EP1934247A1 - Neuartiger seelaus-impfstoff - Google Patents

Neuartiger seelaus-impfstoff

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Publication number
EP1934247A1
EP1934247A1 EP06806932A EP06806932A EP1934247A1 EP 1934247 A1 EP1934247 A1 EP 1934247A1 EP 06806932 A EP06806932 A EP 06806932A EP 06806932 A EP06806932 A EP 06806932A EP 1934247 A1 EP1934247 A1 EP 1934247A1
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EP
European Patent Office
Prior art keywords
protein
acid sequence
nucleic acid
immunogenic fragment
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06806932A
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English (en)
French (fr)
Inventor
Petter Frost
Frank Nilsen
Lars Are Hamre
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Intervet International BV
Original Assignee
Intervet International BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Intervet International BV filed Critical Intervet International BV
Priority to EP06806932A priority Critical patent/EP1934247A1/de
Publication of EP1934247A1 publication Critical patent/EP1934247A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a 200 kD protein, a 180 kD protein, a 100/85 kD protein and a 79 kD protein.
  • the invention further relates to nucleic acid sequences encoding a 200 kD protein, a 180 kD protein, a 100/85 kD protein and a79 kD protein, to vaccines comprising these proteins or a nucleic acid sequences encoding the proteins, to DNA fragments, recombinant DNA molecules, live recombinant carriers and to host cells comprising such nucleic acid sequences, to vaccines comprising such DNA fragments, recombinant DNA molecules, live recombinant carriers and to host cells comprising such nucleic acid sequences, to methods for the preparation of such vaccines and to the use of such protein or nucleic acid sequences encoding such protein in vaccines and for the manufacture of a vaccine for combating sea lice infection in salmonids.
  • the salmon louse Lepeophtheirus salmonis (Kr ⁇ yer)
  • Kr ⁇ yer is host specific in that it infects salmonids only.
  • most sea lice are to a variable degree not host specific, infecting different marine fish including salmonids and cods.
  • Caligus rogercresseyi is mostly found on salmonids while Caligus curtus is mostly found on non-salmonids.
  • One of the least host specific sea lice, Caligus elongatus has been isolated from more than 80 different species of fish.
  • the salmon louse Lepeophtheirus salmonis is a marine ectoparasitic copepod feeding on skin, mucus and blood of salmonid hosts.
  • the louse has ten developmental stages of which two stages are free living in the water, one is infectious and seven stages are parasitic (reviewed in Pike, A.W. and Wadsworth, S.L., Adv. Parasitol. 44: 233-337
  • a first embodiment of the present invention relates to this 200 kD protein.
  • a second embodiment of the present invention relates to this 180 kD protein.
  • a third embodiment of the present invention relates to this 100/85 kD protein.
  • a fourth embodiment of the present invention relates to this 79 kD protein.
  • nucleic acid sequences can encode one and the same protein. This phenomenon is commonly known as wobble in the second and especially the third base of each triplet encoding an amino acid. This phenomenon can result in a heterology of about 30% for two nucleic acid sequences still encoding the same protein. Therefore, two nucleic acid sequences having an overall sequence identity as low as 70 % can still encode one and the same protein.
  • the protein according to the invention comprises those proteins and immunogenic fragments thereof that have an amino acid sequence that is at least 70% identical to the amino acid sequence of the 200 kD protein as depicted in SEQ ID NO: 2, the 180 kD protein as depicted in SEQ ID NO: 4, the 100/85kD protein as depicted in SEQ ID NO: 6 or the 79 kD protein as depicted in SEQ ID NO: 8.
  • one embodiment of the present invention relates to a 200 kD protein or an immunogenic fragment thereof, said protein or immunogenic fragment thereof having an amino acid sequence that is at least 70% identical to the amino acid sequence as depicted in SEQ ID NO: 2.
  • An immunogenic fragment is a fragment that has a length of at least 50 amino acids.
  • the concept of immunogenic fragments will be defined below.
  • the amino acid sequence of a 200 kD protein or an immunogenic fragment of that protein has at least 75%, or more preferably 80% identity with the amino acid sequence of SEQ ID NO: 2. Even more preferred is a identity level of 85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of preference.
  • a preferred form of this embodiment relates to a 200 kD protein or an immunogenic fragment of said protein according to the invention wherein said protein or immunogenic fragment thereof has a sequence identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the amino acid sequence of SEQ ID NO: 2.
  • a most preferred form of this embodiment relates to a 200 kD protein or an immunogenic fragment of said protein, according to the invention as encoded by a nucleic acid sequence described in SEQ ID NO: 1.
  • the level of protein identity can e.g. be determined with the computer program "BLAST 2 SEQUENCES” by selecting sub-program: “BLASTP”, that can be found at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html.
  • Amino acid sequences that comprise tandem arrays of the sequences according to the invention are also within the scope of the invention.
  • a second embodiment of the present invention relates to a 180 kD protein or an immunogenic fragment thereof, said protein or immunogenic fragment thereof having an amino acid sequence that is at least 70% identical to the amino acid sequence as depicted in SEQ ID NO: 4.
  • the amino acid sequence of the 180 kD protein or an immunogenic fragment of that protein has at least 75%, or more preferably 80% identity with the amino acid sequence of SEQ ID NO: 4. Even more preferred is a identity level of 85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of preference.
  • a preferred form of this embodiment relates to a 180 kD protein or an immunogenic fragment of said protein according to the invention wherein said protein or immunogenic fragment thereof has a sequence identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the amino acid sequence of SEQ ID NO: 4.
  • a most preferred form of this embodiment relates to a 180 kD protein or an immunogenic fragment of said protein, according to the invention as encoded by a nucleic acid sequence described in SEQ ID NO: 3.
  • a third embodiment of the present invention relates to a 100/85 kD protein or an immunogenic fragment thereof, said protein or immunogenic fragment thereof having an amino acid sequence that is at least 70% identical to the amino acid sequence as depicted in SEQ ID NO: 6.
  • the amino acid sequence of the 100/85 kD protein or an immunogenic fragment of that protein has at least 75%, or more preferably 80% identity with the amino acid sequence of SEQ ID NO: 6. Even more preferred is a identity level of 85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of preference.
  • a preferred form of this embodiment relates to a 100/85 kD protein or an immunogenic fragment of said protein according to the invention wherein said protein or immunogenic fragment thereof has a sequence identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the amino acid sequence of SEQ ID NO: 6.
  • a most preferred form of this embodiment relates to a 100/85 kD protein or an immunogenic fragment of said protein, according to the invention as encoded by a nucleic acid sequence described in SEQ ID NO: 5.
  • a fourth embodiment of the present invention relates to a 79 kD protein or an immunogenic fragment thereof, said protein or immunogenic fragment thereof having an amino acid sequence that is at least 70% identical to the amino acid sequence as depicted in SEQ ID NO: 8.
  • the amino acid sequence of the 79 kD protein or an immunogenic fragment of that protein has at least 75%, or more preferably 80% identity with the amino acid sequence of SEQ ID NO: 8.
  • Even more preferred is a identity level of 85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of preference.
  • a preferred form of this embodiment relates to a 79 kD protein or an immunogenic fragment of said protein according to the invention wherein said protein or immunogenic fragment thereof has a sequence identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the amino acid sequence of SEQ ID NO: 8.
  • a most preferred form of this embodiment relates to a 79 kD protein or an immunogenic fragment of said protein, according to the invention as encoded by a nucleic acid sequence described in SEQ ID NO: 7.
  • antibodies raised against e.g. the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein as isolated from L. salmonis react strongly in a Western blot with the homologous protein of e.g. Caligus curtus or Caligus rogercresseyi. This already indicates that the epitopes against which the antibodies are directed, are well- conserved within the copepod ectoparasites.
  • copepod ectoparasitic proteins from the eggs of adult egg producing sea lice that react in a Western blot with antiserum raised against a protein having the amino acid sequence as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 are also considered to fall within the scope of the invention.
  • Specific examples of such ectoparasitic copepods are of course the species Lepeophtheirus salmonis, Caligus curtus, Caligus elongatus and Caligus rogercresseyi as mentioned above.
  • nucleic acid sequences encoding the novel 200 kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD protein according to the present invention are disclosed here, it is now feasible to obtain this protein in sufficient quantities. This can e.g. be done by using expression systems to express the whole or parts of the gene encoding the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein.
  • An essential requirement for the expression of the nucleic acid sequence is an adequate promoter functionally linked to the nucleic acid sequence, so that the nucleic acid sequence is under the control of the promoter. It is obvious to those skilled in the art that the choice of a promoter extends to any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells used as host cells for protein expression.
  • Functionally linked promoters are promoters that are capable of controlling the transcription of the nucleic acid sequences to which they are linked. Constructs comprising the nucleic acid sequences encoding the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein according to the invention under the control of a functionally linked promoter will be further referred to as recombinant DNA molecules.
  • Such a promoter can be the native promoter of the protein gene or another promoter, provided that that promoter is functional in the cell used for expression. It can also be a heterologous promoter.
  • useful expression control sequences which may be used include the Trp promoter and operator (Goeddel, et al, Nucl. Acids Res., 8, 4057, 1980); the lac promoter and operator (Chang, et al., Nature, 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M., EMBO J., 1, 771-775, 1982); the bacteriophage lambda promoters and operators (Remaut, E. et al., Nucl. Acids Res., 11, 4677-4688, 1983); the ⁇ -amylase (B. subtilis) promoter and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell.
  • useful expression control sequences include, e.g., ⁇ - mating factor.
  • the polyhedrin or plO promoters of baculo viruses can be used (Smith, G.E. et al., MoI. Cell. Biol. 3, 2156-65, 1983).
  • useful expression control sequences include the (human) cytomegalovirus immediate early promoter (Seed, B. et al., Nature 329, 840- 842, 1987; Fynan, E.F. et al., PNAS 90, 11478-11482,1993; Ulmer, J.B.
  • Rous sarcoma virus LTR Rous sarcoma virus LTR (RSV, Gorman, CM. et al., PNAS 79, 6777-6781, 1982; Fynan et al., supra; Ulmer et al., supra), the MPSV LTR (Stacey et al., J. Virology 50, 725-732, 1984), SV40 immediate early promoter (Sprague J. et al., J. Virology 45, 773 ,1983), the SV-40 promoter (Berman, P. W. et al., Science, 222, 524-527, 1983), the metallothionein promoter (Brinster, R.L.
  • the regulatory sequences may also include terminator and poly-adenylation sequences. Amongst the sequences that can be used are the well known bovine growth hormone poly-adenylation sequence, the SV40 poly-adenylation sequence, the human cytomegalovirus (hCMV) terminator and poly-adenylation sequences.
  • Bacterial, yeast, fungal, insect and vertebrate cell expression systems are very frequently used systems. Such systems are well-known in the art and generally available, e.g. commercially through Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, California 94303-4607, USA. Next to these expression systems, parasite- based expression systems are attractive expression systems. Such systems are e.g. described in the French Patent Application with Publication number 2 714 074, and in US NTIS Publication No US 08/043109 (Hoffman, S. and Rogers, W.: Public. Date 1 December 1993).
  • LRCs Live Recombinant Carriers
  • the LRC comprises a DNA fragment that in turn comprises a nucleic acid sequence encoding the 200 kD protein, a 180 kD protein, a 100/85 kD protein or a 79 kD protein according to the invention or an immunogenic part thereof.
  • the nucleic acid sequence encoding the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic part of any of said proteins is brought under the control of a functionally linked promoter.
  • LRCs are micro-organisms or viruses in which additional genetic information, in this case a nucleic acid sequence encoding the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein, or an immunogenic fragment of any of said proteins as described above has been cloned.
  • LRCs Fish infected with such LRCs will produce an immunological response not only against the immunogens of the carrier, but also against the immunogenic parts of the protein(s) for which the genetic code is additionally cloned into the LRC, e.g. the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein described in the invention.
  • bacteria such as Vibrio anguillarum known in the art can attractively be used. (Singer, J.T. et al, New Developments in Marine Biotechnology, p. 303-306, Eds. Le Gal and Halvorson, Plenum Press, New York, 1998).
  • LRC viruses may be used as a way of transporting the nucleic acid sequence into a target cell.
  • Viruses suitable for this task are e.g. alphavirus- vectors. A review on alphavirus- vectors is given by Sondra Schlesinger and Thomas W. Dubensky Jr., Current opinion in Biotechnology, 10:434-439 (1999).
  • Preferred viral LRCs are viruses from the genus Novirhabdo viruses, especially the species viral hemorrhagic septicemia virus, and infectious hematopoietic necrosis virus (IHNV).
  • IHNV infectious hematopoietic necrosis virus
  • IHNV infectious hematopoietic necrosis virus
  • a preferred construct is a recombinant IHNV carrying a nucleic acid construct capable of encoding a polypeptide or protein according to the invention. Such an LRC is then administered to target fish for instance by immersion vaccination
  • the technique of in vivo homologous recombination can be used to introduce a recombinant nucleic acid sequence into the genome of a bacterium, parasite or virus of choice, capable of inducing expression of the inserted nucleic acid sequence according to the invention in the host animal.
  • a host cell may be a cell of bacterial origin, e.g. Escherichia coli, Bacillus subtilis and Lactobacillus species, in combination with bacteria-based plasmids as pBR322, or bacterial expression vectors as pGEX, or with bacteriophages.
  • the host cell may also be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific vector molecules, or higher eukaryotic cells like insect cells (Luckow et al; Bio -techno logy 6: 47-55 (1988)) in combination with vectors or recombinant baculo viruses, plant cells in combination with e.g.
  • Ti-plasmid based vectors or plant viral vectors Barton, K.A. et al; Cell 32: 1033 (1983), mammalian cells like HeIa cells, Chinese Hamster Ovary cells (CHO) or Crandell Feline Kidney-cells, also with appropriate vectors or recombinant viruses.
  • the 200 kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD protein each induce a degree of protection against sea lice species. Therefore, the 200 kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD protein each constitute a major compound of a vaccine for the protection of fish against fish sea lice species. Therefore, another embodiment relates to vaccines for combating sea lice infection, comprising at least one or, preferably, more of these proteins.
  • an immunogenic fragment is understood to be a fragment of the full-length protein that still has retained its capability to induce an immune response in a vertebrate host, i.e. comprises a B- or T-cell epitope.
  • an immunogenic fragment is a fragment that is capable of inducing antibodies that react with the full length protein, i.e.
  • the method is used world- wide and as such well-known to man skilled in the art. This (empirical) method is especially suitable for the detection of B-cell epitopes. Also, given the sequence of the gene encoding any protein, computer algorithms are able to designate specific protein fragments as the immunologically important epitopes on the basis of their sequential and/or structural agreement with epitopes that are now known. The determination of these regions is based on a combination of the hydrophilicity criteria according to Hopp and Woods (Proc. Natl. Acad. Sci. 78: 38248-3828 (1981)), and the secondary structure aspects according to Chou and Fasman (Advances in Enzymology 47: 45- 148 (1987) and US Patent 4,554,101).
  • T-cell epitopes can likewise be predicted from the sequence by computer with the aid of Berzofsky's amphiphilicity criterion (Science 235, 1059-1062 (1987) and US Patent application NTIS US 07/005,885).
  • a condensed overview is found in: Shan Lu on common principles: Tibtech 9: 238-242 (1991), Good et al on Malaria epitopes; Science 235: 1059-1062 (1987), Lu for a review; Vaccine 10: 3-7 (1992), Berzofsky for HIV-epitopes; The FASEB Journal 5:2412-2418 (1991).
  • one form of this embodiment of the invention relates to vaccines for combating sea lice infection, that comprise a 200 kD protein, a 180 kD protein, a 100/85 kD protein or a 79 kD protein according to the invention or an immunogenic fragment of any of said proteins as described above, together with a pharmaceutically acceptable carrier.
  • Still another embodiment relates to the use of a 200 kD protein, a 180 kD protein, a 100/85 kD protein or a 79 kD protein according to the invention or an immunogenic fragment of any of said proteins for the manufacturing of a vaccine for combating sea louse infections.
  • Vaccines based upon the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein, or an immunogenic fragment of any of said proteins can easily be made by admixing the protein or immunogenic fragments thereof with a pharmaceutically acceptable carrier as described below.
  • vaccines comprising a host cell as described above, and a pharmaceutically acceptable carrier.
  • a vaccine according to the invention can comprise live recombinant carriers as described above, capable of expressing the protein according to the invention or immunogenic fragments thereof.
  • Such vaccines e.g. based upon a Vibrio carrier or a viral carrier e.g. an alphavirus vector have the advantage over subunit vaccines that they better mimic the natural way of infection of sea lice.
  • their self-propagation is an advantage since only low amounts of the recombinant carrier are necessary for immunization.
  • All vaccines described above contribute to active vaccination, i.e. they trigger the host's defense system.
  • antibodies can be raised in e.g. rabbits or can be obtained from antibody-producing cell lines as described below. Such antibodies can then be administered to the fish.
  • This method of vaccination, passive vaccination is the vaccination of choice when an animal is already infected, and there is no time to allow the natural immune response to be triggered. It is also the preferred method for vaccinating animals that are prone to sudden high infection pressure.
  • the administered antibodies against the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein according to the invention or immunogenic fragments thereof can in these cases bind directly to the protein of the sea lice. This has the advantage that it decreases the load of the sea lice infection.
  • one other form of this embodiment of the invention relates to a vaccine for combating sea lice infection that comprises antibodies against the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein, or an immunogenic fragment of any of said proteins, and a pharmaceutically acceptable carrier.
  • proteins or immunogenic fragments thereof e.g. expressed as indicated above can be used to produce antibodies, which may be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are well-known in the art (e.g. Mayer and Walter, eds.
  • Monoclonal antibodies reactive against the protein according to the invention or an immunogenic fragment thereof according to the present invention, can be prepared by immunizing inbred mice by techniques also known in the art (Kohler and Milstein,
  • Still another embodiment of this invention relates to antibodies against the sea lice protein described in the invention or against an immunogenic fragment of that protein.
  • Still another embodiment relates to a method for the preparation of a vaccine according to the invention that comprises the admixing of antibodies according to the invention and a pharmaceutically acceptable carrier.
  • DNA fragments that are suitable for use in a DNA vaccine according to the invention are conventional cloning or expression plasmids for bacterial, eukaryotic and yeast host cells, many of said plasmids being commercially available.
  • Well- known examples of such plasmids are pBR322 and pcDNA3 (Invitrogen).
  • the DNA fragments or recombinant DNA molecules should be able to induce protein expression of the nucleic acid sequences.
  • the DNA fragments or recombinant DNA molecules may comprise one or more protein-encoding nucleic acid sequences.
  • DNA fragments or recombinant DNA molecules may comprise other nucleic acid sequences such as the immune-stimulating oligonucleotides having unmethylated CpG di-nucleotides, or nucleic acid sequences that code for other antigenic proteins or adjuvating cytokines.
  • nucleic acid sequences encoding an 200 kD protein or an immunogenic fragment of that protein comprising a nucleic acid sequence that has a identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the the nucleic acid sequence depicted in SEQ ID NO: 1.
  • the percentage of identity between any nucleic acid and a nucleic acid according to the invention can be determined with the computer program "BLAST 2 SEQUENCES” by selecting sub-program: “BlastN” (T. Tatusova & T. Madden, 1999, FEMS Microbiol. Letters, vol. 174, p. 247-250), that can be found at the internet address www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html. Parameters that are to be used are the default parameters: reward for a match: +1; penalty for a mismatch: -2; open gap penalty: 5; extension gap penalty: 2; and gap x dropoff: 50.
  • the BlastN program does not list similarities, only identities: the percentage of nucleotides that are identical is indicated as "Identities”.
  • Tm [81.5°C + 16.6(log M) + 0.41(%GC) - 0.61(%formamide) - 500/L] - 1°C/1% mismatch
  • M is the molarity of monovalent cations
  • %GC is the percentage of guanosine and cytosine nucleotides in the DNA
  • L is the length of the hybrid in base pairs
  • mismatch is the lack of an identical match.
  • Washing conditions subsequent to the hybridization can also be made more or less stringent, thereby selecting for higher or lower percentages of identity respectively.
  • stringent washing conditions are conditions of 1 x SSC, 0.1% SDS at a temperature of 65°C; highly stringent conditions refer to a reduction in SSC concentration towards 0.3 x SSC.
  • nucleic acid sequences encoding an 180 kD protein or an immunogenic fragment of that protein comprising a nucleic acid sequence that has a identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the the nucleic acid sequence depicted in SEQ ID NO: 3.
  • nucleic acid sequences encoding an 100/85 kD protein or an immunogenic fragment of that protein comprising a nucleic acid sequence that has a identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the the nucleic acid sequence depicted in SEQ ID NO: 5.
  • nucleic acid sequences encoding an 79 kD protein or an immunogenic fragment of that protein comprising a nucleic acid sequence that has a identity of at least 70%, preferably 75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity with the the nucleic acid sequence depicted in SEQ ID NO: 7.
  • stringent conditions are those conditions under which a nucleic acid still hybridises if it has a mismatch of 30 %; i.e. if it is 70 % identical to the (relevant part of the) nucleotide sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.
  • nucleic acid hybridises under stringent conditions to the nucleic acid having a nucleotide sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, it is considered a nucleic acid according to the invention.
  • the invention relates to DNA fragments comprising a nucleic acid sequence encoding the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic part of any of said proteins.
  • a DNA fragment is a stretch of nucleotides that functions as a carrier for a nucleic acid sequence according to the invention.
  • Such DNA fragments can e.g. be plasmids, into which a nucleic acid sequence according to the invention is cloned.
  • Such DNA fragments are e.g. useful for enhancing the amount of DNA for use as a primer and for expression of a nucleic acid sequence according to the invention, as described below.
  • the nucleic acid sequence according to the present invention or the DNA plasmid comprising a nucleic acid sequence according to the present invention, preferably operably linked to a transcriptional regulatory sequence, to be used in the vaccine according to the invention can be naked or can be packaged in a delivery system.
  • Suitable delivery systems are lipid vesicles, iscoms, dendromers, niosomes, polysaccharide matrices and the like, (see further below) all well-known in the art.
  • Also very suitable as delivery system are attenuated live bacteria such as Vibrio species, and attenuated live viruses such as alphavirus vectors, as mentioned above.
  • a more preferred form of this embodiment of the present invention relates to a recombinant DNA molecule comprising a nucleic acid sequence encoding a 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic part of any of said proteins wherein the nucleic acid sequence is placed under the control of a functionally linked promoter.
  • recombinants can be obtained by means of e.g. standard molecular biology techniques. (Maniatis/Sambrook (Sambrook, J. Molecular cloning: a laboratory manual, 1989. ISBN 0-87969-309-6).
  • Still other forms of this embodiment relate to recombinant DNA molecules, live recombinant carriers and host cells comprising a nucleic acids sequence as described above for use in a vaccine.
  • Another embodiment of the present invention relates to vaccines comprising recombinant DNA molecules, live recombinant carriers and host cells comprising a nucleic acids sequence as described above.
  • Another embodiment of the present invention relates to the use of a nucleic acid sequence, a DNA fragment, a recombinant DNA molecule, a Live Recombinant Carrier or a host cell as described above, for the manufacturing of a vaccine for combating sea louse infections.
  • DNA vaccines can easily be administered through intradermal application e.g. using a needle-less injector. This way of administration delivers the DNA directly into the cells of the animal to be vaccinated. Amounts of DNA in the range between 10 pg and 1000 ⁇ g provide good results. Preferably, amounts in the microgram range between 1 and 100 ⁇ g are used. Alternatively, animals can be dipped in solutions comprising e.g. between 10 pg and 1000 ⁇ g per ml of the DNA to be administered.
  • the vaccine according to the present invention additionally comprises one or more antigens derived from fish pathogenic organisms such as sea lice, micro-organisms and viruses, antibodies against those antigens or genetic information encoding such antigens.
  • Such antigens can be e.g. other sea lice antigens.
  • Such an antigen can also be an antigen selected from other fish pathogenic organisms, micro-organisms and viruses.
  • Such organisms and viruses are preferably selected from the group of aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV), aquatic nodaviruses such as striped jack nervous necrosis virus (SJNNV), aquatic rhabdo viruses such as infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV), Pancreas Disease virus (SPDV) and aquatic orthomyxoviruses such as infectious salmon anaemia virus and the group of fish pathogenic bacteria such as
  • IPNV infectious pancreatic necrosis virus
  • SJNNV striped jack nervous necrosis virus
  • VHSV viral haemorrhagic septicaemia virus
  • SPDV Pancreas Disease virus
  • All vaccines according to the present invention comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can be e.g. sterile water or a sterile physiological salt solution.
  • the carrier can e.g. be a buffer.
  • Methods for the preparation of a vaccine comprise the admixing of the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic part of any of said proteins and/or antibodies against that protein or an immunogenic fragment thereof, and/or a nucleic acid sequence and/or a DNA fragment, a recombinant DNA molecule, a live recombinant carrier or host cell according to the invention, and a pharmaceutically acceptable carrier.
  • Vaccines according to the present invention may in a preferred presentation also contain an immunostimulatory substance, a so-called adjuvant.
  • Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner.
  • a number of different adjuvants are known in the art. Examples of adjuvants frequently used in fish and shellfish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans and CarbopolW (a homopolymer).
  • An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1996)).
  • the vaccine may also comprise a so-called "vehicle”.
  • a vehicle is a compound to which the protein adheres, without being covalently bound to it.
  • Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the art.
  • a special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM (EP 109.942, EP 180.564, EP 242.380)
  • the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.
  • the vaccine is mixed with stabilisers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze- drying efficiency.
  • Useful stabilisers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.
  • the vaccines according to the invention are in a freeze-dried form.
  • Freeze-dried proteins and DNA have a much longer shelf-live, especially at room temperature, than when they are in a liquid form.
  • the process of freeze-drying as such is extensively known in the art.
  • the vaccine may be suspended in a physiologically acceptable diluent. It goes without saying, that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilizing a protein are also embodied in the present invention.
  • Vaccines according to the invention that are based upon the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic part of any of said proteins can very suitably be administered in amounts ranging between 1 and 100 micrograms of protein per animal, although smaller doses can in principle be used. A dose exceeding 100 micrograms will, although immunologically very suitable, be less attractive for commercial reasons.
  • Vaccines based upon live attenuated recombinant carriers, such as the LRC-viruses and bacteria described above can be administered in much lower doses, because they multiply themselves during the infection. Therefore, very suitable amounts would range between 10 3 and 10 9 CFU/PFU for bacteria and viruses.
  • the protein-based vaccines according to the invention are preferably administered to the fish via injection, immersion, dipping or per oral.
  • the administration protocol can be optimized in accordance with standard vaccination practice.
  • the vaccine is administered via immersion or per oral, especially in case of commercial aqua culture farms.
  • the vaccine is preferably mixed with a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin.
  • a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin.
  • an attractive way of administration is administration of the vaccine to high concentrations of live- feed organisms, followed by feeding the live-feed organisms to the target animal, e.g. the fish.
  • Particularly preferred food carriers for oral delivery of the vaccine according to the invention are live-feed organisms which are able to encapsulate the vaccine.
  • Suitable live-feed organisms include plankton-like non-selective filter feeders preferably members of Rotifera, Artemia, and the like.
  • Highly preferred is the brine shrimp Artemia sp..
  • a very elegant way of administration would be the following: bacteria, yeast cells or any other cell in which the protein according to the invention has been synthesised are directly fed to plankton-like non-selective filter feeders preferably members of Rotifera, Artemia, and the like.
  • plankton-like non-selective filter feeders can then be administered orally to the crustaceans to be protected against viral infection.
  • plankton-like non-selective filter feeders can then be administered orally to the crustaceans to be protected against viral infection.
  • Salmon lice eggs were hatched in incubators with flowing seawater (34,5%o , 2OuM filtered). After development to the infectious copepodid stage, lice were added to tanks containing Atlantic salmon (S. salar) and left natural development.
  • Atlantic salmon (S. salar) Atlantic salmon
  • Ls L. salmonis
  • oocytes harvested by puncturing the gonade section.
  • Water-soluble proteins were extracted by resuspending oocytes from 50 lice in 2.5 ml of cold sonication buffer (50 mM Tris-HCl pH 7.5, 5OmM NaCl and ImM EDTA) and eggs were disrupted by sonication using a micro ultrasonic cell disrupter.
  • the sonicated extract was clarified by centrifugation (13000g for 20 min at 4 0 C), pellet and lipids discarded and the supernatant stored at -2O 0 C.
  • the supernatant total protein content was analyzed by SDS-PAGE and Coomassie staining and quantified relative to known quantities of BSA.
  • the Ls protein extract were diluted to app. 5mg/ml (BSA equivalents) with dd ⁇ tO and added 0,1% (vol/vol) of 37% formaldehyde.
  • a 5mg/ml BSA control antigen was prepared simultaneously.
  • Vaccines were prepared by emulsification of 27% (w/w) of antigen in 67% ISA763 oil (Seppic).
  • Atlantic salmon pre-smolt (app 4Og) were vaccinated by individually intraperitonal injection of 150ul vaccine (approximately 200ug protein).
  • the experiment was terminated 11 weeks post challenge, three weeks after the first egg string was observed on adult female lice.
  • Vaccine effect was evaluated by comparing prevalence (percentage of fish infected) and the abundance (number of lice/fish).
  • fish-pathology (external wounds) was compared between tank 1 (control) and tank 2 (vaccine) only. All fish were anaesthetized before handling and sampling of animals were conducted in accordance with national legislation.
  • the protein content of the vaccine antigen was analyzed by SDS-PAGE, using 10- 20% Linear Gradient Ready Gel (BioRad) and commercially prepared running buffer and Coomassie stain. Protein band A was excised from the gel and internal amino acid sequence analysis was performed by EuroSequence bv, essentially as described by Rosenfeld et al. (Annal. Biochem. 203:173-179 (1992)). This includes in-situ tryptic digestion of the protein band, extraction of the peptides and RP-HPLC separation of the generated fragments. On purified fragments, identification of the step-wise released PTH-amino acid (Hewick et al, J. Biol. Chem.
  • the 200 kD protein, the 180 kD protein, the 100 and 85 kD proteins and the 79 kD protein were purified by preparative SDS-PAGE using a Prep Cell model 491
  • Vaccine antigen from L. salmonis was prepared as described above (5mg/ml) mixed 1:1 with Freud's complete adjuvant and vortexed until homogeneity.
  • 5 Atlantic salmon (app 25Og) were immunised by intraperitoneal injection of 150ul (app. 375ug protein). Blood were collected 9 weeks post immunisation and left at 4 0 C overnight for coagulation. Antisera were aliquoted and stored at -2O 0 C until used.
  • Nitrocellulose was incubated with rabbit antiserum and pre-serum control (1:1000) for 2 hours at room temperature followed by 3 washes with TBS-Tween. Secondary, nitrocellulose was incubated with horseradish peroxidase conjugated goat anti rabbit antibody (1:2000, BioRad) at room temperature for 1 hour. Following 3 washes with TBS-Tween and 1 with TBS, colorimetric detection was performed using premixed HRP-4CN substrates according to the manufacturer (BioRad).
  • ELISA was performed by coating Nunc immunosorbent plate wells with lOOul of the 200 kD protein, the 180 kD protein, the 100 kD protein, the 85 kD protein or the 79 kD protein purified from eggs (using the egg 35 kD protein as control) diluted to app 2ug/ml in coating buffer (15mM Na 2 CO 3 , 35mM NaHCO 3 , pH 9.6), and incubate at 4 0 C over night. Wells were then washed twice with PBS-Tween, blocked with 5% non-fat dry milk (Nestle) in PBS-Tween (200ul/well) for 1 hour at room temperature and washed again.
  • coating buffer 15mM Na 2 CO 3 , 35mM NaHCO 3 , pH 9.6
  • Antisera from 5 Atlantic salmons immunised with the vaccine antigens were analysed by ELISA using either purified egg 200 kD protein, 180 kD protein, 100 kD protein, 85 kD protein or 79 kD protein as antigen. Briefly, this was performed as described for rabbit antisera analysis but diluting the antisera from 1 :25 and incubating at 4 0 C over night. Furthermore, an incubation step with rabbit anti-salmon-Ig O206 (1:6000, 1 hour at room temperature) was added between the salmon antisera and the horseradish peroxidase conjugated goat-anti-rabbit antibody.
  • a skew sex ratio of lice has never been observed in the lab before independent of density of fish.
  • a skew sex ratio was also observed in tank 2 were abundance and prevalence demonstrate vaccine effect. This indicates that lice in tank 3 have jumped between hosts and therefore all lice in tank 3 may have fed partly on vaccinated fish.
  • SEQ ID NO: 7 encodes a protein of unknown function with molecular weight of 79 KDa. This correlates well with Northern blot transcript analysis (data not shown), indicating that egg-band E (79 kDa) has been processed.
  • the ORF encoded protein has a signal peptide and 3 fasciclin (FASl) domains, an extracellular domain suggested to represent an ancient cell adhesion domain common to plants and animals.
  • Each of the purified egg protein A, B, C, D and E (Fig 4-7) used for the immunization of rabbits induced high levels of specific antibodies, as can be seen from figure 8-11. These antibodies bind only and specifically to either the 200 kD protein, the 180 kD protein, the 100 kD protein, the 85 kD protein or the 79 kD protein on a Western blot of gels comprising all proteins as antigen (Fig 12-15).
  • the vaccine antigens (all egg proteins A-E) induced, when used together for the immunization of Atlantic salmon, production of antibodies against the purified 200 kD protein, the 180 kD protein, the 100 kD protein, the 85 kD protein or the 79 kD protein egg-band, as can be clearly seen from figure 16-19.
  • Figure 1 Prevalence (A) and abundance (B) of adult salmon lice on vaccinated Atlantic salmon smolt 11 weeks post challenge (26 weeks post vaccination).
  • FIG. 1 SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis of purified egg-band A (lane 2) and crude vaccine antigen preparation (lane 3). Molecular weight standard is to the left (lane 1).
  • FIG. 7 SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis of purified egg-band E (lane 1) and crude vaccine antigen preparation (lane 3). Molecular weight standard is in lane 2.
  • FIG. 8 Level of antibodies against egg-band A protein, in antisera from rabbits immunised with egg-band A or egg-band E (control serum), analysed by ELISA. ELISA plates were coated with purified egg-band A protein.
  • Figure 9 Level of antibodies against egg-band B protein, in antisera from rabbits immunised with eggband B or egg-band E (controlserum), analysed by ELISA. ELISA plates were coated with purified egg-band B protein .
  • FIG. 10 Level of antibodies aginst egg proteins C and D (figure A and B, respectively), in antisera from rabbits immunised with egg band C, D or egg band E (controlserum). Sera were analysed by ELISA with plates coated with purified eggband C or D (figure A and B, respectively).
  • FIG. 1 Level of antibodies against egg-band E protein, in antisera from rabbits immunised with egg-band E or egg-band D (control serum), analysed by ELISA. ELISA plates were coated with purified egg-band E protein.
  • FIG. 12 Antigen-specificity of rabbit anti egg-band A antiserum (A2) analyzed by Western blotting using all vaccine proteins as antigen. A3 was incubated with pre- serum. The vaccine antigen used in the Western blotting (egg-band A-E) is shown in lane B2, a total protein stain of the SDS-PAGE. Identical molecular weight standards are shown in lane Al and Bl .
  • FIG 13 Antigen-specificity of rabbit anti egg-band B antiserum (A2) analyzed by Western blotting using all vaccine proteins as antigen. A3 was incubated with pre- serum. The vaccine antigen used in the Western blotting (egg-band A-E) is shown in lane B2, a total protein stain of the SDS-PAGE. Identical molecular weight standards are shown in lane Al and Bl .
  • FIG. 14 Antigen-specificity of rabbit anti egg band C and anti egg band D antiserum (A2 and A3 respectively) analyzed by Western blotting using all vaccine proteins as antigen. A4 was incubated with pre-serum. The vaccine antigen used in the Western blotting (egg band A-E) is shown in lane B2, a total protein stain of the SDS- PAGE. Identical molecular weight standards are shown in lane Al and Bl.
  • FIG. 15 Antigen-specificity of rabbit anti egg-band E antiserum (A2) analyzed by Western blotting using all vaccine proteins as antigen. A3 was incubated with pre- serum. The vaccine antigen used in the Western blotting (egg-band A-E) is shown in lane B2, a total protein stain of the SDS-PAGE. Identical molecular weight standards are shown in lane Al and Bl .
  • FIG. 1 Level of antibodies against egg-band A protein, in antisera from 5 Atlantic salmon immunised with the vaccine antigens. ELISA plates were coated with purified egg-band A protein. Control sera are from un-immunised salmon.
  • FIG. 1 Level of antibodies against egg-band B protein, in antisera from 5 Atlantic salmon immunised with the vaccine antigens. ELISA plates were coated with purified egg-band B protein. Control sera are from un-immunised salmon.
  • FIG. 1 Level of antibodies against egg band C protein (a) and egg band D protein (b), in antisera from 5 Atlantic salmon immunised with the vaccine antigens. ELISA plates were coated with purified egg band C protein (a) and purified egg band D protein (b). Control sera are from un-immunised salmon.
  • FIG. 19 Level of antibodies against egg-band E protein, in antisera from 5 Atlantic salmon immunised with the vaccine antigens. ELISA plates were coated with purified egg-band E protein. Control sera are from un-immunised salmon.
  • FIG. 20 Analysis of egg protein A in Caligus curtus (C. c) and Caligus rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).
  • C. c Caligus curtus
  • C. r Caligus rogercresseyi
  • L. s Lepeophtheirus salmonis
  • FIG. 21 Analysis of egg proteins (A-E) in Caligus curtus (C. c) and Caligus rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).
  • FIG 22 Analysis of egg proteins C and D in Caligus curtus (C. c) and Caligus rogercresseyi (C. r), compared to Lepeophtheirus salmonis (L. s).
  • FIG. 23 Analysis of egg protein E in Caligus curtus (C. c) and Caligus rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).
  • C. c Caligus curtus
  • C. r Caligus rogercresseyi
  • L. s Lepeophtheirus salmonis

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