WO1993014118A1 - Peptide fragments of hsp71 of m. tuberculosis and their use in diagnosis of tuberculosis - Google Patents
Peptide fragments of hsp71 of m. tuberculosis and their use in diagnosis of tuberculosis Download PDFInfo
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- WO1993014118A1 WO1993014118A1 PCT/GB1993/000087 GB9300087W WO9314118A1 WO 1993014118 A1 WO1993014118 A1 WO 1993014118A1 GB 9300087 W GB9300087 W GB 9300087W WO 9314118 A1 WO9314118 A1 WO 9314118A1
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- tuberculosis
- peptide according
- hsp70
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
Definitions
- the present invention relates to peptides which are useful in diagnosing paucibacillary mycobacterial infections.
- the paucibacillary stage of tuberculosis is very hard to detect or diagnose due to the effective absence of clinical signs and symptoms and the lack of detectable pathogen-originating antigens. Diagnosis in the multibacillary stage is relatively easy, for instance using well known smear test. Diagnosis at the paucibacillary (or smear-negative) stage is important in order that appropriate treatment and containment measures can be implemented to avoid transmission to persons at the greatest risk infection. To date efforts to improve the diagnosis of paucibacillary TB have been unsuccessful.
- the present inventors have now identified certain peptides which may be used in detecting paucibacillary TB and other mycobacterial diseases.
- the peptides are related to the carboxy terminal of the stress protein Hsp71 of Mycobacterium tuberculosis and corresponding heat shock proteins of other mycobacterial pathogens.
- the present inventors consider that the carboxy terminal regions of Hsp71 and corresponding mycobacterial heat shock proteins contain species-specific determinants having linear epitopes. These linear epitopes generate an antibody response during the paucibacillary stage of infection and peptides having corresponding sequences can be used to detect the antibodies in the serum or other body fluids of the patient.
- Peptides of the invention may be used for detecting or diagnosing a variety of paucibacillary mycobacterial diseases especially, but not limited to, tuberculosis of humans and wild or domestic mammals such as cattle, donkeys, deer and badgers.
- the peptides may also be used in detecting or diagnosing multibacillary mycobacterial infections.
- the present invention therefore provides a peptide comprising a sequence the same as or immunologically equivalent to a linear epitope of the carboxy terminal region of M. tuberculosis Hsp71 or a corresponding heat shock protein of any other mycobacterial pathogen.
- the peptide comprises a sequence the same as or immunologically equivalent to a linear epitope of the carboxy terminal of M. tuberculosis Hsp71.
- the invention will be illustrated with reference to the figures of the accompanying drawings in which:- Figure l shows the aligned carboxy terminal region sequences of M. tuberculosis Hsp71 and corresponding heat shock proteins using the internationally-accepted 1-letter code for amino acid residues.
- Hu Human hsp70-l
- Ec Escherichia coli dnaK
- Lp Mvcobacterium leprae Hsp71
- - gaps.
- FIG. 2 shows an analysis of antigens by SDS PAGE and Western blots.
- b Western blots developed with tuberculosis patient's serum.
- Figure 3 gives individual serum antibody titers to mycobacterial hsp70 (M-hsp70) and human hsp70(H-hsp70 in groups of patients and controls.
- Groups active TB(A); "self-healedā inactive TB(B); past treated inactive TB (C) ; lung fibrosis (D) ; diseases other than tuberculosis (E) ; healthy BCG-vaccinated individuals (F) (see also table 1) .
- Group means are indicated by the horizontal bars. Boxed symbols in group B: Patients given chemoprophylaxis due to severity of chest X-ray and clinical evaluation.
- Figure 5 gives the ratios between anti-mycobacterial and anti-human hsp70 antibody levels. Groups of patients:see legend to table 1.
- Figure 6 Shows antibody binding to octamer peptides derived from the carboxy-terminal sequence of the hsp7i protein M.tuberculosis. Serum from a patient with sputum smear-negative pulmonary tuberculosis, diluted 1:400 was reacted with solid-phase bound peptides using the ELISA method.
- Figure 7 Shows localization of epitopes within the carboxy-terminal hsp71 sequence. Mean absorbance values from eight adjacent octamer peptides each containing the individual amino acid residues listed on the horizontal scale. Sera tested at 1:400 dilution were collected from patients with smear-negative pulmonary tuberculosis (PT, see binding to individual peptides in figure 6) , lung disease caused by M.avium(AM) and lepromatous leprosy (LL) .
- PT smear-negative pulmonary tuberculosis
- A-D localisation of epitope core hexamers.
- Figure 8 Shows semi-quantitive evalution of antibodies to four (A-D) epitopes.
- Sera diluted 1:400 were collected from patients with smear- positive (full circle) or smear-negative
- tuberculosis and from non- tuberculosis controls (open circles) represented by one serum each from lepromatous leprosy, lung mycobacteriosis, Crohn's disease and a healthy subject. All sera were pre ā selected to contain high antibody levels to the whole hsp71 antigen. Absorbances (vertical scale) are mean values from adjacent peptides corresponding to each of the four epitopes (bottom horizontal scale) .
- the sequences of the carboxy terminal regions of M. tuberculosis Hsp71 and the corresponding human Hsp70 and Escherichia coli dnaK molecules and the Hsp71 of ______ leprae are set out in Figure 1.
- Four linear epitopes within the Hsp71 of M. tuberculosis are indicated by the core sequences A to D underlined in Figure 1. It is presently preferred that the peptide of the invention comprises at least one of these linear epitope core sequences or an immunological equivalent thereof.
- Core sequences A and C, especially the latter, which appear to be more highly specific to M. tuberculosis. are preferred for selective diagnosis of paucibacillary TB in humans.
- Core sequences B and D appear to be more immunogenic than sequences A and C but have some cross reaction between M. tuberculosis and M. leprae; these core sequences are therefore preferred for more highly sensitive diagnosis of paucibacillary TB where infection by M. leprae can be ruled out on other grounds.
- linear epitopes may be longer or shorter than the 6-residue core sequences given in Figure 1 but are unlikely to be less than five residues in length. Accordingly, preferred peptides of the invention comprise at least any five and preferably all six amino acid residues of one of the core sequences indicated in Figure
- More preferred peptides are octamers containing five or preferably all six of the amino acid residues of one of the core sequences and especially preferred peptides are octamers having the whole core sequence and two other residues corresponding to the two flanking residues at either the N- or C- flanks or one residue at each flank in the M. tuberculosis Hsp71 sequence given in Figure 1.
- Other preferred peptides of the invention contain a sequence immunologically equivalent to one of the aforementioned linear epitopes, i.e. peptides which cross- react with antibodies detected by peptides containing a linear epitope sequence; more preferably such immunological equivalents contain sequences which cross-react with one. of the aforementioned core sequences.
- the peptides of the invention may also contain sequences unrelated to Hsp7l or corresponding molecules, at either or both flanks of the core sequence or linear epitope sequence.
- the peptide comprises a branched polylysine bearing multiple copies of a linear epitope sequence or immunological equivalent thereof.
- Peptides of the present invention may be produced by well known peptide synthetic techniques using solid or liquid-phase reactions to assemble the peptide one amino acid residue at a time or by covalently binding preformed sub-sequences, e.g. by linking linear epitope sequences or immunological equivalents thereof to a branched polylysine or other branched structure.
- Branched polylysines and other suitable branched structures are commercially available or described in the literature and linking of peptides to these may be achieved by conventional techniques. Techniques for identifying linear epitopes and immunological equivalents thereof are readily available to those skilled in the art.
- the peptides of the invention may be used in any conventional immunodiagnostic test technique for detecting antibodies in a body fluid such as blood or serum.
- the invention further provides a diagnostic test process comprising contacting a peptide as hereinbefore defined with a body fluid sample taken from an individual suspected of having a mycobacterial infection.
- the peptide may be contacted in the liquid phase or the solid phase, in which case the peptide will be bound to a solid carrier such as the test vessel, for instance a microtitre plate, or latex beads. Binding of antibody from the sample to the peptide may be detected by any conventional technique.
- the peptide For use in certain diagnostic procedures it may be advantageous to bind the peptide to a detectable label such as an enzyme label, a chromophore, a fluorophore or a radio-isotope or to a solid support. Binding of the peptide to a label or solid supports may be effected by conventional techniques. Peptides bound to such labels and solid supports form a further aspect of the invention. Although it is presently envisaged that the peptides of the invention will be used in in vitro diagnost i c procedures, it is possible that in. vivo techniques will be used. Accordingly, the invention further provides a peptide as hereinbefore defined, optionally bound to a label or solid support, for use in a diagnostic process practised on the human or animal body.
- a detectable label such as an enzyme label, a chromophore, a fluorophore or a radio-isotope or to a solid support.
- the invention further provides a process for producing a peptide as hereinbefore defined; a process for linking a peptide as hereinbefore defined to a detectable label or solid support; and tests kits containing the peptide optionally bearing a label or linked to a solid support and optionally also comprising buffer, positive or negative control materials, labelling reagents, reagents for detecting a label and other conventional components.
- the invention will now be illustrated by the following Examples:-
- tuberculin PPD tuberculin PPD
- active tuberculosis 1,2,3
- antigenic epitopes with both species-specificity and strong immunogenicity would be required.
- mycobacterial antigens In smear-positive (multibacillary) tuberculosis (TB) , this requirement is best met by the 38 kDa protein antigen (reviewed in 4) .
- This protein originally identified by monoclonal antibodies (10) has been sequenced by cloning its gene from the DNA of M. leprae and M tuberculosis (11,12). It belongs to the family of heat shock proteins with broadly conserved structure between prokaryotes and eukaryotes. At this is a strong structural basis for autoimmunization, it was of interest to compare serum antibody levels to both mycobacterial and human hsp 70 molecules in groups of patients and controls.
- Serum samples All 198 tested sera were obtained from patients at the Northwick Park Hospital. (Informed consent was obtained from all patients and controls whose blood was sampled) . On the basis of clinical diagnosis, they were classified to groups listed in table 1. Patients with active tuberculosis (group A:90 total) were further classified in respect of pulmonary localization, sputum smear microscopy and bacteriological culture. The clinical parameters of patients with extrapulmonary disease have previously been described in greater detail (7) . Patients with "inactive tuberculosis" (36 total) were represented by patients with radiological evidence of old pulmonary tuberculosis without history of drug treatment (group B "self-healed") , and those with a history of drug treatment (group C "past-treatedā) .
- Groups without active or past TB were represented by patients with clinical and radiologically demonstrable lung fibrosis (group D) and by patients with other diseases such as pneumonia, sarcoidosis, bronchiactasis, carcinoma of the lung, viral infections, liver diseases or heart failure (group E) .
- Healthy subjects were age matched adult BCG-vaccinated individuals (group F) .
- hsp70 protein was prepared from the stress-inducible hsp70 gene (14) kindly provided by Dr Rick Morimoto, Northwestern University, Evanston, IL. , USA. It was subcloned in plasmid PET-3 (15) and expressed in E Coli using bacteriophage T 7 RNA polymerase system for selective high level expression (16) . Hsp70 protein was purified from the crude protein lysates (Jindal and Young, in preparation) .
- Enzyme Linked Immunoassav ELISA
- Polyvinyl microtitre plates (Nunc-Im uno plate MaxiSorp F96) were incubated with 50 ā l of either M-h ā p70 or H-hsp70 at concentration of 1 ā g/ml or 2 ā g/ml respectively at 4°C overnight. After washing once with phosphate-buffered saline containing 0.05% (w/v) "Tween 20" (PBST) , the wells were incubated with 200 ā l of 2% dried milk (w/v) in PBST (PBSTM) at 37°C for 1 hr.
- PBST phosphate-buffered saline containing 0.05%
- the 71kDa protein of M tuberculosis has biochemical and functional features consistent with other members of the hsp family (13) and has been found in supernatants from short-term culture of tubercle bacilli (19) .
- the extracellular location may enable contact of the molecule in its native conformation with B cells which is a requirement for effective stimulation of antibody formation.
- hsp70 Despite the greatly conserved amino acid sequence composition of hsp70, it is of interest that existing monoclonal antibodies have been found restricted in binding mainly to pathogenic mycobacteria of the M. tuberculosis complex. M. leprae and M. scrofulaceum (10) . Following infection with Schistosoma mansoni, antibodies also exhibited only limited cross-reactivity in respect of species specific epitopes of hsp70 and arose early in the course of infection (20,21). However, sera from humans infected with malaria contained autoantibodies which reacted with human hsp70 (22) . Increased antibody levels to mycobacterial hsp70 have been found in sera of patients with active pulmonary tuberculosis (23, 24), but these studies did not show individual values and did not evaluate antibody levels in relation to the stage of disease.
- the tips of the pins have been chemically derivatised to provide functional groups to which amino acids may be coupled in an appropriate form in peptide synthesis. All peptides have received N-terminal acetylation. Positive and negative control peptide sequences were synthesised on the same block as the test peptides to give an indication of the sensitivity being achieved in the ELISA assay.
- the pins were incubated overnight at 4°C in a microtitre plate containing 175 ā l/well of human serum diluted 1/400 in pre-coat buffer.
- the pins were removed from the primary antibody preparation and washed four times 10 min each in a bath of 0.01 M PBS (pH 7.2) at room temperature with agitation.
- the pins were placed in a microtitre plate containing 175 ā l/well of peroxidase labelled goat anti-human IgG diluted 1/1000 in conjugated diluent (1% v/v sheep serum, 0.1% v/v Tween 20,0.1% w/v sodium casemate and 0.01 M PBS pH 7.2) and incubated at 20°C for 60 min with agitation.
- conjugated diluent 1% v/v sheep serum, 0.1% v/v Tween 20,0.1% w/v sodium casemate and 0.01 M PBS pH 7.2
- the pins were washed as above and placed in a microtitre plate containing 150 ā l/well of substrate solution, 0.5 mg/ml of 2,2'-azino- di[3-ethyl-benthiazoline sulfonate] in citrate buffer pH 4.0 and 0.01% w/v hydrogen peroxide.
- the reaction was stopped by removing the pins from the plate after 30 min and immediately the optical density was measured at 405 n in a Titertek "Multiscan MCC II" spectrophotometer.
- each of the four identified epitopes have been alocated to six residues graded with the highest mean OD values. They are represented by sequences DAAVAE for epitope A, WRIGYF for epitope B, GEAGPG for epitope C and CVTGHW for epitope D ( Figure 1) . Alignment of these hexamers within the sequence of the hsp71 carboxy terminus from M.
- tuberculosis (10) , with corresponding ssequences of M.leprae hsp70 (9), E.coli dnaK, (12) and of the human HSP70-1 gene (13) revealed, that the core of epitope A is overlapping in 5 out of the 6 core residues with the M.leprae sequence.
- epitopes D, B and C shared only 2, 1 or no residue respectively with M.leprae.
- the immunogenicity of protein antigens in terms of antibody formation is largely based on the recognition of epitopes displayed on native molecules by the immunoglobulin receptors of B cells (14) .
- the epitopes which react with the bulk of antibodies in patients with tuberculosis are of confor ational nature (1, 15, 16) .
- the need for direct contact with B cells by the native antigens is corroborated by the fact that both 38kDa and 19kDa antigens are lipoproteins, secreted from live mycobacteria (17) and resulting in the highest antibody titres in multibacillary disease (2,3).
- hsp71 The pronounced immunogenicity of hsp71 during paucibacillary infection could be attributed to enhanced stress-induced synthesis during intracellular replication (19) , or to surface expression (20,21) enabling recognition by the Ig receptors of B cells or merely to earlier priming by multiple cross-reacting commensal microbial organisms and autologous hsp70.
- the hsp71 protein is a predominantly intracellular constituent (8)
- its presence in media from short-term mycobacterial cultures (22) indicates some form of secretion from live bacteria which could be contributory to its immunogenicity for B cells.
- Ashbridge K.R. Pre ā tidge R.K., Booth R.J. and Watson J.D. (1990) The mapping of a serodominant region of the Mycobacterium tuberculosis 19 kilodalton antigen. J. Immuno. 144:3137-3142.
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Abstract
Peptides comprising a sequence the same as or immunologically equivalent to a linear epitope of the carboxy terminal region of Mycobacteria tuberculosis heat shock proteins such as Hsp71 useful in the diagnosis of paucibacillary tuberculosis (TB). Methods of diagnosis of TB using such peptides and test kits including such peptides are also provided.
Description
PEPTIDE FRAGMENTS OF HSP71 OF M. TUBERCULOSIS AND THEIR USE IN DIAGNOSIS OF TUBERCULOSIS
The present invention relates to peptides which are useful in diagnosing paucibacillary mycobacterial infections. The paucibacillary stage of tuberculosis is very hard to detect or diagnose due to the effective absence of clinical signs and symptoms and the lack of detectable pathogen-originating antigens. Diagnosis in the multibacillary stage is relatively easy, for instance using well known smear test. Diagnosis at the paucibacillary (or smear-negative) stage is important in order that appropriate treatment and containment measures can be implemented to avoid transmission to persons at the greatest risk infection. To date efforts to improve the diagnosis of paucibacillary TB have been unsuccessful.
The present inventors have now identified certain peptides which may be used in detecting paucibacillary TB and other mycobacterial diseases. The peptides are related to the carboxy terminal of the stress protein Hsp71 of Mycobacterium tuberculosis and corresponding heat shock proteins of other mycobacterial pathogens.
Without wishing to be bound by this theory the
present inventors consider that the carboxy terminal regions of Hsp71 and corresponding mycobacterial heat shock proteins contain species-specific determinants having linear epitopes. These linear epitopes generate an antibody response during the paucibacillary stage of infection and peptides having corresponding sequences can be used to detect the antibodies in the serum or other body fluids of the patient.
Peptides of the invention may be used for detecting or diagnosing a variety of paucibacillary mycobacterial diseases especially, but not limited to, tuberculosis of humans and wild or domestic mammals such as cattle, donkeys, deer and badgers. The peptides may also be used in detecting or diagnosing multibacillary mycobacterial infections.
The present invention therefore provides a peptide comprising a sequence the same as or immunologically equivalent to a linear epitope of the carboxy terminal region of M. tuberculosis Hsp71 or a corresponding heat shock protein of any other mycobacterial pathogen.
Preferably the peptide comprises a sequence the same as or immunologically equivalent to a linear epitope of the carboxy terminal of M. tuberculosis Hsp71. The invention will be illustrated with reference to the figures of the accompanying drawings in
which:- Figure l shows the aligned carboxy terminal region sequences of M. tuberculosis Hsp71 and corresponding heat shock proteins using the internationally-accepted 1-letter code for amino acid residues. Hu=Human hsp70-l, Ec=Escherichia coli dnaK, Lp=Mvcobacterium leprae Hsp71, Tb=Mycobacterium tuberculosis Hsp71 .=sequences identical with M.tuberculosis. -=gaps.
Figure 2 shows an analysis of antigens by SDS PAGE and Western blots. Ec=extract from E coli a=coomassie blue stain; b=Western blots developed with tuberculosis patient's serum.
Figure 3 gives individual serum antibody titers to mycobacterial hsp70 (M-hsp70) and human hsp70(H-hsp70 in groups of patients and controls. Groups:active TB(A); "self-healed" inactive TB(B); past treated inactive TB (C) ; lung fibrosis (D) ; diseases other than tuberculosis (E) ; healthy BCG-vaccinated individuals (F) (see also table 1) . Group means are indicated by the horizontal bars. Boxed symbols in group B: Patients given
chemoprophylaxis due to severity of chest X-ray and clinical evaluation.
Figure 4 gives antibody levels in active tuberculosis divided according to sputum bacillary status. S=smear microscopy; C=culture of M tuberculosis. Group means are indicated by horizontal bars.
Figure 5 gives the ratios between anti-mycobacterial and anti-human hsp70 antibody levels. Groups of patients:see legend to table 1.
Figure 6 Shows antibody binding to octamer peptides derived from the carboxy-terminal sequence of the hsp7i protein M.tuberculosis. Serum from a patient with sputum smear-negative pulmonary tuberculosis, diluted 1:400 was reacted with solid-phase bound peptides using the ELISA method.
Figure 7 Shows localization of epitopes within the carboxy-terminal hsp71 sequence. Mean absorbance values from eight adjacent octamer peptides each containing the individual amino acid residues listed on the horizontal scale.
Sera tested at 1:400 dilution were collected from patients with smear-negative pulmonary tuberculosis (PT, see binding to individual peptides in figure 6) , lung disease caused by M.avium(AM) and lepromatous leprosy (LL) .
A-D=localisation of epitope core hexamers.
Figure 8 Shows semi-quantitive evalution of antibodies to four (A-D) epitopes. Sera diluted 1:400 were collected from patients with smear- positive (full circle) or smear-negative
(shaded circle) tuberculosis and from non- tuberculosis controls (open circles) represented by one serum each from lepromatous leprosy, lung mycobacteriosis, Crohn's disease and a healthy subject. All sera were pre¬ selected to contain high antibody levels to the whole hsp71 antigen. Absorbances (vertical scale) are mean values from adjacent peptides corresponding to each of the four epitopes (bottom horizontal scale) .
The sequences of the carboxy terminal regions of M. tuberculosis Hsp71 and the corresponding human Hsp70 and Escherichia coli dnaK molecules and the Hsp71 of ______ leprae are set out in Figure 1. Four linear epitopes within the Hsp71 of M. tuberculosis are indicated by the
core sequences A to D underlined in Figure 1. It is presently preferred that the peptide of the invention comprises at least one of these linear epitope core sequences or an immunological equivalent thereof. Core sequences A and C, especially the latter, which appear to be more highly specific to M. tuberculosis. are preferred for selective diagnosis of paucibacillary TB in humans. Core sequences B and D appear to be more immunogenic than sequences A and C but have some cross reaction between M. tuberculosis and M. leprae; these core sequences are therefore preferred for more highly sensitive diagnosis of paucibacillary TB where infection by M. leprae can be ruled out on other grounds.
The linear epitopes may be longer or shorter than the 6-residue core sequences given in Figure 1 but are unlikely to be less than five residues in length. Accordingly, preferred peptides of the invention comprise at least any five and preferably all six amino acid residues of one of the core sequences indicated in Figure
1. More preferred peptides are octamers containing five or preferably all six of the amino acid residues of one of the core sequences and especially preferred peptides are octamers having the whole core sequence and two other residues corresponding to the two flanking residues at either the N- or C- flanks or one residue at each flank in
the M. tuberculosis Hsp71 sequence given in Figure 1. Other preferred peptides of the invention contain a sequence immunologically equivalent to one of the aforementioned linear epitopes, i.e. peptides which cross- react with antibodies detected by peptides containing a linear epitope sequence; more preferably such immunological equivalents contain sequences which cross-react with one. of the aforementioned core sequences.
The peptides of the invention may also contain sequences unrelated to Hsp7l or corresponding molecules, at either or both flanks of the core sequence or linear epitope sequence. In a particularly preferred embodiment the peptide comprises a branched polylysine bearing multiple copies of a linear epitope sequence or immunological equivalent thereof.
Peptides of the present invention may be produced by well known peptide synthetic techniques using solid or liquid-phase reactions to assemble the peptide one amino acid residue at a time or by covalently binding preformed sub-sequences, e.g. by linking linear epitope sequences or immunological equivalents thereof to a branched polylysine or other branched structure. Branched polylysines and other suitable branched structures are commercially available or described in the literature and linking of peptides to these may be achieved by conventional techniques.
Techniques for identifying linear epitopes and immunological equivalents thereof are readily available to those skilled in the art.
The peptides of the invention may be used in any conventional immunodiagnostic test technique for detecting antibodies in a body fluid such as blood or serum.
The invention further provides a diagnostic test process comprising contacting a peptide as hereinbefore defined with a body fluid sample taken from an individual suspected of having a mycobacterial infection. The peptide may be contacted in the liquid phase or the solid phase, in which case the peptide will be bound to a solid carrier such as the test vessel, for instance a microtitre plate, or latex beads. Binding of antibody from the sample to the peptide may be detected by any conventional technique.
For use in certain diagnostic procedures it may be advantageous to bind the peptide to a detectable label such as an enzyme label, a chromophore, a fluorophore or a radio-isotope or to a solid support. Binding of the peptide to a label or solid supports may be effected by conventional techniques. Peptides bound to such labels and solid supports form a further aspect of the invention. Although it is presently envisaged that the peptides of the invention will be used in in vitro
diagnostic procedures, it is possible that in. vivo techniques will be used. Accordingly, the invention further provides a peptide as hereinbefore defined, optionally bound to a label or solid support, for use in a diagnostic process practised on the human or animal body.
The invention further provides a process for producing a peptide as hereinbefore defined; a process for linking a peptide as hereinbefore defined to a detectable label or solid support; and tests kits containing the peptide optionally bearing a label or linked to a solid support and optionally also comprising buffer, positive or negative control materials, labelling reagents, reagents for detecting a label and other conventional components. The invention will now be illustrated by the following Examples:-
EXAMPLE 1 INTRODUCTION
In the past, hemagglutinating antibodies to tuberculin PPD were demonstrated in the serum of patients with active tuberculosis (1,2,3). However, in order to achieve satisfactory specificity and sensitivity of serology, antigenic epitopes with both species-specificity and strong immunogenicity would be required. Recent advances in the molecular definition of mycobacterial antigens has been exploring possibilities for developing a clinically useful test for the serodiagnosis of tuberculosis. In smear-positive (multibacillary) tuberculosis (TB) , this requirement is best met by the 38 kDa protein antigen (reviewed in 4) . However, in the case of smear-negative (paucibacillary) disease where laboratory diagnostic help is most required, the search for an "ideal" overlap in specificity and immunogenicity within a single epitope has not as yet been forthcoming. Antibody levels to the 38kDa protein which represents the most specific and immunogenic antigen in multibacillary tuberculosis have been found much lower in smear-negative pulmonary tuberculosis (5,6); nevertheless, useful diagnostic discrimination of paucibacillary patients with this antigen was achieved using a more sensitive modification of the competition test (7) . Of the other candidate antigens, about one half of smear-negative patients had a specific rise of antibodies against the l9kDa and to a lesser extent to the 14 kDa antigens (6) , the
latter being compromised in its discriminatory potential by raised antibody levels in exposed healthy subjects (8) . Consequently a combination of tests also involving antibody levels to the M tuberculosis-specific TB78-epitope of the hsp65 protein and lipoarabinomannan have been advocated (9) . In order to expand further the serological evaluation of mycobacteriai antigens of defined structure, we examined in this paper antibody levels in patients and controls in respect of the hsp70 antigen. This protein, originally identified by monoclonal antibodies (10) has been sequenced by cloning its gene from the DNA of M. leprae and M tuberculosis (11,12). It belongs to the family of heat shock proteins with broadly conserved structure between prokaryotes and eukaryotes. At this is a strong structural basis for autoimmunization, it was of interest to compare serum antibody levels to both mycobacterial and human hsp 70 molecules in groups of patients and controls.
MATERIALS AND METHODS
Serum samples. All 198 tested sera were obtained from patients at the Northwick Park Hospital. (Informed consent was obtained from all patients and controls whose blood was sampled) . On the basis of clinical diagnosis, they were classified to groups listed in table 1. Patients with active tuberculosis (group A:90 total) were further classified in respect of pulmonary localization, sputum smear microscopy and bacteriological culture. The clinical parameters of patients with extrapulmonary disease have
previously been described in greater detail (7) . Patients with "inactive tuberculosis" (36 total) were represented by patients with radiological evidence of old pulmonary tuberculosis without history of drug treatment (group B "self-healed") , and those with a history of drug treatment (group C "past-treated") . Groups without active or past TB were represented by patients with clinical and radiologically demonstrable lung fibrosis (group D) and by patients with other diseases such as pneumonia, sarcoidosis, bronchiactasis, carcinoma of the lung, viral infections, liver diseases or heart failure (group E) . Healthy subjects were age matched adult BCG-vaccinated individuals (group F) .
Preparation of antigens: The gene coding the 71kDa protein of M tuberculosis inserted into the PUC8 multicopy plasmid was expressed in TGI cells and purified by ATP-agarose chromatography (13) . The material used in the study (M- hsp70) was prepared and kindly supplied from the WHO IMMTUB/IMMLEP Bank by Dr. Jan van Embden (Bilthoven, The Netherlands) .
Recombinant human hsp70 protein was prepared from the stress-inducible hsp70 gene (14) kindly provided by Dr Rick Morimoto, Northwestern University, Evanston, IL. , USA. It was subcloned in plasmid PET-3 (15) and expressed in E Coli using bacteriophage T7 RNA polymerase system for selective high level expression (16) . Hsp70 protein was purified from the crude protein lysates (Jindal and Young, in preparation) .
The purity of both recombinant hsp70 antigens was
examined by SDS PAGE and Western blot analysis (figure 2) . The results showed a single major band of 70 kDa for both the mycobacterial and human recombinant antigens. Only a few minor contaminating bands of lower molecular weight were detected in the H-hsp70 preparation with the human serum which reacted with several proteins in the crude E coli extract.
Table 1. Clinical Classification Of Patients
* - Corresponding with designations in figure 12
° - Number of patients
S = smear microscopy (+ or -)
C = culture of M .tuberculosis (+ or -)
Enzyme Linked Immunoassav (ELISA) . Polyvinyl microtitre plates (Nunc-Im uno plate MaxiSorp F96) were incubated with 50μl of either M-hεp70 or H-hsp70 at concentration of 1 μg/ml or 2μg/ml respectively at 4°C overnight. After washing once with phosphate-buffered saline containing 0.05% (w/v) "Tween 20" (PBST) , the wells were incubated with 200μl of 2% dried milk (w/v) in PBST (PBSTM) at 37°C for 1 hr. Four fourfold dilutions of human sera (1/50-1/3200) using 5°μl volume were added to duplicate wells and incubated at 37°C for 2 h. After 5 washings with PBST, 50μl of affinity purified goat antihu an IgG peroxidase conjugate (diluted 1/1000) was added to each well and the plates were further incubated for 1 h. After washing with PBST, 75μl tetramethyl benzidine plus hydrogen peroxide in citrate buffer at pH 5.1 was added to each well; the reaction was stopped in 10 min. time with 25μl per well 0.5 mol/L sulphuric acid. The absorbance at 450 nm was read in the "Titertek Multiskan II" spectrophotometer. Antibody titers were expressed as the dilution of serum giving 30% of the plateau binding value of a standard positive serum (ABT30) .
Statistical analysis: Means and standard deviations of the log10 values of antibody titers were calculated for each group of patients and controls. Groups were compared with each other by the student t-test for unpaired values. The proportion of patients with increased antibody levels in groups A-D was calculated with 98% specificity of the cut¬ off limit in comparison with values in group E.
RESULTS
Patients with diseases other than TB (group E) had antibody levels to both M-hsp70 and H-hsp70 significantly IĀ£ < .001) elevated above those found in healthy individuals (group F) (Figure 3) . Therefore antibody levels in group E rather than those of the healthy controls (group F) were used as the "baseline" for evaluating the extent and significance of rise in antibody levels in groups with either active or inactive TB.
Patients with active tuberculosis (group A, Figure 3) had IgG antibody titers to M-hsp70 (mean ABT303.2+0.4) significantly higher £P < .001) than that of non-TB patients (group E) (mean ABT302.310.4). When taking the anti-M-hsp70 logI0 titer 2.75 as the "cut-off" value 78% out of the 70 tested patients with active TB and significantly raised antibody levels. Corresponding evaluation of anti-H-hsp70 antibody levels using the log10 titer 2.5 as the discriminatory cut-off value in respect of group E, produced a somewhat lower, 50% positivity rate in patients with active TB.
Analysis of titers in groups of patients with "inactive tuberculosis" in comparison with group E deomonstrated that antibody levels have been increased to a significantly greater extent in self-healed (group B:80% for M-hsp70 and 67% for H-hsp70) than in the past treated (group C:53% for M-hsp70 and 47% for M-hsp70) individuals. In view of the large individual variations, we examined the possible association of antibody levels with several clinical
criteria such as age, race, duration of illness, chest X- ray, weight loss, fever and history of tuberculosis. However, none of these parameters were significantly correlated with antibody levels. Interestingly, the clinical evaluation revealed that both anti-M and anti-H hsp70 titers were the highest in five patients of group B (boxed in figure 3) who were on basis of prior independent clinical and radiographic evaluation considered to be at risk of relapse and were therefore put on "secondary" rifampicin/isoniazid chemoprophylaxis. However, irrespective of the tuberculosis process, uniformly high antibody levels were also observed in all five tested patients with lung fibrosis (group D) . Since most of the non-tuberculosis pulmonary and extrapulmonary diseases had "chronic inflammation" but did not have raised antibody levels, it would seem that anti- hsp70 antibodies are not stimulated merely by inflammation but rather represent a marker of the next stage, ensuing fibrosis. It was of further interest to break down the antibody levels within the group with active TB (column A in figure 3) in relation to bacterial smear and culture positivity. The values expressed in Figure 4 showed no significant differences between the multibacillary (S+C+) , intermediary (5'C+) and paucibacillary (S'C") sub-groups in respect of either anti-M or anti-H- hsp70 antibody titers. Comparison of antibody titers to M- and H- hsp70 antigen in individual patients showed highly significant correlation coefficients for all tested groups (mean R value
= 0.66) . Nevertheless, it was noted that patients with active TB, had a relatively high ratio of titres to mycobacterial compared to human hsp70 (M/H ratio 4.2). The form of tuberculosis of the 11 patients with > 10 M/H ratio was pulmonary (3x) , lymph node (4x) , bone & skin (3x) and pericardial (lx) . In contrast, a higher proportion of anti- human hsp70 titres (M/H ratio = 1.8) was found in patients with lung fibrosis and other non-TB diseases (figure 5) .
DISCUSSION
Advances in the structural definition of mycobacterial antigens enabled a systematic study of specificity of the immune repertoire (4) . Since, the relationships between the structure of antigenic molecules and their immune functions following infection with pathogenic mycobacteria are being established. The antibody repertoire of mice has been found to be distinctly more selective during infection with M. tuberculosis than following artificial immunization procedures (17) . Hence, potential serodiagnostic antigens with high immunogenicity and specifically for the infecting pathogen need to be selected on the basis of detailed knowledge of the human immune system to pathogenic mycobacterial infection. The hsp70 protein, chosen for the present study, represents a well known major stress protein which is highly inducible under stress conditions (e.g. heat shock) and is one of the HLA linked hsp70 genes (18) . Proteins of this family "chaperone" the folding and transport of intracellular
polypeptides. The 71kDa protein of M tuberculosis has biochemical and functional features consistent with other members of the hsp family (13) and has been found in supernatants from short-term culture of tubercle bacilli (19) . The extracellular location may enable contact of the molecule in its native conformation with B cells which is a requirement for effective stimulation of antibody formation.
Despite the greatly conserved amino acid sequence composition of hsp70, it is of interest that existing monoclonal antibodies have been found restricted in binding mainly to pathogenic mycobacteria of the M. tuberculosis complex. M. leprae and M. scrofulaceum (10) . Following infection with Schistosoma mansoni, antibodies also exhibited only limited cross-reactivity in respect of species specific epitopes of hsp70 and arose early in the course of infection (20,21). However, sera from humans infected with malaria contained autoantibodies which reacted with human hsp70 (22) . Increased antibody levels to mycobacterial hsp70 have been found in sera of patients with active pulmonary tuberculosis (23, 24), but these studies did not show individual values and did not evaluate antibody levels in relation to the stage of disease.
The results in this study demonstrated significant elevation of anti-hsp 70 antibody levels in the majority (78%) of patients with active pulmonary TB. Furthermore, about 40% (based on the ratio between titres) of the antibodies appeared to be cross-reactive with the human hsp70 molecule. Despite its highly conserved aminoacid structure, the stimulation of anti-hsp70 antibodies appears
to be strongly associated with the tuberculosis infection since patients with a broad range of other diseases (excluding tuberculosis) had much less raised antibodies that TB patients, when compared with healthy controls. In this respect the anti-hsp70 response follows a quite different pattern than that to hsp65 which showed a very broad range of titres in non-TB controls which prevented any meaningful discrimination from tuberculosis patients (6) . However, the persistence of raised hsp70 antibody levels for several years following active tuberculosis, which is known to apply also to other antigenic specificities, represents a caveat for diagnostic consideration.
The most striking finding has been the lack of any difference in the serological response to hsp70 between multibacillary and paucibacillary cases of tuberculosis. This finding is in contrast with the much more pronounced response of smear-positive TB patients to the previously examined M. tuberculosis specific epitopes (5,6). Obviously there may be a limitation for diagnostic application due to the reported elevation of anti-hsp70 levels in some autoimmune diseases such as systemic lupus erythematosis and rheumatoid arthritis (24,25). However, in autoimmune patients and also in the inactive TB cases, the antibodies appear to be predominantly cross-reactive with human hsp70. On the other hand it is encouraging that the anti- mycobacterial titres were approximately four times higher in the diagnostically difficult smear-negative and extrapulmonary cases as often as in smear-posi ive pulmonary TB. In fact, of the 11 sera with < 10 M/H ratio, 73% were
from patients with extrapulomanry TB. Hence, there would would be scope for improving further the mycobacterial specificity of the serology by using synthetic peptides or truncated recombinant segments (26) from the carboxy- terminal portion of the molecule which contains amino acid sequences with the most pronounced specificity for M.tuberculosis (12) .
The mechanisms, which cause the apparently unique immunogenicity of hsp70 in the early stages of tuberculosis need further study. Proliferative responses of peripheral blood CD4 T cells to mycobacterial hsp70 were observed to be somewhat stronger than those to hsp65 (27) and CD8 T cell clones of hsp70 specificity were obtained from the peripheral blood of tuberculosis patients (28) . The reported role of host hsp70 in processing of antigenic peptides (29) and expression on the surface of stimulated macrophages (30) could play a role for enhancing antibody responses to low levels of antigenic stimulation. The reported induction of synthesis of bacterial hsp70 together with other stress proteins during intracellular replication (31) could promote bacterial persistence via the ATP binding activity or by the special genetic regulatory elements (12) of hsp70. The pronounced serological response to hsp70 may indeed be signalling a distinct yet unrecognised function of this protein in the pathogenic host-parasite relationship.
REFERENCES FOR EXAMPLE 1
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Britton WJ, Asbur, L, Trent RJ Basten A. Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein
70 of eucaryotes. Infect Immun 1989;57:204-212.
12. Lathihgra R, Alexander W. Stover K, Coadwell J, Young R, Young D. Genetic Characterization of the dnaK-grpE-dnaJ heat shock gene cluster in
Mycobacterium tuberculosis in press.
13. Mehlert A, Young D.B. Biochemical and antigenic characterisation of the Mycobacterium tuberculosis 71 kD antigen, a member of the 70kD heat-shock protein family. Mol Microbiol 1989; 3:125-130.
14. Hunt C, Morimoto RI. Conserved features of eukarotic HSP70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc.
Natl. Acd. Sci. USA 1985; 82:6455-6459.
15. Rosenberg AH, Lade BN, Cui DS, Lin S-W, Dunn J,J Studier FW. Gene 1987;56:125-135.
16. Studie, FW, Moffatt BA. Use of bacteriophage T7 RNA Polymerase to direct selective high level expression of cloned genes. J Mol Biol 1986;189:113-130.
17. Brett SJ, Ivanyi J. Genetic influences on the immune repertoire following tuberculosis infection in mice. Immunology 1990; 71:113-119.
18. Sargent CA, Dunham I, Trowsdale J, Cambell RD, Human major histocompatibility complex contains genes for the major heat shock protein HSP70. Proc Natl Acad Sci USA 1989;86;1968-1972.
19. Abou-Zeid C, Smith I, Grange JM, Ratliff TL Steele J, Rook GA.W. The secreted antigens of M. tuberculosis and their relationship to those recognised by the available antibodies. J. Gen Microbiol 1988;134;531-538.
20. Hedstrom R, Culpepper J, Harrison RA, Agabian N, Newport G, A major immunogen in Schistosoma Mansoni infections is homologous to the heat-shock protein Hsp70. J Exp Med 1987;165:1430-1435.
21. Newport GR, Hedstrom RC, Kallestad J, Tarr P, Klebanoff S, Agabian N. Identification, molecular cloning and expression of a schistosome antigen displaying diagnostic potential. Am. J. Trop Med
Hyg 1988; 540:540-546.
22. Mattei D, Scherf A, Bensaude 0, da Silva LP, A heat shock-like protein from the human malaria parasite Plasmodium falciparum induces autoantibodies. Eur J Immunol 1989;19:1823-1828.
23 Britton WJ, Hellqvist L, Basten A, Inglis AS,
Immunoreactivity of a 70 kDa protein purified from
Mycobacterium bovis bacillus Calmette-Guerin by monoclonal antibody affinity chromatograph J Exp Med 1986,-164695-708.
24. Tsoulf, G, Rook GAW, Bahr, GM, Sattar, MA,
Behbehani K, Young DB, Mehlert A, van-Embden JDA, Hay FC, Isenberg DA, Lydyard PM. Elevated IgG antibody levels to the mycobacterial 65-kDa heat shock protein are chaacteristic of patients with rheumatoid arthritis. Scand J Immunol 1989;30:519-
527.
25. Minota S, Cameron B, Welch WJ, Winfield JB, Autoantibodies to the constitutive 73-kD member of the hsp70 family of heat shock proteins in systemic lupus erythematosus. J Exp Med 1988;168:1475-1880.
26. Mehra V, Sweetser D, Young RA, Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci USA 1986;83:7013-7017.
27. Adams E, Garsia RJ, Hellqvist L, Holt P, Basten A T cell reactivity to the purified mycobacterial antigens p65 and p70 in leprosy patients and their housefhold contacts. Clin Exp Immunol 1990;80:206-
212.
28. Rees A, Scoging A, Mehlert A, Young DB, Ivanyi J. Specificity of proliferative response of human CD8
clones to mycobacterial antigens. Eur J Immunol 1988;18:1881-1887.
29. Vanbuskirk A, Crump BL, Margoliash E, Pierce SJ. A peptide protein having a role in antigen presentation is a member of the Hsp70 heat shock family. J Exp Med 1989: 170:1799-1809.
30. Jarjour W, Tsai V. Woods V, Welch W, Pierce S, Shaw M, Mehita H, Dillmann W, Zvaifler N, Winfield J.
Cell surface expression of heatshock proteins. Arthritis Rheum 1989:32-S44.
31. Buchemeier N.A, Heffron F, Induction of Salmonella stress proteins upon infection of macrophages.
Science 1990;248:730-732.
EXAMPLE 2
INTRODUCTION
The specificity of the immune repertoire in mycobacterial infections has been of interest from several aspects of the immunopathogenesis as well as for potentials towards diagnostic and vaccine development. Serological studies suggested that the antibody response in respect of species-specific protein antigens is restricted to relatively few constituents represented by the 38kDa, 19kDa and 14kDa antigens in tuberculosis and by the 35 kDa antigen
in leprosy (1) . Antibody levels to all these antigens have been found most pronounced in patients with multibacillary forms of the disease which are clinically the most apparent and can be confirmed by bacteriological techniques. However, the sensitivity of detection of paucibacillary infection i.e. smear/culture negative tuberculosis using single or several antigenic probes has been less satisfactory (2-4) .
A new important advancement in this direction has been achieved recently, by the demonstration that antibody levels to the mycobacteriaIs hsp71 protein have been elevated to the same extent in patients with either smear- positive or smear-negative tuberculosis (5) . The hsp71 antigen, originally identified by monoclonal antibodies is a stress protein constituent of mycobacterial (6-8) . In view of this new opportunity for early serological diagnosis of paucibacillary disease it has been of further interest to identify those hsp71 epitopes which are both immunodominant as well as M.tuberculosis-specific. The need for focusing on the selection of species specific epitopes has been mandatory, to avoid the detection of cross-reactive antibodies to those parts of the hsp70 molecule which carries extensive structural homology with the corresponding proteins contained in all microbial and eukaryotic cells (7) . Thus, we had chosen for epitope scanning the carboxy- terminal part of the molecule which has been found most polymorphic, in alignment of the hsp70 sequences from mycobacteria and other species(9).
MATERIALS AND METHODS
Peptides
The general net synthesis of the peptides purchased from Cambridge Research Biochemicals (CRB) (Cambridge, England) was comprised of 94 peptides based on 101 residues on the basis of the sequence of hsp71 of M. tuberculosis residues 509-621 (10) . Non-cleavable octapeptides, overlapping by one residue starting at residue 509 (VRNQAETL) and ending at residue 621 (GRCPPRLG) were synthesised in solid-phase using the Geysen technique (11) on specially moulded, high-density polyethylene pins which are assembled into a plastic holder designed to hold 96 pins in the format and spacing of a icrotitre plate. The tips of the pins have been chemically derivatised to provide functional groups to which amino acids may be coupled in an appropriate form in peptide synthesis. All peptides have received N-terminal acetylation. Positive and negative control peptide sequences were synthesised on the same block as the test peptides to give an indication of the sensitivity being achieved in the ELISA assay. Sera
Selected sera which had previously been tested and all found as a high titre (1/2000-1/4000) to recombinant mycobacterial hsp70 (5) were collected from 2 cases of smear-positive pulmonary tuberculosis, 6 cases of smear-negative tuberculosis, and 1 case each of lepromatous leprosy, non- tuberculosis pulmonary disease of proven infection with M. avium, Crohn'ε disease and one healthy subject.
ELISA
This was performed following the recommended procedure from CRB. Reactions at the tips of the pins are carried out in polypropylene plates with wells in a standard 8 by 12 matrix matching the pin spacing. Briefly, the pins were precoated by dispensing 200 μl of pre-coat buffer (2% w/v bovine serum albumin), 0.1% v/v Tween 20,0.01 phosphate buffered saline pH 7.2) into each well of a microtitre plate. The pins were placed in the wells and incubated for 60 min at 20°C on a shaker at 100 rpm. Without washing, the pins were incubated overnight at 4°C in a microtitre plate containing 175 μl/well of human serum diluted 1/400 in pre-coat buffer. The pins were removed from the primary antibody preparation and washed four times 10 min each in a bath of 0.01 M PBS (pH 7.2) at room temperature with agitation. Then, the pins were placed in a microtitre plate containing 175 μl/well of peroxidase labelled goat anti-human IgG diluted 1/1000 in conjugated diluent (1% v/v sheep serum, 0.1% v/v Tween 20,0.1% w/v sodium casemate and 0.01 M PBS pH 7.2) and incubated at 20°C for 60 min with agitation. The pins were washed as above and placed in a microtitre plate containing 150 μl/well of substrate solution, 0.5 mg/ml of 2,2'-azino- di[3-ethyl-benthiazoline sulfonate] in citrate buffer pH 4.0 and 0.01% w/v hydrogen peroxide. The reaction was stopped by removing the pins from the plate after 30 min and immediately the optical density was measured at 405 n in a Titertek "Multiscan MCC II" spectrophotometer.
RESULTS
Altogether 13 sera were successively tested for binding to 94 overlapping peptides presented on a single 96- pin polyethylene block. There was no indication of any deterioration in the binding activities of peptides as judged by closely similar binding values to two pins containing control peptides. Optical density values from the ELISA assay of one representative serum from a patient with smear-negative pulmonary tuberculosis showed elevated antibody binding to several discretely separated clusters of adjacent peptides (figure 6) . Four areas of peptides, based on sequences towards the carboxy-terminus and with the most consistent locallisation of increased binding had been designated as A (peptides 33-41) , B (peptides 44-52) , C
(peptides 56-67) and D (peptides 74-85) . Elevated binding values were also observed to peptids located further towards the amino-terminus; however, these presumably cross-reactive epitopes were omitted from evaluation. Peptide No. 77 showed low binding with all tested sera which we considered to be due to defective peptide structure or presentation in view of the consistent performance of adjacent peptides on both sides from peptide 77.
In order to evaluate the contribution of individual amino acids to the epitope structure, we have calculated the mean absorbance values for each residue from all 8 overlapping octapeptides containing the respective amino acid. This analysis was applicable to sequences, starting from the leucine at position 516, which represented the last
residue in peptide number 1. The results shown in Figure 7 demonstrate the values obtained from testing one serum each from patients with pulmonary tuberculosis (PT) , calculated from values shown in figure 1) , lung disease caused by M. avium (AM) and with lepromatous leprosy (LL) . Antibody binding activities towards individual residues closely overlapped in their localisation and were clearly pronounced for epitopes B and D with each of the three tested sera. In contrast, binding to epitopes A and C identified with serum PT was not significant with the AM and LL sera. This individual variation in the binding patterns between sera supports the view that the four identified epitopes are of distinct specificity.
The core structures of each of the four identified epitopes have been alocated to six residues graded with the highest mean OD values. They are represented by sequences DAAVAE for epitope A, WRIGYF for epitope B, GEAGPG for epitope C and CVTGHW for epitope D (Figure 1) . Alignment of these hexamers within the sequence of the hsp71 carboxy terminus from M. tuberculosis (10) , with corresponding ssequences of M.leprae hsp70 (9), E.coli dnaK, (12) and of the human HSP70-1 gene (13) revealed, that the core of epitope A is overlapping in 5 out of the 6 core residues with the M.leprae sequence. In contrast, epitopes D, B and C shared only 2, 1 or no residue respectively with M.leprae. In view of the shared sequence of epitope A, it was surprising to find a lack of binding by the LL serum, selected with a high titre to several mycobacterial antigens. None of the four epitopes had significant
homology with either dnaK or human HSP70-1 structure. Predictive epitope evaluation of the carboxy-terminal sequence by the "Protean" programme (Proteus Biotechnology Ltd, Manchester, UK; kindly performed by J.Edwards at Peptide Technology Ltd. Sydney, Australia) allocated the highest score only to a single broad stretch of residues 565-591 which contains the epitope C (571-576) sequence.
A semi-quantitative evaluation of antibodies in the 13 tested sera against individual epitopes has been done by calculation of mean OD values from the 7 - 11 peptides, corresponding to each of the four epitopes. The results represented in Figure 8, demonstrate significantly higher antibody binding to each of the four epitopes in sera from tuberculosis patients when compared with non-tuberculosis controls. This difference was more significant for epitopes C and D (p<0.001) than for epitopes A and B (p<0.01). However, it should be remembered, that the serum from the healthy subject and the other 3 controls were purposely selected to contain high antibody binding to fulfil the topographic aims of the study. Hence, much better discrimination would be expected between TB patients and randomly selected non-tuberculosis patients and healthy controls. DISCUSSION
The immunogenicity of protein antigens in terms of antibody formation is largely based on the recognition of epitopes displayed on native molecules by the immunoglobulin receptors of B cells (14) . Indeed, the epitopes which react
with the bulk of antibodies in patients with tuberculosis are of confor ational nature (1, 15, 16) . Furthermore, the need for direct contact with B cells by the native antigens is corroborated by the fact that both 38kDa and 19kDa antigens are lipoproteins, secreted from live mycobacteria (17) and resulting in the highest antibody titres in multibacillary disease (2,3). In the paucibacillary stage of infection however, the presentation of those antigens of intracellularly replicating mycobacteria which had been processed within macrophages would stimulate an antibody repertoire of different specificity, directed predominantly to linear epitopes. This view is supported by the recent demonstration that antibody levels to the mycobacterial hsp71 antigen have been raised equally in sera from both bacteriologically positive and negative cases of tuberculosis (5) . Similarly, anti-hsp70 antibodies were detected in the early stages of schistosomiasis (18) . The pronounced immunogenicity of hsp71 during paucibacillary infection could be attributed to enhanced stress-induced synthesis during intracellular replication (19) , or to surface expression (20,21) enabling recognition by the Ig receptors of B cells or merely to earlier priming by multiple cross-reacting commensal microbial organisms and autologous hsp70. Although the hsp71 protein is a predominantly intracellular constituent (8) , its presence in media from short-term mycobacterial cultures (22) indicates some form of secretion from live bacteria which could be contributory to its immunogenicity for B cells.
In view of the extensive homology in the primary
structure of prokaryotic and eukaryotic hsp70 proteins (9) , antibodies are likely to be produced against many cross- reactive epitopes of the hsp71 antigen. Indeed, elevated serum antibody levels to both microbial and human hsp71 have been found in a variety of infectious, chronic inflammatory and autoimmune diseases (5, 6, 23-26) . However, it is notable, that antibodies produced following infection with Schistosoma mansoni were found to be predominantly directed against species-specific epitopes of hsp70 (29) . The present peptide-scan analysis of the most polymorphic carboxy-terminal part of the hsp7l sequence identified four distinct linear epitopes. Localisation of these four epitopes was highly consistent with all 13 tested human sera without demonstrable microheterogeneity in the epitope structure which could have been revealed by antibodies selected from the broad human B cell repertoire.
Despite the limitation in the number of cycles in which the peptide-pins can be re-used, the analysis of a selected collection of sera allowed the unequivocal localiation of four core epitope sequences. Future bulk synthesis of these peptides will enable more extensive serological evaluation of the identified epitopes. It is possible, that the M.tuberculosis epitope aligned sequences from other microbial and eukaryotic species may also carry immunogenic epitopes. Their potential as species-specific probes for the involvement of hsp70 could be valuable in the study of immunopathogenesis of various infectious diseases or autoimmunity and possibly for immunodiagnostic applications.
REFERENCES for Example 2
1. Ivanyii J. , Sharp K. , Jackett P., and Bothamley G. (1988) Immunological study of the defined constituents of mycobacteria Springer Seminars in
Immunopathology 10:279-300.
2. Bothamley G. , Udani P., Rudd R. , Festenstein F. , Ivanyi J. (1988) Humoral response to defined epitopes of tubercle bacilli in adult pulmonary and child tuberculosis Europ J Clin Microbiol 7: (5) 639-545.
3. Jackett P.S., Bothamley G.H., Batra H.V. , Mistry A., Young D.B and Ivanyi J. (1988) Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis. J. Clin Microbiol 26: 2313-2318.
4. Wilkins E.G.L., and Ivanyi J. (1990) the potential of serology for the diagnosis of extrapulmonary tuberculosis. The Lancet 336:641-644.
5. Elsaghler A.A.F., Wilkins E.G.L., Mehrotra P.K., Jindal S. and Ivanyi J. (1991) Elevated antibody levels to stress protein hsp70 in smear-negative tuberculosis. Immunol & Inf. Diseases. In press (Example 1) .
6. Britton, W.J., Hellqvist, L. , Basten, A and Inglis A.S., (1986) Immunoreactivity of a 70 kDa protein purified from Mycobacterium bovis Bacillus Calmette-Guerin by monoclonal antibody affinity chro atograph J. Exp. Med 164: 695-708.
7. Garsia R.J., Hellqvist L. , Booth R.J., Radford A.J., Britton W.J., Astbury L. , Trent R.J., and Basten A. (1989) Homology of the 70-kilodalton Antigens form Mycobacterium leprae and
Mycobacterium tuberculosis 71-kilodalton Antigen and with the Conserved Heat Shock Protein 70 of Eucaryot es. Infection and Immunity,57:204-212.
8. Mehlert A., and Young- D.B. (1989) Biochemical and antigenic characterisation of the Mycobacterium tuberculosis 71 kD antigen a member of the 70kD heat-shock protein family. Molecular Microbiology. 3: (2) :125-130.
9. McKenzie K.R. , Adams E. , Britton W.J., Garsia R.J. and Basten A. (1991) Sequence and immunogenecity of the 70-kDa heat shock protein of Mycobacterium leprae. J. Immunol 147:312-319.
10. Lathihgra R. , Alexander W. , Stover K. , Coadwell J. , Yound R. , and Young D. (19ā_ Genetic characterisation of the dnaK-grpE heat shock gene cluster in Mycobacterium tuberculosis. In press.
11. Geysen HM. , Meloen RH. , and Bartelling SJ. (1984) Use of peptide synthesis to probe viral antiges for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81: 3998-4002.
12. Bardwell J. and Craig E. (1984) Major heat shock genes for Drosophila and the Escherichia Coli heat- inducible dnaK gene are homologous. Proc. Natl Acad.Sci.USA. 81:848-852.
13. Jindal S., Dudani A.K., Singh B., et al (1989) Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen. Mol Cell Biol 248:730-732.
14. Lanzavecchia A. (1990) Receptor-Mediated antigen uptake and its effect on antigen presntation to class II-restricted T lymphocytes. Annu. Rev. Immunokl. 8: 773-793.
15. Andersen A.B. and Hansen E.B. (1989) Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis. Infect Immun. 57: 2481-2488.
16. Ashbridge K.R. , Preεtidge R.K., Booth R.J. and Watson J.D. (1990) The mapping of a serodominant region of the Mycobacterium tuberculosis 19
kilodalton antigen. J. Immuno. 144:3137-3142.
17. Young D.B. and Garbe T.R. (1991) Lipoprotein antigens of Mycobacterium tuberculosis Res. Microbiol. 142:55-65.
18. Hedstrom R. , Culpepper J. , Harrison R.A., Agabian N., and Newport G. , (1987) A major immunogen in Schistosoma Mansoni infections is homologous to the heat-shock protein Hsp-70. J. of Exp. Med. 165:
1430-1435.
19. Buchemeier N.A. , and Heffron F. , (1990) Induction of Salmonella stresε proteins upon infection of macrophages. Science 248:730-732.
20. Vanbuskirk A., Crump B.L. Margollash E. , and Pierce S. J. (1989) A peptide binding protein having a role in antigen presentation is a member of the Hsp70 heat shock family. Exp Med. 170:1799-1809.
21. Jarjour W. , Tsal V., Woods., Welch W. , Pierce S., Shaw M. , Mehita H. , Dillmann W. , Zvalfler N. , Winfield J. (1989) Cell surface expression of heatshock proteins. Arthritis Rheum 32-S44.
22. Abou-Zeid C. , Smith I., Grange J.M. , Ratliff T.L., Steele J. , and Rook G.A.W. (1988) The secreted antigens of M. tuberculoεis and their relationship
to those recognised by the available antibodies. J. Gen Micro. 134:531-538.
23. Minota S., Cameron B. , Welch W.J. and Winfield J.B. (1988) Autoantibodies to the constitutive 73-kD member of the hsp70 family of heat shock proteins in systemic lupus erythematoεus J. Exp. Med. 168: 1475-1880.
24. Tsoulfa G. , Rook G.A.W. , Bahr G.M. , Sattar M.A. , Behbehani K. , Yound D.B., Mehlert A., van-Embden J.D.A., Hay F.C., Isenberg D.A. , and Lydyard, P. M. (1989) Elevated IgG antibody levelε to the mycobacterial 65-kDa heat εhock protein are characteristic of patients with rheumatoid arthritis. Scand. J. Immunol. 30: 519-527.
25. Mattei D. , Scherf A., Bensaude O. , and deed of assignment Silva L.P. (1989) A heat shock-like protein from the human malaria parasite Plasmodium falciparum induces autoantibodies Eur. J. Immunol. 19 1823-1828.
26. Elsaghier A., Prantera C. , Bothamely G. , Wilkins E. , Jindal S. and Ivanyi J. (1991) Disease association of antibodies to human and mycobacterial hsp70 and hsp60 stresε proteins. Submitted for publication.
27. Newport G.R. , Hedstrom R.C., Kallestad J. Tarr P., Klebanoff S., Agabian N. (1988) Identification, molecular cloning and expression of a schistosome antigen displaying diagnostic potential. Am J. Trop. Med. Hyg 540:540-546.
Claims
1. A peptide comprising a sequence the same as or immunologically equivalent to a linear epitope of the carboxy terminal region of Micobacteria tuberculosis Hsp71 or a corresponding heat shock protein or any other mycobacterial pathogen.
2. A peptide according to claim 1 comprising a sequence the same aε or immunologically equivalent to a linear epitope of the carboxy terminal of M.tuberculosis Hsp71.
3. A peptide according to claim 1 containing a sequence comprising at leaεt five residues of one of the following linear epitope core sequenceε:
DAAVAE WRIGYF
GEAGPG CVTGHW
4. A peptide according to claim 3 containing a sequence:
DAAVAE
WRIGYF GEAGPG CVTGHW
5. A peptide according to claim 4 containing one of the following sequenceε:
KVDAAVAE
VDAAVAEA DAAVAEAE
GTWRIGYF TWRIGYFG WRIGYFGH GDGEAGPG DGEAGPGV
GEAGPGVA SGCVTGHW GCVTGHWR CVTGHWRC
6. A peptide according to claim 1 or claim 2 containing a εequence immunologically equivalent to a sequence according to any one of claims 3 to 5.
7. A peptide according to any preceding claim bound to a solid carrier or a detectable label.
8. A peptide according to claim 7 bound to an enzyme label, cromophore, fluorophore or radio-isotope.
9. A peptide according to any preceding claim for use in a diagnostic proceεs practised on the human or animal body.
10. A diagnostic test process comprising contacting a peptide according to any one of the preceding claims with a body fuid sample sample taken from an individual suspected of having a microbacterial infection.
11. A diagnostic test ket comprising a peptide according to any one of claims 1 to 9 optionally bearing a label or linked to a solid εupport and optionally also comprising buffer, positive or negative control materials, labelling reagents, reagents for detecting a label and other conventional components.
Applications Claiming Priority (2)
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GB9200949.7 | 1992-01-17 | ||
GB929200949A GB9200949D0 (en) | 1992-01-17 | 1992-01-17 | Diagnostic peptides |
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WO1993014118A1 true WO1993014118A1 (en) | 1993-07-22 |
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ID=10708733
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Application Number | Title | Priority Date | Filing Date |
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PCT/GB1993/000087 WO1993014118A1 (en) | 1992-01-17 | 1993-01-15 | Peptide fragments of hsp71 of m. tuberculosis and their use in diagnosis of tuberculosis |
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AU (1) | AU3358693A (en) |
GB (1) | GB9200949D0 (en) |
WO (1) | WO1993014118A1 (en) |
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US5750119A (en) * | 1994-01-13 | 1998-05-12 | Mount Sinai School Of Medicine Of The City University Of New York | Immunotherapeutic stress protein-peptide complexes against cancer |
US5830464A (en) * | 1997-02-07 | 1998-11-03 | Fordham University | Compositions and methods for the treatment and growth inhibition of cancer using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy |
US5837251A (en) * | 1995-09-13 | 1998-11-17 | Fordham University | Compositions and methods using complexes of heat shock proteins and antigenic molecules for the treatment and prevention of neoplastic diseases |
US5935576A (en) * | 1995-09-13 | 1999-08-10 | Fordham University | Compositions and methods for the treatment and prevention of neoplastic diseases using heat shock proteins complexed with exogenous antigens |
US5948646A (en) * | 1997-12-11 | 1999-09-07 | Fordham University | Methods for preparation of vaccines against cancer comprising heat shock protein-peptide complexes |
US5961979A (en) * | 1994-03-16 | 1999-10-05 | Mount Sinai School Of Medicine Of The City University Of New York | Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens |
US5985270A (en) * | 1995-09-13 | 1999-11-16 | Fordham University | Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes |
US5997873A (en) * | 1994-01-13 | 1999-12-07 | Mount Sinai School Of Medicine Of The City University Of New York | Method of preparation of heat shock protein 70-peptide complexes |
US6013660A (en) * | 1996-10-02 | 2000-01-11 | The Regents Of The University Of California | Externally targeted prophylactic and chemotherapeutic method and agents |
US6017540A (en) * | 1997-02-07 | 2000-01-25 | Fordham University | Prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with heat shock/stress protein-peptide complexes |
US6752993B1 (en) | 1993-11-23 | 2004-06-22 | The Regents Of The University Of California | Abundant extracellular product vaccines and methods for their production and use |
US6761894B1 (en) | 1993-11-23 | 2004-07-13 | The Regents Of The University Of California | Abundant extracellular products and methods for their production and use |
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US7015309B1 (en) | 1999-06-23 | 2006-03-21 | The Wistar Institute Of Anatomy And Biology | Pyrrhocoricin-derived peptides, and methods of use thereof |
US7300660B2 (en) | 1993-11-23 | 2007-11-27 | The Regents Of The University Of California | Abundant extracellular products and methods for their production and use |
US7449557B2 (en) | 2000-06-02 | 2008-11-11 | University Of Connecticut Health Center | Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy |
US7666581B2 (en) | 2001-08-20 | 2010-02-23 | University Of Connecticut Health Center | Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease |
US8475785B2 (en) | 2008-03-03 | 2013-07-02 | The University Of Miami | Allogeneic cancer cell-based immunotherapy |
US8685384B2 (en) | 1998-02-20 | 2014-04-01 | University Of Miami | Recombinant cancer cell secreting modified heat shock protein-antigenic peptide complex |
US8728170B1 (en) | 2006-12-28 | 2014-05-20 | Boston Scientific Scimed, Inc. | Bioerodible nano-fibrous and nano-porous conductive composites |
US8968720B2 (en) | 2008-03-20 | 2015-03-03 | University Of Miami | Heat shock protein GP96 vaccination and methods of using same |
US10046047B2 (en) | 2015-02-06 | 2018-08-14 | Heat Biologics, Inc. | Vector co-expressing vaccine and costimulatory molecules |
US11548930B2 (en) | 2017-04-04 | 2023-01-10 | Heat Biologics, Inc. | Intratumoral vaccination |
US11666649B2 (en) | 2016-10-11 | 2023-06-06 | University Of Miami | Vectors and vaccine cells for immunity against Zika virus |
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