SEQUENCE

Abstract: The present invention relates to a fish vaccine. More specifically the invention relates to a vaccine to protect salmon against infection by <i>Piscirickettsia salmonis</i>. The invention is based on or derived from the nucleic acid or amino acid sequence of at least a part of the surface antigen present on <i>Piscirickettsia salmonis</i>. Nucleic acid and/or amino acid sequences may be used in the preparation of a vaccine to protect against infection by <i>Piscirickettsia salmonis</i>.

Description: "Sequence" 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) . To date no commercially available vaccine is effective against Piscirickettsia salmonis, the causative agent of SRS. Accordingly there is a need for an effective vaccine against Piscirickettsia salmoni s . It is an object of the present invention to provide a vaccine to protect against SRS. It is an object of the present invention to provide a vaccine to protect against Piscirickettsia salmoni s . The present invention relates to a fish vaccine. More specifically the invention relates to a nucleic acid vaccine for administration to fish to protect against piscirickettsiosis also referred to as salmonid rickettsial septicaemia (SRS) . The invention further provides an amino acid sequence for administration to fish to protect agsint SRS. In a further aspect the present invention provides at least one nucleic acid sequence and / or an amino acid sequence which is derived from Piscirickettsia salmonis or a synthetically prepared analogue thereof or a substantially homologous sequence. A substantially homologous nucleic acid sequence is a sequence which can be transcribed and/or translated to provide an amino acid sequence which is substantially homologous to at least a part of a surface antigen present on Piscirickettsia salmonis . A substantially homologous amino acid sequence encodes at least 70% of a part of a surface antigen and induces an immune response. More preferably the homologous amino acid sequence will encode at least 90% of the amino acid sequence. A typical nucleic acid sequence is chosen from the sequences of Psclone51A plθ.6 (also known as immuno- reactive clone 0110-2-5), IcmE, clone3/original , clone3/3PST-R, clone3.3APA-F, clone7/original , clone7/XbaR, clone7/7MunR, clone7/7MunF, clone20/original, clone20/20VSPF or clone 15. Typically the amino acid sequence is derived from one of the above-mentioned nucleic acid sequences or is chosen from the sequences of p45 or HSP70 antigen. Preferably peptide sequences or nucleic acid sequence identified herein as sequence ID numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 are utilised in a vaccination strategy to induce an immune response to a surface antigen of Pi sciri ckettsia salmoni s and thus to Pi sciri cket tsia salmoni s itself. The invention provides the use of nucleic acid sequences or peptide sequence as defined herein in the preparation of a vaccine for the protection of fish against Pi sciricket tsia salmoni s . The invention further provides a vaccine to protect fish against Pi sciri cket tsia salmoni s wherein the vaccine includes nucleic acid or peptide sequences as defined herein. Sequence ID 1, 2 and 3 A detailed description of the invention, listing the exact nucleotide sequence of Psclone51A and the amino acid sequence deduced for the open reading frame (ORF) , is provided in figures 1, 2 and 3. The genetic sequence has been derived from cloned cDNA wherein the cDNA clones were derived from Piscirickettsia salmonis type strain (LF-89) messenger RNA (mRNA) . The cloned material was sequenced in both directions from the 5' and 3' insertion sites using overlapping amplicons. Veracity of the Psclone51A sequence was confirmed by Polymerase Chain Reaction (PCR) and Reverse- Transcriptase PCR (RT-PCR) of appropriate sized amplicons from Pi scirickettsia salmonis infected Chinook salmon embryonic (CHSE-214) cell line and not from uninfected control material . Expression of the cloned sequence has yielded a protein of approximately 12 kDa. The ORF of Psclone51A described in figure 1 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 Escheri chia coli was found. The ORF has commercial value for the following reasons: There is sufficient reason to believe that the nucleotide corresponding amino acid sequence is of Piscirickettsia salmonis origin. As such its incorporation into nucleic acid vaccines may have an impact on the reduction of mortality of farmed Atlantic salmon caused by Pisciri cket tsia salmoni s . Characterisation of the gene product will lead to the identification of key elements in pathogenesis of infection and to the design of more accurate diagnostic tests which will also aid in epidemiological studies documenting the dissemination of different strains of the disease. The nucleotide sequence Psclone51A and associated derivatives thereof when translated into protein sequence being composed of either identical or equivalent amino acids, should induce a response by the hosts' immune system. This principle can be further expanded to use these proteins in diagnostics tests and vaccination procedures. Sequence ID No 4 and 5 A detailed description of the invention, listing the exact nucleotide sequence of plθ.6 and the amino acid sequence deduced for the open reading frame (ORF) , is provided in figure 4 and figure 5 respectively. The genetic sequence of Pi sciri ckettsia salmoni s (Atcc strain VR-1361) , grown in CHSE-214 cells using homologous anistera from Piscirickettsia salmoni s immunised rabbits, an immuno-reactive clone (0110-2- 5) (plθ.6) was identified from the expression library. This clone was sequenced and polymerase chain reaction (PCR) primers developed. The Pi sciri cket tsia salmoni s origin of the pl0.6 clone was confirmed through PCR amplification of Pi sciri ckettsia salmoni s and Chinook salmon embryonic cell line (CHSE-214) DNA using clone specific primers. Appropriately sized amplicons were amplified from the Pi sciri cket tsia salmoni s DNA, but not from the CHSE-214 DNA confirming that the immuno-reactive clone was of Piscirickettsia salmonis origin and not from the host cell DNA. The open reading frame (ORF) for the full gene was completed by inverse PCR from genomic Pi sciri cket tsia salmoni s DNA. The ORF of pl0.6 described in figure 4 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 17kDa antigen found in Rickettsia of the Spotted Fever Group, where it is considered a group specific, outer membrane protein. Sequence ID No 6 A typical nucleic acid sequence is IcmE (403) . Preferably peptide sequences transcribed or translated from the nucleic acid sequences of IcmE (403) are incorporated into a vaccination strategy to induce an immune response to a surface antigen of Pi sciri cket tsia salmoni s itself. A detailed description of the invention, listing the exact nucleotide sequence of IcmE (403) and the amino acid sequence deduced for the open reading from (OFR) , is provided in figure 6. The genetic sequence has been derived from an inverse polymerase chain reaction (IPCR) product amplified from Pi sciri cket tsia salmoni s type strian (LF-89) genomic DNA (gDNA) . The IPCR product was sequenced in both direction from the 5' and 3' sides using overlapping amplicons. The protein encoded by the ORF of IcmE (403) has a 37% significant homology at the protein level to the IcmE protein of Legionella pneumophi la when compared to previous submissions to databases accessible by BLAST. Sequence ID No 7 A typical amino acid sequenes is p45. Preferably a nucleic acid sequence transcribing the amino acid sequence of p45 are incorporated into a vaccination strategy to induce an immune response to a surface antigen against Pi sciri cket tsia salmoni s and thus to Pi sciri ckettsia salmoni s itself. A detailed description of the invention, listing the exact amino acid sequence of a portion of the p45 major antigen, is provided in figure 7. The amino acid sequence has 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 (SHSE-214) cells infected with Piscirickettsia salmonis and not in infected CHSE-214 cells. The amino acid sequence of p45 has no significant homology to other bacterial proteins when compared to previous submissions to databases accessible by BLAST. Sequence ID No 8, 9 and 10 A typical nucleic acid sequence is clone3/original , clone3/3PST-R, and clone3.3APA-F. Preferably peptide sequences transcribed or translated from the nucleic acid sequence of clone3/original, clone3/3PST-R, and clone3.3APA-F are incorporated into a vaccination strategy to induce an immune response to a surface antigen of Pi sciricket tsia salmoni s genomic DNA (gDNA) . 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 Vijbrio anguillarum (gb AAA81776.1) when compared to previous submissions to databases accessible by BLAST. Sequence ID No 11, 12, 13 and 14 A typical nucleic acid sequence is clone7/original , clone7/7XbaR, clone7/7MunR, and clone 7/7MunF. Preferably peptide sequences transcribed or translated from the nucleic acid sequence of clone7/original, clone7/7XbaR, clone7/7MunR, and clone 7/7MunF are incorporated into a vaccination strategy to induce an immune response to a surface antigen of Piscirickettsia salmonis and thus to Pisciri cket tsia salmoni s itself. A detailed description of the invention, listing the extract nucleotide sequence of clone7/original , clone7/7XbaR, clone7/7MunR, and clone 7/7MunF and the amino acid sequence deduced for their open reading frames (ORF), is provided in figures 11, 12, 13 and 14 respectively. Some of the genetic sequences have been derived from an inverse polymerase chain reaction (IPCR) product amplified from Pi scirickettsia salmonis genomic DNA (gDNA) . The peptides encoded by the ORF of clone7/original , clone7/7XbaR, clone7/7MunR, and clone 7/7MunF 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. 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. Sequence ID No 15 and 16 A typical nucleic acid sequence is clone20/original , and clone20/20VSPF. Preferably peptide sequences transcribed or translated from the nucleic acid sequence of clone20/original, and clone20/20VSPF are incorporated into a vaccination strategy to induce an immune response to a surface antigen of Pi sciri ckettsia salmoni s and thus to Pi sciri cket tsia salmonis itself. A detailed description of the invention, listing the exact nucleotide sequence of clone20/original , and clone20/20VSPF and the amino acid sequence deduced for their open reading frames (ORF) , is provided in figure 15 and figure 16 respectively. Some of the genetic sequences have been derived from an inverse polymerase chain reaction (IPCR) product amplified from Piscirickettsia salmonis genomic DNA (gDNA) . 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. Sequence ID No 17 A typical nucleic acid sequence is clonel5/original . Preferably peptide sequences transcribed or translated from the nucleic acid sequence of clonel5/original are incorporated into a vaccination strategy to induce an immune response to a surface antigen of Piscirickettsia salmonis and thus to Pi sciri cket tsia salmoni s itself. A detailed description of the invention, listing the exact nucleotide sequence of clonel5/original and the amino acid sequence deduced for its open reading frames (ORF), is provided in figure 17. The nucleotide sequence and the peptide encoded by the ORF of clonel5/original have no significant homology to proteins of other bacterial species when compared to previous submissions to databases accessible by BLAST. 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. Sequence ID No 18 A typical amino acid sequence is HSP70. Preferably a nucleic acid sequence transcribing the amino acid sequence HSP70 are incorporated into a vaccination strategy to induce an immune response to a surface antigen of Piscirickettsia salmoni s and thus to Piscirickettsia salmonis itself. A detailed description of the invention, listing the exact amino acid sequence of the major portion of the HSP70 protein is provided in figure 18. 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. The nucleic acid and amino acid sequences disclosed have commercial value for the following reasons: From the previous information there is sufficient reason to believe that the nucleotide and corresponding amino-acid sequences are of Piscirickettsia salmonis origin. Also, several of the ORF were found in immuno-reactive clones of an expression library. Together, this shows the potential of these ORFs to be incorporated into nucleic acid vaccines that may have an impact on the reduction of mortality of farmed salmonids due to Rickettsial Salmonid Septicaemia caused by Pi sciri cket tsia salmoni s . PCR primers derived from the full ORF, used in touchdown PCR are capable of detecting Piscirickettsia salmonis in tissues from infected fish. This will lead to more accurate diagnostic tests to be used for clinical as well as epidemiological study purposes. The nucleotide sequences associated derivatives thereof when translated into protein sequence being composed of either identical or equivalent amino acids, should induce a response by the hosts' immune system. This principle can be further expanded to use these proteins in diagnostic tests and nucleic acid vaccination procedures.

Claims: CLAIMS 1. A nucleic acid which can be transcribed to provide an amino acid sequence which is substantially homologous to at least a part of the surface antigen present on Piscirickettsia salmoni s . 2. A nucleic acid sequence as claimed in claim 1 which encodes a part of a surface antigen of Piscirickettsia salmonis which induces an immune response. 3. A nucleic acid sequence as claimed in claim 1 or claim 2 wherein the nucleic acid sequence is herein chosen from the sequences described as sequence ID numbers 1, 2, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. 4. Use of the nucleic acid sequences set out in any of the preceding claims in the preparation of a vaccine to protect against infection by Pi scirickettsia salmoni s . 5. An amino acid sequence derived from the sequence of any of claims 1 to 4. 6. Use of at least one amino acid sequence chosen from sequence ID numbers 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 in the preparation of a vaccine to protect fish against Pi sciri cket tsia salmoni s . 7. A nucleic acid based vaccine comprising at least one sequence chosen form the sequences described herein under sequence ID numbers 1, 2, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. 8. An amino acid based vaccine against Piscirickettsia salmonis comprising at least one sequence chosen from the amino acid sequences described herein under ID numbers 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

Inventors: BROUWERS HUUB, BURZIO LUIS, GRIFFITHS STEVE, JONES SIMON, SIMARD NATHALIE, VALENZUELA PABLO, BROUWERS, HUUB, BURZIO, LUIS, GRIFFITHS, STEVE, JONES, SIMON, SIMARD, NATHALIE, VALENZUELA, PABLO
Applicants: AQUA HEALTH EUROP LTD, BROUWERS HUUB, BURZIO LUIS, GRIFFITHS STEVE, JONES SIMON, SIMARD NATHALIE, VALENZUELA PABLO, AQUA HEALTH (EUROPE) LIMITED, BROUWERS, HUUB, BURZIO, LUIS, GRIFFITHS, STEVE, JONES, SIMON, SIMARD, NATHALIE, VALENZUELA, PABLO
Application year: 2001
Main classification: C12N15
IPC codes: A61K39, A61P31, C07K14, C12N15