Mycobacterial proteins from culture filtrate or cytosol are disclosed as being useful B cell antigens for early diagnosis of mycobacterial disease, particularly in humans. These proteins include four that had not previously been recognized as B cell antigens (LppZ protein encoded by Mtb gene Rv3006; SodC protein encoded by Mtb gene Rv0432; BfrB protein encoded by Mtb gene Rv3841 and TrxC protein encoded by Mtb gene Rv3914). Antigenic compositions include these proteins and/or peptide fragments thereof, in various combinations with each other or with one or more of a set of 10 additional Mtb proteins known to be antigens (in particular early antigens. Methods and kits for using these antigenic composition for early diagnosis of mycobacterial infection and disease are also disclosed.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is directed to an isolated M. tuberculosis (Mtb) protein or peptide selected from the following group: (a) LppZ protein, encoded by Mtb gene Rv3006, the amino acid sequence of which is SEQ ID NO:1 or a peptide fragment thereof that is recognized by an antibody from a subject infected with Mtb; (b) an isolated peptide fragment of Mtb protein SodC (SEQ ID NO:2) encoded by Mtb gene Rv0432, which peptide fragment is recognized by an antibody from a subject infected with Mtb; (c) BfrB protein, encoded by Mtb gene Rv3841, the amino acid sequence of which is SEQ ID NO:3, or a peptide fragment thereof that is recognized by an antibody from a subject infected with Mtb; and (d) TrxC protein, encoded by Mtb gene Rv3914, the amino acid sequence of which is SEQ ID NO:4 and is, or a peptide fragment thereof that is recognized by an antibody from a subject infected with Mtb.

Examples of preferred peptide fragments of the above four proteins are ones that comprise B cell epitopes, are about 20 amino acids in length and represent 10 amino acid overlaps with their adjacent sequences in the parent protein. Preferred peptides of the invention include: (a) an LppZ peptide the sequence of which is SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51; (b) a peptide of said SodC protein, the sequence of which is SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74; (c) a peptide of said BfrB protein the sequence of which is SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, or SEQ ID NO:92; or (d) a peptide of said TrxC protein the sequence of which is SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, or SEQ ID NO:103.

In one preferred embodiment, the peptide is a BfrB peptide selected from the group consisting of SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82 and SEQ ID NO:92.

In another preferred embodiment, the peptide is a TrxC peptide listed in (d), above.

The present invention is also directed to oligomers or multimers of a peptide as above, which are either fused directly or indirectly via peptide linker or spacer which may be a cleavable linker.

Also provided herein is an antigenic composition useful for detection, preferably early detection, of Mtb disease or infection comprising an Mtb antigen that is a protein or peptide as noted above wherein the protein/peptide includes an epitope recognized by an antibody in a subject infected with Mtb.

In one preferred embodiment, the invention is directed to antigenic composition useful for detection and profiling of Mtb disease or infection comprising two or more, or three or more Mtb antigens which antigens are selected from the above proteins or peptide fragments. The peptides of this antigenic composition may be a fragment of one or more of these proteins.

The antigenic composition of this invention may further comprises one or more additional Mtb proteins, or a peptide fragment thereof that includes an epitope recognized by an antibody in a subject infected with Mtb, the protein being selected from: (a) an isolated PstS1 protein encoded by Mtb gene Rv0934 the amino acid sequence of which is SEQ ID NO:5; (b) an isolated Apa/ModD protein encoded by Mtb gene Rv1860 the amino acid sequence of which is SEQ ID NO:6; (c) an isolated Ag85A protein encoded by Mtb gene Rv3804c the amino acid sequence of which is SEQ ID NO:7; (d) an isolated Ag85B protein encoded by Mtb gene Rv1886c the amino acid sequence of which is SEQ ID NO:8; (e) an isolated GlcB protein encoded by Mtb gene Rv1837c the amino acid sequence of which is SEQ ID NO:9; (f) an isolated protein encoded by Mtb gene Rv3881c the amino acid sequence of which is SEQ ID NO: 10; (g) an isolated SecE2 protein encoded by Mtb gene Rv0379 the amino acid sequence of which is SEQ ID NO: 11; (h) an isolated MPT64 protein encoded by Mtb gene Rv1980c the amino acid sequence of which is SEQ ID NO:12; (i) an isolated HspX protein encoded by Mtb gene Rv2031c the amino acid sequence of which is SEQ ID NO:13; and (j) an isolated LpqH protein encoded by Mtb gene Rv3763 the amino acid sequence of which is SEQ ID NO:14.

Also provided is an immunoassay composition that comprises the LppZ, SodC, BfrB or TrxC protein, a peptide thereof, or an antigenic composition as above, wherein the protein or peptide is immobilized to a solid support and preferably arranged in a format suitable for use in an ELISA or dipstick-based immunoassay.

This invention is directed to a method for the early detection of mycobacterial disease or infection in a subject, comprising assaying a biological fluid sample from a subject having symptoms of active tuberculosis, but before the onset of symptoms identifiable as advanced tuberculosis for the presence of antibodies specific for or reactive with an Mtb antigen selected from the above-noted isolated proteins, peptides or antigenic compositions.

Also included is a method for the early detection of mycobacterial disease or infection in a subject, comprising assaying a biological fluid or cell or tissue sample from a subject having symptoms of active tuberculosis, but before the onset of symptoms identifiable as advanced tuberculosis for the presence of one or more early M. tuberculosis early antigens that are present in the above-noted isolated protein or antigenic composition, using an antiserum or a monoclonal antibody specific for an epitope of the early antigen, wherein the presence of the one or more early antigens is indicative of the presence of the disease or infection.

A related method comprises assaying a biological fluid sample from a subject having symptoms of active tuberculosis, but before the onset of symptoms identifiable as advanced tuberculosis for the presence of immune complexes consisting of one or more early Mtb antigens complexed with an antibody specific for the antigen, which antigen is a protein or peptide as described above.

The above method is preferably an EIA/ELISA or a dipstick-based immunoassay.

The method may further include performance of a test that detects mycobacterial bacilli in a sample of sputum or other body fluid of the subject.

The biological fluid or body fluid used in the method is preferably is serum, urine or saliva.

The above method may further comprise, prior to the assaying step, the step of removing, from the sample, antibodies specific for cross-reactive epitopes or antigens of proteins present in M. tuberculosis and in other bacterial genera, preferably by immunoadsorption of the sample with E. coli antigens.

In the above methods, the subject is preferably a human, and the method is particularly useful for HIV-1.sup.+ subjects and/or those is at high risk for tuberculosis.

The invention includes a kit useful for early detection of Mtb disease or infection as above, the kit comprising:

(a) an early Mtb antigen or antigens in the form of the isolated protein or peptide noted above in combination with

(b) reagents necessary for detection of antibodies which bind to the M. tuberculosis protein or peptide; and

(c) optionally, a set of instructions for carrying out the detection.

In the above kit and method, one or more of the Mtb proteins or peptide antigens is a recombinant protein, glycoprotein, lipoprotein, or a recombinant or synthetic epitope-bearing peptide.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Due to the absence of a complete Mtb open reading frame library, the present inventors produced a first generation Mtb protein microarray based on native proteins. A multi-dimensional separation strategy was devised to efficiently resolve native proteins found in the cytosol (CytPs) and culture filtrate (CFPs) of Mtb. This resulted in 960 relatively simple protein fractions from two highly complex protein pools. These fractions were spotted to nitrocellulose slides and probed with sera from purified-protein-derivative positive (PPD.sup.+) healthy controls, cavitary-TB, noncavitary-TB, and HIV and Mtb co-infected patients. The resulting analyses corroborated the present inventors' earlier two-dimensional (2-D) immunoblot based experiments, confirming that a subset of antigens is recognized early in TB disease progression. Furthermore, four proteins specific for cavitary-TB patients were identified, and four novel antigens previously undetected by other methods were defined as serodiagnostic targets.

In the following description, reference will be made to various methodologies known to those of skill in the art of immunology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of immunology as well as details of mAb production and characterization, and immunoassay procedures, include: Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) and Harlow, E. et al., Using Antibodies: A Laboratory Manual Portable Protocol NO. I, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); A. K. Abbas et al., Cellular and Molecular Immunology (Fourth Ed.), W.B. Saunders Co., Philadelphia, 2000; C. A. Janeway et al., Immunobiology. The Immune System in Health and Disease, Fourth ed., Garland Publishing Co., New York, 1999; Roitt, I. et al., eds, Immunology, C. V. Mosby Co., St. Louis, Mo. (2001); Klein, J., Immunology, 2.sup.nd edition, Blackwell Scientific Publications, Inc., Cambridge, Mass., (1997); Roitt, I et al., eds., Roitt's Essential Immunology, Blackwell Scientific Publications, Oxford (2001);

Immunoassay methods are also described in Coligan, J. E. et al., eds., Current Protocols in Immunology, Sec. 2.4.1, Wiley-Interscience, New York, 1992 (or current edition); Bizollon, Ch. A., ed., Monoclonal Antibodies and New Trends in Immunoassays, Elsevier, N.Y., 1984; Butler, J. E., ELISA (Chapter 29), In: van Oss, C. J. et al., (eds), IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J E, The Behavior of Antigens and Antibodies Immobilized on a Solid Phase (Chapter 11) In: STRUCTURE OF ANTIGENS, Vol. 1 (Van Regenmortel, M., CRC Press, Boca Raton 1992, pp. 209-259; Butler, J E (ed.), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991.

As used herein, definitions of the terms "early" and "late" in reference to (1) Mtb infection or disease, or the subject having the infection or disease, (2) the antibody response to an Mtb antigen, (3) an Mtb antigen itself or (4) a diagnostic assay, are based on terms of the stage of development of TB. Early and late (or advanced) TB are defined in the table below.

Thus, a subject with early TB is asymptomatic or, more typically, has one or more "constitutional symptoms" (e.g., fever, cough, weight loss). In early TB, Mtb bacilli are too few to be detectable microscopically as acid-fast bacilli in smears of sputum or another body fluids, primarily those fluids associated with the lungs (such as bronchial washings, bronchoalveolar lavage, pleural effusion). However, in these subjects, Mtb bacilli are present and culturable, which means that they can be grown in culture from the above body fluids. Finally, early TB subjects may have radiographically evident pulmonary lesions which may include infiltration but are without cavitation. Any antibody that arises by, and is detectable during, such early stages is termed an "early antibody" and any Mtb antigen recognized by such antibodies is termed an "early antigen." The fact that an antibody is characterized as "early" does not mean that this antibody is absent in advanced TB. Rather, such antibodies are expected to persist across the progression of early TB to the advanced stage.

Accordingly, the term "late" or "advanced" is characterized in that the subject has frank clinical disease and more advanced cavitary lesions in the lungs. In late TB, Mtb bacilli are not only culturable from smears of sputum and/or the other body fluids noted above, but also present in sufficient numbers to be detectable as acid-fast bacilli in smears of these fluids. Again, "late TB" or "late mycobacterial disease of infection" is used interchangeably with "advanced TB" or "advanced mycobacterial disease or infection." An antibody that first appears after the onset of diagnostic clinical and other characterizing symptoms (including cavity pulmonary lesions) is a late antibody, and an antigen recognized by such a late antibody (but not by an early antibody) would be termed a late antigen.

To be useful in accordance with this invention, an early diagnostic assay must permit rapid diagnosis of Mtb disease at a stage earlier than that which could have been diagnosed by conventional clinical diagnostic methods, namely, by radiologic examination and bacterial smear and culture or by other laboratory methods available prior to this invention. (Culture positivity is the final confirmatory test but takes two weeks and more.)

An objective of the invention is to define, obtain and characterize the antigens of Mtb expressed by the bacterium in vivo during early infection and TB. These antigens are evaluated for their utility as markers of early disease that may be used to monitor suspected or high-risk individuals to identify those with active, subclinical infection and initiate treatment earlier than would have been done in the absence of such a diagnosis.

Mycobacterial Antigen Compositions

The preferred mycobacterial antigenic composition is a substantially isolated, purified or recombinantly produced preparation of one or more of the Mtb proteins described herein, or a peptide fragment thereof. Alternatively, the antigen composition may be a substantially pure preparation containing one or more Mtb antigens, in the form of the epitope-bearing protein or epitope-bearing peptides which are capable of being recognized by, and bound by, antibodies of an Mtb infected subject. Such epitopes may be in the form of peptide fragments of these proteins or other "functional derivatives" of Mtb proteins as described below.

By "functional derivative" is meant a "fragment," "variant," "analogue," or "chemical derivative" of an early antigen protein, which terms are defined below. A functional derivative retains at least a portion of the function of the protein which permits its utility in accordance with the present invention--primarily the capacity to bind to (which is the equivalent of "be bound by") an early antibody. A "fragment" refers to any subset of the molecule, that is, a shorter peptide. A "variant" refers to a molecule substantially similar to either the entire protein or the peptide fragment thereof. A variant peptide may be conveniently prepared by direct chemical synthesis or by recombinant means. An "analogue" of the protein or peptide refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof. A "chemical derivative" of the antigenic protein or peptide contains additional chemical moieties not normally part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

Several proteins, glycoproteins and lipoproteins in culture filtrates of Mtb organisms, cytosol of Mtb or on the surface of Mtb organisms are the preferred sources of early Mtb antigens. The secreted proteins may also be present in cellular preparations of the bacilli. Thus, these early antigens are not intended to be limited to the secreted protein form. The proteins are characterized at various places below.

The present inventors are the first to have discovered, isolated and used three Mtb proteins LppZ, BfrB and TrxC that were heretofore known only via annotations of the genomic map of Mtb or by gene expression studies. This invention is the first disclosure of these proteins and their peptide fragments as B cell antigens in humans. The inventors are the first to have recognized the utility of a fourth Mtb protein, SodC as a B cell antigen and to consider and delineate peptides thereof for similar uses.

Peptide fragments of the above proteins that comprise an antigenic epitope recognized by an antibody in a subject with TB, preferably early TB, but also including cavitary or late TB, are included herein. Preferred peptides are about 20 residues in length, though they can be as short as 7 residues, or whatever length is required to form a serologically detectable epitope in any type of immunoassay, preferably in ELISA. The peptides listed below are 20-mers, and those derived from a given protein are related to one another with 10 residue overlaps as shown in Tables 1-4 (see Original Patent). For utility in the detection of TB, a peptide should be recognized and bound by an anti-Mtb antibody present in subject infected with Mtb organisms.

In general, preferred diagnostic epitopes and antigens are those recognized by antibodies (or by T cell, preferably Th1 cells) of "early" TB patients as defined above. This does not exclude the possibility that such epitopes are bound by antibodies or recognized by T cells present later in the infectious process. As noted herein, certain of the proteins/peptides are recognized by antibodies arising later in the infectious process, e.g., in cavitary TB, so that the antigens would not be considered "early antigens." In fact, seroreactivity with some of the proteins or peptides or epitopes thereof may detect infection in subjects whose infectious state is not detected by antibodies against the four novel early antigens or additional antigenic compositions disclosed herein.

Table 5 (see Original Patent) presents a list of additional Mtb protein antigens that have been discovered (either as part of this invention or earlier) to be early Mtb antigens. Thus, certain embodiments of the present invention include one or more of these proteins, or an-epitope-bearing peptide fragment thereof in a composition or method.

Although only a subset of possible peptides of the four Mtb proteins antigens described herein are listed in Tables 1-4, the present invention is intended to include any peptide that comprises a B cell epitope that is recognized by an anti-Mtb antibody, such as an antibody in a TB patient, and preferably, an early antibody. One preferred type of peptide combines two of the "adjacent" 20-mers noted in Tables 1-4, while removing one "repeat" of the overlapping sequence. For clarification purposes but not limitation, an example of this approach follows:

Such a peptide combines SEQ ID NO: 75 and 76 of the BfrB protein -- see Original Patent.

(the overlapping portion is underscored) The new "combined" peptide is a 30 mer that combines the N-terminal half of SEQ ID NO:75 with the 10 residue sequence common to both, and the C-terminal half of SEQ ID NO:76, yielding -- see Original Patent.

Such combining can be done along the entire length of the antigenic Mtb proteins and new 30 mer peptides created in this way. Such peptides may include the same epitope that was included in the two initial 20-mers, or may include an additional or different epitope that is useful for serodiagnosis, making the longer peptide useful as a composition and in the methods disclosed herein. Furthermore, there is no reason the combined peptide needs to be limited to 30 residues. If fact, it would require only routine testing to produce all possible peptides and polypeptides derived from the sequences of the proteins of the present invention that large are enough to be antigenic and assess them in studies using, for example, individual patient sera or serum pools (see Example VII).

Also included is an antigenic composition as above which is: (a) A peptide multimer having the formula P.sup.1.sub.n wherein P.sup.1 is any Mtb antigenic peptides as described herein (or a conservative substitution variant thereof), and n=2-8. P.sup.1 may represent different peptides, so that the multimer does not simply comprise repeats of the same peptide. (b) A peptide multimer having the formula (P.sup.1-X.sub.m).sub.n-P.sup.2 wherein P.sup.1 and P.sup.2 are any antigenic Mtb peptide as described herein (or conservative substitution variants thereof), and wherein (i) P.sup.1 and P.sup.2 may be the same or different and each occurrence of P.sup.1 in the P.sup.1-X.sub.m structure may be a different peptide (or variant) from its adjacent neighbor; and (ii) X is an organic linker, including an amino acid linker, for example: C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkenyl, C.sub.1-C.sub.5 alkynyl, C.sub.1-C.sub.5 polyether containing up to 4 oxygen atoms, wherein m=0 or 1 and n=1-7; or and wherein the peptide multimer reacts with an antibody specific for the LppZ, SodC, BfrB or TrxC protein.

The invention is also directed to an antigenic composition as above which is a recombinant peptide multimer having the formula: (P.sup.1-Gly.sub.z).sub.n-P.sup.2 wherein P.sup.1 and P.sup.2 are any of the above peptides (or conservative substitution variants thereof), and wherein (a) P.sup.1 and P.sup.2 may be the same or different and each occurrence of P.sup.1 in the P.sup.1-Gly.sub.z structure may be a different peptide or variant from its adjacent neighbor; (b) n=1-100 and z=0-6, and wherein the peptide multimer reacts with an antibody specific for the LppZ, SodC, BfrB or TrxC protein.

The preferred immunoassays of the present invention are enzyme immunoassays (EIA) such as ELISA, or dipstick-based immunoassays, both types of which are well-known in the art. An assay typically comprises incubating a biological fluid, preferably serum or urine, from a subject suspected of having TB, in the presence of an Mtb antigen-containing composition or reagent which includes one or more Mtb early antigens as described herein and detecting the binding of antibodies in the sample to the mycobacterial antigen(s). By the term "biological fluid" is intended any fluid derived from the body of a normal or diseased subject which may contain antibodies, such as blood, serum, plasma, lymph, urine, saliva, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, pleural fluid, bile, ascites fluid, pus and the like. Also included within the meaning of this term as used herein is a tissue extract, or the culture fluid in which cells or tissue from the subject have been incubated.

In a preferred embodiment, the Mtb antigen composition is brought in contact with, and allowed to bind to, a solid support or carrier, such as nitrocellulose or polystyrene or any other of a myriad of solid supports known in the art (see below), allowing the antigens to adsorb and become immobilized to the solid support. This immobilized antigen is then allowed to interact with the biological fluid sample which is being tested for the presence of anti-Mtb antibodies, such that any antibodies in the sample will bind to the immobilized antigen. The support to which the antibody is now bound may then be washed with suitable buffers after which a detectably labeled binding partner for the antibody is introduced. The binding partner binds to the immobilized antibody. Detection of the label is a measure of the immobilized antibody.

A preferred binding partner for this assay is an anti-immunoglobulin antibody ("second antibody") produced in a different species. Thus to detect a human antibody, a detectably labeled goat anti-human immunoglobulin "second" antibody may be used. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support may then be detected by conventional means appropriate to the type of label used (see below).

Such a "second antibody" may be specific for epitopes characteristic of a particular human immunoglobulin isotype, for example IgM, IgG.sub.1, IgG.sub.2a, IgA and the like, thus permitting identification of the isotype or isotypes of antibodies in the sample which are specific for the Mtb antigen. Alternatively, the second antibody may be specific for an idiotype of the ant-Mtb antibody of the sample.

As alternative binding partners for detection of the sample antibody, other known binding partners for human immunoglobulins may be used. Examples are the staphylococcal immunoglobulin binding proteins, the best know of which is protein A. Also intended is staphylococcal protein G, or a recombinant fusion protein between protein A and protein G. Protein G of group G and group C streptococci binds to the Fc portion of Ig molecules as well as to IgG Fab fragment at the V.sub.H3 domain. Protein C of Peptococcus magnus binds to the Fab region of the immunoglobulin molecule. Any other microbial immunoglobulin binding proteins, for example from Streptococci, are also intended (for example, Langone, J. J., Adv. Immunol 32:157 (1982)).

In another embodiment of this invention, a biological fluid suspected of containing antibodies specific for a Mtb antigen may be brought into contact with a solid support or carrier which is capable of immobilizing soluble proteins. The support may then be washed with suitable buffers followed by treatment with a Mtb antigen reagent, which may be detectably labeled. Bound antigen is then measured by measuring the immobilized detectable label. If the Mtb antigen reagent is not directly detectably labeled, a second reagent comprising a detectably labeled binding partner for the Mtb antigen, generally a second anti-Mtb antibody such as a murine mAb, is allowed to bind to any immobilized antigen. The solid phase support may then be washed with buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means.

By "solid phase support" or carrier is intended any support capable of binding a proteinaceous antigen or antibody molecules or other binding partners according to the present invention. Well-known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, polyvinylidene difluoride, dextran, nylon, magnetic beads, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as it is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads, 96-well polystyrene microplates and test strips, all well-known in the art. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

Using any of the assays described herein, those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Furthermore, other steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

As noted, a preferred type of immunoassay to detect an antibody specific for a Mtb antigen according to the present invention is an enzyme-linked immunosorbent assay (ELISA) or more generically termed an enzyme immunoassay (EIA). In such assays, a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme will react in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label the reagents useful in the present invention include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, .beta.-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, .DELTA.-5-steroid isomerase, yeast alcohol dehydrogenase, .alpha.-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. For descriptions of EIA procedures, see reference cited above or, additionally, Voller, A. et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980.

In another embodiment, the detectable label may be a radiolabel, and the assay termed a radioimmunoassay (RIA), as is well known in the art. The radioisotope can be detected by a gamma counter, a scintillation counter or by autoradiography. Isotopes which are particularly useful for the purpose of the present invention are .sup.125I, .sup.131I, .sup.35S, .sup.3H and .sup.14C.

It is also possible to label the antigen or antibody reagents with a fluorophore. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence of the fluorophore. Among the most commonly used fluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine or fluorescence-emitting metals such as .sup.152Eu or other lanthanides. These metals are attached to antibodies using metal chelators.

The antigen or antibody reagents useful in the present invention also can be detectably labeled by coupling to a chemiluminescent compound. The presence of a chemiluminescent-tagged antibody or antigen is then determined by detecting the luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound such as a bioluminescent protein may be used to label the antigen or antibody reagent useful in the present invention. Binding is measured by detecting the luminescence. Useful bioluminescent compounds include luciferin, luciferase and aequorin.

Detection of the detectably labeled reagent according to the present invention may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorophore. In the case of an enzyme label, the detection is accomplished by colorimetry to measure the colored product produced by conversion of a chromogenic substrate by the enzyme. Detection may also be accomplished by visual comparison of the colored product of the enzymatic reaction in comparison with appropriate standards or controls.

The immunoassay of this invention may be a "two-site" or "sandwich" assay. The fluid containing the antibody being assayed is allowed to contact a solid support. After addition of the Mtb antigen(s), a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody. Sandwich assays are described by Wide, Radioimmune Assay Method, Kirkham et al, Eds., E. & S. Livingstone, Edinburgh, 1970, pp 199-206.

Alternatives assays are various types of agglutination assays, both direct and indirect, which are well known in the art. In these assays, the agglutination of particles containing the antigen (either naturally or by chemical coupling) indicates the presence or absence of the corresponding antibody. Any of a variety of particles, including latex, charcoal, kaolinite, or bentonite, as well as microbial cells or red blood cells, may be used as agglutinable carriers (Mochida, U.S. Pat. No. 4,308,026; Gupta et al, J. Immunol. Meth. 80:177-187 (1985); Castelan et al., J. Clin. Pathol. 21:638 (1968); Molinaro, U.S. Pat. No. 4,130,634). Traditional particle agglutination or hemagglutination assays are generally faster, but much less sensitive than RIA or EIA. However, agglutination assays have advantages under field conditions and in less developed countries.

In addition to detection of antibodies, the present invention provides methods to detect and enumerate cells secreting an antibody specific for a Mtb antigen. Thus, for example, any of a number of plaque or spot assays may be used wherein a sample containing lymphocytes, such as peripheral blood lymphocytes, is mixed with a reagent containing the antigen of interest. As the antibody secreting cells of the sample secrete their antibodies, the antibodies react with the antigen, and the reaction is visualized in such a way that the number of antibody secreting cells (or plaque forming cells) may be determined. The antigen may be coupled to indicator particles, such as erythrocytes, preferably sheep erythrocytes, arranged in a layer. As antibodies are secreted from a single cell, they attach to the surrounding antigen-bearing erythrocytes. By adding complement components, lysis of the erythrocytes to which the antibodies have attached is achieved, resulting in a "hole" or "plaque" in the erythrocyte layer. Each plaque corresponds to a single antibody-secreting cell. In a different embodiment, the sample containing antibody-secreting cells is added to a surface coated with an antigen-bearing reagent, for example, a Mtb antigen alone or conjugated to bovine serum albumin, attached to polystyrene. After the cells are allowed to secrete the antibody which binds to the immobilized antigen, the cells are gently washed away. The presence of a colored "spot" of bound antibody, surrounding the site where the cell had been, can be revealed using modified EIA or other staining methods well-known in the art. (See, for example, Sedgwick, J. D. et al., J. Immunol. Meth. 57:301-309 (1983); Czerkinsky, C. C. et al., J. Immunol. Meth. 65:109-121 (1983); Logtenberg, T. et al., Immunol. Lett. 9:343-347 (1985); Walker, A. G. et al., J. Immunol. Meth. 104:281-283 (1987).

The present invention is also directed to a kit or reagent system useful for practicing the methods described herein. Such a kit will contain a reagent combination comprising the essential elements required to conduct an assay according to the disclosed methods. The reagent system is presented in a commercially packaged form, as a composition or admixture (where the compatibility of the reagents allow), in a test device configuration, or more typically as a test kit. A test kit is a packaged combination of one or more containers, devices, or the like holding the necessary reagents, and usually including written instructions for the performance of assays. The kit may include containers to hold the materials during storage, use or both. The kit of the present invention may include any configurations and compositions for performing the various assay formats described herein.

For example, a kit for determining the presence of anti-Mtb early antibodies or antibodies to early or even later stage Mtb antigens may contain one or more of the proteins disclosed herein or another early Mtb antigen either in immobilizable form or already immobilized to a solid support, and a detectably labeled binding partner capable of recognizing the sample anti-Mtb antibody to be detected, for example. a labeled anti-human Ig or anti-human Fab antibody. A kit for determining the presence of an Mtb antigen may contain an immobilizable or immobilized "capture" antibody which reacts with one epitope of the target Mtb antigen, and a detectably labeled second ("detection") antibody which reacts with a different epitope of the Mtb antigen than that recognized by the (capture) antibody. Any conventional tag or detectable label may be part of the kit, such as a radioisotope, an enzyme, a chromophore or a fluorophore. The kit may also contain a reagent capable of precipitating immune complexes.

A kit according to the present invention can additionally include ancillary chemicals such as the buffers and components of the solution in which binding of antigen and antibody takes place.

The present invention permits isolation of an Mtb early antigen which is then used to produce one or more epitope-specific mAbs, preferably in mice. Screening of these putative early Mtb-specific mAbs is done using known patient sera which have been characterized for their reactivity with the early antigen of interest. The murine mAbs produced in this way are then employed in a highly sensitive epitope-specific competition immunoassay for early detection of TB. Thus, a patient sample is tested for the presence of antibody specific for an early epitope of Mtb by its ability to compete with a known mAb for binding to a purified early antigen. For such an assay, the Mtb preparation may be less than pure because, under the competitive assay conditions, the mAb provides the requisite specificity for detection of patient antibodies to the epitope of choice (for which the mAb is specific).

In addition to the detection of early Mtb antigens or early antibodies, the present invention provides a method to detect immune complexes containing early Mtb antigens in a subject using an EIA as described above. Circulating immune complexes have been suggested to be of diagnostic value in TB. (See, for example, Mehta, P. K. et al, 1989, Med. Microbiol. Immunol. 178:229-233; Radhakrishnan, V. V. et al., 1992, J. Med. Microbiol. 36:128-131). Methods for detection of immune complexes are well-known in the art. Complexes may be dissociated under acid conditions and the resultant antigens and antibodies detected by immunoassay. See, for example, Bollinger, R. C. et al, 1992, J. Infec. Dis. 165:913-916. Immune complexes may be precipitated for direct analysis or for dissociation using known methods such as polyethylene glycol precipitation.

Purified Mtb early antigens as described herein are preferably produced using recombinant methods. See the Examples. Conventional bacterial expression systems include Gram negative bacteria such as E. coli or Salmonella species. However, such systems are not believed to be ideally suited for production of Mtb antigens (Burlein, J E, In: Tuberculosis: Pathogenesis, Protection and Control, B. Bloom, ed., Amer. Soc. Microbiol., Washington, D.C., 1994, pp. 239-252). Rather, it is preferred to utilize homologous mycobacterial hosts for recombinant production of early Mtb antigenic proteins or glycoproteins. Methods for such manipulation and gene expression are provided in Burlein, supra. Expression in mycobacterial hosts, in particular M. bovis (strain BCG) or M. smegmatis are well-known in the art. Two examples, one of mycobacterial genes (Rouse, D A et al., 1996, Mol. Microbiol. 22:583-592) and the other of non mycobacterial genes, such as HIV-1 genes (Winter, N et al., 1992, Vaccines 92, Cold Spring Harbor Press, pp. 373-378) expressed in mycobacterial hosts are examples.

Urine-Based Antibody Assay

The present invention also provides a urine based diagnostic method for TB that can be used either as a stand-alone test, or as an adjunct to the serodiagnostic methods described herein. Such a method enables the practitioner to (1) determine the presence of anti-Mtb antibodies in urine from TB patients with early disease (non-cavitary, smear negative TB patients) and from HIV-infected TB patients; (2) determine the profile of specific Mtb antigens, such as those in the culture filtrate, that are consistently and strongly reactive with the urine antibodies; and (3) obtain the antigens that are recognized by the urine antibodies.

Smear positive (=late) cases constitute only about 50% of the TB cases, and patients with relatively early disease are generally defined as being smear negative. Moreover, as the HIV-epidemic spreads in developing countries, the numbers and proportions of HIV-infected TB patients increases.

Serum and urine samples from non-cavitary and/or smear negative, culture positive TB patients and from HIV-infected TB patients are obtained Cohorts comprising PPD-positive and PPD-negative healthy individuals, non-tuberculous HIV-infected individuals, or close contacts of TB patients can serve as negative controls.

The reactivity of the serum samples with culture filtrate proteins of Mtb, and the purified antigens (as described herein) is preferably determined by ELISA as described herein. All sera are preferably depleted of cross-reactive antibodies prior to use in ELISA.

The following description is of a preferred assay method and approach, and is not intended to be limiting to the particular steps (or their sequence), conditions, reagents and amounts of materials.

Briefly, 200 .mu.l of E. coli lysates (suspended at 500 .mu.g/ml) are coated onto wells of ELISA plates (Immulon 2, Dynex, Chantilly, Va.) and the wells are blocked with 5% bovine serum albumin (BSA). The serum samples (diluted 1:10 in PBS-Tween-20) are exposed to 8 cycles of absorption against the E. coli lysates. The adsorbed sera are then used in the ELISA assays.

Fifty .mu.l of the individual antigens, suspended at 2 .mu.g/ml in coating buffer (except for the total culture filtrate proteins which is used at 5 .mu.g/ml), are allowed to bind overnight to wells of ELISA plates. After 3 washes with PBS (phosphate buffered saline), the wells are blocked with 7.5% FBS (fetal bovine serum, Hyclone, Logan, Utah.) and 2.5% BSA in PBS for 2.5 hr at 37.degree. C. Fifty .mu.l of each serum sample are added per well at predetermined optimal dilutions (e.g., dilutions of about 1:50-1:200). The antigen-antibody binding is allowed to proceed for 90 min at 37.degree. C. The plates are washed 6 times with PBS-Tween 20 (0.05%) and 50 .mu.l/well of alkaline phosphatase-conjugated goat anti-human IgG (Zymed, Calif.), diluted 1:2000 in PBS/Tween 20 is added. After 60 min the plates are washed 6 times with Tris buffered saline (50 mM Tris, 150 mM NaCl) and the Gibco BRL Amplification System (Life Technologies, Gaithersburg, Md.) used for development of color. The absorbance is read at 490 nm after stopping the reaction with 50 .mu.l of 0.3M H.sub.2SO.sub.4. The cutoff in all ELISA assays is determined by using mean absorbance (.dbd.Optical Density O.D.)+3 standard deviations (SD) of the negative control group comprising PPD positive and PPD negative healthy individuals.

The reactivity of the urine samples with the various antigens is determined initially with undiluted urine samples as described above. For the urine ELISA, results obtained by the present inventors showed that the optimal concentration of the culture filtrate protein preparation is about 125 .mu.l/well of 4 .mu.g/ml suspension, and for certain proteins, 125 .mu.l/well of about 2 .mu.g/ml. Also, the urine is left overnight in the antigen coated wells. However, if urine antibody titers of smear-negative and HIV-infected patients are lower than those observed in smear positive patients, it may be necessary to first concentrate the urine samples. For concentration, Amicon concentrators with a molecular weight cut off of 30 kDa is preferred. Concentrated urine samples are evaluated for the presence of antibodies to the above mentioned antigens. Optimal conditions for these assays are determined readily. The sensitivity and specificity of antibody detection by use of one or more of the antigens, with both urine and serum samples is also readily determined.

Claim 1 of 33 Claims

1. A peptide of a Mycobacteria tuberculosis (Mtb) protein selected from the group consisting of: (a) an isolated peptide that is a fragment of Mtb SodC protein (SEQ ID NO:2) encoded by Mtb gene Rv0432, which peptide is recognized by an antibody from a subject infected with Mtb, the sequence of which peptide is SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74; (b) an isolated peptide that is a fragment of Mtb BfrB protein (SEQ ID NO:3), which peptide is recognized by an antibody from a subject infected with Mtb, the sequence of which peptide is SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, or SEQ ID NO:92; and (c) an isolated peptide that is a fragment of Mtb TrxC protein (SEQ ID NO:4), which peptide is recognized by an antibody from a subject infected with Mtb, the sequence of which peptide is SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, or SEQ ID NO:103.
 

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