General Background Human tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The world-wide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent years this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis.

The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy re- mains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of TB, but several human trials in developing countries have failed to demonstrate significant protection. Notably, BCG is not approved by the FDA for use in the United States because BCG vaccination impairs the specificity of the Tuberculin skin test for diagnosis of TB infection.

This makes the development of a new and improved vaccine against TB an urgent matter, which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and different investigators have reported in- creased resistance after experimental vaccination. However, the demonstration of a spe- cific long-term protective immune response with the potency of BCG has not yet been achieved.

Immunity to M. tuberculosis is characterized by some basic features ; specifically sensi- tized T lymphocytes mediates protection, and the most important mediator molecule seems to be interferon gamma (IFN-y).

M. tuberculosis holds, as well as secretes, several proteins of potential relevance for the generation of a new TB vaccine. For a number of years, a major effort has been put into

the identification of new protective antigens for the development of a novel vaccine against TB. The search for candidate molecules has primarily focused on proteins re- leased from dividing bacteria. Despite the characterization of a large number of such pro- teins only a few of these have been demonstrated to induce a protective immune re- sponse as subunit vaccines in animal models, most notably ESAT-6 and Ag85B (Brandt et al 2000).

In 1998 Cole et al published the complete genome sequence of M. tuberculosis and pre- dicted the presence of approximately 4000 open reading frames (Cole et al 1998). Among others, nucleotide sequences comprising Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878 and Rv3879c are described, and putative protein sequences for the above sequences are suggested. However important, this sequence information can- not be used to predict if the DNA is translated and expressed as proteins in vivo. More importantly, it is not possible on the basis of the sequences to predict whether a given se- quence will encode an immunogenic or an inactive protein. The only way to determine if a protein is recognized by the immune system during or after an infection with M. tuberculo- sis is to produce the given protein and test it in an appropriate assay as described herein.

Diagnosing M. tuberculosis infection in its earliest stage is important for effective treat- ment of the disease. Current diagnostic assays to determine M. tuberculosis infection are expensive and labour-intensive. In the industrialized part of the world the majority of pa- tients exposed to M. tuberculosis receive chest x-rays and attempts are made to culture the bacterium in vitro from sputum samples. X-rays are insensitive as a diagnostic assay and can only identify infections in a very progressed stage. Culturing of M. tuberculosis is also not ideal as a diagnostic tool, since the bacteria grows poorly and slowly outside the body, which can produce false negative test results and take weeks before results are obtained. The standard tuberculin skin test is an inexpensive assay, used in third world countries, however it is far from ideal in detecting infection because it cannot distinguish M. tuberculosis-infected individuals from M. bovis BCG-vaccinated individuals and there- fore cannot be used in areas of the world where patients receive or have received child- hood vaccination with bacterial strains related to M. tuberculosis, e. g. a BCG vaccination.

Animal tuberculosis is caused by Mycobacterium bovis, which is closely related to M. ti- berculosis and within the tuberculosis complex. M. bovis is an important pathogen that can infect a range of hosts, including cattle and humans. Tuberculosis in cattle is a major

cause of economic loss and represents a significant cause of zoonotic infection. A number of strategies have been employed against bovine TB, but the approach has generally been based on government-organized programs by which animals deemed positive to de- fined screening test are slaughtered. The most common test used in cattle is Delayed- type hypersensitivity with PPD as antigen, but alternative in vitro assays are also devel- oped. However, investigations have shown the both the in vivo and the in vitro tests have a relative low specificity, and the detection of false-positive is a significant economic problem (Pollock et al 2000). There is therefore a great need for a more specific diagnos- tic reagent, which can be used either in vivo or in vitro to detect M. bovis infections in animals.

Summary of the invention The invention is related to preventing, treating and detecting infections caused by species of the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) by the use of a polypeptide comprising a M. tuberculosis antigen or an immunogenic portion or other variant thereof, or by the use of a DNA sequence encoding a M. tuberculosis antigen or an immunogenic portion or other variant thereof.

Detailed disclosure of the invention The present invention discloses a substantially pure polypeptide, which comprises an amino acid sequence selected from (a) Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1 ; (b) an immunogenic portion, e. g. a T-cell epitope, of any one of the sequences in (a) ; and/or (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic.

Preferably, the amino acid sequence analogue has at least 80%, more preferred at least 90% and most preferred at least 95% sequence identity to any one of the sequences in (a) or (b).

The invention further discloses a fusion polypeptide, which comprises an amino acid se- quence selected from

(a) Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1 (b) an immunogenic portion, e. g. a T-cell epitope, of any one of the sequences in (a) ; and/or (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic ; and at least one fusion partner.

Preferably, the fusion partner comprises a polypeptide fragment selected from (a) a polypeptide fragment derived from a virulent mycobacterium, such as ESAT-6, MPB64, MPT64, TB10. 4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, Ag85A, Ag85B, Ag85C, 19kDa lipoprotein, MPT32, MPB59 and alpha-crystallin ; (b) a polypeptide according to the invention and defined above and/or (c) at least one immunogenic portion, e. g. a T-cell epitope, of any of such polype- tides in (a) or (b) The invention further relates to a polypeptide, which comprises an amino acid sequence selected from (a) Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1 (b) an immunogenic portion, e. g. a T-cell epitope, of any one of the sequences in (a) ; and/or (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic ; which is lipidated so as to allow a self-adjuvating effect of the polypeptide.

Further, the invention relates to a polypeptide, which comprises an amino acid sequence selected from (a) Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1 (b) an immunogenic portion, e. g. a T-cell epitope, of any one of the sequences in (a) ; and/or (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic ; for use as a vaccine, as a pharmaceutical or as a diagnostic reagent.

In another embodiment, the invention relates to the use of a polypeptide as defined above for the preparation of a pharmaceutical composition for diagnosis, e. g. for diagnosis of tuberculosis caused by virulent mycobacteria, e. g. by Mycobacterium tuberculosis, Myco- bacterium africanum or Mycobacterium bovis, and the use of a polypeptide as defined above for the preparation of a pharmaceutical composition, e. g. for the vaccination against infection caused by virulent mycobacteria, e. g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.

In a still further embodiment, the invention relates to an immunogenic composition com- prising a polypeptide as defined above, preferably in the form of a vaccine or in the form of a skin test reagent.

In another embodiment, the invention relates to a nucleic acid fragment in isolated form which (a) comprises a nucleic acid sequence which encodes a polypeptide as defined above, or comprises a nucleic acid sequence complementary thereto ; or (b) has a length of at least 10 nucleotides and hybridizes readily under stringent hy- bridization conditions with a nucleotide sequence selected from Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1 nu- cleotide sequences or a sequence complementary thereto, or with a nucleotide sequence selected from a sequence in (a) The nucleic acid fragment is preferably a DNA fragment. The fragment can be used as a pharmaceutical.

In one embodiment, the invention relates to a vaccine comprising a nucleic acid fragment according to the invention, optionally inserted in a vector, the vaccine effecting in vivo ex- pression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially in- creased resistance to tuberculosis caused by virulent mycobacteria, e. g. by Mycobacteri- um tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, includ- ing a human being.

In a further embodiment, the invention relates to the use of a nucleic acid fragment ac- cording to the invention for the preparation of a composition for the diagnosis of tubercu- losis caused by virulent mycobacteria, e. g. by Mycobacterium tuberculosis, Mycobacte- rium africanum or Mycobacterium bovis, and the use of a nucleic acid fragment according to the invention for the preparation of a pharmaceutical composition for the vaccination against tuberculosis caused by virulent mycobacteria, e. g. by Mycobacterium tuberculo- sis, Mycobacterium africanum or Mycobacterium bovis.

In a still further embodiment, the invention relates to a vaccine for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e. g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, com- prising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined above has been incorporated into the microorganism (e. g. placed on a plasmid or in the genome) in a manner allowing the microorganism to express and optionally secrete the polypeptide.

In another embodiment, the invention relates to a repliable expression vector, which comprises a nucleic acid fragment according to the invention, and a transformed cell har- bouring at least one such vector.

In another embodiment, the invention relates to a method for producing a polypeptide as defined above, comprising (a) inserting a nucleic acid fragment according to the invention into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide, and recovering the polypeptide from the host cell or culture medium ; (b) isolating the polypeptide from a whole mycobacterium, e. g. Mycobacterium tuber- culosis, Mycobacterium africanum or Mycobacterium bovis, from culture filtrate or from lysates or fractions thereof ; or (c) synthesizing the polypeptide e. g. by solid or liquid phase peptide synthesis.

The invention also relates to a method of diagnosing tuberculosis caused by virulent my- cobacteria, e. g. by Mycobacterium tuberculosis, Mycobacterium africanum or Myco-

bacterium bovis, in an animal, including a human being, comprising intradermally inject- ing, in the animal, a polypeptide as defined above or an immunogenic composition as de- fined above, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis.

In another embodiment, the invention relates to a method for immunizing an animal, in- cluding a human being, against tuberculosis caused by virulent mycobacteria, e. g. by My- cobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the polypeptide as defined above, the immunogenic composi- tion according to the invention, or the vaccine according to the invention.

Another embodiment of the invention relates to a monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide as defined above in an immuno assay, or a specific binding fragment of said antibody. Preferably, said antibody is for use as a di- agnostic reagent, e. g. for detection of mycobacterial antigens in sputum, urine or other body fluids of an infected animal, including a human being.

In a further embodiment the invention relates to a pharmaceutical composition which comprises an immunologically responsive amount of at least one member selected from the group consisting of : (a) a polypeptide selected from Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1, or an immunogenic portion thereof ; (b) an amino acid sequence which has a sequence identity of at least 70% to any one of said polypeptides in (a) and is immunogenic ; (c) a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner ; (d) a nucleic acid sequence which encodes a polypeptide or amino acid sequence according to (a), (b) or (c) ; (e) a nucleic acid sequence which is complementary to a sequence according to (d) ; a nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e) ; and

(g) a non-pathogenic micro-organism which has incorporated (e. g. placed on a plas- mid or in the genome) therein a nucleic acid sequence according to (d), (e) or (f) in a manner to permit expression of a polypeptide encoded thereby.

In a still further embodiment the invention relates to a method for stimulating an immuno- genic response in an animal which comprises administering to said animal an immu- nologically stimulating amount of at least one member selected from the group consisting of : (a) a polypeptide selected from Rv0284, Rv0285, Rv0455c, Rv0569, Rv1195, Rv1386, Rv3477, Rv3878, Rv3879c or MT3106. 1, or an immunogenic portion thereof ; (b) an amino acid sequence which has a sequence identity of at least 70% to any one of said polypeptides in (a) and is immunogenic ; (c) a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner ; (d) a nucleic acid sequence which encodes a polypeptide or amino acid sequence according to (a), (b) or (c) ; (e) a nucleic acid sequence which is complementary to a sequence according to (d) ; a nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e) ; and (g) a non-pathogenic micro-organism which has incorporated therein (e. g. placed on a plasmid or in the genome) a nucleic acid sequence according to (d), (e) or (f) in a manner to permit expression of a polypeptide encoded thereby.

The vaccine, immunogenic composition and pharmaceutical composition according to the invention can be used prophylactically in a subject not infected with a virulent mycobacte- rium ; or therapeutical in a subject already infected with a virulent mycobacterium.

The invention also relates to a method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising (a) contacting a sample, e. g. a blood sample, with a composition comprising an anti- body according to the invention, a nucleic acid fragment according to the inven- tion and/or a polypeptide as defined above, or

(b) contacting a sample, e. g. a blood sample comprising mononuclear cells (e. g. T- lymphocytes), with a composition comprising one or more polypeptides as de- fined above in order to detect a positive reaction, e. g. proliferation of the cells or release of cytokines such as IFN-y.

Finally, the invention relates to a method of diagnosing Mycobacterium tuberculosis infec- tion in a subject comprising : (a) contacting a polypeptide as defined above with a bodily fluid of the subject ; (b) detecting binding of a antibody to said polypeptide, said binding being an indica- tion that said subject is infected by Mycobacterium tuberculosis or is susceptible to Mycobacterium tuberculosis infection.

Definitions The word"polypeptide"in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptides, short pep- tides and fragments thereof, wherein the amino acid residues are linked by covalent pep- tide bonds.

The polypeptide may be chemically modified by being glycosylated, by being lipidated (e. g. by chemical lipidation with palmitoyloxy succinimide as described by Mowat et al.

1991 or with dodecanoyl chloride as described by Lustig et al. 1976), by comprising prosthetic groups, or by containing additional amino acids such as e. g. a his-tag or a sig- nal peptide.

Each polypeptide may thus be characterised by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include ana- logues and variants produced by recombinant or synthetic methods wherein such poly- peptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide and still be immunogenic in any of the biological assays described herein. Substitutions are preferably"conserva- tive". These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code. ALIPHATIC Non-polar GAP ILV Polar-uncharged CSTM NQ Polar-charged DE KR AROMATIC HFWY

A preferred polypeptide within the present invention is an immunogenic antigen from M. tuberculosis. Such antigen can for example be derived from M. tuberculosis and/or M. tu- berculosis culture filtrate. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. tuber- culosis antigen or be heterologous and such sequences may, but need not, be immuno- genic.

Each polypeptide is encoded by a specific nucleic acid sequence. It will be understood that such sequences include analogues and variants hereof wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acid. Substitutions are preferably silent substitutions in the codon usage which will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein.

In the present context the term"substantially pure polypeptide fragment"means a poly- peptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e. g. at most 4%, at most 3%, at most 2%, at most 1%, and at most % %). It is preferred that the substantially pure polypeptide is at least 96% pure, i. e. that the poly- peptide constitutes at least 96% by weight of total polypeptide material present in the pre- paration, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99, 25%, at least 99, 5%, and at least 99, 75%. It is especially preferred that the polypeptide fragment is in"essentially pure form", i. e. that the polypeptide frag- ment is essentially free of any other antigen with which it is natively associated, i. e. free of

any other antigen from bacteria belonging to the tuberculosis complex or a virulent myco- bacterium. This can be accomplished by preparing the polypeptide fragment by means of recombinant methods in a non-mycobacterial host cell as will be described in detail below, or by synthesizing the polypeptide fragment by the well-known methods of solid or liquid phase peptide synthesis, e. g. by the method described by Merrifield or variations thereof.

By the term"virulent mycobacterium"is understood a bacterium capable of causing the tuberculosis disease in an animal or in a human being. Examples of virulentmycobacteria are M. tuberculosis, M. africanum, and M. bovis. Examples of relevant animals are cattle, possums, badgers and kangaroos.

By"a TB patient"is understood an individual with culture or microscopically proven infec- tion with virulent mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known by any person skilled in the art.

By the term"PPD-positive individual"is understood an individual with a positive Mantoux test or an individual where PPD induces a positive in vitro recall response determined by release of FN-y.

By the term"delayed type hypersensitivity reaction" (DTH) is understood a T-cell medi- ated inflammatory response elicited after the injection of a polypeptide into, or application to, the skin, said inflammatory response appearing 72-96 hours after the polypeptide in- jection or application.

By the term"IFN-y"is understood interferon-gamma. The measurement of IFN-y is used as an indication of an immunological response.

By the terms"nucleic acid fragment"and"nucleic acid sequence"are understood any nu- cleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length, e. g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e. g. in DNA therapy, or for use in a method for producing a polype- tide according to the invention, a molecule encoding at least one epitope is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being op-

tionally inserted into a vector. When the nucleic acid molecule is used as a probe, as a primer or in antisense therapy, a molecule having a length of 10-100 is preferably used.

According to the invention, other molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nu- cleotides (or nucleotide derivatives). It should be understood that these numbers can be freely combined to produce ranges.

The term"stringent"when used in conjunction with hybridization conditions is as defined in the art, i. e. the hybridization is performed at a temperature not more than 15-20°C un- der the melting point Tm, cf. Sambrook et al, 1989, pages 11. 45-11. 49. Preferably, the conditions are"highly stringent", i. e. 5-10°C under the melting point Tm.

Throughout this specification, unless the context requires otherwise, the word"comprise", or variations thereof such as"comprises"or"comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclu- sion of any other element or integer or group of elements or integers.

The term"sequence identity"indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to best possible fit possible with the insertion of gaps or alternatively truncation at the ends of the protein sequences. The sequence identity can be calculated as <BR> <BR> <BR> <BR> ', wherein Nd, f is the total number of non-identical residues in the two sequences<BR> <BR> <BR> Nif when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndj-2 and Nre=8). A gap is counted as non-identity of the specific residue (s), i. e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA se- quence AGTCAGTC (Ndj=2 and Nre=8). Sequence identity can alternatively be calculated by the BLAST program e. g. the BLASTP program (Pearson W. R. and D. J. Lipman (1988)) (www. ncbi. nlm. nih. gov/cgi-bin/BLAST). In one aspect of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as de- scribed by Thompson J., et a/1994, available at http ://www2. ebi. ac. uk/clustalw/.

A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99. 5%.

In a preferred embodiment of the invention, the polypeptide comprises an immunogenic portion of the polypeptide, such as an epitope for a B-cell or T-cell. The immunogenic por- tion of a polypeptide is a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biologi- cal assays described herein. The immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope. Immunogenic portions can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide. For a few polypeptides epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full se- quence (Ravn et al 1999).

In order to identify relevant T-cell epitopes which are recognised during an immune re- sponse, it is possible to use a"brute force"method : Since T-cell epitopes are linear, dele- tion mutants of the polypeptide will, if constructed systematically, reveal what regions of the polypeptide are essential in immune recognition, e. g. by subjecting these deletion mutants e. g. to the IFN-y assay described herein. Another method utilises overlapping oli- gopeptides for the detection of MHC class 11 epitopes, preferably synthetic, having a length of e. g. 20 amino acid residues derived from the polypeptide. These peptides can be tested in biological assays (e. g. the IFN-y assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide. For the detection of MHC class I epitopes it is possible to predict peptides that will bind (Stryhn et al. 1996) and hereafter produce these peptides synthetic and test them in relevant biological assays e. g. the IFN-y assay as de- scribed herein. The peptides preferably having a length of e. g. 8 to 11 amino acid resi- dues derived from the polypeptide. B-cell epitopes can be determined by analysing the B cell recognition to overlapping peptides covering the polypeptide of interest as e. g. de- scribed in Harboe et al 1998.

Although the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids. Hence, it is preferred that the polypeptide fragment of the invention has a length of at

least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues. Hence, in important embodiments of the inventive method, it is preferred that the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues. It should be understood that these numbers can be freely combined to produce ranges.

It is expected that the peptides having a length of between 10 and 20 amino acid residues will prove to be most efficient as MHC class 11 epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acid residues. It is expected that the peptides having a length of between 7 and 12 amino acid residues will prove to be most efficient as MHC class I epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 11, such as 10, 9, 8 and even 7 amino acid residues.

Immunogenic portions of polypeptides may be recognised by a broad part (high fre- quency) or by a minor part (low frequency) of the genetically heterogenic human popula- tion. In addition some immunogenic portions induce high immunological responses (domi- nant), whereas others induce lower, but still significant, responses (subdominant). High frequencyxtow frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type) or even by multiple MHC molecules (Kilgus et al.

1991, Sinigaglia et al 1988).

In the context of providing candidate molecules for a new vaccine against tuberculosis, the subdominat epitopes are however as relevant as are the dominat epitopes since it has been show (Olsen et al 2000) that such epitopes can induce protection regardless of be- ing subdominant.

A common feature of the polypeptides of the invention is their capability to induce an im- munological response as illustrated in the examples. It is understood that a variant of a polypeptide of the invention produced by substitution, insertion, addition or deletion is also immunogenic determined by any of the assays described herein.

An immune individual is defined as a person or an animal, which has cleared or controlled an infection with virulent mycobacteria or has received a vaccination with M. bovis BCG.

An immunogenic polypeptide is defined as a polypeptide that induces an immune re- sponse in a biological sample or an individual currently or previously infected with a viru- lent mycobacterium. The immune response may be monitored by one of the following methods : An in vitro cellular response is determined by release of a relevant cytokine such as IFN-y, from lymphocytes withdrawn from an animal or human being currently or previously infected with virulent mycobacteria, or by detection of proliferation of these T cells. The induction being performed by the addition of the polypeptide or the immunogenic portion to a suspension comprising from 1x105 cells to 3x105 cells per well. The cells being isolated from either the blood, the spleen, the liver or the lung and the addition of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 ug per ml suspension and the stimulation being performed from two to five days. For monitoring cell proliferation the cells are pulsed with radioactive labeled Thymidine and after 16-22 hours of incubation detecting the proliferation by liquid scintillation counting. A positive response be- ing a response more than background plus two standard derivations. The release of IFN-y can be determined by the ELISA method, which is well known to a person skilled in the art. A positive response being a response more than background plus two standard derivations. Other cytokines than IFN-y could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF-a, IL-4, 1L-5, IL-10, IL-6, TGF-ß. Another and more sensitive method for determining the presence of a cytokine (e. g. IFN-y) is the ELISPOT method where the cells isolated from either the blood, the spleen, the liver or the lung are diluted to a con- centration of preferable of 1 to 4 x 106 cells/ml and incubated for 18-22 hrs in the presence of of the polypeptide or the immunogenic portion resulting in a concen- tration of not more than 20 ug per ml. The cell suspensions are hereafter diluted to 1 to 2 x 106/ml and transferred to Maxisorp plates coated with anti-IFN-y and in- cubated for preferably 4 to 16 hours. The IFN-y producing cells are determined by the use of labeled secondary anti-IFN-y antibody and a relevant substrate giving rise to spots, which can be enumerated using a dissection microscope. It is also a possibility to determine the presence of mRNA coding for the relevant cytokine by the use of the PCR technique. Usually one or more cytokines will be measured

utilizing for example the PCR, ELISPOT or ELISA. It will be appreciated by a per- son skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological activity of the polypeptide.

An in vitro cellular response may also be determined by the use of T cell lines de- rived from an immune individual or an M. tuberculosis infected person where the T cell lines have been driven with either live mycobacteria, extracts from the bacte- rial cell or culture filtrate for 10 to 20 days with the addition of IL-2. The induction being performed by addition of not more than 20 polypeptide per ml suspension to the T cell lines containing from 1x105 cells to 3x105 cells per well and incubation being performed from two to six days. The induction of IFN-y or release of another relevant cytokine is detected by ELISA. The stimulation of T cells can also be monitored by detecting cell proliferation using radioactively labeled Thymidine as described above. For both assays a positive response being a response more than background plus two standard derivations.

An in vivo cellular response which may be determined as a positive DTH response after intradermal injection or local application patch of at most 100ig of the poly- peptide or the immunogenic portion to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at least 5 mm 72-96 hours after the injection or application.

An in vitro humoral response is determined by a specific antibody response in an immune or infected individual. The presence of antibodies may be determined by an ELISA technique or a Western blot where the polypeptide or the immunogenic portion is absorbed to either a nitrocellulose membrane or a polystyrene surface.

The serum is preferably diluted in PBS from 1 : 10 to 1 : 100 and added to the ab- sorbed polypeptide and the incubation being performed from 1 to 12 hours. By the use of labeled secondary antibodies the presence of specific antibodies can be determined by measuring the OD e. g. by ELISA where a positive response is a re- sponse of more than background plus two standard derivations or alternatively a visual response in a Western blot.

Another relevant parameter is measurement of the protection in animal models in- duced after vaccination with the polypeptide in an adjuvant or after DNA vaccina- tion. Suitable animal models include primates, guinea pigs or mice, which are challenged with an infection of a virulent Mycobacterium. Readout for induced protection could be decrease of the bacterial load in target organs compared to non-vaccinated animals, prolonged survival times compared to non-vaccinated animals and diminished weight loss compared to non-vaccinated animals.

In general, M. tuberculosis antigens, and DNA sequences encoding such antigens, may be prepared using any one of a variety of procedures. They may be purified as native proteins from the M. tuberculosis cell or culture filtrate by procedures such as those de- scribed above. Immunogenic antigens may also be produced recombinantly using a DNA sequence encoding the antigen, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli. The polypeptides or immunogenic portion hereof can also be produced synthetically having fewer than about 100 amino acids, and generally fewer than 50 amino acids and may be generated using techniques well known to those ordinarily skilled in the art, such as commercially available solid-phase techniques where amino acids are sequentially added to a growing amino acid chain.

In the construction and preparation of plasmid DNA encoding the polypeptide as defined for DNA vaccination a host strain such as E. coli can be used. Plasmid DNA can then be prepared from overnight cultures of the host strain carrying the plasmid of interest, and purified using e. g. the Qiagen Giga-Plasmid column kit (Qiagen, Santa Clarita, CA, USA) including an endotoxin removal step. It is essential that plasmid DNA used for DNA vacci- nation is endotoxin free.

The immunogenic polypeptides may also be produced as fusion proteins, by which meth- ods superior characteristics of the polypeptide of the invention can be achieved. For in- stance, fusion partners that facilitate export of the polypeptide when produced recombi- nantly, fusion partners that facilitate purification of the polypeptide, and fusion partners which enhance the immunogenicity of the polypeptide fragment of the invention are all interesting possibilities. Therefore, the invention also pertains to a fusion polypeptide comprising at least one polypeptide or immunogenic portion defined above and at least one fusion partner. The fusion partner can, in order to enhance immunogenicity, be an-

other polypeptide derived from M. tuberculosis, such as of a polypeptide fragment derived from a bacterium belonging to the tuberculosis complex, such as ESAT-6, TB10. 4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59,, Ag85C, 19kDa lipoprotein, MPT32 and alpha-crystallin, or at least one T-cell epitope of any of the above mentioned antigens ( (Skjot et al 2000 ; Danish Patent applica- tion PA 2000 00666 ; Danish Patent application PA 1999 01020 ; US patent application 09/0505, 739 ; Rosenkrands et al 1998 ; Nagai et al 1991). The invention also pertains to a fusion polypeptide comprising mutual fusions of two or more of the polypeptides (or im- munogenic portions thereof) of the invention.

Other fusion partners, which could enhance the immunogenicity of the product, are lym- phokines such as IFN-y, IL-2 and IL-12. In order to facilitate expression and/or purification, the fusion partner can e. g. be a bacterial fimbrial protein, e. g. the pilus components pilin and papA ; protein A ; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden) ; the maltose binding protein ; gluthatione S-transferase ; ß-galactosidase ; or poly-histidine.

Fusion proteins can be produced recombinantly in a host cell, which could be E. coli, and it is a possibility to induce a linker region between the different fusion partners.

Other interesting fusion partners are polypeptides, which are lipidated so that the immu- nogenic polypeptide is presented in a suitable manner to the immune system. This effect is e. g. known from vaccines based on the Borrelia burgdorferi OspA polypeptide as de- scribed in e. g. WO 96/40718 A or vaccines based on the Pseudomonas aeruginosa Oprl lipoprotein (Cote-Sierra J 1998). Another possibility is N-terminal fusion of a known signal sequence and an N-terminal cystein to the immunogenic polypeptide. Such a fusion re- sults in lipidation of the immunogenic polypeptide at the N-terminal cystein, when pro- duced in a suitable production host.

Another part of the invention pertains to a vaccine composition comprising a polypeptide (or at least one immunogenic portion thereof) or fusion polypeptide according to the in- vention. In order to ensure optimum performance of such a vaccine composition itis pre- ferred that it comprises an immunologically and pharmaceutical acceptable carrier, vehi- cle or adjuvant.

An effective vaccine, wherein a polypeptide of the invention is recognized by the animal, will in an animal model be able to decrease bacterial load in target organs, prolong sur-

vival times and/or diminish weight loss after challenge with a virulent Mycobacterium, compared to non-vaccinated animals.

Suitable carriers are selected from the group consisting of a polymer to which the poly- peptide (s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e. g. polystyrene, or a polymer to which the polypeptide (s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e. g. bovine serum albumin, albumin or keyhole limpet haemocyanin. Suitable vehicles are selected from the group consisting of a diluent and a suspending agent. The adjuvant is preferably selected from the group consisting of di- methyldioctadecylammonium bromide (DDA), Quil A, poly I : C, aluminium hydroxide, Freund's incomplete adjuvant, IFN-y, IL-2, IL-12, monophosphoryl lipid A (MPL), Tre- holose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP).

Preparation of vaccines which contain peptide sequences as active ingredients is gen- erally well understood in the art, as exemplified by U. S. Patents 4, 608, 251 ; 4, 601, 903 ; 4, 599, 231 and 4, 599, 230, all incorporated herein by reference.

Other methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), ag- gregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emul- sion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Other possibilities involve the use of immune modulating sub- stances such as cytokines or synthetic IFN-y inducers such as poly l : C in combination with the above-mentioned adjuvants.

Another interesting possibility for achieving adjuvant effect is to employ the technique de- scribed in Gosselin et al., 1992 (which is hereby incorporated by reference herein). In brief, a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fcy receptors on mono- cytes/macrophages.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutical effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e. g., the capacity of the in- dividual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0. 1 ug to 1000 p. g, such as in the range from about 1 pg to 300 wog, and especially in the range from about 101l9 to 50 9. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other admini- strations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated.

The vaccines are conventionally administered parenterally, by injection, for example, ei- ther subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral for- mulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides ; such suppositories may be formed from mixtures containing the active ingredient in the range of 0. 5% to 10%, preferably 1-2%. Oral for- mulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, sus- pensions, tablets, pills, capsules, sustained release formulations or powders and advan- tageously contain 10-95% of active ingredient, preferably 25-70%.

In many instances, it will be necessary to have multiple administrations of the vaccine.

Especially, vaccines can be administered to prevent an infection with virulent mycobacte- ria and/or to treat established mycobacterial infection. When administered to prevent an infection, the vaccine is given prophylactically, before definitive clinical signs or symptoms of an infection are present.

Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response. The vaccine may comprise two or more polypeptides or immunogenic portions, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from virulent mycobacteria. In the latter example, the polypeptides not necessarily fulfilling the criteria set forth above for polypeptides may either act due to their own immunogenic- ity or merely act as adjuvants.

The vaccine may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fu- sion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.

The invention also pertains to a method for immunising an animal, including a human be- ing, against TB caused by virulent mycobacteria, comprising administering to the animal the polypeptide of the invention, or a vaccine composition of the invention as described above, or a living vaccine described above.

The invention also pertains to a method for producing an immunologic composition accor- ding to the invention, the method comprising preparing, synthesising or isolating a poly- peptide according to the invention, and solubilizing or dispersing the polypeptide in a me- dium for a vaccine, and optionally adding other M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.

The nucleic acid fragments of the invention may be used for effecting in vivo expression of antigens, i. e. the nucleic acid fragments may be used in so-called DNA vaccines as re- viewed in Ulmer et al 1993, which is included by reference.

Hence, the invention also relates to a vaccine comprising a nucleic acid fragment ac- cording to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of ex- pressed antigen being effective to confer substantially increased resistance to infections caused by virulent mycobacteria in an animal, including a human being.

The efficacy of such a DNA vaccine can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide which has the capability of modulating an immune response.

One possibility for effectively activating a cellular immune response for a vaccine can be achieved by expressing the relevant antigen in a vaccine in a non-pathogenic microor- ganism or virus. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomona and examples of viruses are Vaccinia Virus and Ad- enovirus.

Therefore, another important aspect of the present invention is an improvement of the liv- ing BCG vaccine presently available, wherein one or more copies of a DNA sequence en- coding one or more polypeptide as defined above has been incorporated into the genome of the micro-organism in a manner allowing the micro-organism to express and secrete the polypeptide. The incorporation of more than one copy of a nucleotide sequence of the invention is contemplated to enhance the immune response Another possibility is to integrate the DNA encoding the polypeptide according to the in- vention in an attenuated virus such as the vaccinia virus or Adenovirus (Rolph et al 1997).

The recombinant vaccinia virus is able to replicate within the cytoplasma of the infected host cell and the polypeptide of interest can therefore induce an immune response, which is envisioned to induce protection against TB.

The invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described in the literature exemplified by D.

Lowry (Lowry et al 1999). Antigens with therapeutic properties may be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals or prevent reactivation of previous infection, when administered as a vaccine. The composi- tion used for therapeutic vaccines can be prepared as described above for vaccines.

The invention also relates to a method of diagnosing TB caused by a virulent mycobacte- rium in an animal, including a human being, comprising intradermally injecting, in the ani- mal, a polypeptide according to the invention, a positive skin response at the location of injection being indicative of the animal having TB, and a negative skin response at the lo- cation of injection being indicative of the animal not having TB.

When diagnosis of previous or ongoing infection with virulent mycobacteria is the aim, a blood sample comprising mononuclear cells (i. e. T-lymphocytes) from a patient could be contacted with a sample of one or more polypeptides of the invention. This contacting can be performed in vitro and a positive reaction could e. g. be proliferation of the T-cells or release of cytokines such as IFN-y into the extracellular phase. It is also conceivable to contact a serum sample from a subject with a polypeptide of the invention, the demonstra- tion of a binding between antibodies in the serum sample and the polypeptide being in- dicative of previous or ongoing infection.

The invention therefore also relates to an in vitro method for diagnosing ongoing or previ- ous sensitisation in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and con- tacting the sample from the animal with the polypeptide of the invention, a significant re- lease into the extracellular phase of at least one cytokine by mononuclear cells in the blood sample being indicative of the animal being sensitised. A positive response being a response more than release from a blood sample derived from a patient without the TB diagnosis plus two standard derivations. The invention also relates to the in vitro method for diagnosing ongoing or previous sensitisation in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and by contacting the sample from the animal with the polypeptide of the invention demonstrating the presence of antibodies recognizing the polypeptide of the in- vention in the serum sample.

The immunogenic composition used for diagnosing may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different poly- peptides or fusion polypeptides.

The nucleic acid probes encoding the polypeptide of the invention can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sam- ple. A method of determining the presence of mycobacterial nucleic acids in an animal, including a human being, or in a sample, comprising administering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary thereto, and detecting the pres- ence of hybridised nucleic acids resulting from the incubation (by using the hybridisation

assays which are well-known in the art), is also included in the invention. Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide se- quences in a sample from the animal or human being to be tested which hybridise with the nucleic acid fragment (or a complementary fragment) by the use of PCR technique.

A monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide of the invention in an immuno assay, or a specific binding fragment of said antibody, is also a part of the invention. The antibodies can be produced by methods known to the person skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an ad- juvant. The monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein (1975), or may be produced by recombinant DNA methods such as described in U. S. Pat. No.

4, 816, 567. The monoclonal antibodies may also be isolated from phage libraries gener- ated using the techniques described by McCafferty et al (1990), for example. Methods for producing antibodies are described in the literature, e. g. in US 6, 136, 958.

A sample of a potentially infected organ may be contacted with such an antibody recog- nizing a polypeptide of the invention. The demonstration of the reaction by means of methods well known in the art between the sample and the antibody will be indicative of an ongoing infection. It is of course also a possibility to demonstrate the presence of anti- mycobacterial antibodies in serum by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualising the reaction between the antibody and antigen.

In diagnostics, an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition. Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid frag- ment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e. g. a label (e. g. radioactive or fluorescent) or a carrier molecule.

Concordance list Protein SEQ ID NO : DNA SEQ ID NO : Synonyms Rv0284 2 1 Rv0284ct 4 3 Rv0285 6 5 Rv0455c 8 7 TB13. 7 Rv0569 10 9 TB9. 5 Rv1195 12 11 Rv1386 14 13 Rv3477 16 15 Rv3878 18 17 ORF13A 20 19 Rv3879c 22 21 Rv0285-P1 23 Rv0285-P2 24 Rv0285-P3 25 Rv0285-P4 26 Rv0285-P5 27 Rv0285-P6 28 Rv0285-P7 29 Rv0285-P8 30 Rv0285-P9 31 Rv0285-P10 32 <BR> <BR> <BR> <BR> Rv1386-P1 33<BR> <BR> <BR> <BR> <BR> Rv1386-P2 34 Rv1386-P3 35 Rv1386-P4 36 Rv1386-P5 37 Rv1386-P6 38 Rv1386-P7 39 Rv1386-P8 40 Rv1386-P9 41 Rv1386-P10 42 TB9. 5-1 43 TB9. 5-2 44 TB9. 5-3 45 TB9. 5-4 46 TB13. 7-1 47 TB13. 7-2 48 TB13. 7-3 49 TB13. 7-4 50 TB13. 7-5 51 MT3106. 1 53 52 <BR> <BR> <BR> <BR> Rv0284-P1 54<BR> <BR> <BR> <BR> Rv0284-P2 55 Rv0284-P3 56 Rv0284-P4 57 <BR> <BR> <BR> <BR> Rv0284-P5 58<BR> <BR> <BR> <BR> Rv0284-P6 59 Rv0284-P7 60 Rv0284-P8 61 Rv0284-P9 62 Rv0284-P10 63 Rv0284-P11 64 Rv0284-P12 65 Rv0284-P13 66 Rv0284-P14 67 Rv0284-P15 68 Rv0284-P16 69 Rv0284-P17 70 Rv0284-P18 71 Rv0284-P19 72 Rv0284-P20 73 Rv0284-P21 74 Rv0284-P22 75 Rv0284-P23 76 Rv0284-P24 77 Rv0284-P25 78 Rv0284-P26 79 Rv0284-P27 80 Rv0284-P28 81 Rv0284-P29 82 Rv0284-P30 83 Rv0284-P31 84 Rv0284-P32 85 Rv0284-P33 86 <BR> <BR> <BR> <BR> Rv0284-P34 87<BR> <BR> <BR> <BR> <BR> Rv0284-P35 88<BR> <BR> <BR> <BR> Rv0284-P36 89<BR> <BR> <BR> <BR> <BR> Rv0284-P37 90<BR> <BR> <BR> <BR> Rv0284-P38 91 Rv0284-P39 92 Rv0284-P40 93 <BR> <BR> <BR> <BR> <BR> Rv0284-P41 94 Rv0284-P42 95 Rv0284-P43 96 <BR> <BR> <BR> <BR> <BR> Rv0284-P44 97<BR> <BR> <BR> <BR> <BR> <BR> Rv0284-P45 98 Rv0284-P46 99 Rv0284-P47 100 Rv0284-P48 101 Rv0284-P49 102 Rv0284-P50 103 Rv0284-P51 104 Rv0284-P52 105 <BR> <BR> <BR> <BR> <BR> Rv0284-P53 106 Rv0284-P54 107 <BR> <BR> <BR> <BR> <BR> Rv0284-P55 108 Rv0284-P56 109 <BR> <BR> <BR> <BR> <BR> Rv0284-P57 110<BR> <BR> <BR> <BR> <BR> <BR> Rv0284-P58 111<BR> <BR> <BR> <BR> <BR> <BR> <BR> Rv0284-P59 112 Rv0284-P60 113 Rv0284-P61 114 Rv0284-P62 115 Rv0284-P63 116 Rv0284-P64 117 Rv0284-P65 118 Rv0284-P66 119 Rv0284-P67 120 Rv0284-P68 121 Rv0284-P69 122 Rv3878-P1 123 Rv3878-P2 124 Rv3878-P3 125 Rv3878-P4 126 Rv3878-P5 127 Rv3878-P6 128 Rv3878-P7 129 Rv3878-P8 130 Rv3878-P9 131 <BR> <BR> <BR> <BR> Rv3878-P10 132<BR> <BR> <BR> <BR> <BR> <BR> Rv3878-P11 133 Rv3878-P12 134 <BR> <BR> <BR> <BR> Rv3878-P13 135<BR> <BR> <BR> <BR> Rv3878-P14 136<BR> <BR> <BR> <BR> Rv3878-P15 137 Rv3878-P16 138 Rv3878-P17 139 Rv3878-P18 140 Rv3878-P19 141 Rv3878-P20 142 Rv3878-P21 143 Rv3878-P22 144 Rv3878-P23 145 MT3106. 1-pl 146 MT3106. 1-p2 147 MT3106. 1-p3 148 MT3106. 1-p4 149 MT3106. 1-p5 150 MT3106. 1-p6 151 MT3106. 1-p7 152 MT3106. 1-p8 153 MT3106. 1-p9 154 MT3106. 1-p10 155 MT3106. 1-p11 156 <BR> <BR> <BR> Rv0284-F 157<BR> <BR> <BR> <BR> Rv0284-R 158<BR> <BR> <BR> <BR> Rv0285-F 159 Rv0285-R 160 Rv3878-F 161 Rv3878-R 162 ORF13A-F 163 ORF13A-R 164 <BR> <BR> <BR> <BR> Rv1195-F 165 Rv1195-R 166 <BR> <BR> <BR> <BR> Rv1386-F 167<BR> <BR> <BR> <BR> Rv1386-R 168 Rv3477-F 169 <BR> <BR> <BR> <BR> Rv3477-R 170 TB9. 5 15AA from 171 sequencing TB13. 7 15AA from 172 sequencing

Legends to figures Figure 1 : Stimulation of IFN-y production by synthetic peptides in PBMC from PPD posi- tive healthy donors. Single peptides were tested at concentrations of 10pg, 5 g and 2. 5 pLg/ml in 200 il of cell culture. Pools of peptides were tested at 1 g, 0. 5 p9 and 0. 25 pg/ml of each peptide. Results are presented as pg IFN-y/ml of the maximum stimulation.

Recombinant antigens were included for comparison.

Figure 2A : The antibody response of 48 TB patients to ORF13A evaluated by ELISA. The OD indicated is the mean of two wells coated with 1 ug/ml ORF13A and the serum is di- luted 1 : 100 in PBS.

Figure 2B : The antibody response of 15 BCG vaccinated healthy donors to ORF13A evaluated by ELISA. The OD indicated is the mean of two wells coated with 1 gg/ml ORF13A and the serum is diluted 1 : 100 in PBS.

Figure 2C : The antibody response of 19 non BCG-vaccinated healthy donors to ORF13A evaluated by ELISA. The OD indicated is the mean of two wells coated with 1 pg/ml ORF13A and the serum is diluted 1 : 100 in PBS.

Figure 3 : Stimulation of T-cell proliferation by synthetic peptides derived from Rv3878. T- cell lines against STCF were derived from PBMC isolated from PPD positive donors.

Peptides were tested at 10 ug and 5 pg/ml. Results are presented as cpm of the maxi- mum stimulation. n. d = not determined.

Examples Example 1 : Cloning and expression of Rv0284, Rv0285, Rv3878, Rv1195, Rv1386, Rv3477 and ORF13A The coding region of Rv0285, Rv3878, the 3'-part (380 bp) of Rv0284 and 5'-part of ORF13A (543 bp of Rv3879c) were amplified by PCR using following primer sets : Rv0284-F : CTG AGA TCT CAG GTA CCG GAT TCG CCG Bgl I I Rv0284-R : CTC CCA TGG TCA TGA CTG ACT CCC CTT NcoI

Rv0285-F : CTG AGA TCT ATG ACG TTG CGA GTG GTT BglII Rv0285-R : CTC CCA TGG TCA GCC GCC CAC GAC CCC NcoI Rv3878-F : CTG AGA TCT GCT ACT GTT AAC AGA TCG BglII Rv3878-R : CCG CTC GAG CTA CAA CGT TGT GGT TGT XhoI ORF13A-F : CCC AAG CTT ATG AGT ATT ACC AGG CCG HindIII ORF13A-R : CTC CCA TGG TCA CGA CTT CTG CTG AAG CAA Nicol PCR reactions contained 10 ng of M. tuberculosis H37Rv DNA in 1x low salt Taq+ buffer from Stratagene supplemented with 250 uM of each of the four nucleotides (Boehringer Mannheim), 0. 5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each primer and 0. 5 unit Taq+ DNA polymerase (Stratagene) in 10 jli reaction volume. Reac- tions were initially heated to 94°C for 15 sec, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec, and finally by 72°C for 5 min.

The PCR fragments were cloned into the TA cloning vector pCR2. 1 (Invitrogen) and then transferred to the pMCT3 expression vector at the restriction sites indicated by the prim- ers above. The coding regions of Rv1195, Rv1386 and Rv3477 were amplified by PCR using the following primer sets : Rv1195-F : gggg ACA AgT TTg TAc AAA AAA gCA ggC TTA gTgTCTTTCgTgATggCATACC Rv1195-R : gggg AC CAC TTT gTA CAA gAA AgC Tgg gTC CTA TTAgCTggCCgCCgC Rv1386-F : gggg ACA AgT TTg TAc AAA AAA gCA ggC TTA gTgACgTTgCgAgTCgTTCC Rv1386-R : gggg AC CAC TTT gTA CAA gAA AgC Tgg gTC CTA TAgCCCACCgCTgAgATACg Rv3477-F : gggg ACA AgT TTg TAc AAA AAA gCA ggC TTA gTgTCTTTCACTgCgCAACCg Rv3477-R : gggg AC CAC TTT gTA CAA gAA AgC Tgg gTC CTA gCCggTgACCACAgCgTT

PCR reactions were carried out by Platinums Tag DNA Polymerase (GIBCOBRL@) in 50gui reaction volume containing 60 mM Tris-S04 (pH 8. 9), 18 mM Ammonium Sulfate, 0. 2 mM of each of the four nucleotides, 0. 2pM of each primer and 10 ng of M. tuberculosis H37Rv DNA. The reaction mixtures were initially heated to 95°C for 5 min, followed by 35 cycles of 95°C for 45 sec, 60°C for 45 sec and 72°C for 2 min, and finally by 72°C for 15 min.

The PCR products were precipitated by PEG/MgCl2, and then dissolved in 50 PLI of TE buffer. DNA fragments were then cloned and expressed in Gateway Cloning system (GIBCOBRL@). First, to create Entry Clones, 5 jli of each DNA fragment was mixed with 1 111 of pDONR201, 2 gl of BP CLONASE Enzyme Mix and 2 pl of BP Reaction Buffer. The recombination reactions were carried out at 25°C for 60 min. After degrading the Enzymes by Proteinase K at 37°C for 10 min, 5 jli of each sample was used to transform E. coli DH5a competent cells. The transformants were selected on LB plates containing 50 ug/ml kanamycin. Second, to create Expression clones, 2 jli of each Entry Clone DNA was mixed with 1 gl of the expression vector, pDest17, 2 jli LR reaction buffer and 2p1 LR CLONASE Enzyme Mix in a total volume of 10 joli. After the recombination reaction at 25°C for 60 min and proteinase K treatment at 37°C for 10 min, 5 ul of the samples were used to transform E. coli BL21-SI competent cells. The transformants were selected on LBON (LB without NaCI) plates containing 100 pg/ml ampicillin. The resulting recombinant antigens carried 6-histine residues at the N-terminal. All clones were confirmed by DNA sequencing.

To express his-tagged recombinant antigens in pMCT3 vector, 100 ml of an overnight culture of XL-1 blue carrying the plasmid construct was added to 900 ml of LB-media containing 100 zg/ml ampicillin, grown at 37°C with shaking. 1 mM IPTG was added at OD600 =0. 4-0. 6 and the culture was incubated for additional 3-16 hours before harvesting of cells.

To express his-tagged recombinant antigens in pDest17, BL21-SI cells were cultured in LBON medium at 30°C and the induction of recombinant antigen synthesis was achieved by adding 0. 3 M NaCI to the medium at OD600 =0. 4-0. 6, and cells were harvested 3 hours later.

For purification, the cell pellet was resuspended in 20 ml of Sonication buffer (20 mM Tris- Cl, pH 8. 0, 0. 5 M NaCI, 10% Glycerol, 5 mM 3-ME, 0. 01% Tween 20 and 1 mM imida-

zole). Cells were lysed and DNA was digested by treating with lysozyme (0. 1 mg/ml) and DNase I (2. 5 pLg/ml) at room temperature for 20 min with gentle agitation. The recombi- nant protein was bring to solution by adding 80 ml of Sonication Buffer containing 8 M urea and sonicated the sample 5 x 30 sec, with 30 sec pausing between the pulses.

After centrifugation, the lysate was applied to a 5 ml TALON column (Clonetech). The col- umn was then washed with 25 ml of urea containing Sonication buffer, and the bound protein was eluted by imidazole steps (5, 10, 20, 40 and 100 mM) in the same buffer. The fractions were analyzed by silver stained SDS-PAGE, and recombinant protein containing fractions were pooled. Further purifications were achieved either by anion-and cation- exchange chromatography on Hitrap columns (Pharmacia, Uppsala, Sweden) or by elec- troelution as described below : The pooled TALON fractions were dialyzed against 3 x 1 L of 10 mM Tris-CI (pH 8. 0), 0. 15 M NaCI and 0. 1% SDS. Two mg of TALON purified re- combinant antigen was subjected to SDS-PAGE on a 16 x 16 cm gel. After separation, the recombinant antigen band was cut out and the protein was eluted by a Model 422 Electro-Eluter (Bio-Rad). SDS was removed from eluted protein by Chloroform/Methanol extraction.

Example 2 : Biological activity of the recombinant antigens.

The purified recombinant proteins were screened for the ability to induce a T cell re- sponse measured as IFN-y release and/or cell proliferation. A preliminary screening in- volved testing of the IFN-y induction and/or cell proliferation of T cell lines generated from PPD positive donors. This test was followed by measuring the response in PBMC prepa- rations obtained from TB patients, PPD positive as well as negative healthy donors.

Interferon-y induction and cell proliferation of T cell lines : Human donors : PBMC were obtained from healthy donors with a positive in vitro re- sponse to PPD.

T cell line preparation : T cell lines were prepared by culturing 5 x 106 freshly isolated PBMC/ml with viable M. tuberculosis at a ratio of 5 bacteria per macrophage in a total vol- ume of 1 ml. The cells were cultured in RPMI 1640 medium (Gibco, Grand Island, N. Y) supplemented with HEPES, and 10% heat-inactivated NHS. After 7 days in culture at 37°C and 5% C02, T cells were supplemented with 50 U/ml of r-IL-2 (Boehringer Mann- heim) for approximately 7 days. Finally, in one experiment (Table 1), the T cell lines were

tested for reactivity against the recombinant antigens by stimulating 1-5 x 105 cells/ml with 5 tg/ml of PPD, 3 ig/mi of rRv0284ct (C-terminal part), 5 pg/ml of rRv0285, and 2. 5 ug/ml of rRv3878 in the presence of 5 x 105 autologous antigen-presenting cells/ml. In another experiment (Table 1 a), T cells were stimulated with 5 ug/ml and 1 lg/ml of each recombi- nant antigen indicated in the table. No ag and PHA were used as negative and positive controls, respectively. The supernatants were harvested after 4 days of culture and stored at-80°C until the presence of IFN-y were analysed.

Cytokine analysis : Interferon-y (IFN-y) was detected with a standard sandwich ELISA technique using a commercially available pair of monoclonal antibodies (Endogen, MA, US) and used according to the manufacturer's instructions. Recombinant IFN-y (Endogen, MA, US) was used as a standard. All data are means of duplicate wells and the variation between the wells did not exceed 10 % of the mean. Responses obtained with five T cell lines are shown in Table 1 and Table 1 a.

T-cell proliferation assays : After removal of supernatant for IFN-γ assays, 0. 5pCi of [methyl-3H] thymidine were added to the same wells supplemented with 10% NHS in RPMI for another 16-20 hours. The cells were thereafter harvested with a Skatron cell harvester onto filter mats, dried, and immersed in scintillation fluid before reading the in- corporation of thymidine on a beta liquid scintillation counter (Wallac). Results from 3 T cell lines are shown in Table 1 b.

As shown in Table 1, high levels of IFN-y release are observed after stimulation with the recombinant antigens ranging from 33% (rRv0284ct) to 83% (rRv3878) of the response seen after stimulation with PPD. The antigenicity of the recombinant antigens was con- firmed by three additional T-cell lines as shown in Table 1a and Table 1b.

Table 1. Stimulation of two T cell lines with recombinant rRv0284ct, rRv0285, and rRv3878. Responses to PHA and PPD are shown for comparison. Results are presented as pg IFN-y/ml.

T cell line Donor No ag PHA PPD rRv0284ct rRv0285 rRv3878 (1 ug/ml) (5 lg/ml) (3 Rg/m 1) (5 pg/ml) (2. 5 µg/ml) 1 50 2975 2742 914 2019 1072 2 50 1482 803 352 548 667

Table 1a. Stimulation of three T cell lines with rRv0285 and rRv3878. Responses to PHA and PPD are shown for comparison. Results are presented as pg IFN-y/ml of the maxi- mum stimulation in the presence of either 5 llg/ml or 1 pg/ml of recombinant antigens.

T cell line Donor No ag PHA PPD rRv0285 rRv3878 g/ml) (5 pg/ml) 3 136 4467 2425 1189 504 4 2 1996 1175 626 413 5 4 5410 4490 2804 2034 Table 1 b. Stimulation of T cell proliferation by rRv0285 and rRv3878. Results are pre- sented as Stimulation Index (SI). The maximum stimulation in the presence of either 5 llg/ml or 1 llg/ml of recombinant antigens is given. Donor rRv0285 rRv3878 38.4 N.D 4 5. 8 4. 3 31. 3 16. 1 Interferonry release from PBMC isolated from human TB patients and PPD positive and negative healthy donors Human donors : PBMC were obtained from healthy donors with a positive in vitro re- sponse to purified protein derivative (PPD) or non-vaccinated healthy donors with a nega- tive in vitro response to PPD. PBMC were also obtained from TB patients with microscopy or culture proven infection. Blood samples were drawn from TB patients 0-6 months after diagnosis.

Lymphocyte preparations and cell culture : PBMC were freshly isolated by gradient centrifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway) and stored in liquid nitrogen until use. The cells were resuspended in complete RPMI 1640 medium (Gibco BRL, Life Technologies) supplemented with 1% penicillin/streptomycin (Gibco BRL, Life Technologies), 1% non-essential-amino acids (FLOW, ICN Biomedicals, CA, USA), and 10% heat-inactivated normal human AB serum (NHS). The viability and num- ber of the cells were determined by Nigrosin staining. Cell cultures were established with 1. 25 x 105 PBMCs in 100 lli in microtitre plates (Nunc, Roskilde, Denmark) and stimulated with 5 Fg/ml PPD or rRv0284ct and rRv3878 in a final concentration of 2. 5 and 5 pg/ml, respectively ; or with 2. 5 and 10 pg/ml of rRv0285, Rv1195, rRv1386 and Rv3477. No an- tigen (No ag) was used as a negative control, whereas phytohaemagglutinin (PHA) was

used as a positive control. Moreover, the response to a well-known TB-specific protein, ESAT-6, was included for comparison. Supernatants for the analysis of secreted cytoki- nes were harvested after 5 days of culture, pooled, and stored at-80 °C until use.

Cytokine analysis : IFN-y was detected as above. Responses obtained with PBMCs from 14 individual donors are shown in Table 2.

As shown in Table 2, stimulation of PBMC from TB patients as well as PPD positive do- nors with rRv0284ct and rRv3878 resulted in a marked release of IFN-y with 55% of the donors recognizing the recombinant antigens at a level of more than 500 pg/ml. As ex- pected, none of the recombinant antigens gave rise to FN-y release in PPD negative do- nors. The effects of stimulating with rRv0285, rRv1386, rRv1195 and rRv3477 on IFN-y release in PBMC are demonstrated in Table 2a.

Table 2. Stimulation of PBMCs from 4 TB patients, 7 PPD positive healthy donors, and 3 PPD negative healthy donors with recombinant antigen. Responses to PHA, PPD, and ESAT6 are shown for comparison. Results are given as pg IFN-γ/ml.

TB patients Donor No ag PHA PPD ESAT-6 rRv0284ct rRv3878 (1 µg/ml) (5 rg/ml) (5 µg/ml) (2. 5 ug/ml) (5 zg/ml) 1 3 4541 4074 2154 809 3 2 92 3408 4891 611 236 2029 3 5 5282 4647 2827 308 149 4 10 4531 2077 38 140 287 PPD positive healthy donors Donor No ag PHA PPD ESAT-6 rRv0284ct rRv3878 (1 µg/ml) (5 µg/ml) (5 zg/ml) (2. 5 µg/ml) (5 µg/ml) 1 74 5413 3339 0 382 77 2 14 5614 3852 198 1324 633 3 7 6165 5808 4 2951 2732 4 63 6532 6314 1567 3009 3482 5 43 4733 6195 1272 5166 2589 6 5 3809 2582 15 5 71 7 31 6716 2275 424 1449 832 PPD negative healthy donors Donor No ag PHA PPD ESAT-6 rRv0284ct rRV3878 (1 pg/ml) (5 Fg/ml) (5 µg/ml) (2. 5 µg/ml) (5 pg/ml) 1 0 3354 113 0 269 17 2 0 3803 563 0 22 0 3 0 3446 525 10 203 34 Table 2a. Stimulation of IFN-γ production by rRv0285, rRv1386, rRv1195 and rRv3477 in PBMC from PPD negative controls, PPD positive healthy donors as well as TB patients.

TB10. 4 was included for comparison. Donor No ag PPD Rv0285 Rv1386 Rv1195 Rv3477 TB10.

PPD negative healthy donors' K150 12 265 0 5 2 3 K151 22 50 0 nd nd nd 1 K156 17 522 0 166 86 71 K159 27 155 1 16 12 19 K160 16 242 6 62 9 26 K161 35 510 2 40 23 33 K162 31 352 89 71 nd 0 B-patients 8-160 5 >5549 nd 2885 nd nd n 9-203 0 2232 914 nd nd nd 90 9-208 2 4098 317 186 nd 11 0-199 11 2592 456 nd nd nd 311 0-211 0 10633 2533 2862 1814 1243 416 0-217 22 4140 124 57 nd 66 53 0-218 0 1578 21 28 nd 13 3 0-220 18 9476 77 106 437 34 306 0-222 28 9824 2226 1071 226 44 360 0-223 89 10412 2458 nd nd nd 453 PPD positive healthy donors K119 0 7464 227 296 nd 111 58 K131 29 1730 1777 20 17 31 K147 86 4520 18 79 47 26 1 K148 52 8293 78 11 86 58 384 K149 72 12730 932 243 nd 489 3 K152 96 6120 0 946 40 517 130 K153 5 12391 2 467 nd 622 70 K155 5 9397 0 9 15 37 97 K167 105 15770 3531 1811 nd nd 4881 k172 10 18811 21420 4 3717 10 11 K174 3 1443 492 44 56 17 16 KTB1 34 13748 3067 1307 nd nd 9431 KTB2 23 8104* 391 nd nd nd 223 KTB10 4 2394* 292 nd nd nd 4 L 46 7832* 949 nd nd nd 34 33 6538 303 3 255 116 IFN-Y median=13294 pg/ml on stimulation with PHA. * FN-y on stimulation with STCF BMC from 6 additional TB patients were obtained, and the T-cell stimulatory effect of rRv1195 was also tested in these PBMCs. The results are shown in Table 2b.

Table 2b Stimulation of IFN-y production by rRv1195 in PBMCs from six TB patients.

Donor No ag PPD Rv1195 97-83 42 >3531 1060 97-138 13 >3366 231 98-149 256 >3449 2855 99-163 45 >2303 422 01-226 68 >3994 2133 PT36 342 1510 411 Together, these analyses using PBMC and T cell lines, respectively, indicate that rRv0284ct, rRv0285, rRv1386 and rRv3878 are highly biologically active and frequently recognized by PPD positive donors and TB patients. Though less frequently recognized by these donors rRv1195 and rRv3477 are additionally highly biologically active.

As is expected, due to the genetical heterogeneity of the human population some of the recombinant antigens are recognized more frequently and to a higher level than others are.

Skin test reaction in TB infected guinea pigs The skin test reactivity of the recombinant antigens was tested in M. tuberculosis infected guinea pigs. A group of 5 female outbred guinea pigs of the Dunkin Hartley strains (Mol- legaard Breeding and Research Center A/S, Lille Skensved, Denmark) were infected by the aerosol route in an exposure chamber of a Glas-ColO Inhalation Exposure System, which was calibrated to deliver approximately 20-25 M. tuberculosis Erdman bacilli into the lungs of each animal. As a control, the skin test reactivity of uninfected guinea pigs was tested. Skin tests were performed 28 days after infection with injection of 5 g of rRv0284ct, rRv0285, and rRv3878. As a positive control, the guinea pigs were sensitised with 10 tuberculin units (TU) of PPD (1TU = 0. 02 p9) whereas injection of phosphate- buffered saline (PBS) was used as a negative control. Skin test responses (diameter of erythema) were read 24 h later by two experienced examinators and the results were ex- pressed as the mean of the two readings. The variation between the two readings was less than 10%. Skin test responses larger than 5 mm were regarded as positive.

As seen in Table 3, injection of rRv3878 induced a marked Delayed Type Hypersensitivity (DTH) reaction at the same level as after injection with PPD. rRv0284ct and rRv0285 re- sulted in a highly significant DTH reaction (P < 0. 005 ; Tukey test). As expected, none of the antigens induced non-specific response in uninfected guinea pigs (Table 4).

Table 3. DTH erythema diameter (shown in mm) in guinea pigs aerosol infected with M. tuberculosis after stimulation with recombinant antigens.

Antigen Skin reaction (mm) D SEM PBS 3. 10 0. 30 PPD 13. 10 1. 18 rRv0284ct 8. 40 0. 45 rRv0285 7. 00 1. 08 rRv3878 14. 56 1. 05 a The recombinant antigens were tested in a concentration of 5 ; ng, whereas 10 TU of PPD were used. b The skin reactions are measured in mm erythema 24 h after intradermal injection. The values are the mean of erythema diameter of five animals and the SEM are indicated. The values for rRv3878 are the mean of four animals.

Table 4. DTH erythema diameter (shown in mm) in non-infected guinea pigs after stimula- tion with recombinant antigens.

Antigen Skin reaction (mm) SEM PBS 2. 60 0. 36 PPD 3. 00 0. 44 rRv0284ct 2. 5 0. 18 rRv0285 3. 45 0. 74 rRv3878 2. 5 0. 18 a The recombinant antigens were tested in a concentration of 5 p9, whereas 10 TU of PPD were used. b The skin reactions are measured in mm erythema 24 h after intradermal injection. The values are the mean of erythema diameter of five animals and the SEM are indicated.

Example 3 : Immunological response to synthetic polypeptides Peptide synthesis : Ten overlapping peptides to Rv0285 and Rv1386 respectively, were synthesized. Synthetic polypeptides were purchased from Mimotopes Pty Ltd. The pep- tides were synthesized by Fmoc solid phase strategy. No purification steps were per- formed. Lyophilised peptides were stored dry until use.

Rv0285 peptides : Rv0285-P1 TLRWPEGLAAASAAVEA Rv0285-P2 ASAAVEALTARLAAAHAS Rv0285-P3 TARLAAAHASAAPVITAV Rv0285-P4 AAPVITAWPPAADPVSL Rv0285-P5 PAADPVSLQTAAGFSAQG Rv0285-P6 AAGFSAQGVEHAVVTAEG Rv0285-P7 HAWTAEGVEELGRAGVG Rv0285-P8 GVEELGRAGVGVGESGAS Rv0285-P9 GVGESGASYLAGDAAAAA Rv0285-P10 SYLAGDAAAAATYGWGG Rv1386 peptides :

Rv1386-P1 TLRWPESLAGASAAIEA<BR> <BR> Rv1386-P2 ASAAIEAVTARLAAAHAA<BR> <BR> <BR> Rv1386-P3 TARLAAAHAAAAPFIAAV<BR> <BR> <BR> Rv1386-P4 AAPFIAAVIPPGSDSVSV Rv1386-P5 PGSDSVSVCNAVEFSVHG <BR> <BR> Rv1386-P6 AVEFSVHGSQHVAMAAQG<BR> <BR> <BR> Rv1386-P7 HVAMAAQGVEELGRSGVG Rv1386-P8 GVEELGRSGVGVAESGAS <BR> <BR> Rv1386-P9 GVAESGASYAARDALAAA<BR> <BR> Rv1386-P10 SYAARDALAAASYLSGGL PBMC culture and IFN-y assay : PBMC were isolated and cultured as described in Ex- ample 2. Single peptides were tested at concentrations of 10 p9, 5g and 2. 5pg/ml in 200 pl of cell culture. Pools of peptides were tested at 1 pg, 0. 5 g and 0. 25 pg/ml of each peptide. IFN-y levels were measured by the method described in Example 2.

PBMC recognition of peptides from Rv0285 and Rv1386 The ability of these peptides to induce IFN-y production in PBMC was assayed. The re- sults from three PPD positive healthy donors (referred to as KTB1, KTB10 and K172, re- spectively) are shown in Fig. 1. The pools of peptides from Rv0285 (referred to as Rv0285 p1-Rv0285 p10) stimulated IFN-y production in PBMC from all three donors. This is consistent with the results obtained with recombinant Rv0285 (Table 2a and Fig. 1). When tested singly, seven peptides were recognized by the three donors, indicating the pres- ence of multiple immunogenic portions scattered through out the protein sequence of Rv0285.

The pools of peptides from Rv1386 and recombinant Rv1386 stimulated FN-y production in PBMC from two of the three donors. Four of the peptides were also positive when tested as single peptides. The synthetic peptides were also tested in PBMC from two PPD negative controls ; as expected, no stimulation of IFN-y production was detected for these donors (results not shown).

Example 3a : PBMC recognition of peptides derived from MT3106. 1 A BLAST-P search of the GMT. pep database at TIGR CMR revealed an open reading frame which is highly related to Rv0285. This ORF is designated MT3106. 1, and the pre-

dicted initiation codon is 33 codons upstream of the corresponding initiation codon in Rv0285. Amino acid sequence alignment revealed that the Rv0285-corresponding part of MT3106. 1 has 80% sequence identity to the former, and a peptide fragment spanning residues 2-29 on Rv0285 is 100% conserved on Mt3106. 1. This segment of peptide contains at least 2 distinct T-cell epitopes as demonstrated by the results in Fig. 1 (Rv0285-p1 and Rv0285-p2, respectively). Eleven additional overlapping peptides of MT3106. 1 (MT3106. 1-p1-MT3106. 1-p11, SEQ ID NO 146-156) were synthesized and analyzed for their ability to induce IFN-y production in PBMCs from donor K172. Peptide MT3106. 1-p7 was highly reactive and stimulated IFN-y production to a level of 12079 pg/ml, which corresponds to 87% of the activity obtained with PPD.

PBMC from 6 additional TB patients were obtained, and the T-cell stimulatory effect of rRv1195 was also tested in these PBMCs. The results are shown in Table 2b.

Example 3b. Recognition of synthetic peptides by T-cell lines derived from PBMC of PPD positive subjects.

Non-overlapping peptides (Rv0284-p1-Rv0284-p69, SEQ ID NO 54-122) were synthe- sized for the part of Rv0284 that was not included in rRv0284ct. Peptides were tested as pools consisting of 2 or 3 peptides each. T-cell stimulatory effects were seen in a number of peptide pools. The largest effects on stimulation of IFN-y release were obtained with peptide pools containing Rv0284-p3, Rv0284-p4, Rv0284-p7, Rv0284-p8, Rv0284-p9, Rv0284-p13, Rv0284-p17, Rv0284-p18, Rv0284-p19, Rv0284-p27, Rv0284-p37, Rv0284-p41, Rv0284-p42, Rv0284-p43, Rv0284-p47, Rv0284-p50, Rv0284-p51, Rv0284-p52, and Rv0284-p53.

Twenty-three overlapping peptides were synthesised for Rv3878 (Rv3878-p1-Rv3878- p23, SEQ ID NO 123-145). An initial screening of the peptides in four T-cell lines revealed a number of T-cell epitopes (Fig. 3).

Example 4 : Identification of TB9. 5 and TB13. 7 Short-time culture filtrate (ST-CF) was produced from living Mycobacterium tuberculosis as previously described and used as an antigen source (Andersen, P. et al 1991). In brief, ST-CF was produced by growing M. tuberculosis H37Rv (4 x 106 CFU/ml) on modified

Sauton medium in an incubator at 37 °C at gentle agitation for 7 days. The culture super- natant was steril-filtered and concentrated on a Amicon YM3 membrane. The culture fil- trate was hereafter precipitation with 80 % ammonium sulphate and the precipitated pro- teins were removed by centrifugation and after washing resuspended in buffer containing 8 M urea, CHAPS 0. 5% (w/v) and 5% glycerol. 250 mg of protein was separated on the Rotofor Isoelectrical Cell (Bio-Rad) in a pH gradient with 3% Biolyt 3/5 and 1 % Biolyt 4/6.

Fraction 3-8 were pooled, concentrated and buffer exchanged to PBS on a Centriprep concentrator with a 3 kDa cut off membrane. 100 ug of protein as separated by two- dimensional electrophoresis by applying the sample on immobilized pH 4-7 linear gradient 13 cm strips (Amersham Pharmacia Biotech) and the focusing was performed at 500 V for 1 hour, 1000 V at 1 hour followed by 2 hours at 8000 V in a IPGphor unit. The second di- mension was performed in 10-20% SDS-PAGE gradient gels in the protean xi system (Bio-Rad). The proteins were transferred to a PVDF membrane which was stained for by Coomassie brilliant Blue and two spots was excised and subjected to N-terminal se- quencing analysis by automated Edman degradation using a Procise 494 sequencer (Ap- plied Biosystems) as described by the manufacturer.

Sequence analysis and peptide synthesis The two spots were named TB9. 5 and TB13. 7. For each of the two protein spots a se- quence of 15 amino acids was obtained.

ForTB9. 5 : MKAKVGDILVIKGAT (SEQ ID NO 171) For TB13. 7 : DSTEDFPIPXRMXAT (SEQ ID NO 172) "X"denotes an amino acid, which could not be determined.

The two sequences were used for a homology search using the BLAST program on the M. tuberculosis database : http ://genolist. pasteur. fr/TubercuList/. For TB9. 5 the 15 deter- mined amino acids was 100% identical to the sequence of Rv0569, which is an 88 amino acids long protein. For TB13. 7 the 13 determined amino acids was 100% identical to the sequence of Rv0455c. The 13 N-terminally determined amino acids starts at amino acids 31 in the predicted sequence of Rv0455c, indication the presence of a signal peptide, which has been cleaved off. This is in agreement with the prediction of a signal peptide in Rv0455c by database analysis of the amino acids sequence using the program Signal P at http ://www. cbs. dtu. dk/services/SignaIP/, which also predicts the most likely cleavage site between position 30 and 31.

Overlapping peptides was produced for the mature version of each of the two proteins by Schafer-N, Copenhagen, Denmark as indicated below. The peptides were synthesized on polyamide resins using Fmoc-strategy and purified by reverse phase HPLC on C18- columns in water/acetonitrile gradients containing 0. 1 % TFA (trifluoracetic acid). Purified peptides were lyophilized and stored dry until reconstitution in PBS.

TB9. 5-1 : MKAKVGDWLVIKGATIDQPDHRGLIIEVRS TB9. 5-2 : HRGLIIEVRSSDGSPPYWRWLETDHVATV TB9. 5-3 : VRWLETDHVATVIPGPDAWVTAEEQNAAD TB9. 5-4 : VTAEEQNAADERAQHRFGAVQSAILHARGT TB13. 7-1 : DSTEDFPIPRRMIATTCDAEQYLAAVRDTS TB13. 7-2 : QYLAAVRDTSPVYYQRYMIDFNNHANLQQA TB13. 7-3 : FNNHANLQQATINKAHWFFSLSPAERRDYS TB13. 7-4 : LSPAERRDYSEHFYNGDPLTFAWVNHMKIF TB13. 7-5 : FAWVNHMKIFFNNKGWAKGTEVCNGY Immunological activity of TB9. 5 and TB13. 7 The immunological relevance of the peptides in TB patients was tested by analysing the ability of the peptides to induce an IFN-y production or a cell proliferation on PBMC iso- lated from human TB patients and PPD negative healthy controls (table 5 and table 7).

The TB9. 5 peptides were in addition tested for ability to induce IFN-y and cell proliferation on T cell lines generated from TB patients driven by ST-CF or M. tuberculosis sonicate (table 6). Lymphocyte preparation and T-cell lines generation were performed as de- scribed in example 2.

Table 5 : Stimulation of PBMC from three TB patients and three PPD negative healthy controls with pools of synthetic peptides from TB9. 5 and TB. 13. 7 in total of 10 ug/ml. 2. 5 ug/ml of each peptide TB9. 5-1, TB9. 5-2, TB9. 5-3 and TB9. 5-4 were pooled and tested as TB9. 5. 2 ug/ml of each peptide TB13. 7-1, TB13. 7-2, TB13. 7-3, TB13. 7-4 and TB13. 7-5 were pooled and tested as TB13. 7. The response to 5 ug/ml ST-CF is shown for compari- son. Results are presented as pg IFN-y/ml. TB patients Healthy controls Antigen PT1 PT2 PT3 H1 H2 H3 Control 0 0 0 9 10 0 ST-CF 4803 11810 3221 28 10 0 TB9. 5 38 59 479 39 0 2 10ug/ml TB9. 5 37 56 115 9 7 40 2. 5ug/ml TB13. 7 160 36 29 5 15 13 10ug/ml TB13. 7 131 54 70 15 O 2. 5ug/ml

Pools of the peptides are tested on PBMC purified from human TB patients and healthy controls as seen in table 5. The pools of peptides from TB9. 5 were recognized more fre- quently by TB patients than by the healthy controls. This demonstrates that a positive re- sponse is specific for TB patients. TB13. 7 was also recognized more frequently by the tested TB patients compared to the healthy controls. It is to be expected that not all of the patients recognized each of the peptides pools, due to the genetically heterogeneity of the human population.

Interestingly, it was not the same patient recognizing the two peptide pools indication that the use of a combination of two peptide pools could be superior compared to using the single peptide pools.

The peptides from TB9. 5 was in addition tested for ability to induce an IFN-y response or cell proliferation on five T cell lines derived from TB patients (table 6). TB9. 5-1 was posi- tive in most of the tested T-cell lines demonstrating the presence of one or more broadly recognized T cell epitope within this sequence (table 6). Furthermore, TB9. 5-2, TB9. 5-3 and T9. 5-4 were positive in at least one out of the five T cell lines tested demonstrating that these sequences also contains at least one T cell epitope. The presence of multiple epitopes in the TB9. 5 protein makes the full-length protein or peptides derived hereof an attractive candidate for a TB vaccine.

Tabel 6 : Stimulation of five T cell lines derived from TB patients with synthetic overlapping peptides from TB9. 5. Results are presented as pg IFN-y/ml and cell proliferation. The peptides are tested in 1 ug/ml and 1 Oug/ml and results are shown for the concentration given the highest response. The response to 5 ug/ml ST-CF is shown for comparison. Antigen T-cell line 1 T-cell line 2 T-cell line 3 T-cell line 4 T-cell line 5 IFN-y CPM IFN-y CPM IFN-y CPM IFN-y CPM IFNy CPM Control 133 1359 0 184 120 397 62 2550 9 333 ST-CF 4581 26296 3552 21239 2748 12118 2860 18624 4294 29736 TB9. 5-1 1438 9116 407 3987 512 1749 42 2033 17 1252 TB9. 5-2 3 919 341 3395 69 606 20 1718 10 322 TB9. 5-3 26 1145 120 1859 88 537 49 2410 1 331 TB9. 5-4 86 2556 519 3887 219 839 28 2860 3 1036 TB9. 5-pool 208 3544 52 1825 127 831 6 1738 2 626 Table 7 : Stimulation of PBMCs from two TB patients and two healthy controls with syn- thetic peptides from the TB13. 7 protein. Responses to PPD are given for comparison.

Control is stimulation without antigen. Results are given as pg IFN-y/ml

Antigen/conc. TB patients Healthy controls Control PT1 PT2 H1 H2 PPD 5 ug/ml 5549 1269 1570 11 13. 7-1 10 ug/ml 20 2 26 42 13. 7-1 2. 5 ug/ml 6 1 23 47 13. 7-2 10 ug/ml 21 55 13. 7-2 2. 5 ug/ml 5 3 21 49 13. 7-3 10 ug/ml 11 20 54 13. 7-3 2.5 ug/ml 10 2 28 45 13. 7-4 10 ug/ml 8 7 15 24 13. 7-4 2.5 ug/ml 8 6 16 30 13. 7-5 10 ug/ml 648 5 18 27 13. 7-5 2. 5 ug/ml 205 22 29 The 13. 7 peptides were tested on PBMC isolated from two TB patients and two healthy controls. As seen in table 7 one of the two TB patients recognized peptide TB13. 7-5 while

no of the healthy controls recognized any of the peptides tested. This demonstrates that an epitope is presence in peptide TB13. 7-5, but does not rule out the presence of epi- topes in any of the other peptides. To demonstrate this it would be necessary to test a higher number of TB patients due to the genetically heterogeneity of the human popula- tion.

The expression of TB 9. 5 is induced under low oxygen conditions Immunogenic proteins may be identified by the means of their upregulation in vivo or in environments which reflects the in vivo situation. This may be different stress situations such as low oxygen. To investigate the upregulation of M. tuberculosis proteins during low oxygen conditions the following experiments were performed : M. tuberculosis H37Rv (ATCC 27240) was cultured in Sauton medium enriched with 0. 5 % sodium pyruvate and 0. 5 % glucose. Sterile 10 ml (Nunc, Roskilde, Denmark) polystyrene tubes or 125 ml poly- carbonate Erlenmeyer flasks (Corning, Acton, MA, USA) containing 6. 7 ml or 20 ml of medium, respectively, was inoculated with 2x106 bacteria per ml. Erlenmeyer flasks were placed in a standard 37°C shaking incubator (normal cultures), whereas tubes with tightly screwed caps (low oxygen cultures) were placed at 37°C under magnetic stirring at 100 rpm. After 3 h metabolic labelling was performed by addition of 10 uCi/ml of L- [35S]- methionine and L- [35S] cysteine (Redivue Promix, Amersham Pharmacia Bioctech, Buck- inghamshire, United Kingdom). After 19 h, bacteria were harvested by centrifugation, and the medium was collected. The bacterial pellet was washed once in PBS, pH 7. 4, and re- suspended in 300 ut of a suspension containing equal volumes of 0. 1 mm glass beads and PBS, pH 7. 4, added 0. 1 % SDS and 1 mM PMSF. The bacteria were lysed for 5 min at maximum speed on a MS2 minishaker (IKA Works inc., Wilmington, NC). 20 NI of the lysates was analysed by two-dimensional gel electrophoresis (2-D PAGE) : Samples were applied to 13 cm IPG pH 4-7L strips (Amersham Pharmacia Bioctech, Uppsala, Sweden) during rehydration according to the manufacturer's instructions. Focusing started at 500 V (1 h), was increased to 1000 V (1 h), and finally to 8000 V (2 h) in an IPGphor unit (Amer- sham Pharmacia Biotech). The second dimensional separation was performed in 10-20 % SDS-PAGE gradient gels in the Protean lixi system (Bio-Rad, Richmond, CA, USA). The gel was blotted to PVDF membrane, and the membrane was exposed to Biomax MR film (Kodak, Rochester, NY, USA) for 3-21 days. The autoradiographs were scanned and analysed by the Phoretix 2D gel analysis software (Non Linear Dynamics, Newcastle upon Tyne, United Kingdom). Spots which showed more than two-fold induction under low oxygen conditions compared to normal cultures were selected. A spot with observed

mass of approx. 12 kDa and pl of 6. 3 was found to be induced under low oxygen condi- tions. For identification of this spot, 35 ul of the low oxygen lysate was analysed by 2-D PAGE as described above and the gel was silver stained. The relevant spot was excised and identified by MALDI-MS peptide mass fingerprinting. Four fragments corresponding to the peptides 23-29, 30-40, 75-86 and 75-88 of TB9. 5 (Rv0569) were matched, giving a sequence coverage of 36 % for this protein. This result demonstrates that the TB9. 5 pro- tein is upregulated under conditions that mimics the in vivo situation, which indicates that this protein may be a good vaccine candidate or a therapeutic vaccine candidate.

Example 5 : ORF13A is a serological target in TB patients To test the potential of ORF13A as a serological antigen, sera were collected from 48 TB patients (all proven culture positive for M. tuberculosis) and 15 healthy BCG vaccinated controls and 19 non-BCG vaccinated healthy controls. The sera were assayed for anti- bodies recognizing the recombinantly produced ORF13A in an ELISA assay as follows : Each of the sera was absorbed with Promega E. coli extract (S37761) for 4 hours at room temperature and the supernatants collected after centrifugation. 1 ug/ml of ORF13A in Carbonatbuffer pH 9. 6 were absorbed over night at 5 °C to a polystyrene plate (Maxisorp, Nunc). The plates were washed in PBS-0. 05% Tween-20 and the sera applied in a dilu- tion of 1 : 100. After 1 hour of incubation the plates were washed 3 times with PBS-0. 05% Tween-20 and 100 ul per well of peroxidase-conjugated Rabbit Anti-Human IgA, IgG, IgM was applied in a dilution of 1 : 8000. After 1 hour of incubation the plates were washed 3 times with PBS-0. 05% Tween-20. 100 ul of substrate (TMB PLUS, Kem-En-Tec) was added per well and the reaction stopped after 30 min with 0. 2 M Sulphuric acid and the absorbance was read at 405 nm. The results are shown in figure 2A, 2B and 2C.

56% of the TB patients recognized ORF13A with an absorbance more than OD 0. 3. The mean for all 48 patients was OD 0. 44. In contrast only one BCG vaccinated individual recognized ORF13A slightly above the cutoff and three of the non BCG-vaccinated healthy donors recognized ORF13A, only one significant above the cutoff. The mean for BCG vaccinated individuals were OD 0. 18 and for non BCG-vaccinated OD 0. 3. Table 8 : Serological responses to ORF13A and the 38kDa antigen evaluated by ELISA on 48 TB patients, 15 BCG vaccinated and 19 non BCG vaccinated individuals. TB patients BCG vaccinated Non BCG vaccinated Antigen Percent (n) Mean of OD Percent (n) Mean of OD Percent (n) Mean of OD responders responders responders ORF13A 56% (27) 0. 44 7% (1) 0. 18 16% (3) 0. 3 38 kDa 50% (24) 0. 38 20% (3) 0. 21 26% (5) 0. 24

In table 8 the response to ORF13A is compared to an antigen which is known as one of the best serological antigens ; the 38kDa phosphate binding proteins (Luashchenko, K. P., et al J Immunological Methods 242 (2000) 91-100). The two proteins were tested in par- allel on the same donors. The 38 kDa antigens is recognized by 50% of these TB patients and 20% of the BCG vaccinated and 26% of the non BCG-vaccinated in this study popu- lation. Thus ORF13A is recognized by more TB patients and by less of the healthy con- trols (both BCG vaccinated and non-vaccinated) than the 38 kDa antigen. This clearly demonstrates the potential of ORF13A as a serological antigen for the diagnosis of TB, and demonstrates that ORF13A has the potential to differentiate between BCG vacci- nated and M. tuberculosis infected individuals something, which is not possible with the current diagnostic reagent PPD. It is well known that the antibody repertoire of TB patients is very heterogeneous and it is therefore not likely that all patients will recognized the same mycobacterial antigen, as also demonstrated by these results. It is therefore most likely that a serological kit for the diagnosis of M. tuberculosis infection will consist of more than one component and in this respect it will be obvious to combine ORF13A with other antigens, which are recognized by TB patients. This could be the 38 kDa antigens, but also other proteins could be included.

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