WO2003000900A1 - White spot syndrome virus vaccine - Google Patents
White spot syndrome virus vaccine Download PDFInfo
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- WO2003000900A1 WO2003000900A1 PCT/EP2002/006746 EP0206746W WO03000900A1 WO 2003000900 A1 WO2003000900 A1 WO 2003000900A1 EP 0206746 W EP0206746 W EP 0206746W WO 03000900 A1 WO03000900 A1 WO 03000900A1
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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- C12N2710/18011—Nimaviridae
- C12N2710/18022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- 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).
- 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.
- Taiwan 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 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
- WSSV virions circulate ubiquitously in the haemo lymph 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).
- ORF open reading frame
- 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. ORF 167 is thought to encode a protein involved in the tail-like appendage at one end of the virion. Analysis of the sequence of ORF 167 revealed several immunogenic sites that are suitable for use in a vaccine.
- 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.
- 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 %.
- 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 prefe ⁇ ed 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 a ⁇ ays 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.
- 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.
- 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.
- 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.
- 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 prefe ⁇ ed 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.
- Functionally linked promoters are promoters that are capable of controlling the transcription of the nucleic acid sequences to which they are linked.
- a promoter can be the ORF 167 promoter or another promoter of the WSS Virus, provided that that promoter is functional in the cell used for expression. It can also be a heterologous promoter.
- useful expression control sequences include the T ⁇ promoter and operator (Goeddel, et al., Nucl.
- useful expression control sequences include, e.g., ⁇ -mating factor.
- the polyhedrin or plO promoters of baculoviruses can be used (Smith, G.E. et al., Mol. Cell. Biol. 3, 2156-65, 1983).
- useful expression control sequences include the (human) cytomegalovirus immediate early promoter (Seed, B. et al., Nature 329, 840-842, 1987; Fynan, E.F. et al., PNAS 90, 11478-11482,1993; Ulmer, J.B.
- Rous sarcoma virus LTR Rous sarcoma virus LTR (RSV, Gorman, CM. et al., PNAS 79, 6777-6781, 1982; Fynan et al., supra; Ulmer et al., supra), the MPSV LTR (Stacey et al., J. Virology 50, 725-732, 1984), SV40 immediate early promoter (Sprague J. et al., J. Virology 45, 773 ,1983), the SV-40 promoter (Berman, P.W. et al., Science, 222, 524-527, 1983), the metallothionein promoter (Brinster, R.L.
- the regulatory sequences may also include terminator and poly-adenylation sequences. Amongst the sequences that can be used are the well known bovine growth hormone poly-adenylation sequence, the SV40 poly-adenylation sequence, the human cytomegalovirus (hCMV) terminator and poly-adenylation sequences.
- Bacterial, yeast, fungal, insect and vertebrate cell expression systems are very frequently used systems. Such systems are well-known in the art and generally available, e.g. commercially through Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, California 94303-4607, USA. Next to these expression systems, parasite-based expression systems are attractive expression systems. Such systems are e.g. described in the French Patent Application with Publication number 2 714 074, and in US NTIS Publication No US 08/043109 (Hoffman, S. and Rogers, W.: Public. Date 1 December 1993).
- 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.
- LRCs Live Recombinant Carriers
- 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.
- 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 ORF 167 gene.
- bacteria such as Vibrio anguillarum known in the art can attractively be used. (Singer, J.T. et al., New Developments in Marine Biotechnology, p. 303-306, Eds. Le Gal and Halvorson, Plenum Press, New York, 1998).
- LRC viruses may be used as a way of transporting the nucleic acid sequence into a target cell. Viruses suitable for this task are e.g.
- 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 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.
- 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.
- 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.
- 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:
- 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.
- 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.
- 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.
- 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
- 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.
- 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).
- 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 (DOMAINl), 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 S
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ g provide very good results. Preferably, amounts in the microgram range between 1 and 100 ⁇ g are used. Alternatively, animals can be dipped in solutions comprising e.g. between 10 pg and 1000 ⁇ g per ml of the DNA to be administered.
- the vaccine according to the present invention additionally comprises one or more antigens derived from other shrimp pathogenic organisms and viruses, antibodies against those antigens or genetic information encoding such antigens.
- 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.
- BP Baculovirus penaei
- MBV Monodon baculovirus
- BMNV Baculoviral midgut gland necrosis virus
- IHHNV hematopoietic necrosis virus
- Vibrio alginolyticus V. parahaemo
- 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.
- 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( ⁇ ) (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.
- the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.
- the vaccine is mixed with stabilisers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency.
- Useful stabilisers are i.a. SPGA (Bovamik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.
- the 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 10 3 and 10 9 CFU/PFU for respectively bacteria and viruses.
- 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.
- the vaccine is administered to the crustaceans via immersion or per oral, especially in case of commercial aqua culture farms.
- the vaccine is preferably mixed with a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin.
- a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin.
- an attractive 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 prefe ⁇ ed is the brine shrimp Artemia sp.
- 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.
- 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.
- a standard ELISA 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.
- 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 inte ⁇ reted as limitations of the invention.
- 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.
- FIG. 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.
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- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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EP01202418 | 2001-06-22 | ||
NL01202418.8 | 2001-06-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2003000900A1 true WO2003000900A1 (en) | 2003-01-03 |
Family
ID=8180526
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2002/006746 WO2003000900A1 (en) | 2001-06-22 | 2002-06-18 | White spot syndrome virus vaccine |
Country Status (1)
Country | Link |
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WO (1) | WO2003000900A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005093068A1 (en) * | 2004-03-29 | 2005-10-06 | Dainippon Sumitomo Pharma Co., Ltd. | Novel protein and promoter |
WO2006005222A1 (en) * | 2004-07-09 | 2006-01-19 | Green Life Laboratory Limited | Gene engineering protein p40 and use thereof |
WO2005023992A3 (en) * | 2003-09-09 | 2007-07-05 | Aqua Bounty Technologies Inc | Compositions and methods for inhibiting white spot syndrome virus (wssv) infection |
CN110540996A (en) * | 2019-08-29 | 2019-12-06 | 华中农业大学 | Procambarus clarkii i-type lysozyme gLysi2 gene, gLysi2 protein coded by same and application thereof |
CN111363834A (en) * | 2020-04-30 | 2020-07-03 | 华中农业大学 | SNP molecular marker related to resistance of white spot syndrome of procambarus clarkii |
CN113647516A (en) * | 2021-07-26 | 2021-11-16 | 中农科生物工程技术(苏州)有限公司 | Preparation method and application of streptozochytrium induced resistance protein LiiP1 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0785255A2 (en) * | 1996-01-17 | 1997-07-23 | Guang-Hsiung Kou | Identification, purification and detection of WSBV (baculovirus associated with white spot syndrome) |
WO2001009340A1 (en) * | 1999-08-03 | 2001-02-08 | Akzo Nobel N.V. | Proteins derived from white spot syndrome virus and uses thereof |
WO2001038351A2 (en) * | 1999-11-24 | 2001-05-31 | Pe Corporation (Ny) | Nucleotide sequence of the shrimp white spot syndrome bacilliformvirus (wsbv), systems containing this sequence and detection kits |
-
2002
- 2002-06-18 WO PCT/EP2002/006746 patent/WO2003000900A1/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0785255A2 (en) * | 1996-01-17 | 1997-07-23 | Guang-Hsiung Kou | Identification, purification and detection of WSBV (baculovirus associated with white spot syndrome) |
WO2001009340A1 (en) * | 1999-08-03 | 2001-02-08 | Akzo Nobel N.V. | Proteins derived from white spot syndrome virus and uses thereof |
WO2001038351A2 (en) * | 1999-11-24 | 2001-05-31 | Pe Corporation (Ny) | Nucleotide sequence of the shrimp white spot syndrome bacilliformvirus (wsbv), systems containing this sequence and detection kits |
Non-Patent Citations (1)
Title |
---|
VAN HULTEN MARIELLE C W ET AL: "The white spot syndrome virus DNA genome sequence.", VIROLOGY, vol. 286, no. 1, 20 July 2001 (2001-07-20), pages 7 - 22, XP002184108, ISSN: 0042-6822 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005023992A3 (en) * | 2003-09-09 | 2007-07-05 | Aqua Bounty Technologies Inc | Compositions and methods for inhibiting white spot syndrome virus (wssv) infection |
WO2005093068A1 (en) * | 2004-03-29 | 2005-10-06 | Dainippon Sumitomo Pharma Co., Ltd. | Novel protein and promoter |
WO2006005222A1 (en) * | 2004-07-09 | 2006-01-19 | Green Life Laboratory Limited | Gene engineering protein p40 and use thereof |
CN110540996A (en) * | 2019-08-29 | 2019-12-06 | 华中农业大学 | Procambarus clarkii i-type lysozyme gLysi2 gene, gLysi2 protein coded by same and application thereof |
CN111363834A (en) * | 2020-04-30 | 2020-07-03 | 华中农业大学 | SNP molecular marker related to resistance of white spot syndrome of procambarus clarkii |
CN111363834B (en) * | 2020-04-30 | 2021-08-03 | 华中农业大学 | SNP molecular marker related to resistance of white spot syndrome of procambarus clarkii |
CN113647516A (en) * | 2021-07-26 | 2021-11-16 | 中农科生物工程技术(苏州)有限公司 | Preparation method and application of streptozochytrium induced resistance protein LiiP1 |
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