GB2345061A - Protein and nucleic acid sequences of groES and groEL from B.pseudomallei and their use as a vaccine - Google Patents

Abstract

The nucleic acid sequence of the operon that includes the sequences encoding the proteins of the groES and groEL and the deduced protein sequence of said proteins. Said nucleic and protein sequences derived from Burkholderia psuedomallei. The sequences are suitable for use in preventative treatment for infections caused by Burkholderia species, in particular Edinburgh/Toronto strain of Brukholderia cepacia and as part of a diagnostic kit. The sequences may be incorporated in a vaccine.

Description

Protein and Nucleic Acid Sequence This invention relates to a polypeptide sequence and to nucleic acid sequence encoding it, which sequences can be useful, for example in diagnosis as well as in prophylactic and therapeutic vaccine. In particular, the sequences are used in the diagnosis and prophylactic or therapeutic treatment of diseases caused by Burkholderia spp.

The diseases associated with Burkholderia spp. are numerous and include B. pseudomallei (the causative agent of melioidosis), B. mallei (the causative agent of glanders), and B. cepacia. Infection by B malleosis is endemic in Thailand. Glanders is a relatively widespread disease in many arecs and. is problematic for both military and civilian personnel, although it is becoming rarer in civilian personnel. Infection by B. cepacia is a problem for sufferers of cystic fibrosis who are prone to infection with Pseudomonas aeruginosa, Burkholderia cepacia or both.

Recently, a new strain of B. cepacia has emerged, the Edinburgh/Toronto strain, which is proving difficult to treat and is resulting in death of normally healthy cystic fibrosis sufferers. New treatments for B. cepacia infection in general as well as for the Edinburgh/Toronto strain, are desirable, as is a vaccine for use against melioidosis and/or glanders.

The present invention relates to treatments for these diseases and other diseases associated with infection by Burkholderia spp.

It is therefore an object of the present invention to provide diagnostic kits, vaccines or other treatments against diseases caused by Burkholderia.

In arriving at treatments for these diseases, the present inventors have sequenced an operon and have deduced protein sequences from B. pseudomallei.

Accordingly, the present invention provides a polypeptide of sequence ID NO 1 or a fragment thereof, or SEQ ID NO 2 or a fragment thereof, or a variant of any of these, which polypeptide produces an immunogenic response when administered to a mammal.

Fragments of the polypeptides of SEQ ID Nos 1 and 2 include deletion mutants and polypeptides where small regions of the polypeptides of SEQ ID Nos 1 and 2 are joined together.

They fragments should contain at least one antigenic region however so that they continue to produce an immunogenic response.

Suitable fragments of the protein or peptide have one or more amino acids deleted from the sequence and may be as small as 6 amino acids in length, provided they contain at least one antigenic determinant of the GROES or GROEL protein of Burkholderia spp.. Suitably the fragments will comprise at least 15, more suitably at least 30 and preferably at least 60 amino acids The expression'variant"refers to sequences of amino acids which differ from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as ^conservative"where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.

By'conservative substitution'is meant the substitution of an amino acid by another one of the same class; the classes being as follows: CLASS EXAMPLES OF AMINO ACID Nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, Glu Basic: Lys, Arg, His As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when'the substitution is in a region which is critical in determining the peptides conformation.

Non-conservative substitutions are possible provided that these do not interupt with the immunogenicity of the polypeptide.

Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably variants will be at least 85% homologous, preferably at least 90% homologous to the base sequence. Variants include allelic variants as well as proteins derived from other species which are encoded by nucleic acid sequences which hybridise to DNA sequences which encode SEQ ID NO 1 or NO 2 under stringent conditions as explained hereinafter.

The term mhomologous"as used herein means that two sequences, when aligned, have similar (identical or conservatively replaced) amino acids in like positions or regions, and where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein.

For example, two amino acid sequences which are at least 85% homologous to each other have at least 85% similar (identical or conservatively replaced amino residues) in a like position when aligned. Suitably there will be no more than 5 suitably no more than 3 gaps in the alignment, and preferably, each gap would affect no more than about 25, and preferably no more than 15 amino acid residues.

Homology may be determined using methods well known in the art (see, for example, Deveraux et al. 1984, Nucleic Acids Research 12,387-395, Wilbur, W. J. and Lipman, D. J."Rapid Similarity Searches of Nucleic Acid and Protein Data Banks." Proceedings of the National Academy of Sciences USA 80,726730 (1983) and Myers E. and Miller W."Optimal Alignments in Linear Space". Comput. Appl. Biosci. 4: 11-17 (1988)). One programme which may be used in determining homology is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1228, Selfpark Street, Madison, Wisconsin, 53715, USA as part of the Lasergene system.

The term ^polypeptide"as used herein includes long chain peptides including proteins.

SEQ ID No 1 is that of a protein, derived from B. pseudomallei, which has been designated GROEL, and this forms a preferred aspect of the invention.

SEQ ID No 2 is that of a protein, derived from B. pseudomallei, which has been designated GROES, and this forms a further preferred aspect of the invention.

In a further aspect, the invention provides a nucleic acid which encodes a polypeptide as described above. The nucleic acid may be DNA or and RNA, and where it is aDNA molecule, it may comprise a cDNA or a genomic DNA.

The present inventors have obtained a genomic DNA sequence encoding the polypeptides identified in SEQ ID NO 1 and SEQ ID NO 2, which is illustrated hereinafter as SEQ ID NO 3.

Thus, this sequence, and fragments thereof which encode polypeptides as described above form a further aspect of the invention. In this sequence, SEQ ID NO 1 (GROEL) is encoded by bases 477 to 2127, and SEQ ID NO 2 (GROES) is encoded by bases 136 to 429.

The present invention also includes DNA which hybridises to the DNA of SEQ ID NO 3 and which codes for a polypeptide of the invention. Preferably, such hybridisation occurs at high stringency conditions, for example those illustrated in Molecular Cloning", A Laboratory Manual"by Sambrook, Fritsch and Maniatis, Cold Spring Habor Laboratory Press, Cold Spring Harbor, N. Y.

Examples of high stringency conditions include hybridisation in 0.1 x SSC at about 65 C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate.

Examples of nucleic acids of the invention will have at least 85% homology to SEQ ID NO 3 or a fragment thereof.

SEQ ID NO 3 has been found to have some homology with heat shock proteins, HSP 10 and HSP 60.

The nucleic acid of SEQ ID NO 3 is very rich in guanine and cytosine residues (i. e. they are GC rich). Since these residues bind together very strongly, it is extremely difficult to separate the DNA strands in order to sequence them. By amplifying the product for cloning, based on a hypothetical protein sequence, the present inventors have been able to overcome these difficulties and have determined the actual genomic sequence.

Suitably the polypeptide of the invention produces an immunogenic response in a mammal to which it is administered which is protective against infection by Burkholderia spp. as such proteins can be used as a basis for a vaccine.

The present invention further provides a prophylactic or therapeutic vaccine against Burkholderia related diseases, the vaccine comprising a polypeptide as defined above, in combination with a pharmaceutically acceptable carrier or diluent.

Due to the high levels of conservation of the genetic code of this operon of these organisms, it is likely vaccines to the above identified diseases will give good crossprotection to other Burkholderia diseases.

The vaccine may comprise the protein or peptide itself, suitably formulated as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or excipient. Such compositions form a further aspect of the invention. The compositions may be in a form suitable for intra-muscular, intra-venous, mucosal or parenteral application. Mucosal applications include intra-nasal or oral applications.

Suitable carriers are well known in the art and include solid and liquid diluents, for example, water, saline or aqueous ethanol. The liquid carrier is suitably sterile and pyrogen free.

The compositions may be in the form of liquids suitable for infusion or injection, or syrups, suspensions or solutions, as well as solid forms such as capsules, tablets, or reconstitutable powders.

Proteins or peptides as described above may be prepared by various means including chemical synthesis, isolation from natural sources followed by any chemical modification if required, or more preferably, using recombinant DNA technology. Thus in a preferred embodiment, there is provided a method for preparing a peptide as described above, which method comprises including nucleotide sequence which encodes said peptide into a recombinant expression vector, transforming a host cell with said vector, and culturing said cell and recovering the peptide from the culture. The host cell may be eukaryotic or prokaryotic, but is conveniently a prokaryotic cell such as E. coli.

In an alternative embodiment, the vaccine may be based upon a nucleic acid, and so comprise a DNA or cDNA vaccine. Such a vaccine may be constructed using cells. Preferably, the cell is a eucaryotic cell, such as J774 or a recombinant bacterial cell, such as recombinant Salmonella although other systems are available. The vaccine may comprise a sequence encoding functional fragments of the polypeptides of the invention.

Examples of such a vaccine may be in the form of a vector, which is used to transform a cell, preferably a eucaryotic cell, which is adapted to express the protein or peptide of the invention in situ. Such vectors form a further aspect of the invention. The vector may contain the usual expression control functions such as promoters, enhancers and signal sequences, as well as a selection marker in order to allow detection of successful transformants. The selection of these will depend upon the precise nature of the vector chosen and will be known to or readily determinable by a person skilled in the art.

The vaccine may alternatively be in the form of a so-called "naked DNA"vaccine or in DNA vaccine where the vector consists of a DNA plasmid which is adapted to express the protein or peptide in situ.

Where the vector is a viral vector, it is suitably attenuated to minimise any harmful effects associated with the virus on the host.

Preferably, the viral vector is derived from vaccinia virus, as it has many properties which make it a suitable vector for vaccination, including its ability to efficiently stimulate humoral as well as cell-mediated immune responses.

Where the vaccine of the present invention may comprise an adjuvant, for example a cytokine, particularly IL-12. This is particularly useful where the vaccine is a protein vaccine ; the vaccine may use whole protein or functional fragments of protein.

The vaccine of the invention may be incorporated into a multivalent vaccine in order to increase the benefit-to-risk ratio of vaccination.

The dosage of the vaccines of the invention will depend upon the nature of the mammal being immunised as well as the precise nature and form of the vaccine. This will be determined by the clinician responsible. However in general, when using a virus vector such as a vaccinia virus vectors, dosages of the vector may be in the range of from 104-1012pfu (pfu = particle forming units).

The vaccines of the invention will produce an immune response in test animals, preferably a T-cell response.

Preferably, the proteins or genes of the invention are used to form a pharmaceutical composition useful in the treatment of melioidosis, glanders or cepacia-related diseases. Most preferably, they are used to form a vaccine.

In the light of the homology with heat shock proteins, and in particular HSP 10 and HSP 60, these proteins themselves may provide a protective immune response. Thus in an alternatively embodiment, the invention provides the use of a heat shock protein or a fragment or variant thereof in the diagnosis, prevention or therapy of infection by Burkholderia spp.. In particular, the invention provides a vaccine which comprises heat shock protein 10 or 60 of Burkholderia or modified heat shock proteins 10 or 60 derived from one or more of the sequences.

The polypeptides of the invention may also be suitable for use in the diagnosis of infection by Burkholderia spp. A sample of biological fluid such as serum, taken from a mammal suspected of suffering from such an infection may be contacted with a polypeptide of the invention, and the presence of antibodies which bind to said polypeptides can be determined. Techniques for determining such interaction are well known in the art. They include direct and competitive assays. In general, the polypeptide of the invention will be immobilised on a surface, for example in a well of an assay plate or on a dipstick device. Signal generation can be produced using known techniques such as enzyme-linked immunoassay techniques (ELISA).

Alternatively, the polypeptides of the invention will give rise to antibodies which form a further aspect of the invention. Antibodies may be polyclonal or monoclonal and are preferably monoclonal. These also may be used in diagnostic assays to those described above. These antibodies may be useful, for example, in diagnosis of melioidosis, glanders or infection with cepacia. For diagnostic purposes, the antibodies may form part of a kit as is conventional in the art.

In this instance, the presence of polypeptides of the invention, which are characteristic of the disease state would be detected. There is a possibility that the antibodies themselves may be useful in a passive immunisation program and thus the invention further comprises vaccines which include said antibodies. These may be formulated in a similar manner to those described above.

Thus in yet a further aspect, the invention provides a diagnostic kit which comprises either a polypeptide as described above or an antibody as described above.

In determining the gene sequence, the present inventor has determined the existence of an intergenic spacer between the regions encoding GROES and GROEL ; this spacer may be useful in identifying the various strains of Burkholderia, that is, it is useful as a"fingerprint"for each of the various strains and/or species. The intergenic spacer lies between base pairs 429 and 477 of Sequence ID 3.

Thus in a further aspect, the invention provides a nucleic acid which corresponds to bases 429 to 477 of SEQ ID NO 3 or a sequence which hybridises to said sequence under stringent hybridisation conditions. Said nucleic acid may form the basis for a diagnostic kit.

Thus in yet a further aspect, the invention provides a method of characterising a Burkholderia species, which method comprises contacting a nucleic acid sequence which corresponds to bases 429 to 477 of SEQ ID NO 3, or a sequence which hybridises to said sequence under stringent hybridisation conditions, with a sample suspected to contain said species or DNA extracted therefrom, and determining hybridisation between said sequences. For ease of operation, the sequence used in the hybridisation experiment is suitably labelled, for example with a fluorescent label.

Alternatively the nucleic acid sequences of the invention, and particularly those based upon SEQ ID NO 3 or fragments thereof, may be used in diagnosis. For example, the nucleic acid may be used in hybridisation assays or amplification assays in order to determine the presence of Burkholderia spp. in a sample suspected of containing such. Probes for example labelled or immobilised probes and/or amplification primers may be designed based upon said sequence, and these may be used in the above-mentioned assays in a conventional manner. Thus the invention further provides a diagnostic kit comprising a nucleic acid sequence as described above.

Embodiments of the invention will now be described, with reference to the following non-limiting examples and with reference to the accompanying drawings in which: Figure 1 shows SEQ ID NO 1 which is the amino acid sequence of the GROEL peptide; Figure 2 shows SEQ ID NO 2 which is the amino acid sequence of the GROES peptide ; and Figure 3 show SEQ ID NO 3 which is the genomic sequence of a Burkholderia pseudomallei operon.

Example 1 Sequences were amplified using the polymerase chain reaction (PCR) using degenerate primers based on conserved regions of the proteins from other bacterial species. PCR product were sequenced using degenerate primers initially, then specific sequence primers once some sequence was known. Standard sequencing technology including use of an automated sequencer were employed.

Computer analysis of the nucleotide sequence obtained was used to determine the polypeptide sequences of GROEL and GROES.

Claims (24)

1. CLAIMS 1. A polypeptide of SEQ ID NO 1 or a fragment thereof, or SEQ ID NO 2 or a fragment thereof, or a variant of any of these, which polypeptide produces an immunogenic response when administered to a mammal.
2. 2. A polypeptide according to claim 1 which comprises SEQ ID NO 1 or a sequence which is at least 85% homologous thereto.
3. 3. A polypetide according to claim 3 which comprises SEQ ID NO 1.
4. 4. A polypeptide according to claim 1 which comprises SEQ ID NO 2 or a sequence which is at least 85% homologous thereto.
5. 5. A polypetide according to claim 4 which comprises SEQ ID NO 2.
6. 6. A polypeptide according to any one of claims 1 to 5 wherein the immunogenic response is protective against at least one Burkholderia spp.
7. 7. A nucleic acid which encodes a polypeptide as claimed in any one of claims 1 to 6.
8. 8. A nucleic acid according to claim 6 which comprises SEQ ID NO 3 or a coding region thereof.
9. 9. A pharmaceutical composition which comprises a polypeptide according to claim 6 or a nucleic acid sequence which encodes said polypeptide in combination with a pharmaceutically acceptable carrier or diluent.
10. 10. A pharmaceutical composition according to claim 9 which is a prophylactic vaccine.
11. 11. A pharmaceutical composition according to claim 10 which is a vaccine against Edinburgh/Toronto Burkholderia cepacia and/or glanders.
12. 12. A pharmaceutical composition according to any one of claims 9 to 11 which comprises a polypeptide according to claim 6.
13. 13. A pharmaceutical composition according to any one of claims 9 to 11 which comprises a nucleic acid which encodes a polypeptide according to claim 6.
14. 14. The use of a polypeptide according to any one of claims 1 to 6 in the prevention or treatment of infection by Burkholderia spp..
15. 15. The use of a polypeptide according to any one of claims 1 to 6 in the preparation of a medicament for the prevention or treatment of infection by Burkholderia spp..
16. 16. A diagnostic kit comprising a polypeptide according to any one of claims 1 to 6.
17. 17. A diagnostic kit comprising a nucleic acid according to claim 7.
18. 18. An antibody to a polypeptide according to any one of claims 1 to 6.
19. 19. An antibody according to claim 18 which is a monoclonal antibody.
20. 20. A diagnostic kit which comprises an antibody according to claim 18 or claim 19.
21. 21. A nucleic acid which corresponds to bases 429 to 477 of SEQ ID NO 3 or a sequence which hybridises to said sequence under stringent hybridisation conditions.
22. 22. A diagnostic kit comprising a nucleic acid according to claim 21.
23. 23. A diagnostic kit according to claim 16 or claim 17 for the diagnosis of Edinburgh/Toronto Burkholderia cepacia and/or glanders.
24. 24. The use of a heat shock protein in the preparation of a medicament for the prevention or therapy of infection by Burkholderia spp..