CA2033042A1

CA2033042A1 - Methods for diagnosis of tuberculosis

Info

Publication number: CA2033042A1
Application number: CA002033042A
Authority: CA
Inventors: Chinnaswamy Jagannath; Meenakshi Balganesh; Bachally R. Srinivasa
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1989-12-22
Filing date: 1990-12-21
Publication date: 1991-06-23
Also published as: IE904536A1; GB9027267D0; AU6807390A; GB2239246A

Abstract

Abstract Disclosed are the immunochemical properties of a 17 kDa protein antigen fractionated, purified and sequenced for amino acids from Mycobacterium tuberculosis (South Indian isolate, SII 1) which causes human tuberculosis worldwide among 16 million patients. The 17 kDa protein antigen which has an N-terminus of A T T L P V Q R (aa 1-8) has atleast three specific antibody binding epitopes located on linear peptides of sequences, R A T Y D K R Y E V R (aa 91-101) and S E F A Y G S F V R (aa 68-77) which were useful in a micro ELISA for the early diagnosis of human tuberculosis by the detection of specific antibodies. The 17 kDa protein antigen which was mitogenic for human tuberculous peripheral blood lymphocytes was found to carry three predicted T-cell epitopes on linear peptides of sequences, S E F A Y G S F V R
(aa 68-77) and A E L P G V D P D C D V C I T R (aa 107-122).
The 17 kDa antigen of M. tuberculosis (SII 1) which thus had both B and T cell reactive properties was found to contain 131 amino acids and potentially applicable in the immuno diagnosis, immuno therapy and immuno prophylaxis of human tuberculosis.

Description

2 ,~ ~3 3 ~

NEW METHODS FOR DIAGNOSIS OF TUBERCUL~SIS

1. Su~mary of the invention Disclosed are the immunochemical properties of a 17 kDa protein antigen fractionated, purifi~d and sequenced for amino acids from Mycobacterium _uberculosis ~South Indian isolate, SII 1) which causes human tuberculosis worldwide among 16 million patients. The 17 kDa protein antigen which has an N-terminus of A T T L P V Q R (aa ~-8) has atleast three specific antibody binding epitope~ located on linear peptides of sequences, R A T Y D K R Y E V R ~aa 91-101) and S E F A Y G S F V R (aa 68-77) which were useful in a micro ELISA for the early diagnosis of human tuberculosis by the detection of specific antibodies. The 17 kDa protein antigen which was mitogenic for human tuberculous peripheral blood lymphocytes was found to carry three predicted T-cell epitopes on linear peptides of sequences, S E F A Y G S F V R
(aa 68-77) and A E L P G V D P D C ~ V C I T R (aa 107-122).
The 17 kDa antigen of M. tuberculos-s (SII 1) which thus had both B and T cell reactive properties vas found to contain 131 amino acids and potentially applicable in the immuno diagnosis, immuno therapy and i~muno prophylaxis of human tuberculosis.

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2. Field of invention .
The present inventi~n relates to a novel 17 kDa protein antigen of MYcobacterium tuberculosis (South Indian Isolate SII 1) and certain pept-de fragments derived therefrom, and to the use of the said antigen, and of the peptide fragments derived therefrom, in immuno diagnosis, immunotherapy and immuno prophylaxis of human and experimental tuberculosis.
The invention also relates to the DNA sequence coding for the said 17 kDa antigen, to DNA sequences coding for the said peptide fragments derived from the 17 kDa antigen and to the DNA and RNA probes constructed on the basis of the protein sequence of the 17 kDa ~nti~en includins the sequences of the peptide fragments of the 17 kDa antigen. A major field of use is the use of 17 k~a antigen and peptide fragments thereof in immuno diagnosis of tuberculosis. A further field of use is the use of the 17 kDa antigen or of the said peptide sub structuras thereof for the preparation of a vaccine against tuberculosis. A further field of use is the use of the 17 kDa antigen cr its sub structure peptides for the detection of T cell proliferation by skin tests or invitro tests in man. This last mentioned field of use is of importance in the possible treatment of human cancer by the boosting of cellular i~munity. A further field of use is the use of the 17 kDa antigen or its sub struc~ure peptides for the laboratory production of cellular growth factors and enzymes.

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3. Backqround _ invention Human tuberculosis caused by ~.tuberculosis is an important chronic debilitating disease occuring worldwide affecting about 16 million people. While it is prevalent ~ostly in under developed and developing countries, the recent epidemic of Acquired Immuno Deficiency Syndrome (AIDS) among developed countries has posed a problem of secondary infection with mycobacteria including M.tuberculosis.

Precise and confirmative diagnosis of human TB enables early treatment for which powerful antibiotics are available, although in many countries drug resistance has become a problem. Nevertheless, early diagnosis of TB means effective chemotherapy and thus elimination of the transmissiGn of live bacilli from pulmonary TB patients.

Conventional diagnosis of TB depends upon the clinical and radiological findings, microscopy of tuberculous specimen for bacilli, and bacteriological isolation in culture of M.tuberculosic.

Global control of TB has not yet been establi~hed bec2use of the inadequacy of the presently available methods of diagnosis~

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Thus many clinlcal features of TB are not specific to TB
alone and a ~tudy in India (sour~e: N~tional Tuberculosis Insitutute, Bangalore, India) revealed that only 30% of the X-ray suspect~ eventually developed TB although, in many countries including India, all X-ray sus~ects are put on anti tuberculous chemotherapy.

The microscopy of tuberculous specimen is not easy under field conditions and atleast 104 bacilli~ml are required for effective screening. Many tuberculous specimens like cerebro spinal fluids from tuberculous meningitis infrequently contain bacilli. Further, bacteriological culture generally take~ 6 to 8 weeks and is expensive as a routine diagnostic mea3ure .

The widely used tuberculin skin test lacks sensitivity and specificity and taXes about 3 days for compl~tion. Since chemotherapy of TB requires co~pliance for atleast 6 months, many patients who are irregular in treatment develop drug resistance and transmit live bacilll. Finally, the traditional BCG vaccination has now been found to give varying levels of protection depending upon geographic regions.

Consequent to these factors, many inve~tigators including the WHO recommend that early diagnosis of TB should be considered as a priority area of research and develop~ent.

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Mycobacteria are powerful immunogens for man and animals, as evidenced by their use ;r. i~nuno adjuvants to boost immune responses. Thus, many antigens derived from M.tuberculosis have been known to induce the formation of specific antibodies and proliferative lymphocytes among TB patients (Ivanyi et al., 1988! arld experimental animal models. The detection of an antibody response in TB has a potential application in early diagr.osis. Likewise, the study of M.tuberculosis specific T lymphocytes has application in early diagnosis by skin tests and developmPnt of protective vaccines.

Thus the focus of research in TB pertaining to these aspects lies in the identification and synthesis of M.tuberculosis specific antigens.

4. Prior Art Many investigators have identified protein antigens of M.tuberculosis which have potential immuno diagnostic or immuno prophylatic application. The N-terminal and internal amino acid se~uences o~ many of these antigens have been published. The N-terminal amino acid sequences of some of these are presented in Table 1.

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able I: M.tuberculosis protein antigens identified by N-terminal amino acid sequences.

Investigators Antigens and N-terminus ~~~~~~~__~,~, ~~~~~~_~~~~._~~~~~~~~~~~~~_~~~~~~~.~~~~~~~~~~~~~~___ Shinnick et al, 1987 65 kDa: R G C R H P V
Yamaguchi et al, 1987 MPB 57: M A K F N I K P L
Pattorroyo et al, 1987 13 kDa: A K V N I
18 kDa: G D L V G P G A E
23 kDa: A P K T Y
30 kDa: F S X P G L
68 kDa: W M T M T
77 kDa: G K X I A Y D G A A
Matsuo et al, 1988 30 k~a: F S R P G L P
Ashbridge et al, 1989 19 kDa: E H R V X R G L T V
Baird et al, 1989 10 kDa: A K V N I P K P
Garcia et al, 1989 70 kDa: F Q R I T R Q D L L
Borremans et al, 1989 32 kDa: F S R P G L P
~_~________~___..__________~-_~_____________________________~__ To our knowledge, none of these antigens have been introduced as immuno diagnostic test products. Two of these antigens (10 kDa of Baird et al, 1989 and a homologue of 65 kDa antigen of Shinnick et al, 1987) have been tested for vaccine potency among experimental animal models where they showed poor p.otection against M.tuberculosis (D.W. Smith, University of Wisconsin, USA, personal communication).

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An ELISA kit for diagnosing TE~ using A60 antigen present in all mycobacteria has been imtroduced by ANDA diagnostics (Franca). The test is thus not specific for human TB alone.

~n alternative method of diagnosis of TB has been the use of DNA probes. The commercially available GEN PROBE kit (1988) is used to confirm the identity of members of M.tuberculosis complex isolated in culture and has not been used directl~ on clinical specimen. A positive result with this test does not rule out other mycobacteria. The DNA probe devised by En o Biochem (J.Clin Microbiol.1988, Dec) used specific ~NA
sequences of lO00 bases or more in length and claimed to be more specificO A major handicap of all DNA based probes is that whether a true positive reaction can be obtained ~-ith specimens of patients so as to avoid the laborious culture of bacilli.

Phenotypic variation in virulence is known among the ~; ~uberculosis strains isolated in South India (Naganathan .
_t al 1987), the molecular basis for which is not yet established. Abou Zeid et al, (1988) found that a 13 kDa protein antigen was present in phaqe type II virulent M.tuberculosis and absent in phage type I South Indian low virulent M.tuberculosis. However, the relationship between this antigen and virulence is yet to be investigated in detail.

5. The Invention :

A 17 kDa protein antigen was found to be present among the isolates of M.tuberculosis. The immuno chemical features of this antigen are disclosed under this invention.

Thus the present invention relates to:

1. The 17 kDa protein antigen of M.tuberculosls (SII 1) as defined below and certain sub structures (peptides) of 17 kDa protein antigen as defined below.

2. A DNA sequence coding for the 17 kDa antigen from M.tuberculosis (SII 1).

3. A DNA sequence coding for the sub structures (peptides) of the 17 kDa antigen from M.tuberculosis (SII ').

~ 2 4. The use of the said 17 kDa protein or the said sub structures (peptides) for the preparation of ~onoclonal or polyclonal antisera which react with the said 17 kDa protein antigen or sub structures thereof. The said antibodies can be raised in mammals like mice, rabbits and goats for polyclonals and in mice for monoclonals.

5. The use of the said 17 kDa protein antigen of the said sub structures (peptides) for the detection of antibodies among human and animal specimen for immuno-diagnosis. Detection methods are known in the art one employs such as ELISA, radio immuno assay (RIA) and reverse passive hemagglutination (RPHA).

6. The use of the 17 k~a protein antigen or sub structures (peptides) thereof for the treatment of tuberculosis.

7. The use of the 17 kDa protein antigen or substructures peptides thereof for the preparation of a vaccine against l tuberculosis.

8. The use of the 17 kDa antigen or sub structures (peptides) thereof for the preparation of a reagent for the skin test in immuno diagnosis for tubercuiosis.

9. DNA or RNA probes constructled on the basis of the protein sequence of the 17 kDa antigen or sub structures (peptides) thereof for diagnosis of tuberculosis. Such probes ran be constructed by methods known in the art. Labelling of such probes can be done by known methods such as radioisotope incorporation or by non radio-active labelling use for example, biotin.

10. A method of diagnosis of human tuberculosis by interacting body fluids such as serum, CSF, pleural fluids from a patient to be diagnosed with a monoclonal antibody to the 17 kDa antigen or its sub structures (peptides) as defined in paragraph 3 above.

11. A method for diagnosis of human tuberculosis by interacting a body fluid such as serum from a patient to b~
diagnosed with a 17 kDa protein as defined in paragraph 5 above.

12. A method of diagnosing human tuberculosis by interacting body fluids such as sputum, serum, CSF and pleural fluids from a patient to be diagnosed with a DNA or RNA probe as defined in paragraph 9 above.

13. A method for in vitro detec~ion of human tuberculos~s which comprises contacting a sample of a body fluid such as 12 ~ 2 sputu~, CSF, pleural fluid ~r serum fro~ a patient with a monoclonal antibodY as d~fined in paragraph 10 in labelled form .

14. A method for in ~itro detection of hu~an tuberculosis, which co~prise~ contacting a sample of a body fluid such as sputum, CSF, pleural fluid or serum from a patient with a ~onoclonal anti~ody as defined in paragraph 10 in labelled form.

15. A method for in vitro detection of human tuberculosis, which comprises contacting a sample of a body fluid such as sputum, CSF, plental fluid or serum from a patient with a monoclonal antibody a~ defined in paragraph 10 in labelled form .

16. A kit for perfor~ing i~muno diagnosis of tuberculosis tilizing a ~onoclonal antibody to 17 kDa antigen as disclosed in paragraph 3 abov~.

1~. A kit for im~uno diagnosis of tuberculosis utilizing 17 kDa antigen or peptide sub structures as disclosed in paragraph 5 above.

18. A kit for di~gnosinq tuberculosis utilizing a DNA or RNA
probes as disclosed in paragraph 9 above.

19. A microorganism expressing a 17 kDa protein or sub struc-tures thereof as disclosed in paragraph 1 above. The term microorganism is intended to be a generic term and reference be made by example to bacterial microorganisms such as E.
coli, Bacillus subtilus, etc.

20. A vaccine against tuberculosis developed on the basis of the 17 kDa antigen or sub stL~cture peptides thereof as disclosed in paragraph l aboYe. Such a vaccine can be a product of genetically engir:eered organisms such as Salmonella, Vaccinia virus etc.

The present invention is exemplified by but nct limited to the diagnosis, therapy or prophylaxis of diseases, especially diagnosis of M.tuberculosis nfection. Epidemiological screening, forensic investigations, determination of food contaminations, public health surveys, preventive medicine, veterinary and agricultural applications with regard to the diagnosis of infectious agents may be covered by this disclosure.

5.l Fractionation and purification of l7 kDa antiqen.
5.l.l Crude sonicate antige~.
M.tuberculosis (SII l) was cultured at 37 C for 2 weeks in Kirchner's medium and harvested bacilli were killed in cold acetone for 18 h at 4 C. The bacilli were washed thrice with saline and a suspension of lO mg bacilli in 5 ml saline was sonicated at 40 watts output using a miniprobe of a Branson sonifier. The sonicate ~-as centrifuged at 20,000 x g for 30 min and the supernatant was estimated for protein content (Lowry's method) prior to rreeze storage at -70 C.

14 ~ 'J ~ 2 5.1.2 Fractionation and purification.
500 ug of cruda sonicate wa~ fractionated on 12.5~ sodium dodecyl sulfate polyacrylamide gel as described by Hunkapiller and Lujan (1986). The protein bands were visualized by brief staining with coomassie brilliant blue and the 17 kDa antigen was electroeluted against 0.05 M
ammonium bicarbonate with 0.1% SDS followed by electro-dialysis againEt 0.01 M ammonium bicarbonate with 0.02 % SDS.
The eluted protein was then extracted with chloroform-methanol to remove SDS and the precipitate was dried~

The purity of the eluted protein was analysed by subjecting 5 ug of this preclpitate to HPLC using Lichrosorb RP 18 column (LKB). A single pcak eluted at 45% B and 26 min was found to contain immuno reactive antigen. Fig. 1 shows the HPLC
profile of 17 kDa antigen.

5.2 Amino aci~ sequence analYsis of 17 kDa antiqen 5.2.1 Peptide mapping of 17 kDa antigen Tryptic map: 30 uy of 17 kDa protein was digested with TPCK
treated trypsin with an enzyme to substrate ratio of 1:50 in 0.1 M ammonium bicarbonate buffer, pH 7.8 at 37 C for 5 h. The tryptic digest was fractionated by HPLC on RP 18 column (0.46 x 25 cm~ equilibrated with solvent A (0.1% TFA
in water) and t~le peptides were eluted with a gradient of solvent B (70% acetonitrile containing 0.085% TFA) from 0 to ~ J

65% in 60 min. The tryptic map is shown in Fig. 2.
V8 protease map: 30 ug of 17 kDa antigen was treated with staphylococcal V8 protease for 48 h in 0.07% ammonia at 37 C. The molar ratio of the enzyme to substrate was 1:25. The various peptides in the enzyme digest were purified on an HPI,C column uder the conditions used for tryptic mapping.
The peptide profile is shown in Fig. 3.

5.~.2 Sequence analysis of 17 kDa antigen The amino acid sequence analysis of the protein and the pe~tide was done using protein sequencer model 477A (Applie~
Biosystems Inc., USA) with an on line PTH amino acid analyser. The sample was solubilized in 10% formic acid and fixed onto a polybrene coated (1 mg) TFA treated glass fibre disc and used for sequencing.

The first 18 amino acids from the N-terminal was determined using the whole protein. Based on the amino acid sequences of the tryp~ic peptides, V8 protease was selected to generate the peptides that could give the overlaps for the tryptic peptides. The alignment of both tryptic and V8 protease peptides gave the complete sequence for the 17 kDa antiger..
The details of the overlaps are given in Fig. 4.

5.2.3 Amino acid composition of 17 kDa antigen The protein has A9, C3, Dll, E10, F9, G8, H2, 17, K4, L11.

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M2, P9, Q2, R12, S8, T9, Vll and Y4. It is significant in not having tryptophan and asparagine. The protein is acidic in nature since it has 5 acidic amino acids (~E=21) in excess of the total number of basic amino aci~s (R+K=16).
The protein has 131 amino acids that account for a molecular weight of 14,762.

5.3 Immuno reactivitY of 17 kDa antiqen 5.3.1 Demonstration that 17 kDa antigen is i~nunodominant in M.tuberculosis strains.

M.tuberculosis (SII 1), M.tuberculosis ATCC 272~4, M.phlei, ~.
smeqmatis, M.kanasasii, M.avium intracellulare and M.scrofulaceum were cultured in Kirchner's mediu~, for 2 weeks and harvested bacilli were killed in cold acetone. Sonicate antigens were prepared from each of these species as described in section 5.1.1. S~S PAGE analyis of these antigens was then carried out in 12.5% gel. Coomassie blue stained gels showed that the 17 kDa antigen was present only in M.tuberculosis strains. A rabbit antiserum raised to M.tuberculosis SII 1 was also used to probe these sonicates in Western blotting. The dominant 17 kDa band was found in the Wes-tern blot.

5.3.2 Demonstration that 17 kDa elicits ar.tibodies amongexperimental animals.

Electro eluted 17 kDa antigen (10 u~ in 100 ul saline) was emulsified with an equal volume of Freund'S incomplete adjuvant (FICA) and used for intra peritoneal immunization of BALB/c mice. Serum collected from these mice 30 days after immunization recognized a '7 kDa band in the sonicate antigen of M.tuberculosis SII 1.

Thus this experiment confirms that a polyclonal or monoclonal antibody can be produced in the mouse which recognize the protein structure of 17 kDa antiyen or sub structures (peptides) thereof. Such antibodi~s, in particular the monoclonal antibodies can be used in an antigen detection method like the sandwich ELISA fs_ the detection of the 17 kDa antigen or sub structures thereof among human tuberculosis specimen leading to immuno diagnosis of tuberculosis.

5.3.3 Demonstration that 17 kDa antigen reacts with human TB
patient sera.
Sera derived from 24 healthy persons and 20 culturè proven TB
patients were titrated against the 17 kDa antigen as follows.
PVC Dynatech plates were coated with 1 ug/ml PBS of electro eluted 17 kDa antigen for 24 h at 22 C. PBS-BSA blocked plates were then titrated against duplicate (1/200) dilutions of sera which were incubated at G2 C for 2 . 5 h. Washed plates received anti human IgG ~P conjugate for 1~5 h.
Washed plates were then assayed with 0-phenylene diamine substrate and read at 492 nm. Table II shows that the 17 kDa antigen had a sensit.ivity of 70% and specificity of 85%

Table II. Micro EL~SA with 17 kDa antigen on sera from TB
patients and controls Serum groups n ELISA + ELISA- Sensitivity Specifici Healthy people 24 4 20 -- 85%
TB patients 20 14 6 70% --~~~~_~~~~~~~~_~__~~~~_~~~~~~~~~~~~~~~~~~~~_~~~~~~~~~~~~~~~~~~___~
Sensitivity: Known positivity among TB patients Specificity: Known negativity among healthy controls LISA + : OD 4g2 nm >=0.3 at 1/20G dilution (=mean + 2S~ ofOD 492 nm for healthy controls, n=24].
Thu~ this experimen. confirms that the 17 kDa antigen from M.tuberculosis strains can be used in a micro ELISA system for the immuno diagnosis of tuberculosis in man.

5.3.4 Demonstration that the 17-kDa protein antigen has defined antibod~ epitopes The peptide frag~ents derived from the 17 kDa antigen by tryptic digestion (section 5.2.1, Fig.2) were individually titrated against sera from healthy persons and TB patients as in section 5.3~3. O' the 14 peptides tested, peptides with the sequences RATYDK, YEVR, LEDEMK, LMR, DFDGR and SEFAYGSFVR
showed antibody binding activity with sensitivity levels between 1~ to 36%. To determine whether these peptides ~ ~J ? ~

formed linear or conformational antibody epitope~, an inhibition of ELISA was carried out in which each of the peptide was assayed against the other five using the mouse antiserum to 17 kDa antibody. The peptides YEVR and ATYDK
were mutually inhibitive thus indicating that they were a part of a complete antibody epitope, which was confirmed also by the determination of the complete structure of the 17 kDa antigen as in section 5.2.2, Fig 4. The other four peptides were linear and probably conormational in the presentation of the antibody epitopeO Of the said six antibody epitope bearing peptides, the peptides of the following amino acid se~uences were synthesized by solid phase m2thod of Merrifield and were found to contain specific and sensitive antibody binding activities :
RATYDXRY~VR : Sansitivity 65%; Specificity 95~
SEFAYGSFVR : Sensitivity 66%; Specificity 95%

The antibody epitope mapping as described has thus indicated that defined sub structures or peptides of 17 kDa ant.gen can be synthesized and used for the immunodiagnosis of human tuberculosis in micro ELISA.

5.3.5 Demonstration that 17 kDa antigen is lympho proliferative Periph~ral blood lymphocytes (PBL) from healty dor,ors and TB
patient~ were fractionated and 2 x 10 cells w~re cultured in ~ $ r~J

presence or absence (control~ of 1 ug of 17 kDa antigen for 3 days in RPMl 1640 mediu~ with 10~ autologous serum. 24 h before harvesting cultures wlere pulsed with 1 uCi of 3H
thymidine. Table III shows that the 17 kDa antigen was lympho proliferative to the lymphocytes of TB patients (data shown for 2 persons only)O

Table III. Lympho proliferative assay with 17 kDa antigen.
~________~.,~~~_~~__~~~~~~_~_~___~ _.,_ ____ ______~__~_~~~_~~~___ ource (PBL) 3~ thymidine incorporation (cpm, mean of triplicate cultures) ~~______~~~_~~~~~~________________~~_~~~~~_,_~~~____~_________~~~~
Healthy Control : 90 Antigen ~ 121 TB patient Control : 110 Antigen : 650 ___~__~~~~~~~~~__~~~__~~~~~~~~~~~_~__~~~~~~_~~~~~~~__~~~ ~_~~___~

In addition to the lympho proliferative property of the 17 kDa antigen, a method of prediction of T cell stimulatory epitopes (Rothbard and Taylor, 1988) was used to map the probable sites in the structure of the 17 kDa antigen. These T cell epitopes were located on peptides of the following sequences:
SEFAYGSFVR
AELPGVDPDCDVCITR

Thus this experiment indicatss that th~ 17 kDa antigen or sub structures (peptides~ can be used to stimulate human peripheral blood lymphocytes. Since stimulated l~mphocytes ~ ~ ~ 6~ 2 elaborate several cellular growth and differentiation factors which contribute to the vaccinle effect of 17 kDa a~tigen or its sub structures, the 17 kDa antigen can be used at the first instance as a vaccine against TB and also for non-specifically boosting cellular immunity.
Figure 5 shows the primary structure of the 17 kDa antigen from M.tuberculosi~ containing the biologically active regions, ~lthough similar activity need not be ruled out in the unmarked re~ions.
6. Discussion and summary of test results The present invention describes the immunochemical properties of a novel 17 kDa protein antigen from M.tuberculosis (SII 1 strain). M.tuberculosis causes tuberculosis worldwide among 16 million people. Because of inadequacy of the diagnostic procedures available now the disease has not yet been eradicated. The focus of research in recent years has been the development of immuno diagnostic methods for detecting T~
at an early stage as well identification of suitabl~
candidates for vaccination since the traditional BCG vaccine has given only a partial protection against TB.

The studies described in this invention show that a novel 17 ~Da antigen derived from M.tuberculosis has antigenic activity. Firstly, it was found to be unique for the South Indian strains of M.tuberculosis. Secondly, its antigenic 22 ~J ~q ~ 2 nature and chemistry were investigatad. Thirdly, the biologically active proteins of the protein antigen were mapped and immuno diagnostic ~ethods were developed for the early detection of TB.

Thus the 17 kDa protein antigen which had 131 amino acids was found to contain two peptides of sequence RATYDKRYEVR and SEFAYGSFVR which carried antibody binding epitopes for diagnosis of TB. Further, it contained two peptides which carried predicted T cell stimulating regions in sequences, SEFAYGSFVR and AELPGVDPDCDVCITR. The latter two peptides presumably contributed to the T cell stimulating property of the whole 17 kDa antigen described in this invention. The T
cell stimulating property of the 17 kDa antigen and its sub structure peptides means that they could be used in therapy and vaccination for T~.
The M. tuberculosis (SII 1) strain is freely available from the ATCC. It is coded as ATCC 35811 (Indian 79157) deposited by Tuberculosis Chemotherapy Centre, Madras, (see ATCC
Catalogue of sacteria, 17th edition (1989), page 143).

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7. Figure le~ends.

Fig. 1. HPLC analysis of the electro eluted 17 kDa protein antigen from M. tuberculosis (SII 1).
HPLC conditions: RP 18 column (LKB, 10 um pore size), A: 0.1%
TFA in water, B: 0.085% TFA in 70~ aceto nitrile, Gra~ient: 0 to 65% B in 40 min, Sensitivity: 0.08,220 nm.

Fig. 2~ Tryptic peptides of 17 kDa protein antigen lrom M.tuberculosis (SII 1) fractionated by HPLC on RP 18 (LKB,10 um pore size).
Fractionation: A: 0.1% TFA in water, B: 0.085% TFA in 70%
aceto nitrile, Gradient: 0 to 65% B in 60 min, Sensitivity:
O.08, 220 nm.
Amino acid sequence: Sequence determined are drawn against each peptide.

Fig. 3. V8 protease peptides of 17 kDa protein antiyen from M.tuberculosis (SII 1) fractionated by HPLC on RP 18 (LKB, pore size 10 um).
Fractionation: A: 0.1% TFA in water; B: 0.085% TFA in 70%
aceto nitrile, Gradient: 0 to 6S% in 60 min, Sensitivity:
0.082,220 nm.

Fig. 4. The primary structure of the 17 kDa antigen of M.tuberculosis ~SII 1) showing the alignment of pep.ides. Trp:
Trypsin, V8: Staphylococcus aureus V8 protease. Superseript arrows denote the amino acid sequence obtained with t~e whole protein.

(Single letter code used for amino acids).

Fig. 5. The primary structure of the 17kDa antigen from M.tuberculosis (SII 1) showing the biologically acti~e regions.
AA 68 to 77: Antibody and T cell epitopes present.
AA 91 to 101: Antibody epitope present.
AA 107 to 122: Two T cell epitopes present.

Claims

1. The use of the 17 kDa protein antigen derived from M.tuberculosis (SII 1) which has the following structure for the immuno diagnosis, therapy and vaccination in relation to human tuberculosis :

2. Use of the 17 kDa antigen or peptides thereof of structure as in claim 1 for producing either mammalian polyclonal antibodies or monoclonal antibodies in mice for further use in an immunodiagnostic method like sandwich ELISA
which detects 17 kDa antigen among human tuberculous specimen.

3. Use of the 17 kDa antigen or peptides thereof of structure as in claim 1 for the detection of antibodies among tuberculous specimen by serological methods for early detection of tuberculosis.

4. Use of the 17 kDa antigen or peptides thereof of structure as in claim 1, for the detection of T cell proliferation in tuberculous specimen for obtaining immuno diagnosis through skin tests or obtaining a candidate for vaccine against tuberculosis.

5. Use of 17 kDa antigen or peptides thereof of structure as in claim 1, for developing a reagent for therapy of tuberculous inflammation by T cell proliferation.

6. Use of the 17 kDa antigen or peptides thereof of structure as in claim 1, for the production of growth and differentiation factors using T cell proliferation assay.

7. The use of DNA or RNA probes constructed on the basis of the protein sequence of the 17 kDa antigen or peptides thereof of structure as in claim 1, for detection of M.tuberculosis DNA among human tuberculous specimen for early detection of tuberculosis.

8. The use of DNA or RNA probes constructed as in claim 7, for the purposes of identification of mycobacterial DNA for example among cultural isolates and for laboratory research.

9. A protein of the structure as given in claim 1.

10. Peptides of the structure given below :
SEFAYGSFVR
RATYDKRYEVR
AELPGVDPDCDVCITR

11. A DNA sequence coding for the protein defined in claim 1.

12. A DNA sequence coding for peptides defined in claim 10.

13. A hybridization probe, either DNA or RNA constructed on the basis of the proteins or peptides defined in claim 1 and or on the basis of DNA sequences according to claims 11 and 12.

14. A method of diagnosis of human tuberculosis which comprises interacting body fluids such as sputum, CSF, pleural fluid or serum from a patient with monoclonal antibody as defined in claim 2 in labelled form.

15. A method of diagnosis of human tuberculosis which comprises interacting body fluids such as serum from a patient with a 17 kDa protein as defined in claim 1 or with peptides as defined in claim 10.

16. A method of diagnosis of human tuberculosis which comprises interacting body fluids such as sputum, CSF, plural fluid or serum from a patient with a DNA probe as defined in claim 13 in labelled form.

17. A method for in vitro detection of human tuberculosis which comprises contacting a sample of a body fluid such as sputum, CSF, pleural fluid or serum from a patient with a monoclonal antibody as defined in Claim 2 in labelled form.

18. A method for in vitro detection of human tuberculosis, which comprises contacting a sample of a body fluid such as sputum, CSF, pleural fluid or serum from a patient with a monoclonal antibody as defined in Claim 2 in labelled form.

19. A method for in vitro detection of human tuberculosis, which comprises contacting a sample of a body fluid such as sputum, CSF, pleural fluid or serum from a patient with a polyclonal antibody as defined in Claim 2 in labelled form.

20. A kit for performing immuno diagnosis of tuberculosis utilizing a protein defined in any of claims 1 and 10.

21. A kit for performing immuno diagnosis of tuberculosis utilizing a monoclonal antibody as defined in claim 2.

22. A kit for performing DNA based diagnosis of tuberculosis utilizing a hybridization probe as defined in claim 13.

23. A microorganism expressing a protein as defined in claims 1 and 10.

24. A vaccine against tuberculosis on the basis of a protein as defined in claims 1 and 10.