Note: Descriptions are shown in the official language in which they were submitted.
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.
2 ~ t ~
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.
4 ~ J~
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~
~ 6~ ,3 ~
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.
2 ~
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.
7 ~ 3~ ?~
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).
8 J ' ~
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.
13
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.
~ $ ~ s~J
16
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.
17
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
18
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 controlsLISA + : 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 ? ~
19
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
21
23940-676
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
23940-676
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).
z3 ~ ?
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.
