92, at least or exactly or at most 93 contiguous amino acid residues.
The number of contiguous amino acids can be higher, for all of SEQ ID NOs: 2-20. Another way to phrase this is that for each of SEQ ID NOs: 1-20, the number of the contiguous amino acid residues is at least or exactly or at most N-n, where N is the length of the particular sequence ID in question and n is 1, 2, 3, 4, or 5; 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. Consequently: Insofar as embodiment b relates to SEQ ID NOs: 2-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 94, at least or exactly or at most 95, at least or exactly or at most 96, at least or exactly or at most 97, at least or exactly or at most 98, at least or exactly or at most 99, at least or exactly or at most 100, at least or exactly or at most 101, at least or exactly or at most 102, at least or exactly or at most 103, at least or exactly or at most 104, at least or exactly or at most 105, at least or exactly or at most 106, at least or exactly or at most 107, at least or exactly or at most 108, at least or exactly or at most 109, at least or exactly or at most 110, at least or exactly or at most 111, at least or exactly or at most 112, at least or exactly or at most 113, at least or exactly or at most 114, at least or exactly or at most 115, at least or exactly or at most 116, at least or exactly or at most 117, at least or exactly or at most 118, at least or exactly or at most 119, at least or exactly or at most 120, at least or exactly or at most 121, at least or exactly or at most 122, at least or exactly or at most 123, at least or exactly or at most 124, at least or exactly or at most 125, at least or exactly or at most 126, at least or exactly or at most 127, at least or exactly or at most 128, at least or exactly or at most 129, at least or exactly or at most 130, at least or exactly or at most 131, at least or exactly or at most 132, at least or exactly or at most 133, at least or exactly or at most 134, at least or exactly or at most 135, at least or exactly or at most 136, at least or exactly or at most 137, at least or exactly or at most 138, at least or exactly or at most 139, at least or exactly or at most 140, at least or exactly or at most 141, at least or exactly or at most 142, at least or exactly or at most 143, at least or exactly or at most 144, at least or exactly or at most 145, at least or exactly or at most 146, at least or exactly or at most 147, at least or exactly or at most 148, at least or exactly or at most 149, at least or exactly or at most 150, at least or exactly or at most 151, at least or exactly or at most 152, at least or exactly or at most 153, at least or exactly or at most 154, at least or exactly or at most 155, at least or exactly or at most 156, at least or exactly or at most 157, at least or exactly or at most 158, at least or exactly or at most 159, at least or exactly or at most 160, at least or exactly or at most 161, at least or exactly or at most 162, at least or exactly or at most 163, at least or exactly or at most 164, at least or exactly or at most 165, at least or exactly or at most 166, at least or exactly or at most 167, at least or exactly or at most 168, at least or exactly or at most 169, at least or exactly or at most 170, at least or exactly or at most 171, at least or exactly or at most 172, at least or exactly or at most 173, at least or exactly or at most 174, at least or exactly or at most 175, at least or exactly or at most 176, at least or exactly or at most 177, at least or exactly or at most 178, at least or exactly or at most 179, at least or exactly or at most 180, at least or exactly or at most 181, at least or exactly or at most 182, at least or exactly or at most 183, at least or exactly or at most 184, at least or exactly or at most 185, at least or exactly or at most 186, at least or exactly or at most 187, at least or exactly or at most 188, at least or exactly or at most 189, at least or exactly or at most 190, at least or exactly or at most 191, at least or exactly or at most 192, at least or exactly or at most 193, at least or exactly or at most 194, at least or exactly or at most 195, at least or exactly or at most 196, at least or exactly or at most 197, at least or exactly or at most 198, at least or exactly or at most 199, at least or exactly or at most 200, at least or exactly or at most 201, at least or exactly or at most 202, at least or exactly or at most 203, at least or exactly or at most 204, at least or exactly or at most 205, at least or exactly or at most 206, at least or exactly or at most 207, at least or exactly or at most 208, at least or exactly or at most 209, at least or exactly or at most 210, at least or exactly or at most 211, at least or exactly or at most 212, at least or exactly or at most 213, at least or exactly or at most 214, at least or exactly or at most 215, at least or exactly or at most 216, at least or exactly or at most 217, at least or exactly or at most 218, at least or exactly or at most 219, at least or exactly or at most 220, at least or exactly or at most 221, at least or exactly or at most 222, at least or exactly or at most 223, at least or exactly or at most 224, at least or exactly or at most 225, at least or exactly or at most 226, at least or exactly or at most 227, at least or exactly or at most 228, at least or exactly or at most 229, at least or exactly or at most 230, at least or exactly or at most 231, at least or exactly or at most 232, at least or exactly or at most 233, at least or exactly or at most 234, at least or exactly or at most 235, at least or exactly or at most 236, at least or exactly or at most 237, at least or exactly or at most 238, at least or exactly or at most 239, at least or exactly or at most 240, or at least or exactly or at most 241 contiguo us amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 3-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 242, at least or exactly or at most 243, at least or exactly or at most 244, at least or exactly or at most 245, at least or exactly or at most 246, at least or exactly or at most 247, at least or exactly or at most 248, at least or exactly or at most 249, at least or exactly or at most 250, at least or exactly or at most 251, at least or exactly or at most 252, at least or exactly or at most 253, at least or exactly or at most 254, at least or exactly or at most 255, at least or exactly or at most 256, at least or exactly or at most 257, at least or exactly or at most 258, at least or exactly or at most 259, at least or exactly or at most 260, at least or exactly or at most 261, at least or exactly or at most 262, at least or exactly or at most 263, at least or exactly or at most 264, at least or exactly or at most 265, at least or exactly or at most 266, at least or exactly or at most 267, at least or exactly or at most 268, at least or exactly or at most 269, at least or exactly or at most 270, at least or exactly or at most 271, at least or exactly or at most 272, at least or exactly or at most 273, at least or exactly or at most 274, at least or exactly or at most 275, at least or exactly or at most 276, at least or exactly or at most 277, at least or exactly or at most 278, at least or exactly or at most 279, at least or exactly or at most 280, at least or exactly or at most 281, at least or exactly or at most 282, at least or exactly or at most 283, at least or exactly or at most 284, at least or exactly or at most 285, at least or exactly or at most 286, at least or exactly or at most 287, at least or exactly or at most 288, at least or exactly or at most 289, at least or exactly or at most 290, at least or exactly or at most 291, at least or exactly or at most 292, at least or exactly or at most 293, at least or exactly or at most 294, at least or exactly or at most 295, at least or exactly or at most 296, at least or exactly or at most 297, at least or exactly or at most 298, at least or exactly or at most 299, at least or exactly or at most 300, at least or exactly or at most 301, at least or exactly or at most 302, at least or exactly or at most 303, at least or exactly or at most 304, at least or exactly or at most 305, at least or exactly or at most 306, at least or exactly or at most 307, at least or exactly or at most 308, at least or exactly or at most 309, at least or exactly or at most 310, at least or exactly or at most 311, at least or exactly or at most 312, at least or exactly or at most 313, at least or exactly or at most 314, at least or exactly or at most 315, at least or exactly or at most 316, at least or exactly or at most 317, at least or exactly or at most 318, at least or exactly or at most 319, at least or exactly or at most 320, at least or exactly or at most 321, at least or exactly or at most 322, at least or exactly or at most 323, at least or exactly or at most 324, at least or exactly or at most 325, at least or exactly or at most 326, at least or exactly or at most 327, at least or exactly 328, at least or exactly or at most 329, at least or exactly or at most 330, at least or exactly or at most 331, at least or exactly or at most 332, at least or exactly or at most 333, at least or exactly or at most 334, at least or exactly or at most 335, at least or exactly or at most 336, at least or exactly or at most 337, at least or exactly or at most 338, at least or exactly or at most 339, at least or exactly or at most 340, at least or exactly or at most 341, at least or exactly or at most 342, at least or exactly or at most 343, at least or exactly or at most 344, at least or exactly or at most 345, at least or exactly or at most 346, at least or exactly or at most 347, at least or exactly or at most 348, at least or exactly or at most 349, at least or exactly or at most 350, at least or exactly or at most 351, at least or exactly or at most 352, at least or exactly or at most 353, at least or exactly or at most 354, at least or exactly or at most 355, at least or exactly or at most 356, at least or exactly or at most 357, at least or exactly or at most 358, at least or exactly or at most 359, at least or exactly or at most 360, at least or exactly or at most 361, at least or exactly or at most 362, at least or exactly or at most 363, at least or exactly or at most 364, at least or exactly or at most 365, at least or exactly or at most 366, at least or exactly or at most 367, at least or exactly or at most 368, at least or exactly or at most 369, at least or exactly or at most 370, at least or exactly or at most 371, at least or exactly or at most 372, at least or exactly or at most 373, at least or exactly or at most 374, at least or exactly or at most 375, at least or exactly or at most 376, at least or exactly or at most 377, at least or exactly or at most 378, at least or exactly or at most 379, at least or exactly or at most 380, at least or exactly or at most 381, at least or exactly or at most 382, at least or exactly or at most 383, at least or exactly or at most 384, at least or exactly or at most 385, at least or exactly or at most 386, at least or exactly or at most 387, or at least or exactly or at most 388 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NOs: 4-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 389, at least or exactly or at most 390, at least or exactly or at most 391, at least or exactly or at most 392, at least or exactly or at most 393, at least or exactly or at most 394, at least or exactly or at most 395, at least or exactly or at most 396, at least or exactly or at most 397, at least or exactly or at most 398, at least or exactly or at most 399, at least or exactly or at most 400, at least or exactly or at most 401, or at least or exactly or at most 402 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 5-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 403, at least or exactly or at most 404, at least or exactly or at most 405, at least or exactly or at most 406, at least or exactly or at most 407, at least or exactly or at most 408, at least or exactly or at most 409, at least or exactly or at most 410, at least or exactly or at most 411, at least or exactly or at most 412, at least or exactly or at most 413, at least or exactly or at most 414, at least or exactly or at most 415, at least or exactly or at most 416, at least or exactly or at most 417, at least or exactly or at most 418, at least or exactly or at most 419, at least or exactly or at most 420, at least or exactly or at most 421, at least or exactly or at most 422, at least or exactly or at most 423, at least or exactly or at most 424, at least or exactly or at most 425, at least or exactly or at most 426, at least or exactly or at most 427, at least or exactly or at most 428, at least or exactly or at most 429, at least or exactly or at most 430, at least or exactly or at most 431, at least or exactly or at most 432, at least or exactly or at most 433, at least or exactly or at most 434, at least or exactly or at most 435, at least or exactly or at most 436, at least or exactly or at most 437, at least or exactly or at most 438, at least or exactly or at most 439, at least or exactly or at most 440, at least or exactly or at most 441, at least or exactly or at most 442, at least or exactly or at most 443, at least or exactly or at most 444, at least or exactly or at most 445, at least or exactly or at most 446, at least or exactly or at most 447, at least or exactly or at most 448, at least or exactly or at most 449, at least or exactly or at most 450, at least or exactly or at most 451, at least or exactly or at most 452, at least or exactly or at most 453, at least or exactly or at most 454, at least or exactly or at most 455, at least or exactly or at most 456, at least or exactly or at most 457, at least or exactly or at most 458, at least or exactly or at most 459, at least or exactly or at most 460, at least or exactly or at most 461, at least or exactly or at most 462, at least or exactly or at most 463, at least or exactly or at most 464, at least or exactly or at most 465, at least or exactly or at most 466, at least or exactly or at most 467, or at least or exactly or at most 468 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NOs: 6-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 469, at least or exactly or at most 470, at least or exactly or at most 471, at least or exactly or at most 472, at least or exactly or at most 473, at least or exactly or at most 474, at least or exactly or at most 475, at least or exactly or at most 476, at least or exactly or at most 477, at least or exactly or at most 478, at least or exactly or at most 479, or at least or exactly or at most 480 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 7-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 481, at least or exactly or at most 482, at least or exactly or at most 483, at least or exactly or at most 484, at least or exactly or at most 485, at least or exactly or at most 486, at least or exactly or at most 487, at least or exactly or at most 488, at least or exactly or at most 489, at least or exactly or at most 490, at least or exactly or at most 491, at least or exactly or at most 492, at least or exactly or at most 493, at least or exactly or at most 494, at least or exactly or at most 495, at least or exactly or at most 496, at least or exactly or at most 497, at least or exactly or at most 498, at least or exactly or at most 499, at least or exactly or at most 500, at least or exactly or at most 501, at least or exactly or at most 502, at least or exactly or at most 503, at least or exactly or at most 504, at least or exactly or at most 505, at least or exactly or at most 506, at least or exactly or at most 507, at least or exactly or at most 508, at least or exactly or at most 509, at least or exactly or at most 510, at least or exactly or at most 511, at least or exactly or at most 512, at least or exactly or at most 513, at least or exactly or at most 514, at least or exactly or at most 515, at least or exactly or at most 516, at least or exactly or at most 517, at least or exactly or at most 518, at least or exactly or at most 519, at least or exactly or at most 520, at least or exactly or at most 521, at least or exactly or at most 522, at least or exactly or at most 523, at least or exactly or at most 524, at least or exactly or at most 525, at least or exactly or at most 526, at least or exactly or at most 527, at least or exactly or at most 528, at least or exactly or at most 529, at least or exactly or at most 530, at least or exactly or at most 531, at least or exactly or at most 532, at least or exactly or at most 533, at least or exactly or at most 534, at least or exactly or at most 535, at least or exactly or at most 536, at least or exactly or at most 537, at least or exactly or at most 538, at least or exactly or at most 539, at least or exactly or at most 540, at least or exactly or at most 541, at least or exactly or at most 542, at least or exactly or at most 543, at least or exactly or at most 544, at least or exactly or at most 545, at least or exactly or at most 546, at least or exactly or at most 547, at least or exactly or at most 548, at least or exactly or at most 549, at least or exactly or at most 550, at least or exactly or at most 551, at least or exactly or at most 552, at least or exactly or at most 553, at least or exactly or at most 554, at least or exactly or at most 555, or at least or exactly or at most 556 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NOs: 8-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 557, at least or exactly or at most 558, at least or exactly or at most 559, at least or exactly or at most 560, at least or exactly or at most 561, at least or exactly or at most 562, at least or exactly or at most 563, at least or exactly or at most 564, at least or exactly or at most 565, at least or exactly or at most 566, at least or exactly or at most 567, at least or exactly or at most 568, at least or exactly or at most 569, at least or exactly or at most 570, at least or exactly or at most 571, at least or exactly or at most 572, at least or exactly or at most 573, at least or exactly or at most 574, at least or exactly or at most 575, at least or exactly or at most 576, at least or exactly or at most 577, or at least or exactly or at most 578 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 9-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 579, at least or exactly or at most 580, at least or exactly or at most 581, at least or exactly or at most 582, at least or exactly or at most 583, at least or exactly or at most 584, at least or exactly or at most 585, at least or exactly or at most 586, at least or exactly or at most 587, at least or exactly or at most 588, at least or exactly or at most 589, at least or exactly or at most 590, at least or exactly or at most 591, at least or exactly or at most 592, at least or exactly or at most 593, at least or exactly or at most 594, at least or exactly or at most 595, at least or exactly or at most 596, at least or exactly or at most 597, at least or exactly or at most 598, at least or exactly or at most 599, at least or exactly or at most 600, at least or exactly or at most 601, at least or exactly or at most 602, at least or exactly or at most 603, at least or exactly or at most 604, at least or exactly or at most 605, at least or exactly or at most 606, at least or exactly or at most 607, at least or exactly or at most 608, at least or exactly or at most 609, at least or exactly or at most 610, at least or exactly or at most 611, at least or exactly or at most 612, at least or exactly or at most 613, at least or exactly or at most 614, at least or exactly or at most 615, at least or exactly or at most 616, at least or exactly or at most 617, at least or exactly or at most 618, at least or exactly or at most 619, at least or exactly or at most 620, at least or exactly or at most 621, at least or exactly or at most 622, at least or exactly or at most 623, at least or exactly or at most 624, at least or exactly or at most 625, at least or exactly or at most 626, at least or exactly or at most 627, at least or exactly or at most 628, at least or exactly or at most 629, at least or exactly or at most 630, at least or exactly or at most 631, at least or exactly or at most 632, at least or exactly or at most 633, at least or exactly or at most 634, at least or exactly or at most 635, at least or exactly or at most 636, at least or exactly or at most 637, at least or exactly or at most 638, or at least or exactly or at most 639 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NOs: 10-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 640 contiguous amino acid residues
Insofar as embodiment b relates to SEQ ID NOs: 11-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 641, at least or exactly or at most 642, at least or exactly or at most 643, at least or exactly or at most 644, at least or exactly or at most 645, at least or exactly or at most 646, at least or exactly or at most 647, at least or exactly or at most 648, at least or exactly or at most 649, at least or exactly or at most 650, at least or exactly or at most 651, at least or exactly or at most 652, at least or exactly or at most 653, at least or exactly or at most 654, at least or exactly or at most 655, at least or exactly or at most 656, at least or exactly or at most 657, at least or exactly or at most 658, at least or exactly or at most 659, at least or exactly or at most 660, or at least or exactly or at most 661, at least or exactly or at most 662, at least or exactly or at most 663, at least or exactly or at most 664, at least or exactly or at most 665, at least or exactly or at most 666, at least or exactly or at most 667, at least or exactly or at most 668, at least or exactly or at most 669, at least or exactly or at most 670, at least or exactly or at most 671, at least or exactly or at most 672, at least or exactly or at most 673, at least or exactly or at most 674, at least or exactly or at most 675, at least or exactly or at most 676, at least or exactly or at most 677, at least or exactly or at most 678, at least or exactly or at most 679, or at least or exactly or at most 680 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 12-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 681, at least or exactly or at most 682, at least or exactly or at most 683, at least or exactly or at most 684, at least or exactly or at most 685, at least or exactly or at most 686, at least or exactly or at most 687, at least or exactly or at most 688, at least or exactly or at most 689, at least or exactly or at most 690, at least or exactly or at most 691, at least or exactly or at most 692, at least or exactly or at most 693, at least or exactly or at most 694, at least or exactly or at most 695, at least or exactly or at most 696, or at least or exactly or at most 697 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 13-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 698, at least or exactly or at most 699, at least or exactly or at most 700, at least or exactly or at most 701, at least or exactly or at most 702, at least or exactly or at most 703, at least or exactly or at most 704, at least or exactly or at most 705, at least or exactly or at most 706, at least or exactly or at most 707, at least or exactly or at most 708, at least or exactly or at most 709, at least or exactly or at most 710, at least or exactly or at most 711, or at least or exactly or at most 712 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 14-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 713, at least or exactly or at most 714, at least or exactly or at most 715, at least or exactly or at most 716, at least or exactly or at most 717, at least or exactly or at most 718, at least or exactly or at most 719, at least or exactly or at most 720, at least or exactly or at most 721, at least or exactly or at most 722, at least or exactly or at most 723, at least or exactly or at most 724, at least or exactly or at most 725, at least or exactly or at most 726, at least or exactly or at most 727, at least or exactly or at most 728, at least or exactly or at most 729, at least or exactly or at most 730, at least or exactly or at most 731, at least or exactly or at most 732, at least or exactly or at most 733, at least or exactly or at most 734, at least or exactly or at most 735, at least or exactly or at most 736, at least or exactly or at most 737, at least or exactly or at most 738, at least or exactly or at most 739, at least or exactly or at most 740, at least or exactly or at most 741, at least or exactly or at most 742, at least or exactly or at most 743, at least or exactly or at most 744, at least or exactly or at most 745, at least or exactly or at most 746, at least or exactly or at most 747, at least or exactly or at most 748, at least or exactly or at most 749, at least or exactly or at most 750, at least or exactly or at most 751, at least or exactly or at most 752, at least or exactly or at most 753, or at least or exactly or at most 754 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 15-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 755, at least or exactly or at most 756, at least or exactly or at most 757, at least or exactly or at most 758, at least or exactly or at most 759, at least or exactly or at most 760, at least or exactly or at most 761, at least or exactly or at most 762, at least or exactly or at most 763, at least or exactly or at most 764, at least or exactly or at most 765, at least or exactly or at most 766, at least or exactly or at most 767, at least or exactly or at most 768, at least or exactly or at most 769, at least or exactly or at most 770, at least or exactly or at most 771, at least or exactly or at most 772, at least or exactly or at most 773, at least or exactly or at most 774, at least or exactly or at most 775, at least or exactly or at most 776, at least or exactly or at most 777, at least or exactly or at most 778, at least or exactly or at most 779, at least or exactly or at most 780, at least or exactly or at most 781, at least or exactly or at most 782, at least or exactly or at most 783, at least or exactly or at most 784, at least or exactly or at most 785, at least or exactly or at most 786, at least or exactly or at most 787, at least or exactly or at most 788, at least or exactly or at most 789, at least or exactly or at most 790, at least or exactly or at most 791, at least or exactly or at most 792, at least or exactly or at most 793, at least or exactly or at most 794, at least or exactly or at most 795, at least or exactly or at most 796, at least or exactly or at most 797, at least or exactly or at most 798, at least or exactly or at most 799, at least or exactly or at most 800, at least or exactly or at most 801, at least or exactly or at most 802, at least or exactly or at most 803, at least or exactly or at most 804, at least or exactly or at most 805, at least or exactly or at most 806, at least or exactly or at most 807, at least or exactly or at most 808, at least or exactly or at most 809, at least or exactly or at most 810, at least or exactly or at most 811, at least or exactly or at most 812, at least or exactly or at most 813, at least or exactly or at most 814, at least or exactly or at most 815, at least or exactly or at most 816, at least or exactly or at most 817, at least or exactly or at most 818, at least or exactly or at most 819, at least or exactly or at most 820, at least or exactly or at most 821, at least or exactly or at most 822, at least or exactly or at most 823, at least or exactly or at most 824, at least or exactly or at most 825, at least or exactly or at most 826, at least or exactly or at most 827, at least or exactly or at most 828, at least or exactly or at most 829, at least or exactly or at most 830, at least or exactly or at most 831, at least or exactly or at most 832, at least or exactly or at most 833, at least or exactly or at most 834, at least or exactly or at most 835, at least or exactly or at most 836, at least or exactly or at most 837, at least or exactly or at most 838, at least or exactly or at most 839, at least or exactly or at most 840, at least or exactly or at most 841, at least or exactly or at most 842, at least or exactly or at most 843, at least or exactly or at most 844, at least or exactly or at most 845, at least or exactly or at most 846, at least or exactly or at most 847, at least or exactly or at most 848, at least or exactly or at most 849, at least or exactly or at most 850, at least or exactly or at most 851, at least or exactly or at most 852, at least or exactly or at most 853, at least or exactly or at most 854, at least or exactly or at most 855, at least or exactly or at most 856, at least or exactly or at most 857, at least or exactly or at most 858, at least or exactly or at most 859, at least or exactly or at most 860, at least or exactly or at most 861, at least or exactly or at most 862, at least or exactly or at most 863, at least or exactly or at most 864, at least or exactly or at most 865, at least or exactly or at most 866, at least or exactly or at most 867, at least or exactly or at most 868, at least or exactly or at most 869, at least or exactly or at most 870, at least or exactly or at most 871, at least or exactly or at most 872, at least or exactly or at most 873, at least or exactly or at most 874, at least or exactly or at most 875, at least or exactly or at most 876, or at least or exactly or at most 877 contiguous amino acic I residues.
Insofar as embodiment b relates to SEQ ID NOs: 16-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 878, at least or exactly or at most 879, at least or exactly or at most 880, at least or exactly or at most 881, at least or exactly or at most 882, at least or exactly or at most 883, at least or exactly or at most 884, at least or exactly or at most 885, at least or exactly or at most 886, at least or exactly or at most 887, at least or exactly or at most 888, at least or exactly or at most 889, at least or exactly or at most 890, at least or exactly or at most 891, at least or exactly or at most 892, at least or exactly or at most 893, at least or exactly or at most 894, at least or exactly or at most 895, at least or exactly or at most 896, at least or exactly or at most 897, at least or exactly or at most 898, at least or exactly or at most 899, at least or exactly or at most 900, at least or exactly or at most 901, at least or exactly or at most 902, at least or exactly or at most 903, at least or exactly or at most 904, at least or exactly or at most 905, at least or exactly or at most 906, at least or exactly or at most 907, at least or exactly or at most 908, at least or exactly or at most 909, at least or exactly or at most 910, at least or exactly or at most 911, at least or exactly or at most 912, at least or exactly or at most 913, at least or exactly or at most 914, or at least or exactly or at most 915 contiguo IUS amino acid residues.
Insofar as embodiment b relates to SEQ ID N Os: 17-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 916, at least or exactly or at most 917, at least or exactly or at most 918, at least or exactly or at most 919, at least or exactly or at most 920, at least or exactly or at most 921, at least or exactly or at most 922, at least or exactly or at most 923, at least or exactly or at most 924, at least or exactly or at most 925, at least or exactly or at most 926, at least or exactly or at most 927, at least or exactly or at most 928, at least or exactly or at most 929, at least or exactly or at most 930, at least or exactly or at most 931, at least or exactly or at most 932, at least or exactly or at most 933, at least or exactly or at most 934, at least or exactly or at most 935, at least or exactly or at most 936, at least or exactly or at most 937, at least or exactly or at most 938, at least or exactly or at most 939, at least or exactly or at most 940, at least or exactly or at most 941, at least or exactly or at most 942, at least or exactly or at most 943, at least or exactly or at most 944, at least or exactly or at most 945, at least or exactly or at most 946, at least or exactly or at most 947, at least or exactly or at most 948, at least or exactly or at most 949, at least or exactly or at most 950, at least or exactly or at most 951, at least or exactly or at most 952, at least or exactly or at most 953, at least or exactly or at most 954, at least or exactly or at most 955, at least or exactly or at most 956, at least or exactly or at most 957, at least or exactly or at most 958, at least or exactly or at most 959, at least or exactly or at most 960, at least or exactly or at most 961, at least or exactly or at most 962, at least or exactly or at most 963, at least or exactly or at most 964, at least or exactly or at most 965, at least or exactly or at most 966, at least or exactly or at most 967, at least or exactly or at most 968, at least or exactly or at most 969, at least or exactly or at most 970, at least or exactly or at most 971, at least or exactly or at most 972, at least or exactly or at most 973, at least or exactly or at most 974, at least or exactly or at most 975, at least or exactly or at most 976, at least or exactly or at most 977, at least or exactly or at most 978, at least or exactly or at most 979, at least or exactly or at most 980, at least or exactly or at most 981, at least or exactly or at most 982, at least or exactly or at most 983, at least or exactly or at most 984, at least or exactly or at most 985, at least or exactly or at most 986, at least or exactly or at most 987, at least or exactly or at most 988, at least or exactly or at most 989, at least or exactly or at most 990, at least or exactly or at most 991, at least or exactly or at most 992, at least or exactly or at most 993, at least or exactly or at most 994, at least or exactly or at most 995, at least or exactly or at most 996, at least or exactly or at most 997, at least or exactly or at most 998, at least or exactly or at most 999, at least or exactly or at most 1000, at least or exactly or at most 1001, at least or exactly or at most 1002, at least or exactly or at most 1003, at least or exactly or at most 1004, at least or exactly or at most 1005, at least or exactly or at most 1006, at least or exactly or at most 1007, at least or exactly or at most 1008, at least or exactly or at most 1009, at least or exactly or at most 1010, at least or exactly or at most 1011, at least or exactly or at most 1012, at least or exactly or at most 1013, at least or exactly or at most 1014, at least or exactly or at most 1015, at least or exactly or at most 1016, at least or exactly or at most 1017, at least or exactly or at most 1018, at least or exactly or at most 1019, or at least or exactly or at most 1020 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NOs: 18-20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 1021, at least or exactly or at most 1022, at least or exactly or at most 1023, at least or exactly or at most 1024, at least or exactly or at most 1025, at least or exactly or at most 1026, at least or exactly or at most 1027, at least or exactly or at most 1028, at least or exactly or at most 1029, at least or exactly or at most 1030, at least or exactly or at most 1031, at least or exactly or at most 1032, at least or exactly or at most 1033, at least or exactly or at most 1034, at least or exactly or at most 1035, at least or exactly or at most 1036, at least or exactly or at most 1037, at least or exactly or at most 1038, at least or exactly or at most 1039, at least or exactly or at most 1040, at least or exactly or at most 1041, at least or exactly or at most 1042, at least or exactly or at most 1043, at least or exactly or at most 1044, at least or exactly or at most 1045, at least or exactly or at most 1046, at least or exactly or at most 1047, at least or exactly or at most 1048, at least or exactly or at most 1049, at least or exactly or at most 1050, at least or exactly or at most 1051, at least or exactly or at most 1052, at least or exactly or at most 1053, at least or exactly or at most 1054, at least or exactly or at most 1055, at least or exactly or at most 1056, at least or exactly or at most 1057, at least or exactly or at most 1058, at least or exactly or at most 1059, at least or exactly or at most 1060, at least or exactly or at most 1061, at least or exactly or at most 1062, at least or exactly or at most 1063, at least or exactly or at most 1064, at least or exactly or at most 1065, at least or exactly or at most 1066, at least or exactly or at most 1067, at least or exactly or at most 1068, at least or exactly or at most 1069, at least or exactly or at most 1070, at least or exactly or at most 1071, at least or exactly or at most 1072, at least or exactly or at most 1073, at least or exactly or at most 1074, at least or exactly or at most 1075, at least or exactly or at most 1076, at least or exactly or at most 1077, at least or exactly or at most 1078, at least or exactly or at most 1079, at least or exactly or at most 1080, at least or exactly or at most 1081, at least or exactly or at most 1082, at least or exactly or at most 1083, at least or exactly or at most 1084, at least or exactly or at most 1085, at least or exactly or at most 1086, at least or exactly or at most 1087, at least or exactly or at most 1088, at least or exactly or at most 1089, at least or exactly or at most 1090, at least or exactly or at most 1091, at least or exactly or at most 1092, at least or exactly or at most 1093, at least or exactly or at most 1094, at least or exactly or at most 1095, at least or exactly or at most 1096, at least or exactly or at most 1097, at least or exactly or at most 1098, at least or exactly or at most 1099, at least or exactly or at most 1100, at least or exactly or at most 1101, at least or exactly or at most 1102, at least or exactly or at most 1103, at least or exactly or at most 1104, at least or exactly or at most 1105, at least or exactly or at most 1106, at least or exactly or at most 1107, at least or exactly or at most 1108, at least or exactly or at most 1109, at least or exactly or at most 1110, at least or exactly or at most 1111, at least or exactly or at most 1112, at least or exactly or at most 1113, at least or exactly or at most 1114, at least or exactly or at most 1115, at least or exactly or at most 1116, at least or exactly or at most 1117, at least or exactly or at most 1118, at least or exactly or at most 1119, at least or exactly or at most 1120, at least or exactly or at most 1121, at least or exactly or at most 1122, at least or exactly or at most 1123, at least or exactly or at most 1124, at least or exactly or at most 1125, at least or exactly or at most 1126, at least or exactly or at most 1127, at least or exactly or at most 1128, at least or exactly or at most 1129, at least or exactly or at most 1130, at least or exactly or at most 1131, at least or exactly or at most 1132, at least or exactly or at most 1133, at least or exactly or at most 1134, at least or exactly or at most 1135, at least or exactly or at most 1136, at least or exactly or at most 1137, at least or exactly or at most 1138, at least or exactly or at most 1139, at least or exactly or at most 1140, at least or exactly or at most 1141, at least or exactly or at most 1142, at least or exactly or at most 1143, at least or exactly or at most 1144, at least or exactly or at most 1145, at least or exactly or at most 1146, at least or exactly or at most 1147, at least or exactly or at most 1148, at least or exactly or at most 1149, at least or exactly or at most 1150, at least or exactly or at most 1151, at least or exactly or at most 1152, at least or exactly or at most 1153, at least or exactly or at most 1154, at least or exactly or at most 1155, or at least or exactly or at most 1156 contiguous amino acid residues. Insofar as embodiment b relates to SEQ ID NOs: 19 or 20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 1157, at least or exactly or at most 1158, at least or exactly or at most 1159, at least or exactly or at most 1160, at least or exactly or at most 1161, at least or exactly or at most 1162, at least or exactly or at most 1163, at least or exactly or at most 1164, at least or exactly or at most 1165, at least or exactly or at most 1166, at least or exactly or at most 1167, at least or exactly or at most 1168, at least or exactly or at most 1169, at least or exactly or at most 1170, at least or exactly or at most 1171, at least or exactly or at most 1172, at least or exactly or at most 1173, at least or exactly or at most 1174, at least or exactly or at most 1175, at least or exactly or at most 1176, at least or exactly or at most 1177, at least or exactly or at most 1178, at least or exactly or at most 1179, at least or exactly or at most 1180, at least or exactly or at most 1181, at least or exactly or at most 1182, at least or exactly or at most 1183, at least or exactly or at most 1184, at least or exactly or at most 1185, at least or exactly or at most 1186, at least or exactly or at most 1187, at least or exactly or at most 1188, at least or exactly or at most 1189, at least or exactly or at most 1190, at least or exactly or at most 1191, at least or exactly or at most 1192, at least or exactly or at most 1193, at least or exactly or at most 1194, at least or exactly or at most 1195, at least or exactly or at most 1196, at least or exactly or at most 1197, at least or exactly or at most 1198, at least or exactly or at most 1199, at least or exactly or at most 1200, at least or exactly or at most 1201, at least or exactly or at most 1202, at least or exactly or at most 1203, at least or exactly or at most 1204, at least or exactly or at most 1205, at least or exactly or at most 1206, at least or exactly or at most 1207, at least or exactly or at most 1208, at least or exactly or at most 1209, at least or exactly or at most 1210, at least or exactly or at most 1211, at least or exactly or at most 1212, at least or exactly or at most 1213, at least or exactly or at most 1214, at least or exactly or at most 1215, at least or exactly or at most 1216, at least or exactly or at most 1217, at least or exactly or at most 1218, at least or exactly or at most 1219, at least or exactly or at most 1220, at least or exactly or at most 1221, at least or exactly or at most 1222, at least or exactly or at most 1223, at least or exactly or at most 1224, at least or exactly or at most 1225, at least or exactly or at most 1226, at least or exactly or at most 1227, at least or exactly or at most 1228, at least or exactly or at most 1229, at least or exactly or at most 1230, at least or exactly or at most 1231, at least or exactly or at most 1232, at least or exactly or at most 1233, at least or exactly or at most 1234, at least or exactly or at most 1235, at least or exactly or at most 1236, at least or exactly or at most 1237, at least or exactly or at most 1238, at least or exactly or at most 1239, at least or exactly or at most 1240, at least or exactly or at most 1241, at least or exactly or at most 1242, at least or exactly or at most 1243, at least or exactly or at most 1244, at least or exactly or at most 1245, at least or exactly or at most 1246, at least or exactly or at most 1247, at least or exactly or at most 1248, at least or exactly or at most 1249, at least or exactly or at most 1250, at least or exactly or at most 1251, at least or exactly or at most 1252, at least or exactly or at most 1253, at least or exactly or at most 1254, at least or exactly or at most 1255, at least or exactly or at most 1256, at least or exactly or at most 1257, at least or exactly or at most 1258, at least or exactly or at most 1259, at least or exactly or at most 1260, at least or exactly or at most 1261, at least or exactly or at most 1262, at least or exactly or at most 1263, at least or exactly or at most 1264, or at least or exactly or at most 1265 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NO 20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute at least or exactly or at most 1266, at least or exactly or at most 1267, at least or exactly or at most 1268, at least or exact y or at most 1269, at least or exactly or at most 1270, at least or exactly or at most 1271, at least or exactly or at most 1272, at least or exactly or at most 1273, at least or exact y or at most 1274, at least or exactly or at most 1275, at least or exactly or at most 1276, at least or exactly or at most 1277, at least or exactly or at most 1278, at least or exact y or at most 1279, at least or exactly or at most 1280, at least or exactly or at most 1281, at least or exactly or at most 1282, at least or exactly or at most 1283, at least or exact y or at most 1284, at least or exactly or at most 1285, at least or exactly or at most 1286, at least or exactly or at most 1287, at least or exactly or at most 1288, at least or exact y or at most 1289, at least or exactly or at most 1290, at least or exactly or at most 1291, at least or exactly or at most 1292, at least or exactly or at most 1293, at least or exact y or at most 1294, at least or exactly or at most 1295, at least or exactly or at most 1296, at least or exactly or at most 1297, at least or exactly or at most 1298, at least or exact y or at most 1299, at least or exactly or at most 1300, at least or exactly or at most 1301, at least or exactly or at most 1302, at least or exactly or at most 1303, at least or exact y or at most 1304, at least or exactly or at most 1305, at least or exactly or at most 1306, at least or exactly or at most 1307, at least or exactly or at most 1308, at least or exact y or at most 1309, at least or exactly or at most 1310, at least or exactly or at most 1311, at least or exactly or at most 1312, at least or exactly or at most 1313, at least or exact y or at most 1314, at least or exactly or at most 1315, at least or exactly or at most 1316, at least or exactly or at most 1317, at least or exactly or at most 1318, at least or exact y or at most 1319, at least or exactly or at most 1320, at least or exactly or at most 1321, at least or exactly or at most 1322, at least or exactly or at most 1323, at least or exact y or at most 1324, at least or exactly or at most 1325, at least or exactly or at most 1326, at least or exactly or at most 1327, at least or exactly or at most 1328, at least or exact y or at most 1329, at least or exactly or at most 1330, at least or exactly or at most 1331, at least or exactly or at most 1332, at least or exactly or at most 1333, at least or exact y or at most 1334, at least or exactly or at most 1335, at least or exactly or at most 1336, at least or exactly or at most 1337, at least or exactly or at most 1338, at least or exactly or at most 1339, at least or exactly or at most 1340, at least or exactly or at most 1341, at least or exactly or at most 1342, at least or exactly or at most 1343, or at least or exactly or at most 1344 contiguous amino acid residues.
Insofar as embodiment b relates to SEQ ID NO: 20, the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also constitute, at least or exactly or at most 1345, at least or exactly or at most 1346, at least or exactly or at most 1347, at least or exact y or at most 1348, at least or exactly or at most 1349, at least or exactly or at most 1350, at least or exactly or at most 1351, at least or exactly or at most 1352, at least or exact y or at most 1353, at least or exactly or at most 1354, at least or exactly or at most 1355, at least or exactly or at most 1356, at least or exactly or at most 1357, at least or exact y or at most 1358, at least or exactly or at most 1359, at least or exactly or at most 1360, at least or exactly or at most 1361, at least or exactly or at most 1362, at least or exact y or at most 1363, at least or exactly or at most 1364, at least or exactly or at most 1365, at least or exactly or at most 1366, at least or exactly or at most 1367, at least or exact y or at most 1368, at least or exactly or at most 1369, at least or exactly or at most 1370, at least or exactly or at most 1371, at least or exactly or at most 1372, at least or exact y or at most 1373, at least or exactly or at most 1374, at least or exactly or at most 1375, at least or exactly or at most 1376, at least or exactly or at most 1377, at least or exact y or at most 1378, at least or exactly or at most 1379, at least or exactly or at most 1380, at least or exactly or at most 1381, at least or exactly or at most 1382, at least or exact y or at most 1383, at least or exactly or at most 1384, at least or exactly or at most 1385, at least or exactly or at most 1386, at least or exactly or at most 1387, at least or exact y or at most 1388, at least or exactly or at most 1389, at least or exactly or at most 1390, at least or exactly or at most 1391, at least or exactly or at most 1392, at least or exact y or at most 1393, at least or exactly or at most 1394, at least or exactly or at most 1395, at least or exactly or at most 1396, at least or exactly or at most 1397, at least or exact y or at most 1398, at least or exactly or at most 1399, at least or exactly or at most 1400, at least or exactly or at most 1401, at least or exactly or at most 1402, at least or exact y or at most 1403, at least or exactly or at most 1404, at least or exactly or at most 1405, at least or exactly or at most 1406, at least or exactly or at most 1407, at least or exact y or at most 1408, at least or exactly or at most 1409, at least or exactly or at most 1410, at least or exactly or at most 1411, at least or exactly or at most 1412, at least or exact y or at most 1413, at least or exactly or at most 1414, at least or exactly or at most 1415, at least or exactly or at most 1416, at least or exactly or at most 1417, at least or exact y or at most 1418, at least or exactly or at most
1419, at least or exactly or at most 1420, at least or exactly or at most 1421, at least or exactly or at most 1422, at least or exactly or at most 1423, at least or exactly or at most 1424, at least or exactly or at most 1425, at least or exactly or at most 1426, at least or exactly or at most 1427, at least or exactly or at most 1428, at least or exactly or at most 1429, at least or exactly or at most 1430, at least or exactly or at most 1431, at least or exactly or at most 1432, at least or exactly or at most 1433, at least or exactly or at most 1434, at least or exactly or at most 1435, at least or exactly or at most 1436, at least or exactly or at most 1437, at least or exactly or at most 1438, at least or exactly or at most 1439, at least or exactly or at most 1440, at least or exactly or at most 1441, at least or exactly or at most 1442, at least or exactly or at most 1443, at least or exactly or at most 1444, at least or exactly or at most 1445, at least or exactly or at most 1446, at least or exactly or at most 1447, at least or exactly or at most 1448, at least or exactly or at most 1449, at least or exactly or at most 1450, at least or exactly or at most 1451, at least or exactly or at most 1452, at least or exactly or at most 1453, at least or exactly or at most 1454, at least or exactly or at most 1455, at least or exactly or at most 1456, at least or exactly or at most 1457, at least or exactly or at most 1458, at least or exactly or at most 1459, at least or exactly or at most 1460, at least or exactly or at most 1461, at least or exactly or at most 1462, at least or exactly or at most 1463, at least or exactly or at most 1464, at least or exactly or at most 1465, at least or exactly or at most 1466, at least or exactly or at most 1467, at least or exactly or at most 1468, at least or exactly or at most 1469, at least or exactly or at most 1470, at least or exactly or at most 1471, at least or exactly or at most 1472, at least or exactly or at most 1473, at least or exactly or at most 1474, at least or exactly or at most 1475, at least or exactly or at most 1476, at least or exactly or at most 1477, at least or exactly or at most 1478, at least or exactly or at most 1479, at least or exactly or at most 1480, at least or exactly or at most 1481, at least or exactly or at most 1482, at least or exactly or at most 1483, at least or exactly or at most 1484, at least or exactly or at most 1485, at least or exactly or at most 1486, at least or exactly or at most 1487, at least or exactly or at most 1488, at least or exactly or at most 1489, at least or exactly or at most 1490, at least or exactly or at most 1491, at least or exactly or at most 1492, at least or exactly or at most 1493, at least or exactly or at most 1494, at least or exactly or at most 1495, at least or exactly or at most 1496, at least or exactly or at most 1497, at least or exactly or at most 1498, at least or exactly or at most 1499, at least or exactly or at most 1500, at least or exactly or at most 1501, at least or exactly or at most 1502, at least or exactly or at most 1503, at least or exactly or at most 1504, at least or exactly or at most 1505, at least or exactly or at most 1506, at least or exactly or at most 1507, at least or exactly or at most 1508, at least or exactly or at most 1509, at least or exactly or at most 1510, at least or exactly or at most 1511, at least or exactly or at most 1512, at least or exactly or at most 1513, at least or exactly or at most 1514, at least or exactly or at most 1515, at least or exactly or at most 1516, at least or exactly or at most 1517, at least or exactly or at most 1518, at least or exactly or at most 1519, at least or exactly or at most 1520, at least or exactly or at most 1521, at least or exactly or at most 1522, at least or exactly or at most 1523, at least or exactly or at most 1524, at least or exactly or at most 1525, at least or exactly or at most 1526, at least or exactly or at most 1527, at least or exactly or at most 1528, at least or exactly or at most 1529, at least or exactly or at most 1530, at least or exactly or at most 1531, at least or exactly or at most 1532, at least or exactly or at most 1533, at least or exactly or at most 1534, at least or exactly or at most 1535, at least or exactly or at most 1536, at least or exactly or at most 1537, at least or exactly or at most 1538, at least or exactly or at most 1539, at least or exactly or at most 1540, at least or exactly or at most 1541, at least or exactly or at most 1542, at least or exactly or at most 1543, at least or exactly or at most 1544, at least or exactly or at most 1545, at least or exactly or at most 1546, at least or exactly or at most 1547, at least or exactly or at most 1548, at least or exactly or at most 1549, at least or exactly or at most 1550, at least or exactly or at most 1551, at least or exactly or at most 1552, at least or exactly or at most 1553, at least or exactly or at most 1554, at least or exactly or at most 1555, at least or exactly or at most 1556, at least or exactly or at most 1557, at least or exactly or at most 1558, at least or exactly or at most 1559, at least or exactly or at most 1560, at least or exactly or at most 1561, at least or exactly or at most 1562, at least or exactly or at most 1563, at least or exactly or at most 1564, at least or exactly or at most 1565, at least or exactly or at most 1566, at least or exactly or at most 1567, at least or exactly or at most 1568, at least or exactly or at most 1569, at least or exactly or at most 1570, at least or exactly or at most 1571, at least or exactly or at most 1572, at least or exactly or at most 1573, at least or exactly or at most 1574, at least or exactly or at most 1575, at least or exactly or at most 1576, at least or exactly or at most 1577, at least or exactly or at most 1578, at least or exactly or at most 1579, at least or exactly or at most 1580, at least or exactly or at most 1581, at least or exactly or at most 1582, at least or exactly or at most 1583, at least or exactly or at most 1584, at least or exactly or at most 1585, at least or exactly or at most 1586, at least or exactly or at most 1587, at least or exactly or at most 1588, at least or exactly or at most 1589, at least or exactly or at most 1590, at least or exactly or at most 1591, at least or exactly or at most 1592, at least or exactly or at most 1593, at least or exactly or at most 1594, at least or exactly or at most 1595, at least or exactly or at most 1596, at least or exactly or at most 1597, at least or exactly or at most 1598, at least or exactly or at most 1599, at least or exactly or at most 1600, at least or exactly or at most 1601, at least or exactly or at most 1602, at least or exactly or at most 1603, at least or exactly or at most 1604, at least or exactly or at most 1605, at least or exactly or at most 1606, at least or exactly or at most 1607, at least or exactly or at most 1608, at least or exactly or at most 1609, at least or exactly or at most 1610, at least or exactly or at most 1611, at least or exactly or at most 1612, at least or exactly or at most 1613, at least or exactly or at most 1614, at least or exactly or at most 1615, at least or exactly or at most 1616, at least or exactly or at most 1617, at least or exactly or at most 1618, at least or exactly or at most 1619, at least or exactly or at most 1620, at least or exactly or at most 1621, at least or exactly or at most 1622, at least or exactly or at most 1623, at least or exactly or at most 1624, at least or exactly or at most 1625, at least or exactly or at most 1626, at least or exactly or at most 1627, at least or exactly or at most 1628, at least or exactly or at most 1629, at least or exactly or at most 1630, at least or exactly or at most 1631, at least or exactly or at most 1632, at least or exactly or at most 1633, at least or exactly or at most 1634, at least or exactly or at most 1635, at least or exactly or at most 1636, at least or exactly or at most 1637, at least or exactly or at most 1638, at least or exactly or at most 1639, at least or exactly or at most 1640, at least or exactly or at most 1641, at least or exactly or at most 1642, at least or exactly or at most 1643, at least or exactly or at most 1644, at least or exactly or at most 1645, at least or exactly or at most 1646, at least or exactly or at most 1647, at least or exactly or at most 1648, at least or exactly or at most 1649, at least or exactly or at most 1650, at least or exactly or at most 1651, at least or exactly or at most 1652, at least or exactly or at most 1653, at least or exactly or at most 1654, at least or exactly or at most 1655, at least or exactly or at most 1656, at least or exactly or at most 1657, at least or exactly or at most 1658, at least or exactly or at most 1659, at least or exactly or at most 1660, at least or exactly or at most 1661, at least or exactly or at most 1662, at least or exactly or at most 1663, at least or exactly or at most 1664, at least or exactly or at most 1665, at least or exactly or at most 1666, at least or exactly or at most 1667, at least or exactly or at most 1668, at least or exactly or at most 1669, at least or exactly or at most 1670, at least or exactly or at most 1671, at least or exactly or at most 1672, at least or exactly or at most 1673, at least or exactly or at most 1674, at least or exactly or at most 1675, at least or exactly or at most 1676, at least or exactly or at most 1677, at least or exactly or at most 1678, at least or exactly or at most 1679, at least or exactly or at most 1680, at least or exactly or at most 1681, at least or exactly or at most 1682, at least or exactly or at most 1683, at least or exactly or at most 1684, at least or exactly or at most 1685, at least or exactly or at most 1686, at least or exactly or at most 1687, at least or exactly or at most 1688, at least or exactly or at most 1689, at least or exactly or at most 1690, at least or exactly or at most 1691, at least or exactly or at most 1692, at least or exactly or at most 1693, at least or exactly or at most 1694, at least or exactly or at most 1695, at least or exactly or at most 1696, at least or exactly or at most 1697, at least or exactly or at most 1698, at least or exactly or at most 1699, at least or exactly or at most 1700, at least or exactly or at most 1701, at least or exactly or at most 1702, at least or exactly or at most 1703, at least or exactly or at most 1704, at least or exactly or at most 1705, at least or exactly or at most 1706, at least or exactly or at most 1707, at least or exactly or at most 1708, at least or exactly or at most 1709, at least or exactly or at most 1710, at least or exactly or at most 1711, at least or exactly or at most 1712, at least or exactly or at most 1713, at least or exactly or at most 1714, at least or exactly or at most 1715, at least or exactly or at most 1716, at least or exactly or at most 1717, at least or exactly or at most 1718, at least or exactly or at most 1719, at least or exactly or at most 1720, at least or exactly or at most 1721, at least or exactly or at most 1722, at least or exactly or at most 1723, at least or exactly or at most 1724, at least or exactly or at most 1725, at least or exactly or at most 1726, at least or exactly or at most 1727, at least or exactly or at most 1728, at least or exactly or at most 1729, at least or exactly or at most 1730, at least or exactly or at most 1731, at least or exactly or at most 1732, at least or exactly or at most 1733, at least or exactly or at most 1734, at least or exactly or at most 1735, at least or exactly or at most 1736, at least or exactly or at most 1737, at least or exactly or at most 1738, at least or exactly or at most 1739, at least or exactly or at most 1740, at least or exactly or at most 1741, at least or exactly or at most 1742, at least or exactly or at most 1743, at least or exactly or at most 1744, at least or exactly or at most 1745, at least or exactly or at most 1746, at least or exactly or at most 1747, at least or exactly or at most 1748, at least or exactly or at most 1749, at least or exactly or at most 1750, at least or exactly or at most 1751, at least or exactly or at most 1752, at least or exactly or at most 1753, at least or exactly or at most 1754, at least or exactly or at most 1755, at least or exactly or at most 1756, at least or exactly or at most 1757, at least or exactly or at most 1758, at least or exactly or at most 1759, at least or exactly or at most 1760, at least or exactly or at most 1761, at least or exactly or at most 1762, at least or exactly or at most 1763, at least or exactly or at most 1764, at least or exactly or at most 1765, at least or exactly or at most 1766, at least or exactly or at most 1767, at least or exactly or at most 1768, at least or exactly or at most 1769, at least or exactly or at most 1770, at least or exactly or at most 1771, at least or exactly or at most 1772, at least or exactly or at most 1773, at least or exactly or at most 1774, at least or exactly or at most 1775, at least or exactly or at most 1776, at least or exactly or at most 1777, at least or exactly or at most 1778, at least or exactly or at most 1779, at least or exactly or at most 1780, at least or exactly or at most 1781, at least or exactly or at most 1782, at least or exactly or at most 1783, at least or exactly or at most 1784, at least or exactly or at most 1785, at least or exactly or at most 1786, at least or exactly or at most 1787, at least or exactly or at most 1788, at least or exactly or at most 1789, at least or exactly or at most 1790, at least or exactly or at most 1791, at least or exactly or at most 1792, at least or exactly or at most 1793, at least or exactly or at most 1794, at least or exactly or at most 1795, at least or exactly or at most 1796, at least or exactly or at most 1797, at least or exactly or at most 1798, at least or exactly or at most 1799, at least or exactly or at most 1800, at least or exactly or at most 1801, at least or exactly or at most 1802, at least or exactly or at most 1803, at least or exactly or at most 1804, at least or exactly or at most 1805, at least or exactly or at most 1806, at least or exactly or at most 1807, at least or exactly or at most 1808, at least or exactly or at most 1809, at least or exactly or at most 1810, at least or exactly or at most 1811, at least or exactly or at most 1812, at least or exactly or at most 1813, at least or exactly or at most 1814, at least or exactly or at most 1815, at least or exactly or at most 1816, at least or exactly or at most 1817, at least or exactly or at most 1818, at least or exactly or at most 1819, at least or exactly or at most 1820, at least or exactly or at most 1821, at least or exactly or at most 1822, at least or exactly or at most 1823, at least or exactly or at most 1824, at least or exactly or at most 1825, at least or exactly or at most 1826, at least or exactly or at most 1827, at least or exactly or at most 1828, at least or exactly or at most 1829, at least or exactly or at most 1830, at least or exactly or at most 1831, at least or exactly or at most 1832, at least or exactly or at most 1833, at least or exactly or at most 1834, at least or exactly or at most 1835, at least or exactly or at most 1836, at least or exactly or at most 1837, at least or exactly or at most 1838, at least or exactly or at most 1839, at least or exactly or at most 1840, at least or exactly or at most 1841, at least or exactly or at most 1842, at least or exactly or at most 1843, at least or exactly or at most 1844, at least or exactly or at most 1845, at least or exactly or at most 1846, at least or exactly or at most 1847, at least or exactly or at most 1848, at least or exactly or at most 1849, at least or exactly or at most 1850, at least or exactly or at most 1851, at least or exactly or at most 1852, at least or exactly or at most 1853, at least or exactly or at most 1854, at least or exactly or at most 1855, at least or exactly or at most 1856, at least or exactly or at most 1857, at least or exactly or at most 1858, at least or exactly or at most 1859, at least or exactly or at most 1860, at least or exactly or at most 1861, at least or exactly or at most 1862, at least or exactly or at most 1863, at least or exactly or at most 1864, at least or exactly or at most 1865, at least or exactly or at most 1866, at least or exactly or at most 1867, at least or exactly or at most 1868, at least or exactly or at most 1869, at least or exactly or at most 1870, at least or exactly or at most 1871, at least or exactly or at most 1872, at least or exactly or at most 1873, at least or exactly or at most 1874, at least or exactly or at most 1875, at least or exactly or at most 1876, at least or exactly or at most 1877, at least or exactly or at most 1878, at least or exactly or at most 1879, at least or exactly or at most 1880, at least or exactly or at most 1881, at least or exactly or at most 1882, at least or exactly or at most 1883, at least or exactly or at most 1884, at least or exactly or at most 1885, at least or exactly or at most 1886, at least or exactly or at most 1887, at least or exactly or at most 1888, at least or exactly or at most 1889, at least or exactly or at most 1890, at least or exactly or at most 1891, at least or exactly or at most 1892, at least or exactly or at most 1893, at least or exactly or at most 1894, at least or exactly or at most 1895, at least or exactly or at most 1896, at least or exactly or at most 1897, at least or exactly or at most 1898, at least or exactly or at most 1899, at least or exactly or at most 1900, at least or exactly or at most 1901, at least or exactly or at most 1902, at least or exactly or at most 1903, at least or exactly or at most 1904, at least or exactly or at most 1905, at least or exactly or at most 1906, at least or exactly or at most 1907, at least or exactly or at most 1908, at least or exactly or at most 1909, at least or exactly or at most 1910, at least or exactly or at most 1911, at least or exactly or at most 1912, at least or exactly or at most 1913, at least or exactly or at most 1914, at least or exactly or at most 1915, at least or exactly or at most 1916, at least or exactly or at most 1917, at least or exactly or at most 1918, at least or exactly or at most 1919, at least or exactly or at most 1920, at least or exactly or at most 1921, at least or exactly or at most 1922, at least or exactly or at most 1923, at least or exactly or at most 1924, at least or exactly or at most 1925, at least or exactly or at most 1926, at least or exactly or at most 1927, at least or exactly or at most 1928, at least or exactly or at most 1929, at least or exactly or at most 1930, at least or exactly or at most 1931, at least or exactly or at most 1932, at least or exactly or at most 1933, at least or exactly or at most 1934, at least or exactly or at most 1935, at least or exactly or at most 1936, at least or exactly or at most 1937, at least or exactly or at most 1938, at least or exactly or at most 1939, at least or exactly or at most 1940, at least or exactly or at most 1941, at least or exactly or at most 1942, at least or exactly or at most 1943, at least or exactly or at most 1944, at least or exactly or at most 1945, at least or exactly or at most 1946, at least or exactly or at most 1947, at least or exactly or at most 1948, at least or exactly or at most 1949, at least or exactly or at most 1950, at least or exactly or at most 1951, at least or exactly or at most 1952, at least or exactly or at most 1953, at least or exactly or at most 1954, at least or exactly or at most 1955, at least or exactly or at most 1956, at least or exactly or at most 1957, at least or exactly or at most 1958, at least or exactly or at most 1959, at least or exactly or at most 1960, at least or exactly or at most 1961, at least or exactly or at most 1962, at least or exactly or at most 1963, at least or exactly or at most 1964, at least or exactly or at most 1965, at least or exactly or at most 1966, at least or exactly or at most 1967, at least or exactly or at most 1968, at least or exactly or at most 1969, at least or exactly or at most 1970, at least or exactly or at most 1971, at least or exactly or at most 1972, at least or exactly or at most 1973, at least or exactly or at most 1974, at least or exactly or at most 1975, at least or exactly or at most 1976, at least or exactly or at most 1977, at least or exactly or at most 1978, at least or exactly or at most 1979, at least or exactly or at most 1980, at least or exactly or at most 1981, at least or exactly or at most 1982, at least or exactly or at most 1983, at least or exactly or at most 1984, at least or exactly or at most 1985, at least or exactly or at most 1986, at least or exactly or at most 1987, at least or exactly or at most 1988, at least or exactly or at most 1989, at least or exactly or at most 1990, at least or exactly or at most 1991, at least or exactly or at most 1992, at least or exactly or at most 1993, at least or exactly or at most 1994, at least or exactly or at most 1995, at least or exactly or at most 1996, at least or exactly or at most 1997, at least or exactly or at most 1998, at least or exactly or at most 1999, at least or exactly or at most 2000, at least or exactly or at most 2001, at least or exactly or at most 2002, at least or exactly or at most 2003, at least or exactly or at most 2004, at least or exactly or at most 2005, at least or exactly or at most 2006, at least or exactly or at most 2007, at least or exactly or at most 2008, at least or exactly or at most 2009, at least or exactly or at most 2010, at least or exactly or at most 2011, at least or exactly or at most 2012, at least or exactly or at most 2013, at least or exactly or at most 2014, at least or exactly or at most 2015, at least or exactly or at most 2016, at least or exactly or at most 2017, at least or exactly or at most 2018, at least or exactly or at most 2019, at least or exactly or at most 2020, at least or exactly or at most 2021, at least or exactly or at most 2022, at least or exactly or at most 2023, at least or exactly or at most 2024, at least or exactly or at most 2025, at least or exactly or at most 2026, at least or exactly or at most 2027, at least or exactly or at most 2028, at least or exactly or at most 2029, at least or exactly or at most 2030, at least or exactly or at most 2031, at least or exactly or at most 2032, at least or exactly or at most 2033, at least or exactly or at most 2034, at least or exactly or at most 2035, at least or exactly or at most 2036, at least or exactly or at most 2037, at least or exactly or at most 2038, at least or exactly or at most 2039, at least or exactly or at most 2040, at least or exactly or at most 2041, at least or exactly or at most 2042, at least or exactly or at most 2043, at least or exactly or at most 2044, at least or exactly or at most 2045, at least or exactly or at most 2046, at least or exactly or at most 2047, at least or exactly or at most 2048, at least or exactly or at most 2049, at least or exactly or at most 2050, at least or exactly or at most 2051, at least or exactly or at most 2052, at least or exactly or at most 2053, at least or exactly or at most 2054, at least or exactly or at most 2055, at least or exactly or at most 2056, at least or exactly or at most 2057, at least or exactly or at most 2058, at least or exactly or at most 2059, at least or exactly or at most 2060, at least or exactly or at most 2061, at least or exactly or at most 2062, at least or exactly or at most 2063, at least or exactly or at most 2064, at least or exactly or at most 2065, at least or exactly or at most 2066, at least or exactly or at most 2067, at least or exactly or at most 2068, at least or exactly or at most 2069, at least or exactly or at most 2070, at least or exactly or at most 2071, at least or exactly or at most 2072, at least or exactly or at most 2073, at least or exactly or at most 2074, at least or exactly or at most 2075, at least or exactly or at most 2076, at least or exactly or at most 2077, at least or exactly or at most 2078, at least or exactly or at most 2079, at least or exactly or at most 2080, at least or exactly or at most 2081, at least or exactly or at most 2082, at least or exactly or at most 2083, at least or exactly or at most 2084, at least or exactly or at most 2085, at least or exactly or at most 2086, at least or exactly or at most 2087, at least or exactly or at most 2088, at least or exactly or at most 2089, at least or exactly or at most 2090, at least or exactly or at most 2091, at least or exactly or at most 2092, at least or exactly or at most 2093, at least or exactly or at most 2094, at least or exactly or at most 2095, at least or exactly or at most 2096, at least or exactly or at most 2097, at least or exactly or at most 2098, at least or exactly or at most 2099, at least or exactly or at most 2100, at least or exactly or at most 2101, at least or exactly or at most 2102, at least or exactly or at most 2103, at least or exactly or at most 2104, at least or exactly or at most 2105, at least or exactly or at most 2106, at least or exactly or at most 2107, at least or exactly or at most 2108, at least or exactly or at most 2109, at least or exactly or at most 2110, at least or exactly or at most 2111, at least or exactly or at most 2112, at least or exactly or at most 2113, at least or exactly or at most 2114, at least or exactly or at most 2115, at least or exactly or at most 2116, at least or exactly or at most 2117, at least or exactly or at most 2118, at least or exactly or at most 2119, at least or exactly or at most 2120, at least or exactly or at most 2121, at least or exactly or at most 2122, at least or exactly or at most 2123, at least or exactly or at most 2124, at least or exactly or at most 2125, at least or exactly or at most 2126, at least or exactly or at most 2127, at least or exactly or at most 2128, at least or exactly or at most 2129, at least or exactly or at most 2130, at least or exactly or at most 2131, at least or exactly or at most 2132, at least or exactly or at most 2133, at least or exactly or at most 2134, at least or exactly or at most 2135, at least or exactly or at most 2136, at least or exactly or at most 2137, at least or exactly or at most 2138, at least or exactly or at most 2139, at least or exactly or at most 2140, at least or exactly or at most 2141, at least or exactly or at most 2142, at least or exactly or at most 2143, at least or exactly or at most 2144, at least or exactly or at most 2145, at least or exactly or at most 2146, at least or exactly or at most 2147, at least or exactly or at most 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2175, at least or exactly or at most 2176, at least or exactly or at most 2177, at least or exactly or at most 2178, at least or exactly or at most 2179, at least or exactly or at most 2180, at least or exactly or at most 2181, at least or exactly or at most 2182, at least or exactly or at most 2183, at least or exactly or at most 2184, at least or exactly or at most 2185, at least or exactly or at most 2186, at least or exactly or at most 2187, at least or exactly or at most 2188, at least or exactly or at most 2189, at least or exactly or at most 2190, at least or exactly or at most 2191, at least or exactly or at most 2192, at least or exactly or at most 2193, at least or exactly or at most 2194, at least or exactly or at most 2195, at least or exactly or at most 2196, at least or exactly or at most 2197, at least or exactly or at most 2198, at least or exactly or at most 2199, at least or exactly or at most 2200, at least or exactly or at most 2201, at least or exactly or at most 2202, at least or exactly or at most 2203, at least or exactly or at most 2204, at least or exactly or at most 2205, at least or exactly or at most 2206, at least or exactly or at most 2207, at least or exactly or at most 2208, at least or exactly or at most 2209, at least or exactly or at most 2210, at least or exactly or at most 2211, at least or exactly or at most 2212, at least or exactly or at most 2213, at least or exactly or at most 2214, at least or exactly or at most 2215, at least or exactly or at most 2216, at least or exactly or at most 2217, at least or exactly or at most 2218, at least or exactly or at most 2219, at least or exactly or at most 2220, at least or exactly or at most 2221, at least or exactly or at most 2222, at least or exactly or at most 2223, at least or exactly or at most 2224, at least or exactly or at most 2225, at least or exactly or at most 2226, at least or exactly or at most 2227, at least or exactly or at most 2228, at least or exactly or at most 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2256, at least or exactly or at most 2257, at least or exactly or at most 2258, at least or exactly or at most 2259, at least or exactly or at most 2260, at least or exactly or at most 2261, at least or exactly or at most 2262, at least or exactly or at most 2263, at least or exactly or at most 2264, at least or exactly or at most 2265, at least or exactly or at most 2266, at least or exactly or at most 2267, at least or exactly or at most 2268, at least or exactly or at most 2269, at least or exactly or at most 2270, at least or exactly or at most 2271, at least or exactly or at most 2272, at least or exactly or at most 2273, at least or exactly or at most 2274, at least or exactly or at most 2275, at least or exactly or at most 2276, at least or exactly or at most 2277, at least or exactly or at most 2278, at least or exactly or at most 2279, at least or exactly or at most 2280, at least or exactly or at most 2281, at least or exactly or at most 2282, at least or exactly or at most 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2310, at least or exactly or at most 2311, at least or exactly or at most 2312, at least or exactly or at most 2313, at least or exactly or at most 2314, at least or exactly or at most 2315, at least or exactly or at most 2316, at least or exactly or at most 2317, at least or exactly or at most 2318, at least or exactly or at most 2319, at least or exactly or at most 2320, at least or exactly or at most 2321, at least or exactly or at most 2322, at least or exactly or at most 2323, at least or exactly or at most 2324, at least or exactly or at most 2325, at least or exactly or at most 2326, at least or exactly or at most 2327, at least or exactly or at most 2328, at least or exactly or at most 2329, at least or exactly or at most 2330, at least or exactly or at most 2331, at least or exactly or at most 2332, at least or exactly or at most 2333, at least or exactly or at most 2334, at least or exactly or at most 2335, at least or exactly or at most 2336, at least or exactly or at most 2337, at least or exactly or at most 2338, at least or exactly or at most 2339, at least or exactly or at most 2340, at least or exactly or at most 2341, at least or exactly or at most 2342, at least or exactly or at most 2343, at least or exactly or at most 2344, at least or exactly or at most 2345, at least or exactly or at most 2346, at least or exactly or at most 2347, at least or exactly or at most 2348, at least or exactly or at most 2349, at least or exactly or at most 2350, at least or exactly or at most 2351, at least or exactly or at most 2352, at least or exactly or at most 2353, at least or exactly or at most 2354, at least or exactly or at most 2355, at least or exactly or at most 2356, at least or exactly or at most 2357, at least or exactly or at most 2358, at least or exactly or at most 2359, at least or exactly or at most 2360, at least or exactly or at most 2361, at least or exactly or at most 2362, at least or exactly or at most 2363, at least or exactly or at most 2364, at least or exactly or at most 2365, at least or exactly or at most 2366, at least or exactly or at most 2367, at least or exactly or at most 2368, at least or exactly or at most 2369, at least or exactly or at most 2370, at least or exactly or at most 2371, at least or exactly or at most 2372, at least or exactly or at most 2373, at least or exactly or at most 2374, at least or exactly or at most 2375, at least or exactly or at most 2376, at least or exactly or at most 2377, at least or exactly or at most 2378, at least or exactly or at most 2379, at least or exactly or at most 2380, at least or exactly or at most 2381, at least or exactly or at most 2382, at least or exactly or at most 2383, at least or exactly or at most 2384, at least or exactly or at most 2385, at least or exactly or at most 2386, at least or exactly or at most 2387, at least or exactly or at most 2388, at least or exactly or at most 2389, at least or exactly or at most 2390, at least or exactly or at most 2391, at least or exactly or at most 2392, at least or exactly or at most 2393, at least or exactly or at most 2394, at least or exactly or at most 2395, at least or exactly or at most 2396, at least or exactly or at most 2397, at least or exactly or at most 2398, at least or exactly or at most 2399, at least or exactly or at most 2400, at least or exactly or at most 2401, at least or exactly or at most 2402, at least or exactly or at most 2403, at least or exactly or at most 2404, at least or exactly or at most 2405, at least or exactly or at most 2406, at least or exactly or at most 2407, at least or exactly or at most 2408, at least or exactly or at most 2409, at least or exactly or at most 2410, at least or exactly or at most 2411, at least or exactly or at most 2412, at least or exactly or at most 2413, at least or exactly or at most 2414, at least or exactly or at most 2415, at least or exactly or at most 2416, at least or exactly or at most 2417, at least or exactly or at most 2418, at least or exactly or at most 2419, at least or exactly or at most 2420, at least or exactly or at most 2421, at least or exactly or at most 2422, at least or exactly or at most 2423, at least or exactly or at most 2424, at least or exactly or at most 2425, at least or exactly or at most 2426, at least or exactly or at most 2427, at least or exactly or at most 2428, at least or exactly or at most 2429, at least or exactly or at most 2430, at least or exactly or at most 2431, at least or exactly or at most 2432, at least or exactly or at most 2433, at least or exactly or at most 2434, at least or exactly or at most 2435, at least or exactly or at most 2436, at least or exactly or at most 2437, at least or exactly or at most 2438, at least or exactly or at most 2439, at least or exactly or at most 2440, at least or exactly or at most 2441, at least or exactly or at most 2442, at least or exactly or at most 2443, at least or exactly or at most 2444, at least or exactly or at most 2445, at least or exactly or at most 2446, at least or exactly or at most 2447, at least or exactly or at most 2448, at least or exactly or at most 2449, at least or exactly or at most 2450, at least or exactly or at most 2451, at least or exactly or at most 2452, at least or exactly or at most 2453, at least or exactly or at most 2454, at least or exactly or at most 2455, at least or exactly or at most 2456, at least or exactly or at most 2457, at least or exactly or at most 2458, at least or exactly or at most 2459, at least or exactly or at most 2460, at least or exactly or at most 2461, at least or exactly or at most 2462, at least or exactly or at most 2463, at least or exactly or at most 2464, at least or exactly or at most 2465, at least or exactly or at most 2466, at least or exactly or at most 2467, at least or exactly or at most 2468, at least or exactly or at most 2469, at least or exactly or at most 2470, at least or exactly or at most 2471, at least or exactly or at most 2472, at least or exactly or at most 2473, at least or exactly or at most 2474, at least or exactly or at most 2475, at least or exactly or at most 2476, at least or exactly or at most 2477, at least or exactly or at most 2478, at least or exactly or at most 2479, at least or exactly or at most 2480, at least or exactly or at most 2481, at least or exactly or at most 2482, at least or exactly or at most 2483, at least or exactly or at most 2484, at least or exactly or at most 2485, at least or exactly or at most 2486, at least or exactly or at most 2487, at least or exactly or at most 2488, at least or exactly or at most 2489, at least or exactly or at most 2490, at least or exactly or at most 2491, at least or exactly or at most 2492, at least or exactly or at most 2493, at least or exactly or at most 2494, at least or exactly or at most 2495, at least or exactly or at most 2496, at least or exactly or at most 2497, at least or exactly or at most 2498, at least or exactly or at most 2499, at least or exactly or at most 2500, at least or exactly or at most 2501, at least or exactly or at most 2502, at least or exactly or at most 2503, at least or exactly or at most 2504, at least or exactly or at most 2505, at least or exactly or at most 2506, at least or exactly or at most 2507, at least or exactly or at most 2508, at least or exactly or at most 2509, at least or exactly or at most 2510, at least or exactly or at most 2511, at least or exactly or at most 2512, at least or exactly or at most 2513, at least or exactly or at most 2514, at least or exactly or at most 2515, at least or exactly or at most 2516, at least or exactly or at most 2517, at least or exactly or at most 2518, at least or exactly or at most 2519, at least or exactly or at most 2520, at least or exactly or at most 2521, at least or exactly or at most 2522, at least or exactly or at most 2523, at least or exactly or at most 2524, at least or exactly or at most 2525, at least or exactly or at most 2526, at least or exactly or at most 2527, at least or exactly or at most 2528, at least or exactly or at most 2529, at least or exactly or at most 2530, at least or exactly or at most 2531, at least or exactly or at most 2532, at least or exactly or at most 2533, at least or exactly or at most 2534, at least or exactly or at most 2535, at least or exactly or at most 2536, at least or exactly or at most 2537, at least or exactly or at most 2538, at least or exactly or at most 2539, at least or exactly or at most 2540, at least or exactly or at most 2541, at least or exactly or at most 2542, at least or exactly or at most 2543, at least or exactly or at most 2544, at least or exactly or at most 2545, at least or exactly or at most 2546, at least or exactly or at most 2547, at least or exactly or at most 2548, at least or exactly or at most 2549, at least or exactly or at most 2550, at least or exactly or at most 2551, at least or exactly or at most 2552, at least or exactly or at most 2553, at least or exactly or at most 2554, at least or exactly or at most 2555, at least or exactly or at most 2556, at least or exactly or at most 2557, at least or exactly or at most 2558, at least or exactly or at most 2559, at least or exactly or at most 2560, at least or exactly or at most 2561, at least or exactly or at most 2562, at least or exactly or at most 2563, at least or exactly or at most 2564, at least or exactly or at most 2565, at least or exactly or at most 2566, at least or exactly or at most 2567, at least or exactly or at most 2568, at least or exactly or at most 2569, at least or exactly or at most 2570, at least or exactly or at most 2571, at least or exactly or at most 2572, at least or exactly or at most 2573, at least or exactly or at most 2574, at least or exactly or at most 2575, at least or exactly or at most 2576, at least or exactly or at most 2577, at least or exactly or at most 2578, at least or exactly or at most 2579, at least or exactly or at most 2580, at least or exactly or at most 2581, at least or exactly or at most 2582, at least or exactly or at most 2583, at least or exactly or at most 2584, at least or exactly or at most 2585, at least or exactly or at most 2586, at least or exactly or at most 2587, at least or exactly or at most 2588, at least or exactly or at most 2589, at least or exactly or at most 2590, at least or exactly or at most 2591, at least or exactly or at most 2592, at least or exactly or at most 2593, at least or exactly or at most 2594, at least or exactly or at most 2595, at least or exactly or at most 2596, at least or exactly or at most 2597, at least or exactly or at most 2598, at least or exactly or at most 2599, at least or exactly or at most 2600, at least or exactly or at most 2601, at least or exactly or at most 2602, at least or exactly or at most 2603, at least or exactly or at most 2604, at least or exactly or at most 2605, at least or exactly or at most 2606, at least or exactly or at most 2607, at least or exactly or at most 2608, at least or exactly or at most 2609, at least or exactly or at most 2610, at least or exactly or at most 2611, at least or exactly or at most 2612, at least or exactly or at most 2613, at least or exactly or at most 2614, at least or exactly or at most 2615, at least or exactly or at most 2616, at least or exactly or at most 2617, at least or exactly or at most 2618, at least or exactly or at most 2619, at least or exactly or at most 2620, at least or exactly or at most 2621, at least or exactly or at most 2622, at least or exactly or at most 2623, at least or exactly or at most 2624, at least or exactly or at most 2625, at least or exactly or at most 2626, at least or exactly or at most 2627, at least or exactly or at most 2628, at least or exactly or at most 2629, at least or exactly or at most 2630, at least or exactly or at most 2631, at least or exactly or at most 2632, at least or exactly or at most 2633, at least or exactly or at most 2634, at least or exactly or at most 2635, at least or exactly or at most 2636, at least or exactly or at most 2637, at least or exactly or at most 2638, at least or exactly or at most 2639, at least or exactly or at most 2640, at least or exactly or at most 2641, at least or exactly or at most 2642, at least or exactly or at most 2643, at least or exactly or at most 2644, at least or exactly or at most 2645, at least or exactly or at most 2646, at least or exactly or at most 2647, at least or exactly or at most 2648, at least or exactly or at most 2649, at least or exactly or at most 2650, at least or exactly or at most 2651, at least or exactly or at most 2652, at least or exactly or at most 2653, at least or exactly or at most 2654, at least or exactly or at most 2655, at least or exactly or at most 2656, at least or exactly or at most 2657, at least or exactly or at most 2658, at least or exactly or at most 2659, at least or exactly or at most 2660, at least or exactly or at most 2661, at least or exactly or at most 2662, at least or exactly or at most 2663, at least or exactly or at most 2664, at least or exactly or at most 2665, at least or exactly or at most 2666, at least or exactly or at most 2667, at least or exactly or at most 2668, at least or exactly or at most 2669, at least or exactly or at most 2670, at least or exactly or at most 2671, at least or exactly or at most 2672, at least or exactly or at most 2673, at least or exactly or at most 2674, at least or exactly or at most 2675, at least or exactly or at most 2676, at least or exactly or at most 2677, at least or exactly or at most 2678, at least or exactly or at most 2679, at least or exactly or at most 2680, at least or exactly or at most 2681, at least or exactly or at most 2682, at least or exactly or at most 2683, at least or exactly or at most 2684, at least or exactly or at most 2685, at least or exactly or at most 2686, at least or exactly or at most 2687, at least or exactly or at most 2688, at least or exactly or at most 2689, at least or exactly or at most 2690, at least or exactly or at most 2691, at least or exactly or at most 2692, at least or exactly or at most 2693, at least or exactly or at most 2694, at least or exactly or at most 2695, at least or exactly or at most 2696, at least or exactly or at most 2697, at least or exactly or at most 2698, at least or exactly or at most 2699, at least or exactly or at most 2700, at least or exactly or at most 2701, at least or exactly or at most 2702, at least or exactly or at most 2703, at least or exactly or at most 2704, at least or exactly or at most 2705, at least or exactly or at most 2706, at least or exactly or at most 2707, at least or exactly or at most 2708, at least or exactly or at most 2709, at least or exactly or at most 2710, at least or exactly or at most 2711, at least or exactly or at most 2712, at least or exactly or at most 2713, at least or exactly or at most 2714, at least or exactly or at most 2715, at least or exactly or at most 2716, at least or exactly or at most 2717, at least or exactly or at most 2718, at least or exactly or at most 2719, at least or exactly or at most 2720, at least or exactly or at most 2721, at least or exactly or at most 2722, at least or exactly or at most 2723, at least or exactly or at most 2724, at least or exactly or at most 2725, at least or exactly or at most 2726, at least or exactly or at most 2727, at least or exactly or at most 2728, at least or exactly or at most 2729, at least or exactly or at most 2730, at least or exactly or at most 2731, at least or exactly or at most 2732, at least or exactly or at most 2733, at least or exactly or at most 2734, at least or exactly or at most 2735, at least or exactly or at most 2736, at least or exactly or at most 2737, at least or exactly or at most 2738, at least or exactly or at most 2739, at least or exactly or at most 2740, at least or exactly or at most 2741, at least or exactly or at most 2742, at least or exactly or at most 2743, at least or exactly or at most 2744, at least or exactly or at most 2745, at least or exactly or at most 2746, at least or exactly or at most 2747, at least or exactly or at most 2748, at least or exactly or at most 2749, at least or exactly or at most 2750, at least or exactly or at most 2751, at least or exactly or at most 2752, at least or exactly or at most 2753, at least or exactly or at most 2754, at least or exactly or at most 2755, at least or exactly or at most 2756, at least or exactly or at most 2757, at least or exactly or at most 2758, at least or exactly or at most 2759, at least or exactly or at most 2760, at least or exactly or at most 2761, at least or exactly or at most 2762, at least or exactly or at most 2763, at least or exactly or at most 2764, at least or exactly or at most 2765, at least or exactly or at most 2766, at least or exactly or at most 2767, at least or exactly or at most 2768, at least or exactly or at most 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2796, at least or exactly or at most 2797, at least or exactly or at most 2798, at least or exactly or at most 2799, at least or exactly or at most 2800, at least or exactly or at most 2801, at least or exactly or at most 2802, at least or exactly or at most 2803, at least or exactly or at most 2804, at least or exactly or at most 2805, at least or exactly or at most 2806, at least or exactly or at most 2807, at least or exactly or at most 2808, at least or exactly or at most 2809, at least or exactly or at most 2810, at least or exactly or at most 2811, at least or exactly or at most 2812, at least or exactly or at most 2813, at least or exactly or at most 2814, at least or exactly or at most 2815, at least or exactly or at most 2816, at least or exactly or at most 2817, at least or exactly or at most 2818, at least or exactly or at most 2819, at least or exactly or at most 2820, at least or exactly or at most 2821, at least or exactly or at most 2822, at least or exactly or at most 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2850, at least or exactly or at most 2851, at least or exactly or at most 2852, at least or exactly or at most 2853, at least or exactly or at most 2854, at least or exactly or at most 2855, at least or exactly or at most 2856, at least or exactly or at most 2857, at least or exactly or at most 2858, at least or exactly or at most 2859, at least or exactly or at most 2860, at least or exactly or at most 2861, at least or exactly or at most 2862, at least or exactly or at most 2863, at least or exactly or at most 2864, at least or exactly or at most 2865, at least or exactly or at most 2866, at least or exactly or at most 2867, at least or exactly or at most 2868, at least or exactly or at most 2869, at least or exactly or at most 2870, at least or exactly or at most 2871, at least or exactly or at most 2872, at least or exactly or at most 2873, at least or exactly or at most 2874, at least or exactly or at most 2875, at least or exactly or at most 2876, at least or exactly or at most 2877, at least or exactly or at most 2878, at least or exactly or at most 2879, at least or exactly or at most 2880, at least or exactly or at most 2881, at least or exactly or at most 2882, at least or exactly or at most 2883, at least or exactly or at most 2884, at least or exactly or at most 2885, at least or exactly or at most 2886, at least or exactly or at most 2887, at least or exactly or at most 2888, at least or exactly or at most 2889, at least or exactly or at most 2890, at least or exactly or at most 2891, at least or exactly or at most 2892, at least or exactly or at most 2893, at least or exactly or at most 2894, at least or exactly or at most 2895, at least or exactly or at most 2896, at least or exactly or at most 2897, at least or exactly or at most 2898, at least or exactly or at most 2899, at least or exactly or at most 2900, at least or exactly or at most 2901, at least or exactly or at most 2902, at least or exactly or at most 2903, at least or exactly or at most 2904, at least or exactly or at most 2905, at least or exactly or at most 2906, at least or exactly or at most 2907, at least or exactly or at most 2908, at least or exactly or at most 2909, at least or exactly or at most 2910, at least or exactly or at most 2911, at least or exactly or at most 2912, at least or exactly or at most 2913, at least or exactly or at most 2914, at least or exactly or at most 2915, at least or exactly or at most 2916, at least or exactly or at most 2917, at least or exactly or at most 2918, at least or exactly or at most 2919, at least or exactly or at most 2920, at least or exactly or at most 2921, at least or exactly or at most 2922, at least or exactly or at most 2923, at least or exactly or at most 2924, at least or exactly or at most 2925, at least or exactly or at most 2926, at least or exactly or at most 2927, at least or exactly or at most 2928, at least or exactly or at most 2929, at least or exactly or at most 2930, at least or exactly or at most 2931, at least or exactly or at most 2932, at least or exactly or at most 2933, at least or exactly or at most 2934, at least or exactly or at most 2935, at least or exactly or at most 2936, at least or exactly or at most 2937, at least or exactly or at most 2938, at least or exactly or at most 2939, at least or exactly or at most 2940, at least or exactly or at most 2941, at least or exactly or at most 2942, at least or exactly or at most 2943, at least or exactly or at most 2944, at least or exactly or at most 2945, at least or exactly or at most 2946, at least or exactly or at most 2947, at least or exactly or at most 2948, at least or exactly or at most 2949, at least or exactly or at most 2950, at least or exactly or at most 2951, at least or exactly or at most 2952, at least or exactly or at most 2953, at least or exactly or at most 2954, at least or exactly or at most 2955, at least or exactly or at most 2956, at least or exactly or at most 2957, at least or exactly or at most 2958, at least or exactly or at most 2959, at least or exactly or at most 2960, at least or exactly or at most 2961, at least or exactly or at most 2962, at least or exactly or at most 2963, at least or exactly or at most 2964, at least or exactly or at most 2965, at least or exactly or at most 2966, at least or exactly or at most 2967, at least or exactly or at most 2968, at least or exactly or at most 2969, at least or exactly or at most 2970, at least or exactly or at most 2971, at least or exactly or at most 2972, at least or exactly or at most 2973, at least or exactly or at most 2974, at least or exactly or at most 2975, at least or exactly or at most 2976, at least or exactly or at most 2977, at least or exactly or at most 2978, at least or exactly or at most 2979, at least or exactly or at most 2980, at least or exactly or at most 2981, at least or exactly or at most 2982, at least or exactly or at most 2983, at least or exactly or at most 2984, at least or exactly or at most 2985, at least or exactly or at most 2986, at least or exactly or at most 2987, at least or exactly or at most 2988, at least or exactly or at most 2989, at least or exactly or at most 2990, at least or exactly or at most 2991, at least or exactly or at most 2992, at least or exactly or at most 2993, at least or exactly or at most 2994, at least or exactly or at most 2995, at least or exactly or at most 2996, at least or exactly or at most 2997, at least or exactly or at most 2998, at least or exactly or at most 2999, at least or exactly or at most 3000, at least or exactly or at most 3001, at least or exactly or at most 3002, at least or exactly or at most 3003, at least or exactly or at most 3004, at least or exactly or at most 3005, at least or exactly or at most 3006, at least or exactly or at most 3007, at least or exactly or at most 3008, at least or exactly or at most 3009, at least or exactly or at most 3010, at least or exactly or at most 3011, at least or exactly or at most 3012, at least or exactly or at most 3013, at least or exactly or at most 3014, at least or exactly or at most 3015, at least or exactly or at most 3016, at least or exactly or at most 3017, at least or exactly or at most 3018, at least or exactly or at most 3019, at least or exactly or at most 3020, at least or exactly or at most 3021, at least or exactly or at most 3022, at least or exactly or at most 3023, at least or exactly or at most 3024, at least or exactly or at most 3025, at least or exactly or at most 3026, at least or exactly or at most 3027, at least or exactly or at most 3028, at least or exactly or at most 3029, at least or exactly or at most 3030, at least or exactly or at most 3031, at least or exactly or at most 3032, at least or exactly or at most 3033, at least or exactly or at most 3034, at least or exactly or at most 3035, at least or exactly or at most 3036, at least or exactly or at most 3037, at least or exactly or at most 3038, at least or exactly or at most 3039, at least or exactly or at most 3040, at least or exactly or at most 3041, at least or exactly or at most 3042, at least or exactly or at most 3043, at least or exactly or at most 3044, at least or exactly or at most 3045, at least or exactly or at most 3046, at least or exactly or at most 3047, at least or exactly or at most 3048, at least or exactly or at most 3049, at least or exactly or at most 3050, at least or exactly or at most 3051, at least or exactly or at most 3052, at least or exactly or at most 3053, at least or exactly or at most 3054, at least or exactly or at most 3055, at least or exactly or at most 3056, at least or exactly or at most 3057, at least or exactly or at most 3058, at least or exactly or at most 3059, at least or exactly or at most 3060, at least or exactly or at most 3061, at least or exactly or at most 3062, at least or exactly or at most 3063, at least or exactly or at most 3064, at least or exactly or at most 3065, at least or exactly or at most 3066, at least or exactly or at most 3067, at least or exactly or at most 3068, at least or exactly or at most 3069, at least or exactly or at most 3070, at least or exactly or at most 3071, at least or exactly or at most 3072, at least or exactly or at most 3073, at least or exactly or at most 3074, at least or exactly or at most 3075, at least or exactly or at most 3076, at least or exactly or at most 3077, at least or exactly or at most 3078, at least or exactly or at most 3079, at least or exactly or at most 3080, at least or exactly or at most 3081, at least or exactly or at most 3082, at least or exactly or at most 3083, at least or exactly or at most 3084, at least or exactly or at most 3085, at least or exactly or at most 3086, at least or exactly or at most 3087, at least or exactly or at most 3088, at least or exactly or at most 3089, at least or exactly or at most 3090, at least or exactly or at most 3091, at least or exactly or at most 3092, at least or exactly or at most 3093, at least or exactly or at most 3094, at least or exactly or at most 3095, at least or exactly or at most 3096, at least or exactly or at most 3097, at least or exactly or at most 3098, at least or exactly or at most 3099, at least or exactly or at most 3100, at least or exactly or at most 3101, at least or exactly or at most 3102, at least or exactly or at most 3103, at least or exactly or at most 3104, at least or exactly or at most 3105, at least or exactly or at most 3106, at least or exactly or at most 3107, at least or exactly or at most 3108, at least or exactly or at most 3109, at least or exactly or at most 3110, at least or exactly or at most 3111, at least or exactly or at most 3112, at least or exactly or at most 3113, at least or exactly or at most 3114, at least or exactly or at most 3115, at least or exactly or at most 3116, at least or exactly or at most 3117, at least or exactly or at most 3118, at least or exactly or at most 3119, at least or exactly or at most 3120, at least or exactly or at most 3121, at least or exactly or at most 3122, at least or exactly or at most 3123, at least or exactly or at most 3124, at least or exactly or at most 3125, at least or exactly or at most 3126, at least or exactly or at most 3127, at least or exactly or at most 3128, at least or exactly or at most 3129, at least or exactly or at most 3130, at least or exactly or at most 3131, at least or exactly or at most 3132, at least or exactly or at most 3133, at least or exactly or at most 3134, at least or exactly or at most 3135, at least or exactly or at most 3136, at least or exactly or at most 3137, at least or exactly or at most 3138, at least or exactly or at most 3139, at least or exactly or at most 3140, at least or exactly or at most 3141, at least or exactly or at most 3142, at least or exactly or at most 3143, at least or exactly or at most 3144, at least or exactly or at most 3145, at least or exactly or at most 3146, at least or exactly or at most 3147, at least or exactly or at most 3148, at least or exactly or at most 3149, at least or exactly or at most 3150, at least or exactly or at most 3151, at least or exactly or at most 3152, at least or exactly or at most 3153, at least or exactly or at most 3154, at least or exactly or at most 3155, at least or exactly or at most 3156, at least or exactly or at most 3157, at least or exactly or at most 3158, at least or exactly or at most 3159, at least or exactly or at most 3160, at least or exactly or at most 3161, at least or exactly or at most 3162, at least or exactly or at most 3163, at least or exactly or at most 3164, at least or exactly or at most 3165, at least or exactly or at most 3166, at least or exactly or at most 3167, at least or exactly or at most 3168, at least or exactly or at most 3169, at least or exactly or at most 3170, at least or exactly or at most 3171, at least or exactly or at most 3172, at least or exactly or at most 3173, at least or exactly or at most 3174, at least or exactly or at most 3175, at least or exactly or at most 3176, at least or exactly or at most 3177, at least or exactly or at most 3178, at least or exactly or at most 3179, at least or exactly or at most 3180, at least or exactly or at most 3181, at least or exactly or at most 3182, at least or exactly or at most 3183, at least or exactly or at most 3184, at least or exactly or at most 3185, at least or exactly or at most 3186, at least or exactly or at most 3187, at least or exactly or at most 3188, at least or exactly or at most 3189, at least or exactly or at most 3190, at least or exactly or at most 3191, at least or exactly or at most 3192, at least or exactly or at most 3193, at least or exactly or at most 3194, or at least or exactly or at most 3195 contiguous amino acid residues.
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 disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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, 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, or 89 in any one of SEQ ID NOs: 1-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 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, or 237 in any one of SEQ ID NOs: 2-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 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, or 384 in any one of SEQ ID NOs: 3-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, or 398 in any one of SEQ ID NOs: 4-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, or 464 in any one of SEQ ID NOs: 5-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, or 476 in any one of SEQ ID NOs: 6-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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, 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, or 552 in any one of SEQ ID NOs: 7-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, or 574 in any one of SEQ ID NOs: 8-20, if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635 in any one of SEQ ID NOs: 9-20, if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to amino acid residue 636 in any one of SEQ ID NOs: 10-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 637, 638, 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, or 676 in any one of SEQ ID NOs: 11-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, or 693 in any one of SEQ ID NOs: 12-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, or 708 in any one of SEQ ID NOs: 13-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 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, or 750 in any one of SEQ ID NOs: 14-20, if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 828, 829, 830, 831, 832, 833, 834, 835, 836, 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, or 873 in any one of SEQ ID NOs: 15-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, or 911 in any one of SEQ ID NOs: 16-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 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, 956, 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, or 1016 in any one of SEQ ID NOs: 17-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 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, 1072, 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, or 1152 in any one of SEQ ID NOs: 18-20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 1336, 1337, 1338, 1339, or 1340 in any one of SEQ ID NOs: 19
20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
In the embodiments disclosed for option b) above, the polypeptide used in the invention is also one that has at least or exactly or at most 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 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, 1603
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, or 3191 in SEQ ID NO: 20,
if the number ("n") of the at least or exactly or at most 5 contiguous amino acid residues so permits - the number ("N") of the N-terminal first residue will thus not be higher than L-n+ 1, where "L" is the total number of amino acid residues in the amino acid sequence from which the residue is selected.
Particularly preferred polypeptides of the invention are the following : Each and every of SEQ ID NO: 1-20.
ASA_0744-30-388 (SEQ ID NO: 3, residues 30-388) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-29 of SEQ ID NO: 3.
ASA_4042-19-402 (SEQ ID NO: 4, residues 19-402) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-18 of SEQ ID NO: 4.
ASA_4105-35-468 (SEQ ID NO: 5, residues 35-468) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-34 of SEQ ID NO: 5.
ASA_2321-22-480 (SEQ ID NO: 6, residues 22-480) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-21 of SEQ ID NO: 6. ASA_3943-18-556 (SEQ ID NO: 7, residues 18-556) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-17 of SEQ ID NO: 7.
ASA_3320-24-640 (SEQ ID NO: 10, residues 24-640) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-23 of SEQ ID NO: 10.
ASA_3883-25-680 (SEQ ID NO: 11, residues 25-680) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-24 of SEQ ID NO: 11.
ASA_3328-25-697 (SEQ ID NO: 12, residues 25-697) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-24 of SEQ ID NO: 12.
ASA_2532-23-334 (SEQ ID NO: 13, residues 23-334) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-22 and/or do not include the amino acid sequence constituted by residues 335-712 of SEQ ID NO: 13.
ASA_3455-25-754 (SEQ ID NO: 14, residues 25-754) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-24 of SEQ ID NO: 14.
ASA_P5G035-24-629 (SEQ ID NO: 15, residues 24-629) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-23 and/or do not include the amino acid sequence constituted by residues 630-877 of SEQ ID NO: 15.
ASA_3723-210-915 (SEQ ID NO: 16, residues 210-915) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-209 of SEQ ID NO: 16.
ASA_1675-310-1156 (SEQ ID NO: 18, residues 310-1156) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-309 of SEQ ID NO: 18. ASA_0826-2340-3195 (SEQ ID NO: 20, residues 2340-3195) as well as fragments or sequence variants thereof as disclosed herein, in particular those that do not include the amino acid sequence constituted by residues 1-2340 of SEQ ID NO: 20.
The polypeptide of the invention is in certain embodiments also fused or conjugated to an immunogenic carrier molecule; or, phrased otherwise, the polypeptide of the invention also comprises such an immunogenic carrier molecule in addition to the amino acid sequence derived from SEQ ID NOs: 1-20. The immunogenic carrier molecule is typically a polypeptide that induces cellular immune responses in freshwater fish in a manner similar to induction of T helper lymphocyte responses in mammals; since it has been demonstrated (See Nakanishi T et al. 2015, Biology (Basel) 4(4), 640-663) that fish harbour T cell populations that are equivalent to Th cells (a subset of CD4+ cells) and CTLs (CD8+ cells) the present specification and claims will generally refer to these cell types in a manner analogous to that used for Th cells and CTLs in mammals.
An immunogenic carrier protein can be selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, diphtheria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra. One further fusion partner which is preferably incorporated is a "His tag", i.e. a stretch of amino acids, which is rich in or only consists of histidinyl residues so as to facilitate protein purification subsequent to recombinant production.
In preferred embodiments, the polypeptide of the invention detailed above is capable of inducing an adaptive immune response against the polypeptide in fish, in particular in Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, and Halibut, and where the polypeptide in particular is capable of inducing a prophylactic immune response that reduces the risk of furunculosis in fish.
Epitopes derived from SEP ID NOs 1-20
SEQ ID NOs: 1-20 include antigenic determinants (epitopes) that are as such recognized by antibodies and/or T cells in fish. 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 very simple approach entails that antibodies raised against A.salmonicida or A. salmonidia derived proteins disclosed herein are tested for binding to overlapping oligomeric peptides derived from any one of SEQ ID NO: 1-20. Thereby, the regions of the A.salmonicida 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 . version where each single non-alanine residue in any one of SEQ ID NOs: 1- 20 are point mutated to alanine - this method also assists in identifying complex assembled B-cell epitopes; this is the case when binding of the same antibody is modified by exchanging 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 is provided in Petersen B et al. (November 2010), Plos One 5(11) : el 5079; prediction of linear B-cell epitopes, cf: Larsen J E P 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.
Methods of the invention : modes, routes and timing of administration
The administration of a vaccine to induce an immune response in fish - whether this is done according to the first or second aspect of the invention - is typically via oral administration (providing the polypeptide or the composition as an ingredient in food), immersion (such as by bathing, dipping or spraying), or injection (such as intraperitoneal or intramuscular) . These methods are well-known to the skilled person and are for instance detailed in Gudding R and van Muiswinkel WB, 2013, Fish & Shellfish Immunology 35(6), 1683-1688, and in Sommerset I et al . 2005, Expert Rev. Vaccines 4(1), 89-101.
A reference for that details state of the art methods for vaccination of fish is "Fish
Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470-67455-0. All disclosures in this reference that relate to generally applicable vaccine agents for fish, adjuvants and other formulation technology details, routes of administration, dosages, dosage regimens, and vaccine vectors are hereby incorporated by reference herein. In particular chapters 6, 10, are 12 are relevant in connection with disclosures relating to routes of administration, modes and timing as well as formulation.
Nucleic acids used in the present invention / vectors
The nucleic acid fragment used in the second aspect of the invention referred to above is preferably a DNA fragment (of a sequence such as SEQ ID NOs: 21-40) or an RNA fragment (of a sequence such as SEQ ID NOs 1-60) . The nucleic acid may vary compared to SEQ ID NOs: 21-60 (e.g . by being a fragment since it encodes a fragment of any one of SEQ ID NOs: 1-20 disclosed above) and may have nucleotide sequence with at least 60% sequence identity with any one of SEQ ID NOs: 21-60. Certain of these sequence variants are particularly useful due to choice of codons that optimize expression of the nucleic acid in a particular host cell. These nucleic acids are considered patentable in their own right and are termed nucleic acids of the invention.
The nucleic acid fragment of the invention discussed above typically has a sequence identity with the nucleotide sequence defined for i) or ii) above, which 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%.
The nucleic acid fragment of the invention discussed above may also have a sequence identity with the nucleotide sequence defined for iii) above, which 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%.
The nucleic acid fragment of the invention described above comprises in certain embodiments at least or exactly or at most X distinct nucleic acid sequences, which each encode a polypeptide of the invention, where each of said X distinct nucleic acid sequences encodes at least or exactly or at most one immunogenic amino acid sequence present in or derived from any one of SEQ ID NOs: 1-20 and wherein said X distinct nucleic acid sequences together encode immunogenic amino acid sequences present in or derived from at least or exactly or at most X of SEQ ID NOs: 1-20, wherein X is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In other words, such a nucleic acid fragment encodes several polypeptides of the invention. In some embodiments, the X nucleic acid sequences are expressed as separate encoded proteins and in other embodiments as "pearls on a string", i.e. fused proteins. In some embodiments, immunogenic amino acid sequences from any one of SEQ ID NOs: 1-20 are only present in one of said X nucleic acid sequences.
Vectors used in the invention
It will be understood that the nucleic acid fragments of the invention may be used for both production, carrier and vaccine purposes - the latter will require that the sequences are included in expression vectors that may lead to production of immunogenic proteins in the fish receiving the vector. Or put differently, the nucleic acid is comprised in a vector capable of expressing the nucleic acid in the freshwater fish upon administration. Such a vector of the invention often 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, an optional signal peptide coding sequence, a nucleotide sequence of the invention, 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 protozoan of origin, recombinant production has to be effected in host cells that can express the coding nucleic acid. Bacterial host cells may be used. However, if the vector is to drive expression in eurkaryotic cell (as would be the case for a nucleic acid vaccine vector), the expression control region should be adapted to this particular use.
For production purposes it is therefore often convenient that the expression control region drives expression in a prokaryotic cell such as a bacterium, e.g. in E. coli, or in a eukaryotic cell such as a plant cell, an insect cell, or a mammalian cell. For vaccine purposes, the expression control region has to be able to drive expression in a fish cell.
Also, for production purposes, it is practical that the vector is capable of integrating the nucleic acid into the genome of a host cell - this is particularly useful if the vector is use 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 piscine host cell are useful in e.g . nucleic acid vaccination.
An interesting production system is the use of plants. For instance, proteins can be produced at low cost in plants using an Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest. One commercially available platform are those provided by iBio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovatoin Way, Suite 100, Newark, DE 19711, USA) and disclosed in e.g. EP 2 853 599, EP 1 769 068, and EP 2 192 172. Hence, in such systems the vector is an Agrobacterium vector or other vector suitable for transfection of plants.
The vector is typically selected from the group consisting of a virus, such as a virus which is non-pathogenic in fish, a bacterium such as a bacterium which is non-pathogenic in fish, a plasmid, a minichromosome, and a cosmid.
Interesting vectors are viral vectors (in particular those useful as vaccine agents in fish). These may be selected from the group consisting of a retrovirus vector, such as a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a pox virus vector. Certain pox virus vectors are preferred, in particular vaccinia virus vectors. A particularly preferred vaccinia virus vector is a modified vaccinia Ankara (MVA) vector. Polypeptides of the invention may as indicated be encoded by a nucleic acid molecule comprised in a vector. A nucleic acid sequence can be "heterologous," which means that it is in a context foreign to the cell in which the vector is being introduced, which includes a sequence homologous to a sequence in the cell but in a position within the host cell where it is ordinarily not found.
Vectors include naked 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. 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, 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 or the vector may be purified for direct administration for expression of the protein (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 polymerase chain reaction in connection with the compositions disclosed herein.
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. 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, Immunoglobulin Light Chain, T Cell Receptor, HLA DQa and/or DQ , β- Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRa, β- Actin, Muscle Creatine Kinase (MCK), Prealbumin (Transthyretin), Elastase I, Metallothionein (MTII), Collagenase, Albumin, a-Fetoprotein, γ-Globin, β-Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), al-Antitrypain, H2B (TH2B) Histone, Mouse and/or Type I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived Growth Factor (PDGF), Duchenne Muscular Dystrophy, SV40, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus (CMV) IE, and Gibbon Ape Leukemia Virus. Inducible Elements include MT II - Phorbol Ester (TFA)/Heavy metals; MMTV (mouse mammary tumor virus) - Glucocorticoids; β-Interferon - poly(rl)x/poly(rc); Adenovirus 5 E2 - EIA; Collagenase - Phorbol Ester (TPA); Stromelysin - Phorbol Ester (TPA); SV40 - Phorbol Ester (TPA); Murine MX Gene - Interferon, Newcastle Disease Virus; GRP78 Gene - A23187; α-2-Macroglobulin - IL-6; Vimentin - Serum; MHC Class I Gene H-2Kb - Interferon; HSP70 - E1A/SV40 Large T Antigen; Proliferin - Phorbol Ester/TPA; Tumor Necrosis Factor - PMA; and Thyroid Stimulating Hormonea Gene - Thyroid Hormone.
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 peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell. Where a piscine cell is targeted (as is the case in nucleic acid vaccination), 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 piscine cell. Generally speaking, such a promoter might include either a bacterial, piscine or viral promoter as long as the promoter is effective in piscine cells.
In various embodiments - in particular those where recombinant production of the polypeptide of the invention is the aim - 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 fish 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 fish 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 the fish counterparts of dendritic cells and macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters in man and it is contemplated that corresponding piscine promoters will be effective.
2. Initiation Signals and Internal Ribosome Binding Sites (IRES)
A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent 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. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well an IRES from a mammalian message. 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. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable 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.
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 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 terminator of transcription 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 expression, particularly eukaryotic expression (as is relevant in nucleic acid vaccination), one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
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 "on"), 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 including 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.
Further general details pertaining to vaccine vectors are set forth in the above-referenced "Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470-67455-0, see in particular chapters 4 and 5. Transformed cells
Transformed cells are useful as organisms 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 1) of the second aspect of the invention. Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment defined for option 1).
For recombinant production it is convenient, but not a prerequisite that the transformed cell according is prokaryotic, such as a bacterium, but generally 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 stably integrated into its genome, and 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.
As noted above, stably transformed cells are preferred - these i.a. allows that cell lines comprised of transformed cells as defined herein may be established - such cell lines are partilucarly preferred aspects of the invention.
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 prokaryotes and eukaryotes. 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 Ps.
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, and has been, 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, and 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 replication of many vectors or expression of encoded proteins. Bacterial cells used as host cells for vector replication and/or expression include Staphylococcus strains, DH5a, JMI 09, and KC8, as well as a number of commercially available bacterial hosts such as SURE(R) Competent Cells and SOLOP ACK(TM) Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Appropriate yeast cells 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 from 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.
Cells (in particular bacterial cells) for use directly in vaccination are discussed generally in "Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470-67455-0, see chapter 3 (non-replicating vaccines, e.g. inactivated bacteria) and chapter 4 (replicating vaccines).
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, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ Baculovirus expression system from CLONTECH®
In addition to the disclosed expression systems of the invention, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is 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.
"Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470-67455-0 provides details in chapters 10 and 11 about production of fish vaccines.
Methods of Gene Transfer
Suitable methods for nucleic acid delivery to effect expression of compositions of the present invention are believed to 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), including microinjection (U.S. Patent 5,789,215); by electroporation (U.S. Patent No. 5,384,253); by calcium phosphate precipitation; by using DEAE dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection; 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); by agitation with silicon carbide fibers (U.S. Patents 5,302,523 and 5,464,765) ; by
Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055); or by PEG mediated transformation of protoplasts (U.S. Patents 4,684,611 and 4,952,500); by desiccation/inhibition mediated DNA uptake. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently
transformed. See also "Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470-67455-0, chapter 5, which deals specifically with DNA vaccination of fish. Antibodies binding specifically to the polypeptides of the invention; and their production/isolation
Antibodies directed against the proteins of the invention are useful for affinity
chromatography, immunoassays, and for distinguishing/identifying A.salmonicida proteins 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 x 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. 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 nonspecifically 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 I aedium (elg. hypexanthine, aminopterin, thymidine medium, "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 1251), 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 and avidin or streptavidin, IgG and protein A, and the 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, 1151 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, 1251, 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.
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, or which is an antibody analogue selected from a fragment of an antibody such as an Fab or an F(ab')2, an scFV; cf. also the definition of the term "antibody" presented above.
Antibodies binding the polypeptides of the invention are in their own right useful as treatment agents, but will not be able to confer lasting immunity if administered to fish. Nevetheless, it may be of relevance to also - e.g. as a supplement - immunize fish passively by
administration of such antibodies.
Compositions of the invention; vaccines
Compositions, in particular vaccines, according to the invention are prophylactic. Generic disclosures relating to such vaccines are provided in "Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470- 67455-0, in particular chapters 3-7, 10 and 11. 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.
In some embodiments of the invention, the pharmaceutical compositions such as vaccines include merely one single antigen, immunogen, polypeptide, protein, nucleic acid or vector of the invention, but in other embodiments, the pharmaceutical compositions comprise
"cocktails" of the antigens or of the immunogens or of the polypeptides or of the protein or of the nucleic acids or of the vectors of the invention.
In particularly interesting embodiments, the pharmaceutical composition is a vector mentioned herein, which encodes and can effect expression of at least 2 nucleic acid fragments of the invention.
Another interesting embodiment of a pharmaceutical composition comprises RNA as the active principle, i.e. at least one mRNA encoding a polypeptide of the invention.
An embodiment of a pharmaceutical composition of the invention comprises Y or at least Y or at most Y distinct polypeptides of the invention described above, where each of said Y or at least Y or at most Y distinct polypeptides comprises an immunogenic amino acid sequence present in or derived from any one of SEQ ID NOs: 1-20 and wherein said Y or at least Y or at most Y distinct polypeptides together comprise immunogenic amino acid sequences present in or derived from Y or at least Y or at most Y of SEQ ID NOs: 1-20, wherein Y is an integer selected from 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, and 30.
Another embodiment of the pharmaceutical composition of the invention comprises Z or at least Z or at most Z distinct nucleic acid molecules (such as DNA and RNA) each encoding a polypeptide of the invention, where each of said Z or at least Z or at most Z distinct nucleic acid molecules encodes an immunogenic amino acid sequence present in or derived from any one of SEQ ID NOs: 1-20 and wherein said at Z or least Z distinct nucleic acid molecules together encode immunogenic amino acid sequences present in or derived from Z or at least Z or at most Z of SEQ ID NOs: 1-20, wherein Z is an integer selected from 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, and 30.
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, H. pylori, etc. pathogen, cf. the description of immunogenic carriers supra.
Adjuvants for fish vaccines are discussed in details in "Fish Vaccination", 2014, edited by Gudding R., Lillehaug A, and Evensen 0, published by Wiley Blackwell, ISBN 978-0-470- 67455-0, chapter 7.
The pharmaceutical compositions of the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants 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 (DetoxTM) ; (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 (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg.
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. Alum and MF59™ adjuvants are preferred together with CFA and IFA.
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.
Another possibility for a polypeptide vaccine formulation is to include the vaccine
polypeptide(s) of the present invention in a virus-like particle, i.e. a non-infectious self- assembling structure composed of envelope or capsid proteins, where the protein(s) of the invention are incorporated. The effect is multiple presentations of the polypeptides of the invention on the surface of the VLP, which in turn provides for improved immune recognition of the polypeptides. Hence, VLPs exert immunological adjuvant effects, too.
The immunogenic compositions (eg. 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 under pharmaceutically acceptable carriers.
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 "immunollogically 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 (eg. nonhuma 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 treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount of immunogen 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 (however, often not higher than 5,000 μg). The amount of polypeptide of the invention can therefore be between 1 and 400 μg, between 2 and 350 μg, between 4 and 300 μg, between 5 and 250 μg, and between 10 and 200 μg. Hence, the composition will typically contain between 0.1-500 μg of protein of the invention per g of vaccine composition. The immunogenic compositions are conventionally administered parenterally, eg, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. W098/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination, also the intravenous or intraarterial routes may be applicable.
Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may 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 [eg. Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al. (1997) Avnu Rev Innnunol 15 : 617-648; later herein] .
A further aspect of the invention is as mentioned above the recognition that combination vaccines can be provided, wherein 2 or more antigens disclosed herein are combined to enhance the immune response by the vaccinated fish, 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, longer protein can also be used, such as the use of a combination of different lengths of polypeptide sequence fragments from one protein.
Thus, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 1 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 2 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 3 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 4 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 5 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 6 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 7 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 8 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 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, and 30 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 9 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 10 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 11 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 or in combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 12 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 13 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 14 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 15 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 16 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 17 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 18 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 19 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Also, embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 20 or a variant or fragment thereof disclosed herein in combination with a proteinaceous immunogen selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 or in
combination with a variant or fragment disclosed herein of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
Immunization methods
The method of the first and second aspects of the invention generally relate to induction of immunity and as such also entails methods that are prophylactic.
When immunization methods entail that a polypeptide of the invention or a composition comprising such a polypeptide is administered the animal (e.g. the human) typically receives between 0.5 and 5,000 μg of the polypeptide of the invention per administration, cf. the above indications concerning dosages.
In preferred embodiments, the immunization scheme includes that the fish receives one single administration of the polypeptide(s) or composition(s) of the invention, but it may be necessary to follow up with one or more booster administrations. Preferred embodiments comprise that the administration is for the purpose of inducing protective immunity against A.salmonicida. In this embodiment it is particularly preferred that the protective immunity is effective in reducing the risk of attracting infection with A.salmonicida.
As mentioned herein, the preferred vaccines of the invention induce humoral immunity, so it is preferred that the administration is for the purpose of inducing antibodies specific for A.salmonicida.
But, as also mentioned the immunization method may also be useful in antibody production, so in other embodiments the administration is for the purpose of inducing antibodies specific for A.salmonicida wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies.
Compositions for immunization can as mentioned above comprise polypeptides, antibodies, nucleic acids, or vectors of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount thereof.
The term "therapeutically effective amount" or "prophylactically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable preventative effect in a group of fish. The effect can be detected by, for example, chemical markers or antigen levels. Reference is 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 fish 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, 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 excipients 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.
Further embodiments
The disclosures generally relating to fish vaccination, fish vaccine compositions, and vector construction detailed in US patent 7,026,156 and US patent publication no. 2007/00771776 are applicable within the ambit of the present invention - both these patents are included by reference herein.
EXAMPLE
Vaccination study
Protein selection and tailoring
14 proteins (see details in table A below) were picked based on rationale selection considering functionality, subcellular location and conservation:
Functional analysis
InterPro (available at http://www.ebi.ac.uk/interpro/) provides functional analysis of proteins by classifying them into families, predicting domains and important sites. The functions motility, adhesion, protein binding, enzymatic activity, receptor activity, metal
binding/acquisition have been seen in protective B-cell protein antigens, hence these are targets of selection. Subcellular location
The subcellular localization of the individual proteins was predicted by the Wolf pSORT prediction server (available at http://www.genscript.com/wolf-psort.html). It is common that protective B-cell protein antigens are located in the outer membrane and extracellular environment; consequently, these predicted subcellular locations are a target of selection.
Conservation
The conservation study was done by local sequence alignment (using the Smith-Waterman algorithm) based on the amino-acid sequence of the protein antigens tested for conservation across the public available NCBI chromosome data. The pair-wise alignment values (% similarity and % coverage) were calculated and the most similar protein to a given genome was selected and classified as homologous if the % similarity >75% on at least 75% of the total protein length (% coverage). Protein conservation correlates with an increased probability of success, due to the ability to elicit protection across different bacterial strains. Therefore, we performed a conservation analysis of the initial selected A. salmonicida proteins across the identified incomplete chromosome genome sequences available in the NCBI database. The primary strain (complete chromosome from NCBI) was strain A449. The 4 sub strains (incomplete chromosome from NCBI) were strain: 01-B526, CBA100,
NBRC_13784 and pectinolytica_34mel.
Recombinant construct design
Fragments of the 14 proteins were constructed based on conventional in silico analysis such as prediction of signal peptides (SignalP-4.1, see: Petersen T. N., et al., SignalP 4.0:
discriminating signal peptides from transmembrane regions. Nat Methods, 2011. 8(10) : p. 785-6), transmembrane regions (TmHmm-2.0, see: Krogh A, Larsson B, von Heijne G, Sonnhammer EL. (2001); Predicting transmembrane protein topology with a hidden Markov model : application to complete genomes; Mol Biol. Jan 19;305(3) : 567-80), non-classical secretion proteins (SecretomeP-2.0, see: Bendtsen JD, Kiemer L, Fausboll A, Brunak S. (2005); Non-classical protein secretion in bacteria; BMC Microbiol. Oct 7; 5: 58. PMID:
16212653) and functional and structural domains (InterPro; and DomCut, see: Suyama M. and Ohara O., DomCut: prediction of inter-domain linker regions in amino acid sequences. Bioinformatics, 2003. 19(5) : p. 673-4). KAN I INDS ETTE REFERENCERNE TIL SOFTWAREN HER? (se I kommentar feltet)
The rationale of expressing the protein as fragments was due to: 1) enhanced probability of expressing the native protein structure, 2) expressing the protective part of the protein only, and 3) establishing a successful recombinant expression. Predicted signal peptide, transmembrane and intracellular regions were removed. Proteins larger than 1000 amino acid residues were split in N- and C-terminal fragments and only the fragment comprising the predicted active functional site domains were expressed. The splitting point of these fragments where decided based on structural domain predictor aiming at keeping the native structure of each structural domain. The rationale of splitting the > 1000 amino acid residue protein was to increase the probability of a successful recombinant expression in E. coli.
Protein expression
All recombinant proteins were expressed in E. coli by Creative Biomart Shirley, NY, USA. Vaccine preparation
The 14 protein fragments derived from the proteins listed in Table A were divided into 3 groups as follows: VacA and VacB comprised of 5 proteins and vacC comprised of 4 proteins. See Table B below.
The volume of each protein solution in vaccine formulations was calculated considering each fish to receive 25 μg of an individual protein in the mixture corresponding to a total of 200- 225 μg of mixed protein per fish.
First, to remove the urea buffer and enable the proteins to bind to AI(OH)3, aluminum hydroxide gel adjuvant (Alhydrogel, Brenntag) was added to each vial of protein mixture. The vaccine control containing pure adjuvant only was made accordingly without the proteins.
A volume of 100 μΙ AI(OH)3 was added per 160 μg protein and incubated at room
temperature with end-over-end rotation for 1 hour. The vials were then centrifuged (Heraeus multifuge 3 L-R) at room temperature at 1000 rpm (216 G) for 2 minutes. The supernatant was removed and the absence of proteins verified in NanoDrop 2000 (Thermo Scientific). The vials containing proteins bound to AIOH were washed twice with 0.9 % sterile NaCI. Each time all the vials were centrifuged at 1000 rpm (216 G) for 2 minutes at room temperature and the supernatant was removed. After the last wash an equal volume of Freund's
Incomplete Adjuvant (FIA) (Sigma, F5506) was added and the vials were vortexed at room temperature for 1 hour. The vaccines were stored at 4°C until further use. Fish
Disinfected eyed rainbow trout eggs originating from Fousing Trout Farm, Jutland, Denmark were translocated and hatched in a pathogen-free rearing facility at Bornholm Salmon Hatchery (AquaBaltic, Nexo, Denmark). Fish were vaccinated and then reared for 6 weeks (568 degree-days) in the system containing recirculated municipal water at 14°C in 700 I (1 m3) tanks and fed 1% biomass per day with dry pellet feed (BioMar A/S, Brande, Denmark). Fish were then transported to the fish keeping facility at the University of Copenhagen, Frederiksberg, Denmark and acclimatized by gradually raising the water temperature to 19°C for 1 week before challenge (exposure to AS) was performed. The studies were approved under the license no. 2015-15-0201-00655 issued by the Animal Experiments Inspectorate, Ministry of Environment and Food, Denmark.
Vaccination and challenge experiment
A total of 360 rainbow trout (average 30 g) were randomly divided into 6 groups of 60 fish and each group was further subdivided into duplicate tanks each containing 30 fish. The fish were anaesthetized (75 mg MS222/I) and i.p. injected with 0.1 ml of the different vaccine formulations (room temperature) (Table B). During the immunization period 10 fish from each group (5 fish per duplicate tank) were sampled for ELISA. At 7 wpv (708 degree-days) the remaining 25 fish (now 40 g) in all the duplicate tanks were challenged with 5.4 x 108 CFU/ml AS strain 090710-1/23 by a challenge method using a multi-puncture device.
Morbidity was monitored every second hour during 3 weeks post-challenge (wpc). Moribund fish were immediately removed for euthanasia (300 mg MS222/I) and recorded as mortalities. Swabs from head kidney of all freshly euthanized fish were plated onto 5% blood agar plate (SSI Diagnostica, Denmark) and subsequently subjected for bacteriological analysis. The re-isolated bacteria from all the dead fish were confirmed as Aeromonas salmonicida subsp. salmonicida. The challenge trial was terminated 3 weeks (24 d) post- challenge when 10 fish from each group (5 per replicate tank) was sampled for ELISA.
ELISA
Blood samples for ELISA were collected by caudal vein puncture from euthanized (300 mg MS222/I) fish at 7 wpv and 3 wpc. Blood was allowed to clot at 4 °C overnight whereafter serum was separated by centrifugation at 3000G and 4°C for 10 min and stored at -80° C until further analysis.
Enzyme-linked immunosorbent assay (ELISA) was performed according to previously established protocol (Chettri JK et al.; Comparative evaluation of administration methods for a vaccine protecting rainbow trout against Yersinia ruckeri 01 biotype 2 infections; Vet Immunol Immunopathol. 2013 Jul 15; 154(l-2) :42-7. doi: 10.1016/j.vetimm.2013.04.001. Epub 2013 Apr 8). In brief, the 96-well microtiter plates (MaxiSorp™, Nunc, Denmark) were coated either with sonicated lysate of AS bacteria or proteins. Due to time and labour constraints only 9 proteins comprised in VacB and VacC subunit vaccines were chosen for plate coating in ELISA analysis as these proteins provided the best protection. Bacteria used for coating was AS strain 090710-1/23 (protein cone 5 μg/ml) and 9 proteins were coated individually per plate (protein cone 1 μg/ml).
Working dilutions were chosen based on the calibration trials/preliminary results. Dilutions 1 : 5 X 102 and 1 : 5 X 103 were chosen for testing specific antibodies against sonicated bacteria and dilutions 1 : 102 - 1 : 104 were chosen for testing specific antibodies against individual proteins. All serum samples were diluted with assay diluent (wash buffer with 0.1% BSA) whereafter 100 μΙ_ of sample was added to each duplicate ELISA plate well and incubated at 4 °C overnight. After a washing step the plates were incubated for 1 h with 100 μί mouse anti- salmonid Ig (cat. no. MCA2182 AbD Serotec, Germany, diluted 1 : 500). After an additional wash 100 μL HRP-conjugated rabbit anti-mouse IgG was added and reacted for 1 h (cat. no. STAR13B, AbD Serotec, Germany, diluted 1 : 500). The color reaction was developed with 100 μL tetramethylbenzidine (TMB) PLUS substrate (cat. no. BUF042A, AbD Serotec, Germany) and stopped after 10 min by adding 100 μί IN HCL. All ELISA plates included a pure assay diluent for the background absorbance measurement. The optical density (OD) was measured at 450 nm in an Epoch spectrophotometer (BioTek, USA) in duplicate wells.
Statistical analysis
The relative percentage of survival (RPS) was calculated at 3 wpc (24 d) as proposed by Amend 1981 (ref) : RPS = [1- (% mortality in vaccinated fish / % mortality in control fish)] x 100. All statistical tests were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, USA, www.graphpad.com) and P-values < 0.05 were considered statistically significant. Mortality data was analyzed using Kaplan-Meier survival analysis and log-rank test. Duplicate groups were pooled after survival curve comparison showed no significant difference in mortalities.
ELISA results were compared using one-way ANOVA and followed by Tukey's Multiple Comparison post hoc test. Fish from duplicate tanks were pooled after a t-test had confirmed that there was no significant difference between them Correlation analysis was calculated as Pearson correlation coefficient (r). RESULTS - Challenge experiment
All fish were challenged at 7 wpv by multi-puncture of the upper caudal fin and local exposure to a known number of AS, see above. The mortality post-challenge (pc) is shown in Fig. 1 and Table 1.
Table 1. Group size, mortality and relative percentage of survival (RPS). All Vac groups were immunized with proteins mixed with an adjuvant. The adjuvant consisted of equal volume of Freunds Incomplete Adjuvant (FIA) and aluminium hydroxide gel adjuvant (Alhydrogel). The individual proteins included in the vaccines are presented in table B.
Figure imgf000085_0001
Fish died after anaesthesia during challenge infection.
As a positive control, fish were also vaccinated with a commercially available vaccine
(AlhpaJect® 3000), according to the manufacturer's (Pharmaq/Zoetis) instructions. This positive control is an injectable formulation of inactivated AS in admixture with inactivated Listonella angullarum serotype 01 and Listonella anguillarum serotype 02a, see.
Mortality started at day 3 pc in all fish groups and subsided by day 6 pc. Thereafter minor mortality still occurred until day 15 pc. The highest cumulative mortality was recorded in the saline control group (56 %) followed by the adjuvant only control group (48 %). Mortalities in the experimental subunit vaccine groups VacA (30 %, RPS 46), VacB (20 %, RPS 64) and VacC (17%, RPS 70) (p<0.001, p<0.0001 and p<0.0001 respectively) were significantly lower than in the saline control group. The lowest cumulative mortality of 8 % (RPS 86) occurred in the group vaccinated with the commercially available vaccine (p<0.0001).
RESULTS - ELISA
ELISA analyses were conducted to measure the antibody response towards sonicated bacteria and individual proteins in different experimental groups. We compared antibody responses showing significant differences (p<0.05) between the vaccinated groups (Commercial vaccine, VacA, VacB, VacC) and control groups (saline control and adjuvant only control). Antibodies against AS
Antibody levels in fish vaccinated with the commercial vaccine were significantly higher compared to all other groups after vaccination (7 wpv) and challenge infection (3wpc) when serum samples were diluted 500 times (Fig 2). A slight but non-significant antibody increase in all the groups vaccinated with subunit vaccines was seen when compared to control groups.
Antibodies against proteins used for immunization.
Fish immunized with the commercial vaccine exhibited significantly higher antibody response against all individual proteins compared to control groups at 7 wpv and 3 wpc. Fish immunized with different proteins (Vac groups) showed different degrees of elevated antibody levels after vaccination and challenge (Fig. 2). In some cases the differences were significant compared to control groups.
Antibodies against proteins included in VacB vaccine
Significantly higher antibody levels were seen fish immunized with proteins from VacB vaccine compared to saline injected fish at 7 wpv when serum samples (1 : 100) were tested on plates coated with following proteins from the VacB vaccine: #7, #8, #9, #10. Fish immunized with proteins from VacA vaccine also exhibited significantly higher antibody levels compared to saline injected group at 7 wpv but only against proteins #7 and #10 from VacB vaccine (Fig. 3A).
Significantly higher antibody levels against proteins in VacB vaccine were found in vaccinated fish compared to saline control also after challenge infection (3 wpc). Fish immunized with proteins from VacB vaccine showed significantly elevated antibody levels against #10 from VacB vaccine and fish immunized with proteins from VacA group against #6 from VacB vaccine (Fig. 3B).
Proteins included in VacC vaccine
Fish immunized with proteins from VacC vaccine showed significantly elevated antibody titers at 7 wpv and 3 wpc (d) compared to control groups when serum samples (1 : 100) were tested against protein #11 (Fig. 4A and 4B) . Correlation between ELISA and survival
The correlation between antibody levels (102 DIL) at 7 wpv and the survival at day 24 pc for each group was calculated as Pearson correlation coefficient (r). A significant positive correlation was found for VacB proteins #8 (Pearson r=0.86; R2=0.73, #10 (Pearson r=0.81; R2=0.66 and VacC protein #11 (Pearson r=0.81; R2=0.67).
Conclusions
Rainbow trout was vaccinated with subunit vaccines containing recombinant produced protein antigens. Fourteen protein constructs were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. Proteins were tested for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent AS 7 weeks post-vaccination by applying a multi-puncture challenge method. The immune response in fish was evaluated following vaccination and challenge by measuring antibody levels and recording survival. The control group showed 56% mortality whereas the groups of fish vaccinated with experimental subunit vaccines exhibited significantly lower mortalities (17-30%). These results imply that the antigens of AS disclosed herein have significant protective properties.
Table A
RATIONALE CONSERVATION
PROTEINi PIDtroen ID FUNCTION LOCATION
OF SELECTION (NCBIs incomplete chromosomes) (i) A449tsran: ili Fttunonayc i Ltocaon i Ctonservaon
ASA_2532 motility unknown periplasmic 100% [4/4] 96% 5%
ASA_0744 pathogenesis, membrane cytoplasmic periplasmic + cytoplasmic 100% [4/4] 97% 2%
ASA_3320 extracellular enzyme activity unknown periplasmic 100% [4/4] 98% 1%
ASA_4042 - periplasmic periplasmic 100% [4/4] 92% 9%
ASA_3883 iron sideophore receptor outer membrane outer membrane 100% [4/4] 98% 2%
ASA_4105 zinc metallopeptidase outer membrane outer membrane 100% [4/4] 98% 2%
ASA_3328 haemoglobin/transferrin/lactoferrin receptor outer membrane outer membrane 100% [4/4] 98% 3%
ASA_3723 collagenase extracellular outer membrane + cytoplasmic 100% [4/4] 99% 2%
ASA_2321 heme binding IPtnerro server periplasmic periplasmic 100% [4/4] 98% 2%
ASA_1342 motility extracellular periplasmic 75% [3/4] 97% 3%
ASA_P5G035 adhesion, pilin assembly outer membrane extracellular + outer membrane 50% [2/4] 97% 1%
ASA_0826 virulent enzymatic activity extracellular extracellular 50% [2/4] 99% 1%
ASA_1675 virulent enzymatic activity extracellular extracellular 50% [2/4] 98% 1%
ASA 3455 extracellular enzyme activity extracellular extracellular 50% [2/4] 99% 1%
Table X. Rationale protein selection: These 14 proteins were selected as potential protective B-bc PSORT serverell antigens. The column "RATIONAL OF SELECTION" is showing which area that were responsible for the selection. E.g. protein ASA 2532 was selected due to its predicted motility function domain by InterPro and its strong conservation. That is beside being present in the primary strain A449 (complete chromosome), it was represented in all NCBPs incomplete chromosomes. The "%Library coverage" of protein ASAJ2532 is 100% meaning that this protein is 100% conserved across all the 4 sub strains. The "Mean of %Similiarites to Homologs" is 96% meaning this protein have an average %identity of 96% to the homologes of the 4 sub strains.
CELLO server bi % L Craryoverage / [flfi ] N H N Sto oomoogso orans
fll Miiii H %St tmean omaresoooogs dd diif Sttanarevaon o iilil %Si Ht tmaresoomoogs Table B
Figure imgf000089_0001
Table X— Recombinant construct design, protein type and vaccine formulation: The column "ACTION" lists which part of the sequence have been removed. The column "aa-region" illustrates which amino acid region of the protein has been expressed. E.g. the signal peptide, transmembrane
SEQUENCE INFORMATION
The full length polypeptides disclosed herein having SEQ ID NOs: 1-20 also have the following designations, which correspond to their accession numbers in the KEGG database (http://www.genome.jp/), where also the corresponding DNA sequences are available:
SEQ ID NO: Acc. No. SEQ ID NO: Acc. No
ASA 2861 11 ASA 3883
2 ASA_4127 12 ASA_3328
3 ASA_0744 13 ASA_2532
4 ASA_4042 14 ASA_3455
5 ASA_4105 15 ASA_P5G035
6 ASA_2321 16 ASA_3723
7 ASA_3943 17 ASA_2064
8 ASA_2955 18 ASA_1675
9 ASA_1342 19 ASA_0628
10 ASA 3320 20 ASA 0826
If a sequence is identified by a designation such as ASA_3883_25-680, this means that the sequence is constituted by amino acid residues 25-680 in ASA_3883 (amino acid residues in SEQ ID NO: 11).

Claims

1. A method of inducing immunity in fish against Aeromonas salmonicida (A.
salmonicida) comprising administering an effective amount of a polypeptide comprising a) an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 13, 1-12, and 14-20, or
b) an amino acid sequence consisting of at least or exactly or at most 5 contiguous amino acid residues from any one of SEQ ID NOs: 13, 1-12, and 14-20, 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: 13, 1-12, and 14-20, 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.
2. The method according to claim 1, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) are at least or exactly or at most 6, such as at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or at most 33, at least or exactly or at most 34, and at least or exactly or at most 35 contiguous amino acid residues.
3. The method according to clam 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%.
4. The method according to clam 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%.
5. The method according to any one of the preceding claims wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N- terminus at amino acid residue 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, 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, or 89 in any one of SEQ ID NOs: 1-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
6. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 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, or 237 in any one of SEQ ID NOs: 2-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
7. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 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, or 384 in any one of SEQ ID NOs: 3-20, with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
8. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, or 398 in any one of SEQ ID NOs: 4-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
9. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, or 464 in any one of SEQ ID NOs: 5-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
10. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, or 476 in any one of SEQ ID NOs: 6-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+ 1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
11. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, or 552 in any one of SEQ ID NOs: 7-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where
N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
12. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, or 574 in any one of SEQ ID NOs: 8-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
13. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635 in any one of SEQ ID NOs: 9-20, with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
14. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 636 in any one of SEQ ID NOs: 10-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues
15. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 637, 638, 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, or 676 in any one of SEQ ID NOs: 11-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where
N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
16. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, or 693 in any one of SEQ ID NOs: 12-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
17. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, or 708 in any one of SEQ ID NOs: 13-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
18. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 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, or 750 in any one of SEQ ID NOs: 14-20, with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+ 1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
19. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, 828, 829, 830, 831, 832, 833, 834, 835, 836, 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, or 873 in any one of SEQ ID NOs: 15-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
20. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, or 911 in any one of SEQ ID NOs: 16-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
21. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 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, 956, 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, or 1016 in any one of SEQ ID NOs: 17-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+ 1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
22. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, 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, 1072, 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, or 1152 in any one of SEQ ID NOs: 18-20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
23. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, 1336, 1337, 1338, 1339, or 1340 in any one of SEQ ID NOs: 19-20, with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is the number of amino acid residues in the sequence from which the residue is selected, and n is the number of consecutive amino acid residues.
24. The method according to any one of claims 1-4, wherein the at least or exactly or at most 5 contiguous amino acid residues in option b) commences in its N-terminus at amino acid residue 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, 1603, 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, or 3191 in SEQ ID NO: 20,
with the proviso that the selected amino acid residue satisfies the formula N≤ L-n+1, where N is the number of the selected residue, L is 3195, and n is the number of consecutive amino acid residues.
25. The method according to any one of the preceding claims, wherein the polypeptide is fused or conjugated to an immunogenic carrier molecule.
26. The method according to claim 25, wherein the immunogenic carrier molecule is a polypeptide that induces T-helper lymphocyte responses in fish, 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.
27. The method according to any one of the preceding claims, wherein the polypeptide is capable of inducing an adaptive immune response against the polypeptide in fish, in particular in Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, and Halibut, and where the polypeptide in particular is capable of inducing a prophylactic immune response that reduces the risk of furunculosis in fish.
28. The method according to claim 27, wherein the polypeptide is capable of inducing, in the freshwater fish, a protective adaptive immune response against infection with A.
Salmonidia.
29. The method according to claim 27 or 28, wherein the polypeptide induces a humoral and/or a cellular immune response.
30. The method according to any one of the preceding claims, wherein the polypeptide is comprised in a composition comprising a pharmaceutically acceptable carrier or vehicle.
31. The method according to claim 30, wherein the composition further comprises an immunological adjuvant.
32. The method according to any one of the preceding claims wherein the polypeptide is administered via oral administration, immersion (such as bathing, dipping or spraying), or injection (such as intraperitoneal or intramuscular).
33. A method of inducing immunity in freshwater fish against A. salmonidia comprising administering an effective amount of 1) a nucleic acid encoding a polypeptide defined in any one of claims 1-29 or 2) a vector comprising and being capable of expressing the nucleic acid in fish.
34. The method according to claim 33, wherein the nucleic acid is DNA or RNA.
35. The method according to claim 31 or 42, wherein the nucleic acid is identical to, or is a fragment of, a nucleic acid which consists of
- a nucleotide sequence selected from any one of SEQ ID NOs: 21-60, or
- a nucleotide sequence with at least 60% sequence identity with any one of SEQ ID NOs: 21-60.
36. The method according to any one of claims 33-35, wherein the nucleic acid is comprised in a vector capable of expressing the nucleic acid in the freshwater fish upon administration.
37. The method according to claim 36, wherein the vector 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, an optional signal peptide coding sequence, a nucleotide sequence defined in any one of claims 33-35, and optionally a terminator.
38. The method according to claim 37, wherein the expression control region drives expression in a prokaryotic cell such as a bacterium, e.g. in E. coli.
39. The method according to claims 36 or 38, wherein the vector is capable of integrating the nucleic acid into the genome of a non-piscine host cell.
40. The method according to any one of claims 36-39, wherein the vector is incapable of being integrated into the genome of a piscine host cell.
41. The method according to any one of claims 36-50, wherein the vector is selected from the group consisting of a virus, such as a virus which is non-pathogenic in fish, a bacterium such as a bacterium which is non-pathogenic in fish, a plasmid, a minichromosome, and a cosmid.
42. A composition comprising a polypeptide as defined in any one of claims 1-29 in admixture with a diluent, carrier, excipient or vehicle.
43. The composition according to claim 42, further comprising an immunological adjuvant.
44. The composition according to claim 42 or 43, wherein the polypeptide is present in a concentration between 0.1-500 [ig/g .
45. A polypeptide as defined in any one of claims 1-29.
46. A polypeptide according to claim 45 for use as a pharmaceutical.
47. A polypeptide according to claim 45 for use in a method according to any one of claims 1-32.
48. A nucleic acid fragment as defined in any one of claims 33-35 or a vector as defined in any one of claims 36-41 for use as a pharmaceutical.
PCT/EP2017/065557 2016-06-24 2017-06-23 Vaccines against aearomonas salmonicida infection WO2017220787A1 (en)

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