NZ519395A - Techniques for identifying genes that are important for virulence in Mycobacterial vaccines - Google Patents

Abstract

Attenuated strains of Mycobacterium tuberculosis complex, which comprise at least one inactive gene, are described. Also described are methods for producing and screening the attenuated strains, methods for assessing their vaccine potential and the use of such strains and derivatives thereof as vaccines against tuberculosis in humans and animal medical practice.

Description

519395 PATENTS FORM NO. 5 Fee No. 4: $250.00 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional No: 519395 Dated: 7 June 2003 MYCOBACTERIAL VACCINES WE AGRESEARCH LIMITED a New Zealand Company of East Street, Ruakura Campus, Hamilton, New Zealand hereby declare the invention for which I/We pray that a patent may be granted to me/us, and the method by which it is to be performed to be particularly described in and by the following statement: INTELLECTUAL PROPERTY OFFICE OF N.Z. " 9 JUN 2003 received James & Wells Ref: 31147 MYCOBACTERIAL VACCINES Technical Fieid This invention relates to attenuated strains of the Mycobacterium tuberculosis complex, methods for producing and screening these, methods for assessing their 5 effectiveness as vaccines and the use of such strains and derivatives of them as vaccines against tuberculosis in human and animal medical practice.

Background Art Mycobacteria are rod-shaped, acid-fast, aerobic bacilli that do not form spores. A moderate number of mycobacterial species are pathogenic to humans and/or 10 animals and determining improved methods of preventing or treating the diseases they cause is of prime importance. For example, tuberculosis is a worldwide human health problem which causes 2-3 million deaths per year but no highly effective vaccine is available for its prevention. Tuberculosis in animals is also very widespread, particularly in developing countries.

Tuberculosis in mammals is caused by species of the Mycobacterium tuberculosis complex. These species are so closely related genetically that it has been proposed that they be combined into a single species (van Soolingen et al., 1997) and they are now generally treated at least informally in this way (Brosch et al., 2002). Three important members of the complex are M. tuberculosis, the major cause of 20 human tuberculosis; Mycobacterium africanum, a major cause of human tuberculosis in some populations; and Mycobacterium bovis, the cause of bovine tuberculosis. Other members of the complex include Mycobacterium microti, Mycobacterium bovis subsp. caprae (Niemann et al., 2002), Mycobacterium canettii (Pfyffer et al., 1998) and a discrete group of isolates from seals (Brosch et 25 al., 2002). None of the species of the M. tuberculosis complex is restricted to 2 James & Wells Ref: 31147 being pathogenic for a single host species. For example, M. bovis causes tuberculosis in a wide range of animals including humans in which it causes a disease that is clinically indistinguishable from that caused by M. tuberculosis. Human tuberculosis is a major cause of mortality throughout the world, 5 particularly in less developed countries. It accounts for approximately eight million new cases of clinical disease and two million deaths each year. Bovine tuberculosis, as well as causing a small percentage of these human cases, is a major cause of animal suffering and large economic costs in the animal industries.

There is a long-established vaccine for tuberculosis that is an attenuated form of M. bovis known as BCG. This is very widely used but it provides incomplete protection (Bloom and Fine, 1994). Recently, gene deletions that may contribute to the avirulence of BCG have been identified (Mahairas et al., 1996; Behr et al., 1999; Gordon et al., 1999) and in one case a gene in one of these deleted regions has been shown to be important for virulence (Wards et al., 2000). There are undoubtedly other genes associated with virulence of the M. tuberculosis complex that have also been mutated in BCG but whose identity is not yet known or not yet reported. For example, we have identified a nonsense mutation in BCG in the phoT gene and showed that specific inactivation of this gene in a virulent M. bovis strain caused it to become attenuated in guinea pigs (results not published). Despite this developing knowledge of gene mutations and deletions in BCG that cause loss of virulence, the detailed mechanisms which enable BCG to have some vaccine effectiveness have not been determined.

In the last ten years, there has been intense interest in producing better vaccines against tuberculosis. A large range of possible types of both living and non-living 25 vaccines are being actively researched (Collins, 2000; Snewin et al., 2000) and there is already some information on the ability of different types to stimulate the immune system in different ways. There is general agreement that a successful 3 James & Wells Ref: 31147 vaccine will need to induce cell mediated immunity, but since no convincing correlates of protective immunity have yet been identified, it is not clear which features of a particular type of vaccine are most important (Snewin et al., 2000). Live vaccine candidates include genetically modified forms of BCG, genetically 5 attenuated strains of the M. tuberculosis complex, and genetically engineered vaccinia virus and Salmonella strains. Non-living vaccine candidates include killed mycobacterial species, protein sub-units and DNA vaccines. It may be that different types of vaccine will be useful for different purposes.

Thirteen years ago, we embarked on a programme to produce a new tuberculosis vaccine based on attenuated strains of the M. tuberculosis complex. Until the recent development of molecular genetic techniques for producing mutant strains of the M. tuberculosis complex, the only attenuated strains available were either those which had been available for many years such as M. bovis BCG, M. bovis ATCC35721, Mycobacterium microti and M. tuberculosis H37Ra or new strains made in the laboratory by mutagenic techniques which produced genetic lesions that were not easily identifiable even after the application of modern molecular genetic approaches. In early work in our laboratory, we identified an attenuating mutation in M. bovis ATCC35721 (Collins et al., 1995), isolated avirulent strains of M. bovis by Isoniazid selection (Banajee et al., 1994; Wilson et al., 1995) and produced avirulent strains of M. bovis by chemical mutagenesis (Buddie et al., 2002). For a variety of reasons, none of these mutant strains were suitable candidates for a new vaccine, although two of them in vaccination experiments did provide better protection than BCG against tuberculosis in cattle (Buddie et al, 2002). Nevertheless, the work was useful because it clearly demonstrated that a better live vaccine than BCG could be produced.

The advent of a range of mycobacterial molecular genetic techniques in the last ten years has made it possible to produce both random and specific mutants of the 4 James & Wells Ref: 31147 tuberculosis complex in which the genetic lesions can be easily identified (Collins, 2000; Collins and Gicquel 2000). These techniques are being used in two broadly different ways as the basis for identifying genes that are important for virulence. In the first approach, random mutants are produced by transposon mutagenesis or 5 illegitimate recombination, screened in some way to identify those that have lost virulence, checked for their loss of virulence, and the causative mutation for this loss of virulence is identified. In the second approach, a gene that is thought likely to be important for virulence is inactivated by allelic exchange techniques and the virulence of the mutated strain then determined. Any strains found to be avirulent 10 by either approach can then be tested for their vaccine potential in an appropriate animal vaccine model. Many avirulent strains have now been produced by us and most have been tested for their vaccine ability.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any 15 reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common 20 general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the 25 listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

James & Wells Ref: 31147 It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

Summary of Invention The present invention relates to the identification, production, screening and use of attenuated mycobacterial species as live vaccines against mycobacterial diseases in man and animals. More particularly, it relates to mutant strains of the M. tuberculosis complex, that are sufficiently attenuated so that they do not cause 10 progressive disease when they are inoculated into animals that are susceptible to progressive disease when infected with wildtype species of the M. tuberculosis complex. The mutant strains are produced by several methods including illegitimate recombination, transposon mutagenesis and allelic exchange and are screened by a variety of methods including ability of 5-10 week-old cultures to 15 grow when inoculated into minimal medium, visual changes in morphology, severe limitation of growth in bovine alveolar macrophages, and inability to be recovered from guinea pig spleens in a Signature Tag Mutagenesis approach. Mutants from the screening approaches are then tested individually for their ability to cause disease in guinea pigs and if they achieve a standard of attenuation, for 20 their ability to protect guinea pigs against tuberculosis in a guinea pig vaccine model. Strains that show similar or better protection in guinea pigs than BCG are in some cases then tested for their ability to protect Australian brushtailed possums (Trichosurus vulpecula) against infection. The site of mutation and the identity of the gene or genes affected by the mutation are also identified. The 25 invention also claims the use of these strains, derivatives of these strains, and other strains with mutations in one or more identical or similar genes to these 6 James & Wells Ref: 31147 strains as vaccines against mycobacterial disease and more particularly as vaccines against tuberculosis in human and animal medical practice.

Disclosure of Invention The practice of the present invention will employ, unless otherwise indicated, 5 conventional techniques of molecular biology, microbiology, recombinant DNA, immunology and vaccinology which are within the ordinary knowledge of a person skilled in the art. Such techniques are explained fully in the literature.

According to a first aspect of the present invention there is provided an isolated Mycobacterium species of the M. tuberculosis complex which is attenuated and comprises at least one inactivated gene selected from the group consisting of: -ppiA ; - glnA2; - Rv0097; any of the genes between and including Rv0445c (sigK) - Rv0453 (PPE); any of the genes between and including Rv 0175 - Rv 0186 (bglS); any of the genes between and including Rv 0111 - Rv 0115; -Rv 3545c - any of the genes between and including Rv3757c (proW) - Rv3760 .

According to a second aspect there is provided an isolated Mycobacterium species 20 of M. tuberculosis complex which is attenuated and comprises inactivated or deleted genes selected from group consisting of: 7 James & Wells Ref: 31147 all genes between and including Rv0445c (sigK) - Rv0453 (PPE); all genes between and including RvOl 75 - Rv0186 (bglS); all genes between and including RvOlll - Rv0115; Preferably, the Mycobacterium species of the M. tuberculosis complex may be selected from existing or new members of the M. tuberculosis complex currently comprising: M. tuberculosis; M. ajricanum; M. microti; M. bovis subsp. caprae; M. canettii and M. bovis.

Most preferably the species selected from the M. tuberculosis complex may be M. bovis.

According to a third aspect of the present invention there is provided an isolated Mycobacterium species substantially as described above wherein the strain is selected from the group consisting of: WAg759: M. bovis mutant with an inactivated ppiA gene; WAg530: M. bovis mutant with an inactivated glnA2 gene; WAg533: M. bovis mutant with an inactivated Rv0097 gene; WAg539: M. bovis mutant with inactivation of the genes Rv0445c (sigK) -Rv0453 (PPE); WAg526: M. bovis mutant with inactivation of the genes RvOl 75 - Rv0186 (bglS); WAg569: M. bovis mutant with inactivation of the genes RvOlll - RvOl 15; WAg570: M. bovis mutant with an inactivated Rv3545c gene; 8 James & Wells Ref: 31147 WAg537: M. bovis mutant with an inactivated Rv0097 gene; WAg566: M. bovis mutant with inactivation of the genes Rv3757c (proW) -Rv3760.

According to a fourth aspect of the present invention there is provided an isolated 5 Mycobacterium species substantially as described above which also comprises an inactivated esat6 gene.

According to a fifth aspect of the present invention there is provided an isolated Mycobacterium species substantially as described above wherein the strain comprises an inactivated glnA2 gene and also comprises one or more inactivated 10 genes selected from the group consisting of Rv3874, Rv3875 and Rv3876.

According to a sixth aspect of the present invention there is provided an isolated Mycobacterium species substantially as described above wherein the strain is WAg530.1.

According to a seventh aspect of the present invention there is provided an 15 isolated Mycobacterium species wherein the strain comprises an inactivated Rv2136 gene and also comprises one or more inactivated genes selected from the group consisting of Rv3874, Rv3875 and Rv3876.

According to an eighth aspect of the present invention there is provided an isolated Mycobacterium species substantially as described above wherein the 20 strain is WAg520.4.

According to a ninth aspect of the present invention there is provided an isolated Mycobacterium species which is attenuated and comprises one or more inactivated genes that are identical or have at least 70%-99% nucleotide sequence homology to that of the inactivated genes as claimed above. 9 James & Wells Ref: 31147 According to a tenth aspect of the present invention there is provided an isolated Mycobacterium species of the M. tuberculosis complex which is attenuated and comprises one or more inactivated gene(s) which is the same gene(s) as that inactivated by polar effects of any of the inactivated genes as claimed above.

According to an eleventh aspect of the present invention there is provided an isolated Mycobacterium species of the M. tuberculosis complex which is attenuated and comprises at least one inactivated gene selected from any one of the inactivated genes referred to above.

According to a twelfth aspect of the present invention there is provided an isolated 10 Mycobacterium species of the M. tuberculosis complex which is attenuated and comprises at least one inactivated gene that is identical or has at least 70-99% nucleotide sequence homology to any one of the inactivated genes referred to above.

According to a thirteenth aspect of the present invention there is provided an 15 isolated Mycobacterium species substantially as described above wherein the mutant is further attenuated by inactivation of an additional gene.

According to a fourteenth aspect of the present invention there is provided a method of producing a live attenuated vaccine strain of any Mycobacterium species including M. avium and its subsp. avium, paratuberculosis and silvaticum 20 and M. ulcerans comprising steps of inactivating in these species one or more genes that are identical or have at least 50%-99% nucleotide sequence homology to that of the inactivated genes referred to above.

According to a fifteenth aspect of the present invention there is provided a vaccine to prevent or treat tuberculosis infection in mammals wherein the vaccine 25 comprises an isolated Mycobacterium species substantially as described above.

James & Wells Ref: 31147 Preferably, the vaccine also includes a pharmaceutical^ or veterinarily suitable carrier or diluent.

Most preferably, the vaccine may also include an adjuvant or other immuno-stimulant.

According to a sixteenth aspect of the present invention there is provided a vaccine substantially as described above wherein the Mycobacterium species comprises one or more foreign genes that are capable of enhancing the ability of the vaccine to stimulate the immune system of the diseased mammal to increase the effectiveness of said vaccine.

Preferably, the foreign gene may encode a polypeptide antigen, a cytokine or other immuno-stimulant.

According to a seventeenth aspect of the present invention there is provided a composition to prevent or treat tuberculosis infection in mammals wherein the composition comprises an isolated Mycobacterium species substantially as described above together with a pharmaceutically or veterinarily suitable carrier or diluent.

According to an eighteenth aspect of the present invention there is provided a method of screening mutants of a Mycobacterium species of the M. tuberculosis complex for attenuation comprising the steps of: a) isolating a Mycobacterium species to be screened; b) growing the isolated mutants for 5-10 weeks in a complete medium; c) transferring the 5-10 week old mutants to a minimal media; and d) selecting mutants based on their inability to grow in a minimal media. 11 James & Wells Ref: 31147 According to a nineteenth aspect of the present invention there is provided a method of producing and screening mutants of a Mycobacterium species of the M. tuberculosis complex for attenuation comprising the steps of: a) constructing a number of unique tagged suicide vectors containing a marker gene (e.g. antibiotic resistance gene); b) introducing the vectors to a culture of a Mycobacterium species and optionally leaving a predetermined amount of time; c) plating onto a suitable medium to select for marker gene; d) selecting mutants which contain marker gene, combining mutants together, and probing with DNA to identify the presence of all mutants in an 'input pool; e) injecting the 'input pool' into a test mammal; f) collecting mutant colonies from test mammals to create an 'output pool'; g) probing 'output pool' to identify mutants which are no longer present (ie. attenuated mutants).

The methods for introducing vectors into a mycobacterium may be performed by using methods well known in the art. Such methods may include but should not be limited to electroporation and phage transduction.

The test mammal may generally be selected from the groups consisting of; mice, guinea pigs, rats, possums, cattle, deer, ferrets, badgers and rabbits.

Preferably the test mammal may be a guinea pig. 12 James & Wells Ref: 31147 According to a twentieth aspect of the present invention there is provided a use of mycobacterium substantially as described above in the manufacture of a vaccine or composition to treat or prevent tuberculosis infections in mammals.

According to a twenty-first aspect of the present invention there is provided a method of determining whether a candidate drug is capable of inhibiting a polypeptide involved in mycobacterial infection comprising the steps of: a) Providing an isolated polypeptide encoded by one of the genes claimed above; b) Providing a candidate drug; c) Use of an assay which measures biological activity of the polypeptide in a); d) Measuring inhibition of biological activity of the polypeptide in a).

According to a twenty-second aspect of the present invention there is provided a method of determining whether a compound is an effective drug candidate against tuberculosis comprising the steps of: a) producing a stationary phase culture of a strain of the M. tuberculosis complex b) subculturing it in a minimal medium with the addition of a compound that is a potential drug c) comparing its growth in b) to that of a similar subculture in the same medium without the added compound The strains may be subcultured by means well known to those skilled in the art. 13 James & Wells Ref: 31147 As used herein "stationary phase" refers to the plateau of the growth curve after log growth in a culture, during which cell number remains substantially constant such that new cells are produced at a generally commensurate rate to that at which cell death occurs or in which cell numbers slowly decline as cell death occurs 5 faster than new cells are produced.

According to a twenty-third aspect of the present invention there is provided a method of detecting whether a drug is effective against mycobacterial infection comprising the steps of: a) administering to an animal having a mycobacterial infection, a candidate 10 drug as selected by the methods of the present invention; and b) assessing effectiveness of the drug at reducing or eliminating the clinical symptoms of said infection.

The present invention provides methods to screen for attenuated mutants of the Mycobacterium tuberculosis complex some of which will have utility as vaccines 15 against tuberculosis. The present invention also provides the attenuated mutants detailed in Table 1 that have been shown to have vaccine utility as well as any strains of the M. tuberculosis complex with similar or related mutations that also have vaccine utility.

In addition, the present invention also provides the attenuated mutants in Table 2 20 that have not yet been shown to have vaccine utility.

As used herein the term "attenuated mutants" denotes strains that have been tested for their ability to cause disease in animals and have been shown not to cause any disease or to cause a much diminished disease than wildtype strains of the M. tuberculosis complex. 14 James & Wells Ref: 31147 More particularly, these attenuated mutants have been shown not to cause any grossly visible tuberculosis lesions in the spleens of guinea pigs when 105 - 106 colony forming units (CFU) of the mutants were inoculated sub-cutaneously on one occasion into three guinea pigs each and the animals were autopsied 8-10 5 weeks later (except in the case of WAg570, where one of the three guinea pigs had one very small lesion in the spleen). This does not necessarily imply that such strains would never cause any lesions in guinea pigs but was a convenient criterion by which to identify those mutants that were highly attenuated relative to wildtype strains. In comparison, moderately virulent strains such as M. bovis 10 ATCC35723 and M. tuberculosis H37Rv will cause a mean of 3-15 grossly visible tuberculosis lesions per spleen when inoculated into 3 guinea pigs and recent wildtype isolates of M. bovis such as WAg200, WAg201, WAg203 and AF2122/97 will cause a mean of greater than 30 such lesions per spleen when inoculated into 3 guinea pigs. If a mutant strain is attenuated, this indicates that 15 the expression of one or more genes that are associated with virulence of the wildtype organism has become altered and most likely that the insertion of foreign DNA that occurred into the chromosome has disrupted and prevented the expression of a gene or genes at or near where the inserted DNA integrated. These genes whose expression is disrupted or prevented are associated with one or more 20 virulence properties of the microorganism. These properties are listed in Table 3.

All the strains listed in Tables 1 and 2 contain kanamycin and/or hygromycin resistance genes as a result of the selection methods used in their production. In order to licence a vaccine for use it may be desirable to remove these antibiotic resistance genes or to produce vaccines strains of the M. tuberculosis complex 25 without using such genes. This patent covers the production of vaccines without such antibiotic genes, however that is accomplished. Approaches could include use of other selection genes such as heavy metal resistance (Silver and Phung, James & Wells Ref: 31147 1996; Baulard et al., 1995), phage incompatibility factors (Hatfull et al., 1994), phage excision or resolution factors (Lewis and Hatfull, 2000; Steyn et al., 2002) or counter-selection techniques (Parish and Stoker, 2000; Reyrat et al., 1998).

This patent covers the further modification of vaccines claimed in this patent by 5 over expressing one or more genes of the M. tuberculosis complex. Such an approach has been shown to improve the protection provided by M. bovis BCG (Horwitz et al., 2000) and a similar approach could be applied to the strains claimed here.

Apart from antibiotic resistance genes, none of the strains listed in Tables 1 and 2 10 contain foreign genes. This patent covers the further modification of vaccines claimed in this patent by incorporating into them foreign genes that enhance their ability to stimulate protection against tuberculosis or enable the vaccine to have additional desirable properties such as the ability to induce protection against other disease organisms or enable specific products of the M. tuberculosis 15 complex to be under-expressed, over-expressed or expressed in a modified form. An extensive range of foreign genes have already been expressed in M. bovis BCG with this intention and one or more of the vaccines claimed here may offer advantages over BCG for these purposes. Such foreign genes could be antigens, cytokines, chemokines or other immuno-stimulants or other proteins whose 20 expression modifies the functional properties of strains of the M. tuberculosis complex (Murray and Young, 1998; Hess and Kaufinann, 1999; Ohara and Yamada, 2001). Such foreign genes are at present commonly incorporated using antibiotic resistance genes but the presence of these antibiotic resistance genes in the final vaccine could be avoided by use of the methods in the previous paragraph 25 or specific gene integration (Knipfer et al., 1997). 16 James & Wells Ref: 31147 This patent also covers methods for screening compounds for their use as drugs against mycobacterial infection. In one approach, a stationary phase culture of a strain of the M. tuberculosis complex is subcultured in a minimal medium with the addition of a compound that is a potential drug and the inhibition of growth of 5 the strain under these conditions is compared to that which occurs in minimal medium without the added compound. The concept of screening compounds for their potential use as drugs is well established (Global Alliance for TB Drug Development, 2001). The discovery in the work described here, that stationary phase cultures of random mutants that fail to grow on minimal medium are often 10 avirulent, indicates that genes important for virulence that are at a range of different loci must be active in order for this growth to occur. If a compound that specifically inhibits one of these important genes or its product is added at the right concentration to a subculture in minimal medium of a stationary phase strain of the M. tuberculosis complex, it will inhibit the said gene or product and the 15 organism will not grow. Importantly, this approach targets genes and their products in the M. tuberculosis complex that are involved in enabling organisms to recommence cell division after being in stationary phase. This group of genes and their products are regarded as a particularly important group of targets against which to produce new drugs (Young, 2001; McKinney et al., 2000). In another 20 approach, this patent also provides a method for identifying polypeptides and using these to produce an assay for determining the activity of candidate drugs (Dessen et al., 1995; Global Alliance for TB Drug Development, 2001). Isolating the polypeptide enables its structure to be determined and analysis of this structure assists in the design of new drugs and the development of said assays (Sharma et 25 al., 2000; Huang et al., 2002). 17 James & Wells Ref: 31147 The assays suitable for measuring the biological activity of a polypeptide from M. tuberculosis will be readily apparent to a person skilled in the art and will generally be selected dependant on: - the actually or putative biological function of the polypeptide; 5 - the actual or putative 3-D shape of the polypeptide; - target molecules known to be associated with the polypeptide.

The term "inactivated" means that the nucleotide sequence of the gene(s) has been disrupted for example mutated or removed from the chromosome such that a functional gene product (e.g. protein) normally produced by the gene or a group of 10 genes is no longer produced.

The term "gene" as used herein refers to a nucleic acid molecule comprising an ordered series of nucleotides that encodes a gene product (i.e. specific protein).

The term "protein (or polypeptide or peptide)" refers to a protein encoded by the nucleic acid molecule of the invention, including fragments, mutations and 15 homologs or analogs having the same biological activity i.e. ovulation manipulation activity. The polypeptide of the invention can be isolated from a natural source, produced by the expression of a recombinant nucleic acid molecule, or can be chemically synthesized.

The designation Rvxxxx(c) used to denote genes of the M. tuberculosis complex 20 where xxxx is a number varying from 0001 to 3924 and c refers to genes encoded on the complementary strand refers to the annotation of the complete genome sequence of M. tuberculosis H37Rv (at http://www.sanger.ac.uk). Essentially the same genes are designated in different ways in the sequences of other strains of 18 James & Wells Ref: 31147 the M. tuberculosis complex e.g. M. tuberculosis CDC 1551 (at http://www.tigr.org/).

The term "polar effect" refers to a mutation which in addition to affecting the gene in which it occurs, reduces partially or completely the expression of any gene(s) in 5 the same operon on the "promoter" distal (downstream) side of the mutation.

The term "operon" refers to a controllable unit of transcription consisting of a number of structural genes that are transcribed together.

The term "transcription" and "transcribed" refer to the first step in gene expression - i.e. the synthesis of an RNA copy from a sequence of DNA (a gene).

The term "promoter" refers to a site on DNA to which RNA polymerase (an enzyme) will bind and initiate transcription of DNA (a gene) to produce mRNA.

The term ' Immuno-stimulant' refers to any class of compounds capable of eliciting or otherwise activating or helping to activate/elicit an immune response in a mammal. An immuno-stimulant may include cytokines, chemokines, peptide-15 containing and non-peptide-containing products produced in strains of the M. tuberculosis complex, and peptide-containing and non-peptide-containing bacterial, protozoan or metazoan products but this list should not be seen as limiting. An immuno-stimulant may include: polypeptides: polypeptide-including chemicals, or other non- protein based chemicals: but an immuno-20 stimulant should not be limited to this list.

The term "isolated" means substantially separated or purified away from contaminating sequences in the cell or organism in which the nucleic acid naturally occurs and includes nucleic acids purified by standard purification techniques as well as nucleic acids prepared by recombinant technology, including 25 PCR technology, and those chemically synthesised. Preferably, the nucleic acid 19 James & Wells Ref: 31147 molecule is derived from genomic DNA or the mRNA of the Bovicola ovis chewing louse.

The preparation of pharmaceutical compositions including pharmaceutical carriers are well known in the art, and are set out in textbooks such as Remington's 5 Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.

The compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at 10 the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.

The carrier or diluent, and other excipients, will depend on the route of 15 administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case.

The homology between two nucleotide sequences can be assessed by a variety of methods. Preferably, the nucleotide sequences may be assessed by programs such as BLASTN as available from NCBI http://www.ncbi.nlm.nih. gov or GAP and 20 BESTFIT programmes of GCG (Wisconsin Package Version 10.2, Genetics Computer Group, Madison, Wisconsin) Thus, the homology of the nucleotide sequences may be assessed by comparing on BLASTN or other programmes the nucleotide sequence of interest against those of the relevant gene of the invention as can be determined from the complete genome James & Wells Ref: 31147 of M. tuberculosis (Cole et al, 1998) available at http://www.Sanger.ac.uk/ or at http.//www.pasteur.fr/mycdb/.

The unique tagged suicide vectors may be made from a variety of different vectors provided they are compatible with species of the M. tuberculosis complex. In 5 particular, suitable vectors may include any vector that replicates in Escherichia coli but either cannot replicate in mycobacterial species or does not replicate in mycobacterial species under specified conditions such as growth temperature or the presence of a compound in the growth medium and which contains a variable region of nucleotides called a "tag" such as pUHA604.

Brief Description of Drawings Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 Shows a plasmid pYUB553.1 used for transposon mutagenesis. 15 Transposon Tn5367 is composed of TnpR, aph (kanamycin resistance), and TnpA; Figure 2 Shows the sites of transposon mutagenesis in M. bovis WAg200 to produce WAg533 and in M. bovis WAg201 to produce WAg537; Figure 3A Shows a DNA fragment of the ahpC locus of M. bovis containing a 20 kanamycin resistance gene (neo); Figure 3B Shows a DNA fragment of Rv3844 locus containing a kanamycin resistance gene (neo); Figure 4 Shows a Southern blot hybridization after EcoRl digestion and probing with neo of 8 recombinants that were unable to grow in 21 James & Wells Ref: 31147 minimal medium (lanes 1-5, 7-9) and one recombinant that failed the screen and grew in minimal medium (lane 6); Figure 5A Shows the site of illegitimate recombination and size of deletion in M. bovis ATCC35723 to produce WAg526; Figure 5B Shows the site of illegitimate recombination and size of deletion in M. bovis ATCC35723 to produce WAg530; Figure 5C Shows the site of illegitimate recombination and size of deletion in M. bovis ATCC35723 to produce WAg569; Figure 6A Shows pUHA600 used as intermediate construct in signature tag 10 experiment; Figure 6B Shows linearised pUHA600 after digestion with ZfcrBI; positions of selected restriction sites in the plasmid are given in brackets; Figure 7 Shows plasmid pUHA604 which is comprised of pUHA601used as intermediate construct in signature tag experiment together with 15 inserted tag; Figure 8A Shows the 89 bp DNA fragment made for producing signature tags; Figure 8B Shows the 81 bp DNA product produced by ifrsHII and Bglll digestion of the 89 bp fragment in Fig 8A and inserted into pUHA601 to produce pUHA604 plasmids containing signature 20 tags; Figure 9A Shows a site of illegitimate recombination and size of deletion in M. bovis WAg200 to produce WAg539; 22 James & Wells Ref: 31147 Figure 9B Shows a site of illegitimate recombination and size of deletion in M. bovis WAg201 to produce WAg566; Figure 9C Shows a site of illegitimate recombination in M. bovis WAg201 to produce WAg570; Figure 10 Shows a linearised diagram of plasmid pUHA772 used for allelic exchange of ppiA in M. bovis ATCC35723. pUHA9 = pBluescript KS 11+ with a Pacl-Bcll-Pacl cassette inserted at the BamRl site; hyg is a hygromycin resistance gene; Figure 11A Shows a Southern blot hybridization of PvwII-digested M. bovis 10 recombinants with a probe that spans across the ppiA gene sequence into which the hyg gene was inserted in the suicide vector pUHA772 (Fig. 10), with the size of the wildtype fragment (2.6 kb) and the pUHA772 fragment (4.1 kb) in the recombinants indicated by arrows. Lane 1, ATCC35723 parent strain; lanes 2-6, 15 8, 10-16, illegitimate mutants and single homologous recombinants; lanes 7, 9, allelic exchange mutants; Figure 11B Shows a picture of an agarose gel electrophoresis separation of DNA products from M. bovis ATCC35723 and selected M. bovis recombinants subjected to PCR with DMC312 and DMC313 20 (Table 6) which amplify a DNA fragment of 0.4 kb in wildtype strains and 1.9 kb in strains where ppiA has been interrupted with the 1.5 kb hyg gene. Lanel, DNA standards, lane 2, ATCC35723 parent strain; lanes 3-6, four ppiA allelic exchange mutants (WAg759); lane 7, single homologous recombinant; lane 8, 25 illegitimate recombinant; and 23 James & Wells Ref: 31147 Figure 12 Shows alignment of the M. tuberculosis genome at the esat6 locus with the DNA construct used for esat6 knockout by homologous recombination. The deleted fragment included all of the esat6 gene and small parts of the ilp and Rv3876 genes.

BEST MODES FOR CARRYING OUT THE INVENTION Experimental Non-limiting examples illustrating the invention will now be provided. It will be appreciated that the above description is provided by way of example only and variations in the materials and technique used which are known to those skilled in 10 the art are contemplated.

Bacterial culture All bacteria were cultured at 37°C-38°C. The M. bovis strains used for this work and the carbon sources routinely used for their liquid and solid culture are listed in Table 4. Antibiotics were added where appropriate at the concentrations given in 15 Table 5. Strains were cultured on a variety of different liquid and solid mycobacterial media. Standard liquid culture was in 3 - 100 ml media in static sealed glass containers. Two media were used: Tween-albumin broth (Kent and Kubica, 1985) which does not require an additional carbon source, and Middlebrook 7H9 (Difco) supplemented with albumin/dextrose complex (ADC; 20 Difco), 0.05% Tween 80 and the appropriate carbon source (Table 4). The standard solid medium used was Middlebrook 7H11 (Difco) supplemented with ADC and the appropriate carbon source (Table 4), sometimes with the addition of 2.5% bovine serum. In some cases, M. bovis mutants were isolated on Middlebrook 7H10 (Difco) medium supplemented with 0.085% NaCl, 0.2% 25 glucose, and 0.2% casamino acids. Solid culture was performed in standard plastic 24 James & Wells Ref: 31147 Petri dishes. M. bovis being prepared for electroporation was cultured in roller bottles at 1 rpm in 100 ml Middlebrook 7H9 (Difco) supplemented with albumin/dextrose complex (ADC; Difco), 0.05% Tween 80, 0.4% sodium pyruvate and 0.5% glycerol. For selection of mutants with impaired growth in 5 minimal medium, 5 |_il of a 5 - 10 week old culture in supplemented Middlebrook 7H9 medium was inoculated into supplemented Middlebrook 7H9 medium and also into minimal Proskauer and Beck liquid medium (Darzins, 1958) to which 0.05% Tween 80 and 0.2% sodium pyruvate had been added.

Escherichia coli strains used were XLl-Blue-MR, JM109 and DH10B. They were 10 grown in Luria-Bertani liquid medium or solid agar with the appropriate antibiotic concentrations (Table 5).

Production and screening of M. bovis mutants The following four methods were successfully used to produce mutants of M. bovis, some of which were subsequently found after screening, virulence testing 15 and vaccine testing to have vaccine efficacy against tuberculosis in experimental animals. Those mutant strains of M. bovis that showed vaccine efficacy in guinea pigs that was similar or better than BCG under the standard vaccine challenge conditions used are given in Table 1. In the first three methods, random mutagenesis is used to make a large number of mutants that are subsequently 20 screened to select a few candidates while in the fourth method, allelic exchange, a specific gene to be inactivated is chosen and then deleted. The advantages of random mutation approaches are that they make few assumptions about what genes are important for virulence and only reveal those genes that are amenable to inactivation. Its major disadvantages are that the mutation methods may not be 25 sufficiently random to inactivate all virulence genes and because many thousands and even millions of mutants can be made, there is great difficulty in selecting out James & Wells Ref: 31147 all of the mutants that have become attenuated. Some of the mutants claimed herein were produced by illegitimate recombination. This can produce mutants comparable to those produced by transposon mutagenesis in which a single gene is deleted but in many cases, illegitimate recombination causes deletion of several 5 genes. This may have advantages for producing a live vaccine that cannot revert. In the experiments reported here, highly-attenuated M. bovis recombinants with good vaccine efficacy were obtained by screening transposon mutants for morphological changes, and by screening illegitimate recombinants for survival in macrophages and for impaired ability of 5-10 week-old cultures to grow in 10 minimal medium. While all screening methods were not used on either transposon mutants or illegitimate recombinants this should not be taken to imply that they cannot be so used. One skilled in the art would expect that any of the screening methods used here as well as other screening methods not used such as standard auxotrophic selection (Hondalus et al., 2000) could potentially be successfully 15 applied to mutants of the M. tuberculosis complex produced by different methods in order to acquire highly attenuated mutants with some degree of vaccine efficacy. One skilled in the art would also expect that mutants of the M. tuberculosis complex with identical or related genes inactivated to those strains claimed in this patent would in many cases also have vaccine efficacy and that 20 they could be developed by random mutagenesis or by the use of any of a large number of different methods to identify one or more genes associated with virulence, immune modification or essential metabolic pathways (Collins and Gicquel, 2000; Collins, 2000,2001; DesJardin and Schlesinger, 2000) followed by specific gene inactivation, for example by allelic exchange. 1. Transposon mutagenesis and morphological screening Transposon mutagenesis Strain M. bovis WAg200 was mutated by transposon mutagenesis using the 26 James & Wells Ref: 31147 transposon Tn5367 in plasmid pYUB553.1 (Fig. 1) obtained from Professor W. R. Jacobs, Albert Einstein College of Medicine, New York. The technique followed was similar to that described by McAdam et al. (1995) except that the suicide plasmid used in this work was electroporated into M bovis WAg200 using a high 5 efficiency electroporation technique as described by Wards and Collins (1996).

Morphological screening Ten thousand individual kanamycin resistant colonies were plated at approximately 102 CFU / plate on both 7H10 and 7H11 media and after 4-6 weeks culture were assessed by eye for colony morphology. Colonies that appeared 10 different from the norm were recultured on two separate occasions and compared again to colonies with normal morphology. Three M. bovis mutants appeared to show stable, small differences from normal morphology and these were tested for virulence in guinea pigs. One of these mutants, WAg533, was found to be avirulent and subsequently was shown to have vaccine efficacy at least 15 comparable to that of BCG (Table 7A).

Determination of site of mutation To determine the site of insertion of the transposon in the M. bovis chromosome, chromosomal DNA was digested with £eoRI, a restriction enzyme that does not have a site within Tn5367, ligated into the £coRI site of pBluescript KSII+, 20 electroporated into E. coli XL 1-Blue MR and plated on Luria-Bertani medium containing 25 fag/ml kanamycin and 50 ng/ml ampicillin. The junction regions of the fragment and chromosome were sequenced, using primers DMC153 and DMC154 (Table 6) directed outwards from Tn5367 into the mycobacterial sequence. DNA sequences were analysed using the programmes of the Genetics 25 Computer Group and compared to GenBank (www.ncbi.nlm.nih.gov) and Sanger (www.sanger.ac.uk) databases. 27 James & Wells Ref: 31147 Similar mutant Subsequently, a transposon mutant (WAg537) from a different parent, M. bovis WAg201, was selected by screening for its inability to grow in microaerophilic conditions and to be sensitive to levels of cycloserine that do not inhibit the parent 5 strain. WAg537 was found to be avirulent in guinea pigs and on analysis was found to have a transposon insertion in Rv0097 (Fig. 2), the same gene that is inactivated in WAg533 (Fig. 2). The chromosomal site of inactivation in Rv0097 is different in the two strains; Mtcy251 position 14610 for WAg537 and 14790 for WAg533 (Tables 1 and 2). WAg537 was not originally screened for different 10 colonial morphology and WAg533 was not originally screened for its ability to grow in microaerophilic conditions or its cycloserine sensitivity. However, when WAg533 and WAg537 were compared using all three screening methods they were found to have identical phenotypes. This clearly indicates that in at least some cases, different screening methods will identify different mutants of the M. 15 tuberculosis complex in which the same gene is inactivated.

Vaccine testing The standard vaccine challenge method used for assessing the protective effect induced by WAg533 and the other mutants detailed below was essentially as described in de Lisle et al., (1999) except that instead of the animals being 20 challenged by intratracheal inoculation they were challenged by aerosol inoculation. Briefly, 4-6 guinea pigs were inoculated subcutaneously in the flank with approximately 105 CFU of the M. bovis mutant to be tested. At the same time, a group of 4-6 guinea pigs was inoculated in the same manner with M. bovis BCG and a control group of 4-6 guinea pigs was not inoculated. The number of 25 CFU inoculated was approximated by appropriate dilution following estimation of turbid cultures. The exact number of CFU used was determined retrospectively by 28 James & Wells Ref: 31147 plating 10-fold dilutions on supplemented Middlebrook 7H11 agar. Animals were fed food and water ad libitum and housed in a controlled environment biocontainment unit. Approximately 8 weeks after inoculation, the guinea pigs were challenged with an aerosol containing a single cell suspension of M. bovis 5 WAg201 with a known quantity of organisms. Single cell suspensions of the isolate were prepared using a modification of a method described by Grover et al. (1967) and stored at -70°C. For preparing these suspensions, the bacterial cells were dispersed by sonication for 30 sec and filtered through an 8 [am membrane filter. Guinea pigs were infected via the respiratory route by using an aerosol 10 chamber which produces droplet nuclei of the size appropriate for entry into alveolar spaces (McMurray et al., 1985, Wiegeshaus et al., 1970). The concentration of viable M. bovis in the nebuliser fluid was empirically adjusted to result in the inhalation and retention of 2-10 viable organisms per guinea pig (Buddie and de Lisle, unpublished). The final solution that was aerosolised 15 contained approximately 0.048 x 106 CFU/ml. This challenge dose had previously been estimated from the number of primary tubercles observed grossly in the lungs of non-vaccinated guinea pigs at 4 weeks post-infection. A similar procedure had been reported previously to result in reproducible, uniform infection of the lungs of guinea pigs (Wiegeshaus et al., 1970; Smith et al., 1970). 20 The aerosol infection and subsequent maintenance and manipulation of infected guinea pigs were performed under strict isolation conditions in a biohazard facility. Approximately 5 weeks after challenge, the animals were euthanased and autopsied and body weight and gross pathology was recorded. Samples of spleen and lungs were subjected to mycobacterial culture and enumeration. Delayed type 25 hypersensitivity to tuberculin was measured immediately before vaccination and prior to the animals being sacrificed. Bovine purified protein derivative (4 units; MAF, Central Animal Health Laboratory, New Zealand) was injected intradermally and the diameter of the erythema was measured 24 h later. For 29 James & Wells Ref: 31147 WAg533 and all the other M. bovis recombinants described in this patent, no delayed type hypersensitivity reactions were observed when the animals were tested prior to being inoculated with mycobacteria. All animals had positive responses immediately prior to sacrifice. The vaccine efficacy of the mutants and 5 of BCG was determined by comparison of the gross lesions and CFU in the test animals to those in the control animals.

When WAg533 was tested for its ability to protect guinea pigs against tuberculosis using the above method it provided protection that was comparable to that provided by BCG (Table 7 A). 2. Illegitimate recombination and screening in minimal medium and macrophages Previous work Illegitimate recombination in M. bovis BCG and M. tuberculosis was first described by Kalpana et al. (1991) and occurs when there is recombination 15 between an introduced DNA fragment and a region of the chromosome with which it has little homology. We adapted this finding to produce mutants of M bovis (Wilson et al., 1997). In that study, we used a linear DNA fragment of the ahpC locus of M. bovis with a kanamycin resistance gene (neo) inserted into it (Fig. 3A). In contrast to transposon mutagenesis which produces mutants in which 20 insertion at a single point without any deletion occurs, a variable degree of deletion usually occurs with illegitimate recombination at the site of insertion in the chromosomal DNA and sometimes also at either end of the inserting DNA. In our earlier work, the deletion in the chromosomal DNA varied from 2 bp to 64.6 kb. Four illegitimate mutants that were selected from their inability to grow in 25 minimal medium were shown to be attenuated in guinea pigs. When challenged with virulent M. bovis, two of these mutants induced a level of protection that was James & Wells Ref: 31147 similar to that induced by BCG (de Lisle et al., 1999). In one of these mutants, a putative undecaprenol kinase gene was interrupted by illegitimate insertion of a DNA fragment that accompanied a 2 bp chromosomal deletion. In the other mutant, DNA fragment insertion was accompanied by a large DNA deletion of 15 5 kb containing 12 genes. In none of the four mutants were any of the interrupted genes identified as being involved or having close homology to genes encoding enzymes for common metabolic pathways that are often associated with auxotrophy such as amino acid, purine, pyrimidine or co-factor synthesis. Nevertheless, at the time we reported these experiments, the mutants selected on 10 minimal medium were described as auxotrophs.

Stationary growth phase screening Subsequently, we discovered that these mutants were not auxotrophs in the strict sense as 2-week-old actively growing cultures of the mutants in complete medium were able to grow when inoculated into minimal medium. Re-examination of our 15 procedures revealed that the mutants in the study had been subcultured into minimal medium from 5-10 week old cultures in complete medium, at a stage when they would have been in stationary growth. Since the ability of strains of the M. tuberculosis complex to survive in the host in a stationary or lowered metabolic state is regarded as a crucial facet of tuberculosis pathogenesis, we 20 reasoned that it might be desirable to have a vaccine strain without this ability, with the added consideration that if that was the only ability it had lost, it might provide a better stimulation of host immune systems than a true auxotrophic mutant which required a specific metabolite to achieve adequate growth. Regardless of this explanation, since the use of 5-10 week old cultures had 25 produced two mutants with vaccine effectiveness at least equal to that of BCG, we continued using similar approaches to produce avirulent mutants. In one new study (Collins et a., 2002), we used either an ahpC fragment interrupted with neo 31 James & Wells Ref: 31147 as in the earlier work or a second fragment in which Rv3844, a gene of unknown function, was interrupted with neo (Fig. 3B). A Southern blot hybridisation including DNA from each of the 8 mutants obtained from this experiment is given in Fig. 4. Five of these mutants (WAg526, WAg527, WAg529, WAg530 and 5 WAg531) were found to be highly attenuated when tested for virulence in guinea pigs.

Screening for severe limitation ofgrowth in bovine alveolar macrophages Alveolar macrophages were obtained by lavage from freshly excised lungs of tuberculosis-free cattle and dispensed into flat-bottomed 96-well microtitre plates 10 (Nalge Nunc International) as described previously (Aldwell et al., 1996). After washing to remove non-adherent cells, they were cultured at 37°C under a 5% CC>2-95% air atmosphere in RPMI 1640 supplemented with 4 mM L-glutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate, 2.9 mM sodium bicarbonate, 50 U/ml penicillin G and 10% bovine serum. Sixty illegitimate 15 recombinants of M. bovis ATCC35723 were cultured in TAB medium. The bacteria were serially diluted and 5 |j.l and 10 (il aliquots of each culture containing 1-5 x 107 CFU/ml were placed in duplicate into microtitre wells containing macrophages. Since each well contained approximately lxlO5 macrophages per well, the multiplicity of infection ranged from 0.5:1 - 5:1. An 20 aliquot of each bacterial culture was also subcultured in a microtitre well containing TAB. After 5 days, the cells in both the TAB and macrophage cultures were labelled overnight with [3H]uracil, harvested as described previously (Aldwell et al., 1996) and the level of radioactivity was counted in a Trilux MicroBeta 1450 scintillation counter. The level of radioactivity was used as an 25 approximate measure of bacterial cell numbers based on previous comparisons (Aldwell et al., 1996). The ratio of 3H counts in macrophages compared to the same strain in TAB were calculated and three strains with the lowest ratios were 32 James & Wells Ref: 31147 tested for virulence in guinea pigs. One of these strains, WAg569, was found to be highly attenuated in guinea pigs.

Vaccination results The highly attenuated strains developed in these experiments were tested for their 5 ability to protect guinea pigs against a standard vaccine challenge as described earlier and three of these (WAg526, WAg530 and WAg569) were found to provide comparable protection to that provided by BCG (Table 7A, 7B).

Identification ofsites of mutation The sites of mutation and the size of the deletions that had occurred during illegitimate recombination were determined for WAg526, WAg530 and WAg569 using previously described methods (Wilson et al., 1997) and are shown in Fig. 5A, Fig. 5B and Fig. 5C respectively. Briefly, chromosomal DNA was digested with a restriction enzyme that did not cut within the inserted fragment (Xhol for the Rv3844 fragment and EcoRl or BamRl for the ahpC fragment), ligated into the appropriate site of pBluescript KSII+, electroporated into E. coli XL 1-Blue MR and plated on Luria-Bertani medium containing 25 fig/ml kanamycin and 50 jag/ml ampicillin. The junction regions of the fragment and chromosome were sequenced, using appropriate primers directed outwards from the vector sequence into the mycobacterial sequence. DNA sequences were analysed using BLAST (http://www.ncbi.nlm.nih.gov) and the programmes of the Genetics Computer Group and compared to GenBank (www.ncbi.nlm.nih.gov) and Sanger (www.sanger.ac.uk) databases. 3. Signature Tag Mutagenesis based on illegitimate recombination Approach used in this study 33 James & Wells Ref: 31147 Signature Tag Mutagenesis (STM) combines the production of mutant libraries with the direct identification of individual mutants that have become attenuated. Unlike the other random mutant approaches that are used in this patent to produce attenuated strains of the M. tuberculosis complex, in which mutants are produced, 5 screened in various ways and then tested individually for loss of virulence; STM combines these procedures in a co-ordinated way. In STM, pools of mutants are produced in which each member of the pool is tagged with a unique DNA sequence. Susceptible hosts are infected with a pool of mutants called the "input" pool. Subsequently, an "output" pool of disease-causing organisms is recovered 10 from the infected host and the absence of a member(s) of the input pool in the output pool indicates which particular mutant(s) was attenuated. The method was first applied to Salmonella typhimurium infection in mice (Hensel et al., 1995) and since then has been applied to a considerable number of different pathogens (Mecsas, 2002). Two groups used the technique to identify transposon mutants of 15 M. tuberculosis that are unable to replicate in the lungs of mice (Cox et al., 1999; Camacho et al., 1999). One group (Camacho et al., 1999) identified 14 different genetic loci whose interruption caused attenuation and both groups found that many of the affected loci are involved in lipid metabolism. In particular, a 50 kb region of the chromosome that contains 13 genes involved in biosynthesis or 20 transport of phthiocerol and phenolphthiocerol and in mycocerosic acid synthesis was identified in several mutants by both groups.

A substantially different approach was used in this study. First, we made a tagged suicide vector (pUHA604, Fig. 7) as described below and produced mutants of M. bovis by illegitimate recombination instead of by transposon mutagenesis. Second, 25 we used a guinea pig model in which pools of mutants were inoculated sub-cutaneously and mutants that could not be isolated subsequently from pooled colonies from spleens of three animals were identified and reinoculated singly into 34 James & Wells Ref: 31147 three guinea pigs each to identify those that were most attenuated. This contrasts with both the reported studies with M. tuberculosis mutants where the bacterial pools were inoculated intravenously into two mice and the mutants were tested for their ability to survive in mouse lungs for three weeks.

Preparation of tagged suicide plasmid pUHA604 1. A 2039 bp plasmid (pUHA600, Fig. 6A) was derived from the 5011 bp integrating mycobacterial shuttle plasmid pYUB178 (Pascopella et al., 1994) by digestion with BamUl and BelI which each cut once in this vector followed by self-ligation of the smaller fragment which contains a kanamycin resistance gene (aph) and an origin of replication in E. coli (oriE). 2. Plasmid pUHA600 was digested with BsrBl which cuts the plasmid once between the end of oriE and the start of the aph gene to produce a blunt-end linear full-length plasmid pUHA600 as shown in Fig. 6B. 3. A short blunt-ended double-stranded piece of DNA was made by allowing DMC125 and DMC126 (Table 6) to anneal by heating both primers to 90°C and allowing them to cool slowly over 30 min to 40°C to form a linker that contained ifosHII (italicised) and BglII (lower case) sites as shown below: TTG GCG CGC CAA GAa gat ctT C AAC CGC GCG GTT CTt eta gaA G 4. Linearised and phosphatased pUHA600 (500 ng) was ligated with 1 |ag of this double-strand DNA and the ligation mixture was electroporated into E. coli and plated onto solid medium containing kanamycin. Plasmid DNA James & Wells Ref: 31147 extraction was performed on selected colonies and a plasmid of the expected size that gave a linear fragment of approximately 2.1 kb on digestion with either Bglll or ifosHII was designated pUHA601.

. Plasmid pUHA601 (5 (ig) was digested with an excess of ifasHII and Bglll and the large linear fragment was separated from uncut vector and the tiny (7-11 bp) linking piece between the two restriction sites by electrophoresis and gel extraction (Geneclean, Biol01). 6. Microgram quantities of an 89 bp DNA fragment (Fig. 8A) were produced by polymerase chain amplification (PCR) of DMC120 with DMC121 and DMC123 (Table 6). The size of the product was checked on a 4% Nusieve + 1% normal agarose gel. This DNA fragment contained a variable 40 bp sequence that was used for uniquely tagging individual strains of M. bovis. 1. The 89 bp DNA product (Fig. 8A) was digested with .RssHII and Bglll to produce a sticky-ended 81 bp DNA product (Fig. 8B) that was then dephosphorylated. 8. The sticky-ended 81 bp product was ligated to the large pUHA601 fragment prepared in step 5 by digestion with 2?ssHII + Bglll and the mixture was electroporated into E. coli and plated on solid medium containing kanamycin to produce a cloned family of pUHA604 plasmids each with a different 40 bp tag (Fig. 7).

First experimental approach In the first experimental approach, plasmid DNA was extracted from a liquid culture made from a combined pool of 10,000 CFU of recombinant E. coli containing pUHA604 plasmids. The plasmid DNA was digested with Haell or Spel and electroporated into M. bovis WAg200 which was then plated onto solid 36 James & Wells Ref: 31147 medium containing kanamycin. Two thousand individual colonies were recovered, cultured in liquid medium and stored at room temperature until needed. Restriction digestion of DNA from 20 individual clones, followed by electrophoresis, Southern blotting and hybridisation with a probe of pUHA600, 5 indicated that insertion had occurred into different parts of the chromosome. DNA from 20-50 clones was digested individually with Hindlll, subjected to electrophoresis (1.5% agarose, 4.5 V/cm, 90 min) and transferred to nylon membrane (Zeta Probe, Biorad) by Southern Blotting. A probe of DNA pooled from all the M. bovis recombinants on the membrane was prepared by PCR using 10 the primers DMC122 and DMC124 in a Geneamp 9600 PCR system (Perkin-Elmer Cetus) under the following temperature conditions: 94°C for 3 min initial denaturation followed by 30 cycles of 62°C for 30 sec, 72°C for 10 sec, and 94°C for 30 sec The standard PCR reaction mixture consisted of 50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 15 0.5 |im each of oligonucleotide primers and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer Cetus). The DNA product was separated by electrophoresis on a 1.6% agarose gel (Seaplaque GTG, FMC Bio-Products) and extracted using a QIAquick gel extraction kit (Qiagen). If a strong 81 bp product was not obtained, the PCR was repeated using a different concentration of MgCl2. 20 The probe was labelled with 32P by nick translation. Input pools of sizes ranging from 20-50 recombinants were constructed by separately culturing recombinants, estimating their concentration based on their turbidity and adding equal numbers of each recombinant to make the pool. Each pool was inoculated into three guinea pigs. After 7-9 weeks, the animals were euthanased and in all cases had multiple 25 grossly-visible tuberculous lesions in their spleens from which M. bovis was isolated. Between 40, 000 and 200, 000 colonies from the spleens of animals infected with a pool of recombinants were combined together and extracted for DNA using cetyl-trimethyl ammonium bromide (van Soolingen et al., 1991). A 37 James & Wells Ref: 31147 labelled probe was made from this output-pool DNA in the same way as the probe for the input-pool DNA and hybridised to a Southern blot of DNA from each member of the input pool in the same way as described earlier. Recombinants whose DNA hybridised much less strongly to the output-pool probe than to the 5 input-pool probe were tested individually for their attenuation in three guinea pigs. Considerable technical difficulty was encountered with this approach because DNA from many recombinants hybridised poorly to a pooled DNA probe and the presence of mycobacterial DNA in both the probe and the Southern blot gave significant background hybridisation. Nevertheless, 145 recombinants in various 10 size pools were inoculated into guinea pigs and two attenuated recombinants were detected (WAg539 which gave no lesions in guinea pig spleens and a second strain, WAg540, which gave a small number of lesions in the spleen of one guinea Pig)- Second experimental approach In the second experimental approach, a total of 132 plasmids in three batches of 39, 40 and 53 plasmids each containing a different tag were prepared, subjected individually without restriction enzyme digestion to agarose gel electrophoresis (1% agarose, 60 min, 9 V/cm) and transferred to nylon membranes (Zeta Probe, Biorad) by Southern blotting. Forty-five of the plasmids gave an acceptable 20 positive signal when hybridised to a probe made from their respective combined pools and did not cross-react when half the pool was labelled and hybridised against the other half. The probe was prepared in the same way as in the first experimental approach except that the template DNA used was plasmid DNA instead of DNA from M. bovis recombinants. To make M. bovis recombinants 25 using these plasmids, each plasmid was digested with HaeII, and electroporated separately into M. bovis WAg201 which was then plated on solid medium containing kanamycin. Twenty-seven individual colonies of recombinant M. bovis 38 James & Wells Ref: 31147 from each electroporation were cultured in liquid medium and stored until needed at -70°C. Pools of 42 - 45 M. bovis recombinants containing approximately 106 CFU were inoculated into three guinea pigs each. A pool was constructed by separately culturing 42-45 recombinants each with a different tag, estimating their 5 concentration based on their turbidity and adding equal numbers of each recombinant to make the pool. After 7-9 weeks, the animals were euthanased and in all cases had multiple grossly-visible tuberculous lesions in their spleens from which M. bovis was isolated. Between 40, 000 and 200, 000 colonies from the spleens of animals infected with a pool of recombinants were combined 10 together and extracted for DNA using cetyl-trimethyl ammonium bromide (van Soolingen et al., 1991). Labelled probes were made from both the input-pool DNA and the output-pool DNA in the same way as in the first experimental approach and used to probe a Southern blot of DNA samples from the original 45 plasmids. This gave less background than in the first experimental approach where 15 Southern blots of DNA from M. bovis recombinants were used. Recombinants whose DNA hybridised much less strongly to the output-pool probe than to the input-pool probe were tested individually for their attenuation in three guinea pigs. A total of 1110 M. bovis recombinants were tested. Those that were found to have reduced virulence are given in Table 8. Two of these strains are named in Claim 3: 20 viz. WAg566 and WAg570.

Identification of Sites of mutation The sites of mutation and the size of the deletions that had occurred during illegitimate recombination were determined for WAg539, WAg566, and WAg570 using an approach similar to that described above for other illegitimate 25 recombinants (Wilson et al., 1997) and are shown in Fig. 9A, Fig. 9B, and Fig. 9C respectively and summarised in Tables 1 and 2. In some cases, there were tandem copies of the inserting suicide plasmid at the mutation site which necessitated 39 James & Wells Ref: 31147 subcloning of the recovered constructs containing the kanamycin resistance gene and chromosomal junction fragments in order to get unique sites for the sequencing primers.

Vaccination results One (WAg539) of the three highly attenuated strains (WAg539, WAg566, WAg570) developed in these STM experiments was tested for its ability to protect guinea pigs against a standard vaccine challenge and was found to provide comparable protection to that provided by BCG (Table 7A). 4. Allelic exchange Allelic exchange in the M. tuberculosis complex Allelic exchange has two important roles in the development of attenuated strains of the M. tuberculosis complex with vaccine potential. First, there are a large range of different methods that can be applied to strains of the M. tuberculosis complex in order to identify genes that may be involved in the pathogenesis of 15 tuberculosis or whose inactivation might be expected to affect the virulence of these mycobacteria (Collins and Gicquel, 2000; Collins, 2000, 2001; DesJardin and Schlesinger, 2000). These genes can then be inactivated by allelic exchange techniques and the virulence and vaccine properties of the mutants formed can be determined. This approach was used to produce WAg759 in this present study. 20 Second, allelic exchange can be used in a wide variety of ways to modify strains of the M. tuberculosis complex that have already been mutated. For example, it can be used to produce inactivation of a second gene in a mutant and this was done for WAg520.4 and WAg530.1 in this present study, or it can be used to eliminate a previously inserted selection gene or to modify a mycobacterial gene 25 in a way that alters the gene's properties but does not inactivate the gene. 40 James & Wells Ref: 31147 Initial attempts to perform allelic exchange in the M. tuberculosis complex were unsuccessful and resulted in the production of illegitimate recombinants (Kalpana et al., 1991; Aldovini et al., 1993). More recently, different groups have developed a variety of methods to perform allelic exchange (Pelicic et al., 1997; 5 Hinds et al., 1999; Wards et al., 2000; Raman et al., 2001) and a relatively large number of genes have now been inactivated.

Method of allelic exchange used in this study The method of allelic exchange used in this study has been used to inactivate a number of genes in strains of M. bovis including esat6 (Wards et al., 2000), ahpC 10 (Collins, 2001,) and Rv2136 (Collins and Gicquel, 2000). Briefly, a 2-3 kb DNA sequence incorporating the gene of interest is cloned into an E. coli vector and an antibiotic resistance gene is inserted into the coding sequence of the gene so that normal expression will be prevented. The vector is denatured and electroporated as a suicide vector into a strain of the M. tuberculosis complex and antibiotic 15 resistant clones are recovered and analysed to detect an allelic exchange mutant.

Production ofWAg759 In the case of the ppiA gene, a 2219 bp DNA fragment incorporating the coding sequence of ppiA at position 482-1191 of the fragment was amplified by PCR from chromosomal DNA of M. bovis ATCC35723 by using the primers DMC330 20 and DMC331 (Table 6) which contain a restriction site near their 5' ends for Pacl, an enzyme that has no sites in the chromosome of M. tuberculosis. The fragment was digested with Pacl and cloned into pUHA9 (a derivative of the cloning vector pBluescript II KS (Stratagene) in which a DNA cassette containing the restriction enzyme sites Pacl-Bcll-Pacl replaces the BamRl site) to form pUHA774. 25 pUHA774 was digested with Sfil which cuts once in the plasmid, at 658 bp in the 2219 bp M. bovis DNA fragment. The Sfil digest was treated with T4 DNA 41 James & Wells Ref: 31147 polymerase to blunt end the plasmid by removing the three-nucleotide 3' overhanging ends due to the Sfil digest, and was dephosphorylated. A 1.5 kb Notl - Pstl DNA fragment containing a hygromycin resistance gene was blunt-ended using Klenow fragment, ligated to blunt-ended pUHA774 and transformed into E. coli to produce pUHA772 (shown in linear form in Fig. 10). This plasmid was denatured in 0.2 M NaOH for 5 min, neutralized with half its volume of 1 M sodium acetate pH 5.0, and precipitated and washed with ethanol. The denatured plasmid was electroporated into M. bovis ATCC35723 using a high efficiency electroporation technique (Wards and Collins, 1996) and cells were plated onto 10 medium containing hygromycin. DNA was isolated from liquid subcultures of 72 hygromycin-resistant colonies (van Soolingen et al., 1991), digested with PvwII and characterized by Southern blot hybridization with a probe that spans across the mycobacterial DNA site containing the inserted antibiotic resistance gene. Four recombinants gave Southern blot hybridisation patterns indicating that allelic 15 exchange had occurred. An autoradiogram of one Southern blot hybridisation revealing two of the allelic exchange mutants is shown in Fig. 11 A. All four allelic exchange mutants were confirmed by PCR using DMC312 and DMC313 (Table 6) which amplify a DNA fragment of 0.4 kb in wildtype strains and 1.9 kb in strains where ppiA has been interrupted with the 1.5 kb hyg gene (Fig. 1 IB). In 20 both the Southern blot hybridisations and the PCR product gel the presence of two fragments indicates single homologous recombination or illegitimate recombination has occurred, the presence of a single fragment with a size 1.5 kb more than the wildtype fragment indicates that allelic exchange has occurred. One of these allelic exchange mutants WAg759 was shown to be avirulent in guinea 25 pigs. 42 James & Wells Ref: 31147 Production ofWAg520.4 and WAg530.1 The production of M. bovis WAg520 has been described previously (Wilson et al., 1997) and the production of M. bovis WAg530 is described earlier in this patent. To delete the esat6 gene locus in WAg520 and WAg530 a similar approach was 5 used to that described in Wards et al. (2000) except that the suicide plasmid contained a hygromycin resistance gene inserted into the deleted esat6 locus (consisting of all the esat6 gene and parts of the two adjacent genes, Rv3874 and Rv3876) instead of a kanamycin resistance gene. An outline of the construct used for allelic exchange of the esat6 locus is given in Fig. 12. Both WAg520.4 and 10 WAg530.1 were shown to be highly attenuated in guinea pigs and, using the vaccine testing method described earlier, were found to provide comparable protection to that provided by BCG and by their respective parent strains (Table 7B).

Advantages of deleting esat6 locus in already attenuated strains of the M. 15 tuberculosis complex The advantages of eliminating the esat6 locus as has been done for WAg520.4 and WAg530.1 are threefold. First, the region deleted prevents the production by the strains of two proteins, ESAT6 and CFP10 (also called MTSA10, the product of Ihp also known as Rv3874), which together stimulate strong cell-mediated 20 immune responses in infected hosts (Renshaw et al., 2002). The removal of these proteins from a live vaccine enables the use of testing regimes for tuberculosis infection that combine tests of cell immune responses to ESAT6 and/or CFP10 as well as other tests for tuberculosis infection (Colangeli et al., 2000; van Pinxteren V et al., 2000; Vordermeier et al., 2001). The results of these tests can distinguish 25 hosts infected with wildtype strains of the M. tuberculosis complex from hosts vaccinated with attenuated strains of the M. tuberculosis complex lacking 43 James & Wells Ref: 31147 production of ESAT6 and CFP10. Second, removal of the esat6 locus greatly reduces virulence (Wards et al., 2000) and therefore provides an added safety factor against reversion to virulence of any live vaccine strain that like WAg520.4 and WAg530.1 also contains another attenuating mutation. Third, removal of the 5 esat6 locus may directly improve the vaccine properties of an already attenuated strain of the M. tuberculosis complex if the absence of ESAT6 and/or CFP10 from the vaccine strain allows a more protective or long-lived immune response to be established in the host.

Future use of live vaccines It is anticipated that the vaccine strains described here or vaccine strains with improved properties based on inactivation of genes described here will have wide utility for protecting against tuberculosis and other mycobacterial diseases in mammalian hosts. Already some of these vaccine strains have been tested for their ability to protect Australian brush-tailed possums (Trichosurus vulpecula) and in some cases have demonstrated protection that is at least as good as BCG (Table 9). This demonstrates that the approach being used with guinea pigs is likely to produce attenuated strains of the M. tuberculosis complex with vaccine effectiveness against tuberculosis in many different mammalian hosts including humans.

Table 1. Attenuated mutants of M. bovis shown to have vaccine efficacy that is not significantly different from BCG Mutant Parent strain Method of production Primary screen Genes inactivated (*) Position of insertion and, where applicable, deletion relative to Sanger sequence of M. tuberculosis H37Rv Size of deletion (bp) WAg759 ATCC35723 Allelic exchange Southern blotting ppiA 9390 - 9392 of MtcylOH4 3 WAg530 ATCC35723 Illegitimate recombination Growth in glnA2 5330 - 5359 of Mtcy427 44 James & Wells Ref: 31147 minimal medium WAg533 WAg200 Transposon mutagenesis Morphol ogy Rv0097 14790 ofMtcy251 0 WAg539 WAg200 Illegitimate recombination Signature Tag Rv0445 0SigK)-Rv0453 (PPE) 7900- 18040 of Mtv037 ,141 WAg526 ATCC35723 Illegitimate recombination Growth in minimal medium RvOl 75- RvOJ86 {bglS) 16862-28088 of MtcI28 11,227 WAg569 ATCC35723 Illegitimate recombination Poor growth in macroph ages RvOlll-RvOl15 5325 - 8662 of Mtv031 3,338 WAg520 .4 ATCC35723 Allelic exchange Southern blotting Rv2136 and Rv3874 -Rv3876 3877-3878 of Mtcy270 9754- 10329 of Mtv027 2 575 WAg530 .1 ATCC35723 Allelic exchange Southern blotting gin A 2 and Rv3874 -Rv3876 5330- 5359 of Mtcy427 9754-10329 of Mtv027 575 * Gene designations are those used in annotating the complete genome sequence of M. tuberculosis H37Rv (Cole et al., 1998) and can be found at http://www.sanger.ac.uk and in MycDB at http://www.pasteur.fr/mycdb/ Table 2. Attenuated mutants of M. bovis whose vaccine efficacy has not yet been determined Mutant Parent strain Method of production Primary screen Genes inactivated Position of insertion and, where applicable, deletion relative to Sanger sequence of M. tuberculosis H37Rv Deletion size (bp) WAg537 WAg201 Transposon mutagenesis Cycloserine, Reduced oxygen Rv0097 14610 of Mtcy251 0 WAg566 WAg201 Illegitimate recombination Signature Tag Rv3757c (proW) -Rv3760 110334- 112676 of Mtv025 2,343 WAg570 WAg201 Illegitimate recombination Signature Tag Rv3545c 25861 of Mtcy3C7 0 Table 3. Properties associated with virulence of the M. tuberculosis complex 1 Infectious; capable of being spread from one individual mammal to another 2 Capable of entering mammalian host cells 3 Capable of surviving or escaping phagocyte cellular defences 45 James & Wells Ref: 31147 4 Capable of multiplying in host cells Capable of spreading from an infected cell to an uninfected cell 6 Capable of causing or enabling cell injury that results in pathology 7 Capable in some circumstances of killing the infected host Table 4. Origin and properties of M. bovis strains used in these experiments M. bovis strain Virulence in guinea pigs Origin Carbon source for culture ATCC35723 Moderate Originally isolated from infected bovine. Obtained from American Type Culture Collection. 0.5% Glycerol WAg200 Full AgResearch Wallaceville strain 89/5276. Isolated in this laboratory from infected bovine. 0.4% Pyruvate WAg201 Full AgResearch Wallaceville strain 86/5701. Isolated in this laboratory from infected bovine. 0.4% Pyruvate BCG Avirulent Pasteur strain 1173P2; Statens Seruminstitut, Copenhagen, Denmark 0.5% Glycerol Table 5. Concentration of antibiotics used for selection Antibiotic E. coli M. bovis Kanamycin -50 ng/ml -25 ng/ml Hygromycin 200 |ag/ml 50 ng/ml Ampicillin 100 ng/ml Not used Table 6. DNA oligonucleotides used in this work Oligonucleotide Sequence 5' - 3' DMC120 TTGGCGCGCTACAACCTCAAGCTT[NW]20AAGCT TGGTTAGAATGAGATCTTCA DMC121 TTGGCGCGCTACAACCT DMC122 CGCTACAACCTCAAGCT DMC123 TGAAGATCTCATTCTAACC DMC124 ATCTCATTCTAACCAAGCT DMC125 TTGGCGCGCCAAGAAGATCTTC DMC126 GAAGATCTTCTTGGCGCGCCAA DMC153 CACAGCGCGAAAGCAGC DMC154 CTATCCCGGCACCGACG DMC312 AACAGCCCCCTTGCGAC DMC313 GGTTCAGGTGCGGAGTC DMC330 GCGTTAATTAACCCCGCGAAGATCCAGAGGTG DMC331 GCGTTAATTAACCAGCAGCAGCCCCGCCAG 46 James & Wells Ref: 31147 Table 7A. Vaccination results for some illegitimate recombinants M. bovis recombinant Spleen weight mean (g) ± SE Spleen lesions mean No. ± SE Spleen M. bovis log to CFU ± SE Non-vaccinated 2.50 ±0.33" 46 ± 10a 4.97 ± 0.1 T BCG 1.29 ±0.09b 0.25 ±0.18" 1.53 ± 0.53b WAg526 1.17 ± 0.10b 0.0 ± 0.0b 1.21 ±0.21b WAg530 1.15 ± 0.10b 0.0 ± 0.0b ND WAg533 1.23 ± 0.24b 0.0 ± 0.0b 1.00±0.00b WAg539 1.08 ± 0.12b 0.0 ± 0.0" 1.40 ± 0.30b WAg569 1.07 ± 0.06b 0.33 ± 0.33b ND WAg759 1.18 ± 0.07b 0.33 ± 0.33b 1.62 ± 0.52b SE, standard error a'b, values within the same column with the same suffix are not significantly 5 different ND, not determined Significance for spleen lesions calculated on logio transformed data Table 7B Vaccination results for some illegitimate recombinants M. bovis strain Spleen weight mean (g) ± SE Spleen lesions mean No. ± SE Spleen M. bovis logio CFU ± SE LungM bovis logio CFU ±SE Non-vaccinated 2.15 ±0.30" 59 ± 28a 4.96 ± 0.30a .18 ± 0.14a BCG 1.35 ± 0.10b 0.17 ± 0.00b 2.33 ± 0.36b 3.55 ± 0.54b WAg520 1.28 ± 0.13b 0.00 ± 0.00b 1.46 ± 0.46b 3.49 ±0.12" WAg520.4 1.53 ±0.11" 0.00 ± 0.00b 1.54 ± 0.43b 4.02 ± 0.26" Non-vaccinated 3.57 ± 0.43a 86± 21" .63 ± 0.09a .41 ±0.13a BCG 1.45 ± 0.1 lb 0.67 ± 0.49b 2.55 ± 0.49b 3.71 ±0.30" WAg530 1.12 ± 0.07b 1.0 ± 0.5b 2.69 ± 0.62b 4.73 ± 0.28a WAg530.1 1.58 ±0.09" 2.3 ± 0.7b 3.81 ± 0.12b 4.47 ± 0.38ab SE, standard error a'b Values within the same column with the same suffix are not significantly different Table 8. M. bovis recombinants with greatly reduced virulence from Signature 15 Tag Mutagenesis experiments Numbers and sizes of tuberculous lesions * Parent Strain Guinea pi«l Guinea pig 2 Guinea pig 3 Livers Lungs Degree of virulence WAg201 WAg566 NVL NVL NVL NVL NVL - WAg201 WAg567 NVL NVL NVL NVL NVL - WAg201 WAg568 NVL NVL NVL NVL NVL - WAg200 WAg539 NVL NVL NVL NVL NVL - WAg201 WAg570 NVL NVL 1 x 1mm NVL NVL + WAg201 WAg571 NVL NVL 3 x 3mm NVL NVL + WAg201 WAg572 NVL NVL 2 x <lmm NVL NVL + WAg201 WAg573 2 x 1mm NVL 3 x l-2mm NVL NVL + WAg201 WAg574 3 x l-2mm NVL x 1mm NVL NVL + WAg200 WAg540 100 x 1 NVL NVL many in 1 animal NVL ++ 47 James & Wells Ref: 31147 *NVL: No visible lesions Table 9. Protection of possums against bovine tuberculosis by vaccination.

Vaccine Change in body weight after challenge (g)a Lung weight (g)b Spleen bacterial count0 WAg520 -92 ±146 44 ± 7.6 1.78 ±0.38 WAg526 -225 ± 134 44 ± 7.0 1.64 ±0.25 WAg530 8 ±85* 32 ± 4.4* 0.87 ±0.17* WAg533 42 ± 49* 36 ± 8.2 0.83 ±0.13* WAg539 33 ± 69* 31 ±5.6* 1.13 ±0.14 BCG -108 ±77 31 ±5.3* 1.14 ±0.20 Non- vaccinated -267 ±106 51 ± 7.8 1.95 ±0.43 a Mean change in body weight ± SE b Mean lung weight ± SE c Mean bacterial count, log 10 colony forming units/g spleen ± SE * Significantly different to non-vaccinated group Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 48 James & Wells Ref: 31147 References Aldovini, A., Husson, R.N., Young, R.A. (1993) The uraA locus and homologous recombination in Mycobacterium bovis BCG. JBacteriol 175: 7282-7289.

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Claims (2)

1. WHAT WE CLAIM IS: 1. An isolated Mycobacterium species of the M. tuberculosis complex which is attenuated and comprises at least one inactivated gene selected from the group consisting of: 5 - ppiA; glnA2; Rv0097; any of the genes between and including Rv0445c (sigK) - Rv0453 (PPE); 10 - any of the genes between and including Rv 0175 - Rv 0186 (bglS); any of the genes between and including Rv 0111 - Rv 0115; Rv 3545c any of the genes between and including Rv3757c (pro W) - Rv3760.
2. 2. An isolated Mycobacterium species of M. tuberculosis complex which is 15 attenuated and comprises inactivated or deleted genes selected from group consisting of: all genes between and including Rv0445c (sigK) - Rv0453 (PPE); all genes between and including RvOl 75 - RvOl86 (bglS); all genes between and including RvOlll - RvOl 15; intellectual property office of n.z - 3 NOV 2003 received 59 James & Wells Ref: 31147 An isolated Mycobacterium species as claimed in claims 1-2 wherein the Mycobacterium species of the M. tuberculosis complex is selected from existing or new members of the M. tuberculosis complex currently comprising: M. tuberculosis; M. ajricanum; M. microti; M. bovis subsp. Caprae; M. canettii and M. bovis. An isolated Mycobacterium species as claimed in claim 3 wherein the species selected from the M. tuberculosis complex is M. bovis. An isolated Mycobacterium species as claimed in claim 3 wherein the strain is selected from the group consisting of: WAg759: M. bovis mutant with an inactivated ppiA gene; WAg530: M. bovis mutant with an inactivated glnA2 gene; WAg533: M. bovis mutant with an inactivated Rv0097 gene; WAg539: M. bovis mutant with inactivation of the genes Rv0445c (sigK) - Rv0453 (PPE); WAg526: M. bovis mutant with inactivation of the genes RvOl 75 -RvOl 86 (bglS); WAg569: M. bovis mutant with inactivation of the genes RvOlll -RvOllS; WAg570: M. bovis mutant with an inactivated Rv3545c gene; WAg537: M. bovis mutant with an inactivated Rv0097 gene; WAg566: M. bovis mutant with inactivation of the genes Rv3757c (proW) - Rv3760. 60 James & Wells Ref: 31147 intellectual property office of n.z - 3 NOV 2003 received 7. 5 8. 9. 10 10. 15 11. 12. 20 An isolated Mycobacterium species as claimed in claim 5 which also comprises an inactivated esat6 gene. An isolated Mycobacterium species as claimed in claim 3 wherein the strain comprises an inactivated glnA2 gene and also comprises one or more inactivated genes selected from the group consisting of Rv3874, Rv3875 and Rv3876. An isolated Mycobacterium species as claimed in claim 7 wherein the strain is WAg530.1. An isolated Mycobacterium species as claimed in claim 3 wherein the strain comprises an inactivated Rv2136 gene and also comprises one or more inactivated genes selected from the group consisting of Rv3874, Rv3875 and Rv3876. An isolated Mycobacterium species as claimed in claim 9 wherein the strain is WAg520.4. An isolated Mycobacterium species which is attenuated and comprises one or more inactivated genes that are identical to or have at least 70%-99% nucleotide sequence homology to that of the inactivated genes as claimed in claims 1-10. An isolated Mycobacterium species of the M. tuberculosis complex which is attenuated and comprises one or more inactivated gene(s) which is the same gene(s) as that inactivated by polar effects of any of the inactivated genes as claimed in any one of the preceding claims. 13. An isolated Mycobacterium species as claimed in claim 3 wherein the species is attenuated and further comprises at least one inactive gene selected from any one of the preceding claims. 14. An isolated Mycobacterium species of the M. tuberculosis complex which is 5 attenuated and comprises at least one inactivated gene that is identical or has at least 70-99% nucleotide sequence homology to any one of the inactivated genes as claimed in claim 1. 15. An isolated Mycobacterium species as claimed in claims 1-14 wherein the mutant is further attenuated by inactivation of an additional gene. 10 16. A method of producing a live attenuated vaccine strain of any Mycobacterium species including M. avium and its subsp. avium, paratuberculosis and silvaticum and M. ulcerans comprising steps of inactivating in these species one or more genes that are identical to or have at least 70%-99% nucleotide sequence homology to that of the inactivated genes as claimed in any one of 15 the preceding claims. 17. A vaccine to prevent or treat tuberculosis infection in mammals wherein the vaccine comprises an isolated Mycobacterium species as claimed in any one of claims 1-15. 18. A vaccine as claimed in claim 17 wherein the vaccine also includes a 20 pharmaceutically or veterinarily suitable carrier or diluent. 19. A vaccine as claimed in claim 18 wherein the vaccine also includes an adjuvant or other immuno-stimulant. 20. A vaccine as claimed in claims 17-19 wherein the Mycobacterium species comprises one or more foreign genes that are capable of enhancing the ability mTB^MTpwEm7 3 NOV 2003 | 62 James & Wells Ref: 31147 Received of the vaccine to stimulate the immune system of the diseased mammal to increase the effectiveness of said vaccine. 21. A vaccine as claimed in claim 20 wherein the foreign gene may encode a polypeptide antigen, a cytokine or other immuno-stimulant. 22. A composition to prevent or treat tuberculosis infection in mammals wherein the composition comprises an isolated Mycobacterium species as claimed in claims 1-15, together with a pharmaceutically or veterinarily suitable carrier or diluent. 23. The use of an isolated Mycobacterium species as claimed in claims 1-15 in the manufacture of a vaccine or composition to treat or prevent tuberculosis infections in mammals. 24. A method of determining whether a candidate drug is capable of inhibiting a polypeptide involved in mycobacterial infection comprising the steps of: a) providing an isolated polypeptide encoded by one of the genes as claimed in any one of claims 1-15; b) providing a candidate drug; c) use of an assay which measures biological activity of the polypeptide in a); and d) measuring inhibition of biological activity of the polypeptide in a). 25. An isolated Mycobacterium species as described herein with reference to any example thereof. 26. A vaccine to prevent or treat tuberculosis infection in mammals as described herein with reference to any example thereof. 63 James & Wells Ref: 31147 27. A composition to prevent or treat tuberculosis infection in mammals as described herein with reference to any example thereof. 28. Use of an isolated Mycobacterium species in the manufacture of a vaccine or composition to treat or prevent tuberculosis infections in mammals as described herein with reference to any example thereof. 29. A method of determining whether a candidate drug is capable of inhibiting a polypeptide involved in mycobacterial infection as described herein with reference to any example thereof. 30. A method of determining whether a compound is an effective drug candidate against tuberculosis as described herein with reference to any example thereof. 31. A method of determining whether a drug is effective against mycobacterial infection as described herein with reference to any example thereof. AGRESEARCH LIMITED Intellectual Property Office of NZ \ 6 FEB 200*» 64 James & Wells Ref: 31147 ABSTRACT This invention, relates to attenuated strains of the Mycobacterium tuberculosis complex, methods for producing and screening these, methods for assessing their effectiveness as vaccines and the use of such strains and derivatives of them as vaccines against tuberculosis in human and animal medical practice. INTELLECTUAL PROPERTY OFFICE OF N.Z. : 9 JUN 2003 received 67 James & Wells Ref: 31147