Sequences from Piscirickettsia salmonis

FIELD OF THE INVENTION

[0001] The present invention relates to a fish vaccine. More specifically the invention relates to a vaccine to protect salmon against piscirickettsiosis, also referred to as salmonid rickettsial septicaemia (SRS).

BACKGROUND OF THE INVENTION

[0002] To date no commercially available vaccine has succeeded in controlling infections by Piscirickettsia salmonis, the causative agent of SRS. Accordingly there is a need for an effective vaccine against Piscirickettsia salmonis.

[0003] It is an object of the present invention to provide a protein based vaccine or a Nucleic Acid Vaccine (NAV) to protect fish against infection by Piscirickettsia salmonis, and thereby against SRS.

SUMMARY OF THE INVENTION

[0004] In one aspect of the invention, novel nucleic acid sequences from Piscirickettsia salmonis and their encoded amino acid sequences are provided.

[0005] In another aspect of the invention a method is provided for protecting fish against infection by Piscirickettsia salmonis, comprising administering to a fish one or more of the nucleic acid sequences and amino acid sequences of the invention.

[0006] A further aspect of the invention provides nucleic acid vaccines and protein vaccines comprising one or more of the nucleic acid sequences or amino acid sequences of the invention, for administration to fish to protect against SRS.

DESCRIPTION OF THE FIGURES

[0007]
FIG. 1 is the partial nucleotide sequence of P. salmonis Psclone51A, and encoded amino acid sequence.

[0008]
FIG. 2 is the complete coding sequence of Psclone51A.

[0009]
FIG. 3 is the amino acid sequence derived from the ORF of Psclone 51A.

[0010]
FIG. 4 is the nucleotide sequence of P. salmonis p10.6.

[0011]
FIG. 5 is the amino acid sequence of p10.6.

[0012]
FIG. 6 is the nucleic acid sequence of P. salmonis IcmE and encoded protein.

[0013]
FIG. 7 is the amino acid sequence of a portion of P. salmonis p45 antigen.

[0014]
FIG. 8 is the nucleotide sequence of P. salmonis clone3/original (3′-5′), and the encoded protein.

[0015]
FIG. 9 is the nucleotide sequence of P. salmonis clone3/3PST-R, and the encoded protein.

[0016]
FIG. 10 is the nucleotide sequence of P. salmonis clone3/3APA-F, and the encoded protein.

[0017]
FIG. 11 is the nucleotide sequence of P. salmonis clone7/original, and the encoded protein.

[0018]
FIG. 12 is the nucleotide sequence of P. salmonis clone7/Xbar, and the encoded protein.

[0019]
FIG. 13 is the nucleotide sequence of P. salmonis clone7/MunR, and the encoded protein.

[0020]
FIG. 14 is the nucleotide sequence of P. salmonis clone7/MunF (3′ to 5′), and the encoded protein.

[0021]
FIG. 15 is the nucleotide sequence of P. salmonis clone20/original, and the encoded protein.

[0022]
FIG. 16 is the nucleotide sequence of P. salmonis clone20/20VSPF (3′-5′), and the encoded protein.

[0023]
FIG. 17 is the nucleotide sequence of P. salmonis clone15/original, and the encoded protein.

[0024]
FIG. 18 is the amino acid sequence of P. salmonis hsp70.

[0025]
FIG. 19 is the amino acid sequence of another isolate of P. salmonis hsp70 clone B

[0026]
FIG. 20 is the nucleotide sequence of hsp70 clone B.

[0027]
FIG. 21 is the nucleotide sequence of P. salmonis hsp60.

[0028]
FIG. 22 is the amino acid sequence encoded by P. salmonis hsp60.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The novel sequences of the genes of the invention and the encoded proteins are provided in FIGS. 1 to 22. SEQ ID NOS refer to the sequences in the order provided in the Sequence Listing.

[0030] Psclone 51A

[0031]
FIGS. 1 and 2 depict, respectively, the partial (SEQ ID NO:1) and complete (SEQ ID NO:3) nucleotide sequences of Psclone51A, which was cloned as a cDNA molecule from mRNA of Piscirickettsia salmonis type strain (LF-89). The cloned material was sequenced in both directions from the 5′ and 3′ insertion sites using overlapping amplicons. PCR and RT-PCR comparing uninfected Chinook salmon embryonic (CHSE-214) cell lines and cell lines infected with P. salmonis confirmed that Psclone51A is specific to P. salmonis and is not derived from salmon host cells.

[0032] The partial (SEQ ID NO:2) and complete (SEQ ID NO:4) amino acid sequences deduced for the open reading frame (ORF) of Psclone51A are provided in FIGS. 1 and 3, respectively. Expression of the cloned sequence has yielded a protein of approximately 12 kDa.

[0033] The ORF of Psclone51A described in FIG. 3 does not have any significant homology at the nucleotide level with previous submissions to databases accessible by BLAST. At the protein level, a border line similarity with a hypothetical 21.5 kDa protein of Escherichia coli was found.

[0034] p10.6

[0035] An expression library was constructed from Piscirickettsia salmonis (ATCC strain VR-1361) grown in CHSE-214 cells. An immuno-reactive clone, (0110-2-5) (p10.6), was selected using homologous antisera from Piscirickettsia salmonis immunised rabbits and sequenced. FIG. 4 depicts the nucleotide sequence of this clone: p10.6 (SEQ ID NO:5). The open reading frame (ORF) for the full gene was completed by inverse PCR from genomic Piscirickettsia salmonis DNA.

[0036] Polymerase chain reaction (PCR) primers were designed based on the sequence of clone p10.6. Using these primers, PCR and RT-PCR comparing uninfected Chinook salmon embryonic (CHSE-214) cell lines and cell lines infected with P. salmonis confirmed that clone p10.6 is specific to P. salmonis and is not derived from salmon host cells.

[0037] The amino acid sequence deduced for the open reading frame (ORF) is provided in FIG. 5 (SEQ ID NO:6). The ORF of p10.6 does not have any significant homology at the nucleotide level with previous submissions to databases accessible by BLAST. The derived amino acid sequence, but not the nucleotide sequence, shows significant homology to the 17 kDa antigen found in Rickettsia of the Spotted Fever Group, where it is considered a group specific, outer membrane protein.

[0038] IcmE

[0039]
FIG. 6 depicts the nucleic acid sequence of IcmE (SEQ ID NO: 7). The genetic sequence has been derived from an inverse polymerase chain reaction (IPCR) product amplified from Piscirickettsia salmonis type strain (LF-89) genomic DNA (gDNA). The IPCR product was sequenced in both direction from the 5′ and 3′ sides using overlapping amplicons.

[0040] The amino acid sequence deduced for the Open Reading Frame (ORF) is provided in FIG. 6 (SEQ ID NO:8). The protein encoded by the ORF of IcmE (403) has a 37% significant homology at the protein level to the IcmE protein of Legionella pneumophila when compared to previous submissions to databases accessible by BLAST.

[0041] p45

[0042] The amino acid sequence of a portion of the p45 major antigen of P. salmonis is provided in FIG. 7 (SEQ ID NO:9).

[0043] The amino acid sequence was derived from microsequencing of a protein approximately 45 kDa found to be immunoreactive to rabbit anti-P salmonis antibodies. Moreover, p45 was found uniquely in Chinook salmon embryonic (CHSE-214) cells infected with Piscirickettsia salmonis and not in uninfected CHSE-214 cells.

[0044] The amino acid sequence of p45 has no significant homology to other bacterial proteins when compared to previous submissions to databases accessible by BLAST.

[0045] The fragment of p45 provided in FIG. 7 can be used per se in preparation of an antigen-based vaccine. Alternatively, a nucleic acid sequence encoding this fragment can be employed in the context of a DNA vaccine against P. salmonis. The p45 sequence information can also be used to isolate and sequence genomic or cDNA clones from P. salmonis in order to enable production of a vaccine based on the complete nucleic acid sequence or amino acid sequence of p45.

[0046] clone 3

[0047] Related nucleic acid sequences are clone3/original, clone3/3PST-R, and clone3/3APA-F, shown in FIGS. 8, 9, and 10 (SEQ ID NOS: 10, 12, and 14, respectively). The amino acid sequences encoded by these nucleic acid sequences are presented in FIGS. 8, 9, and 10 (SEQ ID NOS: 11, 13, and 15, respectively).

[0048] The proteins encoded by the ORF of clone3/original, clone3/3PST-R and clone3/3APA-F have respectively 40%, 38% and 34% significant homology at the protein level to different portion of the transposase protein of Vibrio anguillarum (gb AAA81776.1) when compared to previous submissions to databases accessible by BLAST.

[0049] clone 7

[0050] Related nucleic acid sequences are clone7/original, clone7/XbaR, clone7/MunR, and clone 7/MunF, shown in FIGS. 11, 12, 13, and 14, respectively (SEQ ID NOS: 16, 18, 20, and 22, respectively). Amino acid sequences encoded by these nucleic acid sequences are presented in FIGS. 11, 12, 13, and 14, respectively (SEQ ID NOS: 17, 19, 21, and 23, respectively).

[0051] The genetic sequences have been derived from an inverse polymerase chain reaction (IPCR) product amplified from Piscirickettsia salmonis genomic DNA (gDNA).

[0052] The peptides encoded by the ORF of clone7/original, clone7/XbaR, clone7/MunR, and clone 7/MunF have a 40% to 44% significant homology at the protein level to different portion of the ABC transporter ATP-binding protein of the other bacterial species when compared to previous submissions to databases accessible by BLAST.

[0053] There is sufficient reason to believe that the nucleotide and corresponding amino acid sequence are of Piscirickettsia salmonis origin. Also, part of the ORFs was found in an immuno-reactive clone of an expression library.

[0054] clone 20

[0055] Related nucleic acid sequences are clone20/original (SEQ ID NO:24), and clone20/20VSPF (SEQ ID NO: 26), shown in FIGS. 15 and 16, respectively. The encoded amino acid sequences are SEQ ID NO:25 (FIG. 15) and SEQ ID NO: 27 (FIG. 16).

[0056] The genetic sequences have been derived from an inverse polymerase chain reaction (IPCR) product amplified from Piscirickettsia salmonis genomic DNA (gDNA).

[0057] The peptides encoded by the ORF of clone20/original, and clone20/20VSPF have a 41% and 51% significant homology at the protein level to an amino acid transporter/permase protein of other organisms when compared to previous submissions to databases accessible by BLAST.

[0058] clone 15

[0059] The nucleic acid sequence of clone15/original (SEQ ID NO: 28) and the encoded amino acid sequence thereof (SEQ ID NO: 29) are presented in FIG. 17.

[0060] The nucleotide sequence and the peptide encoded by the ORF of clone15/original have no significant homology to proteins of other bacterial species when compared to previous submissions to databases accessible by BLAST.

[0061] From the previous information there is sufficient reason to believe that the nucleotide and corresponding amino acid sequence are of Piscirickettsia salmonis origin. Also, the ORF was found in an immuno-reactive clone of an expression library.

[0062] Hsp70

[0063]
FIG. 18 depicts the amino acid sequence of hsp70 (SEQ ID NO: 30). FIG. 19 shows the amino acid sequence (SEQ ID NO:31) obtained from another hsp70 isolate (clone B). FIG. 20 is the nucleic acid sequence (SEQ ID NO:32) encoding the amino acid sequence of FIG. 19.

[0064] The amino acid sequence of hsp70 has 70% significant homology at the protein level to Coxiella burnetti and Legionella pneumophila hsp70 proteins when compared to previous submissions to databases accessible by BLAST.

[0065] Hsp60

[0066]
FIG. 21 depicts the nucleic acid sequence of the hsp60 gene of P. salmonis (SEQ ID NO:33). The amino acid sequence deduced from the nucleic acid sequence is presented in FIG. 22 (SEQ ID NO:34).

[0067] Characterization of the P. salmonis hsp60 gene was initiated by amplifying a 600 bp product from P. salmonis mRNA using universal degenerate primers. The primers are as described in Swee Han Goh et al.(1996) Journal Clin. Microbiol. 34(4): 818-823. The PCR product was sequenced by standard methods (Molecular Cloning: A Laboratory Manual (1989)2nd ed. Sambrook, J. et al., Cold Spring Harbor Laboratory, NY).

[0068] Once this first PCR product was sequenced, the sequence of the ORF was completed by performing several inverse PCR (IPCR) amplifications on genomic DNA from Piscirickettsia salmonis type strain LF-89 (Triglia et al, 1988, Nucl. Acids Res. 16: 8186). The IPCR products were sequenced in both directions from the 5′ and 3′ insertion sites using overlapping amplicons.

[0069] The nucleic acid sequence and protein sequence of P. salmonis hsp60 show similarity to the corresponding gene/protein in other species. The protein encoded by the ORF of P. salmonis hsp60 has a 75-77% significant homology to the GroEL protein of various Pseudomonas and Vibrio species when compared to previous submissions to databases accessible by BLAST.

[0070] Heat shock proteins are abundant molecules which participate in folding, assembly and disassembly of protein complexes. Hsp60 and Hsp70 are major targets of immune responses to a wide variety of pathogens, including bacteria, fungi, helminths and protozoan parasites. It has been demonstrated that immunization with certain pathogen hsps induces strong immune responses and provides protection against disease caused by these pathogens. The strength of these immune responses may reflect the abundance of these proteins, and their immunogenicity: multiple B cell and T cell epitopes are found on mycobacterial hsp60.

[0071] Consequently, there are grounds for believing that P. salmonis hsp70 and hsp60 can form the basis for a successful monovalent or multivalent vaccine targeted against SRS and related diseases.

[0072] The gene and protein sequences of the invention are preferably derived from P. salmonis type strain LF89. The invention encompasses nucleic acid sequences and amino acid sequences which are substantially homologous to the sequences provided in the Figures. “Substantially homologous” in this context means that a sequence, when compared to a reference sequence, has at least 60% homology, preferably at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology to the reference sequence.

[0073] To determine the percent homology of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence and the intervening non-homologous sequence in the gap can be disregarded for comparison purposes). There is no requirement for the two sequences to be the same length. In general, the length of sequence across which the sequences are compared is the entire extent of the alignment. Optionally, the length of a reference sequence aligned for comparison purpose is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least, 70%, 80%, or 90% of the length of the reference sequence. It possible to restrict homology analysis to any particular portion of the reference sequence.

[0074] When a position in the first (reference) sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the sequence, the molecules are homologous at that position (i.e. there is identity at that position). In the case of nucleic acid sequence comparison there is also homology at a certain position where the codon triplet including the nucleotide encodes the same amino acid in both molecules being compared, due to degeneracy of the genetic code.

[0075] The percent homology between two sequences is a function of the number of homologous positions shared by the sequences (i.e., % homology=no. of homologous positions/total no. of positions). Optionally, the comparison of sequences and determination of percent homology can be accomplished using a mathematical algorithm. Suitable algorithms are incorporated in to the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:430-10.

[0076] The definition of homologous sequences provided above embraces fragments of the reference nucleic acid sequence or amino acid sequence. For present purposes a “fragment” of a protein of the invention is understood to mean any peptide molecule having at least 20, optionally at least 30, or at least 40 contiguous amino acids of the reference amino acid sequence. A “fragment” of a nucleic acid reference sequence is any part of that sequence comprising at least 50, optionally at least 75, or at least 100 consecutive nucleotides.

[0077] Also comprised within the nucleic acid sequences of the invention are sequences which hybridize to the reference nucleic acid sequences under stringent conditions. “Stringent” hybridization conditions in the sense of the present invention are defined as those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104, i.e. a positive hybridization signal is still observed after washing for 1 hour with lx SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and most preferably at 68° C., in particular for 1 hour in 0.2×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and most preferably at 68° C.

[0078] In particular, the invention extends to oligonucleotides 15 to 100, preferably 20 to 50, most preferably 25 to 35 nucleotides in length, which are complementary to any of the nucleic acid sequences depicted in the figures or which are capable of hybridising to these sequences under stringent conditions.

[0079] The amino acid sequences of the invention also comprise synthetic analogues or derivatives of the sequences in the Figures, or of homologues of those sequences. A “derivative” of an amino acid sequence is a sequence related to the reference sequence either on the amino acid sequence level (e.g. a homologous sequence wherein certain naturally-occurring amino acids are replaced with synthetic amino acid substitutes) or at the 3D level, i.e. molecules having approximately the same shape and conformation as the reference amino acid sequence. Thus, derivatives include mutants, mimetics, mimotopes, analogues, monomeric forms and functional equivalents. Amino acid sequence derivatives retain the ability to induce the production of antibodies that recognize and (cross)-react with antigens of P. salmonis and/or to induce an immune response in fish that protects against infection with this pathogen.

[0080] The present invention provides the use of any of the nucleic acid sequences or amino acid sequences shown in the Figures, or related sequences, in the manufacture of a vaccine for the protection of fish against infection by Piscirickettsia salmonis.

[0081] The invention further provides a vaccine to protect fish against Piscirickettsia salmonis wherein the vaccine includes at least one nucleic acid or peptide sequence as defined herein, together with a pharmaceutically acceptable carrier.

[0082] A diagnostic test kit is also provided in accordance with the invention, whereby the kit may comprise a nucleic acid sequence or amino acid sequence of the invention, or may comprise an antibody capable of recognising any of the amino acid sequences of the invention.

[0083] The isolated nucleic acid sequences from P. salmonis can be exploited in the conventional manner, by cloning the gene into an expression vector for generation of large quantities of purified or isolated recombinant protein. A purified antigen can also be obtained by non-recombinant techniques, i.e. through extraction from cells by conventional purification methods. Alternatively, the protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques. A vaccine comprising this purified or isolated recombinant or non-recombinant protein can be termed an antigen-based vaccine.

[0084] An “isolated” or “purified” protein is defined as being substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the purified protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of a candidate protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of candidate protein having less than about 30% (by dry weight) of non-candidate protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of contaminating protein, still more preferably less than about 10% of contaminating protein, and most preferably less than about 5% contaminating protein. When the candidate protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0085] Alternatively, the genes of the invention can be incorporated into Nucleic Acid Vaccines (NAVs), whereby the NAV is taken up by host cells of a living animal, and expression of the gene takes place within the cytosol.

[0086] A gene inserted into a DNA vector can be inoculated directly into a fish (e.g. orally, intramuscularly or intraperitoneally) for expression in vivo within fish cells. Thus, in one aspect of the invention there is provided a nucleic acid vaccine comprising a pharmaceutically acceptable carrier and a DNA plasmid in which a nucleic acid sequence encoding a

[0087]

P. salmonis
gene of the invention is operably linked to a transcriptional regulatory sequence. Transcriptional regulatory sequences include promoters, polyadenylation sequences and other nucleotide sequences such as the immune-stimulating oligonucleotides having unmethylated CpG dinucleotides, or nucleotide sequences that code for other antigenic proteins or adjuvanting cytokines. For optimal in vivo expression it may be preferred to select transcriptional regulatory sequences endogenous to the fish to be vaccinated. For instance, endogenous cytokine or actin gene promoters may be considered. The DNA can be present in naked form or it can be administered together with an agent facilitating cellular uptake (e.g. liposomes or cationic lipids). The technology of DNA vaccination of fish is explained in more detail in U.S. Pat. No. 5,780,448, which is incorporated herein by reference.

[0088] Another aspect of the invention pertains to vectors, preferably expression vectors, comprising a nucleic acid sequencing encoding a gene of the invention (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0089] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operatively linked to the nucleic acid sequence to be expressed. Recombinant expression vectors of the invention may be used for expression within the intended recipient of the antigen of the invention (as a DNA vaccine) or for expression within a host organism other than the final recipient (for production of recombinant antigen vaccines).

[0090] Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[0091] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. A host cell can be any prokaryotic or eukaryotic cell. For example, hsp proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Other suitable host cells are known to those skilled in the art (e.g. Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0092] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0093] The present invention also relates to a method of generating monoclonal or polyclonal antibodies to an amino acid sequence of the invention. In this embodiment, an effective amount of the amino acid sequence (i.e., an amount which results in an immune response in the host) is introduced into an animal host which results in production of antibodies to the substance in the host. The antibodies are removed from the host and purified using known techniques (e.g. chromatography), thereby resulting in production of polyclonal antibodies. Procedures for immunizing animals, eg. mice, with proteins and selection of hybridomas producing immunogen-specific monoclonal antibodies are well known in the art (see for example Kohler and Milstein (1975) Nature 256: 495-497). The antibodies of the invention recognize (have an affinity to) at least one of the amino acid sequences disclosed herein. Preferably, the antibodies of the invention are raised against an isolated or purified amino acid sequence of the invention.

[0094] The vaccines manufactured in accordance with the methodology of the invention are suited for administering to any aquatic animal species for preventative or therapeutic purposes. The vaccines of the invention can be employed in treatment of teleosts such as salmon (Chinook, Atlantic, Coho), trout (including rainbow trout), carp, sea bream, sea bass, yellowtail, tilapia, grouper, catfish, halibut, haddock, or optionally for treatment of other aquatic species such as crustaceans and mollusks. Salmonid fish are the preferred species for treatment.

[0095] It is possible to immunize a subject with the neutral or the salt forms of the present purified or isolated proteins, either administered alone or in admixture with a pharmaceutically acceptable vehicle or excipient. Typically, vaccines are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to administration may also be prepared. The preparation may be emulsified or the active ingredient encapsulated in liposome vehicles. The pharmaceutical compositions of the invention may be administered in a form for immediate release or by extended release.

[0096] Pharmaceutically acceptable carriers or vehicles include conventional excipients, and may be, for example, solvents such as water, oil or saline, dextrose, glycerol, wetting or emulsifying agents, bulking agents, coatings, binders, fillers, disintegrants, diluents, lubricants, pH buffering agents, or conventional adjuvants such as muramyl dipeptides, avridine, aluminium hydroxide, oils, saponins, block copolymers and other substances known in the art.

[0097] To immunize a subject, an antigen or gene vector can be administered parenterally, usually by intramuscular injection in an appropriate vehicle, but optionally by the subcutaneous route, by intravenous injection or by intradermal or intranasal delivery. In the case of immunization of fish, the typical routes of administration are by injection into the peritoneal cavity, orally in feed, or by immersion in sea water or fresh water. Consequently it is preferred to administer the amino acid sequences and nucleic acid sequences of the invention in the form of oral or injectable formulations, or as a liquid (for instance a liquid emulsion or emulsifiable concentrate) to be added to a water tank or bath where the fish are held.

[0098] The effective dosage may vary depending on the size and species of the subject, and according to the mode of administration. The optimal dosage can be determined through trial and error by an aquaculture specialist or veterinarian. Typically, a single dose of antigen will be in the range of from about 0.01 to 1000 μg per kg body weight, preferably 0.5 to 500 μg per kg, more preferably 0.1 to 100 μg per kg. For DNA vaccines, a minimum dosage of 10 pg up to dosages of 1000 pg of plasmid per animal should be sufficient for suitable expression of the antigen in vivo.

[0099] The novel antigens disclosed as part of the present invention are also useful in screening for antibodies to pathogenic proteins. The invention additionally includes diagnostic uses of these antigens, for instance in the preparation of a diagnostic kit, useful for testing animals for the presence of disease-causing organisms.

[0100] It is also contemplated that antibodies raised against the purified antigens of the invention can have both diagnostic and therapeutic applications in disease management. Both polyclonal antibodies and monoclonal antibodies may be useful in this respect. Sandwich assays and ELISA may be mentioned as specific examples of diagnostic assays.

EXAMPLES

Example 1

[0101] Efficacy of nucleic acid vaccines comprising P. salmonis antigen sequences

[0102] Coho salmon parr (Oncorhynchus kisutch) less than 6 months old and of average weight 5.4 grams were obtained from a disease free stock and were acclimatised to water at a temperature of 9±1° C., flowing at a rate of 2.5 L/min. Stocking densities were maintained at <20 kg/m3, and the fish were fed a commercial pelleted diet at a daily rate of 1.5% body weight. The weight of fish in all groups was recorded prior to each vaccination and at the end of the trial. Any behavioural changes in the fish were also noted on a daily basis.

[0103] Coho salmon are particularly susceptible to infection with P. salmonis.

1TABLE 1sets out the experimental design:GroupSizeTreatmentDoseRoute1110pUK blank25μgi.m.2110pUK-Psclone51A25μgi.m.3110pUK-Pshsp6025μgi.m.4110pUK-Ps17kD25μgi.m.5110P. salmonis/oil0.1mli.p.6110PBS0.1mli.m.

[0104] The treatments were administered to randomly allocated groups of fish anaesthetized with 50 mg/L benzocaine, by single injection via the intramuscular (i.m.) route or intraperitoneal (i.p.) route.

[0105] pUK is an expression vector backbone which was not expected to induce any protection against SRS. pUK-psclone51A is a NAV construct carrying the entire ORF of Psclone51A. pUK-PsHSP60 is a NAV construct carrying the entire ORF of the P. salmonis hsp60 gene. pUK-Ps17kD is a NAV construct carrying the entire ORF of the P. salmonis p10.6 gene. The positive control group 4 was injected with a preparation of inactivated P. salmonis (strain LF89). This preparation is known to elicit protection against SRS, but is too expensive to produce on a commercial scale.

[0106] Following immunisation the fish were returned to holding tanks and kept there for 600 degree days before challenge. At this time fish from each treatment group were randomly divided into challenge tanks and control tanks. Treated fish were challenged by intraperitoneal injection with 0.1 ml containing approximately 103.5 TCID50 of cultured P. salmonis. Fish in each tank were monitored daily for mortality. Each mortality event was investigated for evidence of P. salmonis infection (by PCR).

Example 2

[0107] Immunogenicity of Antigens

[0108] Hsp60, Hsp70 and p10.6 nucleic acid sequences were inserted into a conventional expression vector. The recombinant proteins expressed in E. coli were purified and injected into mice in conjunction with an adjuvant. 40 days later, the mice were sacrificed and tested by ELISA for production of antibodies specific to the injected antigens. In every case, a specific immune response had been mounted to the antigen. A similar result was obtained when a plasmid NAV construct carrying the hsp70 gene was injected into mice.

[0109] These data provide solid evidence that the recombinant proteins Hsp60, Hsp70 and p10.6 are highly immunogenic, and are likely to be capable of inducing an immune response in fish, specifically targeting P. salmonis and thereby preventing development of SRS.

Number	Date	Country
0005838.8	Mar 2000	GB
0016080.4	Jul 2000	GB
0016082.0	Jul 2000	GB
0018599.1	Jul 2000	GB

	Number	Date	Country
Parent	PCT/GB01/01055	Mar 2001	US
Child	10241602	Sep 2002	US

Sequences from Piscirickettsia salmonis

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Continuation in Parts (1)