DESC:FIELD OF INVENTION

The present invention relates to Mycobacterium tuberculosis polypeptides, polynucleotide that encodes the polypeptides, and methods of using such compositions in the treatment, prevention and diagnosis of M. tuberculosis infection.

BACKGROUND OF INVENTION

Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis (MTB), especially given the increase of resistance worldwide to first and second-line drugs.

It is well established that cellular immunity is critical for protection against TB. Much of the work in this field is focused on defining the antigens of the causative bacterium, MTB that can elicit effective immunity and on understanding the role of various cell populations in host-pathogen interactions.

Antibody responses in tuberculosis have been studied for several decades primarily for the purpose of developing serodiagnostic assays. Although some seroreactive antigens/epitopes have been identified, interest in antibody responses to MTB has waned because of the lack of progress in simple detection of corresponding antibodies. Studies using crude antigen preparations revealed that healthy individuals possess antibodies that cross-react with several mycobacterial antigens. Such antibodies are believed to have been elicited by exposure to commensal and environmental bacteria and vaccinations. MTB specific antibodies not only facilitate an effective way for diagnostic purposes, but can also be used for identification of drug targets against MTB.

MTB bacilli release a number of proteins during growth in surrounding media. These excreted and secreted proteins are responsible for rapid recognition of bacilli by host lymphocytes as an antigen and thus play an important role in developing immune responses. Purification of such mycobacterial antigens is essentially required for defining structural probe and dissecting basic mechanism of the immune response to mycobacteria.

Several mycobacterial antigens have been isolated and characterized in the past including the 71 kDa DnaK, 65 kDa GroEL, 47 kDa elongation factor tu, 44 kDa PstA homologue, 40 kDa L-alanine dehydrogenase, 38 kDa PhoS, 23 kDa superoxide dismutase, 23 kDa outer membrane protein, 12 kDa thioredoxin, and the 14 kDaGroES. However, a majority of the antigens identified so far bear significant homology to the analogous proteins in other mycobacteria and non-mycobacterial prokaryotes. Thus, almost all individuals (healthy or diseased) have antibodies to epitopes of conserved regions of these antigens. These antibodies are responsible for the uninformative (and possibly misleading) cross-reactivity observed with crude MTB antigen preparations.

Many attempts have been made to develop a drug against the mycobacterial proteasome, usually as peptidyl epoxyketones, aldehydes or boronates, but most inhibit the mammalian proteasome more potently than that of M. tuberculosis. Specificity is thought to be conferred by interaction of the drug with residues outside of the active site not conserved in mammalian proteasomes.

A need therefore exists for identification of novel proteins specific to Mycobacterium tuberculosis that serve as effective drug targets and can also be used for diagnostic purposes.

SUMMARY OF INVENTION

In one aspect, the present invention an isolated mycobacterium tuberculosis antigen comprising a polypeptide having SEQ ID NO.2.

Preferably, the polypeptide is protease from mycobacterium tuberculosis.

In a further aspect, the present invention provides an isolated polypeptide having SEQ ID NO.2 for detection MTB infection.

Preferably, the isolated polypeptide is used as a drug target for treatment of tuberculosis.

Preferably, the isolated polypeptide is used individually or in combination with other polypeptides, including fusion proteins.

In a further aspect, the present invention provides a composition comprising an isolated antigen comprising a polypeptide SEQ ID NO.2 from mycobacterium tuberculosis, for use in the detection of mycobacterium tuberculosis infection.

Preferably, the composition is formulated as pharmaceutical compositions for administration into a subject in the prevention
and/or treatment of M. tuberculosis infection.
Preferably, the composition includes an adjuvant.
Preferably, the composition is formulated as a vaccine.
A polyclonal mycobacterium tuberculosis antibody being reactive to an antigen comprising a polypeptide having SEQ ID NO. 2.
In a further aspect, the present invention provides a method of detecting mycobacterium tuberculosis infection comprising:providing a biological sample on a surface;contacting said sample with a composition comprising an antigen encoding a polypeptide having a SEQ ID NO. 2.
In yet a further aspect, the present invention provides an isolated antigen specific to mycobacterium tuberculosis, comprising either one of the following epitopes depicted by SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 wherein the epitopes are specific to the disclosed protease of present invention depicted by SEQ ID NO: 2.
In yet a further aspect, the present invention provides an isolated polypeptide for use as an immunogen to generate anti- M. tuberculosis antibodies in a non-human animal, the polypeptide having SEQ ID NO. 2
Preferably, the antibodies can be used to detect the target antigens in vivo and in vitro.

BRIEF DESCRIPTION OF DRAWINGS

The present invention may be best understood by reference to the following detailed description when read with accompanying drawing in which:

FIG. 1: Inhibition of Anti ES31 pAB raised in goat an Axenic Culture;
FIG. 2: Purification of large scale native 31Kda excretory secretory pool of protein and separating on 2D and identifying the pool of protein;
FIG. 3: Results for binding Assay of GNGTB1 with ES-31Kda Polyclonal Antibody conforming the presence of GNGTB1 protein in 31Kda Pool.
FIG. 4: Results in Characterization of GNG TB1 ( Target against MTB)
FIG. 5: Results for Biochemical Assay: Protease Activity of GNGTB1
FIG. 6: Results for Inhibition Assay: Monospecific anti-GNG-MTB1 IgG on MTB avirulent cells
FIG. 7: Results related to Invitro Inhibition of Mycobacterium H37Rv Culture using mini bodies against selected epitope of the target
FIG. 8: Binding assay with Diabody (ScFV)
FIG. 9: Binding of the antibody to 3H

SEQUENCE LISTING: SEQUENCE ID NO. 2 - 18

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT(S)

The description of a number of specific and alternative embodiments is provided to understand the inventive features of the present invention. It shall be apparent to one skilled in the art, however that this invention may not be practiced without such specific details.

It is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In a first aspect, the present invention relates to Mycobacterium tuberculosis (MTB) specific antigens. More particularly, it relates to MTB specific polypeptides, nucleic acids sequences coding such polypeptides, methods of using such polypeptides for detection, treatment and prevention of MTB infection.

In an embodiment, the present matter discloses a nucleotide sequence depicted by SEQ ID NO:1 encoding the protease depicted by SEQ IDNO: 2.The embodiments also include “Conservatively modified variants” of the protease depicted by SEQ ID NO:2 and nucleic acid sequence depicted by SEQ ID NO:1 . With respect to particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.

Both the coding sequences and their encoded polypeptide products are suitable for a variety of uses. In a specific embodiment of the invention, the M. Tuberculosis polypeptide of the invention may be formulated as pharmaceutical compositions for administration into a subject in the prevention and/or treatment of M. tuberculosis infection.

In that regard, the polypeptide may be used individually or in combination with other polypeptides, including fusion proteins. The immunogenicity of the antigens may be enhanced by the inclusion of an adjuvant.

It is also another aspect of the invention that the polypeptide be used in in vitro assays for detecting humoral antibodies or cell-mediated immunity against M. tuberculosis for diagnosis of infection or for monitoring disease progression. Additionally, the polypeptide of present invention may be used as an in vivo diagnostic agent in the form of an intradermal skin test. Alternatively, the polypeptide may be used as an immunogen to generate anti-M. tuberculosis antibodies in a non-human animal. The antibodies can be used to detect the target antigens in vivo andin vitro.

In another aspect of the invention, the nucleic acid sequence depicted by SEQ ID NO:1 can be used to produce recombinant polypeptide antigens. These antigens may be used as probes for development of specific antibodies that can be used for treatment of tuberculosis.

Alternatively, the polynucleotide may be administered directly into a subject as DNA vaccine to cause antigen expression in the subject, and the subsequent induction of an anti-M. tuberculosis immune response. Additionally, the nucleic acid sequence, portions thereof, or their complements may be used as molecular probes or primers to detect the presence of bacteria in a biologic sample.

Accordingly, the polypeptide sequence depicted by SEQ ID NO: 2 comprises MTB specific protease that can serve as a diagnostic biomarker for detection of MTB infection and also as a drug target for treatment of tuberculosis. The protease disclosed herein has been identified as being vital to growth of MTB and for its survival during stress conditions.

According to another embodiment, the polypeptide disclosed herein presents ideal target for drug development wherein inhibitors to the protease disclosed hereinarrests microbial growth and thus helps control MTB infection.

DNA sequences encoding the inventive polypeptides, expression vectors comprising these DNA sequences, and host cells transformed or transfected with such expression vectors are also provided.

In an aspect, polypeptide derived from Mycobacterium tuberculosis is provided, wherein the polypeptide comprise an antigen or a variant thereof and is depicted by SEQ ID NO: 2.

Specifically, the present invention discloses polypeptides specific to MTB comprising unique epitopes that are highly specific to Mycobacterium tuberculosis and are not found in humans or other organisms. The present invention also discloses 3 unique epitope regions depicted by SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 , wherein the epitopes are specific to the disclosed protease of present invention depicted by SEQ ID NO:2.The unique epitopes depicted by SEQ ID NO: 3, SEQ ID NO:4 and SEQ ID NO:5 can serve as drug targets for treatment of MTB infection . In addition, the epitopes may serve as targets for detection of MTB infection and may be used for development of a diagnostic kit.

Also within the scope of the present invention are antibodies that specifically recognize one or more epitopes of the invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized antibodies, chimeric antibodies, single chain antibodies, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above such as Fab and F(ab')2.

In another aspect, the polypeptide disclosed herein can be used for inducing protective immunity in a patient, comprising administering to a patient an effective amount of the polypeptide with an immune response amplifier.

The invention also provides vaccines comprising the polypeptide depicted by SEQ ID NO:2 and a non-specific immune response amplifier, together with vaccines comprising at least one DNA sequence encoding such polypeptide and a non-specific immune response amplifier.

Alternately, the polypeptides comprising the specific epitopes as disclosed in the present invention can be used to produce vaccines.

In another aspect the polypeptides comprising the specific epitopes as disclosed in the present invention can be used to develop diagnostic methods for detection of MTB in a patient sample.In further aspects of this invention, methods and diagnostic kits are provided for detecting tuberculosis in a patient.

According to an embodiment the present inventive subject matter discloses antibodies specific to the polypeptide depicted by SEQ ID NO:2 as disclosed herein, such that the antibodies act as viable drugs against MTB and also can also be used to develop a method for detection of Mycobacterium tuberculosis in a sample.

In yet another embodiment the present inventive subject matter discloses a series of nano bodies working against the MTB specific protease antigen as depicted by SEQ ID NO: 2.

It is within the scope of the present invention to provide antibodies under accession no A2VKP5 and D5ZJ53, shown in FIG. 13.

In another embodiment, the present inventive subject matter discloses polypeptides comprising a ScFv based antibody depicted by SEQ ID NO: 6 wherein the ScFv antibody acts as a diabody against target polypeptide of SEQ ID NO: 2.

Accordingly, polypeptide sequences of the ScFv based antibody fragments against MTB specific proteases are provided. The antibodies disclosed herein act as diabody against the polypeptide sequence of MTB specific protease antigen as depicted by SEQ ID NO:2.

The present invention also discloses nucleotide sequence encoding active epitopes in the MTB specific protease as given in the present embodiment .In a further embodiment the present invention discloses a protease based antibody library for detection of Mycobacterium tuberculosis in a sample.

Within other aspects, the present invention provides pharmaceutical compositions that comprise the MTB specific polypeptide of SEQ ID NO:2 , or a DNA molecule encoding such a polypeptide, and a physiologically acceptable carrier. The invention also provides vaccines comprising the MTB specific polypeptide of SEQ ID NO:2 and a non-specific immune response amplifier, together with vaccines comprising at least one DNA sequence encoding such polypeptides and a non-specific immune response amplifier.

This invention in one aspect pertains to a solution containing a diabody reactive with a mycobacterial antigen or a fragment thereof. More specifically, it pertains to a solution containing a diabody reactive with a mycobacterial antigen wherein, the antigen is a excretory secretory protease specific to MTB. Antibody solutions provided herein serve as drugs to inhibit MTB growth and infection by binding to the MTB specific proteases as disclosed herein.

This invention also pertains to immortalized, stable antibody producing cell lines, e.g. hybridoma cell lines, capable of producing the above-described antibodies, as well as immunoassays using the antibodies, and therapeutic compositions having the antibodies as a component. The invention also relates to kits useful in aiding in the diagnosis of mycobacterial disease.

The immunoassays of this invention may be any immunoassay in which a diabody as described above may be useful.

MTB excretory secretory antigens were isolated from MTB spent culture and used to raise antiserum containing polyclonal antibodies. The antibodies were tested for inhibitory effect by incubating with macrophages infected with MTB. Since the antiserum was generated against MTB polypeptides that had been purified from bacterial culture supernatants, the antiserum preferentially reacted with bacterial secretory products. Further, the MTB excretory secretory antigens were separated and sequenced. Sequences of the antigens were compared with publicly available sequences and filtered through several parameters including similarity score, molecular weight, isoelectric point (pI) , biochemical nature, protease group, metalloaffinity, membrane domain, culture filtrate presence, essential for growth. Based on above mentioned parameters, a protease depicted by SEQ ID NO: 2 was identified as an effective antigen showing high specificity to MTB.

The following provides experimental examples in relation to the preparation of non-human model and the characterisation of the xenografts in accordance with an embodiment of the present invention. It should be noted that the experimental examples should not be construed as limitations to the scope of protection.

EXPERIMENTAL EXAMPLES

EXAMPLE 1

Establishing and validating the inhibitory effect on axenic culture of mycobacterium tuberculosis as well as in macrophages by 31-kDa protein antibody.
Materials and Method
MTB excretory secretory polypeptides were isolated from the 10-day spent culture. The culture was sterilized of bacilli by sterile filtration through Seitz filter followed by 0.45 µm Maxflow membrane filter. The culture filtrate was concentrated to 100 fold. Ammonium sulphate precipitation at 50% saturation of culture filtrate was carried out at 4°C. Precipitated antigen was fractionated by 10% non-gradient sodium dodesyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The stained and unstained gel overlapped and fractions named 6th and 7th on the basis of relative Rf value were isolated and extracted. Mono S cation exchanger column was used for further purification. Approximately 31-kDa molecular weight prominent protein band was purified. Fractionated, isolated and purified 31-kDa protein was used for raising polyclonal antibody in goat. An MTT assay was standardized for studying the inhibition of bacteria using pAb against 31-kDa along with positive controls such as Isoniazide and Rifampicin.
Results
MTT which is 3(4,5-diamethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide is reduced by mitochondrial dehydrogenase in living cell to produce insoluble purple MTT formazan crystal which is measured spectrophotometrically on solubilization. Cell number validation clearly shows linear relation between absorbance of the formazan solution with bacterial concentration. Hence a validated functional assay using MTT was performed in order to study the inhibition of Mycobacterium tuberculosis with pAb raised against 31-kDa protein extracted from excretory secretory protein supernatant of MTB. Inhibition of axenic culture was seen at around 89% which was comparable to the control (Rifampicin, first line drug, ATT) Table 1. The same Ab was treated to infected macrophage with M. tuberculosis and CFU was done along with control untreated and 95% inhibition was achieved (Figure 1c). Results based on inhibition of anti 31-kDa pAb raised in goat on axenic culture were recorded and plotted in FIG. 1a. The negative control was taken as Goat IgG to check specific activity of the antibody as depicted in Figure 1b. The result showed the inhibition is specifically the subject of polyclonal antibody raised against 31-kDa protein.

EXAMPLE 2

Purification and Characterization
Purification of large scale 31-kDa excretory secretory pool of protein and separating on 2D gel electrophoresis and identifying the specific target.
With reference to FIG. 2, purified approximate molecular weight 31-kDa protein further separated on 2D gel electrophoresis. Identified few spots were excised and gel digested for mass spectrometry. Digested peptides were reconstituted in 15 µL 0.1% formic acid and 3 µL of reconstitute solution injected on column. Digested peptides were subjected to 70 minute RPLC-MS/MS analysis. Generated data was searched for the identity on MASCOT as search engine using Uniprot Swiss-Prot database (non redundant database with reviewed proteins) and Uniprot trEMBL database (database with unreviewed proteins). High confident peptides with prerequisite of minimum two peptides leading into identification of proteins were selected and list was generated. As a control to check the performance of the experiment, 12.5 fmol of BSA digest was also loaded on column. The acquired results were processed in the same way as that of samples. The number of hits obtained (Appendix I) was filtered through the parameters (Score, Molecular weight, pI, Biochemical Nature, Protease group, Metallo affinity, Membrane domain etc.). Filtering through all these parameters of the protease Rv2535c Cytoplasmic Peptidase pepQ was chosen as high priority and synthesized at GeneArt as GNGTB1.

EXAMPLE 3

Developing the recombinant construct of the probable target protein/polypeptides and characterization
The complete sequence of GNGTB1 protein (SEQ ID NO. 2) was taken from the Genbank database and synthesized through GeneArt. GNGTB1 was recombinantly cloned as 6-histidine tag in pET 28 expression vector as full length as well as fragment for different domains. The clones were transformed in Rosetta 2 expression system and protein over expressed by induction with IPTG.
GNGTB1 protein was purified from inclusion bodies using histidine affinity column (His-Trap column, GE) in denaturing conditions (6 M Urea) and increasing imidazole concentration (0 to 300 mM). The molecular weight of the protein according to the nucleotide sequence is 38.8-kDa, and the same was verified on 10% SDS-PAGE gel.

Results
The following TB target proteases construct were obtained from example 3 methods and used for characterization study as shown in TABLE 1 below:
TABLE 1
Protein Constructs MW Vector backbone Expression strain
GNGTB-3 3-372 aa 38.5-kDa pET28a Rosetta-2
GNGTB-104 104-372 aa 27.8-kDa pET28a Rosetta-2
GNGTB-139 137-372 aa 24.5-kDa pET28a Rosetta-2

Sequence of different construct of GNGTB1:

EXAMPLE 4
Binding assay of GNGTB1 with anti 31-kDa protein polyclonal antibody conforming the presence of GNGTB1 protein in 31-kDa pool and characterization of GNGTB1 (Target against MTB) are shown in FIG. 3 and FIG. 4 respectively.

EXAMPLE 5

Protease Activity

With reference to FIG. 5, the protease activity was high at alkaline pH and at higher temperature showing a crucial role in bacterial survival. The activity however was unhindered by the following inhibitors used.

• 1,10-Phenanthroline (Zn metallo protease inhibitor)
• Z-Pro-OH (human prolidase inhibitor)
• P-(hydroxymercuri)-benzoic acid sodium salt (cysteine and
• serene prorotease inhibitor)

The activity was inhibited by monospecific polyclonal antibody. Same Ab was used to do the inhibition assay suggesting that the protease activity is important for bacterial survival and growth.

EXAMPLE 6

Assay Development to showcase the essentiality of protein for mycobacterial survival.

Reference to FIG. 6, polyclonal antibody specific to GNGTB1 shows 92% inhibition on MTB culture (2X109 cells grown in Sauton for 10 days with inhibitors i.e 100 µg of monospecific polyclonal antibody against GNGTB1) showcasing the essentiality of functional protein for mycobacterial survival and growth.

EXAMPLE 7

Study the effect of mAb (minibody) on the target and compare it with pAb as well other chemical modulator.
Identifying the correct epitopic region which could be actual target for drug development
In order to identify the correct epitopic region which could be a potential target for developing some lead candidate and study inhibition, A prediction analysis was performed based on model structure and found peptide (epitope) which could be a potential target. These peptide were screened for all the organism for their conserved region and identified three unique peptide (9-12 aa long) which are not there in human and other organisms.
EXAMPLE 8
Development of minibodies against the target peptide
GNGTB1 is target protein with two domains and gives activity when it is a dimer. The activity of dimeric GNGTB1 is retained even at 50°C temperature and high pH showing a potential molecule in TB bacteria survival in stress.
It is identified that that the target has some essential role in mycobacterium survival and the functional assay showcased that the Monospecific Polyclonal antibody against GNGTB1 which inhibits Proteolytic activity (active site) of target also inhibit the mycobacterium growth.
In order to identify the correct epitopic region which could be a potential target for developing some lead candidate and study inhibition, A structural prediction analysis was performed and found peptide (epitope) which could be a potential target. These peptides were screened for all the organism for their conserved region and identified three unique peptide (9-12 aa long) which are not there in human and other organisms.
These peptides are used to develop a 15-kDa mini body which will have neutralizing affect on the target and would be utilized to showcase anti tubercular activity with growth inhibition assay both in-vitro and in-vivo.

EXAMPLE 9
Inhibition of bacilli using minibodies developed against the specific epitopes of GNGTB1 (Probable Cytoplasmic pepQ) target.
Epitopes that have been identified with nano-bodies as given in IDF
The structure of MRA_2563 (MTB1) was predicted on RaptorX software program analysis and a 3D structure was obtained. Based on 3D structure, peptides stretch for limiting access to the natural substrate. Three promising candidates were chosen and synthesized along with protein to screen the nano bodies. The nanobodies were screened for neutralization of target in protease assay and for inhibition effect on Mycobacterium Tuberculosis culture. In vitro inhibition of mycobacterium H37Rv culture using mini bodies against selected epitope of the target is depicted in FIG. 7.
EXAMPLE 10
ScFv Library generation with Pool of TB1 and TB 2
Balb/C were immunized with GNGTB-3 and GNGTB-139 with 7 days protocol of 50 µg each protein per immunization and were sacrificed for spleen. mRNA was first isolated from the immunized spleen followed by reverse transcribed into cDNA to serve as a template for antibody genes amplification (PCR). A series of Mouse ScFv Primers were used to amplify the VH and VL region and cloned into pSEX81 Vector (Progen) and displayed using phage display method. With 4-5 rounds of panning and infection, single phage was isolated which showed binding to both GNGTB-3 and GNGTB-139. The Sequence of the ScFvis as shown in FIG. 8.
Accordingly, an ScFv library was generated using the spleen of immunized mice from both the target protease and phagemid pSEX81. The library was displayed and plaque was isolated through round of panning and elution. Clone A5.3 phages was showing positive to both the target proteins. This was cloned in expression vector and sequence was retrieved.

EXAMPLE 11
Development of Monoclonal Antibody against GNGTB-3

The monoclonal Antibody was generated using recombinant GNGTB-3 and the antibody was evaluated for binding of the antigen.
Clone 1F3.H1 was developed against GNGTB-3 which has both creatinase domain as well as peptidase domain and was checked for binding. The Clone 1F3.H1 antibody is specific to creatinase domain as it does not bind to 104 and 139 fragment protein. FIG. 9 shows the binding of the antibody to GNGTB-3.

EXAMPLE 10

The following are the CDRs based on the amino acid sequence of the antigen receptor, for both heavy and light chain, with respect to GNGTB-3 Mono-Antibody (1F3.H1). Sequences for Variable Heavy Chain (VH) and Variable Light Chain (VL) are provided in the Sequence List as sequence SEQ ID 14 – 18.

This invention also pertains to kits useful in aiding the diagnosis of mycobacterial disease. The kits contain antibodies as described above and may further contain instructions directing the user on usage of the antibodies in particular immunoassays.

The method and device of the present invention may be deployed in, but not limiting to, evaluation methods or diagnostics in relation to mycobacterium pathogens, as tools for determining the impact of mycobacterium infection, as well as for the discovery of new treatments; vaccines and for the screening of novel treatment or therapeutic agents.

From the foregoing, it would be appreciated that the present invention may be modified in light of the above teachings. It is therefore understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.
,CLAIMS:CLAIMS

1. An isolated mycobacterium tuberculosis antigen comprising a polypeptide having SEQ ID NO.2.

2. The isolated antigen as claimed in Claim 1, wherein the polypeptide is protease from mycobacterium tuberculosis.

3. An isolated polypeptide having SEQ ID NO.2 for detection MTB infection.

4. The isolated polypeptide as claimed in Claim 3 used as a drug target for treatment of tuberculosis.

5. The isolated polypeptide as claimed in Claim 3, wherein the polypeptide is used individually or in combination with other polypeptides, including fusion proteins.

6. A composition comprising an isolated antigen comprising a polypeptide SEQ ID NO.2 from mycobacterium tuberculosis, for use in the detection of mycobacterium tuberculosis infection.

7. The composition as claimed in Claim 6, wherein the composition is formulated as pharmaceutical compositions for administration into a subject in the prevention and/or treatment of M. tuberculosis infection.

8. The composition as claimed in Claim 6, wherein the composition includes an adjuvant.

9. The composition as claimed in Claim 6, wherein the composition is formulated as a vaccine.

10. A polyclonal mycobacterium tuberculosis antibody being reactive to an antigen comprising a polypeptide having SEQ ID NO. 2.

11. A method of detecting mycobacterium tuberculosis infection comprising:

providing a biological sample on a surface;
contacting said sample with a composition comprising an antigen encoding a polypeptide having a SEQ ID NO. 2.
12. An isolated antigen specific to mycobacterium tuberculosis, comprising either one of the following epitope depicted by SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 wherein the epitopes are specific to the disclosed protease of present invention depicted by SEQ ID NO: 2.

13. An isolated polypeptide for use as an immunogen to generate anti- M. tuberculosis antibodies in a non-human animal, the polypeptide having SEQ ID NO. 2.

14. The isolated polypeptide as claimed in Claim 13, wherein the antibodies are identified as accession numbers A2VKP5 and D5ZJ53.

15. A composition comprising an effective amount of M. tuberculosis antibody which reacts with polypeptide having the sequence SEQ ID NO. 2, for the treatment of M tuberculosis infection.

16. The composition as claimed in Claim 15, wherein the antibody targets either one of the following sequences identified as SEQ ID NO 11 -13.

17. Isolated polypeptides comprising a ScFv based antibody depicted by SEQ ID NO:6 wherein the ScFv antibody acts as a diabody against target polypeptide of SEQ ID NO: 2.

18. The isolated polypeptide as claimed in Claim 13 wherein the antibodies can be used to detect the target antigens in vivo and in vitro.

19. An antibody that includes a fragment that is generated against creatinase domain of SEQ ID No. 2.

20. An isolated fragment comprising a VH sequence identified as SEQ ID NO. 14 to 16.

21. An isolated fragment comprising a VL sequence identified as SEQ ID NO. 17 -18.

22. A composition comprising fragments having VH and VL sequences identified as SEQ ID NO 14 – 18.