WO2017216384A1 - Vaccination ciblant ichthyophthirius multifiliis - Google Patents

Vaccination ciblant ichthyophthirius multifiliis Download PDF

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Publication number
WO2017216384A1
WO2017216384A1 PCT/EP2017/064899 EP2017064899W WO2017216384A1 WO 2017216384 A1 WO2017216384 A1 WO 2017216384A1 EP 2017064899 W EP2017064899 W EP 2017064899W WO 2017216384 A1 WO2017216384 A1 WO 2017216384A1
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exactly
amino acid
residue
acid residues
sequence
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PCT/EP2017/064899
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English (en)
Inventor
Niels IVERSEN MØLLER
Andreas HOLM MATTSSON
Louise VON GERSDORFF JØRGENSEN
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Evaxion Biotech Aps
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Publication of WO2017216384A1 publication Critical patent/WO2017216384A1/fr

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    • 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
    • A61K39/002—Protozoa antigens
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine

Definitions

  • the present invention relates to active immunization of freshwater fish.
  • the present invention relates to a vaccine for inducing immunity against Ichthyophthirius multifiliis (I. multifiliis) in order to reduce the incidence of white spot disease in freshwater fish.
  • Ichthyophthirius multifiliis I. multifiliis
  • Ichthyophthirius multifiliis is a protozoan parasite infecting both aqua-cultured freshwater fish and ornamental freshwater fish worldwide.
  • the parasite is the causative agent of
  • ichthyophthiriases also termed "white spot disease”.
  • the disease induces high morbidity and mortality in the fish and as such constitutes an economical burden in fish production.
  • the parasite infects the exterior surfaces of the fish, mainly the skin and gills and to a lesser degree the nasal and buccal cavity. It penetrates the epithelium and settles above the basal layer and in an intra-epidermal space where it feeds on fish cells and cell debris. After a maturation period at this location the parasite can grow up to approximately 1 mm in diameter. Subsequently, it leaves the fish and undergoes multiplication into new infective stages.
  • Freshwater teleosts are known to be able to acquire a high level of immunity against white spot disease, which is the basic requirement when considering vaccine production. Following a first infection surviving fish may endure a secondary infection with only a few parasites. Different vaccines have been experimentally tested against I.
  • multifiliis are excellent vaccine agents that can induce protective immunity in freshwater fish.
  • the present invention relates to a method of inducing immunity in freshwater fish against Ichthyophthirius multifiliis (I. multifiliis) comprising administering an effective amount of a polypeptide comprising
  • the present invention relates to method of inducing immunity in freshwater fish against I. multifiliis 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 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: 1-30, 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 herein meant that the polypeptide is a polypeptide derived from any one of SEQ ID NOs: 1-30, 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 Parasite burden following challenge with I. multifiliis in 4 different vaccine groups of rainbow trout.
  • Fig. 2 Mobility of theronts (I. multifiliis) following treatment with rainbow trout plasma from 4 different vaccine groups 13 wpi (weeks past infection).
  • the Y-axis shows theront mobility with 3 being maximum mobility and 0 being complete immobilisation.
  • Fig. 3 ELISA results from rainbow trout plasma following different vaccine treatments against the parasite I. multifiliis 4 and 13 wpi.
  • the specific antibody response against the zinc-finger-like protein (#5) is shown in panel A- D; peptide #5a is shown in panel A and B, the response against #5b in panel C and D.
  • the growth factor receptor domain-like protein (#10) induces a specific antibody response in the two experimental vaccine groups (panel E-F).
  • the K + -dependent Na + /Ca 2+ exchanger-like protein (#11) also induced a specific antibody response in the sub-unit vaccine group and the parasite homogenate group had produced a significant level of antibodies against this protein (panel G-H). Plasma from the parasite homogenate vaccine group reacted specifically against parasite homogenate on the micro plates and the sub-unit vaccine group had a slightly elevated level of J.
  • amino acid sequences set forth in SEQ ID NOs: 1-30 also can be retrieved from National Center for Biotechnology Information (NCBI) under the following accession numbers:
  • the corresponding wild-type coding sequences are set forth in SEQ ID NOs: 31-60 (DNA) and 61-90 (RNA).
  • 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.
  • 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.
  • 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. Specific embodiments of the invention
  • 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: 2-30. Another way to phrase this is that for each of SEQ ID NOs: 1-30, 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:
  • 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 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, at least or exactly or at most 241, 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
  • 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 329 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 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, or at least or exactly or at most 343 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 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, or at least or exactly or at most 350 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 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
  • 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 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, at least or exactly or at most 388, 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 378, at least or exactly or at most 379,
  • 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 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
  • 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 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, at least or exactly or at most 468, 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, or at least or exactly or at most 474 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 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, at least or exactly or at most 480, 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
  • 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 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, or at least or exactly or at most 539 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 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, or at least or exactly or at most 553 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 554, at least or exactly or at most 555, at least or exactly or at most 556, 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 5
  • 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 660 or 661 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 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, at least or exactly or at most 680, at least or exactly or at most 681, 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 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, or at least or exactly or at most 737 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 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, at least or exactly or at most 754, 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
  • 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 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, or at least or exactly or at most 785 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 786 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 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
  • 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 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, or at least or exactly or at most 844 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 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
  • 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 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
  • 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 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 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 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
  • 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 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, at least or exactly or at most 1156, 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 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 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
  • 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 exactly 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 exactly 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 exactly 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 exactly or at most 1284, at least or exactly or at most 1285, 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 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
  • the at least 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention may also const tute at least or exactly or at most 3573, at least or exactly or at most 3574, at least or exact y or at most 3575, at least or exact ly or at most 3576, at least or exactly or at most 3577, at least or exactly or at most 3578, at least or exactly or at most 3579, at least or exact y or at most 3580, at least or exact ly or at most 3581, at least or exactly or at most 3582, at least or exactly or at most 3583, at least or exactly or at most 3584, at least or exact y or at most 3585, at least or exact ly or at most 3586, at least or exactly or at most 3587, at least or exactly or at most 3588, at least or exactly or at most 3589, at least or exact y or at most 3590, at least or exact ly or at most 3591
  • 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, 89, 90, 91, 92, 93, 94, 95 96, 97,
  • 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 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 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,
  • 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 326 in any one of SEQ ID NOs: 3-30,
  • 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 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, or 340 in any one of SEQ ID NOs: 4-30,
  • 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 341, 342, 343, 344, 345, 346, or 347 in any one of SEQ ID NOs: 5-30, 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 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, or 374 in any one of SEQ ID NOs: 6-30,
  • 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 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, or 427 in any one of SEQ ID NOs: 7-30,
  • 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 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, or 457 in any one of SEQ ID NOs: 8-30,
  • 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 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, or 471 in any one of SEQ ID NOs: 9-30,
  • 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 472, 473, 474, 475, 476, 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, or 519 in any one of SEQ ID NOs: 10-30,
  • 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 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, or 536 in any one of SEQ ID NOs: 11-30,
  • 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 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, or 550 in any one of SEQ ID NOs: 12-30,
  • 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 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617,
  • 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 657 or 658 in any one of SEQ ID NOs: 14-30,
  • 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 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, or 720 in any one of SEQ ID NOs
  • 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 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, or 734 in any one of SEQ ID NOs: 16-30,
  • 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 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, or 772 in any one of SEQ ID NOs: 17-30,
  • 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 773, 774, 775, 776, 777, 778, 779, 780, 781, or 782 in any one of SEQ ID NOs: 18-30, 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 783 in any one of SEQ ID NOs: 19-30,
  • 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 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, or 836 in any one of SEQ ID NOs: 20-30,
  • 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 837, 838, 839, 840, or 841 in any one of SEQ ID NOs: 21-30, 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 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907
  • 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 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,
  • 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 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059,
  • 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 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, or 1140 in any one of SEQ ID NOs: 25-30,
  • 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 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, or 1165 in any one of SEQ ID NOs: 26-30,
  • 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 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
  • 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 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273,
  • 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 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674,
  • 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 3570, 3571, 3572, 3573, 3574, 3575, 3576, 3577, 3578, 3579, 3580, 3581, 3582, 3583, 3584, 3585, 3586, 3587, 3588, 3589, 3590, 3591, 3592, 3593, 3594, 3595, 3596, 3597, 3598, 3599, 3600, 3601, 3602, 3603, 3604, 3605, 3606, 3607, 3608, 3609, 3610, 3611, 3612, 3613, 3614, 3615, 3616, 3617, 3618, 3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630, 3631, 3632, 3633, 3634
  • 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: 1-30.
  • 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
  • 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 freshwater fish, in particular of the family Salmonidae, Cyprinidae, Cichlidae, Chanidae, Siluridae, Characidae, Cobitidae, Poeciliidae, Belontiidae and Percidae.
  • the adaptive immune response is a protective adaptive immune response against infection with I. multifiliis.
  • the polypeptide may in these cases induce a humeral and/or a cellular immune response.
  • SEQ ID NOs: 1-30 include antigenic determinants (epitopes) that are as such recognized by antibodies and/or T cells in fish.
  • B-cell epitopes I.e. antibody binding 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 agains I. multifHiis or I. multifiliis derived proteins disclosed herein are tested for binding to overlapping oligomeric peptides derived from any one of SEQ ID NO: 1-30. Thereby, the regions of the I. multifiliis polypeptide which are responsible for or contribute to binding to the antibodies can be identified.
  • mutated versions of the polypeptides of the invention e.g . version where each single non-alanine residue in any one of SEQ ID NOs: 1- 30 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.
  • silico methods for B-cell epitope prediction can be employed : useful state-of-the-art systems for ⁇ -turn prediction is provided in Petersen B et al.
  • 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: 31-60) or an RNA fragment (of a sequence such as SEQ ID NOs 61-90).
  • the nucleic acid may vary compared to SEQ ID NOs: 31-90 (e.g. by being a fragment since it encodes a fragment of any one of SEQ ID NOs: 1-30 disclosed above) and may have nucleotide sequence with at least 60% sequence identity with any one of SEQ ID NOs: 31-90. 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: 1-30 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: 1-30, 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, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • 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: 1-30 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.
  • 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.
  • 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.
  • a retrovirus vector such as a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a pox virus vector.
  • 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).
  • 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. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • a “promoter” is a control sequence.
  • the promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural state.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including 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 ⁇ (_ ⁇ , ⁇ - 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-Reg
  • CMV Cytomegalovirus
  • 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 Hormone
  • 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.
  • IVS Internal Ribosome Binding Sites
  • 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). 3. Multiple Cloning Sites
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS 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.
  • 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. 6. Polyadenylation Signals
  • 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.
  • the construct will instead include a poly-A tail - in this case it is not possible for a host cell to cause polyadenylation of a transcript because the RNA-based vector takes the place of a transcript.
  • 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
  • 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
  • ATCC American Type Culture Collection
  • 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.
  • 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.
  • 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: 1-30 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: 1-30, 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: 1-30 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: 1-30, 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.
  • 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.
  • Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.
  • 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.
  • 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • compositions or the use as a vaccine thereof
  • a composition comprising 2 distinct (i.e. non-identical) proteinaceaous 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.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 3, 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.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 3, 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.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous 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, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 21 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 22 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 23 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 24 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 25 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 26 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 27 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 28 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 29 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • embodiments of the invention relate to a composition (or the use as a vaccine thereof) comprising 2 distinct (i.e. non-identical) proteinaceaous immunogens disclosed herein wherein the first of said immunogens is SEQ ID NO: 30 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
  • 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 I. multifiliis.
  • the protective immunity is effective in reducing the risk of attracting infection with I.
  • 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 I. multifiliis.
  • 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 i. multifiHis 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.
  • 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.
  • 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.
  • 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.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • 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
  • the vaccine included 40 ⁇ g of each of two peptides (#5a and #5b, i.e. SEQ ID NO: 22 residues 1-230 and residues 232-925, respectively) created from the protein ranked as number 5 (#5) and 40 ⁇ g of a protein ranked as number 11 (#11, i.e. SEQ ID NO: 2) and 25 ⁇ g (due to quantitative limitations) of a protein ranked as number 10 (#10, i.e. SEQ ID NO: 7).
  • the protein ranking was done biotechnologically (see the next section).
  • the fourth group was injected with homogenised parasites (tomonts).
  • the parasite homogenate vaccine included 150 ⁇ _ per fish of equal parts alhydrogel and FIA and 100 ⁇ g of homogenised tomonts bound to the alhydrogel.
  • the last group included controls, which would not receive a challenge with the parasite. Table 1.
  • Control group Adjuvant Sub-unit Parasite Uninfected control vaccine homogenate controls
  • 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 three proteins were constructed based on conventional in silico analyses predicting signal peptides, transmembrane regions, non-classical secretion proteins, and functional and structural domains. Predicted signal peptide, transmembrane and intracellular regions were removed. The fragments were constructed to enhance the probability of expressing the native protein structure, to express the protective part of the protein and establish a successful recombinant expression. Protein expression
  • the bacteria were subsequently plated out on LB agar-plates containing ampicillin (Amp) and kanamycin (Kan) in concentrations of 50 ⁇ g/mL and 25 ⁇ g/mL respectively and incubated at 37°C over night (ON). Up to 8 colonies were
  • the pellet was subsequently disrupted in 50 mL binding buffer (8 M urea, 100 mM NaH 2 (Po) 4 , 100 mM tris (base), 300 mM NaCI, 5 mM imidazole, pH 8.0) and placed at 4°C ON.
  • the disrupted pellet was rotated end-over-end for 60 min at room temperature (lysis) and subsequently centrifuged at 1500 rpm for 20 min at room temperature.
  • the supernatant was filtered with a 0.45 ⁇ filter and 8 mL (4 mL Ni-resin beads) of prewashed Ni-resin
  • binding buffer (Thermofischer Scientific, Denmark) in binding buffer was added to the supernatant and subsequently rotated end-over-end for one hour at room temperature.
  • the recombinant proteins bound to the Ni-resin with a C-terminal His-tag.
  • the resin was washed three times in binding buffer prior to elution of the protein in elution buffer (8 M urea, 100 mM NaH 2 Po 4 , 100 mM tris (base), 300 mM NaCI, 300 mM imidazole, pH 8.0).
  • the eluates were stored at 4°C. Standard SDS PAGE was carried out to evaluate the eluates.
  • the eluates were dialysed twice ON in 5 L of 6 M urea in 0.9% NaCI to get rid of the imidazole without precipitating the peptides and stored at -20°C.
  • the protein #10 which contained 44 cysteines, was expressed in a baculovirus system.
  • the fragment was cloned into the Baculovirus vector, pAcGP67-A (BD Biosciences), which was modified to contain the V5 epitope upstream of a histidine tag in the C-terminal end of the constructs.
  • Linearised Bakpak6 Baculovirus DNA (BD Biosciences) was co-transfected with pAcGP67-A into Spodoptera Sf9 cells for generation of recombinant virus particles.
  • Recombinant protein was purified on Ni ++ metal-chelate agarose columns as secreted histidine-tagged proteins from the supernatant of infected High-Five insect cells.
  • Another experimental vaccine was formulated with homogenised parasites as protein source.
  • Fish infected with mature trophonts of J. multifiHis were killed with an overdose of MS222 (Sigma-Aldrich, Denmark) in a concentration of 300 mg/L and parasites, which subsequently exited the fish were collected in Eppendorf tubes.
  • the parasites were washed three times in phosphate buffered saline (PBS) and stored at -80 °C until use.
  • the day of vaccine preparation (see below) the parasites were thawed and homogenised by sonication (285 W, Heat Systems XL 2020, New York, USA) on ice in 5 pulses of 5 seconds sonication and 5 seconds pause. Protein measurement was conducted with Pierce BCA protein assay kit (Thermo Fisher Scientific, Denmark).
  • the Alhydrogel including proteins was washed twice in 0.9% NaCI and following the last wash an equal volume of FIA was added. The mixture was vigorously vortexed at maximum speed for one hour at room temperature until consistency resembled mayonnaise.
  • the experimental sub-unit vaccine was stored at 4°C ON. The procedure for the parasite homogenate vaccine and the control adjuvant vaccine followed the above.
  • Infected fish were euthanized in 300 mg/L MS222 and left for 4 hours in a container with water from the tank to shed parasites. The dead fish were subsequently removed and the parasites were left for one-two days for development into the infective stage (theronts). The theronts were counted and challenge was conducted with approximately 10,000 theronts per fish. Water containing the appropriate amount of theronts was poured into the tanks.
  • Blood sampling of 4 fish per tank was conducted both at 4 weeks post vaccination (wpv), which equals 435 degree days (dd) and at 25 days post challenge (dpch) corresponding to 13 wpv or 1274 dd.
  • the fish were euthanized in an overdose of MS222 (300 mg/L) and all blood samples were taken from the caudal vein immediately, using heparinized syringes, and kept on ice. Plasma was separated from blood by centrifugation (4000 g for 10 min) and kept at - 80°C until use. Parasite burden
  • a pooled blood sample from 4 fish per replicate group sampled 13 wpi were diluted 1 : 10 in PBS and 100 ⁇ _ were distributed in duplicate on a 96 well non-absorbing round bottomed micro plate. Plasma from a natural immune fish was included (a survivor of several I.
  • the specific trout antibody production was determined by ELISA. Plasma from the individual fish were tested on plates coated with the injected peptides/proteins and plates coated with homogenised parasites as antigens. In brief, parasites were collected, washed in PBS as described above, sonicated and protein concentration was subsequently estimated with Pierce BCA protein assay kit. Microtiter plates were coated with 100 ⁇ _ parasite antigen in coating buffer (C3041, Sigma-Aldrich, Denmark) in a concentration of 5 ⁇ g/mL ON at 4°C. Microtiter plates were also coated with 50 ⁇ _ of each of the peptide antigens in coating buffer in a concentration of 1 ⁇ g/L. Subsequently, the free binding sites were blocked in 1% bovine serum albumin (BSA, Sigma-Aldrich, Denmark) and 0.2% Tween 20 (Sigma-Adrich,
  • Table 2 below shows the rank number of the proteins/fragments, the number of cysteines in the sequence, the length of the sequence, a description of the proteins/fragments and how they function.
  • Rank ProteinlD #5a has zinc-finger domains which are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule.
  • Rank proteinID #5b could have one di-sulphide bridge. Originally the rank proteinID #11 sequence had 4 cysteines but 2 cysteines were converted to serines in order to have one disulphide bridge.
  • the life cycle was completed at least once, before parasites were detected.
  • the control group had a mean of 2.8 parasites per g/cm fish and the adjuvant control group had a statistically significant (p ⁇ 0.05) higher mean of 4.1 compared to the sub-unit vaccine group and the parasite homogenate vaccine group with a mean of 1.3 and 0.8, respectively (see Fig. 1).
  • the replicates within the control and the adjuvant control groups were statistically different from each other with regard to the parasite count.
  • the replicates within the sub-unit vaccine group and the parasite homogenate group were not statistically different from each other. Both the sub-unit vaccine group and the parasite homogenate group were statistically different from the adjuvant control group but were not statistically different from each other.
  • Plasma from a naturally infected fish immobilised all the theronts within 30 min (Fig. 2) .
  • the plasma from the uninfected controls did not immobilise the theronts throughout the two-hour observation period.
  • the parasite homogenate vaccine group performed the best of the experimental groups with regard to immobilisation of theronts. This was characterised by a clear inhibition of theront movements with several deaths at the end of the study.
  • the sub-unit vaccine group also inhibited the theront mobility with the highest effect after 30 min, however, the theronts seemed to recover to some extent during the following hour.
  • the adjuvant group followed the same pattern however with less immobilization of the theronts.
  • the infected control group immobilized the theronts slightly after 30 min and throughout the observation period.
  • homogenate vaccine group differed statistically from all other groups in all dilutions (Fig 3b).
  • the adjuvant control group, the sub-unit vaccine group and the parasite homogenate vaccine group had a higher level of antibodies against peptide #5b than the control group 4 wpi. Significance was observed between the control group and the parasite homogenate vaccine group at dilution 10. At dilution 100, 1,000 and 10,000 significant differences were observed between control and the other groups (Fig 3c). The parasite homogenate vaccine group differed statistically from the other three groups 13 wpi in all dilutions (Fig 3d).
  • the parasite homogenate vaccine group had a significantly elevated level of antibodies against the protein #11 compared the other groups 4 wpi at dilutions 10 and 100.
  • the control group differed statistically from the two experimental vaccine groups and at dilution 10,000 the control group differed statistically from the sub-unit vaccine group (Fig 3g).
  • the two experimental vaccine groups differed statistically both from the control and the adjuvant control group 13 wpi at 10 times dilution. At 13 wpi only the parasite homogenate vaccine group differed statistically from the control groups (Fig 3h).
  • the experimental vaccine group injected with parasite homogenate had a significant higher level of I. multifiliis specific antibodies and differed statistically from the two control groups at 10 times dilution and from all three groups at 100, 1,000 and 10,000 times dilution (Fig 31).
  • the parasite homogenate vaccine group differed statistically from all three groups at all dilutions at 13 wpi.
  • the sub-unit vaccine group also differed statistically from the control group (Fig 3J).
  • a significant difference was observed 4 wpi between the control group and the other three groups when the micro plates were coated with a recombinant protein with no relevance to I. multifiliis (Fig 3K).
  • Immobilisation data showed that only the natural immune fish completely immobilised the parasites in vitro (Fig. 2).
  • the two experimental vaccine groups partially immobilised the free-swimming infective parasites, with the group injected with homogenised parasites performing best.
  • the method used in our study with homogenised parasites bound to alhydrogel injected with FIA induced a medium level of protection, which was not as effective as natural immunity.
  • the uninfected control fish was the only group that did not immobilise I. multifiliis at any level, indicating that the infected fish had started a protective response against the parasites.
  • the homogenised parasite vaccine group reacted with a high level of antibodies against the zinc-finger like peptides (#5a-b) indicating that the recombinant peptides fold at least partly correct and include parasite epitopes.
  • the peptides do not induce a high level of antibodies in this experimental vaccine and may not be immunogenic (Fig. 3 A-D).
  • the homogenised parasite vaccine group had produced antibodies against proteins #10 and #11 indicating at least partly correct folding.
  • the recombinant proteins #10 and #11 induced an antibody response in the fish and may therefore be immunogenic and potential vaccine candidates (Fig. 3 E-H).
  • the level of protection observed for this group was medium (Fig. 1 and Fig. 2) and in order to induce a high level of protection against I.

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Abstract

L'invention concerne des procédés d'induction de l'immunité contre Ichthyophthirius multifiliis (I. multifiliis) chez des poissons d'eau douce par administration d'un immunogène dérivé des protéines présentant l'une quelconque des séquences d'acides aminés présentées dans SEQ ID NO : 1 à 30. L'invention concerne également une vaccination par des acides nucléiques utilisant des acides nucléiques codant pour l'une quelconque des SEQ ID NO : 1 à 30 ou des fragments ou des variants de séquence 5 de celles-ci.
PCT/EP2017/064899 2016-06-17 2017-06-19 Vaccination ciblant ichthyophthirius multifiliis WO2017216384A1 (fr)

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WO2020051274A3 (fr) * 2018-09-05 2020-07-30 The Penn State Research Foundation Protéine capable de liaison avec les métaux et utilisation associée
CN112739711A (zh) * 2018-03-05 2021-04-30 佩普蒂诺夫公司 抗pd-l1疫苗组合物

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CN112739711A (zh) * 2018-03-05 2021-04-30 佩普蒂诺夫公司 抗pd-l1疫苗组合物
WO2020051274A3 (fr) * 2018-09-05 2020-07-30 The Penn State Research Foundation Protéine capable de liaison avec les métaux et utilisation associée

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