WO1997023624A2

WO1997023624A2 - Polynucleotides and polypeptides in pathogenic mycobacteria and their use as diagnostics, vaccines and targets for chemotherapy

Info

Publication number: WO1997023624A2
Application number: PCT/GB1996/003221
Authority: WO
Inventors: John Hermon-Taylor; Tim Doran; Douglas Millar; Mark Tizard; Mark Loughlin; Nazira Sumar; John Ford
Original assignee: St Georges Hospital Medical School
Current assignee: City St George’s, University of London
Priority date: 1995-12-21
Filing date: 1996-12-23
Publication date: 1997-07-03
Anticipated expiration: 1998-06-21
Also published as: EP0870032A2; GB9526178D0; US7541181B2; DE69634918D1; AU735112B2; US20040260078A1; DE69634918T2; WO1997023624A3; US20060204521A1; AU1202797A; EP0870032B1; ATE299182T1; US6156322A

Abstract

The invention provides a nucleotide sequence representing a pathogenicity island found in species of pathogenic mycobacteria. The islands are shown as SEQ ID NOs: 3 and 4 and comprises several open reading frames encoding polypeptides. These polypeptides and their use in diagnosis and therapy form a further aspect of the invention.

Description

Novel polynucleotides andpolypeptides inpathogenic ntvcobacteria and their use as diagnostics. vaccines and targets for chemotherapy.

This invention relates to the novel polynucleotide sequence we have designated "GS" which we have identified in pathogenic mycobacteria. GS is a pathogenicity island within 8 b of DNA comprising a core region of 5.75kb and an adjacent transmissable element within 2.25kb. GS is contained within Myco acterium paratuberculosis, Mycobac erium avium subsp. silvaticum and some 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 My cobac terium 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 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 are required. The importance of M 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.

MycoJbacterium avium subsp. silvaticum (Mavs) is a pathogenic mycobacterium causing diseases of animals and birds, but it can 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. MptJb is associated with other chronic inflammatory diseases of 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, 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. IS500 and IS902 provide convenient highly specific multi-copy DNA targets for the sensitive detection of 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 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 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 trans issable 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 a non-disease-causing to a disease-causing strain.

Description of the Drawings 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 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

Cisolates 0022 from a Guernsey cow and 0021 from a red deer) . 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 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) 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 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 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 H_x H₂ on the complementary DNA strand linked by a programmed frameshifting site so that a single polypeptide is translated from the ORF GSH. The nucleotide 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.

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 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 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. 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 H_x: 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 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 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 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 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 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 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 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 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 polynucleotides, as well as antibodies capable of binding such polypeptides. In an additional aspect, the invention provides 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 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 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 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 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 computer 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 homologous ORFs close to one another. The use of polynucleotides and polypeptides from Mtb (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 processes involved in the preparation and use of these diagnostics, vaccines and new chemotherapeutic agents, comprise further aspects of the invention. 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 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 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 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 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, 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 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 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 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 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 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 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 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 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 a polynucleotide which is at least 80% homologous over 25, preferably 30 nucleotides forms one aspect of the invention, as 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 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 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 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 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 provides polynucleotides which encode the same polypeptide as the above entioned 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. 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 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 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 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 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, F and H, may also be identified. 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 recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair 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 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 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 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 probing genomic DNA libraries made from such isolates or strains 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 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 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 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 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 vi tro 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 using by techniques known per se. 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 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 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. 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 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 WO89/03891 and WO90/13667.

Polynucleotides of the invention or fragments thereof labelled or unlabelled may also be used to identify and characterise 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 probe 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 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 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 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 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 NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 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% 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 NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29 over their entire length. Even more preferably, this homology is 98%. 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 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 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 40 amino acids may also be obtained 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 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. , 2J3_; 709-715 (1986), as well as other techniques known in the art.

The term "an epitope which is specific to the pathogenicity of 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 any proteins which react with the epitope. Epitopes may be linear or conformational. 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 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 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 cr-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 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 biosensor. Such labelled and/or immobilized polypeptides may be 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 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-an igen 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. 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 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 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 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 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, 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 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. 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 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 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 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 β- l - 3-N-acetylglucosamine may be used as substrates in assays.

Candidate chemical compounds which may be used may be natural or 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 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 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 be utilized in further assays or used to design further panels of candidate compounds. For example, a number of assay methods to define peptide interaction with peptides are known. For example, WO86/00991 describes a method for determining mimotopes which comprises making panels of catamer preparations, for example octamers of 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 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.

WO89/03430 describes screening methods which permit the preparation of specific mimotopes which mimic the immunological 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 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 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 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. 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 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 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 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 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 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 vi tro, for example for the production of RNA or used to transfect or transform a host cell. The vector 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 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. 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 adenovirus, the SV40 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 β-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.; 3-50 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 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 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 example WO95/14091, 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, WO95/07994, 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 done by any suitable means, for example oral administration or intravenous injection. From 10⁵ to 10⁸ cf.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 some cases. This will generally be achieved by administering doses, for example from 10⁵ to 10⁸ 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 designed to increase the immune response. Such a oleclue may be for example and interferon, particularly interferon gamma, an interleukin, for example IL-lα, IL-13 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 already infected with a mycobacteria and it is desired to boost the immune response. ) . 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 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 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 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 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 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 under conditions which allow for the formation of an antibody-antigen complex; and

(c) determining whether antibody-antigen complex comprising said antibody is formed. 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 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. 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) 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 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 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 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 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 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 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 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. 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 established protocols. 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 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 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 the art, including the formation of disulfide linkages using N- succinimidyl-3- (2-pyridylthio) propionate (SPDP) andsuccinimidyl 4- (N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) obtained from Pierce Company, Rockford, Illinois, (if the peptide lacks a sulfhydryl group, this can be provided by addition of a 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, Immun Rev (1982) 6_2:185. 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-1-carboxylic acid, and the like. The carboxyl group can be activated by combining them with succinimide or l-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Additional methods of coupling antigens employs the rotavirus/"binding peptide" system described in EPO Pub. No. 259,149, the disclosure of which is incorporated herein by reference. The foregoing list is not meant to be exhaustive, and modifications of the named compounds can clearly be used. 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 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 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, 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, 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 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 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 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 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 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, 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 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 (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 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 injection, for example, either subcutaneously or intramuscularly.

Additional formulations which are suitable for other modes of administration include suppositories, oral formulations or as 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 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 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 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, 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 prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5μg to 250μg, 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. 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 which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals 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 individual and be dependent upon the judgement of the practitioner.

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 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 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 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 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 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. 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 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 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 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 described further in Gene (1993) 129; 123-128. A lesion in the asd gene, encoding the enzyme aspartate β-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 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 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 with DNA from wild-type strains has been minimized. i?ecA 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 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 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 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 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 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 for the production of an essential metabolite or catabolite 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 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 prepared. Concentrations of from about 10⁴ to 10⁹ bacteria per ml will generally be suitable, e.g. from about 10⁵ to 10⁸ such as about 10⁶ per ml. Live attenuated organisms may be administered subcutaneously or intramuscularly at up to 10^β organisms in one or more doses, e.g from around 10^s to 10⁸, e.g about 10⁶ or 10⁷ 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 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.

EXAMPLE 1

Tests for the presence of the GS identifier sequence were performed on 5μl bacterial DNA extracts (25 μg/ml to 500 μg/ml) using polymerase chain reaction based on the oligonucleotide primers 5' -GATGCCGTGAGGAGGTAAAGCTGC-3' (Seq ID No. 40) and 5'- 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 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) , 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 ISS02 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. fortui turn, M.phlei , as well as E. coli , S. areus, Nocardia sp, 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 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 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 a protein of 38.4kDa 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, Y. enterocoli tica, 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 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. 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, IgrtD of H. influenzae, lsi of N . gonorrhoae . In E. coli an equivalent gene galE adds /S-l-3 N-acetylglucosamine to galactose, the latter only found in 'O' and 'M' antigens which are also related to pathogenicity. GSH comprising the ORFs GSH_α 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 insertion sequences is broadly distributed amongst gram negative bacteria and is responsible for mobility and transposition of genetic elements. An IS22- like element in B . fragilis is split either side of the β-lactamase gene controlling its activation and expression. We programmed an E. coli S30 cell-free extract 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 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.l

5'- 1 GATCCAACTA AACCCGATGG AACCCCGCGC AAACTAHGG ACGTCTCCGC GCTACGCAGT

61 TGGGΠGGCG CCCGCGAATC GCACTGAAAG AGGGCATCGA TGCAACGGTG TCGTGGTACC 121 GCACAAATGC CGATGCCGTG AGGAGGTAAA GCTGCGGGCC GGCCGATGH ATCCCTCCGG

181 CCGGACGGGT AGGGCGACCT GCCATCGAGT GGTACGGCAG TCGCCTGGCC GGCGAGGCGC

241 ATGGCCTATG TGAGTATCCC ATAGCCTGGC TTGGCTCGCC CCTACGCAH ATCAGHGAC

301 CGCTTTCGCG CCACGTCGCA GGCTTGCGGC AGCATCCCGT TCAGGTCTCC TCATGGTCCG

361 GTGTGGCACG ACCACGCAAG CTCGAACCGA CTCGTTTCCC AATTTCGCAT GCTAATATCG 421 CTCGATGGAT TTTTTGCGCA ACGCCGGCH GATGGCTCGT AACGHAGCA CCGAGATGCT

481 GCGCCACTCC GAACGAMGC GCCTAHAGT AAACCAAGTC GAAGCATACG GAGTCAACGT

541 TGΠAΠGAT GTCGGTGCTA ACTCCGGCCA GTTCGGTAGC GCTTTGCGTC GTGCAGGAΠ

601 CAAGAGCCGT ATCGTTTCCT HGAACCTCT TTCGGGGCCA TTTGCGCAAC TAACGCGCAA

661 GTCGGCATCG GATC -3'

Seq. ID No.2

5'- 1 GATCCGATGC CGACTTGCGC GTTAGTTGCG CAAATGGCCC CGAAAGAGGT TCAAAGGAAA

61 CGATACGGCT CTTGAATCCT GCACGACGCA AAGCGCTACC GAACTGGCCG GAGTTAGCAC

121 CGACATCAAT AACAACGTTG ACTCCGTATG CTTCGACTTG GTTTACTAAT AGGCGCTTTC

IBI GTTCGGAGTG GCGCAGCATC TCGGTGCTAA CGTTACGAGC CATCAAGCCG GCGTTGCGCA 2 1 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

1 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

1351 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

1801 AAGTGAGAAC CCGGTGAAGC GAGCGCTTAT AACAGGGATC ACGGGGCAGG

1851 ATGGTTCCTA CCTCGCCGAG CTACTACTGA GCAAGGGATA CGAGGTTCAC 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 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 2401 GATCACGCGT GCCGTGGCGC GCATCCGAGC TGGCGTCCAA TCGGAGGTCT

2451 ATATGGGCAA CCTCGATGCG ATCCGCGACT GGGGCTACGC GCCCGAATAT

2501 GTCGAGGGGA TGTGGAGGAT GTTGCAAGCG CCTGAACCTG ATGACTACGT 2551 CCTGGCGACA GGGCGTGGTT ACACCG ACG 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 351 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 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

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

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

7651 GCGATCGTTG GGCACGGCGC AGATCTCCGA GTGGACCGTG GCATTGACCT

7701 CGGCGCACCA TAGTTGCGCC TGGGCGTTGA GGGCACGTAG GTCGACCTGC

7751 TCACCGGCTA ACGCAGCTTC GGTCAGCAGC GGCACCGCAA GGTCGTCCTG

7801 AGCGTAGCCA CAGAGGTTCT CCACGATGCC CTTCGATTGC GGATCCGCAC 7851 CGTGGCAGAA GTCCGGAACG AAGCCATAGT GGGACGCGAA TCGCACATAA 7901 TCCGGTGTTG GAACAACAAC ATTGGCGACG ACACCACCTT TGAGGCAGCC 7951 CATCCGGTCG GCCAGGATCT TGGCCGGAAC CCCACCGATC GCCTC

Seq. ID No.4 1 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 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 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 2701 GAGTCGGCAT CGGATCCACT ATGGGAGTGT CACCAGTATG CCCTAGGCGA CGCCGATGAG 2761 ACGATTACCA TCAATGTGGC AGGCAATGCG GGGGCAAGTA GTTCCGTGCT GCCGATGCTT 2821 AAAAGTCATC AAGATGCCTT TCCTCCCGCG AATTATATTG GCACCGAAGA CGTTGCAATA 2881 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

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 W S A V P T G T V D L S T I T Y R S 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 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 o.7

1 gtgtcatctg ctccaaccgt gtcggtgata acgatttcgc cgaacgatct 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 tcccccggcc gtacggctat

421 atgccgttta agatgcggaa acttctgctc 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 V S S A P T V S V I T I S L N D E G K S T V E S V R A Q

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 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 E A

181 D Q L F I Y R A A 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 W D L H G D Y P G G R R V S 241 W A Y 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 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 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 1021 agagtaagtt ga Seq. ID No.10

1 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 S S I D P D E V Y N 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 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 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 D A I R D W G Y A P E Y V E G M W R M 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 W K A S V H T G E 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 1 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

1 V K R A L I T G I T G Q D G s Y L A E 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 σ T R 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 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 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 D A I R D w G Y A P E Y V E G M w 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 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

331 I D T P M L P G W G G V s 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 W H T M D R H A D V A W L G Q S K T T T P G P D R A

31 T P V Y I A G H R G L V G S A V R R F E A E G F T N 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 H V A T A V G Y I G E T R 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

Seq. ID No.15

1 gtgcgatggc acaccatgga tcgacacgcc gatgttgcct ggttggggcg gagtaagttg

61 acgactacac ctgggcctct ggaccgcgca acgcccgtgt atatcgccgg tcatcggggg

241 acaagaccac aggtgatcat cgatgcggcc gcacgggtcg gcggcatcat ggcgaataac

301 acctatcccg cggacttctt gtccgaaaac ctccgaatcc agaccaattt gctcgacgca

361 gctgtcgccg tgcgtgtgcc gcggctcctt ttcctcggtt cgtcatgcat ctacccgaag

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 1 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

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

Seq. ID No.17

1 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 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 661 acggatccgc gcaatggtcg aatgcttcaa gctgacggca ttttcttccg tggggacgat

721 tga 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 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 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

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

181 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

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 g

Seq. ID No.22

1 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 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 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

1 atgactgcgc cagtgttctc gataattatc cctaccttca atgcagcggt gacgctgcaa

61 gcctgcctcg gaagcatcgt cgggcagacc taccgggaag tggaagtggt ccttgtcgac

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 781 gaacgaagcg cagaaccgta g Seq. ID No.24

1 M T A P V F S 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

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 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 AV 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 L G S G G H E AV P S V V L I L Seq. ID No.27

1 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 L 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 L 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 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 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 W 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

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 W E N V F T R M S K 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 cactgacgag 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 W 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

1 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 361 ttgaaacgac atcaggacgc ctttccacca gccaactacg tgggcgccca acgggtgccg

421 atacatcgac tcgattccgt ggctgcagac gttctgcggc ccaacgatat tgcgttcttg

481 aagatcgacg ttcaaggatt cgagaagcag gtgatcgcgg gtggcgattc aacggtgcac

541 gaccgatgcg tcggcatgca gctcgagctg tctttccagc cgttgtacga gggtggcatg

601 ctcatccgcg aggcgctcga tctcgtggat tcgttgggct ttacgctctc gggattgcaa 661 cccggtttca ccgacccccg caacggtcga atgctgcagg ccgatggcat cttcttccgg

721 ggcagcgatt ga 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 L 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 W 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 G 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 Seq. ID No.38

1 atggtgcaga cgaaacgata cgccggcttg accgcagcta acacaaagaa agtcgccatg

61 gccgcaccaa tgttttcgat catcatcccc accttgaacg tggctgcggt attgcctgcc

121 tgcctcgaca gcatcgcccg tcagacctgc ggtgacttcg agctggtact ggtcgacggc 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 W 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 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

1. A polypeptide in substantially isolated form which comprises any one of the sequences selected from the group consisting of 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.

2. A polypeptide in substantially isolated form which comprises any one of the sequences selected from the group consisting of Seq. ID.No: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 33, 35, 37 and 39.

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

4. A polynucleotide in substantially isolated form which encodes a polypeptide according to any one of claims 1 to 3.

5. A polynucleotide in substantially isolated form which is capable of selectively hybridizing to SEQ ID NO: 3 or 4 or a fragment thereof.

6. A polynucleotide fragment according to claim 5 which comprises a sequence selected from the group consisting 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.

7. A polynucleotide in substantially isolated form comprising a sequence selected from the group consisting of Seq.ID.No: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27.

8. A polynucleotide in substantially isolated form consiting essentially of a sequence selected from the group Seq ID Nos. 30, 32, 34, 36 and 38, or a polynucleotide at least 90% homologous thereto.

9. A polynucleotide in substantially isolated form consiting essentially of a sequence selected from the group Seq ID Nos. 30, 32, 34, 36 and 38.

10. A polynucleotide probe which comprises a fragment of at least 15 nucleotides of a polynucleotide as defined in any one of claims 4 to 8, optionally carrying a revealing label .

11. A recombinant vector carrying a polynucleotide as defined in any one of claims 4 to 8.

12. An antibody capable of binding a polypeptide or fragment thereof as defined in any one of claims l to 3.

13. A test kit for detecting the presence or absence of a pathogenic mycobacterium in a sample which comprises a polynucleotide according to any one of claims 4 to 10, a polypeptide according to any one of claims 1 to 3 , or an antibody according to claim 12.

14. 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 3 comprising an epitope;

(b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and

15. A method of detecting the presence or absence of a polypeptide according to any one of claims 1 to 3 in a biological sample which method which comprises:

(a) providing an antibody according to claim 11;

(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.

16. 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 3 which method comprises

(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.

17. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 3 in a suitable carrier or diluent.

18. A composition according to claim 17 for use in the treatment or prevention of diseases caused by mycobacteria.

19. A method of treating or preventing mycobacterial disease in an animal or human caused by mycobacteria which express a polypeptide according to claims l to 3, which method comprises vaccinating or treating an animal or human with an effective amount of said polypeptide.

20. A method of treating or preventing mycobacterial diseases in animals or humans caused by mycobacteria containing the polynucleotide of SEQ ID NO: 3 or 4, which method comprises vaccinating or treating an animal or human with an effective amount of a polynucleotide according to claims 4 to 9, or a vector according to claim 11.

21. A method according to claims 19 or 20 for increasing the in vivo susceptibility of mycobacteria to antimicrobial drugs.

22. A vaccine comprising a normally pathogenic mycobacteria, which pathogenicity is mediated in all or in part by the presence of the expression of a polypeptide as defined in any one of claims 1 to 3, which mycobacteria harbours an attenuating mutation in any one of said genes.

23. A vaccine according to claim 22 wherein the mycobacteria is selected from Mavs, Mptb and Mtb.