White Spot Syndrome Virus vaccine The present invention relates to nucleic acid sequences encoding a novel White Spot Syndrome Virus protein, to DNA fragments, recombinant DNA molecules and live recombinant carriers comprising these sequences, to host cells comprising such nucleic acid sequences, DNA fragments, recombinant DNA molecules and live recombinant carriers, to a novel White Spot Syndrome Virus protein encoded by these nucleotide sequences, to vaccines for combating White Spot Syndrome Virus infections and methods for the preparation thereof, and to diagnostic tools for the detection of White Spot Syndrome Virus.

White spot syndrome virus (WSSV, also known as Systemic Ectodermal and Mesodermal Baculovirus SEMBV or Bacilliform Baculovirus) is a pathogen of major economic importance in cultured penaeid shrimp. The virus is not only present in shrimp but also occurs in other freshwater and marine crustaceans including crabs and crayfish (Lo et al., 1996). In cultured shrimp WSSV infection can reach a cumulative mortality of up to 100% within 3-10 days (Lightner, 1996) and can cause large economic losses to the shrimp culture industry. The virus was first discovered in Taiwan, from where it quickly spread to other shrimp farming areas in Southeast Asia (Cai et al., 1995). WSSV initially appeared to be limited to Asia until it was found in Texas and South-Carolina in November 1995 (Rosenberry, 1996). In early 1999 WSSV was also reported from Central-and South-America and it has now also been detected in Europe and Australia (Rosenberry, 2000). Intensive shrimp cultivation, inadequate sanitation and world-wide trade has aggravated the disease incidence in crustaceans and enhanced disease dissemination. As such WSS has become an epizootic disease and is not only a major threat to shrimp culture but also to marine ecology (Flegel, 1997).

WSSV virions circulate ubiquitously in the haemolymph of infected shrimp. Electron microscopy studies revealed that WSSV virions are enveloped rod-shaped nucleocapsids with a bacilliform to ovoid shape of about 275 nm in length and 120 nm in width. Most characteristic is the tail-like appendage at one end of the virion (Durand et al., 1997).

WSSV nucleocapsids have a striated appearance and a size of about 300 nm x 70 nm.

The striations are probably the result of stacked ring-like structures consisting of rows of globular subunits of about 10 nm in diameter (Durand et al., 1997; Nadala et al., 1998).

So far, only a diagnostic tool for the detection of the disease has been developed as disclosed in European Patent No. EP 0.785.255. No vaccines have been developed or described yet.

Therefore, it is an objective of the present invention to provide vaccines for combating WSSV infections.

Surprisingly, a very long open reading frame (ORF) of the viral genome has now been found, which is thought to be a structural protein involved in the WSSV infection in shrimp. This large ORF has now been cloned and sequenced and has been designated ORF167. The ORF consists of 18,234 nucleotides. Its full sequence is given in SEQ ID NO 1. ORF167 is located at position 258666 to 276899 of the viral genome (See Fig. 1).

The ORF codes for a structural protein of 6077 amino acids (SEQ ID NO 2) with a theoretical mass of 664 kD. This is the largest ORF to date in viruses. ORFs of about half the size have been identified in herpes viruses and the proteins encoded by these ORFs are located in the tegument. ORFs of similar sizes are found in eukaryotes and are members of the family of giant actin-binding/cytoskeletal cross-linking proteins. ORF167 is thought to encode a protein involved in the tail-like appendage at one end of the virion.

Analysis of the sequence of ORF167 revealed several immunogenic sites that are suitable for use in a vaccine.

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 a sequence homology of about 70 % can still encode one and the same protein.

Thus, one embodiment relates to nucleic acid sequences encoding a WSSV protein that has a molecular weight of 664 kD and to parts of those nucleic acid sequences that encode an immunogenic fragment of that protein, wherein those nucleic acid sequences or those parts thereof have a level of homology with the nucleic acid sequence of SEQ ID NO: 1 of at least 70 %.

Preferably, the nucleic acid sequences encoding this WSSV protein or the parts of those nucleic acid sequences have at least 80 %, preferably 90 %, more preferably 95 % homology with the nucleic acid sequence of SEQ ID NO: 1. Even more preferred is a homology level of 98% or even 100%.

Nucleotide sequences that are complementary to the sequence depicted in SEQ ID NO 1 or nucleotide sequences that comprise tandem arrays of the sequences according to the invention are also within the scope of the invention.

The level of nucleotide homology can be determined with the computer program "BLAST 2 SEQUENCES"by selecting sub-program:"BLASTN"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). Parameters used are the default parameters: Reward for a match: +1. Penalty for a mismatch:-2. Open gap: 5. Extension gap: 2. Gap x_dropoff : 50.

Also, one form of this embodiment of the invention relates to nucleic acid sequences encoding a 664 kD WSSV protein comprising an amino acid sequence that has an homology of at least 70 %, preferably 80%, 90 %, 95 %, 98 % or even 100 % with the amino acid sequence depicted in SEQ ID NO: 2, or an immunogenic fragment of that protein.

In a preferred form of that embodiment, those nucleic acid sequences have a homology of at least 70 %, more preferably 80 %, 90 %, 95 %, 98 % or even 100 % with the nucleic

acid sequence as depicted in SEQ ID NO: 1.

Since the present invention discloses nucleic acid sequences encoding a novel 664 kD WSSV 664 kD protein, it is now for the first time possible 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 protein.

Therefore, in a more preferred form of this embodiment, the invention relates to DNA fragments comprising a nucleic acid sequence according to the invention. A DNA fragment is a stretch of nucleotides that comprises 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, as described below.

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.

Therefore, an even more preferred form of this embodiment relates to a recombinant DNA molecule comprising a DNA fragment or a nucleic acid sequence according to the invention wherein the nucleic acid sequence according to the invention is placed under the control of a functionally linked promoter. This 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).

Functionally linked promoters are promoters that are capable of controlling the transcription of the nucleic acid sequences to which they are linked.

Such a promoter can be the ORF167 promoter or another promoter of the WSSVirus, 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 a-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., a-mating factor. For insect cells the polyhedrin or plO promoters of baculoviruses can be used (Smith, G. E. et al., Mol. 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, C. M. 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 p-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).

A still even more preferred form of this embodiment of the invention relates to Live Recombinant Carriers (LRCs) comprising a nucleic acid sequence encoding the 664 kD protein or an immunogenic fragment thereof according to the invention, a DNA fragment according to the invention or a recombinant DNA molecule according to the invention.

These LRCs are micro-organisms or viruses in which additional genetic information, in this case a nucleic acid sequence encoding the 664 kD protein or an immunogenic fragment thereof according to the invention has been cloned. Shrimp infected with such LRCs will produce an immunologic 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 ORF167 gene.

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. Yellow Head virus and Gill Associated virus, both belonging to the family coronaviridae. (see e. g. Spann, K. M. et al., Dis. Aquat.

Org. 42: 221-225, (2000), and Cowley, J. A. et al., Dis. Aquat. Org. 36: 153-157 (1999) for the virus, or Enjuanes, L. et al., p. 28-31 of the Proceedings of the ESW, Brescia, Italia, 27-30 August 2000 for live recombinant carrier corona viruses).

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 nucleic acid sequence encoding a protein according to the invention, a DNA fragment comprising such a nucleic acid sequence or a recombinant DNA molecule comprising such a nucleic acid sequence under the control of a functionally linked promoter. This form also relates to a host cell containing a live recombinant carrier containing a nucleic

acid molecule encoding a 664 kD protein or a fragment thereof according to the invention.

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-technology 6: 47-55 (1988)) in combination with vectors or recombinant baculoviruses, 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 Hela cells, Chinese Hamster Ovary cells (CHO) or Crandell Feline Kidney-cells, also with appropriate vectors or recombinant viruses.

Another embodiment of the invention relates to the novel protein; the 664 kD WSSV protein and to immunogenic fragments thereof according to the invention.

The concept of immunogenic fragments will be defined below.

One form of this embodiment relates i. a. to WSSV proteins that have an amino acid sequence that is at least 70 % homologous to the amino acid sequence as depicted in SEQ ID NO: 2 and to immunogenic fragments of said protein.

In a preferred form, the embodiment relates to such WSSV proteins that have a sequence homology of at least 80 %, preferably 90 %, more preferably 95 % homology to the amino acid sequence as depicted in SEQ ID NO: 2 and to immunogenic fragments of such proteins.

Even more preferred is a homology level of 98% or even 100%.

The level of protein homology can 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 xdropoff : 50.

It will be understood that, for the particular proteins embraced herein, natural variations can exist between individual WSSV 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 homologous 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 WSSV proteins according to the invention, when isolated from different field isolates, may have homology 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 WSSV or at least against the clinical manifestations of the infection are considered as"not essentially influencing the immunogenicity".

When a protein is used for e. g. vaccination purposes or for raising antibodies, it is however 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. Antibodies raised in a vertebrate host are very suitable as passive means of vaccination in shrimps. At this moment, a variety of techniques is available to easily identify DNA fragments encoding antigenic 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), Berzowsky for HIV-epitopes; The FASEB Journal 5: 2412-2418 (1991).

Therefore, one form of still another embodiment of the invention relates to vaccines capable of protecting shrimp against WSSV infection, that comprise a protein or

immunogenic fragments thereof, according to the invention as described above together with a pharmaceutically acceptable carrier.

A preferred form of this embodiment relates to the protein fragments spanning amino acid residues 4932 to 5333 (DOMAIN1), amino acid residues 3600 to 4056 (DOMAIN2), amino acid residues 4328 to 4666 (DOMAIN3), amino acid residues 160 to 450 (HSP1), amino acid residues 670 to 850 (HSP2), amino acid residues 2100 to 2620 (HSP3), amino acid residues 3200 to 3500 (HSP4), amino acid residues 1 to 160 (A), amino acid residues 450 to 670 (B), amino acid residues 850 to 1270 (C), amino acid residues 1270 to 1685 (D), amino acid residues 1685 to 2100 (E), amino acid residues 2620 to 3200 (F), amino acid residues 3400 to 3600 (G), amino acid residues 4000 to 4330 (H), amino acid residues 4660 to 4935 (I), amino acid residues 5330 to 5705 (J), or amino acid residues 5700 to 6077 (K) of the amino acid sequence depicted in SEQ ID NO 2, or immunogenic fragments thereof.

In a more preferred form of this embodiment, these protein fragments are encoded by the nucleotide sequences given in SEQ ID NO.: 3-20 or by parts of these nucleotide sequences encoding immunogenic fragments of these protein fragments. (These nucleotide sequences are all present in SEQ ID NO.: 1). Protein fragments are understood to be fragments of the 664 kD protein according to the invention, as encoded by ORF 167.

Still another embodiment of the present invention relates to the protein according to the invention or immunogenic fragments thereof for use in a vaccine.

Still another embodiment relates to the use of a protein according to the invention or immunogenic fragments of that protein for the manufacturing of a vaccine for combating WSSV infections.

One way of making a vaccine according to the invention is by growing the white spot syndrome virus in cell culture, followed by biochemical purification of the 664 kD

protein or immunogenic fragments thereof, from the virus. This is however a very time- consuming way of making the vaccine.

It is therefore much more convenient to use the expression products of the genes encoding the protein or immunogenic fragments thereof in vaccines. This is possible for the first time now because the nucleic acid sequence of the gene encoding the 664 kD protein is provided in the present invention.

Vaccines based upon the expression products of these genes can easily be made by admixing the protein according to the invention or immunogenic fragments thereof according to the invention with a pharmaceutically acceptable carrier as described below.

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. Yellow Head virus have the advantage over subunit vaccines that they better mimic the natural way of infection of WSSV. Moreover, their self-propagation is an advantage since only low amounts of the recombinant carrier are necessary for immunisation.

Vaccines can also be based upon host cells as described above, that comprise the proteins or immunogenic fragments thereof according to the invention.

All vaccines described above contribute to active vaccination, i. e. they trigger the host's defence 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 shrimp. 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 protein

according to the invention or immunogenic fragments thereof can in these cases bind directly to WSSV and to cells exposing the WSSV protein according to the invention due to infection with WSSV. This has the advantage that it decreases or stops WSSV replication.

Therefore, one other form of this embodiment of the invention relates to a vaccine for combating WSSV infection that comprises antibodies against the WSSV protein according to the invention or an immunogenic fragment of that protein, and a pharmaceutically acceptable carrier.

Still another embodiment of this invention relates to antibodies against the WSSV protein according to the invention or 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- marburg. de/-rek/aepphage. html., 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 attractive for the vaccination of shrimp against WSSV infection. Therefore, still other forms of this embodiment of the invention relate to vaccines comprising nucleic acid sequences encoding a protein according to the invention or immunogenic fragments thereof, and to vaccines comprising DNA fragments that comprise such nucleic acid sequences.

Examples of DNA plasmids 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 plasmids according to the invention should be able to induce protein expression of the nucleotide sequences. The DNA plasmid can comprise one or more nucleotide sequences according to the invention. In addition, the DNA plasmid can comprise other nucleotide sequences such as the immune-stimulating oligonucleotides having unmethylated CpG di- nucleotides, or nucleotide sequences that code for other antigenic proteins or adjuvating cytokines.

The nucleotide sequence according to the present invention or the DNA plasmid comprising a nucleotide 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 Yellow Head virus, as mentioned above.

Still other forms of this embodiment relate to vaccines comprising recombinant DNA molecules according to the invention.

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 microgram range between 10 pg and 1000 jig provide very good results. Preferably, amounts in the microgram range between 1 and 100 pg are used. Alternatively, animals can be dipped in solutions comprising e. g. between 10 pg and 1000 ig 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 other shrimp pathogenic organisms and viruses, antibodies against those antigens or genetic information encoding such antigens.

Such organisms and viruses are preferably selected from the group of Baculovirus penaei (BP), Monodon baculovirus (MBV), Baculoviral midgut gland necrosis virus (BMNV) hematopoietic necrosis virus (IHHNV), Vibrio alginolyticus, V. parahaemolyticus, V. anguillarum, Pseudomonas spp, Aeromonas spp, Lagendium callinectes, Siropidium sp, Pythium sp.

A vaccine according to the invention can be used to protect crustaceans such as shrimp including but not limited to members from the Penaeidae family such as for example P. monodon, P. vannamei, P. chinensis, P. merguensis, or Metapeaeus spp. ; prawns including but not limited to members from the Palaemonidae family such as for example Macrobrachium spp. or Palaemon spp. ; lobsters including but not limited to members from the Palinuridae and Nephropidae family such as for example Calinectes spp., Palinurus spp., Panuliris spp. or Homarus spp. ; crayfish including but not limited to members from the Astacidae family examples of which are Astacus spp., Procambarus spp., and Oronectes spp. ; and crab including but not limited to members from the Cancridae and Portuidae family, examples of which are Cancer spp., Callinectes spp., Carcinus spp. and Portunus spp.

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 a protein or an immunogenic fragment thereof, according to the invention and/or antibodies against that protein or an immunogenic fragment thereof, and/or a nucleic acid sequence 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 Carbopol (R) (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.

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 stabilising a protein are also embodied in the present invention.

Vaccines according to the invention 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 103 and 109 CFU/PFU for respectively bacteria and viruses.

Many ways of administration, all known in the art can be applied. The vaccines according to the invention are preferably administered to the crustaceans via injection, immersion, dipping or per oral. The administration protocol can be optimised in accordance with standard vaccination practice. Preferably the vaccine is administered to the crustaceans 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 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 shrimp. 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 but are not limited to plankton-like non-selective filter feeders preferably members of Rotifera, Artemia, and the like. Highly preferred is the brine shrimp Artemia sp.

As mentioned above, the virus infection proceeds fast and lethality can be up to 100%.

Therefore, for efficient protection against disease, a quick and correct diagnosis of WSSV infection is important.

Therefore it is another objective of this invention to provide diagnostic tools suitable for the detection of WSSV infection.

A diagnostic test based upon the detection of antigenic material of the specific 664 kD proteins of WSSV and therefore suitable for the detection of WSSV infection can e. g. be a standard ELISA test. In one example of such a test the walls of the wells of an ELISA plate are coated with antibodies directed against the 664 kD protein. After incubation with the material to be tested, labeled anti-WSSV antibodies are added to the wells. A colour reaction then reveals the presence of antigenic material from WSSV.

Therefore, still another embodiment of the present invention relates to diagnostic tests for the detection of antigenic material of WSSV. Such tests comprise antibodies against a protein or a fragment thereof according to the invention.

The proteins or immunogenic fragments thereof according to the invention 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 variants or fragments thereof) according to the present invention, can be prepared by immunising inbred mice by techniques also known in the art (Kohler and Milstein, Nature, 256,495-497,1975).

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

Examples Example 1: WSSV isolation The virus isolate used in this study originates from WSSV-infected Penaeus monodon shrimp imported from Thailand in 1996 and was obtained as described before (Van Hulten et al., 2000c). Crayfish Procambarus clarkii were injected intramuscularly with a lethal dose of WSSV. After one week the haemolymph was withdrawn from moribund crayfish and mixed with modified Alsever solution (Rodriguez et al., 1995) as anticoagulant. The virus was purified by centrifugation at 80,000 x g for 1.5 h at 4°C on a 20-45% continuous sucrose gradient in TN (20 mM Tris, 400 mM NaCl, pH 7.4). The visible virus bands were removed and the virus particles were subsequently sedimented by centrifugation at 45,000 x g at 4°C for 1 h after dilution with TN. The virus pellet was resuspended in TE (pH 7.5).

Example 2 WSSV DNA isolation, cloning, and sequence determination The WSSV DNA was sequenced to a 6-fold genomic coverage using a shotgun approach.

The viral DNA was purified as described in Van Hulten et al. (2000a) and sheared by nebulization into fragments with an average size of 1,200 bp. Blunt repair of the ends was performed with Pfu DNA polymerase (Stratagene) according to the manufacturer's directions. DNA fragments were size-fractionated by gel electrophoresis and cloned into the EcoRV site of pBluescriptSK (Stratagene). After transformation into XL2 blue competent cells (Stratagene) 1510 recombinant colonies were picked randomly. DNA templates for sequencing were isolated using QIAprep Turbo kits (Qiagen) on a QIAGEN BioRobot 9600. Sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready reaction kit with FS AmpliTaq DNA polymerase (Perkin Elmer) and analysed on an ABI 3700 DNA Analyser.

Legend to the Figures.

Figure 1. Linearized map of the circular double-stranded WSSV genome showing the genomic organisation. The A of the ATG initiation codon of VP28 (ORF1) has been arbitrarily designated position 1. Restriction BamHI sites are shown in the black central bar; fragments are indicated A to W according to size from the largest (A) to the smallest (W). ORFs are numbered form left to right. ORFs transcribed forward are located above the genome; ORFs transcribed in the reverse orientation are located below. Genes with similar functions are indicated according to the key given the box in the lower right corner. Repeat regions (hrs) are presented according to the key and numbered (1-9).

Numbers on the right indicate the number of nucleotides in kilobase pairs.

REFERENCES Cai, S. L., Huang, J., Wang, C. M., Song, X. L., Sun, X., Yu, J., Zhang, Y., Yang, C. H.

(1995). Epidemiological studies on the explosive epidemic disease of prawn in 1993- 1994. J. China Fish 19,112-117.

Durand, S., Lightner, D. V., Redman, R. M., and Bonami, J. R. (1997). Ultrastructure and morphogenesis of white spot syndrome baculovirus (WSSV). Dis. Aquat. Org. 29,205- 211.

Flegel, T. W. (1997). Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol. 13,433-442.

Lightner, D. V. (1996). A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. World Aquatic Society, Baton Rouge, LA.

Nadala, E. C. B., Tapay, L. M., and Loh, P. C. (1998). Characterisation of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp. Dis. Aquat. Org. 33,221-229.

Rodriguez, J., Boulo, V., Mialhe, E., and Bachère, E. (1995). Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies. J. Cell Sci. 108,1043- 1050.

Rosenberry, B. (1996). World shrimp farming 1996. Shrimp News International, San Siego, California, p. 29-30.

Rosenberry, B. (2000). World shrimp farming 2000. Shrimp News International, San Siego, California.

Lo, C. F., Ho, C. H., Peng, S. E., Chen, C. H., Hsu, H. C., Chiu, Y. L., Chang, C. F., Liu, K. F., Su, M. S., Wang, C. H., Kou, G. H. (1996a). White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis.

Aquat. Org. 27,215-225.

Van Hulten, M. C. W., Tsai, M. F., Schipper, C. A., Lo, C. F., Kou, G. H., and Vlak, J.

M. (2000c). Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions. J. Gen. Virol. 81,307-316. Van Hulten, M. C. W., Westenberg, M., Goodall, S. D., and Vlak, J. M. (2000a).

Identification of two major virion protein genes of White Spot Syndrome virus of shrimp.

Virology 266,227-236.