NZ502423A - Polynucleotide sequences, designated GS, in pathogenic mycobacteria and their use in vaccines - Google Patents

Abstract

A polynucleotide comprising a gene region designated GS, which is found in Mycobacterium paratuberculosis (Mptb). GS is also defined in Mycobacterium avium. subsp.silvatium (Mavs). The complete DNA sequence incorporating the positive strand of GS from an isolate of Mavs comprises 7995 nucleotides, including the core region of GS and adjacent transposable elements. The DNA sequence of GS from Mptb comprises 4435 bp including the core region of GS (nucleotides 1614 to 6047 of GS in Mavs). The DNA sequence of GS from Mptb is 99.4% homologous to GS in Mptb. There are eight open reading frames (ORFs) in GS. Six of these, designated GSA, GSB, GSC, GSD, GSE and GSF are encoded by the core DNA region of GS which, characteristically for a pathogenicity island, has a different GC content from the rest of the microbial genome. The proteins encoded by the ORFs in GS may be used in immunoassays of antibody or cell mediated immuno-reactivity for diagnosing infections caused by the mycobacteria. Mutation or deletion of all or some of the ORFs in GS may be used to generate attenuated strains of Mptb, Mavs or Mtb with lower pathogenicity for use as living or killed vaccines in humans and animals.

Description

/ \ 4. / Patents Form 5 N.Z. No.

Divided out of Parent Application No. 324777 NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION NOVEL POLYNUCLEOTIDES AND POLYPEPTIDES IN PATHOGENIC MYCOBACTERIA AND THEIR USE AS DIAGNOSTICS, VACCINES AND TARGETS FOR CHEMISTRY We, ST. GEORGE'S HOSPITAL MEDICAL SCHOOL, a British company of, Cranmer Terrace, London SW17 ORE, Great Britain, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 1 - (Followed by 1A) INTELLECTUAL PROPERTY OFFICE OF NZ. 1 7 OCT 2001 RECEIVED - 1A - NOVEL POLYNUCLEOTIDES AND POLYPEPTIDES IN PATHOGENIC MYCOBACTERIA AND THEIR USE AS DIAGNOSTICS, VACCINES AND TARGETS FOR CHEMOTHERAPY The present invention is a divisional of parent, specification No. 324777 and relates to the novel polynucleotide sequence we 5 have designated "GS" which we have identified in pathogenic mycobacteria. GS is a pathogenicity island within 8kb of DNA comprising a core region of 5.75kb and an adjacent transmissable element within 2.25kb. GS is contained within Mycobacterium paratuberculosis, Mycobacterium avium subsp. silvaticum and some 10 pathogenic isolates of M.avium. Functional portions of the core region of GS are also represented by regions with a high degree of homology that we have identified in cosmids containing genomic DNA from Mycobacterium tuberculosis.

Background to the invention Mycobacterium tuberculosis (Mtb) is a major cause of global diseases of humans as well as animals. Although conventional methods of diagnosis including microscopy, culture and skin testing exist for the recognition of these diseases, improved 20 methods particularly new immunodiagnostics and DNA-based detection systems are needed. Drugs used to treat tuberculosis are increasingly encountering the problem of resistant organisms. New drugs targeted at specific pathogenicity determinants as well as new vaccines for the prevention and treatment of tuberculosis 25 are required. The importance of Mtb as a global pathogen is reflected in the commitment being made to sequencing the entire genome of this organism. This has generated a large amount of DNA sequence data of genomic DNA within cosmid and other libraries. Although the DNA sequence is known in the art, the functions of the vast majority of these sequences, the proteins «■ they encode, the biological significance of these proteins, and the overall relevance and use of these genes and their products as diagnostics, vaccines and targets for chemotherapy for tuberculous disease, remains entirely unknown.

Mycobacterium avium subsp. silvaticum (Mavs) is a pathogenic mycobacterium causing diseases of animals and birds, but it can WO 97/2*":24 PCT/GB96/03221 also affect humans . Mycobacterium paratuberculosis (Mptb) causes chronic inflammation of the intestine in many species of animals including primates and can also cause Crohn's disease in humans. Mptb is associated with other chronic inflammatory diseases of 5 humans such as sarcoidosis. Subclinical Mptb infection is widespread in domestic livestock and is present in milk from infected animals. The organism is more resistant to pasteurisation than Mtb and can be conveyed to humans in retail milk supplies. Mptb is also present in water supplies, 10 particularly those contaminated with run-off from heavily grazed pastures. Mptb and Mavs contain the insertion elements IS900 and IS902 respectively, and these are linked to pathogenicity in these organisms. IS900 and IS902 provide convenient highly specific multi-copy DNA targets for the sensitive detection of 15 these organisms using DNA-based methods and for the diagnosis of infections in animals and humans. Much improvement is however required in the immunodiagnosis of Mptb and Mavs infections in animals and humans. Mptb and Mavs are in general, resistant in vivo to standard anti-tuberculous drugs. Although substantial 20 clinical improvements in infections caused by Mptb, such as Crohn's disease, may result from treatment of patients with combinations of existing drugs such as Rifabutin, Clarithromycin or Azithromycin, additional effective drug treatments are required. Furthermore, there is an urgent need for effective 25 vaccines for the prevention and treatment of Mptb and Mavs infections in animals and humans based upon the recognition of specific pathogenicity determinants.

Pathogenicity islands are, in general, 7-9kb regions of DNA comprising a core domain with multiple ORFs and an adjacent 30 transmissable element. The transmissable element also encodes proteins which may be linked to pathogenicity, such as by providing receptors for cellular recognition. Pathogenicity islands are envisaged as mobile packages of DNA which, when they enter an organism, assist in bringing about its convertion from 35 a non-disease-causing to a disease-causing strain.

Description of the Drawings WO 97/2" 4 \ PCT/GB96/0322I Figure 1(a) and (b) shows a linear map of the pathogenicity island GS in Mavs (Fig la) and in Mptb (Fig lb). The main open reading frames are illustrated as ORFs A to H. ORFs A to F are found within the core region of GS. ORFs G and H are encoded by 5 the adjacent transmissable element portion of GS.

Disclosure of the invention Using a DNA-based differential analysis technology we have discovered and characterised a novel polynucleotide in Mptb ("isolates 0022 from a Guernsey cow and 0021 from a red deer) . 10 This polynucleotide comprises the gene region we have designated GS. GS is found in Mptb using the identifier DNA sequences Seq.ID.No 1 and 2 where the Seq.ID No2 is the complementary sequence of Seq.ID No 1. GS is also identified in Mavs. The complete DNA sequence incorporating the positive strand of GS 15 from an isolate of Mavs comprising 7995 nucleotides, including the core region of GS and adjacent transsmissable element, is given in Seq.ID No.3. DNA sequence comprising 4435 bp of the positive strand of GS obtained from an isolate of Mptb including the core region of GS (nucleotides 1614 to 6047 of GS in Mavs) 20 is given in Seq.ID No 4. The DNA sequence of GS from Mptb is highly (99.4%) homologous to GS in Mavs. The remaining portion of the DNA sequence of GS in Mptb, is readily obtainable by a person skilled in the art using standard laboratory procedures. The entire functional DNA sequence including core region and 25 transmisable element of GS in Mptb and Mavs as described above, comprise the polynucleotide sequences of the invention.

There are 8 open reading frames (ORFs) in GS. Six of these designated GSA, GSB, GSC, GSD, GSE and GSF are encoded by the # core DNA region of GS which, characteristically for a 30 pathogenicity island, has a different GC content than the rest of the microbial genome. Two ORFs designated GSG and GSH are encoded by the transmissable element of GS whose GC content resembles that of the rest of the mycobacterial genome. The ORF GSH comprises two sub-ORFs Hx H2 on the complementary DNA strand 35 linked by a programmed frameshif ting site so that a single polypeptide is translated from the ORF GSH. The nucleotide PCT/G B96/03221 sequences of the 8 ORFs in GS and their translations are shown in Seq. ID No 5 to Seq.ID No 29 as follows: ORF A: Seq. ID No 5 Nucleotides 50 to 427 of GS from Mavs Seq. ID No 6 Amino acid sequence encoded by Seq.ID No 5 5.

ORF B: Seq. ID No 7 Nucleotides 772 to 1605 of GS from Mavs Seq. ID No 8 Amino acid sequence encoded by Seq.ID No 7.

ORF C: Seq. ID No 9 Nucleotides 1814 to 2845 of GS from Mavs ^10 Seq. ID No 10 Amino acid sequence encoded by Seq.ID No 9.

Seq. ID No 11 Nucleotides 201 to 1232 of GS from Mptb Seq. ID No 12 Amino acid sequence encoded by Seq.ID No 11 ORF D: Seq. ID No 13 Nucleotides 2785 to 3804 of GS from Mavs Seq. ID No 14 Amino acid sequence encoded by Seq.ID No 13 .

Seq. ID No 15 Nucleotides 1172 to 2191 of GS from Mptb Seq. ID No 16 Amino acid sequence encoded by Seq.ID No 20 15.

ORF E: Seq. ID No 17 Nucleotides 4080 to 4802 of GS from Mavs Seq. ID No 18 Amino acid sequence encoded by Seq.ID No 17.

Seq. ID No 19 Nucleotides 2467 to 3189 of GS from Mptb Seq. ID No 20 Amino acid sequence encoded by Seq.ID No 19.

ORF F: Seq. ID No 21 Nucleotides 4947 to 5747 of GS from Mavs Seq. ID No 22 Amino acid sequence encoded by Seq.ID No 21.

Seq. ID No 23 Nucleotides 3335 to 4135 of GS from Mptb Seq. ID No 24 Amino acid sequence encoded by Seq.ID No 23 .

YV0 97/27 "4 PCT/GB96/0322I ( ORF G: Seq. ID No 25 Nucleotides 6176 to 7042 of GS from Mavs Seq. ID No 26 Amino acid sequence encoded by Seq.ID No 25.

ORF H: Seq.ID No 27 Nucleotides 7953 to 6215 from Mavs.

ORF Hx: Seq.ID No 28 Amino acid sequence encoded by nucleotides 7953 to 7006 of Seq.ID No 27 ORF H2: Seq.ID No 29 Amino acid sequence encoded by nucleotides 7009 to 6215 of Seq.ID No 27 The polynucleotides in Mtb with homology to the ORFs B, C, E and 10 F of GS in Mptb and Mavs, and the polypeptides they are now known to encode as a result of our invention, are as follows: ORF B: Seq.ID No 30 Cosmid MTCY277 nucleotides 35493 to 34705 Seq.ID No 31 Amino acid sequence encoded by Seq.ID 15 No30.

ORF C: Seq.ID No 32 Cosmid MTCY277 nucleotides 31972 to 32994 Seq.ID No 33 Amino acid sequence encoded by Seq.ID No32.

ORF E: Seq.ID No 34 Cosmid MTCY277 nucleotides 34687 to 33956 20 Seq.ID No 35 Amino acid sequence encoded by Seq.ID No34.

ORF E: Seq.ID No 36 Cosmid MT024 nucleotides 15934 to 15203 Seq.ID No 37 Amino acid sequence encoded by Seq.ID No36.

♦ ORF F: Seq.ID No38 Cosmid MT024 nucleotides 15133 to 14306 Seq.ID No 39 Amino acid sequence encoded by Seq.ID No38.

The proteins and peptides encoded by the ORFs A to H in Mptb and Mavs and the amino acid sequences from homologous genes we have WO 97/23624 " ' PCT/GB96/03221 discovered in Mtb given in Seq.ID Nos 31, 33, 35, 37 and 39, as described above and fragments thereof, comprise the polypeptides of the invention. The polypeptides of the invention are believed to be associated with specific immunoreactivity and with the 5 pathogenicity of the host micro-organisms from which they were obtained.

The present invention thus provides a polynucleotide in substantially isolated form which is capable of selectively hybridising to sequence ID Nos 3 or 4 or a fragment thereof. The 10 polynucleotide fragment may alternatively comprise a sequence selected from the group of Seq.ID.No: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27. The invention further provides a polynucleotide in substantially isolated form whose sequence consists essentially of a sequence selected from the group Seq 15 ID Nos. 30, 32, 34, 36 and 38, or a corresponding sequence selectively hybridizable thereto, or a fragment of said sequence or corresponding sequence.

The invention further provides diagnostic probes such as a probe which comprises a fragment of at least 15 nucleotides of a 20 polynucleotide of the invention, or a peptide nucleic acid or similar synthetic sequence specific ligand, optionally carrying a revealing label. The invention also provides a vector carrying a polynucleotide as defined above, particularly an expression vector.

The invention further provides a polypeptide in substantially isolated form which comprises any one of the sequences selected from the group consisting Seq.ID.No: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 33, 35, 37 and 39, or a polypeptide substantially homologous thereto. The invention additionally ♦ provides a polypeptide fragment which comprises a fragment of a polypeptide defined above, said fragment comprising at least 10 amino acids and an epitope. The invention also provides polynucleotides in substantially isolated form which encode polypeptides of the invention, and vectors which comprise such 35 polynucleotides, as well as antibodies capable of binding such polypeptides. In an additional aspect, the invention provides WO 97 >24 PCT/GB96/03221 kits comprising polynucleotides, polypeptides, antibodies or synthetic ligands of the invention and methods of using such kits in diagnosing the presence or absence of mycobacteria in a sample. The invention also provides pharmaceutical compositions 5 comprising polynucleotides of the invention, polypeptides of the invention or antisense probes and the use of such compositions in the treatment or prevention of diseases caused by mycobacteria. The invention also provides polynucleotihe prevention and treatment of infections due to GS-containing 10 pathogenic mycobacteria in animals and humans and as a means of enhacing in vivo susceptibility of said mycobacteria to antimicrobial drugs. The invention also provides bacteria or viruses transformed with polynucleotides of the invention for use as vaccines. The invention further provides Mptb or Mavs in 15 which all or part or the polynucleotides of the invention have been deleted or disabled to provide mutated organisms of lower pathogenicity for use as vaccines in animals and humans. The invention further provides Mtb in which all or part of the polynucleotides encoding polypeptides of the invention have been 20 deleted or disabled to provided mutated organisms or lower pathogenicity for use as vaccines in animals and humans.

A further aspect of the invention is our discovery of homologies between the ORFs B, C and E in GS on the one hand, and Mtb cosmid MTCY277 on the other (data from Genbank database using the 25 fcomputer programmes BLAST and BLIXEM). The homologous ORFs in MTCY277 are adjacent to one another consistent with the form of another pathogenicity island in Mtb. A further aspect of the invention is our discovery of homologies between ORFs E and F in GS, and Mtb cosmid MT024 (also Genbank, as above) with the 30 homologous ORFs close to one another. The use of polynucleotides ♦ and polypeptides from MtJb (Seq. ID Nos 30,31, 32, 33, 34, 35, 36, 37, 38 and 39) in substantially isolated form as diagnostics, vaccines and targets for chemotherapy, for the management and prevention of Mtb infections in humans and animals, and the 35 processes involved in the preparation and use of these diagnostics, vaccines and new chemotherapeutic agents, comprise further aspects of the invention. < * WO 97/' 14 PCT/GB96/0322I Detailed description of the invention.

A. Polynucleotides Polynucleotides of the invention as defined herein may comprise DNA or RNA. They may also be polynucleotides which include 5 within them synthetic or modified nucleotides or peptide nucleic acids. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the 10 molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to couple the said polynucleotide to a solid phase or to enhance the recognition, the in vivo 15 activity, or the lifespan of polynucleotides of the invention.

A number of different types of polynucleotides of the invention are envisaged. In the broadest aspect, polynucleotides and fragments thereof capable of hybridizing to SEQ ID NO:3 or 4 form a first aspect of the invention. This includes the 20 polynucleotide of SEQ ID NO: 3 or 4. Within this class of polynucleotides various sub-classes of polynucleotides are of particular interest.

One sub-class of polynucleotides which is of interest is the class of polynucleotides encoding the open reading frames A, B, 25 C, D, E, F, G and H, including SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27. As discussed below, polynucleotides encoding ORF H include the polynucleotide sequences 7953 to 7006 ♦ and 7009 to 6215 within SEQ ID NO: 27, as well as modified sequences in which the frame-shift has been modified so that the 30 two sub-reading frames are placed in a single reading frame. This may be desirable where the polypeptide is to be produced in recombinant expression systems.

The invention thus provides a polynucleotide in substantially isolated form which encodes any one of these ORFs or combinations WO 97n '4 4 thereof. Combinations thereof includes combinations of 2, 3, 4, 5 or all of the ORFs. Polynucleotides may be provided which comprise an individual ORF carried in a recombinant vector including the vectors described herein. Thus in one preferred 5 aspect the invention provides a polynucleotide in substantially isolated form capable of selectively hybridizing to the nucleic acid comprising ORFs A to F of the core region of the Mptb and Mavs pathogenicity islands of the invention. Fragments thereof corresponding to ORFs A to E, B to F, A to D, B to E, A to C, B 10 to D or any two adjacent ORFs are also included in the invention.

Polynucleotides of the invention will be capable of selectively hybridizing to the corresponding portion of the GS region, or to the corresponding ORFs of Mtb described herein. The term "selectively hybridizing" indicates that the polynucleotides will 15 hybridize, under conditions of medium to high stringency (for example 0.03 M sodium chloride and 0.03 M sodium citrate at from about 50oc to about 60oC) to the corresponding portion of SEQ ID NO:3 or 4 or the complementary strands thereof but not to genomic DNA from mycobacteria which are usually non-pathogenic including 20 non-pathogenic species of M. avium. Such polynucleotides will generally be generally at least 68%, e.g. at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the corresponding DNA of GS. The corresponding portion will be of over a region of at least 20, preferably at 25 least 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.

By "corresponding portion" it is meant a sequence from the GS region of the same or substantially similar size which has been determined, for example by computer alignment, to have the 30, greatest degree of homology to the polynucleotide.

Any combination of the above mentioned degrees of homology and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example 35 a polynucleotide which is at least 80% homologous over 25, preferably 30 nucleotides forms one aspect of the invention, as WO 91 n *624 i does a polynucleotide which is at least 90% homologous over 40 nucleotides.

A further class of polynucleotides of the invention is the class of polynucleotides encoding polypeptides of the invention, the 5 polypeptides of the invention being defined in section B below. Due to the redundancy of the genetic code as such, polynucleotides may be of a lower degree of homology than required for selective hybridization to the GS region. However, when such polynucleotides encode polypeptides of the invention 10 these polynucleotides form a further aspect. It may for example be desirable where polypeptides of the invention are produced recombinantly to increase the GC content of such polynucleotides. This increase in GC content may result in higher levels of expression via codon usage more appropriate to the host cell in 15 which recombinant expression is taking place.

An additional class of polynucleotides of the invention are those obtainable from cosmids MTCY277 and MT024 (containing Mtb genomic sequences), which polynucleotides consist essentially of the fragment of the cosmid containing an open reading frame encoding 20 any one of the homologous ORFs B, C, E or F respectively. Such polynucleotides are referred to below as Mtb polynucleotides. However, where reference is made to polynucleotides in general such reference includes Mtb polynucleotides unless the context is explicitly to the contrary. In addition, the invention 25 provides polynucleotides which encode the same polypeptide as the abovementioned ORFs of Mtb but which, due to the redundancy of the genetic code, have different nucleotide sequences. These form further Mtb polynucleotides of the invention. Fragments of Mtb polynucleotides suitable for use as probes or primers also form a further aspect of the invention.

* The invention further provides polynucleotides in substantially isolated form capable of selectively hybridizing (where selectively hybridizing is as defined above) to the Mtb polynucleotides of the invention.

WO 97/27<24 ' PCT/GB96/03221 The invention further provides the Mtb polynucleotides of the invention linked, at either the 5' and/or 3' end to polynucleotide sequences to which they are not naturally contiguous. Such sequences will typically be sequences found in 5 cloning or expression vectors, such as promoters, 5' untranslated sequence, 3' untranslated sequence or termination sequences. The sequences may also include further coding sequences such as signal sequences used in recombinant production of proteins.

Further polynucleotides of the invention are illustrated in the 10 accompanying examples.

Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels 15 or a probe linked covalently to a solid phase, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 or more nucleotides in length, and are also encompassed by the term polynucleotides 20 of the invention as used herein.

Primers of the invention which are preferred include primers directed to any part of the ORFs defined herein. The ORFs from other isolates of pathogenic mycobacteria which contain a GS region may be determined and conserved regions within each individual ORF may be identified. Primers directed to such conserved regions form a further preferred aspect of the invention. In addition, the primers and other polynucleotides of the invention may be used to identify, obtain and isolate ORFs capable of selectively hybridizing to the polynucleotides of the * invention which are present in pathogenic mycobacteria but which are not part of a pathogenicity island in that particular species of bacteria. Thus in addition to the ORFs B, C, E and F which have been identified in Mtb, similar ORFs may be identified in other pathogens and ORFs corresponding to the GS ORFs C, D, E, 35 F and H, may also be identified.

WO 97/2-X24 PCT/GB96/0322I Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a step-wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using 10 recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair 1 or primers (e.g. of about 15-30 nucleotides) to a region of GS, which it is desired to clone, bringing the primers into contact with genomic DNA from a mycobacterium or a vector carrying the 15 GS sequence, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme 20 recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

Such techniques may be used to obtain all or part of the GS or ORF sequences described herein, as well as further genomic clones containing full open reading frames. Although in general such 25 techniques are well known in the art, reference may be made in particular to Sambrook J., Fritsch EF., Maniatis T (1989). Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory.

Polynucleotides which are not 100% homologous to the sequences ♦ of the present invention but fall within the scope of the invention can be obtained in a number of ways.

Other isolates or strains of pathogenic mycobacteria will be expected to contain allelic variants of the GS sequences described herein, and these may be obtained for example by 35 probing genomic DNA libraries made from such isolates or strains WO 97/23*24 I of bacteria using GS or ORF sequences as probes under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C) .

A particularly preferred group of pathogenic mycobacteria are 5 isolates of M. para tuberculosis. Polynucleotides based on GS regions from such bacteria are particularly preferred. Preferred fragments of such regions include fragments encoding individual open reading frames including the preferred groups and combinations of open reading frames discussed above.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of the GS or ORF sequences or allelic variants thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the 15 polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides of the invention. Such altered property or function will include the addition of 20 amino acid sequences of consensus signal peptides known in the art to effect transport and secretion of the modified polypeptide of the invention. Another altered property will include metagenesis of a catalytic residue or generation of fusion proteins with another polypeptide. Such fusion proteins may be 25 with an enzyme, with an antibody or with a cytokine or other ligand for a receptor, to target a polypeptide of the invention to a specific cell type in vitro or in vivo.

The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement.

* Polynucleotides or primers of the invention may carry a revealing label. Suitable labels include radioisotopes such as 32P or 35S, enzyme labels, other protein labels or smaller labels such as biotin or fluorophores. Such labels may be added to polynucleotides or primers of the invention and may be detected 35 using by techniques known per se.

WO 97/2"'"M Polynucleotides or primers of the invention or fragments thereof labelled or unlabelled may be used by a person skilled in the art in nucleic acid-based tests for the presence or absence of Mptb, Mavs, other GS-containing pathogenic mycobacteria, or Mtb applied 5 to samples of body fluids, tissues, or excreta from animals and humans, as well as to food and environmental samples such as river or ground water and domestic water supplies.

Human and animal body fluids include sputum, blood, serum, plasma, saliva, milk, urine, csf, semen, faeces and infected 10 discharges. Tissues include intestine, mouth ulcers, skin, lymph nodes, spleen, lung and liver obtained surgically or by a biopsy technique. Animals particularly include commercial livestock such as cattle, sheep, goats, deer, rabbits but wild animals and animals in zoos may also be tested.

Such tests comprise bringing a human or animal body fluid or tissue extract, or an extract of an environmental or food sample, into contact with a probe comprising a polynucleotide or primer of the invention under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample. 20 Such detection may be achieved using techniques such as PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridized to the probe, and then detecting nucleic acid which has hybridized to the probe. Alternatively, the sample nucleic acid may be immobilized on a 25 solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this any other formats can be found in for example W089/03891 and W090/13667.

Polynucleotides of the invention or fragments thereof labelled or unlabelled may also be used to identify and characterise 30 different strains of Mptb, Mavs, other GS-containing pathogenic mycobacteria, or Mtb, and properties such as drug resistance or susceptibility.

The probes of the invention may conveniently be packaged in the form of a test kit in a suitable container. In such kits the 35 probe may be bound to a solid support where the assay format for WO 97/2 ""14 PCT/GB96/0322I which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be probed, hybridising the probe to nucleic acid in the sample, control reagents, instructions, and the like.

The use of polynucleotides of the invention in the diagnosis of inflammatory diseases such as Crohn's disease or sarcoidosis in humans or Johne's disease in animals form a preferred aspect of the invention. The polynucleotides may also be used in the prognosis of these diseases. For example, the response of a 10 human or animal subject in response to antibiotic, vaccination or other therapies may be monitored by utilizing the diagnostic methods of the invention over the course of a period of treatment and following such treatment.

The use of Mtb polynucleotides (particularly in the form of 15 probes and primers) of the invention in the above-described methods form a further aspect of the invention, particularly for the detection, diagnosis or prognosis of Mtb infections.

B. Polypeptides.

Polypeptides of the invention include polypeptides in 20 substantially isolated form encoded by GS. This includes the full length polypeptides encoded by the positive and complementary negative strands of GS. Each of the full length polypeptides will contain one of the amino acid sequences set out in Seq ID N0s:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 25 29. Polypeptides of the invention further include variants of such sequences, including naturally occurring allelic variants and synthetic variants which are substantially homologous to said polypeptides. In this context, substantial homology is regarded *■ as a sequence which has at least 70%, e.g. 80%, 90%, 95% or 98% 30 amino acid homology (identity) over 30 or more, e.g 40, 50 or 100 amino acids. For example, one group of substantially homolgous polypeptides are those which have at least 95% amino acid identity to a polypeptide of any one of Seq ID N0s:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29 over their entire length. 35 Even more preferably, this homology is 98%.

WO '23624 PCT/GB96/0322I Polypeptides of the invention further include the polypeptide sequences of the homologous ORFs of Mtb, namely Seq ID Nos. 31, 33, 35, 37 and 39. Unless explicitly specified to the contrary, reference to polypeptides of the invention and their fragments 5 include these Mtb polypeptides and fragments, and variants thereof (substanially homologous to said sequences) as defined herein.

Polypeptides of the invention may be obtained by the standard techniques mentioned above. Polypeptides of the invention also 10 include fragments of the above mentioned full length polypeptides and variants thereof, including fragments of the sequences set out in SEQ ID N0s:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 33, 35, 37 and 39. Such fragments for example of 8, 10, 12, 15 or up to 30 or 4 0 amino acids may also be obtained 15 synthetically using standard techniques known in the art.

Preferred fragments include those which include an epitope, especially an epitope which is specific to the pathogenicity of the mycobacterial cell from which the polypeptide is derived. Suitable fragments will be at least about 5, e.g. 8, 10, 12, 15 20 or 20 amino acids in size, or larger. Epitopes may be determined either by techniques such as peptide scanning techniques as described by Geysen et al, Mol. Immunol., 23.; 709-715 (1986), as well as other techniques known in the art.

The term "an epitope which is specific to the pathogenicity of 25 the mycobacterial cell" means that the epitope is encoded by a portion of the GS region, or by the corresponding ORF sequences of Mtb which can be used to distinguish mycobacteria which are pathogenic by from related non-pathogenic mycobacteria including non-pathogenic species of M. avium. This may be determined using * routine methodology. A candidate epitope from an ORF may be prepared and used to immunise an animal such as a rat or rabbit in order to generate antibodies. The antibodies may then be used to detect the presence of the epitope in pathogenic mycobacteria and to confirm that non-pathogenic mycobacteria do not contain 35 any proteins which react with the epitope. Epitopes may be linear or conformational.

WOr ">.3624 Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as 5 substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be modified to confer a desired property or function for example by the addition of Histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell.

Thus, polypeptides of the invention include fusion proteins which 15 comprise a polypeptide encoding all or part of one or more of an ORF of the invention fused at the N- or C-terminus to a second sequence to provide the desired property or function. Sequences which promote secretion from a cell include, for example the yeast a-factor signal sequence.

A polypeptide of the invention may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. 125I, 35S enzymes, antibodies, polynucleotides and ligands such as biotin. Labelled polypeptides of the 25 invention may be used in diagnostic procedures such as immunoassays in order to determine the amount of a polypeptide of the invention in a sample. Polypeptides or labelled polypeptides of the invention may also be used in serological or cell mediated immune assays for the detection of immune ♦ reactivity to said polypeptides in animals and humans using standard protocols.

A polypeptide or labelled polypeptide of the invention or fragment thereof may also be fixed to a solid phase, for example the surface of an immunoassay well, microparticle, dipstick or 35 biosensor. Such labelled and/or immobilized polypeptides may be WO 0"'23624 packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.

Such polypeptides and kits may be used in methods of detection of antibodies or cell mediated immunoreactivity, to the 5 mycobacterial proteins and peptides encoded by the ORFs of the invention and their allelic variants and fragments, using immunoassay. Such host antibodies or cell mediated immune reactivity will occur in humans or animals with an immune system which detects and reacts against polypeptides of the invention.

The antibodies may be present in a biological sample from such humans or animals, where the biological sample may be a sample as defined above particularly blood, milk or saliva.

Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide of the invention comprising an epitope bindable by an antibody against said mycobacterial polypeptide; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed.

Immunoassay methods for cell mediated immune reactivity in animals and humans are also well known in the art (e.g. as described by Weir et al 1994, J.Immunol Methods 176; 93-101) and will generally comprise (a) providing a polypeptide of the invention comprising an epitope bindable by a lymphocyte or macrophage or * other cell receptor; (b) incubating a cell sample with said polypeptide under conditions which allow for a cellular immune response such as release of cytokines or other mediator to occur; and (c) detecting the presence of said cytokine or mediator in the incubate. i * WOf 3624 PCT/GB96/0322I I Polypeptides of the invention may be made by standard synthetic means well known in the art or recombinantly, as described below.

Polypeptides of the invention or fragments thereof labelled or unlabelled may also be used to identify and characterise 5 different strains of Mptb, Mavs, other GS-containing pathogenic mycobacteria, or Mtb, and properties such as drug resistance or susceptibility.

The polypeptides of the invention may conveniently be packaged in the form of a test kit in a suitable container. In such kits 10 the polypeptide may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be examined, control reagents, instructions, and the like.

The use of polypeptides of the invention in the diagnosis of 15 inflammatory diseases such as Crohn's disease or sarcoidosis in humans or Johne's disease in animals form a preferred aspect of the invention. The polypeptides may also be used in the prognosis of these diseases. For example, the response of a human or animal subject in response to antibiotic or other 20 therapies may be monitored by utilizing the diagnostic methods of the invention over the course of a period of treatment and following such treatment.

The use of Mtb polypeptides of the invention in the above-described methods form a further aspect of the invention, 25 particularly for the detection, diagnosis or prognosis of Mtb infections.

Polypeptides of the invention may also be used in assay methods for identifying candidate chemical compounds which will be useful in inhibiting, binding to or disrupting the function of said 30 polypeptides required for pathogenicity. In general, such assays involve bringing the polypeptide into contact with a candidate inhibitor compound and observing the ability of the compound to disrupt, bind to or interfer with the polypeptide.

WO 97/23624 • * PCT/GB96/03221 There are a number of ways in which the assay may be formatted. For example, those polypeptides which have an enzymatic function may be assayed using labelled substrates for the enzyme, and the amount of, or rate of, conversion of the substrate into a product 5 measured, e.g by chromatograpy such as HPLC or by a colourimetric assay. Suitable labels include 35S, 125I, biotin or enzymes such as horse radish peroxidase.

For example, the gene product of ORF C is believed to have GDP-mannose dehydratase activty. Thus an assay for inhbitors of the 10 gene product may utilise for example labelled GDP-mannose, GDP or mannose and the activity of the gene product followed. ORF D encodes a gene related to the synthesis and regulation of capuslar polysaccharides, which are often associated with invasiveness and pathogenicity. Labelled polysaccharide 15 substrates may be used in assays of the ORF D gene product. The gene product of ORF F encodes a protein with putative glucosyl transferase activity and thus labelled amino sugars such as /3-1-3-N-acetylglucosamine may be used as substrates in assays.

Candidate chemical compounds which may be used may be natural or 20 synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.

Alternatively, the a polypeptide of the invention may be screened against a panel of peptides, nucleic acids or other chemical 25 functionalities which are generated by combinatorial chemistry. This will allow the definition of chemical entities which bind to polypeptides of the invention. Typically, the polypeptide of the invention will be brought into contact with a panel of compounds from a combinantorial library, with either the panel 30* or the polypeptide being immobilized on a solid phase, under conditions suitable for the polypeptide to bind to the panel. The solid phase will then be washed under conditions in which only specific interactions between the polypeptide and individual members of the panel are retained, and those specific members may 35 be utilized in further assays or used to design further panels of candidate ompounds.

WOr "'3624 For example, a number of assay methods to define peptide interaction with peptides are known. For example, W086/00991 describes a method for determining mimotopes which comprises making panels of catamer preparations, for example octamers of 5 amino acids, at which one or more of the positions is defined and the remaining positions are randomly made up of other amino acids, determining which catamer binds to a protein of interest and re-screening the protein of interest against a further panel based on the most reactive catamer in which one or more 10 additional designated positions are systematically varied. This may be repeated throughout a number of cycles and used to build ^ up a sequence of a binding candidate compound of interest.

W089/03430 describes screening methods which permit the preparation of specific mimotopes which mimic the immunological 15 activity of a desired analyte. These mimotopes are identified by reacting a panel of individual peptides wherein said peptides are of systematically varying hydrophobicity, amphipathic characteristics and charge patterns, using an antibody against an antigen of interest. Thus in the present case antibodies 20 against the a polypeptide of the inventoin may be employed and mimotope peptides from such panels may be identified.

C. Vectors.

Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to 25 replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under 30 conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.

D. Expression Vectors.

WO' '23624 PCT/GB96/03221 Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably 5 linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the 10 control sequences. Such vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide of the invention. Thus, in a further aspect the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell 15 transformed or transfected with an expression vector as described above, under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.

A further embodiment of the invention provides vectors for the 20 replication and expression of polynucleotides of the invention, or fragments thereof. The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The 25 vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector 30 may also be adapted to be used in vivo, for example in a method of naked DNA vaccination or gene therapy. A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of polynucleotides of the invention, including the DNA of GS, the 35 open reading frames thereof and other corresponding ORFs particularly ORFs B, C, E and F from Mtb. The cells will be chosen to be compatible with the said vector and may for example be bacterial, yeast, insect or mammalian.

WO 9 3624 Expression vectors are widely available in the art and can be obtained commercially. Mammalian expression vectors may comprise a mammalian or viral promoter. Mammalian promoters include the metallothionien promoter. Viral promoters include promoters from 5 adenovirus, the SV4 0 large T promoter and retroviral LTR promoters. Promoters compatible with insect cells include the polyhedrin promoter. Yeast promoters include the alcohol dehydrogenase promoter. Bacterial promoters include the /?-galactosidase promoter.

The expression vectors may also comprise enhancers, and in the case of eukaryotic vectors polyadenylation signal sequence downstream of the coding sequence being expressed.

Polypeptides of the invention may be expressed in suitable host cells, for example bacterial, yeast, plant, insect and mammalian cells, and recovered using standard purification techniques including, for example affinity chromatography, HPLC or other chromatographic separation techniques.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides or ligands may also be produced by synthetic means. Such antisense polynucleotides may be used in a method of controlling the levels of the proteins encoded by the ORFs of the invention in a mycobacterial cell.

Polynucleotides of the invention may also be carried by vectors suitable for gene therapy methods. Such gene therapy methods include those designed to provide vaccination against diseases , caused by pathogenic mycobacteria or to boost the immune response of a human or animal infected with a pathogenic mycobacteria.

For example, Ziegner et al, AIDS, 1995, 9.;43-50 describes the use of a replication defective recombinant amphotropic retrovirus to boost the immune response in patients with HIV infection. Such a retrovirus may be modified to carry a polynucleotide encoding a polypeptide or fragment thereof of the invention and the WO r~ "13624 PCT/GB96/0322I retrovirus delivered to the cells of a human or animal subject in order to provide an immune response against said polypeptide. The retrovirus may be delivered directly to the patient or may be used to infecte cells ex-vivo, e.g. fibroblast cells, which •5 are then introduced into the patient, optionally after being inactivated. The cells are desirably autologous or HLA-matched cells from the human or animal subject.

Gene therapy methods including methods for boosting an immune response to a particluar pathogen are disclosed generally in for 10 example W095/14091, the disclosure of which is incoporated herein by reference. Recombinant viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors, herpes virus vectors and alphavirus vectors. Alpha virus vectors are described in, for example, 15 W095/07994, the disclosure of which is incorporated herein by reference.

Where direct administration of the recombinant viral vector is contemplated, either in the form of naked nucleic acid or in the form of packaged particles carrying the nucleic acid this may be 20 done by any suitable means, for example oral administration or intravenous injection. From 10s to 108 c.f.u of virus represents a typical dose, which may be repeated for example weekly over a period of a few months. Administration of autologous or HLA-matched cells infected with the virus may be more convenient in 25 some cases. This will generally be achieved by administering doses, for example from 10s to 10s cells per dose which may be repeated as described above.

The recombinant viral vector may further comprise nucleic acid capable of expressing an accessory molecule of the immune system 30 designed to increase the immune response. Such a moleclue may be for example and interferon, particularly interferon gamma, an interleukin, for example IL-la, IL-1|8 or IL-2, or an HLA class I or II moleclue. This may be particularly desirable where the vector is intended for use in the treatment of humans or animals 35 already infected with a mycobacteria and it is desired to boost the immune response.

E. Antibodies.

The invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof. The invention further provides a process for the production of 5 monoclonal or polyclonal antibodies to polypeptides of the invention. Monoclonal antibodies may be prepared by conventional hybridoma technology using the polypeptides of the invention or peptide fragments thereof, as immunogens. Polyclonal antibodies may also be prepared by conventional means which comprise 10 inoculating a host animal, for example a rat or a rabbit, with a polypeptide of the invention or peptide fragment thereof and recovering immune serum.

In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof 15 haptenised to another polypeptide for use as immunogens in animals or humans.

For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a polypeptide of the 20 invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400.

Antibodies may be used in methods of detecting polypeptides of 25 the invention present in biological samples (where such samples include the human or animal body samples, and environmental samples, mentioned above) by a method which comprises: (a) providing an antibody of the invention; (b) incubating a biological sample with said antibody 30 under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed.

WO °7/23624 Antibodies of the invention may be bound to a solid support for example an immunoassay well, microparticle, dipstick or biosensor and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.

Antibodies of the invention may be used in the detection, diagnosis and prognosis of diseases as descirbed above in relation to polypeptides of the invention.

F. Compositions.

The present invention also provides compositions comprising a 10 polynucleotide or polypeptide of the invention together with a carrier or diluent. Compositions of the invention also include compositions comprising a nucleic acid, particularly and expression vector, of the invention. Compositions further include those carrying a recombinant virus of the invention. 15 Such compositions include pharmaceutical compositions in which case the carrier or diluent will be pharmaceutically acceptable.

Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for inhalation as well as oral, parenteral (e.g. intramuscular or intravenous or transcutaneous) 20 administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In 25 general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

For example, formulations suitable for parenteral administration 30 include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening WO 0"7/23624 agents, and liposomes or other microparticulate systems which are designed to target the polynucleotide or the polypeptide of the invention to blood components or one or more organs, or to target cells such as M cells of the intestine after oral administration.

G. Vaccines.

In another aspect, the invention provides novel vaccines for the prevention and treatment of infections caused by Mptb, Mavs, other GS-containing pathogenic mycobacteria and Mtb in animals and humans. The term "vaccine" as used herein means an agent 10 used to stimulate the immune system of a vertebrate, particularly a warm blooded vertebrate including humans, so as to provide protection against future harm by an organism to which the vaccine is directed or to assist in the eradication of an organism in the treatment of established infection. The immune 15 system will be stimulated by the production of cellular immunity antibodies, desirably neutralizing antibodies, directed to epitopes found on or in a pathogenic mycobacterium which expresses any one of the ORFs of the invention. The antibody so produced may be any of the immunological classes, such as the 20 immunoglobulins A, D, E, G or M. Vaccines which stimulate the production of IgA are interest since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals, and the production of such antibodies will help prevent infection or colonization of the intestinal tract. However an 25 IgM and IgG response will also be desirable for systemic infections such as Crohn's disease or tuberculosis.

Vaccines of the invention include polynucleotides of the invention or fragments thereof in suitable vectors and administered by injection of naked DNA using standard protocols. 30 Polynucleotides of the invention or fragments thereof in suitable vectors for the expression of the polypeptides of the invention may be given by injection, inhalation or by mouth. Suitable vectors include M.bovis BCG, M.smegmatis or other mycobacteria, Corynebacteria, Salmonella or other agents according to 35 established protocols. 97/23624 Polypeptides of the invention or fragments thereof in substantially isolated form may be used as vaccines by injection, inhalation, oral administration or by transcutaneous application according to standard protocols. Adjuvants (such as Iscoms or 5 polylactide-coglycolide encapsulation) , cytokines such as IL-12 and other immunomodulators may be used for the selective enhancement of the cell mediated or humoral immunological responses. Vaccination with polynucleotides and/or polypeptides of the invention may be undertaken to increase the susceptibility 10 of pathogenic mycobacteria to antimicrobial agents in vivo.

In instances wherein the polypeptide is correctly configured so as to provide the correct epitope, but is too small to be immunogenic, the polypeptide may be linked to a suitable carrier.

A number of techniques for obtaining such linkage are known in 15 the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) and succinimidyl 4-(N-maleimido-methyl) cyclohexane-l-carboxylate (SMCC) obtained from Pierce Company, Rockford, Illinois, (if the peptide lacks a sulfhydryl group, this can be provided by addition of a 20 cysteine residue). These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. See, for example, 25 Immun Rev (1982) 62:185. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimido-methyl) cyclohexane-l-carboxylic 30, acid, and the like. The carboxyl group can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Additional methods of coupling antigens employs the rotavirus/"binding peptide" system described in EPO Pub. No. 259,14 9, the disclosure of which is incorporated herein 35 by reference. The foregoing list is not meant to be exhaustive, and modifications of the named compounds can clearly be used.

WO 97/23624 ' " PCT/GB96/03221 Any carrier may be used which does not itself induce the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; polysaccharides, such as latex functionalized 5 Sepharose®, agarose, cellulose, cellulose beads and the like; polymeric amino acids, such as polyglutamic acid, polylysine, polylactide-coglycolide and the like; amino acid copolymers; and inactive virus particles. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin 10 molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those skilled in the art.

The immunogenicity of the epitopes may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, 15 that associated with hepatitis B surface antigen. See, e.g., US-A-4,722,840. Constructs wherein the epitope is linked directly to the particle-forming protein coding sequences produce hybrids which are immunogenic with respect to the epitope. In addition, all of the vectors prepared include epitopes specific to HBV, 20 having various degrees of immunogenicity, such as, for example, the pre-S peptide.

In addition, portions of the particle-forming protein coding sequence may be replaced with codons encoding an epitope of the invention. In this replacement, regions which are not required 25 to mediate the aggregation of the units to form immunogenic particles in yeast or mammals can be deleted, thus eliminating additional HBV antigenic sites from competition with the epitope of the invention.

Vaccines may be prepared from one or more immunogenic 30 * polypeptides of the invention. These polypeptides may be expressed in various host cells (e.g., bacteria, yeast, insect, or mammalian cells) , or alternatively may be isolated from viral preparations or made synthetically.

In addition to the above, it is also possible to prepare live 35 vaccines of attenuated microorganisms which express one or more recombinant polypeptides of the invention. Suitable attenuated microorganisms are known in the art and include, for example, viruses (e.g., vaccinia virus), as well as bacteria.

The preparation of vaccines which contain an immunogenic 5 polypeptide(s) as active ingredients, is known to one skilled in the art. Typically, such vaccines are prepared as injectables, or as suitably encapsulated oral preparations and either liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injestion or injection may also 10 be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, 15 dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may 20 be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1' -2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine 25 (CGP 19835A, referred to as MTP-PE) , and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween® 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies 30 directed against an immunogenic polypeptide containing an antigenic sequence resulting from administration of this * * polypeptide in vaccines which are also comprised of the various adjuvants.

The vaccines are conventionally administered parenterally, by 35 injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories, oral formulations or as WO Q7/23624 " ' PCT/GB96/03221 enemas. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% - 2%. Oral 5 formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained 10 release formulations or powders and contain 10% - 95% of active ingredient, preferably 25% - 70%.

The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and 15 which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, 20 or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be 25 prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5/*g to 250/xg, of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, mode of administration and the degree of protection desired. 30 Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.

The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in 35 which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals Wr "*7/23624 PCT/GB96/0322I required to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the 5 individual and be dependent upon the judgement of the practitioner. 0 In a further aspect of the invention, there is provided an attenuated vaccine comprising a normally pathogenic mycobacteria which harbours an attenuating mutation in any one of the genes 10 encoding a polypeptide of the invention. The gene is selected from the group of ORFs A, B, C, D, E, F, G and H, including the homologous ORFs B, C, E and F in Mtb.

The mycobacteria may be used in the form of killed bacteria or as a live attenuated vaccine. There are advantages to a live 15 attenuated vaccine. The whole live organism is used, rather than dead cells or selected cell components which may exhibit modified or denatured antigens. Protein antigens in the outer membrane will maintain their tertiary and quaternary structures. Therefore the potential to elicit a good protective long term 20 immunity should be higher.

The term "mutation" and the like refers to a genetic lesion in a gene which renders the gene non-functional. This may be at either the level of transcription or translation. The term thus envisages deletion of the entire gene or substantial portions 25 thereof, and also point mutations in the coding sequence which result in truncated gene products unable to carry out the normal function of the gene.

, A mutation introduced into a bacterium of the invention will generally be a non-reverting attenuating mutation. Non-reverting 30 means that for practical purposes the probability of the mutated gene being restored to its normal function is small, for example less than 1 in 106 such as less than 1 in 109 or even less than 1 in 1012.

WO' '23624 PCT/G B96/03221 An attenuated mycobacteria of the invention may be in isolated form. This is usually desirable when the bacterium is to be used for the purposes of vaccination. The term "isolated" means that the bacterium is in a form in which it can be cultured, processed 5 or otherwise used in a form in which it can be readily identified and in which it is substantially uncontaminated by other bacterial strains, for example non-attenuated parent strains or unrelated bacterial strains. The term "isolated bacterium" thus encompasses cultures of a bacterial mutant of the invention, for 10 example in the form of colonies on a solid medium or in the form of a liquid culture, as well as frozen or dried preparations of the strains.

In a preferred aspect, the attenuated mycobacterium further comprises at least one additional mutation. This may be a 15 mutation in a gene responsible for the production of products essential to bacterial growth which are absent in a human or animal host. For example, mutations to the gene for aspartate semi-aldehyde dehydrogenase (asd) have been proposed for the production of attenuated strains of Salmonella. The asd gene is 20 described further in Gene (1993) 129; 123-128. A lesion in the asd gene, encoding the enzyme aspartate /3-semialdehyde dehydrogenase would render the organism auxotrophic for the essential nutrient diaminopelic acid (DAP), which can be provided exogenously during bulk culture of the vaccine strain. Since 25 this compound is an essential constituent of the cell wall for gram-negative and some gram-positive organisms and is absent from mammalian or other vertebrate tissues, mutants would undergo lysis after about three rounds of division in such tissues. Analogous mutations may be made to the attenuated mycobacteria of the invention.

* In addition or in the alternative, the attenuated mycobacteria may carry a recA mutation. The recA mutation knocks out homologous recombination - the process which is exploited for the construction of the mutations. Once the recA mutation has been 35 incorporated the strain will be unable to repair the constructed deletion mutations. Such a mutation will provide attenuated strains in which the possibility of homologous recombination to WO 97/?- '14 with DNA from wild-type strains has been minimized. RecA genes have been widely studied in the art and their sequences are available. Further modifications may be made for additional safety.

The invention further provides a process for preparing a vaccine composition comprising an attenuated bacterium according to the invention process comprises (a) inoculating a culture vessel containing a nutrient medium suitable for growth of said bacterium; (b) culturing said bacterium; (c) recovering said 10 bacteria and (d) mixing said bacteria with a pharmaceutically acceptable diluent or carrier.

Attenuated bacterial strains according to the invention may be constructed using recombinant DNA methodology which is known per se. In general, bacterial genes may be mutated by a process of 15 targeted homologous recombination in which a DNA construct containing a mutated form of the gene is introduced into a host bacterium which it is desired to attenuate. The construct will recombine with the wild-type gene carried by the host and thus the mutated gene may be incorporated into the host genome to 20 provide a bacterium of the present invention which may then be isolated.

The mutated gene may be obtained by introducing deletions into the gene, e.g by digesting with a restriction enzyme which cuts the coding sequence twice to excise a portion of the gene and 25 then religating under conditions in which the excised portion is not reintroduced into the cut gene. Alternatively frame shift mutations may be introduced by cutting with a restriction enzyme which leaves overhanging 5' and 3' termini, filling in and/or trimming back the overhangs, and religating. Similar mutations 30 may be made by site directed mutagenesis. These are only examples of the types of techniques which will readily be at the disposal of those of skill in the art.

Various assays are available to detect successful recombination. In the case of attenuations which mutate a target gene necessary 35 for the production of an essential metabolite or catabolite WO 97/23' < compound, selection may be carried out by screening for bacteria unable to grow in the absence of such a compound. Bacteria may also be screened with antibodies or nucleic acids of the invention to determine the absence of production of a mutated 5 gene product of the invention or to confirm that the genetic lesion introduced - e.g. a deletion - has been incorporated into the genome of the attenuated strain.

The concentration of the attenuated strain in the vaccine will be formulated to allow convenient unit dosage forms to be 10 prepared. Concentrations of from about 104 to 109 bacteria per ml will generally be suitable, e.g. from about 105 to 10® such as about 10s per ml. Live attenuated organisms may be administered subcutaneously or intramuscularly at up to 10® organisms in one or more doses, e.g from around 105 to 10®, e.g about 106 or 107 15 organisms in a single dose.

The vaccines of the invention may be administered to recipients to treat established disease or in order to protect them against diseases caused by the corresponding wild type mycobacteria, such as inflammatory diseases such as Crohn's disease or sarcoidosis 20 in humans or Johne's disease in animals. The vaccine may be administered by any suitable route. In general, subcutaneous or intramuscular injection is most convenient, but oral, intranasal and colorectal administration may also be used.

The following Examples illustrates aspects of the invention. 25 EXAMPLE 1 Tests for the presence of the GS identifier sequence were performed on 5/xl bacterial DNA extracts (25 /xg/ml to 500 pg/ml) using polymerase chain reaction based on the oligonucleotide primers 5' -GATGCCGTGAGGAGGTAAAGCTGC-3' (Seq ID No. 40) and 5'-30 GATACGGCTCTTGAATCCTGCACG-3' (Seq ID No. 41) from within the identifier DNA sequences (Seq.ID Nos 1 and 2) . PCR was performed for 40 cycles in the presence of 1.5 mM magnesium and an annealing temperature of 58°C. The presence or absence of the correct amplification product indicated the presence or absence WO 97/^1624 PCT/G B96/03221 of GS identifier sequence in the corresponding bacterium. GS identifier sequence is shown to be present in all the laboratory and field strains of Mptb and Mavs tested. This includes Mptb isolates 0025 (bovine CVL Weybridge), 0021 (caprine, Moredun), 5 0022 (bovine, Moredun), 0139 (human, Chiodini 1984), 0209, 0208, 0211, 0210, 0212, 0207, 0204, 0206 (bovine, Whipple 1990). All Mptb strains were IS900 positive. The Mavs strains include 0010 and 0012 (woodpigeon, Thorel) 0018 (armadillo, Portaels) and 0034, 0037, 0038, 0040 (AIDS, Hoffner). All Mavs strains were 10 IS902 positive. One pathogenic M. avium strain 0033 (AIDS, Hoffner) also contained GS identifier sequence. GS identifier sequence is absent from other mycobacteria including other M. avium, M.malmoense, M.szulgai, M.gordonae, M.chelonei, M. fortuitum, M.phlei, as well as E.coli, S.areus, Nocardia sp, 15 Streptococcus sp. Shigella sp. Pseudomonas sp.

Example 2: To obtain the full sequence of GS in Mavs and Mptb we generated a genomic library of Mavs using the restriction endonuclease EcoRI and cloning into the vector pUC18. This achieved a representative library which was screened with 32P-labelled identifier sequence yielding a positive clone containing a 17kbp insert. We constructed a restriction map of this insert and identified GS as fragments unique to Mavs and Mptb and not occurring in laboratory strains of M.avium. These fragments were sub-cloned into pUC18 and pGEM4Z. We identified GS contained within an 8kb region. The full nucleotide sequence was determined for GS on both DNA strands using primer walking and automated DNA sequencing. DNA sequence for GS in Mptb was obtained using overlapping PCR products generated using PwoDNA polymerase, a proofreading thermostable enzyme. The final DNA sequences were derived using the University of Wisconsin GCG gel assembly software package.

Example 3: The DNA sequence of GS in Mavs and Mptb was found to be more 35 than 99% homologous. The ORFs encoded in GS were identified using GeneRunner and DNAStar computer programmes. Eight ORFs were identified and designated GSA, GSB, GSC, GSD, GSE, GSF, GSG WO 91 r" '24 and GSH. Database comparisons were carried out against the GenEMBL Database release version 48.0 (9/96) , using the BLAST and BLIXEM programmes. GSA and GSB encoded proteins of 13.5kDa and 30.7kDa respectively, both of unknown functions. GSC encoded 5 a protein of 3 8.4kDa with a 65% homology to the amino acid sequence of rfbD of V.cholerae, a 62% amino acid sequence homology to gmd of E.coli and a 58% homology to gca of Ps.aeruginosa which are all GDP-D-mannose dehydratases. Equivalent gene products in H. influenzae, S.dysenteriae, 10 Y. enterocolitica, N. gonorrhoea, K.pneumoniae and rfbD in Salmonella enterica are all involved in '0'-antigen processing known to be linked to pathogenicity. GSD encoded a protein of 37.1kDa which showed 58% homology at the DNA level to wcaG from E.coli, a gene involved in the synthesis and regulation of 15 capsular polysaccharides, also related to pathogenicity. GSE was found to have a > 30% amino acid homology to rfbT of V.cholerae, involved in the transport of specific LPS components across the cell membrane. In V.cholerae the gene product causes a seroconversion from the Inaba to the Ogawa 'epidemic' strain. 20 GSF encoded a protein of 30.2kDa which was homologous in the range 25-40% at the amino acid level to several glucosyl transferases such as rfpA of K.pneumoniae, rfbB of K.pneumoniae, lgtD of H. influenzae, lsi of N. gonorrhoae. In E.coli an equivalent gene galE adds jS-1-3 N-acetylglucosamine to galactose, 25 the latter only found in 'O' and 'M' antigens which are also related to pathogenicity. GSH comprising the ORFs GSHX and GSH2 encodes a protein totalling about 60kDa which is a putative transposase with a 40 - 43% homology at the amino acid level to the equivalent gene product of IS21 in E.coli. This family of 30 insertion sequences is broadly distributed amongst gram negative bacteria and is responsible for mobility and transposition of genetic elements. An IS21- like element in B.fragilis is split either side of the /3-lactamase gene controlling its activation and expression. We programmed an E.coli S30 cell-free extract 35 with plasmid DNA containing the ORF GSH under the control of a lac promoter in the presence of a 3SS-methionine, and demonstrated the translation of an abundant 60kDa protein. The proteins homologous to GS encoded in other organisms are in general highly antigenic. Thus the proteins encoded by the ORFs WO 97/2*^24 PCT/GB96/0322I in GS may be used in immunoassays of antibody or cell mediated immuno-reactivity for diagnosing infections caused by mycobacteria, particularly Mptb, Mavs and Mtb. Enhancement of host immune recognition of GS encoded proteins by vaccination using naked specific DNA or recombinant GS proteins, may be used in the prevention and treatment of infections caused by Mptb, Mavs and Mtb in humans and animals. Mutation or deletion of all or some of the ORFs A to H in GS may be used to generate attenuated strains of Mptb, Mavs or Mtb with lower pathogenicity for use as living or killed vaccines in humans and animals. Such vaccines are particularly relevant to Johne's disease in animals, to diseases caused by Mptb in humans such as Crohn's disease, and to the management of tuberculosis especially where the disease is caused by multiple drug-resistant organisms.

SEQUENCE LISTING Seq. ID No.1 "- 1 GATCCAACTA AACCCGATGG AACCCCGCGC AAACTATTGG ACGTCTCCGC GCTACGCAGT 61 TGGGTTGGCG CCCGCGAATC GCACTGAAAG AGGGCATCGA TGCAACGGTG TCGTGGTACC 5 121 GCACAAATGC CGATGCCGTG AGGAGGTAAA GCTGCGGGCC GGCCGATGTT ATCCCTCCGG 181 CCGGACGGGT AGGGCGACCT GCCATCGAGT GGTACGGCAG TCGCCTGGCC GGCGAGGCGC 241 ATGGCCTATG TGAGTATCCC ATAGCCTGGC TTGGCTCGCC CCTACGCATT ATCAGTTGAC 301 CGCTTTCGCG CCACGTCGCA GGCTTGCGGC AGCATCCCGT TCAGGTCTCC TCATGGTCCG 361 GTGTGGCACG ACCACGCAAG CTCGAACCGA CTCGTTTCCC AATTTCGCAT GCTAATATCG 10 421 CTCGATGGAT mTTGCGCA ACGCCGGCTT GATGGCTCGT AACGTTAGCA CCGAGATGCT 481 GCGCCACTCC GAACGAAAGC GCCTATTAGT AAACCAAGTC GAAGCATACG GAGTCAACGT 541 TGTTATTGAT GTCGGTGCTA ACTCCGGCCA GTTCGGTAGC GCTTTGCGTC GTGCAGGATT 601 CAAGAGCCGT ATCGTTTCCT TTGAACCTCT TTCGGGGCCA TTTGCGCAAC TAACGCGCAA 661 GTCGGCATCG GATC -3' Seq. ID No.2 - 1 GATCCGATGC CGACTTGCGC GTTAGTTGCG CAAATGGCCC CGAAAGAGGT TCAAAGGAAA 61 CGATACGGCT CTTGAATCCT GCACGACGCA AAGCGCTACC GAACTGGCCG GAGTTAGCAC 121 CGACATCAAT AACAACGTTG ACTCCGTATG CTTCGACTTG GTTTACTAAT AGGCGCTTTC 181 GTTCGGAGTG GCGCAGCATC TCGGTGCTAA CGTTACGAGC CATCAAGCCG GCGTTGCGCA 241 AAAAATCCAT CGAGCGATAT TAGCATGCGA AATTGGGAAA CGAGTCGGTT CGAGCTTGCG 301 TGGTCGTGCC ACACCGGACC ATGAGGAGAC CTGAACGGGA TGCTGCCGCA AGCCTGCGAC 361 GTGGCGCGAA AGCGGTCAAC TGATAATGCG TAGGGGCGAG CCAAGCCAGG CTATGGGATA 421 CTCACATAGG CCATGCGCCT CGCCGGCCAG GCGACTGCCG TACCACTCGA TGGCAGGTCG 481 CCCTACCCGT CCGGCCGGAG GGATAACATC GGCCGGCCCG CAGCTTTACC TCCTCACGGC 541 ATCGGCATTT GTGCGGTACC ACGACACCGT TGCATCGATG CCCTCTTTCA GTGCGATTCG 601 CGGGCGCCAA CCCAACTGCG TAGCGCGGAG ACGTCCAATA GTTTGCGCGG GGTTCCATCG 661 GGTTTAGTTG GATC -3' Seq. ID No.3 l GAATTCTGGG TTGGAGACGA CGTCGAACTC CTGGTCGGTC TTGCTTCGAA 51 TGATCGCTGT GATCTGGTCG GCGGTGCCGA CAGGAACCGT CGACTTGTCG 101 ACGATCACCT TGTACCGGTC GATGTATGAC CCAATGTCGT CCGCAACCGA 151 GAAGACGTAC GTCAGGTCCG CCGCCCCGCT TTCACCCATG GGCGTCGGGA 201 CGGCGATGAA AATGACGTCC GCGTGCTCGA TTCCGCGTTG CCGGTCGGTG 251 GTGAAGTCAA TCAGCCCGTT CTCACGGTTC CTCGCAATCA ACTCCCAACC 301 CGGGCTCGAA AATCGGGACA CTGCCTGCGA GGAGCAAATC GATCTTGGCC 351 TGATCGATAT CGACACAGAC GACATCGTTG CCGCTATCCG CGAGACAGGC 401 GCCCGTGACG AGGCCTACAT AGCCTGATCC GACCACCGAA ATTTTCAAGA 451 TGACCCCTTC AAGTCCCCGA TCGGTCGACG ACCATACTGC CGCAACTCTG 501 TACCCTCCGT GGGTAATTCG CATGTCGCGT TCGTAAGGAG CAGCCAGCGA 551 GTCGGGGACG TTCGGTGAGA GAGTCGCAGG ACTACGAGGT TGCCGGTGCG 601 ATACATCACA GTGTTGCGTC TGTCGGCAAC GATGCAGCAA GAACCCACGG 651 GGCAGCCCTG AACTGCGCGC ATGACCGGTC CTTGTCCTGG CACCTTTGAT 701 CGGCCACCGC TTCCATGCGA ACATGACCGG AATCCATAGC GCGTGGTCAA 751 GCAGCGGGGA GGTAGACGTC GGTGTCATCT GCTCCAACCG TGTCGGTGAT 801 AACGATTTCG CTGAACGATC TCGAGGGATT GAAAAGCACC GTGGAGAGCG 851 TTCGCGCGCA GCGCTATGGG GGGCGAATCG AGCACATCGT CATCGACGGT 901 GGATCGGGCG ACGCCGTCGT GGAGTATCTG TCCGGCGATC CTGGCTTTGC 951 ATATTGGCAA TCTCAGCCCG ACAACGGGAG ATATGACGCG ATGAATCAGG 1001 GCATTGCCCA TTCGTCGGGC GACCTGTTGT GGTTTATGCA CTCCACGGAT 1051 CGTTTCTCCG ATCCAGATGC AGTCGCTTCC GTGGTGGAGG CGCTCTCGGG 1101 GCATGGACCA GTACGTGATT TGTGGGGTTA CGGGAAAAAC AACCTTGTCG 1151 GACTCGACGG CAAACCACTT TTCCCTCGGC CGTACGGCTA TATGCCGTTT 1201 AAGATGCGGA AATTTCTGCT CGGCGCGACG GTTGCGCATC AGGCGACATT 1251 CTTCGGCGCG TCGCTGGTAG CCAAGTTGGG CGGTTACGAT CTTGATTTTG 1301 GACTCGAGGC GGACCAGCTG TTCATCTACC GTGCCGCACT AATACGGCCT 13S1 CCCGTCACGA TCGACCGCGT GGTTTGCGAC TTCGATGTCA CGGGACCTGG 1401 TTCAACCCAG CCCATCCGTG AGCACTATCG GACCCTGCGG CGGCTCTGGG 1451 ACCTGCATGG CGACTACCCG CTGGGTGGGC GCAGAGTGTC GTGGGCTTAC 1501 TTGCGTGTGA AGGAGTACTT GATTCGGGCC GACCTGGCCG CATTCAACGC 1551 GGTAAAGTTC TTGCGAGCGA AGTTCGCCAG AGCTTCGCGG AAGCAAAATT 1601 CATAGAAACC AACTTCTACT GCCTGACCTG AGCAGCGCCG AGGCGCGCAG 1651 CGCGATCAGT GCGACCTGAA CGGCCAGGTG GAAAGCGCCA CCGATCCCGG 1701 CACCGAGTGC CTGACGCTTC GGATCCCTTG CACCACAACG AGAGTGAGAG 1751 CGCCATGATG AGGAAATATC GGCTGGGCGG AGTCAACGCC GGAGTGACAA 1601 AAGTGAGAAC CCGGTGAAGC GAGCGCTTAT AACAGGGATC ACGGGGCAGG 1851 ATGGTTCCTA CCTCGCCGAG CTACTACTGA GCAAGGGATA CGAGGTTCAC 40 1901 GGGCTCGTTC GTCGAGCTTC GACGTTTAAC ACGTCGCGGA TCGATCACCT 1951 CTACGTTGAC CCACACCAAC CGGGCGCGCG CTTGTTCTTG CACTATGCAG 2001 ACCTCACTGA CGGCACCCGG TTGGTGACCC TGCTCAGCAG TATCGACCCG * 2051 GATGAGGTCT ACAACCTCGC AGCGCAGTCC CATGTGCGCG TCAGCTTTGA 2101 CGAGCCAGTG CATACCGGAG ACACCACCGG CATGGGATCG ATCCGACTTC 45 2151 TGGAAGCAGT CCGCCTTTCT CGGGTGGACT GCCGGTTCTA TCAGGCTTCC 2201 TCGTCGGAGA TGTTCGGCGC ATCTCCGCCA CCGCAGAACG AATCGACGCC 2251 GTTCTATCCC CGTTCGCCAT ACGGCGCGGC CAAGGTCTTC TCGTACTGGA 2301 CGACTCGCAA CTATCGAGAG GCGTACGGAT TATTCGCAGT GAATGGCATC 2351 TTGTTCAACC ATGAGTCCCC CCGGCGCGGC GAGACTTTCG TGACCCGAAA 50 2401 GATCACGCGT GCCGTGGCGC GCATCCGAGC TGGCGTCCAA TCGGAGGTCT 2451 ATATGGGCAA CCTCGATGCG ATCCGCGACT GGGGCTACGC GCCCGAATAT 2501 GTCGAGGGGA TGTGGAGGAT GTTGCAAGCG CCTGAACCTG ATGACTACGT 40 45 50 WO 97/7 "24 2551 CCTGGCGACA GGGCGTGGTT ACACCGTACG TGAGTTCGCT CAAGCTGCTT 2601 TTGACCATGT CGGGCTCGAC TGGCAAAAGC GCGTCAAGTT TGACGACCGC 2651 TATTTGCGTC CCACCGAGGT CGATTCGCTA GTAGGAGATG CCGACAAGGC 2701 GGCCCAGTCA CTCGGCTGGA AAGCTTCGGT TCATACTGGT GAACTCGCGC 2751 GCATCATGGT GGACGCGGAC ATCGCCGCGT TGGAGTGCGA TGGCACACCA 2801 TGGATCGACA CGCCGATGTT GCCTGGTTGG GGCAGAGTAA GTTGACGACT 2851 ACACCTGGGC CTCTGGACCG CGCAACGCCC GTGTATATCG CCGGTCATCG 2901 GGGGCTGGTC GGCTCAGCGC TCGTACGTAG ATTTGAGGCC GAGGGGTTCA 2951 CCAATCTCAT TGTGCGATCA CGCGATGAGA TTGATCTGAC GGACCGAGCC 3001 GCAACGTTTG ATTTTGTGTC TGAGACAAGA CCACAGGTGA TCATCGATGC 3051 GGCCGCACGG GTCGGCGGCA TCATGGCGAA TAACACCTAT CCCGCGGACT 3101 TCTTGTCCGA AAACCTCCGA ATCCAGACCA ATTTGCTCGA CGCAGCTGTC 3151 GCCGTGCGTG TGCCGCGGCT CCTTTTCCTC GGTTCGTCAT GCATCTACCC 3201 GAAGTACGCT CCGCAACCTA TCCACGAGAG TGCTTTATTG ACTGGCCCTT 3251 TGGAGCCCAC CAACGACGCG TATGCGATCG CCAAGATCGC CGGTATCCTG 3301 CAAGTTCAGG CGGTTAGGCG CCAATATGGG CTGGCGTGGA TCTCTGCGAT 3351 GCCGACTAAC CTCTACGGAC CCGGCGACAA CTTCTCCCCG TCCGGGTCGC 3401 ATCTCTTGCC GGCGCTCATC CGTCGATATG AGGAAGCCAA AGCTGGTGGT 3451 GCAGAAGAGG TGACGAATTG GGGGACCGGT ACTCCGCGGC GCGAACTTCT 3501 GCATGTCGAC GATCTGGCGA GCGCATGCCT GTTCCTTTTG GAACATTTCG 3551 ATGGTCCGAA CCACGTCAAC GTGGGCACCG GCGTCGATCA CAGCATTAGC 3601 GAGATCGCAG ACATGGTCGC TACAGCGGTG GGCTACATCG GCGAAACACG 3651 TTGGGATCCA ACTAAACCCG ATGGAACCCC GCGCAAACTA TTGGACGTCT 3701 CCGCGCTACG CGAGTTGGGT TGGCGCCCGC GAATCGCACT GAAAGACGGC 3751 ATCGATGCAA CGGTGTCGTG GTACCGCACA AATGCCGATG CCGTGAGGAG 3801 GTAAAGCTGC GGGTCGGCCG ATGTTATCCC TCCGGCCGGA CGGGTGGGGC 3851 GACCTGCCGT CGAGTGGTAC GGCAGTCGCC TGGCCGGCGA GGCGCGTGGC 3901 CTATGGGAGT ATCCAATAGC CTGGCTTGGC TCGCCCCTAC GCATTATCAG 3951 TTGACCGCTT TCGCGCCAGC TCGCAGGCTT GCGGCAGCAT CCCGTTCAGG 4001 TCTCCTCATG GTCCGGTGTG GCACGACCAC GCAAGCTCGA ACCGACTCGT 4051 TTCCCAATTT CGCATGCTAA TATCGCTCGA TGGATTTTTT GCGCAACGCC 4101 GGCTTGATGG CTCGTAACGT TAGTACCGAG ATGCTGCGCC ACTTCGAACG 4151 AAAGCGCCTA TTAGTAAACC AATTCAAAGC ATACGGAGTC AACGTTGTTA 4201 TTGATGTCGG TGCTAACTCC GGCCAGTTCG GTAGCGCTTT GCGTCGTGCA 4251 GGATTCAAGA GCCGTATCGT TTCCTTTGAA CCTCTTTCGG GGCCATTTGC 4301 GCAACTAACG CGCAAGTCGG CATCGGATCC ACTATGGGAG TGTCACCAGT 4351 ATGCCCTAGG CGACGCCGAT GAGACGATTA CCATCAATGT GGCAGGCAAT 4401 GCGGGGGCAA GTAGTTCCGT GCTGCCGATG CTTAAAAGTC ATCAAGATGC 4451 CTTTCCTCCC GCGAATTATA TTGGCACCGA AGACGTTGCA ATACACCGCC 4501 TTGATTCGGT TGCATCAGAA TTTCTGAACC CTACCGATGT TACTTTCCTG 4551 AAGATCGACG TACAGGGTTT CGAGAAGCAG GTTATCACGG GCAGTAAGTC 4601 AACGCTTAAC GAAAGCTGCG TCGGCATGCA ACTCGAACTT TCTTTTATTC 4651 CGTTGTACGA AGGTGACATG CTGATTCATG AAGCGCTTGA ACTTGTCTAT 4701 TCCCTAGGTT TCAGACTGAC GGGTTTGTTG CCCGGCTTTA CGGATCCGCG 4751 CAATGGTCGA ATGCTTCAAG CTGACGGCAT TTTCTTCCGT GGGGACGATT 4801 GACATAAATG CTCCGTCGGC ACCCTGCCGG TATCCAAACG GGCGATCTGG 4851 TGAGCCGGCC TCCCGGGCAC CTAATCGACT ATCTAAATTG AGGCGGCCGC 4901 GACGTGCGGC ACGAACAGGT GGCCGGCTGC TAGCGTTACA CACGTCATGA 4951 CTGCGCCAGT GTTCTCGATA ATTATCCCTA CCTTCAATGC AGCGGTGACG 5001 CTGCAAGCCT GCCTCGGAAG CATCGTCGGG CAGACCTACC GGGAAGTGGA 5051 AGTGGTCCTT GTCGACGGCG GTTCGACCGA TCGGACCCTC GACATCGCGA 5101 ACAGTTTCCG CCCGGAACTC GGCTCGCGAC TGGTCGTTCA CAGCGGGCCC 5151 GATGATGGCC CCTACGACGC CATGAACCGC GGCGTCGGCG TGGCCACAGG WO 97^624 5201 CGAATGGGTA CTTTTTTTAG GCGCCGACGA CACCCTCTAC GAACCAACCA 5251 CGTTGGCCCA GGTAGCCGCT TTTCTCGGCG ACCATGCGGC AAGCCATCTT 5301 GTCTATGGCG ATGTTGTGAT GCGTTCGACG AAAAGCCGGC ATGCCGGACC 5351 TTTCGACCTC GACCGCCTCC TATTTGAGAC GAATTTGTGC CACCAATCGA 5401 TCTTTTACCG CCGTGAGCTT TTCGACGGCA TCGGCCCTTA CAACCTGCGC 5451 TACCGAGTCT GGGCGGACTG GGACTTCAAT ATTCGCTGCT TCTCCAACCC 5501 GGCGCTGATT ACCCGCTACA TGGACGTCGT GATTTCCGAA TACAACGACA 5551 TGACCGGCTT CAGCATGAGG CAGGGGACTG ATAAAGAGTT CAGAAAACGG 5601 CTGCCAATGT ACTTCTGGGT TGCAGGGTGG GAGACTTGCA GGCGCATGCT 5651 GGCGTTTTTG AAAGACAAGG AGAATCGCCG TCTGGCCTTG CGTACGCGGT 5701 TGATAAGGGT TAAGGCCGTC TCCAAAGAAC GAAGCGCAGA ACCGTAGTCG 5751 CGGATCCACA TTGGACTTCT TTAACGCGTT TGCGTCCTGA TCCACCTTTC 5801 AAGCCCGTTC CGCGTAACGC GGCGCGCAGA GAGTGGTCGC ATATCGCATC 5851 ACTGTTCTCG TGCCAGTGCT TGGAAAGCGT CGAGCACTCT GGTTCGCGTT 5901 CTTGACGTTC GCGCCCGCTC CTAGAGGTAG CGTGTCACGT GACTGAAGCC 5951 AATGAGTGCA ACTCGGCGTC GCGAAAGGTT TCAGTCGCGG TTGAGCAAGA 6001 CACCGCAAGA CTACTGGAGT GCGTGCACAA GCGCCTCCAG CTCGCGGCTG 6051 AAAGCGGATG CAAAGGGATT CGAAGCTTGA GCAACATGCG AAGGGGAGAA 6101 CGGCCTATGA GGCTGGGACA GGTTTTCGAT CCGCGCGCGA ATGCACTGTC 6151 AATGGCCAAG TAGAAGTCCC CGCTGGTGGC CAGCAGAAGT CCCCACTCCG 6201 CTGCGGGTGG TTGGCTAATT CTTGGCGGCT CCCTTCTTGT GGTCGGCGTG 6251 GCGCATCCGG TAGGACTCGC CGGAGGTGAC GACGATGCTG GCGTGGTGCA 6301 GCAGCCGATC GAGGATGCTG GCGGCGGTGG TGTGCTCGGG CAGGAATCGC 6351 CCCCATTGTT CGAAGGGCCA ATGCGAGGCG ATGGCCAGGG AGCGGCGCTC 6401 GTAGCCGGCA GCCACGAGCC GGAACAACAG TTGAGTCCCG GTGTCGTCGA 6451 GCGGGGCGAA GCCGATCTCG TCCAAGATGA CCAGATCCGC GCGGAGCAGG 6501 GTGTCGATGA TCTTGCCGAC GGTGTTGTCG GCCAGGCCGC GGTAGAGGAC 6551 CTCGATCAGG TCGGCGGCGG TGAAGTAGCG GACTTTGAAT CCGGCGTGGA 6601 CGGCAGCGTG CCCGCAGCCG ATGAGCAGGT GACTTTTGCC CGTACCAGGT 6651 GGGCCAATGA CCGCCAGGTT CTGTTGTGCC CGAATCCATT CCAGGCTCGA 6701 CAGGTAGTCG AACGTGGCTG CGGTGATCGA CGATCCGGTG ACGTCGAACC 6751 CGTCGAGGGT CTTGGTGACC GGGAAGGCTG CGGCCTTGAG ACGGTTGGCG 6801 GTGTTGGAGG CATCGCGGGC AGCGATCTCG GCCTCAACCA ACGTCCGCAG 6851 GATCTCCTCC GGTGTCCAGC GTTGCGTCTT GGCGACTTGC AACACCTCGG 6901 CGGCGTTGCG GCGCACCGTG GCCAGCTTCA ACCGCCGCAG CGCCGCGTCA 6951 AGGTCAGCAG CCAGCGGTGC CGCCGAGGAC GGTGCCACCG GCTTGGCAGC 7001 GGTGGTCATG AGGCCGTCCC GTCGGTGGTG TTGATCTTGT AGGCCTCCAA 7051 CGAGCGGGTC TCGACGGTGG GCAGATCGAG CACGAGTGCG TCGCCGGCGG 7101 GGCGGGGTTG TGGGGTGCCG GCGCCGGCGG CCAGGATCGA GCGCACGTCG 40 7151 GCAGCGCGGA ACCGGCGAAA CGCAACCGCC CGGCGCAGCG CGTCAATCAA 7201 AGCCTGTTCG CCGTGGGCGG CGCCAAGGCC GAGCAGAATG TCGAGTTCGG 7251 ATTTCAGTCG GGTGTTGCCG ATCGCAGCAG CACCGACGAG GAACTGCTGC 7301 GCTTCGGTTC CCAATGCGCA GAATCGTTTC TCTGCTTGGG TTTTCGGGCG * 7351 AGGACCACGC GAGGGTGCGG GTCTGGGTCC GTCGTAGTGT TCATCGAGGA 45 7401 TGGACACCTC ACCTGGGCTG ACGAGCTCGT GCTCGGCCAC GATCACACCG 7451 GTCGCAGGTT CCAACAGGAT CAGGGCGCCA TGATCGACCA CCACCGCCAC 7501 GGTGGCACCG ACGAGCCGCT GAGGCACCGA GTAACGAGCT GAGCCGTAAC 7551 GGATGCACGA GAGGCCGTCG ACCTTACGGC GCACCGACCC CGAGCCGATC 7601 GTCGGCCGCA GCGAGGGCAG CTCCCTCAAG ACGGTGCGCT CGTCAACCAA 50 7651 GCGATCGTTG GGCACGGCGC AGATCTCCGA GTGGACCGTG GCATTGACCT 7701 CGGCGCACCA TAGTTGCGCC TGGGCGTTGA GGGCACGTAG GTCGACCTGC 7751 TCACCGGCTA ACGCAGCTTC GGTCAGCAGC GGCACCGCAA GGTCGTCCTG 7801 AGCGTAGCCA CAGAGGTTCT CCACGATGCC CTTCGATTGC GGATCCGCAC WO 97/- 14 PCT/GB96/03221 7851 CGTGGCAGAA GTCCGGAACG AAGCCATAGT GGGACGCGAA TCGCACATAA 7901 TCCGGTGTTG GAACAACAAC ATTGGCGACG ACACCACCTT TGAGGCAGCC 7951 CATCCGGTCG GCCAGGATCT TGGCCGGAAC CCCACCGATC GCCTC Seq. ID No.4 l TTCTACTGCC TGACCTGAGC AGCGCCGAGG CGCGCAGCGC GATCACTGCG ACCTGAATGG 61 CCAGGTGGAA AGCGCCACCG ATCCCGGCAC CGAGTGCCTG ACGATTCGGA TCCCTTGCAC 121 CACAACGAGA GTGAGACCGC CATGATGACG AAATATCGGC TGGGCGGAGT CAACGCCGGA 181 GTGACAAAAG TGAGAACCCG GTGAAGCGAG CGCTTATAAC AGGGATCACG GGGCAGGATG 241 GTTCCTACCT CGCCGAGCTA CTACTGAGCA AGGGATACGA GGTTCACGGG CTCGTTCGTC 301 GAGCTTCGAC GTTTAACACG TCGCGGATCG ATCACCTCTA CGTTGACCCA CACCAACCGG 361 GCGCGCGCTT GTTCTTGCAC TATGCAGACC TCACTGACGG CACCCGGTTG GTGACCCTGC 421 TCAGCAGTAT CGACCCGGAT GAGGTCTACA ACCTCGCAGC GCAGTCCCAT GTGCGCGTCA 481 GCTTTGACGA GCCAGTGCAT ACCGGAGACA CCACCGGCAT GGGATCGATC CGACTTCTGG 541 AAGCAGTCCG CCTTTCTCGG GTGGACTGCC GGTTCTATCA GGCTTCCTCG TCGGAGATGT 601 TCGGCGCATC TCCGCCACCG CAGAACGAAT CGACGCCGTT CTATCCCCGT TCGCCATACG 661 GCGCGGCCAA GGTCTTCTCG TACTGGACGA CTCGCAACTA TCGAGAGGCG TACGGATTAT 721 TCGCAGTGAA TGGCATCTTG TTCAACCATG AGTCCCCCCG GCGCGGCGAG ACTTTCGTGA 781 CCCGAAAGAT CACGCGTGCC GTGGCGCGCA TCCGAGCTGG CGTCCAATCG GAGGTCTATA 841 TGGGCAACCT CGATGCGATC CGCGACTGGG GCTACGCGCC CGAATATGTC GAGGGGATGT 901 GGAGGATGTT GCAAGCGCCT GAACCTGATG ACTACGTCCT GGCGACAGGG CGTGGTTACA 961 CCGTACGTGA GTTCGCTCAA GCTGCTTTTG ACCACGTCGG GCTCGACTGG CAAAAGCACG 1021 TCAAGTTTGA CGACCGCTAT TTGCGCCCCA CCGAGGTCGA TTCGCTAGTA GGAGATGCCG 1081 ACAGGGCGGC CCAGTCACTC GGCTGGAAAG CTTCGGTTCA TACTGGTGAA CTCGCGCGCA 1141 TCATGGTGGA CGCGGACATC GCCGCGTCGG AGTGCGATGG CACACCATGG ATCGACACGC 1201 CGATGTTGCC TGGTTGGGGC GGAGTAAGTT GACGACTACA CCTGGGCCTC TGGACCGCGC 1261 AACGCCCGTG TATATCGCCG GTCATCGGGG GCTGGTCGGC TCAGCGCTCG TACGTAGATT 1321 TGAGGCCGAG GGGTTCACCA ATCTCATTGT GCGATCACGC GATGAGATTG ATCTGACGGA 1381 CCGAGCCGCA ACGTTTGATT TTGTGTCTGA GACAAGACCA CAGGTGATCA TCGATGCGGC 1441 CGCACGGGTC GGCGGCATCA TGGCGAATAA CACCTATCCC GCGGACTTCT TGTCCGAAAA 1501 CCTCCGAATC CAGACCAATT TGCTCGACGC AGCTGTCGCC GTGCGTGTGC CGCGGCTCCT 1561 TTTCCTCGGT TCGTCATGCA TCTACCCGAA GTACGCTCCG CAACCTATCC ACGAGAGTGC 1621 TTTATTGACT GGCCCTTTGG AGCCCACCAA CGACGCGTAT GCGATCGCCA AGATCGCCGG 1681 TATCCTGCAA GTTCAGGCGG TTAGGCGCCA ATATGGGCTG GCGTGGATCT CTGCGATGCC 1741 GACTAACCTC TACGGACCCG GCGACAACTT CTCCCCGTCC GGGTCGCATC TCTTGCCGGC 1801 GCTCATCCGT CGATATGAGG AAGCCAAAGC TGGTGGTGCA GAAGAGGTGA CGAATTGGGG 1861 GACCGGTACT CCGCGGCGCG AACTTCTGCA TGTCGACGAT CTGGCGAGCG CATGCCTGTT 1921 CCTTTTGGAA CATTTCGATG GTCCGAACCA CGTCAACGTG GGCACCGGCG TCGATCACAG 1981 CATTAGCGAG ATCGCAGACA TGGTCGCTAC GGCGGTGGGC TACATCGGCG AAACACGTTG 2041 GGATCCAACT AAACCCGATG GAACCCCGCG CAAACTATTG GACGTCTCCG CGCTACGCGA 40 2101 GTTGGGTTGG CGCCCGCGAA TCGCACTGAA AGACGGCATC GATGCAACGG TGTCGTGGTA * 2161 CCGCACAAAT GCCGATGCCG TGAGGAGGTA AAGCTGCGGG CCGGCCGATG TTATCCCTCC 2221 GGCCGGACGG GTAGGGCGAC CTGCCATCGA GTGGTACGGC AGTCGCCTGG CCGGCGAGGC 2281 GCATGGCCTA TGGGAGTATC CCATAGCCTG GCTTGGCTCG CCCCTACGCA TTATCAGTTG 2341 ACCGCTTTCG CGCCAGCTCG CAGGCTCGCG GCAGCATCCC GTTCAGGTCT CCTCATGGTC 45 2401 CGGTGTGGCA CGACCACGCA AGCTCGAACC GACTCGTTTC CCAATTTCGC ATGCTAATAT 2461 CGCTCGATGG ATTTTTTGCG CAACGCCGGC TTGATGGCTC GTAACGTTAG CACCGAGATG 2521 CTGCGCCACT TCGAACGAAA GCGCCTATTA GTAAACCAAT TCAAAGCATA CGGAGTCAAC 2581 GTTGTTATTG ATGTCGGTGC TAACTCCGGC CAGTTCGGTA GCGCTTTGCG TCGTGCAGGA 2641 TTCAAGAGCC GTATCGTTTC CTTTGAACCT CTTTCGGGGC CATTTGCGCA ACTAACGCGC 50 2701 GAGTCGGCAT CGGATCCACT ATGGGAGTGT CACCAGTATG CCCTAGGCGA CGCCGATGAG WO 97/1"*624 27G1 ACGATTACCA TCAATGTGGC AGGCAATGCG GGGGCAAGTA GTTCCGTGCT GCCGATGCTT 2821 AAAAGTCATC AAGATGCCTT TCCTCCCGCG AATTATATTG GCACCGAAGA CGTTGCAATA 2681 CACCGCCTTG ATTCGGTTGC ATCAGAATTT CTGAACCCTA CCGATGTTAC TTTCCTGAAG 2941 ATCGACGTAC AGGGTTTCGA GAAGCAGGTT ATCGCGGGCA GTAAGTCAAC GCTTAACGAA 3001 AGCTGCGTCG GCATGCAACT CGAACTTTCT TTTATTCCGT TGTACGAAGG TGACATGCTG 3061 ATTCATGAAG CGCTTGAACT TGTCTATTCC CTAGGTTTCA GACTGACGGG TTTGTTGCCC 3121 GGATTTACGG ATCCGCGCAA TGGTCGAATG CTTCAAGCTG ACGGCATTTT CTTCCGTGGG 3181 GACGATTGAC ATAAATGCTT GCGTCGGCAC CCTGCCGGTA TCCAAACGGG CGATCTGGTG 3241 AGCCGGCCTC CCGGGCACCT AATCGACTAT CTAAATTGAG GCGGCCGCGA CGTGCGGCAC 3301 GAACAGGTGG CCGGCTGCTA GCGTTACACA CGTCATGACT GCGCCAGTGT TCTCGATAAT 3361 TATCCCTACC TTCAATGCAG CGGTGACGCT GCAAGCCTGC CTCGGAAGCA TCGTCGGGCA 3421 GACCTACCGG GAAGTGGAAG TGGTCCTTGT CGACGGCGGT TCGACCGATC GGACCCTCGA 3481 CATCGCGAAC AGTTTCCGCC CGGAACTCGG CTCGCGACTG GTCGTTCACA GCGGGCCCGA 3541 TGATGGCCCC TACGACGCCA TGAACCGCGG CGTCGGCGTA GCCACAGGCG AATGGGTACT 3601 TTTTTTAGGC GCCGACGACA CCCTCTACGA ACCAACCACG TTGGCCCAGG TAGCCGCTTT 3661 TCTCGGCGAC CATGCGGCAA GCCATCTTGT CTATGGCGAT GTTGTGATGC GTTCGACGAA 3721 AAGCCGGCAT GCCGGACCTT TCGACCTCGA CCGCCTCCTA TTTGAGACGA ATTTGTGCCA 3781 CCAATCGATC TTTTACCGCC GTGAGCTTTT CGACGGCATC GGCCCTTACA ACCTGCGCTA 3841 CCGAGTCTGG GCGGACTGGG ACTTCAATAT TCGCTGCTTC TCCAACCCGG CGCTGATTAC 3901 CCGCTACATG GACGTCGTGA TTTCCGAATA CAACGACATG ACCGGCTTCA GCATGAGGCA 3961 GGGGACTGAT AAAGAGTTCA GAAAACGGCT GCCAATGTAC TTCTGGGTTG CAGGGTGGGA 4021 GACTTGCAGG CGCATGCTGG CGTTTTTGAA AGACAAGGAG AATCGCCGTC TGGCCTTGCG 4081 TACGCGGTTG ATAAGGGTTA AGGCCGTCTC CAAAGAACGA AGCGCAGAAC CGTAGTCGCG 4141 GATCCACATT GGACTTCTTT AACGCGTTTG CGTCCTGATC CACCTTTCAA CCCCGTTCCG 4201 CGTGACGCGG CGCGCAGAGA GTGGTCGCAT ATCGCGTCAC TGTTCTCGTG CCAGTGCTTG 4261 GAAAGCGTCG AGCACTCTGG TTCGCGTTCT TGACGTTCGC GCCCGCCCCT AGAGGTAGCG 4321 TGTCACGTGA CTGAAGCCAA TGAGTGCAAC TCGGCGTCGC GAAAGGTTTC AGTCGCGGTT 4381 GAGCAAGACA CCGCAAGACT ACTGGAGTGC GTGCACAAGC GCCTCCAGCT CACGG Seq. ID No.5 1 atgatcgctg tgatctggtc ggcggtgccg acaggaaccg tcgacttgtc gacgatcacc 61 ttgtaccggt cgatgtatga cccaatgtcg tccgcaaccg agaagacgta cgtcaggtcc 121 gccgccccgc tttcacccat gggcgtcggg acggcgatga aaatgacgtc cgcgtgctcg 181 attccgcgtt gccggtcggt ggtgaagtca atcagcccgt tctcacggtt cctcgcaatc 241 aactcccaac ccgggctcga aaatcgggac actgcctgcg aggagcaaat cgatcttggc 301 ctgatcgata tcgacacaga cgacatcgtt gccgctatcc gcgagacagg cgcccgtgac 361 gaggcctaca tagcctga Seq. ID No.6 1 M I A V I H S A V P T G T V D L S T I T L Y R s M Y D P M S 31 S A T E K T y V R S A A P L S P M G V G T A M K M T S A C S 61 I P R C R S V V K S I S P F S R F L A I N S Q P G L E N R D 91 T A C E E Q I D L G L I D I D T D D I V A A I R E T G A R D 121 E A Y I A Seq. ID No. 7 l gtgtcatctg ctccaaccgt gtcggtgata acgatttcgc tgaacgatct cgagggattg 61 aaaagcaccg tggagagcgt tcgcgcgcag cgctatgggg ggcgaatcga gcacatcgtc 121 atcgacggtg gatcgggcga cgccgtcgtg gagtatctgt ccggcgatcc tggctttgca 181 tattggcaat ctcagcccga caacgggaga tatgacgcga tgaatcaggg cattgcccat 241 tcgtcgggcg acctgttgtg gtttatgcac tccacggatc gtttctccga tccagatgca 301 gtcgcttccg tggtggaggc gctctcgggg catggaccag tacgtgattt gtggggttac 361 gggaaaaaca accttgtcgg actcgacggc aaaccacttt tccctcggcc gtacggctat 421 atgccgttta agatgcggaa atttctgctc ggcgcgacgg ttgcgcatca ggcgacattc 481 ttcggcgcgt cgctggtagc caagttgggc ggttacgatc ttgattttgg actcgaggcg 541 gaccagctgt tcatctaccg tgccgcacta atacggcctc ccgtcacgat cgaccgcgtg 601 gtttgcgact tcgatgtcac gggacctggt tcaacccagc ccatccgtga gcactatcgg 661 accctgcggc ggctctggga cctgcatggc gactacccgc tgggtgggcg cagagtgtcg 721 tgggcttact tgcgtgtgaa ggagtacttg attcgggccg acctggccgc attcaacgcg 781 gtaaagttct tgcgagcgaa gttcgccaga gcttcgcgga agcaaaattc atag Seq. ID No.8 1 VSSAPTVSVITI SLNDLEGLKSTVESVRAQ 31 R Y G G R I E H I V I D G G S G D A V V E Y L S G D P G F A 61 Y W Q S Q P D N G R Y D A M N Q G I A H S S G D L h W F M H 91 S T D R F S D P D A V A S V V E A L S G H G P V R D L W G Y 121 G K N N L V G L D G K P L F P R P Y G Y M P F K M R K F L L 151 G A T V A H Q A T F F G A S L V A K L G G Y D L D F G L E A 181 D Q L F I Y R A A L I R P P V T I D R V V C D F D V T G P G 211 S T Q P I R E H Y R T L R R L W D h H G D Y P L G G R R V S 241 H A Y L R V K E Y L I R A D L A A F N A V K F L R A K F A R 271 A S R K Q N S Seq. ID No.9 1 gtgaagcgag cgcttataac agggatcacg gggcaggatg gttcctacct cgccgagcta 61 ctactgagca agggatacga ggttcacggg ctcgttcgtc gagcttcgac gtttaacacg 30 121 tcgcggatcg atcacctcta cgttgaccca caccaaccgg gcgcgcgctt gttcttgcac 181 tatgcagacc tcactgacgg cacccggttg gtgaccctgc tcagcagtat cgacccggat 241 gaggtctaca acctcgcagc gcagtcccat gtgcgcgtca gctttgacga gccagtgcat 301 accggagaca ccaccggcat gggatcgatc cgacttctgg aagcagtccg cctttctcgg 361 gtggactgcc ggttctatca ggcttcctcg tcggagatgt tcggcgcatc tccgccaccg 35 421 cagaacgaat cgacgccgtt ctatccccgt tcgccatacg gcgcggccaa ggtcttctcg 481 tactggacga ctcgcaacta tcgagaggcg tacggattat tcgcagtgaa tggcatcttg t 541 ttcaaccatg agtccccccg gcgcggcgag actttcgtga cccgaaagat cacgcgtgcc 601 gtggcgcgca tccgagctgg cgtccaatcg gaggtctata tgggcaacct cgatgcgatc 661 cgcgactggg gctacgcgcc cgaacatgtc gaggggatgt ggaggatgtt gcaagcgcct 40 721 gaacctgatg actacgtcct ggcgacaggg cgtggttaca ccgtacgtga gttcgctcaa 781 gctgcttttg accatgtcgg gctcgactgg caaaagcgcg tcaagtttga cgaccgctat 841 ttgcgtccca ccgaggtcga ttcgctagta ggagatgccg acaaggcggc ccagtcactc 901 ggctggaaag cttcggttca tactggtgaa ctcgcgcgca tcatggtgga cgcggacatc 961 gccgcgttgg agtgcgatgg cacaccatgg atcgacacgc cgatgttgcc tggttggggc 45 1021 agagtaagtt ga Seq. ID No.10 1 V K R A L I T G I T G Q D G S Y h A E L L L S K G Y E V G 31 L V R R A S T F N T S R I D H L Y V D P H Q P G A R L F L H 61 Y A D L T D G T R L V T L h S S I D P D E V Y N L A A Q S H 91 V R V S F D E P V H T G D T T G M G S I R L L E A V R L S R 121 V D C R F Y Q A S S S E M F G A S P P P Q N E S T P F Y P R 151 S P y G A A K V F S Y W T T R N Y R E A Y G L F A V N G I L 181 F N H E S P R R G E T F V T R K I T R A V A R I R A G V Q S 211 E V Y M G N L D A I R D W G Y A P E Y V E G M H R M L Q A P 241 E P D D Y V L A T G R G Y T V R E F A Q A A F D H V G L D W 271 Q K R V K F D D R Y L R P T E V D S L V G D A D K A A Q S L 301 G H K A S V H T G E L A R I M V D A D I A A L E C D G T P W 331 I D T P M L P G W G R V S Seq. ID No.11 l gtgaagcgag cgcttataac agggatcacg gggcaggatg gttcctacct cgccgagcta 61 ctactgagca agggatacga ggttcacggg ctcgttcgtc gagcttcgac gtttaacacg 121 tcgcggatcg atcacctcta cgttgaccca caccaaccgg gcgcgcgctt gttcttgcac 181 tatgcagacc tcactgacgg cacccggttg gtgaccctgc tcagcagtat cgacccggat 241 gaggtctaca acctcgcagc gcagtcccat gtgcgcgtca gctttgacga gccagtgcat 301 accggagaca ccaccggcat gggatcgatc cgacttctgg aagcagtccg cctttctcgg 361 gtggactgcc ggttctatca ggcttcctcg tcggagatgt tcggcgcatc tccgccaccg 421 cagaacgaat cgacgccgtt ctatccccgt tcgccatacg gcgcggccaa ggtcttctcg 481 tactggacga ctcgcaacta tcgagaggcg tacggattat tcgcagtgaa tggcatcttg 541 ttcaaccatg agtccccccg gcgcggcgag actttcgtga cccgaaagat cacgcgtgcc 601 gtggcgcgca tccgagctgg cgtccaatcg gaggtctata tgggcaacct cgatgcgatc 661 cgcgactggg gctacgcgcc cgaatatgtc gaggggatgt ggaggatgtt gcaagcgcct 721 gaacctgatg actacgtcct ggcgacaggg cgtggttaca ccgtacgtga gttcgctcaa 781 gctgcttttg accacgtcgg gctcgactgg caaaagcacg tcaagtttga cgaccgctat 841 ttgcgcccca ccgaggtcga ttcgctagta ggagatgccg acagggcggc ccagtcactc 901 ggctggaaag cttcggttca tactggtgaa ctcgcgcgca tcatggtgga cgcggacatc 961 gccgcgtcgg agtgcgatgg cacaccatgg atcgacacgc cgatgttgcc tggttggggc 1021 ggagtaagtt ga Seq. ID No.12 l V K R A L I T G I T G Q D G S Y L A E L L L S K G Y E V H G 31 L V R R A S T F N T S R I D H L Y V D P H Q P G A R L F L H 61 Y A D L T D G T R L V T L L S S I D P D E V Y N L A A Q S H * 91 V R V S F D E P V H T G D T T G M G S I R L L E A V R L S R 121 V D C R F Y Q A S S S E M F G A S P P P Q N E S T P F Y P R 151 S P y G A A K V F S Y W T T R N Y R E A Y G L F A V N G I L 40 181 F N H E S P R R G E T F V T R K I T R A V A R I R A G V Q S 211 E V Y M G N L D A I R D H G Y A P E Y V E G M H R M L Q A P 241 E P D D Y V L A T G R G Y T V R E F A Q A A F D H V G L D W 271 Q K H V K F D D R Y L R P T E V D S L V G D A D R A A Q S L 301 G W K A S V H T G E L A R I M V D A D I A A S E C D G T P W 45 331 I D T P M L P G W G G V S WO 97/?'"'24 PCT/G B96/03221 Seq. ID No.13 1 gtgcgatggc acaccatgga tcgacacgcc gatgttgcct ggttggggca gagtaagttg 61 acgactacac ctgggcctct ggaccgcgca acgcccgtgt atatcgccgg tcatcggggg 121 ctggtcggct cagcgctcgt acgtagattt gaggccgagg ggttcaccaa tctcattgtg 181 cgatcacgcg atgagattga tctgacggac cgagccgcaa cgtttgattt tgtgtctgag 241 acaagaccac aggtgatcat cgatgcggcc gcacgggtcg gcggcatcat ggcgaataac 301 acctatcccg cggacttctt gtccgaaaac ctccgaatcc agaccaattt gctcgacgca 361 gctgtcgccg tgcgtgtgcc gcggctcctt ttcctcggtt cgtcatgcat ctacccgaag 421 tacgctccgc aacctatcca cgagagtgct ttattgactg gccctttgga gcccaccaac 481 gacgcgtatg cgatcgccaa gatcgccggt atcctgcaag ttcaggcggt taggcgccaa 541 tatgggctgg cgtggatctc tgcgatgccg actaacctct acggacccgg cgacaacttc 601 tccccgtccg ggtcgcatct cttgccggcg ctcatccgtc gatatgagga agccaaagct 661 ggtggtgcag aagaggtgac gaattggggg accggtactc cgcggcgcga acttctgcat 721 gtcgacgatc tggcgagcgc atgcctgttc cttttggaac atttcgatgg tccgaaccac 781 gtcaacgtgg gcaccggcgt cgatcacagc attagcgaga tcgcagacat ggtcgctaca 841 gcggtgggct acatcggcga aacacgttgg gatccaacta aacccgatgg aaccccgcgc 901 aaactattgg acgtctccgc gctacgcgag ttgggttggc gcccgcgaat cgcactgaaa 961 gacggcatcg atgcaacggt gtcgtggtac cgcacaaatg ccgatgccgt gaggaggtaa Seq. ID No.14 1 V R H H T M D R H A D V A W h G Q S K L T T T P G P L D R A 31 T P V Y I A G H R G L V G S A L V R R F E A E G F T N L I V 61 R S R D E I D h T D R A A T F D F V S E T R P Q V I I D A A 91 A R V G G I M A N N T Y P A D F L S E N L R I Q T N L L D A 121 A V A V R V P R L L F L G S S C I Y P K Y A P Q P I H E S A 151 L L T G P L E P T N D A Y A I A K I A G I L Q V Q A V R R Q 181 Y G L A W I S A M P T N L Y G P G D N F S P S G S H L L P A 211 L I R R Y E E A K A G G A E E V T N w G T G T P R R E L L H 241 V D D L A S A C L F L L E H F D G P N H V N V G T G V D H S 271 I S E I A D M V A T A V G Y I G E T R W D P T K P D G T P R 301 K L L D V S A L R E L G W R P R I A L K D G I D A T V S W Y 331 R T N A D A V R R WO 97/^24 Seq. ID No.15 1 gtgcgatggc acaccatgga tcgacacgcc gatgttgcct ggttggggcg gagtaagttg ei acgactacac ctgggcctct ggaccgcgca acgcccgtgt atatcgccgg tcatcggggg 121 ctggtcggct cagcgctcgt acgtagattt gaggccgagg ggttcaccaa tctcattgtg 181 cgatcacgcg atgagattga tctgacggac cgagccgcaa cgtttgattt tgtgtctgag 241 acaagaccac aggtgatcat cgatgcggcc gcacgggtcg gcggcatcat ggcgaataac 301 acctatcccg cggacttctt gtccgaaaac ctccgaatcc agaccaattt gctcgacgca 361 gctgtcgccg tgcgtgtgcc gcggctcctt ttcctcggtt cgtcatgcat ctacccgaag 421 tacgctccgc aacctatcca cgagagtgct ttattgactg gccctttgga gcccaccaac 4B1 gacgcgtatg cgatcgccaa gatcgccggt atcctgcaag ttcaggcggt taggcgccaa 541 tatgggctgg cgtggatctc tgcgatgccg actaacctct acggacccgg cgacaacttc 601 tccccgtccg ggtcgcatct cttgccggcg ctcatccgtc gatatgagga agccaaagct 661 ggtggtgcag aagaggtgac gaattggggg accggtactc cgcggcgcga acttctgcat 721 gtcgacgatc tggcgagcgc atgcctgttc cttttggaac atttcgatgg tccgaaccac 781 gtcaacgtgg gcaccggcgt cgatcacagc attagcgaga tcgcagacat ggtcgctacg 841 gcggtgggct acatcggcga aacacgttgg gatccaacta aacccgatgg aaccccgcgc 901 aaactattgg acgtctccgc gctacgcgag ttgggttggc gcccgcgaat cgcactgaaa 961 gacggcatcg atgcaacggt gtcgtggtac cgcacaaatg ccgatgccgt gaggaggtaa Seq. ID No.16 l V R W H T M D R H A D V A W L G R S K L T T T P G P L D R A 31 T P V Y I A G H R G L V G s A L V R R F E A E G F T N L I V 61 R S R D E I D L T D R A A T F D F V S E T R P Q V I I D A A 91 A R V G G I M A N N T Y P A D F L S E N L R I Q T N L L D A 121 A V A V R V P R L L F L G S S C I Y P K Y A P Q P I H E S A 151 L L T G P L E P T N D A Y A I A K I A G I L Q V Q A V R R Q 181 Y G L A W I S A M P T N L Y G P G D N F S P S G S H L L P A 211 L I R R Y E E A K A G G A E E V T N W G T G T P R R E L L H 241 V D D L A S A C L F L L E H F D G P N H V N V G T G V D H S 271 I S E I A D M V A T A V G y I G E T R W D P T K P D G T P R 301 K L L D V S A L R E L G W R P R I A L K D G I D A T V S W Y 331 R T N A D A V R R Seg. ID No.17 l atggattttt tgcgcaacgc cggcttgatg gctcgtaacg ttagtaccga gatgctgcgc 61 cacttcgaac gaaagcgcct attagtaaac caattcaaag catacggagt caacgttgtt 121 attgatgtcg gtgctaactc cggccagttc ggtagcgctt tgcgtcgtgc aggattcaag 181 agccgtatcg tttcctttga acctctttcg gggccatttg cgcaactaac gcgcaagtcg * 241 gcatcggatc cactatggga gtgtcaccag tatgccctag gcgacgccga tgagacgatt 301 accatcaatg tggcaggcaa tgcgggggca agtagttccg tgctgccgat gcttaaaagt 40 361 catcaagatg cctttcctcc cgcgaattat attggcaccg aagacgttgc aatacaccgc 421 cttgattcgg ttgcatcaga atttctgaac cctaccgatg ttactttcct gaagatcgac 481 gtacagggtt tcgagaagca ggttatcacg ggcagtaagt caacgcttaa cgaaagctgc 541 gtcggcatgc aactcgaact ttcttttatt ccgttgtacg aaggtgacat gctgattcat 601 gaagcgcttg aacttgtcta ttccctaggt ttcagactga cgggtttgtt gcccggcttt 45 661 acggatccgc gcaatggtcg aatgcttcaa gctgacggca ttttcttccg tggggacgat 721 tga WO 97/2 """>4 Seq. ID No.18 1 M D F L R N A G L M A R N V S T E M L R H F E R K R L L V N 31 Q F K A Y G V N V V I D V G A N S G Q F G S A L R R A G F K 61 S R I V S F E P L S G P F A Q L T R K S A S D P L H E C H Q 91 Y A L G D A D E T I T I N V A G N A G A S S S V L P M L K S 121 H Q D A F P P A N Y I G T E D V A I H R L D S V A S E F L N 151 P T D V T F L K I D V Q G F E K Q V I T G S K S T L N E S C 181 V G M Q L E L S F I P L Y E G D M L I K E A L E L V Y S L G 211 F R L T G L L P G F T D P R N G R M L Q A D G I F F R G D D Seq. ID No.19 1 atggattttt tgcgcaacgc cggcttgatg gctcgtaacg ttagcaccga gatgctgcgc 61 cacttcgaac gaaagcgcct attagtaaac caattcaaag catacggagt caacgttgtt 121 attgatgtcg gtgctaactc cggccagttc ggtagcgctt tgcgtcgtgc aggattcaag 181 agccgtatcg tttcctttga acctctttcg gggccatttg cgcaactaac gcgcgagtcg 241 gcatcggatc cactatggga gtgtcaccag tatgccctag gcgacgccga tgagacgatt 301 accatcaatg tggcaggcaa tgcgggggca agtagttccg tgctgccgat gcttaaaagt 361 catcaagatg cctttcctcc cgcgaattat attggcaccg aagacgttgc aatacaccgc 421 cttgattcgg ttgcatcaga atttctgaac cctaccgatg ttactttcct gaagatcgac 481 gtacagggtt tcgagaagca ggttatcgcg ggcagtaagt caacgcttaa cgaaagctgc 541 gtcggcatgc aactcgaact ttcttttatt ccgttgtacg aaggtgacat gctgattcat 601 gaagcgcttg aacttgtcta ttccctaggt ttcagactga cgggtttgtt gcccggattt 661 acggatccgc gcaatggtcg aatgcttcaa gctgacggca ttttcttccg tggggacgat 721 tga Seq. ID No.20 1 M D F L R N A G L M A R N V S T E M L R H F E R K R L L V N 31 Q F K A Y G V N V V I D V G A N S G Q F G S A L R R A G F K 61 S R I V S F E P L S G P F A Q L T R E S A S D P L W E C H Q 91 Y A L G D A D E T I T I N V A G N A G A S S S V L P M L K S 121 H Q D A F P P A N Y I G T E D V A I H R L D S V A S E F L N 151 P T D V T F L K I D V Q G F E K Q V I A G S K S T L N E S C 161 V G M Q L E L S F I P L Y E G D M L I H E A L E L V Y S L G 211 F R L T G L L P G F T D P R N G R M L Q A D G I F F R G D D WO 91 r "524 Seq. ID No.21 1 atgactgcgc cagtgttctc gataattatc cctaccttca atgcagcggt gacgctgcaa 61 gcctgcctcg gaagcatcgt cgggcagacc taccgggaag tggaagtggt ccttgtcgac 121 ggcggttcga ccgatcggac cctcgacatc gcgaacagtt tccgcccgga actcggctcg 181 cgactggtcg ttcacagcgg gcccgatgat ggcccctacg acgccatgaa ccgcggcgtc 241 ggcgtggcca caggcgaatg ggtacttttt ttaggcgccg acgacaccct ctacgaacca 301 accacgttgg cccaggtagc cgcttttctc ggcgaccatg cggcaagcca tcttgtctat 361 ggcgatgttg tgatgcgttc gacgaaaagc cggcatgccg gacctttcga cctcgaccgc 421 ctcctatttg agacgaattt gtgccaccaa tcgatctttt accgccgtga gcttttcgac 481 ggcatcggcc cttacaacct gcgctaccga gtctgggcgg actgggactt caatattcgc 541 tgcttctcca acccggcgct gattacccgc tacatggacg tcgtgatttc cgaatacaac 601 gacatgaccg gcttcagcat gaggcagggg actgataaag agttcagaaa acggctgcca 661 atgtacttct gggttgcagg gtgggagact tgcaggcgca tgctggcgtt tttgaaagac 721 aaggagaatc gccgtctggc cttgcgtacg cggttgataa gggttaaggc cgtctccaaa 781 gaacgaagcg cagaaccgta S Seq. ID No.22 l M T A P V F S I I I P T F N A A V T L Q A C L G S I V G Q T 31 Y R E V E V V L V D G G S T D R T L D I A N S F R P E L G S 61 R L V V H S G P D D G P Y D A M N R G V G V A T G E W V L F 91 L G A D D T L Y E P T T L A Q V A A F L G D H A A S H L V Y 121 G D V V M R S T K S R H A G P F D L D R L L F E T N L C H Q 151 S I F Y R R E L F D G I G P Y N L R Y R V W A D W D F N I R 181 C F S N P A L I T R Y M D V V I S E Y N D M T G F S M R Q G 211 T D K E F R K R L P M Y F W V A G H E T C R R M L A F L K D 241 K E N R R L A L R T R L I R V K A V S K E R S A E P Seq. ID No.23 l atgactgcgc cagtgttctc gataattatc cctaccttca atgcagcggt gacgctgcaa 61 gcctgcctcg gaagcatcgt cgggcagacc taccgggaag tggaagtggt ccttgtcgac 121 ggcggttcga ccgatcggac cctcgacatc gcgaacagtt tccgcccgga actcggctcg 181 cgactggtcg ttcacagcgg gcccgatgat ggcccctacg acgccatgaa ccgcggcgtc 241 ggcgtagcca caggcgaatg ggtacttttt ttaggcgccg acgacaccct ctacgaacca 301 accacgttgg cccaggtagc cgcttttctc ggcgaccatg cggcaagcca tcttgtctat 361 ggcgatgttg tgatgcgttc gacgaaaagc cggcatgccg gacctttcga cctcgaccgc 421 ctcctatttg agacgaattt gtgccaccaa tcgatctttt accgccgtga gcttttcgac 481 ggcatcggcc cttacaacct gcgctaccga gtctgggcgg actgggactt caatattcgc 541 tgcttctcca acccggcgct gattacccgc tacatggacg tcgtgatttc cgaatacaac ♦ 601 gacatgaccg gcttcagcat gaggcagggg actgataaag agttcagaaa acggctgcca 661 atgtacttct gggttgcagg gtgggagact tgcaggcgca tgctggcgtt tttgaaagac 721 aaggagaatc gccgtctggc cttgcgtacg cggttgataa gggttaaggc cgtctccaaa 40 781 gaacgaagcg cagaaccgta 9 WO 91 r 74 PCT/GB96/0322I Seq. ID No.24 l M T A P V F S I I I P T F N A A V T L Q A C L G S I V G Q T 31 y R E V E V V L V D G G S T D R T L D I A N S F R P E L G S 61 R L V V H S G P D D G P Y D A M N R G V G V A T G E W V L F 91 L G A D D T L Y E P T T L A Q V A A F L G D H A A S H L V Y 121 G D V V M R S T K S R H A G P F D L D R L L F E T N L C H Q 151 S I F y R R E L F D G I G P Y N h R Y R V W A D W D F N I R 181 C F S N P A L I T R Y M D V V I S E Y N D M T G F S M R Q G 211 T D K E F R K R L P M Y F W V A G W E T C R R M L A F L K D 241 K E N R R L A L R T R L I R V K A V S K E R S A E P Seq. ID No.25 1 gtggccagca gaagtcccca ctccgctgcg ggtggttggc taattcttgg cggctccctt 61 cttgtggtcg gcgtggcgca tccggtagga ctcgccggag gtgacgacga tgctggcgtg 121 gtgcagcagc cgatcgagga tgctggcggc ggtggtgtgc tcgggcagga atcgccccca 181 ttgttcgaag ggccaatgcg aggcgatggc cagggagcgg cgctcgtagc cggcagccac 241 gagccggaac aacagttgag tcccggtgtc gtcgagcggg gcgaagccga tctcgtccaa 301 gatgaccaga tccgcgcgga gcagggtgtc gatgatcttg ccgacggtgt tgtcggccag 361 gccgcggtag aggacctcga tcaggtcggc ggcggtgaag tagcggactt tgaatccggc 421 gtggacggca gcgtgcccgc agccgatgag caggtgactt ttgcccgtac caggtgggcc 481 aatgaccgcc aggttctgtt gtgcccgaat ccattccagg ctcgacaggt agtcgaacgt 541 ggctgcggtg atcgacgatc cggtgacgtc gaacccgtcg agggtcttgg tgaccgggaa 601 ggctgcggcc ttgagacggt tggcggtgtt ggaggcatcg cgggcagcga tctcggcctc 661 aaccaacgtc cgcaggatct cctccggtgt ccagcgttgc gtcttggcga cttgcaacac 721 ctcggcggcg ttgcggcgca ccgtggccag cttcaaccgc cgcagcgccg cgtcaaggtc 781 agcagccagc ggtgccgccg aggacggtgc caccggcttg gcagcggtgg tcatgaggcc 841 gtcccgtcgg tggtgttgat cttgtag Seq. ID No.26 1 V A S R S P H S A A G G W L I L G G S L L V V G V A H P V G 31 L A G G D D D A G V V Q Q P I E D A G G G G V L G Q E S P P 61 L F E G P M R G D G Q G A A L V A G S H E P E Q Q L S P G V 91 V E R G E A D L V Q D D Q I R A E Q G V D D L A D G V V G Q 121 A A V E D L D Q V G G G E V A D F E S G V D G S V P A A D E 151 Q V T F A R T R W A N D R Q V L L C P N P F Q A R Q V V E R 181 G C G D R R S G D V E P V E G L G D R E G C G L E T V G G V 211 G G I A G S D L G L N Q R P Q D L L R C P A L R L G D L Q H 241 L G G V A A H R G Q L Q P P Q R R V K V S S Q R C R R G R C 271 H R h G S G G H E A V P S V V L I L PCT/GB96/0322I Seq. ID No.27 l atgggctgcc tcaaaggtgg tgtcgtcgcc aatgttgttg ttccaacacc ggattatgtg 61 cgattcgcgt cccactatgg cttcgttccg gacttctgcc acggtgcgga tccgcaatcg 121 aagggcatcg tggagaacct ctgtggctac gctcaggacg accttgcggt gccgctgctg 181 accgaagctg cgttagccgg tgagcaggtc gacctacgtg ccctcaacgc ccaggcgcaa 241 ctatggtgcg ccgaggtcaa tgccacggtc cactcggaga tctgcgccgt gcccaacgat 301 cgcttggttg acgagcgcac cgtcttgagg gagctgccct cgctgcggcc gacgatcggc 361 tcggggtcgg tgcgccgtaa ggtcgacggc ctctcgtgca tccgttacgg ctcagctcgt 421 tactcggtgc ctcagcggct cgtcggtgcc accgtggcgg tggtggtcga tcatggcgcc 481 ctgatcctgt tggaacctgc gaccggtgtg atcgtggccg agcacgagct cgtcagccca 541 ggtgaggtgt ccatcctcga tgaacactac gacggaccca gacccgcacc ctcgcgtggt 601 cctcgcccga aaacccaagc agagaaacga ttctgcgcat tgggaaccga agcgcagcag 661 ttcctcgtcg gtgctgctgc gatcggcaac acccgactga aatccgaact cgacattctg 721 ctcggccttg gcgccgccca cggcgaacag gctttgattg acgcgctgcg ccgggcggtt 781 gcgtttcgcc ggttccgcgc tgccgacgtg cgctcgatcc tggccgccgg cgccggcacc 841 ccacaacccc gccccgccgg cgacgcactc gtgctcgatc tgcccaccgt cgagacccgc 901 tcgttggagg cctacaagat caacaccacc gacgggacgg cctcatgacc accgctgcca 961 agccggtggc accgtcctcg gcggcaccgc tggctgctga ccttgacgcg gcgctgcggc 1021 ggttgaagct ggccacggtg cgccgcaacg ccgccgaggt gttgcaagtc gccaagacgc 1081 aacgctggac accggaggag atcctgcgga cgttggttga ggccgagatc gctgcccgcg 1141 atgcctccaa caccgccaac cgtctcaagg ccgcagcctt cccggtcacc aagaccctcg 1201 acgggttcga cgtcaccgga tcgtcgatca ccgcagccac gttcgactac ctgtcgagcc 1261 tggaatggat tcgggcacaa cagaacctgg cggtcattgg cccacctggt acgggcaaaa 1321 gtcacctgct catcggctgc gggcacgctg ccgtccacgc cggattcaaa gtccgctact 1381 tcaccgccgc cgacctgatc gaggtcctct accgcggcct ggccgacaac accgtcggca 1441 agatcatcga caccctgctc cgcgcggatc tggtcatctt ggacgagatc ggcttcgccc 1501 cgctcgacga caccgggact caactgttgt tccggctcgt ggctgccggc tacgagcgcc 1561 gctccctggc catcgcctcg cattggccct tcgaacaatg ggggcgattc ctgcccgagc 1621 acaccaccgc cgccagcatc ctcgatcggc tgctgcacca cgccagcatc gtcgtcacct 1681 ccggcgagtc ctaccggatg cgccacgccg accacaagaa gggagccgcc aagaattag Seq. ID No.28 1 M G C I> K G G V V A N V V V P T P D Y V R F A S H Y G F V P 31 D F C H G A D P Q S K G I V E N L C G Y A Q D D L A V P L L 61 T E A A L A G E Q V D L R A L N A Q A Q L w C A E V N A T V 91 H S E I C A V P N D R L V D E R T V L R E h P S L R P T I G 121 S G S V R R K V D G L S C I R Y G S A R Y S V P Q R L V G A 151 T V A V V V D H G A L I L L E P A T G V I V A E H E L V S P 181 G E V S I L D E H Y D G P R P A P S R G P R P K T Q A E K R 211 F C A L G T E A Q Q F L V G A A A I G N T R L K S E L D I L 241 L G L G A A H G E Q A L I D A L R R A V A F R R F R A A D V 271 R S I L A A G A G T P Q P R P A G D A L V L D L P T V E T R 301 S L E A y K I N T T D G T A S WO 97/^24 Seq. ID No.29 1 M T T A A K P V A P S S A A P L A A D L D A A L R R L K L A 31 T V R R N A A E V L Q V A K T Q R W T P E E I L R T L V E A 61 E I A A R D A S N T A N R L K A A A F P V T K T L D G F D V 91 T G S S I T A A T F D Y L S S L E H I R A Q Q N L A V I G P 121 P G T G K S H L L I G C G H A A V H A G F K V R Y F T A A D 151 L I E V L Y R G L A D N T V G K I I D T L L R A D L V I L D 181 E I G F A P L D D T G T Q L L F R L V A A G Y E R R S L A I 211 A S H W P F E Q W G R F L P E H T T A A S I L D R L L H H A 241 S I V V T S G E S Y R M R H A D H K K G A A K N I ^''Seq. ID No.30 1 gtgacgtctg ctccgaccgt ctcggtgata acgatctcgt tcaacgacct cgacgggttg 61 cagcgcacgg tgaaaagtgt gcgggcgcaa cgctaccggg gacgcatcga gcacatcgta 121 atcgacggtg gcagcggcga cgacgtggtg gcatacctgt ccgggtgtga accaggcttc 181 gcgtattggc agtccgagcc cgacggcggg cggtacgacg cgatgaacca gggcatcgcg 241 cacgcatcgg gtgatctgtt gtggttcttg cactccgccg atcgtttttc cgggcccgac 301 gtggtagccc aggccgtgga ggcgctatcc ggcaagggac cggtgtccga attgtggggc 361 ttcgggatgg atcgtctcgt cgggctcgat cgggtgcgcg gcccgatacc tttcagcctg 421 cgcaaattcc tggccggcaa gcaggttgtt ccgcatcaag catcgttctt cggatcatcg 481 ctggtggcca agatcggtgg ctacgacctt gatttcggga tcgccgccga ccaggaattc 541 atattgcggg ccgcgctggt atgcgagccg gtcacgattc ggtgtgtgct gtgcgagttc 601 gacaccacgg gcgtcggctc gcaccgggaa ccaagcgcgg tcttcggtga tctgcgccgc 661 atgggcgacc ttcatcgccg ctacccgttc gggggaaggc gaatatcaca tgcctaccta 721 cgcggccggg agttctacgc ctacaacagt cgattctggg aaaacgtctt cacgcgaatg 781 tcgaaatag Seq. ID No.31 1 M T S A P T V S V I T I S F N D L D G L Q R T V K S V R A Q 31 R Y R G R I E H I V I D G G S G D D V V A Y L S G c E P G F 61 A Y W Q S E P D G G R Y D A M N Q G I A H A S G D L L W F L 91 H S A D R F S G P D V V A Q A V E A L S G K G P V S E L W G 121 F G M D R L V G L D R V R G P I P F S L R K F L A G K Q V V 151 P H Q A S F F G s S L V A K I G G Y D L D F G I A A D Q E F 181 I L R A A L V C E P V T I R C V L C E F D T T G V G S H R E 211 P S A V F G D L R R M G D L H R R y P F G G R R I S H A Y L 241 R G R E F Y A Y N S R F H E N V F T R M S K WO 97P1624 Seq. ID No.32 1 gtgaagcgag cgctcatcac cggaatcacc ggccaggacg gctcgtatct cgccgaactg 61 ctgctggcca aggggtatga ggttcacggg ctcatccggc gcgcttcgac gttcaacacc 121 tcgcggatcg atcacctcta cgtcgacccg caccaaccgg gcgcgcggct gtttctgcac 181 tatggtgacc tgatcgacgg aacccggttg gtgaccctgc tgagcaccat cgaacccgac 241 gaggtgtaca acctggcggc gcagtcacac gtgcgggtga gcttcgacga acccgtgcac 301 accggtgaca ccaccggcat gggatccatg cgactgctgg aagccgttcg gctctctcgg 361 gtgcactgcc gcttctatca ggcgtcctcg tcggagatgt tcggcgcctc gccgccaccg 421 cagaacgagc tgacgccgtt ctacccgcgg tcaccgtatg gcgccgccaa ggtctattcg 481 tactgggcga cccgcaatta tcgcgaagcg tacggattgt tcgccgttaa cggcatcttg 541 ttcaatcacg aatcaccgcg gcgcggtgag acgttcgtga cccgaaagat caccagggcc 601 gtggcacgca tcaaggccgg tatccagtcc gaggtctata tgggcaatct ggatgcggtc 661 cgcgactggg ggtacgcgcc cgaatacgtc gaaggcatgt ggcggatgct gcagaccgac 721 gagcccgacg acttcgtttt ggcgaccggg cgcggtttca ccgtgcgtga gttcgcgcgg 781 gccgcgttcg agcatgccgg tttggactgg cagcagtacg tgaaattcga ccaacgctat 841 ctgcggccca ccgaggtgga ttcgctgatc ggcgacgcga ccaaggctgc cgaattgctg 901 ggctggaggg cttcggtgca c&ctgacgag ttggctcgga tcatggtcga cgcggacatg 961 gcggcgctgg agtgcgaagg caagccgtgg atcgacaagc cgatgatcgc cggccggaca 1021 tga Seq. ID No.33 1 M K R A L I T G I T G Q D G S Y L A E L L L A K G Y E V H G 31 L I R R A S T F N T S R I D H L Y V D P H Q P G A R L F L H 61 Y G D L I D G T R L V T L L S T I E P D E V Y N L A A Q S H 91 V R V S F D E P V H T G D T T G M G S M R L L E A V R L S R 121 V H C R F Y Q A S S S E M F G A S P P P Q N E L T P F Y P R 151 S P Y G A A K V Y S Y W A T R N Y R E A Y G L F A V N G I L 181 F N H E S P R R G E T F V T R K I T R A V A R I K A G I Q S 211 E V Y M G N L D A V R D W G Y A P E Y V E G M W R M L Q T D 241 E P D D F V L A T G R G F T V R E F A R A A F E H A G L D W 271 Q Q Y V K F D Q R Y L R P T E V D S L I G D A T K A A E L L 301 G H R A S V H T D E L A R I M V D A D M A A L E C E G K P W 331 I D K P M I A G R T Seq. ID No.34 l atgaggctgg cccgtcgcgc tcggaacatc ttgcgtcgca acggcatcga ggtgtcgcgc 61 tactttgccg aactggactg ggaacgcaat ttcttgcgcc aactgcaatc gcatcgggtc 121 agtgccgtgc tcgatgtcgg ggccaattcg gggcagtacg ccaggggtct gcgcggcgcg ♦ 181 ggcttcgcgg gccgcatcgt ctcgttcgag ccgctgcccg ggccctttgc cgtcttgcag 241 cgcagcgcct ccacggaccc gttgtgggaa tgccggcgct gtgcgctggg cgatgtcgat 301 ggaaccatct cgatcaacgt cgccggcaac gagggcgcca gcagttccgt cttgccgatg 40 361 ttgaaacgac atcaggacgc ctttccacca gccaactacg tgggcgccca acgggtgccg 421 atacatcgac tcgattccgt ggctgcagac gttctgcggc ccaacgatat tgcgttcttg 4B1 aagatcgacg ttcaaggatt cgagaagcag gtgatcgcgg gtggcgattc aacggtgcac 541 gaccgatgcg tcggcatgca gctcgagctg tctttccagc cgttgtacga gggtggcatg 601 ctcatccgcg aggcgctcga tctcgtggat tcgttgggct ttacgctctc gggattgcaa 45 661 cccggtttca ccgacccccg caacggtcga atgctgcagg ccgatggcat cttcttccgg 721 ggcagcgatt ga WO 97/- ^24 1 Seq. ID No.35 1 M R L A R R A R N I L R R N G I E V S R Y F A E L D W E R N 31 F L R Q L Q S H R V S A V L D V G A N S G Q Y A R G L R G A 61 G F A G R I V S F E P L P G P F A V L Q R S A S T D P L W E 91 C R R C A L G D V D G T I S I N V A G N E G A S S S V L P M 121 L K R H Q D A F P P A N Y V G A Q R V P I H R L D S V A A D 151 V L R P N D I A F L K I D V Q G F E K Q V I A G G D s T V H 181 D R C V G M Q L E L S F Q P L Y E G G M L I R E A L D L V D 211 S L G F T L S G L Q P G F T D P R N G R M L Q A D G I F F R 241 G S D Seq. ID No.36 1 gtgaaatcgt tgaaactcgc tcgtttcatc gcgcgtagcg ccgccttcga ggtttcgcgc 61 cgctattctg agcgagacct gaagcaccag tttgtgaagc aactcaaatc gcgtcgggta 121 gatgtcgttt tcgatgtcgg cgccaactca ggacaatacg ccgccggcct ccgccgagca 181 gcatataagg gccgcattgt ctcgttcgaa ccgctatccg gaccgtttac gatcttggaa 241 agcaaagcgt caacggatcc actttgggat tgccggcagc atgcgttggg cgattctgat 301 ggaacggtta cgatcaatat cgcaggaaac gccggtcaga gcagttccgt cttgcccatg 361 ctgaaaagtc atcagaacgc ttttcccccg gcaaactatg tcggtaccca agaggcgtcc 421 atacatcgac ttgattccgt ggcgccagaa tttctaggca tgaacggtgt cgcttttctc 481 aaggtcgacg ttcaaggctt tgaaaagcag gtgctcgccg ggggcaaatc aaccatagat 541 gaccattgcg tcggcatgca actcgaactg tccttcctgc cgttgtacga aggtggcatg 601 ctcattcctg aagccctcga tctcgtgtat tccttgggct tcacgttgac gggattgctg 661 ccttgtttca ttgatgcaaa taatggtcga atgttgcagg ccgacggcat ctttttccgc 721 gaggacgatt ga Seq. ID No.37 1 M K s L K h A R F I A R S A A F E V S R R Y S E R D L K H Q 31 F V K Q L K S R R V D V V F D F T V G A N S G Q Y A A G L R 61 R A A Y K G R I V s F E P L S G P F T I L E S K A S T D P L 91 H D C R Q H A L G D S D G T V T I N I A G N A G Q s S S V L 121 P M L K S H Q N A F P P A N Y V 6 T Q E A S I H R L D S V A 151 P E F L G M N G V A F L K V D V Q G F E K Q V L A G G K S T 181 I D D H C V G M Q L E L S F L P L Y E G G M L I P E A L D L 211 V Y S L G F T L T G L L P C F I D A N N G R M L Q A D G I F 241 F R E D D 1 Seq. ID No.38 l atggtgcaga cgaaacgata cgccggcttg accgcagcta acacaaagaa agtcgccatg 61 gccgcaccaa tgttttcgat catcatcccc accttgaacg tggctgcggt attgcctgcc 121 tgcctcgaca gcatcgcccg tcagacctgc ggtgacttcg agctggtact ggtcgaccfgc 181 ggctcgacgg acgaaaccct cgacatcgcc aacattttcg cccccaacct cggcgagcgg 241 ttgatcattc atcgcgacac cgaccagggc gtctacgacg ccatgaaccg cggcgtggac 301 ctggccaccg gaacgtggtt gctctttctg ggcgcggacg acagcctgta cgaggctgac 361 accctggcgc gggtggccgc cttcattggc gaacacgagc ccagcgatct ggtatatggc 421 gacgtgatca tgcgctcaac caatttccgc tggggtggcg ccttcgacct cgaccgtctg 481 ttgttcaagc gcaacatctg ccatcaggcg atcttctacc gccgcggact cttcggcacc 541 atcggtccct acaacctccg ctaccgggtc ctggccgact gggacttcaa tattcgctgc 601 ttttccaacc cagcgctcgt cacccgctac atgcacgtgg tcgttgcaag ctacaacgaa 661 ttcggcgggc tcagcaatac gatcgtcgac aaggagtttt tgaagcggct gccgatgtcc 721 acgagactcg gcataaggct ggtcatagtt ctggtgcgca ggtggccaaa ggtgatcagc 781 agggccatgg taatgcgcac cgtcatttct tggcggcgcc gacgttag Seq. ID No.39 1 M V Q T K R Y A G L T A A N T K K V A M A A P M F S I I I P 31 T L N V A A V L P A C L D S I A R Q T C G D F E L V L V D G 61 G S T D E T L D I A N I F A P N L G E R L I I H R D T D Q G 91 V y D A M N R G V D L A T G T W L L F L G A D D S L y E A D 121 T L A R V A A F I G E H E P S D L V Y G D V I M R S T N F R 151 H G G A F D L D R L L F K R N I C H Q A I F Y R R G L F G T 181 I G P Y N L R Y R V L A D W D F N I R C F S N P A L V T R Y 211 M H V V V A S Y N E F G G L S N T I V D K E F L K R L P M S 241 T R L G I R L V I V L V R R W P K V I S R A M V M R T V I S 271 W R R R R Seq 40: GATGCCGTGAGGAGGTAAAGCTGC Seq 41: GATACGGCTCTTGAATCCTGCACG

Claims (36)

1. A polypeptide in substantially isolated form which comprises a sequence selected from the sequences of Seq.ID.No: 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28 and 29, or a polypeptide substantially homologous thereto.

2. A polypeptide in substantially isolated form which comprises a sequence selected from the sequences of Seq.ID.No: 6, 8, 10,12,14, 16,18, 20,22, 24, 26, 28 and 29.

3. A polypeptide according to claim 1 or 2 which comprises the sequence of Seq ID No: 24.

4. A polypeptide which comprises a fragment of a polypeptide defined in claim 1, 2 or 3, said fragment comprising at least 12 amino acids and an epitope.

5. A recombinant expression vector comprising a polynucleotide which: (a) encodes a polypeptide according to any one of claims 1 to 3 (b) is capable of selectively hybridizing to Seq.ID.No: 3 or 4 or a fragment thereof; (c) is a fragment according to (b) which comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9, 11, 13,15,17, 19, 21,23, 25 and 27 or a polynucleotide at least 90% homologous thereto; or (d) comprises a sequence selected from the sequences of Seq.ID.No: 5,7, 9,11, 13, 15,17,19, 21, 23, 25 and 27. and a control sequence capable of providing for the expression of the coding sequence of said polynucleotide.

6. A recombinant expression vector according to claim 5 wherein said polynucleotide comprises the sequence of Seq ID No: 23, a fragment thereof, a polynucleotide at least 90% homologous to either thereto, or a polynucleotide which encodes INTELLECTUAL PROPERTY OFFICE OF N.Z. - 1 MAY 2001 DcrFivpn c7. -58- TPfr (, ./, /7 --N\ y »„ "ir L, a polypeptide according to Seq ID No: 24;

7. An antibody capable of binding a polypeptide or fragment thereof as defined in any one of claims 1 to 4.

8. An antibody capable of binding a polypeptide or fragment thereof wherein the polypeptide is a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or is a peptide substantially homologous thereto.

9. A test kit for detecting the presence or absence of a pathogenic mycobacterium in a sample which comprises a polynucleotide which: (a) encodes a polypeptide according to any one of claims 1 to 3 (b) is capable of selectively hybridizing to Seq.ID.No: 3 or 4 or a fragment thereof; (c) is a fragment according to (b) which comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9,11,13,15,17,19,21,23,25 and 27 or a polynucleotide at least 90% homologous thereto; or (d) comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9, 11, 13,15,17,19, 21,23,25 and 27. a polypeptide according to any one of claims 1 to 4, a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, or an antibody according to claim 7 or 8.

10. A method of detecting the presence or absence of antibodies in an animal or human, against a pathogenic mycobacteria in a sample which comprises: (a) providing a polypeptide according to any one of claims 1 to 4 or a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, which comprises an epitope; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and INTELLECTUAL PROPERTY OFFICE OF N.Z. - 1 MAY 2001 D r (> r i \i r n (c) determining whether antibody-antigen complex comprising said polypeptide is formed.

11. A method of detecting the presence or absence of a polypeptide according to any one of claims 1 to 4 or a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31,33, 35, 37 and 39 or a polypeptide substantially homologous thereto in a biological sample which method which comprises: (a) providing an antibody according to claim 7 or 8; (b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed.

12. A method of detecting the presence or absence of cell mediated immune reactivity in an animal or human, to a polypeptide according to claims 1 to 4 or a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, which method comprises (a) providing a polypeptide according to any one of claims 1 to 4 or a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, which comprises an epitope; (b) incubating a cell sample with said polypeptide under conditions which allow for a cellular immune response such as release of cytokines or other mediator or reaction to occur; and (c) detecting the presence of said cytokine or mediator or cellular response in the incubate.

13. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 4; a polynucleotide which: (a) encodes a polypeptide according to any one of claims 1 to 3 (b) is capable of selectively hybridizing to Seq.ID.No: 3 or 4 or a fragment INTELLECTUAL PROPERTY OFFICE OF N.Z. - 1 MAY 2001 RECEIVED thereof; (c) is a fragment according to (b) which comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9, 11,13, 15,17, 19, 21, 23, 25 and 27 or a polynucleotide at least 90% homologous thereto; or (d) comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9,11, 13,15,17,19,21,23,25 and 27, or an expression vector according to claim 5 in a suitable carrier or diluent.

14. A pharmaceutical composition according to claim 13 which comprises a polypeptide which comprises a sequence according to Seq ID No: 24, a polypeptide substantially homologous thereto or a fragment of either such polypeptide which comprises at least 12 amino acids and an epitope; a polynucleotide which encodes such a polypeptide; a polynucleotide which comprises the sequence of Seq ID No: 23, a fragment thereof or a polynucleotide at least 90% homologous to either thereto; or an expression vector comprising any such polynucleotide.

15. A composition according to claim 13 or 14 or a composition comprising a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, for use in the treatment or prevention of diseases caused by mycobacteria.

16. An isolated normally pathogenic mycobacterium, whose pathogenicity is mediated in all or in part by the presence or the expression of a polypeptide as defined in any one of claims 1 to 4 or a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31,33,35,37 and 39 or a polypeptide substantially homologous thereto, which mycobacterium harbours an attenuating mutation in a gene encoding one of the said polypeptides.

17. A vaccine comprising a mycobacterium as claimed in claim 16.

18. A vaccine according to claim 17 wherein the mycobacteria is selected from INTELLECTUAL PROPERTY OFFICE OF N.Z. - 1 MAY 2001 RECEIVED Mavs, Mptb and Mtb.

19. A vaccine comprising a polynucleotide which: (a) encodes a polypeptide according to any one of claims 1 to 3 (b) is capable of selectively hybridizing to Seq.ID.No: 3 or 4 or a fragment thereof; (c) is a fragment according to (b) which comprises a sequence selected from the sequences of Seq.ID.No: 5,7,9,11,13,15,17,19,21,23,25 and 27 or a polynucleotide at least 90% homologous thereto; or (d) comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9, 11, 13,15,17,19,21,23,25 and 27. or an expression vector according to claim 5.

20. A vaccine according to claim 19 comprising a polynucleotide which comprises the sequence of Seq ID No: 23, a fragment thereof or a polynucleotide at least 90% homologous either thereto; a polynucleotide which encodes a polypeptide according to Seq ID No: 24; or an expression vector comprising any such polynucleotide.

21. Use of a polypeptide according to claims 1 to 4 or a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No: 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, for the manufacture of a medicament for treating or preventing mycobacterial disease in an animal or human caused by mycobacteria which express said polypeptide by vaccinating or treating said animal or human with an effective amount of said polypeptide.

22. Use of a polynucleotide which: (a) encodes a polypeptide according to any one of claims 1 to 3 (b) is capable of selectively hybridizing to Seq.ID.No: 3 or 4 or a fragment thereof; (c) is a fragment according to (b) which comprises a sequence selected from the sequences of Seq.ID.No: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 or a INTELLECTUAL PROPERTY OFFICE OF N.Z. - 1 MAY 2001 RECEIVED „ 50242? polynucleotide at least 90% homologous thereto; or (d) comprises a sequence selected from the sequences of Seq ID No: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27, a vector according to claim 5 or 6 or a polynucleotide which encodes a polypeptide which comprises a sequence selected from the sequences of Seq.ID.No. 31, 33, 35, 37 and 39 or a polypeptide substantially homologous thereto, for the manufacture of a medicament for treating or preventing mycobacterial diseases in animals or humans caused by mycobacteria containing the polynucleotide of Seq.ID No. 3 or 4, by vaccinating or treating an animal or human with an effective amount of a said polynucleotide or vector.

23 A use according to claim 21 or 22 which comprises vaccinating or treating with a polypeptide which comprises a sequence according to Seq ID No: 24, a polypeptide substantially homologous thereto or a fragment of either such polypeptide which comprises at least 12 amino acids and an epitope, a polynucleotide which encodes such a polypeptide, a polynucleotide which comprises the sequence of Seq ID No: 23, a fragment thereof or a polynucleotide at least 90% homologous to either thereto; or an expression vector comprising any such polynucleotide.

24 Use of a vaccine according to any one of claims 17 to 20 for the manufacture of a medicament for treating or preventing mycobacterial diseases in animals or humans caused by pathogenic mycobacteria

25. A polypeptide according to claim 1 substantially as herein described or exemplified

26. A polypeptide according to claim 2 substantially as herein described or exemplified

27. A recombinant expression vector according to claim 5 substantially as herem described or exemplified I ~1 OCT 2001 RECEIVED

28. An antibody according to claim 7 or 8 substantially as herein described or exemplified. '

29. A test kit according to claim 9 substantially as herein described or exemplified.

30. A method according to claim 10 substantially as herein described or exemplified.

31. A method according to claim 11 substantially as herein described or exemplified.

32. A method according to claim 12 substantially as herein described or exemplified.

33. A pharmaceutical composition according to claim 13 substantially as herein described or exemplified.

34. A use according to claim 21 or 22 substantially as herein described or exemplified.

35. A mycobacterium according to claim 16 substantially as herein described or exemplified.

36. A vaccine according to claim 17 or 19 substantially as herein described or exemplified. END OF CLAIMS ST. GEORGF.'S HOSPITAL MF.DTCAL SCHOOL By their attorneys HENRY HUGHES Per: 1 INTELLECTUAL PROPERTY OFFICE OF N.Z. - 1 MAY 2001 RECEIVED