Title of Invention

"AN IN VITRO METHOD FOR DETERMINING THE EFFICACY OF A COMPOUND OR THERAPEUTIC AGENT OR VACCINE"

Abstract

The present invention relates to an in vitro method for determining the efficacy of a compound or therapeutic agent or vaccine having anti-tuberculosis activity, comprising determining in test sample whether the level of T cells specific for an antigen of M. tuberculosis has decreased after the treatment, wherein said antigen is ESAT-6 or CFP10 and wherein said determining is by quantification of said T cells as directly ex vivo cells.

Full Text

Filed of the Invention The present invention relates to an in vitro method for determining the efficacy of a compound or therapeutic agent or vaccine having anti-tuberculosis activity. The invention further relates to a method of monitoring the progress of a mycobacterial infection and to a method of detecting latent mucobacterial infection. Background of the Invention In healthy individuals with latent infection, there is no means of monitoring whether the bacilli remain viable or not. The existing test for latent M. tuberculosis infection is the tuberculin skin test (TST), and this remains positive even after M tuberculosis bacilli have been killed with appropriate preventive drug treatment, e.g. isoniazid preventive therapy (IPT). In patients with active tuberculosis undergoing treatment, there are no good surrogate markers of in vivo bacillary burden. Expectorated bacilli in sputum and bacilli isolated from other disease sites (e.g. pleural fluid or cerebrospinal fluid) represent extracellular bacilli. Although bacilli (whether measured by microscopy, culture, PCR or phage-based assays) gradually disappear from clinical specimens (from disease sites) with effective therapy, these measures are (a) not a good measure of total body bacillary burden and (b) tell us nothing about the remaining intracellular bacterial burden after extracellular bacilli at the disease site have become undetectable. Conventional immunological assays do not provide a useful surrogate marker of the progress of infection. Antibody responses are not sufficiently dynamic, and stay positive fOr long after finishing treatment. All cellular immunological assays used to date, actually rise with treatment because of the ill-understood, non-specific immunosuppressive effect of active TB itself. The invention is a means of monitoring the progress of M. tuberculosis -infection by sequentially measuring the ex vivo frequency of ESAT-6- specific T cells by ex vivo interferon-'y ELISPOT assay. When patients with active tuberculosis or healthy contact with latent infection undergo effective anti-tub erculous drug treatment in vivo bacterial load decreases progressively. After 6-9 months of treatment, it is believed that. in the vast majority of individuals (>95%), all remaining intracellular bacilli have been killed, since these individuals very rarely relapse to develop tuberculosis again (unless they acquire exogenous reinfection). Longitudinal follow-up of patients with active tuberculosis and healthy contacts with latent M tuberculosis infection during anti-tuberculous drug treatment, shows that the frequency of ESAT-6-specific T cells measured ex vivo, unlike other cellular immune responses, actually declines progressively with increasing duration of therapy. After completion of therapy, ESAT-6-specific T cells measured ex vivo, are undetectable in most cases (this potentially offers a means of determining whether mycobacterial infection has been eradicated by an intervention). By comparison, the decay kinetics of ESAT-6-specific T cells in patients receiving 2nd and 3rd line antibiotics for multidrug resistant TB, were slower, and correlated with delayed conversion of sputum to culture negativity (and patients with multidrug resistant TB have a much slower clinical and bacteriological improvement). The decline in ESAT-6-specific T cells with effective chemotherapy, which reduces bacterial load by several orders of magnitude, suggests that, within a given individual, for the frequency of ESAT-6-specific T cells, as enumerated by the ex vivo ELISPOT, is related to antigen load. In biological systems where the in vivo burden of a pathogen and the corresponding antigen load are measurable, it is recognised that the frequency .of antigen-specific T cells enumerated ex vivo, correlates with, and is in part driven by, the antigen load. Although there are no quantitative absolute measures of bacterial or antigen load in TB, they must be directly and closely inter-related, and ex vivo quantitation of ESAT-6-specific T cells thus offers a means for longitudinally tracking changes inM tuberculosis bacterial load within an individual. Thus, the invention covers, the monitoring of the progress of M. tuberculosis infection over time by direct ex vivo quantitation of ESAT-6-specific T cells. The data generated to date are all based on the use of the ex vivo ELISPOT assay, but othei methods for the ex vivo enumeration of antigen-specific T cells may also be used (e.g. MHC teiramer-pepude complexes, intr a cellular cytokine staining) in addition, or alternatively. Thus the inventors have found that after effective treatment is administered to a patient with mycobacterial infection there is a decrease in the level of mycobacterial antigen-specific T cells enumerated directly ex vivo. This is the first time that a decrease in1 the levels of T cells specific for mycobacterial antigens has been found after the treatment of a mycobacteriai infection. The invention thus provides a means of monitoring the progress of a mycobacteriai infection by sequentially measuring the ex vivo frequency of myobacterial antigen specific T cells. The inventors have also found that individuals with a latent mycobacteriai infection have detectable levels of T cells specific for mycobacteriai antigens. Thus the detection of these T cells may be used to diagnose latent infection. Accordingly, the invention provides a method of determining the efficacy of treatment for mycobacteriai infection in an individual comprising determining in samples from the individual whether the level of T cells specific for a mycobacteriai antigen has decreased after treatment, thereby determining the efficacy of the • treatment. The invention also provides a method of determining the presence of a latent infection in an individual comprising determining in a sample from the individual the presence of T cells specific for a mycobacteriai antigen. • The invention additionally provides a method of determining the effect of an intervention on a mycobacteriai infection in an individual comprising measuring the effect on the levels of the T cells in samples from the individual, thereby determining the effect of the intervention. The method of detection of T cells preferably directly ex vivo enumeration The T cells are typically specific for ESAT-6 or CFP10, but may also be specific for any other mycobacteriai antigen. Brief description of the Figures Figure 1 shows frequencies of ESAT-6 peptide-specific IFN-y-secreting T ceils for the 5 different groups of subjects. Each circle represents an individual subject; the frequency of EFN-y-secreting T cells to each peptide was summated to give the total number of ESAT-6 peptide-specific T cells. The vast majority of tuberculosis contacts and patients responded (Table 2) and only the responders are represented here. The horizontal bars represent the median frequency of ESAT-6 peptide-specific T cells for each group. Figure 2 shows frequencies of ESAT-6 peptide-specific IFN-y-secreting T cells decline with anti-tuberculous chemotherapy (p=0.005; n=12). The sum mated number of ESAT-6 peptide-specific T cells for each patient, enumerated by ex vivo ELISPOT, is shown at various time points durrng the course of therapy. The overall frequency of ESAT-6 peptide-specific IFN-y-secreting T cells fell, on average, by a factor of 0,62 (95% (T: 0.37 - 0.76) (that is, to 38% of the initial values) over the time period shown. The average rate of decay was 5.5% (95% CI: 2.4 - 8.4) per week. This decline was observed for all groups of patients followed up: C+PTB (culture positive pulmonary tuberculosis: n=4); TBLN (tuberculous lymphadenitis: n=3) arid EPTB (extrapulmonary tuberculosis: miliary, n=l; pleural, n=2, osteomyelitis, n=l). For one EPTB patient (NPFI232: miliary), the T cell frequency initially rose, then declined. Figure 3 shows CD4 epitope map of ESAT-6 as defined by ex vivo peptide-specific IFN-y ELISPOT responses for all 88 tuberculosis patients and contacts. Th= percentage of subjects that responded to each peptide is shown, and in each case 88 subjects were tested against each peptide. For rESAT-6 antigen, 35 tuberculosis patients and contacts were tested. Figure 4 shows CD4 T cell responses to the immunodominant epitopes ESAT-6,.;, and ESAT-671.S« are HLA-DQ-restricted. IFN-y SFCs specific for pepudes ESAT-6;-i5 (Fig. 4A-C) and ESAT-67MJ (Fig. 4D, E) are markedly diminished by anti-FfLA-DQ antibody but not by antibodies to FILA-DR or F3LA-DP. For ESAT-6i.,5, results are shown for ex vivo PBMC from healthy contact NPH209 (4A), ESAT-6M5-speciiic T cell line from healthy contact GMl 9 (4B) and ESAT-Dj.jj-speciik T eel] line from TBLN patient NPH223 (4C). For ESAT-671.g5, results are shown for ex vivo PBMC from healthy contact.GMl 9 (4D) and ESAT-671. 85-specifk T cell line from TBLN patient NPH223 (4D). In each case, the frequency of IFN-y SFCs shown is the mean for two duplicate wells. Figure 5 shows frequencies of ESAT-6 peptide-specific and PPD-specific IFN-y-secretine T cells enumerated by ex vivo enzyme-linked immunospot (ELISPOT) assay in 50 culture-confirmed tuberculosis patients in India. For each indrvidual, the number of peptide-specific T cells for each of the ESAT-6-derived pepiides was summated. Figure 6 shows frequencies of ESAT-6 and CFP10 peptide-specific. and PPD-specific rplvy-secreting T cells enumerated by ex vivo enzyme-linked ffliimmospot (ELISPOT) assay in 100 healthy adults (with no past history of tuberculosis and normal chest radiography) resident in Mumbai, India (A) and 40 healthy adults resident in the UK (B). For each individual, the number of peptide-specific T cells for each of the ESAT-6-derived peptides was summated. Figure 6 shows frequencies of ESAT-6 and CFP10 peptide-specific, and PPD-specific IFN-γ-secreting T cells enumerated by ex vivo enzyme-linked immunospot (ELISPOT) assay in 100 healthy adults (with no past history of tuberculosis and normal chest radiography) resident in Mumbai, India (A) and 40 healthy adults resident in the UK (B). For each individual, the number of peptide-specific T cells for each of the ESAT-6- or CFP10-derived peptides was summated. Figure 7 shows epitope maps of ESAT-6 and CFP 10. Epitope map of ESAT-6 in tuberculosis patients in India (A), epitope map of ESAT-6 in healthy Indian adults resident in Mumbai (B) and epitope map of CFP 10 in healthy Indian adults resident in Mumbai (C). For each antigen the number of individuals responding to a given peptide is expressed as a percentage of the total number of subjects responding to that antigen. Figure 8 shows the progressive decline in frequency of ESAT-6 peptide-specific IFN-γ-secreting T cells during anti-tuberculosis chemotherapy in a series of 5 culture-confirmed tuberculosis patients followed-up longitudinally. For each individual, the number of peptide-specific T cells for each of the ESAT-6-derived peptides, enumerated by the ex vivo enzyme-linked immunospot (ELISPOT) assay, was summated at each time point. Statement of invention: Accordingly, the present invention relates to an in vitro method for determining the efficacy of a compound or therapeutic agent or vaccine having anti-tuberculosis activity, comprising: (i) measuring the level of ex-vivo effector T cells specific for an antigen of M. tuberculosis in a first reference sample, the sample comprising freshly-isolated T cells from a peripheral blood sample; (ii) obtaining a second or plurality of test samples at one or more later time points after the start of treatment; (iii) measuring the level of ex-vivo effector T cells specific for said antigen of M. tuberculosis in the second or plurality of samples; and (iv) comparing the level of effector T cells in the reference sample and the second or plurality of samples; said antigen being selected from ESAT-6 or CFP10, wherein decrease in effector T cell level is indicative of efficacy of compound or therapeutic agent or vaccine and, wherein all the samples are obtained from the same source. Detailed description of the invention The invention provides a method which can be used to measure in an individual the efficacy of a given treatment or intervention for mycobacterial infection. In the method the effect of a treatment on the level of a particular T cell is detected. The invention also provides a method of diagnosing a latent mycobacterial infection by detecting the presence of the T cell. The invention also provides a method of determining the effect of an intervention on a mycobacterial infection. Herein the term "method" refers to any of these different methods unless the context requires otherwise. and may for example by infection by another disease, such as HIV infection or hepatitis B infection. The method is generally performed on a sample from an individual who is a human or animal, typically an animal which can be naturally or artificially infected by a mycobactenum. Thus the individual may be a mammal, such as a primate, cow. sheep, pig, badger or rodent e.g. a mouse or rat. The individual typically has an active or latent mycobacterial infection, has had such an infection recently, or is suspected of having a latent mycobacterial infection. The individual may test positive or negative .in a Mantoux test. The individual may be at risk of a mycobacterial infection, typically for socio-economic reasons or may have a genetic or acquired predisposition to mycobacterial infection. The individual may be healthy or may have symptoms of a mycobacterial infection. The individual is generally infected with, or suspected of being infected with, a mycobacterium. The mycobacterium typically expresses ESAT-6 or CFP10, and may btM.tuberculosis. The mycobacterium may beM.marinum orM.kansasii. The pattern of clinical symptoms can be used to distinguish between these two organisms and M. tuberculosis. The mycobacterium may beM. bovis (which can. infect humans). In the method of determining the efficacy of a treatment the change in level of T cells specific for a mycobacterial antigen is determined by measuring the level of such T cells in a sample from the individual. In order to determine the change in the level of the T cells the level of the T cells in the sample taken after the treatment is administered (referred to herein as the "subsequent sample") is compared to a reference sample taken previously from the same patient. Typically the reference sample is taken from the patient before the treatment or intervention is administered or before it has had any effect, such as within one or two hours of the treatment being administered. The subsequent sample is typically taken within six months of the treatment administration, such as within a month or 2. weeks of the administration. In the method of detecting the effect of an intervention the sample may be taken at any of the above-mentioned tunes after the intervention. The treatment whose efficacy is being determined is typically a new or old treatment. The treatment may be a natural agent such as a vitamin (e.g. vitamin D), mineral or plant-derived product. The treatment may be a preventative or therapeutic vaccine. The treatment is typically isoniazid, rifampicin, ethambutol, pyrazinamide or streptomycin. If a particular treatment is found to have a low efficacy, i.e. the treatment does not cause a substantial decrease in the level of the T cells then a different treatment may be administered to the individual. The decrease in the level of T cells may be compared with the decrease in the case of a successful treatment. In one embodiment a treatment which has a low efficacy will cause a decrease of a rate of less than 5% (e.g. less man 1%) per week in the levels of the T cells. The different treatment which is typically administered when a first treatment has low efficacy (for example when bacilli are resistant to the first line drug) may be para-amino-salicyclic acid, kanamyin, capreomycin, ethionamide, cycloserine, thiacetazone or a flouroquinolone (e.g. ciprofloxacrn). In certain individuals the T cell levels may- decrease after treatment and then remain detectable at a substantially constant level, say over the period of at least two months. This may indicate the change of the infection from an active one to a latent one, unless re-exposure to a mycobactenum occurs whereupon the level of the T cells may rise. Typically in the method of determining the presence of a latent mycobactenal infection the detection of a frequency of,at least 20 T cells specific for ESAT-6 or 20 T cells specific for CFP-1Q per million peripheral blood mononuclear cells (PBMCs) may be taken to indicate the presence of a latent infection. The frequency of these ceils would be expected to remain relatively constant in an individual with an untreated latent infection. The T cells which are detected in the method recognise a mycobactenal protein antigen. Thus the T cells will be specific for fo'md to peptides from that protein, i.e. will be specific for/hind to particular fragments (or epitopes) of the protein. Typically the T cells are specific for peptides from ESAT-6 or CFP-10. The T cells may be CD4 or CDS T cells. The T cells have been pre-sensitised in vivo to the mycobacterial proteins, and may be present in the peripheral blood of an individual which has been exposed to the mycobacterium at a frequency of 1 in 106 to 3 in 103PBMCs. In a preferred embodiment the level of. or presence of; T cells is detected by contacting cells of the sample with (i) a whole myobacterial protein, or (ii) a peptide which is a fragment of a my cob acted al protein, or (iii) an analogue of (i) or (ii) which is recognised by a T cell receptor that recognises (i) or (ii). Thus the peptide sequence which is recognised may be a fragment of ESAT-6 or CFP-10. The sequence which is recognised is at least S amino acids in length, typically at least 12,' 15, 20 or more amino acids in length.. The peptide which is used in the method is thus typically a peptide which has or comprises a sequence which is the same as the fragment of ESAT-6 or CFP-10 referred to above, e.g. a fusion protein. The peptide typically has a length of at least 8, 10, 15, 20 or more amino acids, and typically has a length of less than 100 amino acids, such as less than 80 or less than 60 amino acids. Determination of whether the T cells recognise the peptide is generally done by detecting a change in the state of the T cells in the presence of the peptide or determining whether the T cells bind the peptide. The change in state is generally caused by antigen specific functional activity of the T cell after the T cell receptor binds the peptide. Generally when binding the T cell receptor the peptide is bound to an MHC class 1 or n molecule, which is typically present on the surface of an antigen presenting cell (APC). The change in state of the T cell may be the start of or increase in secretion of a substance from the T cell, such as a cytokine, especially IFN-'y, IL-2 or TNF-a. Intracellular changes may be detected, for example by using intracellular staining techniques, tvpically mtracelJular cytokrne staining (e.g. for any of the cytokine mentioned herein). After such staining the staining can be detected using a cell sortins technique, for example using a FACS technique. Determination of IFN-'Y secretion is particularly preferred. The substance can typically be detected by allowing it to bind to a specific binding agent and then measuring the presence of the specific binding agent/substance complex. The specific binding agent is typically an antibody, such as polyclonal or monoclonal antibodies. Antibodies to are commercially available, or can be made using standard techniques. Typically the specific_ binding agent is immobilised on a solid support. In one embodiment this allows the actual number of responding T cells to be determined since after binding the agent the substance will remain in the vicinity of the T cell which secreted it Thus 'spots' of sub stance/agent complex may form on the support, each spot representing a T cell which is secreting the substance. Quantifying the spots (and typically comparing against a control) allows determination of recognition, of the peptide. After the substance is allowed to bind the solid support can optionally be washed to remove material which is not specifically bound to the agent. The agent/substance complex may be detected by using a second binding agent which will bind the complex. Typically the second agent binds the substance at a site which is different from the site which binds the first agent. The second agent is preferably an antibody and is labelled directly or indirectly by a detectable label. Thus the second agent may be detected by a third agent which is typically labelled directly or indirectly by a detectable label. For example the second agent may comprise a biotin moiety, allowing detection by a third agent which comprises a streptavidm moiety and typically alkaline phosphatase as a detectable label. In one embodiment the detection system, which is used is the ex-vivo ELISPOT assay described in WO 98/23960. In that assay IFN-y secreted from the T cell is bound by a first IFN-y specific antibody which is imrnobih'sed on a solid support. The bound IFN-y is then detected using a second IFN-y specific antibody which is labelled with a detectable label. Such a labelled antibody can be obtained from MABTECB ('Stockholm. Sweden). Other detectable labels which can be used are discussed below. Generally the T cells which are contacted in the method are taken from the individual in a blood sample, although other types of body sample which contain T ceils can be used. The sample may be added directly to the assay or may be processed first. Typically the processing may comprise diluting of the sample, for example with water or buffer. Typically the sample is diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold. The processing may comprise separation of components of the sample. Typically monormclear cells (MCs)are separated from the samples. The MCs will comprise the T cells and APCs. Thus in the method the APCs present in the separated MCs can present the peptide to the T cells. In another embodiment only T cells, such as only CD4 T cells, can be purified from the sample. PBMCs, MCs and T cells can be separated from the sample using techniques known in the art. such as those described in Lalvani e/ al (1997) J.Exp. Med. 186, p859-865. Preferably the T cells which are detected are in the form of unprocessed or diluted samples. The T cells are preferably directly ex vivo, i.e. they are not cultured before being used in the method. The T cells are typically freshly isolated T cells (such as in the form of freshly isolated MCs or PBMCs). The APC which is typically present in the method may from the same individual as the T cell or from a different individual. The APC may be a naturally occurring APC or an artificial APC. The APC is a cell which is capable of presenting the peptide to a T cell. It is typically a B cell, dendritic cell or macrophage. It is typically separated from the same sample as the T cell and is typically co-purified with the T cell. Thus the APC may be present in MCs or PBMCs. The APC is typically a freshly isolated ex vivo cell or a cultured cell. It may be in the form of a cell line, such as a short term or immortalised cell line. The APC may express empty MHC class II molecules on its surface. In the method one or more peptides (typically at least 2, 5, 10 or more different peptides) may be used. Thus the T cells can be placed into an assay with all the peptides ('i.e. a pool of the peptides) which it is intended to test. 'Alternatively the T cells can be divided and placed into separate assays each of which contain some of the peptides which it is intended to test. In one embodiment peptide per se is added directly to an assay comprising T ceils and APCs. As discussed above the T cells and APCs in such an assay could be in the form of MCs. When peptides which can be recognised by the T cell without the need for presentation by APCs are used then APCs are not required. Analogues which comprise' or mimic the original peptide bound to a MHC molecule are an exaraple of such a peptide. In one embodiment the peptide is provided to the APC in the absence of the T cell. The APC is then provided to the T cell, typically after being allowed to present the peptide on its surface. The peptide may have been taken up inside the APC and presented, or simply be taken up onto the surface without entering inside the APC. The duration for which the peptide is contacted with the T cells will vary depending on the method used for determining recognition of the peptide. Typically 10" to 10", preferably 5xl05 to 10° PBMCs are added to each assay. In the case where peptide is added directly to the assay its concentration is generally from 10"1 to 103ug/ml preferably 0.5 to 5dug/ml or 1 to 10jig/ml. Typically the length of time for which the T cells are incubated with the peptide is from 4 to 24 hours, preferably 6 to 16 hours. When using ex vivo PBMCs it has been found that O.SxlO6 PBMCs can be incubated in lOug/rnl of peptide for 12 hours at 37°C. In the method instead of the peptide(s) an analogue may be used which is recognised by the T cell receptor that recognises the peptide. Therefore generally when the analogue is added to T cells in the presence of the equivalent said peptide, typically also in the presence of an APC, the analogue inhibits the recognition of the equivalent peptide The binding of the analogue to the said T cell receptors can be tested by standard techniques. For example T cell receptors can be isolated from T cells which have been shown to recognise the peptide (e.g. using any of the detection of recognition assays discussed above). Demonstration of the binding of the analogue to the T cell receptors can then shown by detemining whether the T cell receptors inhibit the binding of the analogue to a substance that binds the analogue, e.«. an antibody to the analogue. Typically the analogue is bound jn an MHC class II molecule in such an inhibition of binding assay. Typically the analogue inhibits the binding of the peptide to a T cell receptor. In this case the amount of peptide which can bind the T cell receptor in the presence of the analogue is decreased. This is because the analogue is able to bind the T cell receptor and therefore competes with the peptide for binding to the T cell receptor. T cells for use in the above binding experiments can be isolated from patients . with mycobacterial infection. Other binding characteristics of the analogue are also the same as the peptide, and thus typically the analogue binds to the same MHC class II molecule which the peptide binds. The analogue typically binds to antibodies specific for the peptide, and thus inhibits binding of the peptide to such an antibody. The analogue is typically a peptide. It may have homology with the equivalent original peptide. A peptide which is homologous to another peptide is typically at least 70% homologous to the peptide, preferably at least SO or 90% and ' more preferably at least 95%, 97% or 99% homologous thereto, for example over a region of at .least 15. preferably at least 3 0, for instance at least 40, 60 or 100 or more contiguous ammo acids. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology") For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux el al (1984) Nucleic Acids Research 12, p3S7-395). An analogue which is a peptide typically has any the amino acid lengths mentioned above for the peptide discussed above. Typically the amino acids in the analogue at the equivalent positions to ammo acids in the original peptide which contribute to binding, the MHC molecule or are responsible for the recognition by the T cell receptor, are the same or are conserved. Typically the analogue peptide comprises one or more modifications, which ma}' be natural post-translation modifications or artificial modifications. The modification may provide a chemical moiety (typically by substitution of a hydrogen, e.g. of a C-H bond), such as an aniino; acetyl, hydroxy or halogen (e.g. fluorine) group or carbohydrate group Typically the modification is present on the N or C terminus. The analogue may comprise one or more non-natural ammo acids, for example amino acids with a side chain different from natural amino acids. Generally, the non-natural amino acid will have an N terminus and/or a C terminus. The non-natural amino acid may be an L- or D- ammo acid. The analogue typically has a shape, size, flexibility or electronic configuration which is substantial!}7 similar to the original peptide. It is typically a derivative of the original peptide. In one embodiment the analogue is or mimics the original peptide bound to a MBC class 1 or II molecule. The analogue may be or may mimic the original peptide bound 10 2, 3, 4 or more MHC class I orTJ molecules associated or bound to each other. These MHC molecules may be bound together using a biotin/streptavidin based system.. ID which typically 2. 3 or 4 biotin labelled MHC molecules bind to a streptavidin moiety. This analogue typically inhibits the binding of the peptide/MHC Class J or II complex to a T cell receptor or antibody which is specific for the complex The analogue is typically an antibody or a fragment of an antibody, such as a Fab or (Tab )2 fragment. The analogue may be immobilised on a solid support, particularly an analogue which mimics peptide bound to a MHC molecule. The analogue is typically designed by computational means and then synthesised using methods known in the art. Alternatively the analogue can be selected from a library of compounds. The library may be a combinatorial library or a display library, such as a phage display library. The library of compounds may be expressed in the display library in the form of being bound to a MHC class I or II molecule, such as the MHC molecule which the original peptide binds. Analogues are generally selected from the library based on their ability to mimic the binding characteristics of the original peptides Thus they may be selected based on ability to bind a T cell receptor or antibody which recognises the original peptide. In one embodiment the T cells are detected not based on their response to a substance but based on their ability to bind a specific binding agent. Typically the agent is or comprises any of the proteins, peptides or analogues mentioned herein. The agent ruav be labelled (for example using any of the detectable labels mentioned herein). The .specific binding agent may comprise an MHC molecule, and is preferably an MHC tetramer-peptide complex (for example as mentioned above). The pe.puae or analogue discussed herein can be made using standard synthetic chemistry techniques, such as by use of an automated synthesizer. They can be made from a longer polypeptide, e.g. a fusion protein, which polypeptide typically comprivSes the sequence of the peptide. and may be derived from the polypeptide by for example hydrolysing the porypeptide, such as using a protease; or by physically breaking the polypeptide. The polypeptide is typically ESAT-6 or CSF-10, which may have been expressed recombinantly. The therapeutic agents mentioned herein may be in the form of a pharmaceutical composition which comprises the agent and a pharmaceutically acceptable earner or diluent. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. Typically the agent is administered by parenteral, intravenous, intramuscular, subcutaneous, trans dermal, intradermal, oral intranasal, intravaginal, or intrarectal administration. The dose of agent may be determined according to various parameters, especially according to the particular agent; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A suitable dose may however be from lOug to 10g. for example from 100 ug to Ig of the agent. The invention is illustrated by the following Examples: Example 1 The detection of the correlation between CD4 T cell levels and bacteria] load Methods Subjects Adult patients and contacts were recruited prospectively from London and . Oxford. A heparrmsed blood sample was drawn from all participants. No subjects had features of HIV infection and patients known to be HTV infected were excluded. Demographic characteristics are shown in Table 1. 27 health}' household contacts (HHC) were recruited on the basis of the following criteria. All had lived in the same household as an index case of untreated sputum smear positive pulmonary tuberculosis within the last 6 months, were asymptomatic and had normal chest radiography. In addition, all HHCs had strongly positive TSTs (Heaf grade 3 or 4), which was performed by standardised multiple puncture tuberculin skin testing with a 6 needle disposable head Heaf gun (Bignall 2000, Bignall Surgical Instruments, UK) and concentrated purified protein derivative (PPD) 100,000 tuberculin units/ml (Evans Medical, Liverpool, UK) in accordance with UK. guidelines for evaluation of tuberculosis contacts. 23 HHCs had beenBCG-vaccinated and none developed tuberculosis over a 12 month period of follow up. None had received cbemoprophylaxis at the time of venepuncture. 32 healthy unexposed controls (UC) with no history of tuberculosis and no • known tuberculosis contact were recruited prospectively. Most were laboratory staff ~ and 28 had beenBCG-vaccinated. 25 patients with culture positive PTB (C+PTB) had clinical and radiographic findings consistent with tuberculosis and positive cultures for M. tuberculosis from one or more respiratory specimens. 18/25 (72%) PTB patients had received either no therapy, or less in an 4 weeks therapy at the time of venepuncture. S patients with culture negative PTB (C-PTB) had a clinical diagnosis made on the basis of highly suggestive appearances on chest radiography and positive tuberculin skin tests. All were asymptomatic and had no previous history of treatment for tuberculosis. All respirator}7 samples, which consisted of 3 sputum specimens (or 3 gastric washings in the absence of productive cough) and a bronchoalveolar lavage for each patient, were negative fcrM tuberculosis culture. 6/8 (75%) of this group had received either no therapy, or less than 4 weeks therapy. 11 patients with tuberculous lymphadenitis (TBLN) had consistent clinical findings, positive TSTs and a good response to anti-tuberculous chemotherapy. 7 had lymph node biopsies of which 5 grewM tuberculosis on culture. 5/11 (45%) TBLN patients had received either no therapy, or less than 4 weeks therapy at the time of venepuncture. Seventeen patients with aon-lj-mph node extrapulmonary tuberculosis (EPTB) were recruited on the basis of clinical findings and positive cultures forM. tuberculosis from one or more clinical specimens. 4 patients had tuberculous osteomyelitis, 2 had musculoskeletal tuberculosis (psoas abscess). 4 had pleural disease, 3 had miliary disease, 3 had abdominal disease and 1 had meningitis. The small number of patients precluded statistical comparisons of T cell frequencies between the subgroups of EPTB Peptides 17 peptides spanning the length of the ESAT-6 molecule were synthesised by solid-phase Fmoc chemistry (Research Genetics, Alabama, USA). Each peptide was 15 ammo acids in length and overlapped its adjacent peptide by 10 residues. Identity was confirmed by mass spectrometry and purity by high performance liquid chromatography. Sequence homology searches of the'SwissProt and translated GeaBank protein databases confirmed that these peptides are uniquely restricted to the ESAT-6 protein ofM tuberculosis complex. Ex vivo ELISPOT assay for single cell IFN-y release: enumeration of circulating ESAT-6 peplide-specific T cells from peripheral blood PBMC were separated from 20 mis blood by standard means and suspended in RPMI supplemented with 2 mM L-glutamme, 100 u.g/ml ampicillin, 50 u.g/m.1 gentarmcin, ] mM sodium pyruvate and 1 0% heat-inactivated foetal calf serum (Sigma, St. Louis, M0; USA) (RIO). As previously described (1. 2), 96-well PYDF-backed plates (MAIPS45, Millipore, Bedford. MA, USA), pre-coated with 15 us/ml of anti-IFN-y mAb 1-D IK (Mabtech., Stockholm, Sweden), were blocked with RIO for 2 h. 3 x JO5PBMC were added in 100 jj RlO/well and peptides added individually to single wells at 10 u,g/ml. PPD (Batch RT49, Statens Seruminstitut Copenhagen, Denmark) was also tested at 20 u.g/m.1. Phytohaemagglutmin (PHA) (ICN Biomedicals. Aurora,-OH, USA) at 5 u.g/m.1 was added to duplicate positive control wells and no peptide to duplicate negative control wells. Recombinant ESAT-6 was added to single wells at 10 ug/ml for 35 subjects. .After 14 h incubation ;at 37°C, 5% CO, plates were washed with PBS 0.05% Tween-20 (Sigma, St. Louis, MO, USA). 50 ^1 of 1 u.g/m.1 of biotinylated anti-IFN-y mAb, 7-B6-1 -biotin (Mabtech). was added. After 2 h, plates were washed and streptavidin-alkaline phosphatase conjugate (Mabtech) was added to 1:1000. After 1 h and further washing, 50 ul of diluted chromogenic alkaline phosphatase substrate (Biorad, Hercules, CA. USA) was added. After 20 mins plates were washed and allowed to dry. ELI SPOT assay for single cell IL-4 release These assays were carried out as described above using high affinity IL-4-specific antibodies (IL-4-I catcher and biotinylated IL-4-II detector (Mabtech)) and a 60 h incubation period. Enumeration of IFN-y spot-forming cells (SFCs) IFN-y SFCs were counted using a magnifying glass and responses were scored as positive if test wells contained at least 5 IFN-y SFCs more than negative control wells and this number was al least twice that in negative control wells. This predefined cut-off (5 IFN-y SFCs per 3 x 105 PBMC) translates into a detection threshold of 1 7 specific T cells per million PBMC, or 1/59,000 PBMC. The person performing and reading the assays was blinded to the clinical status of the different groups of patients and HHCs, but not to the UCs. Background numbers of SFCs in negative control wells were below 5. After subtraction of background values, the number of IFN-y SFCs specific for each peptide was sumrnated -and this total number of ESAT-6 peptide-specific SFCs for a given individual was used for comparisons between sroups of subjects and between different time points during treatment of individual patients. In vitro generation of peptide-specific T cell lines T ceil lines were generated as previously described (2). Briefly. PBMC were suspended at 5 x 10i- cells /ml in AB medium (RPMI supplemented with L-giutamine 2 rnM, ampicillin 100 jag/ml, gentamicin 5 ug/ml and 10% heat-inactivated pooled human AB seram {National Blood Transfusion Service, Bristol, UK}) and 200 ul were added per well in a round-bottomed 96-well plate. Peptide was added at 10 fig/ml. 10 U/ml IL-2 in the form of Lymphocult-T (Biotest, Dreiech, Germany) was -added ever}' 3 days. After 12 days, T cell lines were immunomagneticaily depleted and tested against the stimulating peptide in duplicate wells in ELISPOT assays for IFN-y using 1-5 x 1 O4 cells/well. Immunomagnetic cell depletions CD4 and CDS T cells were depleted from T cell lines by 30 mins incubation with anti-CD4 or anti-CDS mAbs conjugated to ferrous beads at a ratio of 10 beads to 1 cell using Dynabeads M-450-(Dynal, Oslo, Norway) in 200 |_d RIO on ice. Following dilution in RIO, the conjugate-coated cells were removed by a magnet (Dynal, Oslo. Norway). These Dynabeads reliably deplete >99% of the target cell population. •'H-thymidine incorporation lymphoproliferation assays 2 x lO" PBMC were seeded per well in 200 u.1 AB medium in 96-well round-bottomed plates. Peptides were added at 10 u.g/ml, negative control wells had medium only and positive control wells contained PPD at 20 u.g/ml. All conditions were set up in triplicate wells. Following incubation at 37°C, 5% CO2 for 5 days. 1 u.Ci.3H-thymidine was added to each well. After 18 h, wells were harvested and 3H-thymidine incorporation measured in a p-counter (Wallac. UK). Results were scored as positive if the stimulation index Avas 3 or more Inhibition of T cell responses in ELISPOT assays with anti-MHC class II antibodies HLA class fl restriction of the CD4 T cell response to two immunodominant peptides ESAT-6,_15 and ESAT-67!_85, was investigated using T cell lines as well as ex vivo PBMC. The murine monoclonal antibodies L243. L2 and B7.21, that block peptide presentation to CD4 T cells by HLA-DR, -DQ and -DP, respectively, were added at 10 u.g/'ml to 3 separate pairs-of duplicate wells in ELISPOT assays followed by 10 jag/ml peptide 90 mins later. Control duplicate wells received peptide only, or neither peptide nor antibody Statistical Methods Surnmated frequencies of ESAT-6-peptide-specific IFN-y SFCs for responders were compared between patient groups using the non-parametric Mann-Whitney test (2-tailed) and between the first and last time points sampled in the treatment course of individual patients using the non-parametric Wilcoxon Signed Rank test (2-taiJed). The proportional decrease in antigen-specific T cell frequencies during therapy was calculated using the Student's t test on log transformed data. Example 2 Freguepcie.s_of ESAT-6 specific T cells It was found that ESAT-6 peptide-specrfic IFN-y-secreting CD4 T cells circulate at high frequencies inM tuberculosis-infected individuals and correlate with clinically defined protective immunity. Such T cells were detected in almost all tuberculosis patients and the majority of contacts. Responses were observed in 10/11 TBLN patients, 7/S C-PTB patients, 23/25 C+PTB patients and 23/27 PTHCs (Table 1). It is possible that the 4 HHCs who did not respond to ESAT-6 were not infected with M. tuberculosis; their positive TSTs may have resulted from prior BCG vaccination. By contrast, none of the 32 UCs (of whom 28 were BCG-vaccrnated) responded to ESAT-6 peptides in the ex vivo ELISPOT assay for IFN-y (Table 1), consistent with ESAT-6-specific responses being M. tuberculosis-specific. Frequencies of ESAT-6 peptide-specific TFN-y-secreting T cells for all responders from each group are shown in Fig. 1 and summarised in Table 1. For the FEHCs that responded to ESAT-6 (n=23), frequencies of ESAT-6-specific T cells were significantly higher (p=0.009) than among the C-PTB patients who responded (n=23) and higher (p=0.044) than the PTB (C+ and C-) group as a whole (n=30). Both groups of • ESAT-6-responsive patients with minimal, paucibacillary disease, TBLN (n=10) and C- PTB (n=7). also had significantly higher frequencies of ESAT-6 peptide-specific T cells than the C+PTB patients (p=0.002 and p=0.029, respectively). It was also observed that frequencies of circulating ESAT-6-specific IFN-y-secreting T cells in tuberculosis patients decline progressively with treatment. If lower frequencies of ESAT-6-specific IFN-y-secreting T cells in patients with active C—PTB were a result of non-specific disease-associated immunosuppression. T cell frequencies would be expected to rise during effective treatment. We therefore longitudinally tracked 12 patients (4 C-rPTB, 3 TBLN and 5 EPTB) on.anti-tuberculous chemotherapy and observed a decline in the overall frequency of ESAT-6 peptide-specific T cells (p—0.005) (Fig. 2). The average decrease over the mean follow-up period of 18.6 weeks was by a factor of 0.62 (95% CI: 0.37 - 0.76), that is, to 38% of the initial values, and the rate of decay was 5.5% (95% CI: 2.4 - 8.4) per week. The frequencies of T cells specific for each of the peptides declined in parallel but only the summated responses to all ESAT-6-derived peptides are shown for clarity. Using the ex vivo ELISPOT assay, each of the overlapping ESAT-6-derived ] 5mer peptides was recognised by IFN-y-secreting T cells from one or more of 88 tuberculosis patients and contacts tested (Fig 3). In this population, peptides ESAT-6,.153 ESAT-6fr_2o and ESAT-67J_85 are particularly widely recognised, by 59%, 45% and 37% of subjects respectively. 59 T cell lines were generated from several patients and HHCs against 15 ESAT-6-derived peptides.' After 12 days culture with IL-2 supplementation, ELISPOT assays were performed before and after irnnrunomagnetic depletion of CD4 or CDS T ceils. For 55/59 T cell lines, peptide-specific responses were abrogated by CD4 depletion (Table 2); 4 T cell lines specific for certain peptides were CDS positive (2. 3). A response in the ex vivo ELISPOT assay for IFN-y indicates T cell-mediated IFN-y-secretion within 14 h of exposure to peptide. To establish how quickly these T cells can release IFN-y upon antigen encounter, we carried out 6 h ex vivo ELISPOT assays in 3 subjects (2 FHiCs and 3 C-PTB) with 13 peptides. In each case, IFN-y SFCs were readily detected at 6 h to all the peptides that gave a response at 14 h. The frequency of pepti.de-specific IFN-y-secreting T cells enumerated at 6 h was 80-90% of that at 14 h, (data not shown), indicating that ESAT-6 peptide-specific T cells are capable of rapid effector-function (1). Using PBMC from 15 subjects, lymphoproliferation assays were performed in parallel to ex vivo IFN-y ELISPOT assays using 16 different peptides. Surprisingly, we did not detect proliferation to peptides ESAT-6M5 and ESAT-67].jj5 m several subjects with IFN-y-secreting T cells specific for these peptides in the ex vivo ELISPOT (Table 2). 12 subjects responded to ESAT-6M5 by ex vivo ELISPOT (mean peptide-specific IFN-y SFCs for res ponders: 77 /106 PBMC [IQ range: 38-] 00]) compared with only 3 by lymphoproliferation. 'For ESAT-671.j[5, 10 subjects responded by ex vivo ELISPOT (mean peptide-specific IFN-y SFCs for responders: 121 / 10" PBMC [IQ range: 65-171]) compared with 5 by lymphoproliferation. However, lymphoproliferative responses to the positive control, PPD, were strong: mean stimulation index: 82 (IQ range: 35-98). Nonetheless, peptide-specific T cell lines were readily generated by peptide stimulation hi vitro with IL-2 supplementation, thus, these ESAT-6 peptide-specific T cells, while displaying rapid effector function upon antigen contact, cannot proliferate in vitro in the absence of exogenous IL-2. For several other peptides. however, there was a broad concordance between the two assays (Table 2). Example 3 HLA restriction of ESAT-6M5 ESAT-67].8 We were interested to determine the MHC restriction of the two peptides that were unmunodominant by e; vivo ELISPOT but gave discrepant results in lymphoproliferauon assays. We used monoclonal antibodies to block presentation of peptide to T cell lines and ex vivo PBMC in ELISPOT assays. For ESAT-6^, in all 3 individuals tested. IFN-y SFCs were markedly diminished by anti-HLA-DQ antibody but not by antibodies to HLA-DR or -DP (Fig. 4A-C). For ESAT-67;.S5, 2 individuals were tested, and in both cases, the response was blocked only by the anti-HLA-DQ antibody (Fig. 4 D, E). In contrast, ex vivo ELISPOT responses to.the other peptides tested (ESAT-66.20, ESAT-651.65, ESAT-666.go and ESAT-676.90) were shown to be HLA-DR-restricted (data not shown). Example 4 Furthex_characterisation of the ESAT-6 specific T cells In 3 5 subjects (28 patients and 7 HHCs) rESAT-6 antigen was tested m the ex vivo ELISPOT assays in parallel with ESAT-6 peptides and 30/35 (86%) responded to rESAT-6 (Fig 3") The 5 subjects who did not respond to the protein were subjects who also failed to respond to any of the 17 peptides..Thus, of 30 patients who responded to one or more peptides, all responded to rESAT-6 antigen, indicating that rESAT-6 is processed and presented to T cells in the ex vivo ELISPOT assay. Freshly isolated PBMC from 10 tuberculosis patients (5 C+PTB and 5 EPTB) and 8 HHCs were tested ex vivo against all 17 ESAT-6 derived peptides in an ELISPOT assay for EL-4 in parallel with the ELISPOT assay for IFN-y. The numbers of SFCs were summated for each peptide. Among the tuberculosis patients 10/10 had ESAT-6 peptide-specific IFN-y-secreting T cells (mean: 668 IFN-y SFCs/million PBMC) while 0/10 had ESAT-6 peptide-specific TL-4-secreting T cells. For the HHCs. 6/S responded in the IFN-y ELISPOT assay (mean frequency in responders: 414 ESAT-6 peptide-specific FN-y SFCs/million PBMC) compared with 2/8 responders in the IL-4 ELISPOT assay (mean frequency in responders: 93 ESAT-6 pepude-specific IFN-y SFCs/million PBMC). Positive controls for IFN-y and EL-4 ELISPOT assays with PHA stimulation were always over 500 IFN-y or IL-4 SFCs per 1 OtJ PBMC, respectively. Discuss ion By direct ex vivo analysis, we have identified a population of antigen-specific IFN-y-secreting CD4 T cells that circulate in the blood of aknost all M. tuberculosis-mfected individuals, but are absent in unexposed, BCG-vaccinated controls. The presence of high frequencies of circulating IFN-y-secreting ESAT-6-specific CD4 T cells in PPD-posmve recently exposed HHCs, who were clinically and radiographically free of disease, indicates that tliese T cells are not necessarily associated with tissue pathology per se. Rather, their presence in these individuals ma}- be consistent with a role in the containment of M tuberculosis in vivo. This hypothesis is supported by the observation that ESAT-6-specific CD4 T cells circulate ai higher frequencies in HHCs than in C+PTB patients (Fig 1). Amongst subgroups of tuberculosis patients, the frequency of these T cells was higher in those groups who manifest relatively more successful containment of M tuberculosis in vivo. Thus TBLN patients (with highly localised disease, minimal symptoms and frequent spontaneous resolution) and C-PTB patients (with'HO symptoms and no evidence of ongoing bacterial replication in multiple clinical specimens') both had higher frequencies of ESAT~6-specific CD4 T cells than C+PTB patients with M. tuberculosis actively growing in respiratory secretions (Table 1 and Fig. 1). Thus, the overall frequency of HSAT-6-specific IFN-y-secreting CD4 T cells appears to correlate with clinically-defined protective immunity. However, active tuberculosis causes an ill-defined non-specific immunosuppression. It is therefore possible that immune responses that are weaker in patients with more extensive active disease than in patients with minimal disease, are merely secondarily suppressed as a consequence of active tuberculosis itself. If this were the case, immune responses should rise with treatment.and, in general, antigen-specific or PPD-speciiic IFN-y secretion in PBMC bulk cultures and lymphoproliferative responses do increase during anti-tuberculous therapy. Thus, previously identified associations of stronger T cell responses with minimal disease did not correlate -with protective immunity because the weaker responses in patients with more extensive disease recover with treatment. It has therefore not been possible tc identify7 correlates of clinically defined protective immunity based on these conventional assays However, longitudinal follow up of tuberculosis patients with the ex vivo ELISPOT assay showed that the frequency of ESAT-6 specific IFN-y-secretrng CD4 T cells actually fell during therapy (p-0.005) (Fig 2). This indicates that the lower frequency of ESAT-6-specific T cells in patients with more extensive disease is not a secondary effect of disease-related komunosuppression nor a result of sequestration ofA-f tuberculosis-specific T cells at sites of active disease. Rather, the decline in ESAT-6-specific T cells with therapy, which reduces bacterial load by several orders of magnitude, suggests that, within a given individual, the frequency of ESAT-6-specific T cells is related to antigen load. This observation was made possible by using an assay that directly quantitates antigen-specific T cells without involving in vitro proliferation. Our finding is somewhat analogous to the decay in virus-specific CDS and CD4 T cell frequencies, directly enumerated from peripheral blood, in HJV-infecied patients during combination antiretroviral therapy and, in general for mtracelluiar pathogens, frequencies of antigen-specific T cells, when directly quantitated ex. vivo, appear to be driven by antigen load. Although there is no quantitative absolute measure of bacterial or antigen load in tuberculosis, they must be directly and closely inter-related. Antigen load is almost certainly much lower in HHCs than in patients and, amongst patients, will obviously be lower in TBLN patients and C-PTB patients than m C+PTB patients. Given that antigen load appears to drive ESAT-6-specifrc CD4 T cell frequencies, it is remarkable thai the M. tuberculosis-infecied subjects with lower antigen loads have higher levels of ESAT-6-specific CD4 T cells. This inverse correlation is similar to thai observed for virus-specific CDS T cell frequencies and plasma viral load in HJV-mfec:ed patients, a finding which provided important support for the protective role for CDS T cells in control of HTV and which depended crucially upon a means to quantify antigen-specific T cells ex vivo directly from peripheral blood. Thus, consideration of the frequencies of ESAT-6-specific T cells in the context of the differms bacterial load ha the different groups ofM. tuberculosis-infected subjects, lends further support to our hypothesis that these T cells mediate containment ofM tuberculosis in vivo. All groups of patients had similar proportions of individuals who had undergone treatment except for the HHCs. none of whom had received chemoprophylaxis. Moreover, the effect of treatment on ESAT-6 peptide-specific T cell frequencies appears to be similar for all groups of patients followed up (Fig. 2). Thus, the differences in ESAT-6-specific CD4 T cell frequencies between the 3 groups of patients are unlikely to result from differences in duration of treatment, although this may not apply to the HHCs. Our interpretation of these findings is that, at the time of initial infection with M. tuberculosis, HBCs mount a strong, high frequency Thl-type CD4 T cell response to M. tuberculosis and, in particular, to ESAT-6, and so limit bacterial replication. Individuals who go on to develop active disease, by contrast, make a weak'CD4 T cell response and the bacteria are allowed to reach a higher equilibrium bacterial load resulting m disease. Thus, HHCs maintain a high frequency of ESAT-6-specific CD4 T cells with limited antigenic stimulation from a low bacterial load, while in C+PTB patients, a high bacterial load stimulates weaker, less efficient proliferation of antigen-specific CD4 T cells in vivo. This model is analogous to that proposed for .certain chronic viral infections, where virus-specific CDS T cells are believed to mediate protective immunity. The difference between HHCs and PTB patients may lie in an individual's T cell responsiveness, which has previously been defined as the rate at which pathogen-specific T cells proliferate in vivo after encountering an infected host cell. Indeed, the early emergence and efficient proliferation of IFN-y-secreting CD4 T cells in vivo has recently been identified as a crucial factor in the early containment of my co bacterial infection m murine models. ESAT-6 contains multiple CD4 T cell epitopes. Figure 3 shows that several ESAT-6-derived peptides are widely recognised by T cells from an ethnically and genetically diverse range of patients and HHCs, suggesting that these peptides may be perrrussively restricted by & wide range of HLA class II haplotypes. The hierarchy of irnniunodominance using the ex vivo ELISPOT assay (Fig 3') is quite different to that reported by others using more conventional assays that depend on in vitro proliferation, hi particular, the striking immunodominance of peptides ESAT-6i_L5 'and ESAT-67I_8; has not been previously recognised to the extent observed here, and might reflect the fact that the ex vivo ELISPOT assay can detect T cells that have lost their in vitro proliferative potential We therefore tested these, and the other ESAT-6-derived peptides, for their ability to stimulate T cells in tymphoproliferation assays. Although for most peptides there was a broad concordance between T cell responses in these assays and in the ex vivo ELISPOT, for peptides ESAT-6M5 and ESAT-67j.85 there was a marked discrepancy (Table 2). The impaired ability of ESAT-6M5 specific and ESAl-67J.S5-specific T cells to proliferate in vitro probably explains why the iinmunodominance of these peptides was not hitherto fully appreciated. Interestingly, however, these T cells did-proliferate in vitro if stimulated with JL-2 as well as peptide. Since peptides ESAT-oj.^ and ESAT-671_85 are recognised ex vivo by T cells from a very high proportion ofM. tuberculosis-infected subjects, their HLA restriction is of special interest. Immunodominant., permissively restricted CD4 epitopes are usually HLA-DR-restricted and this is the case in tuberculosis. Surprisingly, peptides ESAT-Oj..^ and ESAT-671_8i were shown to be HLA-DQ-restricted in EL1SPOT assays (Fig. 4). These are, to our knowledge, the fust immunodominant HLA-DQ-restricted mycobacterial epitopes to be identified. Having found that most M. tuberculosis-infected individuals have high frequencies of ESAT-6-specific IFN-y-secreting CD4 T cells, we asked whether ESAT-6-specmc Th2-tvpe CD4 T cells are also induced to a similar extent. Ex vivo ELISPOT assays for IL-4 in a subset of patients and HHCs showed that, hi contrast to the almost universal presence of ESAT-6-specific IFN-y-secreting T cells mM. tuberculosis-infected subjects. IL-4-secreting T cells specific for ESAT-6 are rare. ESAT-6-specific CD4 T cells induced by natural M tuberculosis infection thus have a highly Thl-polarised pattern of cytokine secretion. We have identified a population of circulating IFN-y-secreting M. tuberculosis antigen-specific CD4 T cells that circulate at high frequencies ID asymptomatic individuals with latent M. tuberculosis infection, as well as tuberculosis patients. Our direct quantitative ex vivo approach led to certain key findings that have not been recognised using previous assays. Unlike other cellular immune responses in tuberculosis, frequencies of ESAT-6-specific CD4 T cells decay progressively with treatment suggesting that these T cell frequencies -are driven, at least in part, by antigen load. Therefore, patients with more extensive disease and nigher bacterial loads would be expected to have the highest antigen-specific T cell frequencies. However, we observed the opposite: between groups of tuberculosis patients, the frequencies of these T cells appear to correlate with clinically-defined protective immunity; that is they correlate inversely with inferred bacterial load. These findings are consistent with a role for this population of Thl-tvpe antigen-specific CD4 T cells in the containment of M. tuberculosis in vivo. These results, together with the fact that ESAT-6 contains multiple CDS T cell epitopes. suggest that this antigen may be a target of protective immune responses in A'/ tiihcrculosis-infected humans; its absence fromM bovis BCG might thus account in part, for the limited efficacy of BCG vaccination. Example5 The detection.of CQ4 T cells in individuals with latent infection Methods Study population All subjects were recruited prospectively in Mumbai and Oxford. A heparinised blood .sample was drawn after obtaining informed consent. 50 patients with clinical and radiographic features consistent with tuberculosis and with positive cultures for M tuberculosis from one or more clinical specimens, were recruited. 45 patients had pulmonary tuberculosis, 4 had tuberculous lymphadenitis and one had tuberculous pleuntis. 6 patients (5 pulmonary and one pleura] tuberculosis) were HIV antibody positive. None of the other 44 patients had clinical features suggestive of HIV infection. The mean age of the patients was 33 years (range: 14 - 69) and the rnale:female ratio was 33:17. The patients were ethnically diverse comprising Maharashtrans (n = 28). Gujaratis (n - 6), Muslims (n = 6), South Indians m = 4). Punjabis (n = 3\ Uttar Pradeshis (n - 2) and Sindhis (n = 1). 35 patients (70%) had received less than one-month's therapy or were untreated at the time of venepuncture for ELISPOT assays; the remaining ] 5 patients were at later rime points m their treatment course ( OS healthy residents of Mumbai were prospective!}' recruited at an executive health check-up clinic, all were corporate executives requested to attend by their employers for msurance_medicals and none had self-referred with symptoms. 7 subjects gave a past medical history of tuberculosis and one was resident in Zambia: these- 8 subjects were excluded The remaining 100 subjects all had normal chest radiography and none had any features of HTY infection. The male:female ratio was 78:22 and the mean age was 47 years (range: 18 - 70). The executives were ethnically diverse comprising Gujaratis (n - 36), Maharashtrans (n = 22), South Indians (n = 14). Smdhis (n = 7), Parsis (n = 6), Punjabis (n = 6), Marwaris (n = 4), Muslims (n - 3 ) and Bengalis (n = 2). 40 healthy adult UK residents were recruited in Oxford. None had lived in a tuberculosis endemic country for more than one month and none had a past medical history of tuberculosis nor any known tuberculosis contact. All were ethnically white Caucasians, the rnale:female ratio was 21:19 and the mean age was 32 years (range: 23 - 49). 33/40 (82%) had a BCG scar or history of BCG vaccination. Five bacteriologically confirmed tuberculosis patients were followed-up in the UK and ESAT-6 peptide-speciftc EFN-y-secreting T cells were enumerated at different time points during anti-tuberculosis chemotherapy. Three of these patients had pulmonary tuberculosis, one had pleural disease (DD1) and one had tuberculous lymphadenitis (TADS'.). ESAT-6 arid CFPlO-derived peptides 1 7 peptides spanning the length of the ESAT-6 molecule and IS peptides spanning the 1 ength of the CFP10 molecule were obtained and their identity tested as described foi the rjeptides of Example 1. Each peptide was 15 amino acids long and overlapped us adjacent peptide by 10 residues. Sequence homology searches of the SwissProt and translated GenBank databases of all known protein sequences confirmed that the sequences of these peptides are uniquely restricted to the ESAT-6 and CFP10 proteins of M. tuberculosis complex. A response to one or more of these 35 peptides was scored as indicative of M tuberculosis infection. Ei vivo enzyme-linked immunospot (ELISPOT) assay for single cell IFN-y release: enumeration of circulating ESAT-6 peptide-specific T cells from peripheral blood PBMC were separated from ISrnls blood.by standard means and suspended in RPM1 supplemented with L-glutamine 2 mM, penicillin 100 iu/rol and 10% heat-inactivated foetal calf serum (Sigma, St. Louis, M0; US A)'(RIO). The ELISPOT assay was earned out as described in Example 1. 2.5 x 10-PBMC (3 x 105 for the UK subjects) were added in 100 pd RIO/well' and each of the 35 ESAT-6 and CFPlO-derived peptides was added individually to single wells at 10 u.g/ml. PPD (Batch RT49, Staatens Seruniinstitut. Copenhagen, Denmark > was also tested at 20 pig/ml in duplicate wells. Phytohaemagglutmin (ICN Biomedicals, Aurora, OH, USA) at 5 jo.g/m.1 was added to duplicate positive control wells and no peptide to duplicate negative control wells: Whole recombinant ESAT-6 was added at 10 ug/ml for ] 1 tuberculosis patients and all healthy adults. Incubation and further washes were performed as in Example 1. Responses were scored as positive if test wells contained at least 5 IFN-7 SFCs more than negative control wells and this number was at least twice that in negative control wells Although the person performing the assays was not blind to the patients' tuberculosis status, the read-out in SFCs is quantitative, criteria for a positive response were stringent and pre-defined and SFCs in negative control wells were always below 15. so that positive responses were clear-cut. Positive and negative responses were immediately recognisable by direct inspection of the plate, prior to precise enumeration with a magnifying lens. ELISPOT assay wells where the number of IFN-y SFCs exceeds 250 cannot be precisely enumerated because the spots coalesce with each other 250 SFCs per well (equivalent to 1000 SFCs per million PBMC) was therefore taken as the upper limit for accurate quantrtation. Tne total number of ESAT-6- and CFPlO-specific T cells in a given individual was calculated by summaung all the IFN-y SFCs specific for each of the different ESAT-6- or CFPlO-derived peptides (after subtraction of the background number of SFCs in the negative control wells), respectively, in that individual. HIV antibody testing HTv7 antibodies were detected by a rapid test with recornbraant protein (Immunocomb EL, Orgenics Ltd. Israel) followed by 2 different microtitre ELISA tests with synthetic peptides from different antigens (Laboratory Systems, Finland and Innogenetics. Innogenetics N.V. Belgium). Tuberculin skin testing 1 TU of PPD (1:10,000) (Span Diagnostics, India) was injected intradermally in the flexor surface of the forearm to raise a pale white bleb. Cutaneous induration was measured with a ruler at 72 hrs and induration >10 mm was taken as positive. Example 6 Resjjjts A high proportion of Indian tuberculosis patients have circulating ESAT-6 peptide-specific IFN-y-secreting T cells and the ESAT-6-based ex vivo ELISPOT also detects tuberculosis patients with HTV infection. Fig. 5 shows that 40/50-bacteriologically confirmed cases of tuberculosis responded in the ESAT-6-based ex vivo ELISPOT assay. All 6 tuberculosis patients with HIV co-infection responded. The median number of ESAT-6 pepiide specific T cells in the 40 patients who responded was 128 (inter-quartile range 73-20S) per million PBMC. Many patients responded to multiple pep tides. Positive responses were cieai-cut when compared with negative control wells and. out of a total of 40 positive responses, only 2 tuberculosis patients gave borderline positive responses (20 peptide-specific T cells per million PBMC). Of the 11 patients tested against whole recombinant ESAT-6, including 4 patients with HIV coinfection, all responded to recornbinani antiyen as well as to one or more ESAT-6-denved peptides. 6 tuberculosis patients (including 3 with HTV infection) underwent TST: all 6 were negative by Mantoux bur positive m the ESAT-6-based ex vivo ELISPOT assay. High frequencies of RDl-encoded antigen-specific IFN-y-secreting T cells in S0% of healthy Indian adults 100 healthy adults with normal chest radiography and no history of tuberculosis were tested by ESAT-6 and CFP10 peptide-based ex vivo ELISPOT assay for IFN-y. 56 had ESAT-6 peptide-specific T cells and 76 had CFP10 peptide-specific T cells detectable in peripheral blood (Fig. 6A). Overall 80% responded to either ESAT-6 or CFP10 (Table ]). For the responders, the median frequency of ESAT-6 peptide-specific T cells was 86 (interquartile range: 43 - 156) per million PBMC and the median frequency of CFP10 peptide-specific T cells was 158 (interquartile range: 84 - 261) per million PBMC. These antigen-specific T cell frequencies are as high as (in the case of ESAT-6) or even higher (in the case of CFP10) than the frequency of PPD-specific TFN-y-secreting T cells among the 98 individuals who responded to PPD: median = 86 (interquartile range: 56 - 222) per million PBMC. CFP10 peptide-specific T ceil frequencies were especially high and 9 individuals had over 1000 CFPlO-specific IFN-y-secreting T cells per million PBMC. The combined frequency of IFN-y-secreting T cells specific for ESAT-6 and CFP10 derived peptides in the 80 individuals who responded to either of these 2 antigens (median = 208 per million PBMC [interquartile range: 116-357]) is higher than the frequency of IFN-y-secreting PPD-specific T cells. Thus, in healthy residents of a tuberculosis endemic area, these two antigens alone are a more potent target of IFN-y-secreting T cells ex vivo than is PPD, which contains over 200 antigens. Of the 56 healthy individuals who responded to ESAT-6-denved peptides, the majority (n=47) also responded to whole recombinant antigen, suggesting that processing and presentation of recombinant antigen occurs when it is added exogenous])' to PBMC-in ELISPOT assays Tins is consistent with our previous observations and supports the fact that the peptide-specific responses are not artefactuai. We have previously observed that M. tuberculosis-infected individuals who respond to'ESAT-6-derived peptides but not whole antigen in the ex vivo ELISPOT assay, have ESAT-6-specific CDS T cells; the lack of recognition of whole antigen is because exogenously added ESAT-6 is not be processed through the MHC class I antigen processing pathway, whereas vaccinia virus recombinant for ESAT-6 will present to CDS T cells. Thus, in the 9 individuals in whom peptide- specific responses were not accompanied by a response to exogenous.lv added antigen, the T cell response to ESAT-6 was probably CDS-mediated. Absence of ESAT-6 and CFPlO-specific T cells in healthy residents of a non-endemic area 40 healthy adult UK residents with no past history of tuberculosis were tested prospective!}- bv ESAT-6 and CFP10 peptide-based ex vivo ELISPOT assay for IFN-y. None of the donors reported any tuberculosis contact. Fig. 6B shows that no donors responded to any of the CFP10 or ESAT-6 peptides. By contrast, 33/40 subjects responded to PPD in the er vivo ELISPOT assay for IFN-y with a median frequency of 83 (interquartile range: 53 - 215) PPD-specific T cells per million PBMC which was very similar to the frequency of PPD-specific T cells seen in the healthy Indian adults. The high prevalence of PPD-specific responses in the unexposed UK residents probably reflects the fact that 33/40 were BCG-vaccinated. Epitope mapping of ESAT-6 and CFP10 in Indian tuberculosis patients and healthy adults Many Indian patients and healthy adults responded to multiple peptides and, as illustrated in Fig. 7 A & 7B, ESAT-6 contains multiple T cell epitopes which are concentrated at the ammo and carboxy termini of the molecule. These highly iirimunogenic peptides are mostly CD4 T cell epitopes because T cell lines have been generated in vitro against peptides ESAT-6M5, ESAT-66.20, ESAT-6I6_30, ESAT-62i.35, ESAT-63M5r ESAT~646.SO, ESAT-651.6i, ESAT-656.70, ESAT-666.80, ESAT-67].8i, ESAT-6-7,:,.9[l and ESAT-6S1.95. and, m each case, peptide-specific responses were abrogated by immunomagnetic depletion of CD4 T cells (data not shown). We have previously identified 8-mer and 9-merHLA class I-restricted CDS epitopes in peptides 4 and 14 but, since these are strictly restricted by HLA-A6802 and HLA-B52 respectively, only a small proportion of infected people respond. In contrast to these CDS epitopes, many of the CD4 epitopes were recognized by T cells from a relatively large percentage of the 150 ethnically diverse Indians in this study. For several of these peptides (ESAT-6M5, ESAT-66.20;, ESAT-651_65 and ESAT-671_85), we have previously used anti-HLA-DR. -DQ and -DP monoclonal antibodies to block presentation to CD4 T cells in ELISPOT assays In all tuberculosis patients and contacts where this \ was tested (n-14), the responses were MHC class Il-restricted. The pattern of ESAT- 6 epitopes to which the tuberculosis patients responded is similar to that for the healthy subjects (Pig. 7A & 7B). Analysis of the T cell response to the 18 overlapping CFPlO pepudes in the hundred residents of Mumbai has generated the first epitope map of CFPlO (Fig. 7C). Tins reveals two strikingly immunodominant domains, CFP51.70 and CFP71.90; almost 50% of healthy Indians responded to peptides from these two regions. 15-20% of Indians responded to CFP16.30 and CFP4].60, identifying these peptides as additional immune a en ic regions. Progressive decline in frequency of ESAT-6 peptide-specific IFN-y-secreting T cells during anti-tuberculosis treatment Figure 8 shows the progressive fall in the frequency of ESAT-6 peptide-specific IFN-y-secreting T cells during anti-tuberculosis treatment in a series of culture-confirmed tuberculosis cases followed up longitudinally in the UK (n=5). All patients had drug-sensitive isolates of A'f tuberculosis and responded well to therapy. Discussion We have identified ESAT-6-specrfic IFN-y-secreting T cells as an accurate marker of M. tuberculosis infection in patients with bacteriologically confirmed active disease and latently infected TST positive healthy household contacts of sputum smear positive tuberculosis cases. CFPlO shares the same species specificity as ESAT-6 and ,;s thus absent from all strains ofM. bovis BCG and the vast majority of environmental mycobactena. Here we demonstrate-that 80% of our sample of healthy Indian adults harbour circulating IFN-y-secreting T cells specific for at least one of these, two RD1 encoded antigens. ESAT-6- and CFPlO. This suggests that at least 80% of residents of Mumbai have been exposed to, or are infected with, M. tuberculosis. The striking absence of ESAT-6 and CFPlO-specific T cells in all the unexposed UK residents supports this conclusion and the fact that 33/40 of these subjects were BCG-vaccinated indicates moreover that this approach is highly specific and not confounded by BCG-vaccination. Given that all the Mumbai residents were asymptomatic.and had normal chest radiography, M. tuberculosis infection in these individuals would, by definition, be latent rather than active. Might the high prevalence of ESAT-6- and CFPlO-specific T cells in this population resuh from exposure to a cross-reactive environmental antigen unrelated to M. tuberculosis that is present in India but absent in the UK? We consider thus unlikely since sequence homology searches of the SwissProt and translated GenBank databases with each of the ESAT-6 and CFPlO-derived peptides indicate that the sequences of all these peptides are uniquely restricted to ESAT-6 and CFPlO ofM. tuberculosis complex. However, the esal-6 gene.(and therefore very likely the gene for CFPlO, which is within the same operon as esat-6) are present in 4 atypical mycobacteria: M, kansasii, szulgai, flavescens and marinimt. It is thus conceivable that the ESAT-6 and CFPlO-specific responses could result from infection with one of these organisms. To account for the striking dichotomy of ESAT-6- and CFP10-specrfic responses between India and the UK, an environmental mycobacterium would have to cause widespread infection in Mumbai but be absent in southern England. However, none of these mycobacteria fit this geographic distribution, nor are they specifically associated with tropical climates. Rather, M. kansasii is the most common non-HTY associated nontuberculous mycobacterium isolated from patients in southern England and is a recognized isolate from tap water in cities in this region. In contrast. M. kansasii disease has not been reported from Mumbai, although this may reflect under-reporting by local laboratories. The absence of RDl-encoded antigen-specific T cells in the 40 UK. residents thus makes M. kansasii exposure an unlikely cause for the difference in responses between the UK and India. M. marimim infection is usually acquired by trauma in a saltwater or freshwater setting or whilst handling fish; these scenarios would be rare among the corporate executives attending for insurance medicals at our institution in Mumbai. M. szulgai and M. flcrvescens are very rare causes of infection in humans. M. szulgai has been isolated from patients throughout the world, including the UK, but environmental isolates have not been reported. M. fla\>escens has been found in water supplies in several European countnes and thus, as withM kansasii. cannot account for the widespread sensitization to ESAT-6 and CFP10 inMumbai residents and the striking absence of sensmzation in the UK donors. The circulating IPN-y-secreting T cells specific for ESAT-6 and CFP10 were' capable of rapid effector function since they released IFN-y in the ex vrvo ELISPOT assay without requixme prior m vitro stimulation. This suggests that the donors are latently infected with tubercle bacilli that continue to secrete ESAT-6 and CFP10. since high frequencies of circulating effector T cells are probably maintained by recent encounter with antigen 777 vivo Since ongoing, active secretion of ESAT-6 and CFP 10 requires metabolically active and viable bacilli, the presence of circulating ESAT-6 and CFP10-specific effector T cells in healthy donors suggests that they are latently infected and not merely exposed at some point in die past. Nonetheless, we cannot rule out the possibility that circulating IFN-y-secreting T cells undergo repeated 277 vivo stimulation as a result of Jong term persistence of ESAT-6 and CFP 10 in dendritic cells in the absence of viable' bacilli. However longitudinal follow-up of tuberculosis patients in the UK shows that the frequency of ESAT-6 peptide-specific IFN-y-secreting CD4 T cells declines progressively during the course of anti-tuberculosis chemotherapy (Fig. 8), suggesting that the frequencies of these T cells are maintained 77? vfvo by ESAT-6 secreted by viable bacilli. TST surveys in India, have generated widely varying results, with the prevalence of TST posmvity in adults ranging from 3S - 81%. This wide variation in part reflects poor standardisation of P.PD preparations used, differences in methodology, inconsistency in reading of skin test results, rnisclassifications and the instability of delayed type hypersensitivity over time. These limitations., together with the intrinsic poor specificity of PPD. render estimates of the prevalence of latent M tuberculosis infection by TST surveys doubtful Our novel, quantitative approach has generated a more objective estimate that is not confounded by prior BCG vaccination and is unlikely to be affected by environmental roycobacterial infection. The 80% prevalence in corporate executives is higher than would have been expected on the basis of past TST surveys, especially considering that the latter have all been carried out in rural areas or urban slums. Interestingly, the prevalence of positive results by RDl encoded antigen-based ELISPOT was higher in men (64/78 = 82%) than in women (16/22 = 73%) which parallels the difference between the sexes seen in TST surveys. This estimate of the prevalence of latent infection is thus unexpectedly high for both men and women, particularly if one takes into account the relative affluence and lack of domestic overcrowding in our i-? population sample. In contrast to the'ELISPOT assay for IFN-y incorporating the two BJD1 encoded antigens; the use of PPD does not differentiate between the Mumbai-resident and UK-resident populations, with 98% and 85% of these populations, respectively, responding to PPD (Figs 6A & 6B). The 83% (33/40) prevalence of PPD-specific responses in the tuberculosis unexposed UK residents very likely results from 33/40 being BCG-vaccinated. Thus, unlike ESAT-6- and CFPlO-specific T cells, PPD-specific IFN-y-secreting T cells do not discriminate between latently infected residents of a tuberculosis endemic area and unexposed, BCG-vaccinated residents of a non-endemic region; this underscores the practical advantage of using defined antigens with a highly restricted species distribution. .Among the 50 patients with active tuberculosis who were tested with ESAT-6-derived peptides only, 80% had ESAT-6 peptide-specific T cells by ex vivo ELISPOT-. It is not clear why this response rate is lower than that found among a group of patients with active tuberculosis recruited in the UK, where 45/47 (96%) patients responded to one or more ESAT-6-derived peptides. but the following 3 factors are likely to be relevant. First, peptide ESAT-6,.,.. was an immunodominant and permissively recognized epitope in the previous UK study, with 60% of patients responding to this peptide alone, whereas in Mumbai, only 25% of patients respond to this peptide (Fig. 7A). This probably reflects the different HL A. backgrounds of the 2 populations. Although 50% of the UK tuberculosis patients were from the Indian Subcontinent, none were Maharashtrans and the remaining 50% were Afro carribeans or whites. Of the tuberculosis patients recruited in India, 56% were Maharashtrans and the remainder from other Indian ethnic groups. Thus, peptide ESAT-G,.^ might be restricted by HLA class II molecules that are rare among Maharashtrans. Second, tie median frequency of total ESAT-6-specific T cells in the Mumbai patients who responded (128 per million PBMC; interquartile range: 72 - 208) was somewhat lower than in the London pulmonary tuberculosis patients (177 per million PBMC; interquartile range: 104 - 392). Mumbai patients would therefore be more likely to have very lew numbers of circulating ESAT-6-specific T cells and if the absolute frequency of ESAT-6-specific T cells in a given patient were below the threshold of our assay (20 per million PBMC), such patients would not be detected by the ex vivo ELISPOT assay. Third, the two UK patients who did not respond had very advanced pulmonary tuberculosis with cachexia and, whereas this was rare in the UK patients, many of the Murabai patients presented at an advanced stage of disease. Interestingly, all 6 of the tuberculosis patients with HTV infection had detectable ESAT-6 peptide-specific T cells by ex vivo ELISPOT. This preliminary result is the first demonstration of ESAT-6-specific T cells in HIV-infected tuberculosis patients and suggests that the clinical and epideniiologic utility of the ex vivo ELISPOT assay for M. tuberculosis antigen-specific T cells may be maintained even in HTV-infected individuals. Finally, the frequency of ESAT-6 and CFPlO-specific T cells among healthy urban Indians, as well as the prevalence of positive responses, was also very high (Table 3). The median frequency of total CFP10 peptide-specific T cells for the CFP10 responders (158 per million PBMC; interquartile range: 84 - 261) was actually higher than the median frequency of PPD-specific T cells for the PPD responders (S6 per million PBMC; interquartile range: 56 - 222), while the median frequency of total ESAT-6 peptide-specific T cells (86 per million PBMC; interquartile range: 43-156) was similar to that for PPD. Many of these responses were moreover directed against: discrete immunodominant regions within the ESAT-6 and CFP10 molecules (Figs. 7A & 7B). Immune responses in patients with active tuberculosis probably contribute to pathogenesis and tissue destruction, as well as protection. This intense and nighly focused T cell response in residents of a tuberculosis endemic region who are free of active tuberculosis indicates that these RL1 -encoded anngen-specmc T cells are not necessarily associated with active disease per se. Rather, the presence of these T cells in latently infected healthy subjects is consistent with a protective role in the long-term control ofM tuberculosis in vivo. In conclusion, we have demonstrated that 80/100 healthy adults in Mumbai have high frequencies of T cells specific for two RD1 encoded M tuberculosis antigens, suggesting that at least 80% of this urban population may be latently infected "withM tuberculosis. This represents the first estimate of the prevalence of latent M. tuberculosis infection in an endemic region using a T cell-based approach with antigens of tightly defined species specificity. References 1. Lalvani, A., R. Brookes, S. Hambleton, WJ. Britton, A.V. Hill, and A.J. McMichael. 1997. Rapid effector function in CD8+ memory T cells. JExp Med. 186:859-65. 2. Lalvani, A,. R. Brookes, RJ. Wilkinson, A.S. Malin, A.A. Pathan, P. Andersen, H. Dockrell, G. Pasvol, and A.V. Hill. 1998. Human cytolytic and interferon gamma-secreting CDS-r T lymphocytes specific for Mycobacterium tuberculosis. ProcNailAcadSci USA. 95:270-5. 3. Pathan, A.A., K.A. Wilkinson, RJ. Wilkinson, M. Latif, H. McShane, G. Pasvol, A.V. Hill, and A. Lalvani. 2000. High frequencies of circulating 3FN- gamma-secreting CDS cytotoxic T cells specific for a novel MHC class 1-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease Eur JJmmunol. 30.2713-21 (Table Remove) Table 1 Demographic characteristics and median frequencies of ESAT-6 peptide-specific IFN-y SFQ / million PBiviC for ai! participants. "Median cf the number of ESAT-6 peptiae-specinc CI'4 T cells ^urLmacad for ail the pepudss) for all the responders within a group (C- = culture Dcsmve; C- = culture negative; PTB = puLmonary- tuberculosis; TBLN = tubercuioiis I'-Tnuhaderutis' HHC = healthy household contact; UC = unsxuosed conrral; IQ range = inter-auamle ran?e; ISC — Indian subcontinent). (Table Remove)Table 2. Most ESAT-6-derived peptides are targets of CD4 T cells: the number of donors for whom a peptide-speciiic T cell line was shown to be CD4 positive by immunomagnetic depletion is shown in proportion to the total number of donors'in whom T cell lines were tested for rhat pepride, T cells specific for certain immunodominant ESAT-6-deriyed peptides, as enumerated by cr vivo ELISPOT, were not detectable in •lymphoproliferation assays: PJ3MC from 15 subjects were tested in naralle! in er vivo ELISPOT assays for IFlN'-y and JH-Lhymidine incorporation assavs. in vvhich a stimulation index of 3 or more was taken as positive. (Table Remove) WE CLAIM: 1. An in vitro method for determining the efficacy of a compound or therapeutic agent or vaccine having anti-tuberculosis activity, comprising: (i) measuring the level of ex-vivo effector T cells specific for an antigen of M. tuberculosis in a first reference sample, the sample comprising freshly-isolated T cells from a peripheral blood sample; (ii) obtaining a second or plurality of test samples at one or more later time points after the start of treatment; (iii) measuring the level of ex-vivo effector T cells specific for said antigen of M. tuberculosis in the second or plurality of samples; and (iv) comparing the level of effector T cells in the reference sample and the second or plurality of samples; said antigen being selected from ESAT-6 or CFP10, wherein decrease in effector T cell level is indicative of efficacy of compound or therapeutic agent or vaccine and, wherein all the samples are obtained from the same source. 2. A method as claimed in claim 1, wherein the T cells are CD4 T cells. 3. A method as claimed in claim 1, wherein said reference sample and said test samples are freshly isolated peripheral blood mononuclear cells comprising T cells from peripheral blood. 4. A method as claimed in claim 1, wherein the level of effector T cells specific for said antigen is determined by contacting the sample with peptide fragments of said antigen, each of said fragments comprising a T cell epitope. 5. A method as claimed in claims 1 and 4, wherein the step of determining is performed by detection of cytokine secreted by the effector T cells specific for said antigen. 6. A method as claimed in claim 5, wherein the cytokine is detected by allowing the cytokine to bind to an immobilized antibody specific to the cytokine and then detecting the presence of the antibody/cytokine complex. 7. A method as claimed in claims 5 and 6, wherein the cytokine is detected by EL1SPOT assay. 8. A method as claimed in claims 5 to 7, wherein the cytokine is IFNγ. 9. A method for determining the efficacy of a compound or therapeutic agent or vaccine substantially such as herein described and illustrated in the accompanying specification.

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