Novel sea lice vaccine.

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.

Sea lice form a group of ectoparasites that have marine fish as host. They are in some cases host specific. Merely as an example; the salmon louse (Lepeophtheirus salmonis (Krøyer)) is host specific in that it infects salmonids only. However, 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

(1999)).

Although under natural conditions both species are usually found in low numbers, L. salmonis can cause significant harm to both the wild Atlantic salmon (Salmo salar) and the sea trout {Salmo trutta). Salmo salar is grown more and more frequently in aquaculture, leading to a high local density of hosts for sea lice. As a consequence, infection with sea lice of salmon in aqua- farming is clearly increasing.

For Caligus infections in cod, the same will happen in the foreseeable future, due to the increasing importance of cod farming.

Newly started cod production amounted to 605 tons only. However, given the fact that only in the UK, cod consumption is 170.000 tons yearly, and given the sharp reduction in European catch limits, it is clear that cod farming commercially becomes more and more attractive.

Louse infection as such is seldom the direct cause of illness and death, since sea lice feed primarily on mucus and skin. This however causes damage to both mucus and skin, making the host more vulnerable to opportunistic infections. These opportunistic infections thus are the direct cause of increasingly significant economic losses. Classical treatment with organophosphate pesticides such as Nevugon and Aquagard is no longer an option. This is due to the fact that lice are developing an increasing resistance to such pesticides, and to the fact that such pesticides are no longer acceptable from an environmental point of view.

Therefore, vaccines against sea lice are highly desirable. Up till now, however, no such vaccines are on the market.

The search for suitable vaccine candidates has been going on for many years already. This approach has however so far not led to vaccines for combating sea lice infection.

It is an objective of the present invention to provide a novel vaccine that is capable of inducing in susceptible fish such as i.a. Salmo salar, Salmo trutta and cod, a degree of protection against sea lice infection and to the effects of the infection.

Surprisingly it was found now, that several proteins could be isolated from the eggs of adult egg producing sea lice, more specifically Lepeophtheirus salmonis, Caligus rogercresseyi, Caligus elongatus and Caligus curtus, that are capable of inducing antibodies in Salmo salar, Salmo trutta and cod to the extent that fish vaccinated with these proteins are to an efficient degree protected against sea lice infection and the effects thereof.

Even more surprisingly, a strong cross-reactivity between antibodies against the 200 kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD protein respectively of one species and the homologous 200 kD protein, the 180 kD protein,

the 100/85 kD protein and the 79 kD protein in other species appered to exist. Antibodies raised against this 200 kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD protein from e.g. Lepeophtheirus salmonis additionally appeared to react strongly in a Western blot with homologous proteins from Caligus curtus and Caligus rogercresseyi.

As will be further clarified below, 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.

It is well-known in the art, that many different 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.

It will also be understood that, for the particular proteins embraced herein, natural variations can exist between individual sea lice strains. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al. in "The Proteins" Academic Press New York (1979). Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D. C, 1978, vol. 5, suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science,227, 1435-1441, 1985) and determining the functional similarity between identical proteins. Such amino acid substitutions of the exemplary

embodiments of this invention, as well as variations having deletions and/or insertions are within the scope of the invention as long as the resulting proteins retain their immune reactivity. This explains why sea lice proteins according to the invention, when isolated from different field isolates, may have identity levels of about 70%, while still representing the same protein with the same immunological characteristics. Those variations in the amino acid sequence of a certain protein according to the invention that still provide a protein capable of inducing an immune response against infection with sea lice or at least against the clinical manifestations of the infection are considered as "not essentially influencing the immunological characteristics".

Therefore, 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.

Thus, 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.

Preferably, 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.

Therefore, 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.

A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174: 247-250 (1999). Matrix used: "blosum62". Parameters used are the default parameters:

Open gap: 11. Extension gap: 1. Gap x dropoff: 50.

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.

Preferably, 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.

Therefore, 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.

Preferably, 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.

Therefore, 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.

Preferably, 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.

Therefore, 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.

As an readily be seen from the Examples, 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. Therefore, 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.

Since the 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. When the host cells are bacteria, 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 α-amylase (B. subtilis) promoter and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell.

When the host cell is yeast, useful expression control sequences include, e.g., α- mating factor. For insect cells the polyhedrin or plO promoters of baculo viruses can be used (Smith, G.E. et al., MoI. Cell. Biol. 3, 2156-65, 1983). When the host cell is of vertebrate origin illustrative 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. et al., Science 259, 1745-1748, 1993), 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. et al., Nature 296, 39-42, 1982), the heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82, 4949-53, 1985), the major late promoter of Ad2 and the β-actin promoter (Tang et al., Nature 356, 152-154, 1992). 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).

In view of this, another embodiment of the invention relates to Live Recombinant Carriers (LRCs) 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. Preferably, 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. More preferably, 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. These 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. 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. As an example of bacterial LRCs, 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).

Also, 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). For instance IHNV is a fish pathogen, for which an attenuated viral expression and delivery system for use in salmonids has been described. (WO 03/097090; Biacchesi et al., 2000, J. of Virol, vol. 74, p. 11247-11253). Deletion of the viral NV protein attenuates the virus and creates room for insertion of a foreign gene. 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, well-known in the art, 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.

Finally another form of this embodiment of the invention relates to a host cell comprising a recombinant DNA molecule or a live recombinant carrier as described above.

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.

As is mentioned above, it is one of the merits of the present invention that it was found that 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.

When a protein is used for vaccination purposes or for raising antibodies, it is not necessary to use the whole protein. It is also possible to use a fragment of that protein that is capable, as such or coupled to a carrier such as e.g. KLH, of inducing an immune response against that protein, a so-called immunogenic fragment. 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. Shortly, an immunogenic fragment is a fragment that is capable of inducing antibodies that react with the full length protein, i.e. the 200 kD protein, the 180 kD protein, the 100/85 kD protein or the 79 D protein according to the invention. At this moment, a variety of techniques is available to easily identify DNA fragments encoding immunogenic fragments (determinants). The method described by Geysen et al (Patent Application WO 84/03564, Patent Application WO 86/06487, US Patent NR. 4,833,092, Proc. Natl Acad. Sci. 81: 3998-4002 (1984), J. Imm. Meth. 102, 259-274 (1987), the so-called PEPSCAN method is an easy to perform, quick and well-established method for the detection of epitopes; the immunologically important regions of the protein. 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).

Thus, 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.

Another possibility for such vaccines is a vaccine comprising a host cell as described above, and a pharmaceutically acceptable carrier.

Alternatively, 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. Moreover, 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. Alternatively, 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.

Therefore, 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.

The 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.

Immunochemical Methods in Cell and Molecular Biology, Academic Press, London,

1987).

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,

Nature, 256, 495-497, 1975).

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.

Methods for large-scale production of antibodies according to the invention are also known in the art. Such methods rely on the cloning of (fragments of) the genetic information encoding the protein according to the invention in a filamentous phage for phage display. Such techniques are described i.a. at the "Antibody Engineering Page" under "filamentous phage display" at http://aximtl .imt.uni- injrbjm^d^/^rck/aeBpJTageJilniL, and in review papers by Cortese, R. et al, (1994) in Trends Biotechn. 12: 262-267., by Clackson, T. & Wells, J.A. (1994) in Trends Biotechn. 12: 173-183, by Marks, J.D. et al., (1992) in J. Biol. Chem. 267: 16007- 16010, by Winter, G. et al., (1994) in Annu. Rev. Immunol. 12: 433-455, and by Little, M. et al., (1994) Biotechn. Adv. 12: 539-555. The phages are subsequently used to screen camelid expression libraries expressing camelid heavy chain antibodies. (Muyldermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12: 131-140 (1999) and Ghahroudi, M.A. et al., FEBS Letters 414: 512-526 (1997)). Cells from the library that express the desired antibodies can be replicated and subsequently be used for large scale expression of antibodies.

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.

An alternative and efficient way of vaccination is direct vaccination with DNA encoding the relevant antigen. Direct vaccination with DNA encoding proteins has been successful for many different proteins. (As reviewed in e.g. Donnelly et al., The Immunologist 2: 20-26 (1993)). This way of vaccination is also attractive for the vaccination of fish against sea lice infection. Therefore, still other forms of the vaccine embodiment of the invention relate to vaccines comprising nucleic acid sequences encoding a protein according to the invention or immunogenic fragments thereof, to vaccines comprising DNA fragments that comprise such nucleic acid sequences and to recombinant DNA molecules comprising such nucleic acid sequences.

Examples of 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. In addition, the 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.

Thus, both in view of their use in expression systems and their use in DNA vaccination, another embodiment of the invention relates to 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.

Unlike the output of the BlastP program described above, the BlastN program does not list similarities, only identities: the percentage of nucleotides that are identical is indicated as "Identities".

Next to using computer algorithms for determining the level of identity/mismatch between any nucleic acid and a nucleic acid according to the invention, experimental

techniques can also be used. Especially by hybridisation under conditions of controlled stringency.

The definition of stringent hybridisation conditions, as a function of the identity between two nucleotide sequences, follows from the formula for the melting temperature Tm of Meinkoth and Wahl (1984, Anal. Biochem, vol. 138, p. 267-284):

Tm = [81.5°C + 16.6(log M) + 0.41(%GC) - 0.61(%formamide) - 500/L] - 1°C/1% mismatch

In this formula: 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; and "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.

In general, higher stringency is obtained by reducing the salt concentration, and increasing the incubation temperature. It is well within the capacity of the skilled person to select hybridisation conditions that match a certain percentage-level of identity as determined by computer analysis.

Merely as an example, and of course depending upon the G/C content and the length of the fragment: "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.

Another embodiment of the invention relates to 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.

Another embodiment of the invention relates to 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.

Another embodiment of the invention relates to 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.

For the purpose of the invention, 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.

Therefore, if a 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.

In a preferred form of these embodiments, 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. Such 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.

Again 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 μg provide good results. Preferably, amounts in the microgram range between 1

and 100 μg are used. Alternatively, animals can be dipped in solutions comprising e.g. between 10 pg and 1000 μg per ml of the DNA to be administered.

In a further embodiment, 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.

Of course, 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

Flexibacter columnaris, Edwardsialla ictaluri, E. tarda, Yersinia ruckeri, Pasteurella piscicida, Vibrio anguillarum, Aeromonas salmonicida and Renibacterium salmoninarum

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. In a more complex form 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) In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.

Often, 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. Preferably, 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. In addition, 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.

Many ways of administration, all known in the art can be applied. 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. Preferably the vaccine is administered via immersion or per oral, especially in case of commercial aqua culture farms. For oral administration 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. Also 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. The pharmaceutical composition so made, and comprising i.e. those bacteria , yeast cells or any other cell ingested by plankton-like non-selective filter feeders can then be administered orally to the crustaceans to be protected against viral infection.

The following examples are illustrative for the invention and should not be interpreted as limitations of the invention.

EXAMPLES

Example 1:

1. Vaccination and challenge

Vaccine preparation

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. Adult egg producing L. salmonis (Ls) females were collected with forceps from anaesthetized fish and 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 ddϊtO 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).

Vaccination and challenge experiment

50 Atlantic salmon pre-smolt (app 4Og) were vaccinated by individually intraperitonal injection of 150ul vaccine (approximately 200ug protein). A control group (n=50) was vaccinated with the control vaccine containing BSA (200ug/dose). All fish were kept

at 1O ⁰ C in one circular 1200 L tank. Clipping of the adipose fin differentiated the two groups. Fish were kept on natural light and were adapted to salt water during week 9- 11 post vaccination. The groups were challenged (15 weeks post vaccination) as one population, by adding approximately 2,600 infective copepodids to the tank, following reduction in water volume and flow. The fish were maintained in the thank for two weeks for parasite development into the immobile physically attached Chalimus stage, and then transferred to three different 250 L tanks; Tank 1 and 2 contained control and vaccinated fish, respectively (n=25) and tank 3 contained a mixed group of vaccinated and control vaccinated fish (n=25+25). 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). Furthermore, 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.

2. Identification of genes encoding proteins in the vaccine

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. 256: 7990-7997 (1981)) fragments was determined using an Applied Biosystems Model 494 Precise Sequencing system, on-line connected to an RP-HPLC unit. The internal peptide sequences were back-translated and used to search an internal database of salmon louse ESTs for gene transcripts encoding the peptide sequences. Following this, overlapping ESTs clones were identified and, supplemented by RACE clones, full-length cDNA sequences were assembled using Vector NTI 9.1.0 (Invitrogen). Blast searches were performed to identify protein domains and to indicate gene function.

3. Evaluation of protein antigenicity

Immunization of rabbits with purified protein

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

(BioRad) according to the manufacturer instructions. Purified protein was analysed by SDS-PAGE and Coomassie staining, and quantified relative to BSA as described above. One rabbit was immunized with the purified 200 kD protein, one with the purified 180 kD protein, one with the purified 100 D protein, one with the purified 85 kD protein and one with the purified 79 kD protein by Eurogentec at their facility using their immunisation protocol (boost at day 14, 28 and 56).

Immunisation of Atlantic salmon with all vaccine antigens

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.

Rabbit antisera analysis

Rabbit anti-200 kD protein antiserum, anti-180 kD protein antiserum, anti-100 kD protein antiserum, anti-85 kD protein antiserum and anti-79 kD protein antiserum were analysed by Western blotting (using a blot comprising all egg proteins) and by ELISA using single purified proteins as antigen. Briefly, Western blotting was performed by SDS-PAGE as described above followed by blotting onto nitrocellulose (150V for 45 min using 25mM Tris, 192mM glycin, 20% methanol and cooling) and blocked for 1 hour at room temperature using 3% non-fat dry milk (Nestle) in TBS-Tween. 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).

Briefly, 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. Twofold dilutions of rabbit antiserum (from 1:2000) were incubated (lOOul/well) for 2 hours at room temperature followed by 3 washes with PBS-Tween. Secondary, horseradish peroxidase conjugated goat-anti- rabbit antibody (1:3000, BioRad) were added (100ul/well) and incubated at room temperature for 1 hour. Following 4 washes with PBS-Tween and 1 with PBS, colour was developed for 20 minutes using 150ul o-phenylenediamine dihydrocloride (0,4 mg/ml in phosphate-citrate buffer pH 5 added fresh H ₂ O ₂ to 0,012%) to each well. Reaction was stopped by adding 50ul 2,5N H ₂ SO ₄ before absorbance was measured at 492nm.

Salmon antisera analysis

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.

4. Identification of homolog antigen in other copepod ectoparasites. Comparison of homolog antigen in other copepod ectoparasites The protein content of external egg strings of L. salmonis (lab strain,), Caligus curtus (4th generation lab strain kept on cod) and Caligus rogercresseyi (collected from salmon on-growing farm in Chile) was compared. Water-soluble egg proteins were extracted from the egg strings (15ul buffer/cm egg string) using sonication as described for vaccine preparation. The supernatant total protein content was analyzed as described above using SDS-PAGE followed by Coomassie staining and by Western

blotting as described above, using the rabbit anti Z. salmonis egg 200 kD protein, 180 kD protein, 100 kD protein, 85 kD protein or 79 kD protein antiserum.

Results 1. Vaccine experiment

No differences in fish weight, length and condition factor were observed between the experimental groups (tanks 1-3), demonstrating equal conditions between the tanks.

Both prevalence and abundance of adult female lice was lower in tank 2 (vaccine) compared to the tank 1 (control). Prevalence and abundance of male lice did not differ significantly between the two tanks, indicating an increased mortality of female lice in the vaccinated group (Fig 1). Furthermore, 80% of the vaccinated fish (tank 2) had no pathology, while 80% of the control fish (tank 1) had erosion or wounds (Fig 2). In tank 3 (mixed groups), prevalence and abundance did not vary significantly between vaccinated and control fish (Fig 1). However, on both vaccinated and control fish a skew sex ratio was observed, with increased number of males for each female, unlike the typical 1:1 ratio as seen in tank 1 (Table 1). 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.

Table 1. Total number of salmon lice and sex ratio in experimental groups of vaccinated Atlantic salmon

Separate tanks (tank 1 and 2) Mixed tank (tank 3)

Control Vaccine Control Vaccine

Male 72 48 40 41

Female 71 16 16 10

Ratio male/female 1,0 2,7 2,5 4,1

2. Identification of genes encoding proteins in the vaccine preparation

SDS-PAGE of unfertilized eggs from L. salmonis revealed 5 major bands with approximate molecular weight of 200 kD, 160 kD, 100 kD, 85 kD and 35K kD, called egg-band A-E respectively (Fig 3).

Three peptide sequences were obtained for egg protein A, the 200 kD protein according to the invention (Table 2) and EST's encoding the peptide sequences were identified in an in-house salmon louse EST database. The full-length cDNA sequence was successfully assembled based on overlapping ESTs and RACE clones. An in silico analysis of the egg-band A encoding cDNA sequence V6x (SEQ ID NO: 1), revealed that it is a protein that possesses key features of a Vitelogenin (Table 2). Band A in the oocyte is approximately 200 kD, which correlates well with the V6X ORF and with Northern blot transcript analysis (data not shown). Table 2. The peptide sequences obtained from isolated egg-band A and the identified gene that encodes egg-band A. Indications of protein-domains are also given. Table 2.

Mw Internal peptide Identified cDNA / aa Signal Protein domain isolated sequences Gene length peptide protein

20O kD 1. YSPSYYGXAPXL V6x* 6,093* / Yes LPD-N + VWD

2. KDETLLEAFVSR 1,965 3. TXGNLFMEYPE

* See SEQ ID NO: 1 for cDNA sequence

Three peptide sequences were obtained for egg protein B, the 180 kD protein according to the invention (Table 3) and EST's encoding the peptide sequences were identified in an in-house salmon louse EST database. The full-length cDNA sequence was successfully assembled based on overlapping ESTs and RACE clones. An in silico analysis of the egg-band B encoding cDNA sequence VIx (SEQ ID NO: 3), revealed that it is a protein that possesses key features of a Vitelogenin. Band B in the oocyte is approximately 180 kD, which correlates well with the V6X ORF and with Northern blot transcript analysis (data not shown).

Table 3. The peptide sequences obtained from isolated egg-band B and the identified gene that encodes egg-band B. Indications of protein-domains are also given.

Mw Internal peptide Identified cDNA / aa Signal Protein domain isolated sequences Gene length peptide protein

18O kD 1 GYGGEYSYXVIGS VIx* 4,747* / Yes LPD-N + VWD 2 EGYLATGQFFEXD 1,521 3 TGLLPYWDIDPEI

Table 3.

* See SEQ ID NO: 3 for cDNA sequence

One peptide sequence was obtained for egg protein C and one peptide sequence was obtained for egg protein D (Table 4) and EST's encoding the peptide sequences were identified in an in-house salmon louse EST database. A full-length cDNA sequence was successfully assembled based on overlapping ESTs and RACE clones and revealed that egg protein C and D are encoded by the same gene. According to in silco analysis this gene sequence encodes a protein that possesses key features of a Vitelogenin. The molecular weight of the egg proteins and C and D together is approximately 185 Kda, which correlates well with the open reading frame of the gene and with Northern blot transcript analysis. However, in the L. salmonis oocyte the protein encoded by this gene is apparently processed into one N-terminal part (band D) and one C-terminal part (band C).

Mw Internal peptide Identified cDNA / aa Signal Protein domain isolated sequences Gene length peptide protein

10O kD QGGSTLXSXMPY C%* 5,948* / Yes LPD-N + VWD

85 kD LLSGIPGLRPHFSGIG 1,903

Table 4. * See SEQ ID NO: 5 for cDNA sequence

One peptide sequences was obtained for egg protein E (Table 5) and EST encoding the peptide sequence was identified in an in-house salmon louse EST database. The full-length cDNA sequence was successfully assembled based on overlapping ESTs and RACE clones. According to in silco analysis the eggband E cDNA sequence

(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.

Table 5. The peptide sequences obtained from isolated egg-band E and the identified gene that encodes egg-band E. Indications of protein-domains are also given.

Mw Internal peptide Identified cDNA / aa Signal Protein domain isolated sequences Gene length peptide protein

79 kD GTWFTPGLISGQSVK Band E* 2,511* / Yes FASl x3 722

Table 5.

* See SEQ ID NO: 7 for cDNA sequence

3. Evaluation of protein antigenicity

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.

Conclusion: it follows from the experiments above that each of the proteins according to the invention; the 200 kD protein, the 180 kD protein, the 100 kD protein, the 85 kD protein or the 79 kD protein of the egg protein preparation are capable of inducing protection against sea lice, are highly immunogenic and are recognized by both antibodies raised against them in rabbits and antibodies raised in fish

4. Identification of homolog antigens in other copepod ectoparasites

SDS-PAGE and Coomassie total protein staining of egg-proteins from L. salmonis, Caligus curtus and Caligus rogercresseyi showed that all three species have a similar egg protein pattern (Fig 20a-23a). Western blotting of the same egg proteins demonstrated that each of the rabbit anti L. salmonis egg-bands A, B, C, D and E, corresponding to the 200 kD, 180 kD, 100 kD, 85 kD and 79 kD proteins specifically bind to a homolog protein in both Caligus species (Fig 20b, 21b, 22b, 22c, 23b). Conclusion: it can be concluded that protection on the basis of the 200 kD, 180 kD, 100 kD, 85 kD and 79 kD protein according to the invention is not only feasible against L. salmonis, but equally well against Caligus spp.

Legend to the figures

Figure 1. Prevalence (A) and abundance (B) of adult salmon lice on vaccinated Atlantic salmon smolt 11 weeks post challenge (26 weeks post vaccination).

Figure 2. Frequency and level of salmon lice induced external pathology on vaccinated (tank 2) and control vaccinated (tank 1) Atlantic salmon, 11 weeks post challenge with salmon louse.

Figure 3. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis of vaccine antigen preparation.

Figure 4. 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).

Figure 5. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis of purified egg-band B (lane 2) and crude vaccine antigen preparation (lane 3). Molecular weight standard is to the left (lane 1).

Figure 6. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis of purified egg band C (lane 2), D (lane 5) and crude vaccine antigen preparation (duplicated in lanes 3 and 6). Molecular weight standards are in lanes 1 and 4.

Figure 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.

Figure 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 .

Figure 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).

Figure 11. 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.

Figure 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 .

Figure 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 .

Figure 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.

Figure 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 .

Figure 16. 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.

Figure 17. 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.

Figure 18. 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.

Figure 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.

Figure 20. Analysis of egg protein A in Caligus curtus (C. c) and Caligus rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s). (a) SDS-PAGE and Coomassie staining was performed for total protein analysis of all egg proteins, (b) Western blotting was performed using rabbit anti L. s egg protein A to analyze for cross binding to homologue gene products in Caligus. (c) Rabbit pre- serum was used as negative Western blotting control. Lanes labeled Std contain identical molecular weight standards.

Figure 21. Analysis of egg proteins (A-E) in Caligus curtus (C. c) and Caligus rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).

(a) SDS-PAGE and Coomassie staining was performed for total protein analysis of all egg proteins, (b) Western blotting was performed using rabbit anti L. s egg protein B to analyze for cross binding to homologue gene products in Caligus. (c) Rabbit pre-

serum was used as negative Western blotting control. Lanes labeled Std contain identical molecular weight standards.

Figure 22. Analysis of egg proteins C and D in Caligus curtus (C. c) and Caligus rogercresseyi (C. r), compared to Lepeophtheirus salmonis (L. s).

(a) SDS-PAGE and Coomassie staining was performed for total protein analysis of all egg proteins. Western blotting was performed using rabbit anti L. s egg protein C (b) and D (c) to analyze for cross binding to homologue gene products in Caligus. (d) Rabbit pre-serum was used as negative Western blotting control. Lanes labeled Std contain identical molecular weight standards.

Figure 23. Analysis of egg protein E in Caligus curtus (C. c) and Caligus rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s). (a) SDS-PAGE and Coomassie staining was performed for total protein analysis of all egg proteins, (b) Western blotting was performed using rabbit anti L. s egg protein E to analyze for cross binding to homologue gene products in Caligus. (c) Rabbit pre- serum was used as negative Western blotting control. Lanes labeled Std contain identical molecular weight standards.